Dragoj, Miodrag

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Authority KeyName Variants
orcid::0000-0001-6214-9220
  • Dragoj, Miodrag (47)
Projects
Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200007 (University of Belgrade, Institute for Biological Research 'Siniša Stanković') Identification of predictive molecular markers for cancer progression, response to therapy and disease outcome
TargetedResponse - Functional diagnostics in non-small cell lung carcinoma: A new concept for the improvement of personalized therapy in Serbian patients Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200042 (University of Belgrade, Institute of Molecular Genetics and Genetic Engineering)
Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200135 (University of Belgrade, Faculty of Technology and Metallurgy) Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200168 (University of Belgrade, Faculty of Chemistry)
Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200287 (Innovation Center of the Faculty of Technology and Metallurgy) COST Action CM1106 Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells
COST Action CM1106 (Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells) COST Action CM1407 Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery
COST Action CM1407 (Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery) COST Action CM1407 - Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery
COST Actions CM1106 - Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells Donnell Foundation 21st Century Science Initiative in Mathematical and Complex Systems Approaches for Brain Cancer (220020560)
ERA.Net RUS plus joint program grant RUS_ST2017-309 (“THIOREDIN”) ERA.Net RUS plus joint program (THIOREDIN)
European Commission (grant 952033) Signaling molecules in diabetes: search for potential targets in intrinsic pathways for prediction and intervention in diabetes
Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200026 (University of Belgrade, Institute of Chemistry, Technology and Metallurgy - IChTM) James S. Mc. Donnell Foundation (USA) 21st Century Science Initiative in Mathematical and Complex Systems Approaches for Brain Cancer (Collaborative award 220020560, doi:10.37717/220020560)
Ministerio de Ciencia e Innovacion and FEDER funds, Spain (grant number PID2019110895RB-I00, doi: 10.13039/501100011033 to VMP-G, and RTI2018093596 and PI21CIII/00002 to PS-G) STSM grant awarded by STRATAGEM COST Action CA17104 “New diagnostic and therapeutic tools against multidrug resistant tumours”.
The European Commission (grant 952033) Universidad de Castilla-La Mancha (grant number 2020-PREDUCLM-15634 to JJ-S)

Author's Bibliography

Long-term three-dimensional glioblastoma cell culture model for drug response studies

Dragoj, Miodrag

(Belgrade: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, 2023)

TY  - CONF
AU  - Dragoj, Miodrag
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6700
AB  - Introduction: Glioblastoma, although not the most common, is one of the deadliest human cancers.
Despite the implementation of the Stupp protocol in clinical practice, which prolongs patients’ survival
for only 2.5 months, there have been no significant advances in glioblastoma treatment. Therefore, it is
imperative to better understand the mechanisms behind glioblastoma behavior for more efficient treat-
ment in the future. Conventional two-dimensional cell cultures provide an affordable and easy-to-
perform in vitro system but fail to recapitulate glioblastoma’s complex tumor structures and
microenvironmental conditions, which often results in a lack of translation to clinical settings. In con-
trast, three-dimensional (3D) cancer models can advance the understanding of cancer biology and have
the potential to revolutionize the development of new drugs and predict their clinical efficacy.
Methods: We have developed a novel long-term 3D glioblastoma model with potential applications in
preclinical studies. Using alginate fibers, this model allows the cultivation of U87 glioblastoma cells for
extended periods, lasting up to 28 days, which corresponds to a clinically relevant treatment cycle.
Results: This model was used to validate hypothesized optimal temozolomide scheduling for glioblas-
tomas generated via mathematical modeling. The results indicated that increasing the time spacing be-
tween doses of TMZ may reduce toxicity, delay the development of drug resistance, and potentially
improve survival outcomes.
Conclusion: In conclusion, the establishment and utilization of advanced 3D glioblastoma models offer
significant opportunities to advance our understanding of glioblastoma biology and improve treatment
outcomes.
PB  - Belgrade: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade
C3  - Abstract Book: CoMBoS2 - the Second Congress of Molecular Biologists of Serbia; 2023 Oct 6-8; Belgrade, Serbia
T1  - Long-term three-dimensional glioblastoma cell culture model for drug response studies
SP  - 32
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6700
ER  - 
@conference{
author = "Dragoj, Miodrag",
year = "2023",
abstract = "Introduction: Glioblastoma, although not the most common, is one of the deadliest human cancers.
Despite the implementation of the Stupp protocol in clinical practice, which prolongs patients’ survival
for only 2.5 months, there have been no significant advances in glioblastoma treatment. Therefore, it is
imperative to better understand the mechanisms behind glioblastoma behavior for more efficient treat-
ment in the future. Conventional two-dimensional cell cultures provide an affordable and easy-to-
perform in vitro system but fail to recapitulate glioblastoma’s complex tumor structures and
microenvironmental conditions, which often results in a lack of translation to clinical settings. In con-
trast, three-dimensional (3D) cancer models can advance the understanding of cancer biology and have
the potential to revolutionize the development of new drugs and predict their clinical efficacy.
Methods: We have developed a novel long-term 3D glioblastoma model with potential applications in
preclinical studies. Using alginate fibers, this model allows the cultivation of U87 glioblastoma cells for
extended periods, lasting up to 28 days, which corresponds to a clinically relevant treatment cycle.
Results: This model was used to validate hypothesized optimal temozolomide scheduling for glioblas-
tomas generated via mathematical modeling. The results indicated that increasing the time spacing be-
tween doses of TMZ may reduce toxicity, delay the development of drug resistance, and potentially
improve survival outcomes.
Conclusion: In conclusion, the establishment and utilization of advanced 3D glioblastoma models offer
significant opportunities to advance our understanding of glioblastoma biology and improve treatment
outcomes.",
publisher = "Belgrade: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade",
journal = "Abstract Book: CoMBoS2 - the Second Congress of Molecular Biologists of Serbia; 2023 Oct 6-8; Belgrade, Serbia",
title = "Long-term three-dimensional glioblastoma cell culture model for drug response studies",
pages = "32",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6700"
}
Dragoj, M.. (2023). Long-term three-dimensional glioblastoma cell culture model for drug response studies. in Abstract Book: CoMBoS2 - the Second Congress of Molecular Biologists of Serbia; 2023 Oct 6-8; Belgrade, Serbia
Belgrade: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade., 32.
https://hdl.handle.net/21.15107/rcub_ibiss_6700
Dragoj M. Long-term three-dimensional glioblastoma cell culture model for drug response studies. in Abstract Book: CoMBoS2 - the Second Congress of Molecular Biologists of Serbia; 2023 Oct 6-8; Belgrade, Serbia. 2023;:32.
https://hdl.handle.net/21.15107/rcub_ibiss_6700 .
Dragoj, Miodrag, "Long-term three-dimensional glioblastoma cell culture model for drug response studies" in Abstract Book: CoMBoS2 - the Second Congress of Molecular Biologists of Serbia; 2023 Oct 6-8; Belgrade, Serbia (2023):32,
https://hdl.handle.net/21.15107/rcub_ibiss_6700 .

Immunofluorescence-Based Assay for High-Throughput Analysis of Multidrug Resistance Markers in Non-Small Cell Lung Carcinoma Patient-Derived Cells

Dinić, Jelena; Podolski-Renić, Ana; Dragoj, Miodrag; Jovanović Stojanov, Sofija; Stepanović, Ana; Lupšić, Ema; Pajović, Milica; Jovanović, Mirna; Petrović Rodić, Dušica; Marić, Dragana; Ercegovac, Maja; Pešić, Milica

(Basel: MDPI, 2023)

TY  - JOUR
AU  - Dinić, Jelena
AU  - Podolski-Renić, Ana
AU  - Dragoj, Miodrag
AU  - Jovanović Stojanov, Sofija
AU  - Stepanović, Ana
AU  - Lupšić, Ema
AU  - Pajović, Milica
AU  - Jovanović, Mirna
AU  - Petrović Rodić, Dušica
AU  - Marić, Dragana
AU  - Ercegovac, Maja
AU  - Pešić, Milica
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6448
AB  - Lung cancer remains the leading cause of cancer death globally, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Multidrug resistance (MDR), often caused by ATP-binding cassette (ABC) transporters, represents a significant obstacle in the treatment of NSCLC. While genetic profiling has an important role in personalized therapy, functional assays that measure cellular responses to drugs are gaining in importance. We developed an automated microplate-based immunofluorescence assay for the evaluation of MDR markers ABCB1, ABCC1, and ABCG2 in cells obtained from NSCLC patients through high-content imaging and image analysis, as part of a functional diagnostic approach. This assay effectively discriminated cancer from non-cancer cells within mixed cultures, which is vital for accurate assessment of changes in MDR marker expression in different cell populations in response to anticancer drugs. Validation was performed using established drug-sensitive (NCI-H460) and drug-resistant (NCI-H460/R) NSCLC cell lines, demonstrating the assay’s capacity to distinguish and evaluate different MDR profiles. The obtained results revealed wide-ranging effects of various chemotherapeutic agents on MDR marker expression in different patient-derived NSCLC cultures, emphasizing the need for MDR diagnostics in NSCLC. In addition to being a valuable tool for assessing drug effects on MDR markers in different cell populations, the assay can complement genetic profiling to optimize treatment. Further assay adaptations may extend its application to other cancer types, improving treatment efficacy while minimizing the development of resistance.
PB  - Basel: MDPI
T2  - Diagnostics
T1  - Immunofluorescence-Based Assay for High-Throughput Analysis of Multidrug Resistance Markers in Non-Small Cell Lung Carcinoma Patient-Derived Cells
IS  - 24
VL  - 13
DO  - 10.3390/diagnostics13243617
SP  - 3617
ER  - 
@article{
author = "Dinić, Jelena and Podolski-Renić, Ana and Dragoj, Miodrag and Jovanović Stojanov, Sofija and Stepanović, Ana and Lupšić, Ema and Pajović, Milica and Jovanović, Mirna and Petrović Rodić, Dušica and Marić, Dragana and Ercegovac, Maja and Pešić, Milica",
year = "2023",
abstract = "Lung cancer remains the leading cause of cancer death globally, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Multidrug resistance (MDR), often caused by ATP-binding cassette (ABC) transporters, represents a significant obstacle in the treatment of NSCLC. While genetic profiling has an important role in personalized therapy, functional assays that measure cellular responses to drugs are gaining in importance. We developed an automated microplate-based immunofluorescence assay for the evaluation of MDR markers ABCB1, ABCC1, and ABCG2 in cells obtained from NSCLC patients through high-content imaging and image analysis, as part of a functional diagnostic approach. This assay effectively discriminated cancer from non-cancer cells within mixed cultures, which is vital for accurate assessment of changes in MDR marker expression in different cell populations in response to anticancer drugs. Validation was performed using established drug-sensitive (NCI-H460) and drug-resistant (NCI-H460/R) NSCLC cell lines, demonstrating the assay’s capacity to distinguish and evaluate different MDR profiles. The obtained results revealed wide-ranging effects of various chemotherapeutic agents on MDR marker expression in different patient-derived NSCLC cultures, emphasizing the need for MDR diagnostics in NSCLC. In addition to being a valuable tool for assessing drug effects on MDR markers in different cell populations, the assay can complement genetic profiling to optimize treatment. Further assay adaptations may extend its application to other cancer types, improving treatment efficacy while minimizing the development of resistance.",
publisher = "Basel: MDPI",
journal = "Diagnostics",
title = "Immunofluorescence-Based Assay for High-Throughput Analysis of Multidrug Resistance Markers in Non-Small Cell Lung Carcinoma Patient-Derived Cells",
number = "24",
volume = "13",
doi = "10.3390/diagnostics13243617",
pages = "3617"
}
Dinić, J., Podolski-Renić, A., Dragoj, M., Jovanović Stojanov, S., Stepanović, A., Lupšić, E., Pajović, M., Jovanović, M., Petrović Rodić, D., Marić, D., Ercegovac, M.,& Pešić, M.. (2023). Immunofluorescence-Based Assay for High-Throughput Analysis of Multidrug Resistance Markers in Non-Small Cell Lung Carcinoma Patient-Derived Cells. in Diagnostics
Basel: MDPI., 13(24), 3617.
https://doi.org/10.3390/diagnostics13243617
Dinić J, Podolski-Renić A, Dragoj M, Jovanović Stojanov S, Stepanović A, Lupšić E, Pajović M, Jovanović M, Petrović Rodić D, Marić D, Ercegovac M, Pešić M. Immunofluorescence-Based Assay for High-Throughput Analysis of Multidrug Resistance Markers in Non-Small Cell Lung Carcinoma Patient-Derived Cells. in Diagnostics. 2023;13(24):3617.
doi:10.3390/diagnostics13243617 .
Dinić, Jelena, Podolski-Renić, Ana, Dragoj, Miodrag, Jovanović Stojanov, Sofija, Stepanović, Ana, Lupšić, Ema, Pajović, Milica, Jovanović, Mirna, Petrović Rodić, Dušica, Marić, Dragana, Ercegovac, Maja, Pešić, Milica, "Immunofluorescence-Based Assay for High-Throughput Analysis of Multidrug Resistance Markers in Non-Small Cell Lung Carcinoma Patient-Derived Cells" in Diagnostics, 13, no. 24 (2023):3617,
https://doi.org/10.3390/diagnostics13243617 . .
1

High-throughput screening of multidrug-resistance markers in non-small cell lung carcinoma patient-derived cells - contribution to personalized treatment

Jovanović Stojanov, Sofija; Podolski-Renić, Ana; Dinić, Jelena; Dragoj, Miodrag; Jovanović, Mirna; Stepanović, Ana; Lupšić, Ema; Pajović, Milica; Petrović Rodić, Dušica; Marić, Dragana; Ercegovac, Maja; Pešić, Milica

(Belgrade: Serbian Association for Cancer Research Belgrade, Serbia, 2023)

TY  - CONF
AU  - Jovanović Stojanov, Sofija
AU  - Podolski-Renić, Ana
AU  - Dinić, Jelena
AU  - Dragoj, Miodrag
AU  - Jovanović, Mirna
AU  - Stepanović, Ana
AU  - Lupšić, Ema
AU  - Pajović, Milica
AU  - Petrović Rodić, Dušica
AU  - Marić, Dragana
AU  - Ercegovac, Maja
AU  - Pešić, Milica
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6449
AB  - Introduction

Cancer remains one of the leading causes of death globally, despite significant advancements in cancer treatment over the past decades. A major challenge in cancer therapy is multidrug resistance (MDR), which is responsible for over 90% of deaths in cancer patients receiving both traditional chemotherapeutics and novel targeted drugs. MDR arises from various mechanisms, including elevated metabolism of foreign substances (xenobiotics), enhanced drug efflux from cells, increased DNA repair capacity, and genetic factors such as gene mutations, amplifications, and epigenetic alterations.1 It is categorized into two types: primary resistance, which exists before initiating therapy and acquired resistance, which develops after the initial treatment. The incidence of primary resistance to cancer treatment can be remarkably high (up to 60%) in certain cancer types.2 Furthermore, the majority of cancer patients are likely to develop resistance at some point during treatment.
Although, the various underlying mechanism for drug resistance development in tumors have been highlighted in the past years, enhanced drug efflux, caused by increased expression of ATP-binding cassette (ABC) membrane transporters, is one of the major contributors to MDR. Among the known ABC transporters, three members, P-glycoprotein (P-gp, encoded by the MDR1 gene), Multidrug Resistance-Associated Protein 1 (MRP1), and Breast Cancer Resistance Protein - BCRP or Placenta ABC Protein - ABC-P), have been linked to chemoresistance to various drugs. P-gp and BCRP regulate various chemical compounds' distribution, absorption, and excretion. However, their overexpression can interfere with drug administration, reducing drug bioavailability and intracellular concentration.3 There is a significant correlation between increased expression of P-gp in cancer cells and enhanced resistance to drugs like paclitaxel, etoposide, DOX, and vinblastine. Overexpression of P-gp has been observed in approximately 50% of all human cancers. While in some tumor types, such as lung, liver, kidney, rectum, and colon, increased P-gp expression has been observed before chemotherapy treatment, in others, such as acute lymphoblastic leukemia and acute myeloid leukemia, it has been noticed after exposure to anticancer agents.4 Overexpression of P-gp and BCRP has been associated with poor clinical response and MDR in patients. Therefore, the pharmacological inhibition of the efflux function of these transporters was pursued as a strategy to overcome resistance to anticancer drugs in the clinic. However, despite showing high efficacy in preclinical studies, none of the P-gp inhibitors have been approved yet by the U.S. Food and Drug Administration (FDA) for clinical use in cancer treatment.5
Taking into account all the above mentioned it is clear that screening and assessment of MDR markers in patient’s cancer cells could play an important role in personalized treatment approaches. Expressing MDR markers in cancer cells could predict a patient's response to specific drugs or drug classes, allowing the selection of the most effective treatment regimen and avoiding using drugs that are likely ineffective due to resistance. Moreover, the presence of MDR markers associated with resistance to multiple drugs could guide the design of personalized treatment regimens with a combination of drugs that have a higher chance of overcoming the patient's specific drug resistance profile. Monitoring the expression level of MDR markers during the course of treatment could provide valuable insights into the development of drug resistance, and would allow healthcare professionals to adjust the treatment plan if drug resistance emerges, ensuring that the patient receives the most effective therapy.
Our team established a promising method for high-throughput screening for MDR markers in non-small cell lung carcinoma (NSCLC) patient-derived cells, which implies pharmacological screening and an ex vivo experimental setting. It enables gaining valuable insights into patient characteristics and drug responses that may not be apparent through conventional sequencing or clinical trials. This strategy has the potential to improve personalized cancer treatment approaches, offering patients more effective and tailored therapies based on their individual characteristics and drug responses.

Methodology

Patient-derived NSCLC cell cultures 
Samples from NSCLC patients are collected from the Thoracic Surgery Clinic at the Clinical Center of Serbia. The histological grade is determined by histopathological analysis of the surgical specimen. Collected NSCLC samples are used to establish patient-derived NSCLC cell cultures comprising cancer and stromal cells (mainly fibroblasts). It is well known that the sensitivity of cancer cells depends on their interaction with the microenvironment including neighboring cells.6 The primary cultures obtained from the samples are grown for 1-2 weeks prior to drug testing.

Fluorescence immunoassay for high-throughput identification of cancers cells and MDR markers in NSCLC patient-derived cell cultures
The fluorescence immunoassay utilizes antibodies against CK8 and CK18, which are expressed in nearly all carcinomas of epithelial origin, to identify epithelial cancer cells. Co-staining of CK8/18 with Hoechst 33342 allows the identification and quantification of two types of cells: CK8/18-negative (non-cancer cells) and CK8/18-positive (cancer cells). This immunoassay is also used to identify and quantify changes in the expression of MDR markers ABCB1, ABCC1, and ABCG2 both in cancer and non-cancer cells in primary NSCLC cultures that may occur during chemotherapy and tyrosine kinase inhibitors (TKIs) treatment.7 Co-staining of ABCB1, ABCC1, and ABCG2 with CK8/18 and Hoechst 33342 enables the identification of four types of cells in NSCLC primary cell cultures: drug-sensitive non-cancer cells, MDR non-cancer cells, drug-sensitive cancer cells, and MDR cancer cells.
For validation of the immunoassay patient-derived cells are seeded in 384 well-plates and treated with 5 different concentrations of 8 chemotherapeutics known to induce overexpression of MDR markers (cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide, and pemetrexed), allowing the ex vivo evaluation of NSCLC MDR profile. Validated immunoassay is further used to evaluate the expression of MDR markers ABCB1, ABCC1, and ABCG2 (MDR profile) in patient-derived cell cultures after treatment with a panel of 10 TKIs (erlotinib, gefitinib, afatinib, osimertinib crizotinib, alectinib, ceritinib, nintedanib, dabrafenib, and trametinib), allowing evaluation of MDR profile in both cancer and stromal cells. The sensitivity of cancer and stromal cells for each individual NSCLC patient to a particular TKI is assessed using a discriminative immunoassay employing CK8/18 antibodies cocktail.
Whole Exome Sequencing (WES)
	Paired patient samples (normal and tumor) were subjected to a DNA isolation procedure using Qiagen genomic DNA extraction kit, recommended for NGS applications. Isolated DNA samples underwent WES analyses by Novogene Company. Bioinformatics and statistics tools were employed to define clinically relevant gene alterations in MDR markers ABCB1, ABCC1, and ABCG2.

Results


In order to understand how NSCLC patient cells respond to chemotherapy and targeted therapy, ex vivo testing was performed. The maximum concentration of drugs in human plasma that the patient is exposed to during therapy (Cmax) was used as an upper limit for drug concentration during testing, with four lower concentrations also used. The results showed that patient-derived cells display individual differences in sensitivity to both chemo and targeted therapeutics. IC50 values, which indicate sensitivity, fell within the concentration range for most chemotherapeutics. Only some chemotherapeutics (cisplatin, etoposide, docetaxel, gemcitabine, and pemetrexed) showed selectivity towards cancer cells with lower IC50 values in cancer than in stromal cells. Among TKIs, only erlotinib was efficient with IC50 below Cmax, showing selectivity towards cancer cells in all investigated patient-derived cell cultures. A number of chemotherapeutics increased the expression of ABCB1, ABCC1, and ABCG2, while TKIs afatinib, alectinib, ceritinib, osimertinib, and trametinib did not affect these transporters. Some TKIs increased the expression of ABC transporters, with nintedanib showing the potential to select cancer cells with higher MDR marker expression. WES showed significant ABCC1 gene instability, while ABCB1 had many SNPs with clinical relevance for drug response. ABCG2 had the lowest number of SNPs, but intron deletions were still identified. However, the clinical significance of these changes is currently unknown.

Conclusion

Screening for multidrug-resistance markers through a high-throughput process provides valuable information about how a patient will respond to therapy. This process can identify if the MDR phenotype is already present or if it can be induced with targeted or chemotherapy. Based on this information, it can provide recommendations for a patient's mono- and combined therapy. This methodology has the potential to greatly impact cancer treatment strategies and improve patient outcomes by tailoring therapies to individual patient profiles. Ultimately, this will benefit a wider range of patients with non-small cell lung carcinoma and other cancers, as it leads to more precise and targeted treatment selections.


References
1.	Bukowski, K., Kciuk, M., & Kontek, R. (2020). Mechanisms of Multidrug Resistance in Cancer Chemotherapy. International Journal of Molecular Sciences, 21(9). 
2.	Sharma, P., Hu-Lieskovan, S., Wargo, J. A., & Ribas, A. (2017). Primary, Adaptive and Acquired Resistance to Cancer Immunotherapy. Cell, 168(4), 707. 
3.	Wang, J. Q., Wu, Z. X., Yang, Y., Teng, Q. X., Li, Y. D., Lei, Z. N., Jani, K. A., Kaushal, N., & Chen, Z. S. (2021). ATP-binding cassette (ABC) transporters in cancer: A review of recent updates. Journal of Evidence-Based Medicine, 14(3), 232–256. 
4.	Wang, X., Zhang, H., & Chen, X. (n.d.). Review Open Access Cancer Drug Resistance Drug resistance and combating drug resistance in cancer.
5.	Nanayakkara, A. K., Follit, C. A., Chen, G., Williams, N. S., Vogel, P. D., & Wise, J. G. (n.d.). Targeted inhibitors of P-glycoprotein increase chemotherapeutic-induced mortality of multidrug resistant tumor cells OPEN. 
6.	 Sazeides, C., & Le, A. (2021). Metabolic Relationship Between Cancer-Associated Fibroblasts and Cancer Cells. Advances in Experimental Medicine and Biology, 1311, 189–204. 
7.	Beretta, G. L., Cassinelli, G., Pennati, M., Zuco, V., & Gatti, L. (2017). Overcoming ABC transporter-mediated multidrug resistance: The dual role of tyrosine kinase inhibitors as multitargeting agents. European Journal of Medicinal Chemistry, 142, 271–289.
PB  - Belgrade: Serbian Association for Cancer Research Belgrade, Serbia
C3  - Proceedings book of The Sixth Congress of The Serbian Association for Cancer Research with international participation: From Collaboration to Innovation in Cancer Research; 2023 Oct 2-4; Belgrade, Serbia
T1  - High-throughput screening of multidrug-resistance markers in non-small cell lung carcinoma patient-derived cells - contribution to personalized treatment
SP  - 37
EP  - 39
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6449
ER  - 
@conference{
author = "Jovanović Stojanov, Sofija and Podolski-Renić, Ana and Dinić, Jelena and Dragoj, Miodrag and Jovanović, Mirna and Stepanović, Ana and Lupšić, Ema and Pajović, Milica and Petrović Rodić, Dušica and Marić, Dragana and Ercegovac, Maja and Pešić, Milica",
year = "2023",
abstract = "Introduction

Cancer remains one of the leading causes of death globally, despite significant advancements in cancer treatment over the past decades. A major challenge in cancer therapy is multidrug resistance (MDR), which is responsible for over 90% of deaths in cancer patients receiving both traditional chemotherapeutics and novel targeted drugs. MDR arises from various mechanisms, including elevated metabolism of foreign substances (xenobiotics), enhanced drug efflux from cells, increased DNA repair capacity, and genetic factors such as gene mutations, amplifications, and epigenetic alterations.1 It is categorized into two types: primary resistance, which exists before initiating therapy and acquired resistance, which develops after the initial treatment. The incidence of primary resistance to cancer treatment can be remarkably high (up to 60%) in certain cancer types.2 Furthermore, the majority of cancer patients are likely to develop resistance at some point during treatment.
Although, the various underlying mechanism for drug resistance development in tumors have been highlighted in the past years, enhanced drug efflux, caused by increased expression of ATP-binding cassette (ABC) membrane transporters, is one of the major contributors to MDR. Among the known ABC transporters, three members, P-glycoprotein (P-gp, encoded by the MDR1 gene), Multidrug Resistance-Associated Protein 1 (MRP1), and Breast Cancer Resistance Protein - BCRP or Placenta ABC Protein - ABC-P), have been linked to chemoresistance to various drugs. P-gp and BCRP regulate various chemical compounds' distribution, absorption, and excretion. However, their overexpression can interfere with drug administration, reducing drug bioavailability and intracellular concentration.3 There is a significant correlation between increased expression of P-gp in cancer cells and enhanced resistance to drugs like paclitaxel, etoposide, DOX, and vinblastine. Overexpression of P-gp has been observed in approximately 50% of all human cancers. While in some tumor types, such as lung, liver, kidney, rectum, and colon, increased P-gp expression has been observed before chemotherapy treatment, in others, such as acute lymphoblastic leukemia and acute myeloid leukemia, it has been noticed after exposure to anticancer agents.4 Overexpression of P-gp and BCRP has been associated with poor clinical response and MDR in patients. Therefore, the pharmacological inhibition of the efflux function of these transporters was pursued as a strategy to overcome resistance to anticancer drugs in the clinic. However, despite showing high efficacy in preclinical studies, none of the P-gp inhibitors have been approved yet by the U.S. Food and Drug Administration (FDA) for clinical use in cancer treatment.5
Taking into account all the above mentioned it is clear that screening and assessment of MDR markers in patient’s cancer cells could play an important role in personalized treatment approaches. Expressing MDR markers in cancer cells could predict a patient's response to specific drugs or drug classes, allowing the selection of the most effective treatment regimen and avoiding using drugs that are likely ineffective due to resistance. Moreover, the presence of MDR markers associated with resistance to multiple drugs could guide the design of personalized treatment regimens with a combination of drugs that have a higher chance of overcoming the patient's specific drug resistance profile. Monitoring the expression level of MDR markers during the course of treatment could provide valuable insights into the development of drug resistance, and would allow healthcare professionals to adjust the treatment plan if drug resistance emerges, ensuring that the patient receives the most effective therapy.
Our team established a promising method for high-throughput screening for MDR markers in non-small cell lung carcinoma (NSCLC) patient-derived cells, which implies pharmacological screening and an ex vivo experimental setting. It enables gaining valuable insights into patient characteristics and drug responses that may not be apparent through conventional sequencing or clinical trials. This strategy has the potential to improve personalized cancer treatment approaches, offering patients more effective and tailored therapies based on their individual characteristics and drug responses.

Methodology

Patient-derived NSCLC cell cultures 
Samples from NSCLC patients are collected from the Thoracic Surgery Clinic at the Clinical Center of Serbia. The histological grade is determined by histopathological analysis of the surgical specimen. Collected NSCLC samples are used to establish patient-derived NSCLC cell cultures comprising cancer and stromal cells (mainly fibroblasts). It is well known that the sensitivity of cancer cells depends on their interaction with the microenvironment including neighboring cells.6 The primary cultures obtained from the samples are grown for 1-2 weeks prior to drug testing.

Fluorescence immunoassay for high-throughput identification of cancers cells and MDR markers in NSCLC patient-derived cell cultures
The fluorescence immunoassay utilizes antibodies against CK8 and CK18, which are expressed in nearly all carcinomas of epithelial origin, to identify epithelial cancer cells. Co-staining of CK8/18 with Hoechst 33342 allows the identification and quantification of two types of cells: CK8/18-negative (non-cancer cells) and CK8/18-positive (cancer cells). This immunoassay is also used to identify and quantify changes in the expression of MDR markers ABCB1, ABCC1, and ABCG2 both in cancer and non-cancer cells in primary NSCLC cultures that may occur during chemotherapy and tyrosine kinase inhibitors (TKIs) treatment.7 Co-staining of ABCB1, ABCC1, and ABCG2 with CK8/18 and Hoechst 33342 enables the identification of four types of cells in NSCLC primary cell cultures: drug-sensitive non-cancer cells, MDR non-cancer cells, drug-sensitive cancer cells, and MDR cancer cells.
For validation of the immunoassay patient-derived cells are seeded in 384 well-plates and treated with 5 different concentrations of 8 chemotherapeutics known to induce overexpression of MDR markers (cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide, and pemetrexed), allowing the ex vivo evaluation of NSCLC MDR profile. Validated immunoassay is further used to evaluate the expression of MDR markers ABCB1, ABCC1, and ABCG2 (MDR profile) in patient-derived cell cultures after treatment with a panel of 10 TKIs (erlotinib, gefitinib, afatinib, osimertinib crizotinib, alectinib, ceritinib, nintedanib, dabrafenib, and trametinib), allowing evaluation of MDR profile in both cancer and stromal cells. The sensitivity of cancer and stromal cells for each individual NSCLC patient to a particular TKI is assessed using a discriminative immunoassay employing CK8/18 antibodies cocktail.
Whole Exome Sequencing (WES)
	Paired patient samples (normal and tumor) were subjected to a DNA isolation procedure using Qiagen genomic DNA extraction kit, recommended for NGS applications. Isolated DNA samples underwent WES analyses by Novogene Company. Bioinformatics and statistics tools were employed to define clinically relevant gene alterations in MDR markers ABCB1, ABCC1, and ABCG2.

Results


In order to understand how NSCLC patient cells respond to chemotherapy and targeted therapy, ex vivo testing was performed. The maximum concentration of drugs in human plasma that the patient is exposed to during therapy (Cmax) was used as an upper limit for drug concentration during testing, with four lower concentrations also used. The results showed that patient-derived cells display individual differences in sensitivity to both chemo and targeted therapeutics. IC50 values, which indicate sensitivity, fell within the concentration range for most chemotherapeutics. Only some chemotherapeutics (cisplatin, etoposide, docetaxel, gemcitabine, and pemetrexed) showed selectivity towards cancer cells with lower IC50 values in cancer than in stromal cells. Among TKIs, only erlotinib was efficient with IC50 below Cmax, showing selectivity towards cancer cells in all investigated patient-derived cell cultures. A number of chemotherapeutics increased the expression of ABCB1, ABCC1, and ABCG2, while TKIs afatinib, alectinib, ceritinib, osimertinib, and trametinib did not affect these transporters. Some TKIs increased the expression of ABC transporters, with nintedanib showing the potential to select cancer cells with higher MDR marker expression. WES showed significant ABCC1 gene instability, while ABCB1 had many SNPs with clinical relevance for drug response. ABCG2 had the lowest number of SNPs, but intron deletions were still identified. However, the clinical significance of these changes is currently unknown.

Conclusion

Screening for multidrug-resistance markers through a high-throughput process provides valuable information about how a patient will respond to therapy. This process can identify if the MDR phenotype is already present or if it can be induced with targeted or chemotherapy. Based on this information, it can provide recommendations for a patient's mono- and combined therapy. This methodology has the potential to greatly impact cancer treatment strategies and improve patient outcomes by tailoring therapies to individual patient profiles. Ultimately, this will benefit a wider range of patients with non-small cell lung carcinoma and other cancers, as it leads to more precise and targeted treatment selections.


References
1.	Bukowski, K., Kciuk, M., & Kontek, R. (2020). Mechanisms of Multidrug Resistance in Cancer Chemotherapy. International Journal of Molecular Sciences, 21(9). 
2.	Sharma, P., Hu-Lieskovan, S., Wargo, J. A., & Ribas, A. (2017). Primary, Adaptive and Acquired Resistance to Cancer Immunotherapy. Cell, 168(4), 707. 
3.	Wang, J. Q., Wu, Z. X., Yang, Y., Teng, Q. X., Li, Y. D., Lei, Z. N., Jani, K. A., Kaushal, N., & Chen, Z. S. (2021). ATP-binding cassette (ABC) transporters in cancer: A review of recent updates. Journal of Evidence-Based Medicine, 14(3), 232–256. 
4.	Wang, X., Zhang, H., & Chen, X. (n.d.). Review Open Access Cancer Drug Resistance Drug resistance and combating drug resistance in cancer.
5.	Nanayakkara, A. K., Follit, C. A., Chen, G., Williams, N. S., Vogel, P. D., & Wise, J. G. (n.d.). Targeted inhibitors of P-glycoprotein increase chemotherapeutic-induced mortality of multidrug resistant tumor cells OPEN. 
6.	 Sazeides, C., & Le, A. (2021). Metabolic Relationship Between Cancer-Associated Fibroblasts and Cancer Cells. Advances in Experimental Medicine and Biology, 1311, 189–204. 
7.	Beretta, G. L., Cassinelli, G., Pennati, M., Zuco, V., & Gatti, L. (2017). Overcoming ABC transporter-mediated multidrug resistance: The dual role of tyrosine kinase inhibitors as multitargeting agents. European Journal of Medicinal Chemistry, 142, 271–289.",
publisher = "Belgrade: Serbian Association for Cancer Research Belgrade, Serbia",
journal = "Proceedings book of The Sixth Congress of The Serbian Association for Cancer Research with international participation: From Collaboration to Innovation in Cancer Research; 2023 Oct 2-4; Belgrade, Serbia",
title = "High-throughput screening of multidrug-resistance markers in non-small cell lung carcinoma patient-derived cells - contribution to personalized treatment",
pages = "37-39",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6449"
}
Jovanović Stojanov, S., Podolski-Renić, A., Dinić, J., Dragoj, M., Jovanović, M., Stepanović, A., Lupšić, E., Pajović, M., Petrović Rodić, D., Marić, D., Ercegovac, M.,& Pešić, M.. (2023). High-throughput screening of multidrug-resistance markers in non-small cell lung carcinoma patient-derived cells - contribution to personalized treatment. in Proceedings book of The Sixth Congress of The Serbian Association for Cancer Research with international participation: From Collaboration to Innovation in Cancer Research; 2023 Oct 2-4; Belgrade, Serbia
Belgrade: Serbian Association for Cancer Research Belgrade, Serbia., 37-39.
https://hdl.handle.net/21.15107/rcub_ibiss_6449
Jovanović Stojanov S, Podolski-Renić A, Dinić J, Dragoj M, Jovanović M, Stepanović A, Lupšić E, Pajović M, Petrović Rodić D, Marić D, Ercegovac M, Pešić M. High-throughput screening of multidrug-resistance markers in non-small cell lung carcinoma patient-derived cells - contribution to personalized treatment. in Proceedings book of The Sixth Congress of The Serbian Association for Cancer Research with international participation: From Collaboration to Innovation in Cancer Research; 2023 Oct 2-4; Belgrade, Serbia. 2023;:37-39.
https://hdl.handle.net/21.15107/rcub_ibiss_6449 .
Jovanović Stojanov, Sofija, Podolski-Renić, Ana, Dinić, Jelena, Dragoj, Miodrag, Jovanović, Mirna, Stepanović, Ana, Lupšić, Ema, Pajović, Milica, Petrović Rodić, Dušica, Marić, Dragana, Ercegovac, Maja, Pešić, Milica, "High-throughput screening of multidrug-resistance markers in non-small cell lung carcinoma patient-derived cells - contribution to personalized treatment" in Proceedings book of The Sixth Congress of The Serbian Association for Cancer Research with international participation: From Collaboration to Innovation in Cancer Research; 2023 Oct 2-4; Belgrade, Serbia (2023):37-39,
https://hdl.handle.net/21.15107/rcub_ibiss_6449 .

Functional diagnostics and ex-vivo screening of erlotinib and nintedanib in non-small cell lung carcinoma: Implications for multidrug resistance and personalized therapy

Dinić, Jelena; Podolski-Renić, Ana; Dragoj, Miodrag; Jovanović Stojanov, Sofija; Stepanović, Ana; Lupšić, Ema; Pajović, Milica; Petrović Rodić, Dušica; Marić, Dragana; Ercegovac, Maja; Pešić, Milica

(Elsevier Inc, 2023)

TY  - CONF
AU  - Dinić, Jelena
AU  - Podolski-Renić, Ana
AU  - Dragoj, Miodrag
AU  - Jovanović Stojanov, Sofija
AU  - Stepanović, Ana
AU  - Lupšić, Ema
AU  - Pajović, Milica
AU  - Petrović Rodić, Dušica
AU  - Marić, Dragana
AU  - Ercegovac, Maja
AU  - Pešić, Milica
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6380
AB  - Background: Multidrug resistance (MDR) hampers tyrosine kinase inhibitor (TKI) efficacy in non-small cell lung carcinoma (NSCLC). ABC transporters ABCB1, ABCC1, and ABCG2 trigger MDR by effluxing drugs from cancer cells. We studied erlotinib and nintedanib effects in patient-derived NSCLC cultures, MDR phenotype impact, and genetic alterations influencing drug response.
Methods: ABC transporter expression in 10 NSCLC patient-derived cell cultures was assessed after TKI treatment via immunofluorescence assay which enables discrimination between cancer and stromal cells. Erlotinib (1 µM – 4 µM) and nintedanib (2.5 µM – 20 µM) were used in clinically relevant concentrations. Whole exome sequencing was employed to analyze genetic alterations in NSCLC samples. 
Results: Erlotinib selectively inhibited cancer cell growth (IC50: 0.25 µM – 3.2 µM). It increased ABCC1 expression in 4/10 cultures and ABCB1/ABCG2 in 2/10 cultures. Erlotinib induced MDR markers expression at all concentrations. Nintedanib stimulated cancer cell growth at lower concentrations (˂10 µM) and caused 90% cell death at higher concentrations (˃15 µM), enriching the culture with cancer cells with high expression of ABCB1, ABCC1, and ABCG2. TKIs had no impact on MDR marker expression in stromal cells. Genetic alterations without clinical relevance for NSCLC were found in EGFR, ALK, ROS1, RET, and BRAF. L858R mutation in EGFR, indicated for erlotinib treatment, was detected in one patient, although all patients were responsive to erlotinib. Genetic alterations related to drug response were found in ABCB1 (7/10 patients) and ABCG2 (1/10 patients). 
Conclusions: The employed functional diagnostics approach can effectively assess how erlotinib and nintedanib influence the MDR phenotype for individual patients. The ex-vivo screening system utilized in this study identifies the sensitivity of cancer and stromal cells and the correlation between response and their MDR profile, as well as the dependence of drug response on genetic alterations. This approach holds great promise for advancing personalized treatment strategies in NSCLC.
PB  - Elsevier Inc
C3  - Abstract book: Molecular Analysis for Precision Oncology Congress 2023; 2023 Oct 4-6; Paris, France
T1  - Functional diagnostics and ex-vivo screening of erlotinib and nintedanib in non-small cell lung carcinoma: Implications for multidrug resistance and personalized therapy
DO  - 10.1016/j.esmoop.2023.101680
SP  - 12
EP  - 12
ER  - 
@conference{
author = "Dinić, Jelena and Podolski-Renić, Ana and Dragoj, Miodrag and Jovanović Stojanov, Sofija and Stepanović, Ana and Lupšić, Ema and Pajović, Milica and Petrović Rodić, Dušica and Marić, Dragana and Ercegovac, Maja and Pešić, Milica",
year = "2023",
abstract = "Background: Multidrug resistance (MDR) hampers tyrosine kinase inhibitor (TKI) efficacy in non-small cell lung carcinoma (NSCLC). ABC transporters ABCB1, ABCC1, and ABCG2 trigger MDR by effluxing drugs from cancer cells. We studied erlotinib and nintedanib effects in patient-derived NSCLC cultures, MDR phenotype impact, and genetic alterations influencing drug response.
Methods: ABC transporter expression in 10 NSCLC patient-derived cell cultures was assessed after TKI treatment via immunofluorescence assay which enables discrimination between cancer and stromal cells. Erlotinib (1 µM – 4 µM) and nintedanib (2.5 µM – 20 µM) were used in clinically relevant concentrations. Whole exome sequencing was employed to analyze genetic alterations in NSCLC samples. 
Results: Erlotinib selectively inhibited cancer cell growth (IC50: 0.25 µM – 3.2 µM). It increased ABCC1 expression in 4/10 cultures and ABCB1/ABCG2 in 2/10 cultures. Erlotinib induced MDR markers expression at all concentrations. Nintedanib stimulated cancer cell growth at lower concentrations (˂10 µM) and caused 90% cell death at higher concentrations (˃15 µM), enriching the culture with cancer cells with high expression of ABCB1, ABCC1, and ABCG2. TKIs had no impact on MDR marker expression in stromal cells. Genetic alterations without clinical relevance for NSCLC were found in EGFR, ALK, ROS1, RET, and BRAF. L858R mutation in EGFR, indicated for erlotinib treatment, was detected in one patient, although all patients were responsive to erlotinib. Genetic alterations related to drug response were found in ABCB1 (7/10 patients) and ABCG2 (1/10 patients). 
Conclusions: The employed functional diagnostics approach can effectively assess how erlotinib and nintedanib influence the MDR phenotype for individual patients. The ex-vivo screening system utilized in this study identifies the sensitivity of cancer and stromal cells and the correlation between response and their MDR profile, as well as the dependence of drug response on genetic alterations. This approach holds great promise for advancing personalized treatment strategies in NSCLC.",
publisher = "Elsevier Inc",
journal = "Abstract book: Molecular Analysis for Precision Oncology Congress 2023; 2023 Oct 4-6; Paris, France",
title = "Functional diagnostics and ex-vivo screening of erlotinib and nintedanib in non-small cell lung carcinoma: Implications for multidrug resistance and personalized therapy",
doi = "10.1016/j.esmoop.2023.101680",
pages = "12-12"
}
Dinić, J., Podolski-Renić, A., Dragoj, M., Jovanović Stojanov, S., Stepanović, A., Lupšić, E., Pajović, M., Petrović Rodić, D., Marić, D., Ercegovac, M.,& Pešić, M.. (2023). Functional diagnostics and ex-vivo screening of erlotinib and nintedanib in non-small cell lung carcinoma: Implications for multidrug resistance and personalized therapy. in Abstract book: Molecular Analysis for Precision Oncology Congress 2023; 2023 Oct 4-6; Paris, France
Elsevier Inc., 12-12.
https://doi.org/10.1016/j.esmoop.2023.101680
Dinić J, Podolski-Renić A, Dragoj M, Jovanović Stojanov S, Stepanović A, Lupšić E, Pajović M, Petrović Rodić D, Marić D, Ercegovac M, Pešić M. Functional diagnostics and ex-vivo screening of erlotinib and nintedanib in non-small cell lung carcinoma: Implications for multidrug resistance and personalized therapy. in Abstract book: Molecular Analysis for Precision Oncology Congress 2023; 2023 Oct 4-6; Paris, France. 2023;:12-12.
doi:10.1016/j.esmoop.2023.101680 .
Dinić, Jelena, Podolski-Renić, Ana, Dragoj, Miodrag, Jovanović Stojanov, Sofija, Stepanović, Ana, Lupšić, Ema, Pajović, Milica, Petrović Rodić, Dušica, Marić, Dragana, Ercegovac, Maja, Pešić, Milica, "Functional diagnostics and ex-vivo screening of erlotinib and nintedanib in non-small cell lung carcinoma: Implications for multidrug resistance and personalized therapy" in Abstract book: Molecular Analysis for Precision Oncology Congress 2023; 2023 Oct 4-6; Paris, France (2023):12-12,
https://doi.org/10.1016/j.esmoop.2023.101680 . .

Osimertinib is selective against NSCLC cells and modulates the multidrug-resistant phenotype in patient-derived cell cultures and co-cultures of NSCLC cells and fibroblasts

Jovanović Stojanov, Sofija; Podolski-Renić, Ana; Dinić, Jelena; Dragoj, Miodrag; Jovanović, Mirna; Stepanović, Ana; Lupšić, Ema; Bajović, Radovan; Glumac, Sofija; Marić, Dragana; Ercegovac, Maja; Pešić, Milica

(Elsevier Inc., 2023)

TY  - CONF
AU  - Jovanović Stojanov, Sofija
AU  - Podolski-Renić, Ana
AU  - Dinić, Jelena
AU  - Dragoj, Miodrag
AU  - Jovanović, Mirna
AU  - Stepanović, Ana
AU  - Lupšić, Ema
AU  - Bajović, Radovan
AU  - Glumac, Sofija
AU  - Marić, Dragana
AU  - Ercegovac, Maja
AU  - Pešić, Milica
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5712
AB  - Background: Osimertinib belongs to the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved for metastatic EGFR-mutant non-small-cell lung carcinoma (NSCLC) patients. Herein, we studied osimertinib selectivity towards NSCLC cells, its efficacy dependence on the EGFR mutation status, and its ability to evade the classical mechanism of multidrug-resistance (MDR) mirrored in the increased expression of main ATP Binding Cassette (ABC) transporters (ABCB1, ABCC1, and ABCG2).
Methods: Primary patient-derived cultures were established from the NSCLC resections. After short-term culturing (2-3 weeks), a mixed population of cancer and non-cancer cells (around a ratio of 1:1) and two co-cultures of NSCLC cell lines (sensitive NCI-H460 and MDR NCI-H460/R) with lung fibroblasts MRC-5 were treated with 8 chemotherapeutics (cisplatin, carboplatin, paclitaxel, docetaxel, etoposide, vinorelbine, gemcitabine, and pemetrexed) as well as osimertinib. The maximum concentration reached in human plasma to which the patient is exposed during therapy (Cmax) was set as an upper limit and four lower concentrations were also applied during the study. Immunofluorescence assay enabling discrimination of epithelial cancer cells positive to a cocktail of antibodies against cytokeratin 8/18 vs. negative mesenchymal non-cancer cells was conducted using high-content imager ImageXpress Pico (Molecular Devices) with CellReporterXpress 2.9 software. Within the same immunoassay, MDR markers (ABCB1, ABCC1, and ABCG2) were analyzed by corresponding antibodies.
Results: Osimertinib showed selectivity against NSCLC cells, particularly in the patient-derived cell culture without EGFR mutations. Other chemotherapeutics were not selective towards cancer cells, on contrary, they showed higher cytotoxicity in non-cancer cells. Osimertinib did not change the expression of ABCB1 in cancer cells, but it significantly decreased the expression of ABCC1 and ABCG2 transporters in cancer and non-cancer cells.
Conclusions: Osimertinib can be valuable as a selective anticancer drug and an MDR modulator even in NSCLC without EGFR mutations.
PB  - Elsevier Inc.
C3  - Abstract Book of the ESMO Targeted Anticancer Therapies Congress (TAT); 2023 Mar 6-8; Paris, France
T1  - Osimertinib is selective against NSCLC cells and modulates the multidrug-resistant phenotype in patient-derived cell cultures and co-cultures of NSCLC cells and fibroblasts
IS  - 69p
DO  - 10.1016/j.esmoop.2023.100927
ER  - 
@conference{
author = "Jovanović Stojanov, Sofija and Podolski-Renić, Ana and Dinić, Jelena and Dragoj, Miodrag and Jovanović, Mirna and Stepanović, Ana and Lupšić, Ema and Bajović, Radovan and Glumac, Sofija and Marić, Dragana and Ercegovac, Maja and Pešić, Milica",
year = "2023",
abstract = "Background: Osimertinib belongs to the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved for metastatic EGFR-mutant non-small-cell lung carcinoma (NSCLC) patients. Herein, we studied osimertinib selectivity towards NSCLC cells, its efficacy dependence on the EGFR mutation status, and its ability to evade the classical mechanism of multidrug-resistance (MDR) mirrored in the increased expression of main ATP Binding Cassette (ABC) transporters (ABCB1, ABCC1, and ABCG2).
Methods: Primary patient-derived cultures were established from the NSCLC resections. After short-term culturing (2-3 weeks), a mixed population of cancer and non-cancer cells (around a ratio of 1:1) and two co-cultures of NSCLC cell lines (sensitive NCI-H460 and MDR NCI-H460/R) with lung fibroblasts MRC-5 were treated with 8 chemotherapeutics (cisplatin, carboplatin, paclitaxel, docetaxel, etoposide, vinorelbine, gemcitabine, and pemetrexed) as well as osimertinib. The maximum concentration reached in human plasma to which the patient is exposed during therapy (Cmax) was set as an upper limit and four lower concentrations were also applied during the study. Immunofluorescence assay enabling discrimination of epithelial cancer cells positive to a cocktail of antibodies against cytokeratin 8/18 vs. negative mesenchymal non-cancer cells was conducted using high-content imager ImageXpress Pico (Molecular Devices) with CellReporterXpress 2.9 software. Within the same immunoassay, MDR markers (ABCB1, ABCC1, and ABCG2) were analyzed by corresponding antibodies.
Results: Osimertinib showed selectivity against NSCLC cells, particularly in the patient-derived cell culture without EGFR mutations. Other chemotherapeutics were not selective towards cancer cells, on contrary, they showed higher cytotoxicity in non-cancer cells. Osimertinib did not change the expression of ABCB1 in cancer cells, but it significantly decreased the expression of ABCC1 and ABCG2 transporters in cancer and non-cancer cells.
Conclusions: Osimertinib can be valuable as a selective anticancer drug and an MDR modulator even in NSCLC without EGFR mutations.",
publisher = "Elsevier Inc.",
journal = "Abstract Book of the ESMO Targeted Anticancer Therapies Congress (TAT); 2023 Mar 6-8; Paris, France",
title = "Osimertinib is selective against NSCLC cells and modulates the multidrug-resistant phenotype in patient-derived cell cultures and co-cultures of NSCLC cells and fibroblasts",
number = "69p",
doi = "10.1016/j.esmoop.2023.100927"
}
Jovanović Stojanov, S., Podolski-Renić, A., Dinić, J., Dragoj, M., Jovanović, M., Stepanović, A., Lupšić, E., Bajović, R., Glumac, S., Marić, D., Ercegovac, M.,& Pešić, M.. (2023). Osimertinib is selective against NSCLC cells and modulates the multidrug-resistant phenotype in patient-derived cell cultures and co-cultures of NSCLC cells and fibroblasts. in Abstract Book of the ESMO Targeted Anticancer Therapies Congress (TAT); 2023 Mar 6-8; Paris, France
Elsevier Inc..(69p).
https://doi.org/10.1016/j.esmoop.2023.100927
Jovanović Stojanov S, Podolski-Renić A, Dinić J, Dragoj M, Jovanović M, Stepanović A, Lupšić E, Bajović R, Glumac S, Marić D, Ercegovac M, Pešić M. Osimertinib is selective against NSCLC cells and modulates the multidrug-resistant phenotype in patient-derived cell cultures and co-cultures of NSCLC cells and fibroblasts. in Abstract Book of the ESMO Targeted Anticancer Therapies Congress (TAT); 2023 Mar 6-8; Paris, France. 2023;(69p).
doi:10.1016/j.esmoop.2023.100927 .
Jovanović Stojanov, Sofija, Podolski-Renić, Ana, Dinić, Jelena, Dragoj, Miodrag, Jovanović, Mirna, Stepanović, Ana, Lupšić, Ema, Bajović, Radovan, Glumac, Sofija, Marić, Dragana, Ercegovac, Maja, Pešić, Milica, "Osimertinib is selective against NSCLC cells and modulates the multidrug-resistant phenotype in patient-derived cell cultures and co-cultures of NSCLC cells and fibroblasts" in Abstract Book of the ESMO Targeted Anticancer Therapies Congress (TAT); 2023 Mar 6-8; Paris, France, no. 69p (2023),
https://doi.org/10.1016/j.esmoop.2023.100927 . .

New anti-glioblastoma strategy with natural compounds sclareol and doxorubicin

Stepanović, Ana; Lupšić, Ema; Dinić, Jelena; Podolski-Renić, Ana; Pajović, Milica; Jovanović Stojanov, Sofija; Dragoj, Miodrag; Terzić Jovanović, Nataša; Opsenica, Igor; Pešić, Milica

(Belgrade: Serbian Neuroscience Society, 2023)

TY  - CONF
AU  - Stepanović, Ana
AU  - Lupšić, Ema
AU  - Dinić, Jelena
AU  - Podolski-Renić, Ana
AU  - Pajović, Milica
AU  - Jovanović Stojanov, Sofija
AU  - Dragoj, Miodrag
AU  - Terzić Jovanović, Nataša
AU  - Opsenica, Igor
AU  - Pešić, Milica
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5905
AB  - Background: Doxorubicin (DOX) has been very effective against glioblastoma in
vitro. Its application in vivo is hampered because it cannot pass the blood–brain
barrier (BBB). Significant research efforts are invested to overcome this limitation.
Sclareol (SC) is an aromatic compound naturally found in clary sage. The
combination of SC and DOX showed promising effects in different tumor types in
vitro and in vivo. Therefore, we tested their combination and innovative hybrid
molecules (SC:DOX) on glioblastoma cells with the expression of P-glycoprotein, a
major component of BBB and cancer multidrug resistance marker. Methods:
Cytotoxicity and selectivity towards glioblastoma cells of SC, DOX, their
combination, and SC:DOX were examined by MTT assay. The effect of SC on DOX
accumulation was determined by flow cytometry. We also studied SC:DOX
accumulation, cellular uptake, localization imaging, and DNA damage induction.
Results: The effects of simultaneous SC and DOX treatments demonstrated the
considerable potential of SC to reverse DOX resistance in glioblastoma cells and
increase DOX accumulation. SC:DOX hybrids, named CON1 and CON2 were less
cytotoxic than DOX, but with reduced resistance and increased selectivity towards
glioblastoma cells. Cellular uptake of CON1 and CON2 was increased in glioblastoma
cells compared to DOX. Perinuclear localization of CON1 and CON2 vs. nuclear
localization of DOX as well as no DNA damaging effects suggest a different
mechanism of action for SC:DOX. Conclusion: The combination of SC and DOX, and
their innovative hybrids, could be considered a promising strategy that can overcome
the limitations of DOX application in glioblastoma.
PB  - Belgrade: Serbian Neuroscience Society
C3  - Book of abstracts: 8th Congress of Serbian neuroscience society with international participation; 2023 May 31 - Jun 2; Belgrade, Serbia
T1  - New anti-glioblastoma strategy with natural compounds sclareol and doxorubicin
SP  - 71
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5905
ER  - 
@conference{
author = "Stepanović, Ana and Lupšić, Ema and Dinić, Jelena and Podolski-Renić, Ana and Pajović, Milica and Jovanović Stojanov, Sofija and Dragoj, Miodrag and Terzić Jovanović, Nataša and Opsenica, Igor and Pešić, Milica",
year = "2023",
abstract = "Background: Doxorubicin (DOX) has been very effective against glioblastoma in
vitro. Its application in vivo is hampered because it cannot pass the blood–brain
barrier (BBB). Significant research efforts are invested to overcome this limitation.
Sclareol (SC) is an aromatic compound naturally found in clary sage. The
combination of SC and DOX showed promising effects in different tumor types in
vitro and in vivo. Therefore, we tested their combination and innovative hybrid
molecules (SC:DOX) on glioblastoma cells with the expression of P-glycoprotein, a
major component of BBB and cancer multidrug resistance marker. Methods:
Cytotoxicity and selectivity towards glioblastoma cells of SC, DOX, their
combination, and SC:DOX were examined by MTT assay. The effect of SC on DOX
accumulation was determined by flow cytometry. We also studied SC:DOX
accumulation, cellular uptake, localization imaging, and DNA damage induction.
Results: The effects of simultaneous SC and DOX treatments demonstrated the
considerable potential of SC to reverse DOX resistance in glioblastoma cells and
increase DOX accumulation. SC:DOX hybrids, named CON1 and CON2 were less
cytotoxic than DOX, but with reduced resistance and increased selectivity towards
glioblastoma cells. Cellular uptake of CON1 and CON2 was increased in glioblastoma
cells compared to DOX. Perinuclear localization of CON1 and CON2 vs. nuclear
localization of DOX as well as no DNA damaging effects suggest a different
mechanism of action for SC:DOX. Conclusion: The combination of SC and DOX, and
their innovative hybrids, could be considered a promising strategy that can overcome
the limitations of DOX application in glioblastoma.",
publisher = "Belgrade: Serbian Neuroscience Society",
journal = "Book of abstracts: 8th Congress of Serbian neuroscience society with international participation; 2023 May 31 - Jun 2; Belgrade, Serbia",
title = "New anti-glioblastoma strategy with natural compounds sclareol and doxorubicin",
pages = "71",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5905"
}
Stepanović, A., Lupšić, E., Dinić, J., Podolski-Renić, A., Pajović, M., Jovanović Stojanov, S., Dragoj, M., Terzić Jovanović, N., Opsenica, I.,& Pešić, M.. (2023). New anti-glioblastoma strategy with natural compounds sclareol and doxorubicin. in Book of abstracts: 8th Congress of Serbian neuroscience society with international participation; 2023 May 31 - Jun 2; Belgrade, Serbia
Belgrade: Serbian Neuroscience Society., 71.
https://hdl.handle.net/21.15107/rcub_ibiss_5905
Stepanović A, Lupšić E, Dinić J, Podolski-Renić A, Pajović M, Jovanović Stojanov S, Dragoj M, Terzić Jovanović N, Opsenica I, Pešić M. New anti-glioblastoma strategy with natural compounds sclareol and doxorubicin. in Book of abstracts: 8th Congress of Serbian neuroscience society with international participation; 2023 May 31 - Jun 2; Belgrade, Serbia. 2023;:71.
https://hdl.handle.net/21.15107/rcub_ibiss_5905 .
Stepanović, Ana, Lupšić, Ema, Dinić, Jelena, Podolski-Renić, Ana, Pajović, Milica, Jovanović Stojanov, Sofija, Dragoj, Miodrag, Terzić Jovanović, Nataša, Opsenica, Igor, Pešić, Milica, "New anti-glioblastoma strategy with natural compounds sclareol and doxorubicin" in Book of abstracts: 8th Congress of Serbian neuroscience society with international participation; 2023 May 31 - Jun 2; Belgrade, Serbia (2023):71,
https://hdl.handle.net/21.15107/rcub_ibiss_5905 .

Novel functional immunoassay for identification of multidrug resistance markers in non-small cell lung carcinoma patient-derived cells

Stepanović, Ana; Dinić, Jelena; Podolski-Renić, Ana; Jovanović Stojanov, Sofija; Dragoj, Miodrag; Jovanović, Mirna; Lupšić, Ema; Milićević, Aleksandar; Glumac, Sofija; Marić, Dragana; Ercegovac, Maja; Pešić, Milica

(John Wiley and Sons Inc, 2023)

TY  - CONF
AU  - Stepanović, Ana
AU  - Dinić, Jelena
AU  - Podolski-Renić, Ana
AU  - Jovanović Stojanov, Sofija
AU  - Dragoj, Miodrag
AU  - Jovanović, Mirna
AU  - Lupšić, Ema
AU  - Milićević, Aleksandar
AU  - Glumac, Sofija
AU  - Marić, Dragana
AU  - Ercegovac, Maja
AU  - Pešić, Milica
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5884
AB  - Introduction: Multidrug resistance (MDR) significantly hampers nonsmall cell lung carcinoma (NSCLC) drugs’ efficacy. To evaluate the contribution of MDR markers to anticancer drugs’ sensitivity, we performed pharmacological screening on patient-derived NSCLC cells ex vivo and assessed the expression of MDR markers in cancer and stromal (non-cancer) cells.
Material and Methods: Primary patient-derived cultures were established from the NSCLC resections. After short-term culturing (2-3 weeks), a mixed population of cancer and non-cancer cells were treated with 8 chemotherapeutics (cisplatin, carboplatin, paclitaxel, docetaxel, etoposide, vinorelbine, gemcitabine, and pemetrexed). The maximum concentration reached in human plasma to which the patient is exposed during therapy (Cmax) was set as an upper limit and four lower concentrations were 
also applied during the study. Immunofluorescence assay enabling discrimination of epithelial cancer cells positive to a cocktail of antibodies against cytokeratin 8/18 vs. negative mesenchymal noncancer cells was conducted using high-content imager ImageXpress Pico (Molecular Devices) with CellReporterXpress 2.9 software. Within the same immunoassay, MDR markers (ABCB1, ABCC1, and ABCG2) were analyzed by corresponding antibodies.
Results and Discussions: Among all tested compounds, only gemcitabine increased the number of positive cancer cells to all MDR markers in all investigated primary cell cultures. Pemetrexed did not
change the number of MDR-positive cancer cells. In a patient sample IIIA stage bearing EGFR mutation
(L858R), the number of positive cancer cells to all MDR markers increased upon treatment with cisplatin, carboplatin, paclitaxel, docetaxel, etoposide, vinorelbine, and gemcitabine. Stromal (non-cancer) cells mainly followed the pattern of MDR observed in cancer cells.
Conclusion: Novel functional immunoassay can provide valuable information about the sensitivity of NSCLC to different drugs and possible treatment outcomes based on the
expression of MDR markers.
PB  - John Wiley and Sons Inc
C3  - EACR 2023 Congress: Innovative Cancer Science; 2023 Jun 12-15; Torino, Italy
T1  - Novel functional immunoassay for identification of multidrug resistance markers in non-small cell lung carcinoma patient-derived cells
DO  - 10.1002/1878-0261.13469
SP  - 461
EP  - 462
ER  - 
@conference{
author = "Stepanović, Ana and Dinić, Jelena and Podolski-Renić, Ana and Jovanović Stojanov, Sofija and Dragoj, Miodrag and Jovanović, Mirna and Lupšić, Ema and Milićević, Aleksandar and Glumac, Sofija and Marić, Dragana and Ercegovac, Maja and Pešić, Milica",
year = "2023",
abstract = "Introduction: Multidrug resistance (MDR) significantly hampers nonsmall cell lung carcinoma (NSCLC) drugs’ efficacy. To evaluate the contribution of MDR markers to anticancer drugs’ sensitivity, we performed pharmacological screening on patient-derived NSCLC cells ex vivo and assessed the expression of MDR markers in cancer and stromal (non-cancer) cells.
Material and Methods: Primary patient-derived cultures were established from the NSCLC resections. After short-term culturing (2-3 weeks), a mixed population of cancer and non-cancer cells were treated with 8 chemotherapeutics (cisplatin, carboplatin, paclitaxel, docetaxel, etoposide, vinorelbine, gemcitabine, and pemetrexed). The maximum concentration reached in human plasma to which the patient is exposed during therapy (Cmax) was set as an upper limit and four lower concentrations were 
also applied during the study. Immunofluorescence assay enabling discrimination of epithelial cancer cells positive to a cocktail of antibodies against cytokeratin 8/18 vs. negative mesenchymal noncancer cells was conducted using high-content imager ImageXpress Pico (Molecular Devices) with CellReporterXpress 2.9 software. Within the same immunoassay, MDR markers (ABCB1, ABCC1, and ABCG2) were analyzed by corresponding antibodies.
Results and Discussions: Among all tested compounds, only gemcitabine increased the number of positive cancer cells to all MDR markers in all investigated primary cell cultures. Pemetrexed did not
change the number of MDR-positive cancer cells. In a patient sample IIIA stage bearing EGFR mutation
(L858R), the number of positive cancer cells to all MDR markers increased upon treatment with cisplatin, carboplatin, paclitaxel, docetaxel, etoposide, vinorelbine, and gemcitabine. Stromal (non-cancer) cells mainly followed the pattern of MDR observed in cancer cells.
Conclusion: Novel functional immunoassay can provide valuable information about the sensitivity of NSCLC to different drugs and possible treatment outcomes based on the
expression of MDR markers.",
publisher = "John Wiley and Sons Inc",
journal = "EACR 2023 Congress: Innovative Cancer Science; 2023 Jun 12-15; Torino, Italy",
title = "Novel functional immunoassay for identification of multidrug resistance markers in non-small cell lung carcinoma patient-derived cells",
doi = "10.1002/1878-0261.13469",
pages = "461-462"
}
Stepanović, A., Dinić, J., Podolski-Renić, A., Jovanović Stojanov, S., Dragoj, M., Jovanović, M., Lupšić, E., Milićević, A., Glumac, S., Marić, D., Ercegovac, M.,& Pešić, M.. (2023). Novel functional immunoassay for identification of multidrug resistance markers in non-small cell lung carcinoma patient-derived cells. in EACR 2023 Congress: Innovative Cancer Science; 2023 Jun 12-15; Torino, Italy
John Wiley and Sons Inc., 461-462.
https://doi.org/10.1002/1878-0261.13469
Stepanović A, Dinić J, Podolski-Renić A, Jovanović Stojanov S, Dragoj M, Jovanović M, Lupšić E, Milićević A, Glumac S, Marić D, Ercegovac M, Pešić M. Novel functional immunoassay for identification of multidrug resistance markers in non-small cell lung carcinoma patient-derived cells. in EACR 2023 Congress: Innovative Cancer Science; 2023 Jun 12-15; Torino, Italy. 2023;:461-462.
doi:10.1002/1878-0261.13469 .
Stepanović, Ana, Dinić, Jelena, Podolski-Renić, Ana, Jovanović Stojanov, Sofija, Dragoj, Miodrag, Jovanović, Mirna, Lupšić, Ema, Milićević, Aleksandar, Glumac, Sofija, Marić, Dragana, Ercegovac, Maja, Pešić, Milica, "Novel functional immunoassay for identification of multidrug resistance markers in non-small cell lung carcinoma patient-derived cells" in EACR 2023 Congress: Innovative Cancer Science; 2023 Jun 12-15; Torino, Italy (2023):461-462,
https://doi.org/10.1002/1878-0261.13469 . .

Alpha-1 antitrypsin expression is upregulated in multidrug-resistant cancer cells

Divac Rankov, Aleksandra; Jovanović Stojanov, Sofija; Dragoj, Miodrag; Ljujić, Mila

(2022)

TY  - JOUR
AU  - Divac Rankov, Aleksandra
AU  - Jovanović Stojanov, Sofija
AU  - Dragoj, Miodrag
AU  - Ljujić, Mila
PY  - 2022
UR  - https://link.springer.com/10.1007/s00418-022-02172-3
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5350
AB  - Identification of the signature molecular profiles involved in therapy resistance is of vital importance in developing new strategies for treatments and disease monitoring. Protein alpha-1 antitrypsin (AAT, encoded by SERPINA1 gene) is an acute-phase protein, and its high expression has been linked with unfavorable clinical outcome in different types of cancer; however, data on its involvement in therapy resistance are still insufficient. We analyzed SERPINA1 mRNA expression in three different multidrug-resistant (MDR) cell lines—U87-TxR, NCI-H460/R, and DLD1-TxR—and in U87 cells grown in alginate microfibers as a 3D cellular model of glioblastoma. Expression of IL-6 as a major modulator of SERPINA1 was also analyzed. Additionally, AAT protein expression in MDR cells was analyzed by immunofluorescence. SERPINA1 gene expression and AAT protein expression were significantly upregulated in all the tested MDR cell lines compared with their sensitive counterparts. Moreover, SERPINA1 was significantly upregulated in 3D models of glioblastoma, previously found to have upregulated drug-resistance-related gene expression compared with 2D cells. With the exception of NCI-H460/R, in all cell lines as well as in a 3D model of U87 cells, increase in SERPINA1 expression correlated with the increase in IL-6 expression. Our results indicate that AAT could be utilized as a biomarker of therapy resistance in cancer; however, further studies are needed to elucidate the mechanisms driving AAT upregulation in therapy resistance and its biological significance in this process.
T2  - Histochemistry and Cell Biology
T1  - Alpha-1 antitrypsin expression is upregulated in multidrug-resistant cancer cells
DO  - 10.1007/s00418-022-02172-3
ER  - 
@article{
author = "Divac Rankov, Aleksandra and Jovanović Stojanov, Sofija and Dragoj, Miodrag and Ljujić, Mila",
year = "2022",
abstract = "Identification of the signature molecular profiles involved in therapy resistance is of vital importance in developing new strategies for treatments and disease monitoring. Protein alpha-1 antitrypsin (AAT, encoded by SERPINA1 gene) is an acute-phase protein, and its high expression has been linked with unfavorable clinical outcome in different types of cancer; however, data on its involvement in therapy resistance are still insufficient. We analyzed SERPINA1 mRNA expression in three different multidrug-resistant (MDR) cell lines—U87-TxR, NCI-H460/R, and DLD1-TxR—and in U87 cells grown in alginate microfibers as a 3D cellular model of glioblastoma. Expression of IL-6 as a major modulator of SERPINA1 was also analyzed. Additionally, AAT protein expression in MDR cells was analyzed by immunofluorescence. SERPINA1 gene expression and AAT protein expression were significantly upregulated in all the tested MDR cell lines compared with their sensitive counterparts. Moreover, SERPINA1 was significantly upregulated in 3D models of glioblastoma, previously found to have upregulated drug-resistance-related gene expression compared with 2D cells. With the exception of NCI-H460/R, in all cell lines as well as in a 3D model of U87 cells, increase in SERPINA1 expression correlated with the increase in IL-6 expression. Our results indicate that AAT could be utilized as a biomarker of therapy resistance in cancer; however, further studies are needed to elucidate the mechanisms driving AAT upregulation in therapy resistance and its biological significance in this process.",
journal = "Histochemistry and Cell Biology",
title = "Alpha-1 antitrypsin expression is upregulated in multidrug-resistant cancer cells",
doi = "10.1007/s00418-022-02172-3"
}
Divac Rankov, A., Jovanović Stojanov, S., Dragoj, M.,& Ljujić, M.. (2022). Alpha-1 antitrypsin expression is upregulated in multidrug-resistant cancer cells. in Histochemistry and Cell Biology.
https://doi.org/10.1007/s00418-022-02172-3
Divac Rankov A, Jovanović Stojanov S, Dragoj M, Ljujić M. Alpha-1 antitrypsin expression is upregulated in multidrug-resistant cancer cells. in Histochemistry and Cell Biology. 2022;.
doi:10.1007/s00418-022-02172-3 .
Divac Rankov, Aleksandra, Jovanović Stojanov, Sofija, Dragoj, Miodrag, Ljujić, Mila, "Alpha-1 antitrypsin expression is upregulated in multidrug-resistant cancer cells" in Histochemistry and Cell Biology (2022),
https://doi.org/10.1007/s00418-022-02172-3 . .
1

Decreased TSPAN14 Expression Contributes to NSCLC Progression

Jovanović, Mirna; Stanković, Tijana; Stojković Burić, Sonja; Banković, Jasna; Dinić, Jelena; Ljujić, Mila; Pešić, Milica; Dragoj, Miodrag

(Basel : MDPI, 2022)

TY  - JOUR
AU  - Jovanović, Mirna
AU  - Stanković, Tijana
AU  - Stojković Burić, Sonja
AU  - Banković, Jasna
AU  - Dinić, Jelena
AU  - Ljujić, Mila
AU  - Pešić, Milica
AU  - Dragoj, Miodrag
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5041
AB  - Tspan14 is a transmembrane protein of the tetraspanin (Tspan) protein family. Different members of the Tspan family can promote or suppress tumor progression. The exact role of Tspan14 in tumor cells is unknown. Earlier, mutational inactivation of the TSPAN14 gene has been proposed to coincide with a low survival rate in NSCLC patients. This study aimed to investigate the correlation of TSPAN14 lack of function with clinicopathological features of NSCLC patients, and to elucidate the role TSPAN14 might have in NSCLC progression. TSPAN14 expression was lower in tumor cells than non-tumor cells in NSCLC patients’ samples. The decreased gene expression was correlated with a low survival rate of patients and was more frequent in patients with aggressive, invasive tumor types. Additionally, the role of decreased TSPAN14 expression in the metastatic potential of cancer cells was confirmed in NSCLC cell lines. The highly invasive NSCLC cell line (NCI-H661) had the lowest TSPAN14 gene and protein expression, whereas the NSCLC cell line with the highest TSPAN14 expression (NCI-H460) had no significant metastatic potential. Finally, silencing of TSPAN14 in these non-metastatic cancer cells caused an increased expression of matrix-degrading enzymes MMP-2 and MMP-9, followed by an elevated capacity of cancer cells to degrade gelatin. The results of this study propose TSPAN14 expression as an indicator of NSCLC metastatic potential and progression.
PB  - Basel : MDPI
T2  - Life
T1  - Decreased TSPAN14 Expression Contributes to NSCLC Progression
IS  - 9
VL  - 12
DO  - 10.3390/life12091291
SP  - 1291
ER  - 
@article{
author = "Jovanović, Mirna and Stanković, Tijana and Stojković Burić, Sonja and Banković, Jasna and Dinić, Jelena and Ljujić, Mila and Pešić, Milica and Dragoj, Miodrag",
year = "2022",
abstract = "Tspan14 is a transmembrane protein of the tetraspanin (Tspan) protein family. Different members of the Tspan family can promote or suppress tumor progression. The exact role of Tspan14 in tumor cells is unknown. Earlier, mutational inactivation of the TSPAN14 gene has been proposed to coincide with a low survival rate in NSCLC patients. This study aimed to investigate the correlation of TSPAN14 lack of function with clinicopathological features of NSCLC patients, and to elucidate the role TSPAN14 might have in NSCLC progression. TSPAN14 expression was lower in tumor cells than non-tumor cells in NSCLC patients’ samples. The decreased gene expression was correlated with a low survival rate of patients and was more frequent in patients with aggressive, invasive tumor types. Additionally, the role of decreased TSPAN14 expression in the metastatic potential of cancer cells was confirmed in NSCLC cell lines. The highly invasive NSCLC cell line (NCI-H661) had the lowest TSPAN14 gene and protein expression, whereas the NSCLC cell line with the highest TSPAN14 expression (NCI-H460) had no significant metastatic potential. Finally, silencing of TSPAN14 in these non-metastatic cancer cells caused an increased expression of matrix-degrading enzymes MMP-2 and MMP-9, followed by an elevated capacity of cancer cells to degrade gelatin. The results of this study propose TSPAN14 expression as an indicator of NSCLC metastatic potential and progression.",
publisher = "Basel : MDPI",
journal = "Life",
title = "Decreased TSPAN14 Expression Contributes to NSCLC Progression",
number = "9",
volume = "12",
doi = "10.3390/life12091291",
pages = "1291"
}
Jovanović, M., Stanković, T., Stojković Burić, S., Banković, J., Dinić, J., Ljujić, M., Pešić, M.,& Dragoj, M.. (2022). Decreased TSPAN14 Expression Contributes to NSCLC Progression. in Life
Basel : MDPI., 12(9), 1291.
https://doi.org/10.3390/life12091291
Jovanović M, Stanković T, Stojković Burić S, Banković J, Dinić J, Ljujić M, Pešić M, Dragoj M. Decreased TSPAN14 Expression Contributes to NSCLC Progression. in Life. 2022;12(9):1291.
doi:10.3390/life12091291 .
Jovanović, Mirna, Stanković, Tijana, Stojković Burić, Sonja, Banković, Jasna, Dinić, Jelena, Ljujić, Mila, Pešić, Milica, Dragoj, Miodrag, "Decreased TSPAN14 Expression Contributes to NSCLC Progression" in Life, 12, no. 9 (2022):1291,
https://doi.org/10.3390/life12091291 . .
1
5
4

Anti-tumorski efekat inhibitora ugljenične anhidraze - derivata kumarina na ćelijama tumora pluća

Jovanović, Mirna; Jovanović Stojanov, Sofija; Dragoj, Miodrag; Stepanović, Ana; Lupšić, Ema; Podolski-Renić, Ana; Dinić, Jelena; Pešić, Milica

(Belgrade: Serbian Biological Society, 2022)

TY  - CONF
AU  - Jovanović, Mirna
AU  - Jovanović Stojanov, Sofija
AU  - Dragoj, Miodrag
AU  - Stepanović, Ana
AU  - Lupšić, Ema
AU  - Podolski-Renić, Ana
AU  - Dinić, Jelena
AU  - Pešić, Milica
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5392
AB  - Експресија  ензима  угљеничне  анхидразе  9  (УА9)  је  често  повишена  код  ћелија 
тумора.1  УА9 има важну улогу у регулацији pH вредности која доприноси расту и 
деоби  туморске  ћелије,2  због  чега  се  инхибитори  овог  ензима  истражују  као кандидати за анти-туморске лекове.3 Овде је описан анти-туморски ефекат новог деривата кумарина, гуанидина АФА-36, за који је показано да инхибира активност УА9.  У  испитивању,  коришћена  је  туморска  ћелијска  линија  неситноћелијског карцинома  плућа  (NCI-H460).  Показано  је  да  АФА-36  инхибира  раст  NCI-H460 ћелија  узгајаних  у  једном  слоју,  у  условима  нормоксије  и  хипоксије,  са  ИЦ50 вредностима око 5 μМ. У тесту ћелијске смрти, 25 μМ АФА-36 селективно доводи до смрти NCI-H460 (50% мртвих ћелија) у поређењу са нормалним фибробластима човека  MRC-5  (без  ефекта  ћелијске  смрти).  Флуоресцентном  бојом  BCECF показано је да 5 μМ АФА-36 смањује унутарћелијску pH, за око 30% у односу на нетретиране NCI-H460 ћелије. Притом, под третманом се експресија УА9 повећава 1,5 пута. Флуоресцентном бојом TMRE показали смо да АФА-36 има инхибиторни ефекат  на  активност  митохондрија.  Инхибиција  раста  NCI-H460  ћелија  са  5  μМ 
АФА-36  узгајаних  у  3Д  систему  алгинатних  влакана  је  израженија  у  хипоксији, него  у нормоксији.  Дериват  кумарина  АФА-36  са  својством  инхибитора  УА9, остварује  значајан  анти-туморски  ефекат  и  има  потенцијал  за  даља  опсежнија преклиничка испитивања посебно код тумора са израженим зонама хипоксије које доприносе већој малигности. 
1.  Ivanov, S., Liao, S.Y., Ivanova, A., et al., 2001, Am. J. Pathol. 158:905-919. 
2.  Sedlakova, O., Svastova, E., Takacova, M., et al., 2014, Front. Physiol. 4:400. 
3.  Supuran, C.T., 2008, Nat. Rev. Drug Discov. 7:168-181.
AB  - Ekspresija enzima ugljenične anhidraze 9 (UA9) je često povišena kod ćelija tumora.1 UA9 ima važnu ulogu u regulaciji pH vrednosti koja doprinosi rastu i deobi tumorske ćelije,2 zbog čega se inhibitori ovog enzima istražuju kao kandidati za anti-tumorske lekove.3 Ovde je opisan anti-tumorski efekat novog derivata kumarina, guanidina AFA-36, za koji je pokazano da inhibira aktivnost UA9. U ispitivanju, korišćena je tumorska ćelijska linija nesitnoćelijskog karcinoma pluća (NCI-H460). Pokazano je da AFA-36 inhibira rast NCI-H460 ćelija uzgajanih u jednom sloju, u uslovima normoksije i hipoksije, sa IC50 vrednostima oko 5 μM. U testu ćelijske smrti, 25 μM AFA-36 selektivno dovodi do smrti NCI-H460 (50% mrtvih ćelija) u poređenju sa normalnim fibroblastima čoveka MRC-5 (bez efekta ćelijske smrti). Fluorescentnom bojom BCECF pokazano je da 5 μM AFA-36 smanjuje unutarćelijsku pH, za oko 30% u odnosu na netretirane NCI-H460 ćelije. Pritom, pod tretmanom se ekspresija UA9 povećava 1,5 puta. Fluorescentnom bojom TMRE pokazali smo da AFA-36 ima inhibitorni efekat na aktivnost mitohondrija. Inhibicija rasta NCI-H460 ćelija sa 5 μM AFA-36 uzgajanih u 3D sistemu alginatnih vlakana je izraženija u hipoksiji, nego u normoksiji. Derivat kumarina AFA-36 sa svojstvom inhibitora UA9, ostvaruje značajan anti-tumorski efekat i ima potencijal za dalja opsežnija preklinička ispitivanja posebno kod tumora sa izraženim zonama hipoksije koje doprinose većoj malignosti.
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - Anti-tumorski efekat inhibitora ugljenične anhidraze - derivata kumarina na ćelijama tumora pluća
T1  - Анти-туморски ефекат инхибитора угљеничне анхидразе – деривата кумарина на ћелијама тумора плућа
SP  - 318
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5392
ER  - 
@conference{
author = "Jovanović, Mirna and Jovanović Stojanov, Sofija and Dragoj, Miodrag and Stepanović, Ana and Lupšić, Ema and Podolski-Renić, Ana and Dinić, Jelena and Pešić, Milica",
year = "2022",
abstract = "Експресија  ензима  угљеничне  анхидразе  9  (УА9)  је  често  повишена  код  ћелија 
тумора.1  УА9 има важну улогу у регулацији pH вредности која доприноси расту и 
деоби  туморске  ћелије,2  због  чега  се  инхибитори  овог  ензима  истражују  као кандидати за анти-туморске лекове.3 Овде је описан анти-туморски ефекат новог деривата кумарина, гуанидина АФА-36, за који је показано да инхибира активност УА9.  У  испитивању,  коришћена  је  туморска  ћелијска  линија  неситноћелијског карцинома  плућа  (NCI-H460).  Показано  је  да  АФА-36  инхибира  раст  NCI-H460 ћелија  узгајаних  у  једном  слоју,  у  условима  нормоксије  и  хипоксије,  са  ИЦ50 вредностима око 5 μМ. У тесту ћелијске смрти, 25 μМ АФА-36 селективно доводи до смрти NCI-H460 (50% мртвих ћелија) у поређењу са нормалним фибробластима човека  MRC-5  (без  ефекта  ћелијске  смрти).  Флуоресцентном  бојом  BCECF показано је да 5 μМ АФА-36 смањује унутарћелијску pH, за око 30% у односу на нетретиране NCI-H460 ћелије. Притом, под третманом се експресија УА9 повећава 1,5 пута. Флуоресцентном бојом TMRE показали смо да АФА-36 има инхибиторни ефекат  на  активност  митохондрија.  Инхибиција  раста  NCI-H460  ћелија  са  5  μМ 
АФА-36  узгајаних  у  3Д  систему  алгинатних  влакана  је  израженија  у  хипоксији, него  у нормоксији.  Дериват  кумарина  АФА-36  са  својством  инхибитора  УА9, остварује  значајан  анти-туморски  ефекат  и  има  потенцијал  за  даља  опсежнија преклиничка испитивања посебно код тумора са израженим зонама хипоксије које доприносе већој малигности. 
1.  Ivanov, S., Liao, S.Y., Ivanova, A., et al., 2001, Am. J. Pathol. 158:905-919. 
2.  Sedlakova, O., Svastova, E., Takacova, M., et al., 2014, Front. Physiol. 4:400. 
3.  Supuran, C.T., 2008, Nat. Rev. Drug Discov. 7:168-181., Ekspresija enzima ugljenične anhidraze 9 (UA9) je često povišena kod ćelija tumora.1 UA9 ima važnu ulogu u regulaciji pH vrednosti koja doprinosi rastu i deobi tumorske ćelije,2 zbog čega se inhibitori ovog enzima istražuju kao kandidati za anti-tumorske lekove.3 Ovde je opisan anti-tumorski efekat novog derivata kumarina, guanidina AFA-36, za koji je pokazano da inhibira aktivnost UA9. U ispitivanju, korišćena je tumorska ćelijska linija nesitnoćelijskog karcinoma pluća (NCI-H460). Pokazano je da AFA-36 inhibira rast NCI-H460 ćelija uzgajanih u jednom sloju, u uslovima normoksije i hipoksije, sa IC50 vrednostima oko 5 μM. U testu ćelijske smrti, 25 μM AFA-36 selektivno dovodi do smrti NCI-H460 (50% mrtvih ćelija) u poređenju sa normalnim fibroblastima čoveka MRC-5 (bez efekta ćelijske smrti). Fluorescentnom bojom BCECF pokazano je da 5 μM AFA-36 smanjuje unutarćelijsku pH, za oko 30% u odnosu na netretirane NCI-H460 ćelije. Pritom, pod tretmanom se ekspresija UA9 povećava 1,5 puta. Fluorescentnom bojom TMRE pokazali smo da AFA-36 ima inhibitorni efekat na aktivnost mitohondrija. Inhibicija rasta NCI-H460 ćelija sa 5 μM AFA-36 uzgajanih u 3D sistemu alginatnih vlakana je izraženija u hipoksiji, nego u normoksiji. Derivat kumarina AFA-36 sa svojstvom inhibitora UA9, ostvaruje značajan anti-tumorski efekat i ima potencijal za dalja opsežnija preklinička ispitivanja posebno kod tumora sa izraženim zonama hipoksije koje doprinose većoj malignosti.",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "Anti-tumorski efekat inhibitora ugljenične anhidraze - derivata kumarina na ćelijama tumora pluća, Анти-туморски ефекат инхибитора угљеничне анхидразе – деривата кумарина на ћелијама тумора плућа",
pages = "318",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5392"
}
Jovanović, M., Jovanović Stojanov, S., Dragoj, M., Stepanović, A., Lupšić, E., Podolski-Renić, A., Dinić, J.,& Pešić, M.. (2022). Anti-tumorski efekat inhibitora ugljenične anhidraze - derivata kumarina na ćelijama tumora pluća. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 318.
https://hdl.handle.net/21.15107/rcub_ibiss_5392
Jovanović M, Jovanović Stojanov S, Dragoj M, Stepanović A, Lupšić E, Podolski-Renić A, Dinić J, Pešić M. Anti-tumorski efekat inhibitora ugljenične anhidraze - derivata kumarina na ćelijama tumora pluća. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:318.
https://hdl.handle.net/21.15107/rcub_ibiss_5392 .
Jovanović, Mirna, Jovanović Stojanov, Sofija, Dragoj, Miodrag, Stepanović, Ana, Lupšić, Ema, Podolski-Renić, Ana, Dinić, Jelena, Pešić, Milica, "Anti-tumorski efekat inhibitora ugljenične anhidraze - derivata kumarina na ćelijama tumora pluća" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):318,
https://hdl.handle.net/21.15107/rcub_ibiss_5392 .

Anticancer effect of new carbonic anhydrase 9 inhibitors in glioblastoma cells

Jovanović, Mirna; Jovanović Stojanov, Sofija; Dragoj, Miodrag; Stepanović, Ana; Lupšić, Ema; Podolski-Renić, Ana; Dinić, Jelena; Pešić, Milica

(2022)

TY  - CONF
AU  - Jovanović, Mirna
AU  - Jovanović Stojanov, Sofija
AU  - Dragoj, Miodrag
AU  - Stepanović, Ana
AU  - Lupšić, Ema
AU  - Podolski-Renić, Ana
AU  - Dinić, Jelena
AU  - Pešić, Milica
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5467
AB  - Introduction: Carbonic anhydrase 9 (CA9) is a membrane enzyme, a regulator of intracellular and extracellular pH, overexpressed in cells in a hypoxic environment [1]. Solid tumors, adapted to hypoxia, have large quantities of the CA9 and the increased expression correlates with tumor patients’ poor prognosis, tumor malignancy, and resistance to drugs [2]. In glioblastoma, hypoxia promotes the spreading of cancer cells into the brain tissue, to evade the environment with low oxygen levels [3]. Inhibitors of CA9 have previously been investigated for anticancer drugs [2]. In the present study, we evaluated the anticancer properties of three CA9 inhibitors (AFA-30, AFA-40 and AFA-49), phosphonium salts derived from coumarin, in sensitive (U87) and chemoresistant (U87-TxR) human glioblastoma cell lines. 
Materials & Methods: The effect of CA9 inhibitors on cell growth, either alone or in combination with tariquidar was determined by sulforhodamine B assay. Flow cytometry was used for the assessment of change in intracellular pH by BCECF staining, and the rhodamine 123 assay of P-gp activity. Changes in the expression of CA9, CA12, and ABCB1 genes were analyzed by qPCR.
Results: The three compounds inhibited cell growth of both sensitive (U87) and resistant (U87-TxR) cells in 48 h treatments, in both hypoxic (1% O2) and normoxic (20% O2) conditions. However, compared to U87 (IC50 range 1 - 5 μM), the U87-TxR were less sensitive to the compounds’ growth inhibition effect (IC50 range 8 – 30 μM). U87-TxR cells are characterized by the increased expression of the P-gp extruding pump. When tariquidar, a P-gp inhibitor, was applied in combination with CA9 inhibitors, U87-TxR cells were sensitized to these compounds. In the P-gp activity assay, we demonstrated that compounds (5 – 50 μM) increase a P-gp substrate accumulation – rhodamine 123. Further, gene expression of ABCB1 was increased 2 – 8 times in U87, following treatment. In 24 h treatments, these CA9 inhibitors decreased intracellular pH. Moreover, the 24 h treatments resulted in decreased expression of CA9 and CA12.
Conclusion: The three CA9 inhibitors here described have significant anticancer effects in glioblastoma cells and show potential for further pre-clinical investigation, especially in tumors with emphasized hypoxic zones contributing to increased malignancy, such as glioblastomas. 
1.	Mussi, S., et al., Antiproliferative effects of sulphonamide carbonic anhydrase inhibitors C18, SLC-0111 and acetazolamide on bladder, glioblastoma and pancreatic cancer cell lines. J Enzyme Inhib Med Chem, 2022. 37(1): p. 280-286.
2.	Kalinin, S., et al., Carbonic Anhydrase IX Inhibitors as Candidates for Combination Therapy of Solid Tumors. Int J Mol Sci, 2021. 22(24).
3.	Monteiro, A.R., et al., The Role of Hypoxia in Glioblastoma Invasion. Cells, 2017. 6(4).
 Funding: This research was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia (ref. number 451-03-68/2020-14/200007).
C3  - Abstract Book: 3rd Symposium in Biomedicine: Basic and Clinical Neuroscience; 2022 Jun 29; Belgrade, Serbia
T1  - Anticancer effect of new carbonic anhydrase 9 inhibitors in glioblastoma cells
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5467
ER  - 
@conference{
author = "Jovanović, Mirna and Jovanović Stojanov, Sofija and Dragoj, Miodrag and Stepanović, Ana and Lupšić, Ema and Podolski-Renić, Ana and Dinić, Jelena and Pešić, Milica",
year = "2022",
abstract = "Introduction: Carbonic anhydrase 9 (CA9) is a membrane enzyme, a regulator of intracellular and extracellular pH, overexpressed in cells in a hypoxic environment [1]. Solid tumors, adapted to hypoxia, have large quantities of the CA9 and the increased expression correlates with tumor patients’ poor prognosis, tumor malignancy, and resistance to drugs [2]. In glioblastoma, hypoxia promotes the spreading of cancer cells into the brain tissue, to evade the environment with low oxygen levels [3]. Inhibitors of CA9 have previously been investigated for anticancer drugs [2]. In the present study, we evaluated the anticancer properties of three CA9 inhibitors (AFA-30, AFA-40 and AFA-49), phosphonium salts derived from coumarin, in sensitive (U87) and chemoresistant (U87-TxR) human glioblastoma cell lines. 
Materials & Methods: The effect of CA9 inhibitors on cell growth, either alone or in combination with tariquidar was determined by sulforhodamine B assay. Flow cytometry was used for the assessment of change in intracellular pH by BCECF staining, and the rhodamine 123 assay of P-gp activity. Changes in the expression of CA9, CA12, and ABCB1 genes were analyzed by qPCR.
Results: The three compounds inhibited cell growth of both sensitive (U87) and resistant (U87-TxR) cells in 48 h treatments, in both hypoxic (1% O2) and normoxic (20% O2) conditions. However, compared to U87 (IC50 range 1 - 5 μM), the U87-TxR were less sensitive to the compounds’ growth inhibition effect (IC50 range 8 – 30 μM). U87-TxR cells are characterized by the increased expression of the P-gp extruding pump. When tariquidar, a P-gp inhibitor, was applied in combination with CA9 inhibitors, U87-TxR cells were sensitized to these compounds. In the P-gp activity assay, we demonstrated that compounds (5 – 50 μM) increase a P-gp substrate accumulation – rhodamine 123. Further, gene expression of ABCB1 was increased 2 – 8 times in U87, following treatment. In 24 h treatments, these CA9 inhibitors decreased intracellular pH. Moreover, the 24 h treatments resulted in decreased expression of CA9 and CA12.
Conclusion: The three CA9 inhibitors here described have significant anticancer effects in glioblastoma cells and show potential for further pre-clinical investigation, especially in tumors with emphasized hypoxic zones contributing to increased malignancy, such as glioblastomas. 
1.	Mussi, S., et al., Antiproliferative effects of sulphonamide carbonic anhydrase inhibitors C18, SLC-0111 and acetazolamide on bladder, glioblastoma and pancreatic cancer cell lines. J Enzyme Inhib Med Chem, 2022. 37(1): p. 280-286.
2.	Kalinin, S., et al., Carbonic Anhydrase IX Inhibitors as Candidates for Combination Therapy of Solid Tumors. Int J Mol Sci, 2021. 22(24).
3.	Monteiro, A.R., et al., The Role of Hypoxia in Glioblastoma Invasion. Cells, 2017. 6(4).
 Funding: This research was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia (ref. number 451-03-68/2020-14/200007).",
journal = "Abstract Book: 3rd Symposium in Biomedicine: Basic and Clinical Neuroscience; 2022 Jun 29; Belgrade, Serbia",
title = "Anticancer effect of new carbonic anhydrase 9 inhibitors in glioblastoma cells",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5467"
}
Jovanović, M., Jovanović Stojanov, S., Dragoj, M., Stepanović, A., Lupšić, E., Podolski-Renić, A., Dinić, J.,& Pešić, M.. (2022). Anticancer effect of new carbonic anhydrase 9 inhibitors in glioblastoma cells. in Abstract Book: 3rd Symposium in Biomedicine: Basic and Clinical Neuroscience; 2022 Jun 29; Belgrade, Serbia.
https://hdl.handle.net/21.15107/rcub_ibiss_5467
Jovanović M, Jovanović Stojanov S, Dragoj M, Stepanović A, Lupšić E, Podolski-Renić A, Dinić J, Pešić M. Anticancer effect of new carbonic anhydrase 9 inhibitors in glioblastoma cells. in Abstract Book: 3rd Symposium in Biomedicine: Basic and Clinical Neuroscience; 2022 Jun 29; Belgrade, Serbia. 2022;.
https://hdl.handle.net/21.15107/rcub_ibiss_5467 .
Jovanović, Mirna, Jovanović Stojanov, Sofija, Dragoj, Miodrag, Stepanović, Ana, Lupšić, Ema, Podolski-Renić, Ana, Dinić, Jelena, Pešić, Milica, "Anticancer effect of new carbonic anhydrase 9 inhibitors in glioblastoma cells" in Abstract Book: 3rd Symposium in Biomedicine: Basic and Clinical Neuroscience; 2022 Jun 29; Belgrade, Serbia (2022),
https://hdl.handle.net/21.15107/rcub_ibiss_5467 .

Functional diagnostics as a new concept for the improvement of personalized targeted therapy

Dragoj, Miodrag; Dinić, Jelena; Podolski-Renić, Ana; Jovanović Stojanov, Sofija; Jovanović, Mirna; Stepanović, Ana; Ercegovac, Maja; Marić, Dragana; Pešić, Milica

(STRATAGEM COST Action, 2022)

TY  - CONF
AU  - Dragoj, Miodrag
AU  - Dinić, Jelena
AU  - Podolski-Renić, Ana
AU  - Jovanović Stojanov, Sofija
AU  - Jovanović, Mirna
AU  - Stepanović, Ana
AU  - Ercegovac, Maja
AU  - Marić, Dragana
AU  - Pešić, Milica
PY  - 2022
UR  - https://stratagem-cost.eu/2022/04/stratagems-5th-co-located-annual-conference-and-wg3-4-training-school-to-be-held-in-coimbra-portugal-june-july-2022/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5371
AB  - Although advances in sequencing technology and target identification enabled the implementation of a
personalized therapy approach, unfortunately, only 3-9% of cancer patients who receive the targeted
therapy show an adequate response. On the other side, there are exceptional responders to targeted
therapy among cancer patients without common genetic alterations. Therefore, current patient
classifications relying only on sequencing are not sufficient to determine optimal treatment. Our
intention is to start in the opposite direction to conventional diagnostics by performing pharmacological
screening on patient-derived cancer cells ex vivo because testing of multiple drugs is not possible in
clinical trials. An incomplete understanding of how tumour genotype reflects on tumour phenotype
limits the efficacy of DNA and mRNA sequencing for personalized therapy. Functional diagnostics using
patient-derived cancer cells is recently implicated to overcome this limitation and it is clinically available
for haematological malignancies. We plan to perform the immunofluorescence-based drug-screening
assay to determine non-small cell lung carcinoma (NSCLC) patients’ cancer cells’ response to targeted
therapeutics, particularly tyrosine kinase inhibitors (TKIs) within the time frame necessary to influence
patient care. The usage of the functional diagnostics approach should be an addition to clinical trials and
complement DNA and mRNA sequencing.
In contrast to similar research efforts [1], we will shorten the cultivation of NSCLC patient-derived
cells to 1-2 weeks because we intend to test drugs on a mixture of cancer and stromal cells (fibroblasts).
It is well-known that the sensitivity of cancer cells depends on their interaction with the
microenvironment including neighbouring cells. In addition, we will examine the changes in the
expression level of ATP Binding Cassette transporters (ABCB1, ABCC1, and ABCG2) in both cancer
and stromal cells that may occur during TKIs and chemotherapy treatment. In such way, we will gain
knowledge about (i) which TKI or chemotherapeutic induces multidrug-resistant (MDR) phenotype in
our NSCLC patients’ cohort, (ii) whether the induction of MDR depends on the ratio between cancer
and stromal cells, (iii) whether the induction of MDR is prevalent in cancer cells, and (iv) whether MDR
induction depends on individual patient’s characteristics (comparison with Whole Exome Sequencing
results).
PB  - STRATAGEM COST Action
C3  - Abstract Book: STRATAGEM’s 5th Annual Meeting: New Diagnostic and Therapeutic Tools against Multidrug Resistant Tumours; 2022 Jun 29 - Jul 1; Coimbra, Portugal
T1  - Functional diagnostics as a new concept for the improvement of personalized targeted therapy
SP  - 94
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5371
ER  - 
@conference{
author = "Dragoj, Miodrag and Dinić, Jelena and Podolski-Renić, Ana and Jovanović Stojanov, Sofija and Jovanović, Mirna and Stepanović, Ana and Ercegovac, Maja and Marić, Dragana and Pešić, Milica",
year = "2022",
abstract = "Although advances in sequencing technology and target identification enabled the implementation of a
personalized therapy approach, unfortunately, only 3-9% of cancer patients who receive the targeted
therapy show an adequate response. On the other side, there are exceptional responders to targeted
therapy among cancer patients without common genetic alterations. Therefore, current patient
classifications relying only on sequencing are not sufficient to determine optimal treatment. Our
intention is to start in the opposite direction to conventional diagnostics by performing pharmacological
screening on patient-derived cancer cells ex vivo because testing of multiple drugs is not possible in
clinical trials. An incomplete understanding of how tumour genotype reflects on tumour phenotype
limits the efficacy of DNA and mRNA sequencing for personalized therapy. Functional diagnostics using
patient-derived cancer cells is recently implicated to overcome this limitation and it is clinically available
for haematological malignancies. We plan to perform the immunofluorescence-based drug-screening
assay to determine non-small cell lung carcinoma (NSCLC) patients’ cancer cells’ response to targeted
therapeutics, particularly tyrosine kinase inhibitors (TKIs) within the time frame necessary to influence
patient care. The usage of the functional diagnostics approach should be an addition to clinical trials and
complement DNA and mRNA sequencing.
In contrast to similar research efforts [1], we will shorten the cultivation of NSCLC patient-derived
cells to 1-2 weeks because we intend to test drugs on a mixture of cancer and stromal cells (fibroblasts).
It is well-known that the sensitivity of cancer cells depends on their interaction with the
microenvironment including neighbouring cells. In addition, we will examine the changes in the
expression level of ATP Binding Cassette transporters (ABCB1, ABCC1, and ABCG2) in both cancer
and stromal cells that may occur during TKIs and chemotherapy treatment. In such way, we will gain
knowledge about (i) which TKI or chemotherapeutic induces multidrug-resistant (MDR) phenotype in
our NSCLC patients’ cohort, (ii) whether the induction of MDR depends on the ratio between cancer
and stromal cells, (iii) whether the induction of MDR is prevalent in cancer cells, and (iv) whether MDR
induction depends on individual patient’s characteristics (comparison with Whole Exome Sequencing
results).",
publisher = "STRATAGEM COST Action",
journal = "Abstract Book: STRATAGEM’s 5th Annual Meeting: New Diagnostic and Therapeutic Tools against Multidrug Resistant Tumours; 2022 Jun 29 - Jul 1; Coimbra, Portugal",
title = "Functional diagnostics as a new concept for the improvement of personalized targeted therapy",
pages = "94",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5371"
}
Dragoj, M., Dinić, J., Podolski-Renić, A., Jovanović Stojanov, S., Jovanović, M., Stepanović, A., Ercegovac, M., Marić, D.,& Pešić, M.. (2022). Functional diagnostics as a new concept for the improvement of personalized targeted therapy. in Abstract Book: STRATAGEM’s 5th Annual Meeting: New Diagnostic and Therapeutic Tools against Multidrug Resistant Tumours; 2022 Jun 29 - Jul 1; Coimbra, Portugal
STRATAGEM COST Action., 94.
https://hdl.handle.net/21.15107/rcub_ibiss_5371
Dragoj M, Dinić J, Podolski-Renić A, Jovanović Stojanov S, Jovanović M, Stepanović A, Ercegovac M, Marić D, Pešić M. Functional diagnostics as a new concept for the improvement of personalized targeted therapy. in Abstract Book: STRATAGEM’s 5th Annual Meeting: New Diagnostic and Therapeutic Tools against Multidrug Resistant Tumours; 2022 Jun 29 - Jul 1; Coimbra, Portugal. 2022;:94.
https://hdl.handle.net/21.15107/rcub_ibiss_5371 .
Dragoj, Miodrag, Dinić, Jelena, Podolski-Renić, Ana, Jovanović Stojanov, Sofija, Jovanović, Mirna, Stepanović, Ana, Ercegovac, Maja, Marić, Dragana, Pešić, Milica, "Functional diagnostics as a new concept for the improvement of personalized targeted therapy" in Abstract Book: STRATAGEM’s 5th Annual Meeting: New Diagnostic and Therapeutic Tools against Multidrug Resistant Tumours; 2022 Jun 29 - Jul 1; Coimbra, Portugal (2022):94,
https://hdl.handle.net/21.15107/rcub_ibiss_5371 .

Autophagy inhibition sensitises glioblastoma cells to Src family kinase inhibitors Si306 and its prodrug

Jovanović Stojanov, Sofija; Stepanović, Ana; Ljujić, Mila; Lupšić, Ema; Podolski-Renić, Ana; Dragoj, Miodrag; Jovanović, Mirna; Schenone, Silvia; Pešić, Milica; Dinić, Jelena

(European Association for Cancer Research, 2022)

TY  - CONF
AU  - Jovanović Stojanov, Sofija
AU  - Stepanović, Ana
AU  - Ljujić, Mila
AU  - Lupšić, Ema
AU  - Podolski-Renić, Ana
AU  - Dragoj, Miodrag
AU  - Jovanović, Mirna
AU  - Schenone, Silvia
AU  - Pešić, Milica
AU  - Dinić, Jelena
PY  - 2022
UR  - http://nwm.covr.be/EACR2022abstracts/data/HtmlApp/main.html#
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5425
AB  - Introduction:
Glioblastoma (GBM) is among the most frequent and aggressive brain tumors characterized by
infiltrating nature, high proliferation, and resistance to chemotherapy and radiation. GBM exhibit high
expression of Src tyrosine kinase which regulates proliferation, survival, and invasiveness of tumor
cells, making Src a potential target for GBM therapy. Numerous Src family kinase inhibitors (SFKI)
were reported to induce autophagy, thus protecting cells from undergoing cell death. However,
inhibition of autophagy was shown to sensitize cells to SFKI in several cancer types.
Material and Methods:
Human GBM cell line U87 and its multidrug-resistant (MDR) counterpart U87-TxR were transfected
with RFP-LC3, an autophagy marker. The ability of two SFKIs, pyrazolo[3,4-d]pyrimidines Si306 and
its prodrug pro-si306, to induce autophagy in RFP-LC3-transfected GBM cells was evaluated by flow
cytometry and fluorescent microscopy. Cell viability was assessed by MTT assay. The autophagy
induction and autophagic flux were evaluated by Acridine orange assay, immunocytochemistry and
immunoblotting. Cell proliferation rate was analyzed by CFSE assay. Cell death was detected by
Annexin/Propidium Iodide assay. PARP-1 cleavage was assessed by immunoblotting.
Results and Discussions:
SFKI treatment resulted in degradation of RFP-LC3 after 3 h treatment as well as in formation of
RFP-LC3 puncta in GBM cells demonstrating autophagy induction. The effect of SFKIs on autophagy
induction persisted after 48 h, as demonstrated by autophagy markers LC3 and p62. Inhibition of
autophagy by Bafilomycin A1 sensitized both U87 and U87-TxR cells to Si306 and its pro-drug after
48 h. The anti-proliferative effect of Si306 and pro-Si306 was additionally increased after autophagy
inhibition by Bafilomycin A1. Furthermore, while single SFKI treatments did not cause significant
cell death, combination treatments with autophagy inhibitor induced necrosis in U87 and U87-TxR
cells after 48 h. Detection of necrotic PARP-1 fragment further confirmed necrotic cell death.
Conclusion:
Taken together, these data suggest that autophagy induced by Si306 and pro-Si306 has a protective
role in GBM cells, and that autophagy modulation may be used to enhance the anticancer effects of
SFKIs. In addition, as the ability of the SFKIs to induce autophagy was not diminished by the
presence of the MDR phenotype makes these compounds promising for treatment of MDR cancers.
PB  - European Association for Cancer Research
C3  - Congress abstracts: Annual Congress of the European Association for Cancer Research EACR 2022: Innovative Cancer Service: Translating Biology to Medicine; 2022 Jun 20-23; Seville, Spain
T1  - Autophagy inhibition sensitises glioblastoma cells to Src family kinase inhibitors Si306 and its prodrug
SP  - P1-135
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5425
ER  - 
@conference{
author = "Jovanović Stojanov, Sofija and Stepanović, Ana and Ljujić, Mila and Lupšić, Ema and Podolski-Renić, Ana and Dragoj, Miodrag and Jovanović, Mirna and Schenone, Silvia and Pešić, Milica and Dinić, Jelena",
year = "2022",
abstract = "Introduction:
Glioblastoma (GBM) is among the most frequent and aggressive brain tumors characterized by
infiltrating nature, high proliferation, and resistance to chemotherapy and radiation. GBM exhibit high
expression of Src tyrosine kinase which regulates proliferation, survival, and invasiveness of tumor
cells, making Src a potential target for GBM therapy. Numerous Src family kinase inhibitors (SFKI)
were reported to induce autophagy, thus protecting cells from undergoing cell death. However,
inhibition of autophagy was shown to sensitize cells to SFKI in several cancer types.
Material and Methods:
Human GBM cell line U87 and its multidrug-resistant (MDR) counterpart U87-TxR were transfected
with RFP-LC3, an autophagy marker. The ability of two SFKIs, pyrazolo[3,4-d]pyrimidines Si306 and
its prodrug pro-si306, to induce autophagy in RFP-LC3-transfected GBM cells was evaluated by flow
cytometry and fluorescent microscopy. Cell viability was assessed by MTT assay. The autophagy
induction and autophagic flux were evaluated by Acridine orange assay, immunocytochemistry and
immunoblotting. Cell proliferation rate was analyzed by CFSE assay. Cell death was detected by
Annexin/Propidium Iodide assay. PARP-1 cleavage was assessed by immunoblotting.
Results and Discussions:
SFKI treatment resulted in degradation of RFP-LC3 after 3 h treatment as well as in formation of
RFP-LC3 puncta in GBM cells demonstrating autophagy induction. The effect of SFKIs on autophagy
induction persisted after 48 h, as demonstrated by autophagy markers LC3 and p62. Inhibition of
autophagy by Bafilomycin A1 sensitized both U87 and U87-TxR cells to Si306 and its pro-drug after
48 h. The anti-proliferative effect of Si306 and pro-Si306 was additionally increased after autophagy
inhibition by Bafilomycin A1. Furthermore, while single SFKI treatments did not cause significant
cell death, combination treatments with autophagy inhibitor induced necrosis in U87 and U87-TxR
cells after 48 h. Detection of necrotic PARP-1 fragment further confirmed necrotic cell death.
Conclusion:
Taken together, these data suggest that autophagy induced by Si306 and pro-Si306 has a protective
role in GBM cells, and that autophagy modulation may be used to enhance the anticancer effects of
SFKIs. In addition, as the ability of the SFKIs to induce autophagy was not diminished by the
presence of the MDR phenotype makes these compounds promising for treatment of MDR cancers.",
publisher = "European Association for Cancer Research",
journal = "Congress abstracts: Annual Congress of the European Association for Cancer Research EACR 2022: Innovative Cancer Service: Translating Biology to Medicine; 2022 Jun 20-23; Seville, Spain",
title = "Autophagy inhibition sensitises glioblastoma cells to Src family kinase inhibitors Si306 and its prodrug",
pages = "P1-135",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5425"
}
Jovanović Stojanov, S., Stepanović, A., Ljujić, M., Lupšić, E., Podolski-Renić, A., Dragoj, M., Jovanović, M., Schenone, S., Pešić, M.,& Dinić, J.. (2022). Autophagy inhibition sensitises glioblastoma cells to Src family kinase inhibitors Si306 and its prodrug. in Congress abstracts: Annual Congress of the European Association for Cancer Research EACR 2022: Innovative Cancer Service: Translating Biology to Medicine; 2022 Jun 20-23; Seville, Spain
European Association for Cancer Research., P1-135.
https://hdl.handle.net/21.15107/rcub_ibiss_5425
Jovanović Stojanov S, Stepanović A, Ljujić M, Lupšić E, Podolski-Renić A, Dragoj M, Jovanović M, Schenone S, Pešić M, Dinić J. Autophagy inhibition sensitises glioblastoma cells to Src family kinase inhibitors Si306 and its prodrug. in Congress abstracts: Annual Congress of the European Association for Cancer Research EACR 2022: Innovative Cancer Service: Translating Biology to Medicine; 2022 Jun 20-23; Seville, Spain. 2022;:P1-135.
https://hdl.handle.net/21.15107/rcub_ibiss_5425 .
Jovanović Stojanov, Sofija, Stepanović, Ana, Ljujić, Mila, Lupšić, Ema, Podolski-Renić, Ana, Dragoj, Miodrag, Jovanović, Mirna, Schenone, Silvia, Pešić, Milica, Dinić, Jelena, "Autophagy inhibition sensitises glioblastoma cells to Src family kinase inhibitors Si306 and its prodrug" in Congress abstracts: Annual Congress of the European Association for Cancer Research EACR 2022: Innovative Cancer Service: Translating Biology to Medicine; 2022 Jun 20-23; Seville, Spain (2022):P1-135,
https://hdl.handle.net/21.15107/rcub_ibiss_5425 .

Inhibicija autofagije senzitizuje ćelije glioblastoma na inhibitore Src tirozin-kinaze, derivate pirazolo[3,4-d]pirimidina Si306 i pro-Si306

Jovanović Stojanov, Sofija; Stepanović, Ana; Ljujić, Mila; Lupšić, Ema; Dragoj, Miodrag; Jovanović, Mirna; Dinić, Jelena; Pešić, Milica

(Belgrade: Serbian Biological Society, 2022)

TY  - CONF
AU  - Jovanović Stojanov, Sofija
AU  - Stepanović, Ana
AU  - Ljujić, Mila
AU  - Lupšić, Ema
AU  - Dragoj, Miodrag
AU  - Jovanović, Mirna
AU  - Dinić, Jelena
AU  - Pešić, Milica
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5394
AB  - Глиобластом је један од најагресивнијих тумора мозга који карактерише инфилтрирајућа природа, интензивна пролиферација и резистенција на терапију. Ћелије глиобластома имају високу експресију Срц тирозин-киназе која регулише пролиферацију, преживљавање и инвазивност туморских ћелија чинећи је потенцијалном метом за терапију. Инхибитори тирозин-киназа могу индуковати аутофагију која делује протективно на туморске ћелије. Способност инхибитора
Срц тирозин-киназе, деривата пиразоло[3,4-д]пиримидина Si306 и његовог пролека pro-Si306, да индукују аутофагију испитана је на ћелијској линији хуманог глиобластома U87 и њеној варијанти са вишеструком резистенцијом на лекове U87-TxR. Третман овим једињењима узроковао је појаву аутофагозома у ћелијама након 3 сата, а ефекат на индукцију аутофагије опстао је и након 48 сати што је утврђено анализом маркера аутофагије LC3 и p62. Инхибиција аутофагног флукса бафиломицином А1 значајно је увећала постојеће анти-пролиферативно дејство Si306 и pro-Si306. Такође, комбиновани третмани Срц инхибитора са бафиломицином А1 довели су до некрозе након 48 сати. Добијени резултати сугеришу да аутофагија индукована овим једињењима има заштитну улогу у ћелијама глиобластома и да се модулација аутофагије може користити за сензитизацију ћелија глиобластома на инхибиторе Срц тирозин-киназе. Поред тога, поменути ефекти Si306 и pro-Si306 нису умањени присуством вишеструкорезистентног фенотипа, што овим једињењима даје потенцијал за лечење резистентних тумора.
AB  - Glioblastom je jedan od najagresivnijih tumora mozga koji karakteriše infiltrirajuća priroda, intenzivna proliferacija i rezistencija na terapiju. Ćelije glioblastoma imaju visoku ekspresiju Src tirozin-kinaze koja reguliše proliferaciju, preživljavanje i invazivnost tumorskih ćelija čineći je potencijalnom metom za terapiju. Inhibitori tirozin-kinaza mogu indukovati autofagiju koja deluje protektivno na tumorske ćelije. Sposobnost inhibitora Src tirozin-kinaze, derivata pirazolo[3,4-d]pirimidina Si306 i njegovog proleka pro-Si306, da indukuju autofagiju ispitana je na ćelijskoj liniji humanog glioblastoma U87 i njenoj varijanti sa višestrukom rezistencijom na lekove U87-TxR. Tretman ovim jedinjenjima uzrokovao je pojavu autofagozoma u ćelijama nakon 3 sata, a efekat na indukciju autofagije opstao je i nakon 48 sati što je utvrđeno analizom markera autofagije LC3 i p62. Inhibicija autofagnog fluksa bafilomicinom A1 značajno je uvećala postojeće anti-proliferativno dejstvo Si306 i pro-Si306. Takođe, kombinovani tretmani Src inhibitora sa bafilomicinom A1 doveli su do nekroze nakon 48 sati. Dobijeni rezultati sugerišu da autofagija indukovana ovim jedinjenjima ima zaštitnu ulogu u ćelijama glioblastoma i da se modulacija autofagije može koristiti za senzitizaciju ćelija glioblastoma na inhibitore Src tirozin-kinaze. Pored toga, pomenuti efekti Si306 i pro-Si306 nisu umanjeni prisustvom višestrukorezistentnog fenotipa, što ovim jedinjenjima daje potencijal za lečenje rezistentnih tumora.
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - Inhibicija  autofagije  senzitizuje  ćelije  glioblastoma  na inhibitore  Src  tirozin-kinaze,  derivate  pirazolo[3,4-d]pirimidina Si306 i pro-Si306
T1  - Инхибиција аутофагије сензитизује ћелије глиобластома на инхибиторе Срц тирозин-киназе, деривате пиразоло[3,4- д]пиримидина Si306 и pro-Si306
SP  - 330
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5394
ER  - 
@conference{
author = "Jovanović Stojanov, Sofija and Stepanović, Ana and Ljujić, Mila and Lupšić, Ema and Dragoj, Miodrag and Jovanović, Mirna and Dinić, Jelena and Pešić, Milica",
year = "2022",
abstract = "Глиобластом је један од најагресивнијих тумора мозга који карактерише инфилтрирајућа природа, интензивна пролиферација и резистенција на терапију. Ћелије глиобластома имају високу експресију Срц тирозин-киназе која регулише пролиферацију, преживљавање и инвазивност туморских ћелија чинећи је потенцијалном метом за терапију. Инхибитори тирозин-киназа могу индуковати аутофагију која делује протективно на туморске ћелије. Способност инхибитора
Срц тирозин-киназе, деривата пиразоло[3,4-д]пиримидина Si306 и његовог пролека pro-Si306, да индукују аутофагију испитана је на ћелијској линији хуманог глиобластома U87 и њеној варијанти са вишеструком резистенцијом на лекове U87-TxR. Третман овим једињењима узроковао је појаву аутофагозома у ћелијама након 3 сата, а ефекат на индукцију аутофагије опстао је и након 48 сати што је утврђено анализом маркера аутофагије LC3 и p62. Инхибиција аутофагног флукса бафиломицином А1 значајно је увећала постојеће анти-пролиферативно дејство Si306 и pro-Si306. Такође, комбиновани третмани Срц инхибитора са бафиломицином А1 довели су до некрозе након 48 сати. Добијени резултати сугеришу да аутофагија индукована овим једињењима има заштитну улогу у ћелијама глиобластома и да се модулација аутофагије може користити за сензитизацију ћелија глиобластома на инхибиторе Срц тирозин-киназе. Поред тога, поменути ефекти Si306 и pro-Si306 нису умањени присуством вишеструкорезистентног фенотипа, што овим једињењима даје потенцијал за лечење резистентних тумора., Glioblastom je jedan od najagresivnijih tumora mozga koji karakteriše infiltrirajuća priroda, intenzivna proliferacija i rezistencija na terapiju. Ćelije glioblastoma imaju visoku ekspresiju Src tirozin-kinaze koja reguliše proliferaciju, preživljavanje i invazivnost tumorskih ćelija čineći je potencijalnom metom za terapiju. Inhibitori tirozin-kinaza mogu indukovati autofagiju koja deluje protektivno na tumorske ćelije. Sposobnost inhibitora Src tirozin-kinaze, derivata pirazolo[3,4-d]pirimidina Si306 i njegovog proleka pro-Si306, da indukuju autofagiju ispitana je na ćelijskoj liniji humanog glioblastoma U87 i njenoj varijanti sa višestrukom rezistencijom na lekove U87-TxR. Tretman ovim jedinjenjima uzrokovao je pojavu autofagozoma u ćelijama nakon 3 sata, a efekat na indukciju autofagije opstao je i nakon 48 sati što je utvrđeno analizom markera autofagije LC3 i p62. Inhibicija autofagnog fluksa bafilomicinom A1 značajno je uvećala postojeće anti-proliferativno dejstvo Si306 i pro-Si306. Takođe, kombinovani tretmani Src inhibitora sa bafilomicinom A1 doveli su do nekroze nakon 48 sati. Dobijeni rezultati sugerišu da autofagija indukovana ovim jedinjenjima ima zaštitnu ulogu u ćelijama glioblastoma i da se modulacija autofagije može koristiti za senzitizaciju ćelija glioblastoma na inhibitore Src tirozin-kinaze. Pored toga, pomenuti efekti Si306 i pro-Si306 nisu umanjeni prisustvom višestrukorezistentnog fenotipa, što ovim jedinjenjima daje potencijal za lečenje rezistentnih tumora.",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "Inhibicija  autofagije  senzitizuje  ćelije  glioblastoma  na inhibitore  Src  tirozin-kinaze,  derivate  pirazolo[3,4-d]pirimidina Si306 i pro-Si306, Инхибиција аутофагије сензитизује ћелије глиобластома на инхибиторе Срц тирозин-киназе, деривате пиразоло[3,4- д]пиримидина Si306 и pro-Si306",
pages = "330",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5394"
}
Jovanović Stojanov, S., Stepanović, A., Ljujić, M., Lupšić, E., Dragoj, M., Jovanović, M., Dinić, J.,& Pešić, M.. (2022). Inhibicija  autofagije  senzitizuje  ćelije  glioblastoma  na inhibitore  Src  tirozin-kinaze,  derivate  pirazolo[3,4-d]pirimidina Si306 i pro-Si306. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 330.
https://hdl.handle.net/21.15107/rcub_ibiss_5394
Jovanović Stojanov S, Stepanović A, Ljujić M, Lupšić E, Dragoj M, Jovanović M, Dinić J, Pešić M. Inhibicija  autofagije  senzitizuje  ćelije  glioblastoma  na inhibitore  Src  tirozin-kinaze,  derivate  pirazolo[3,4-d]pirimidina Si306 i pro-Si306. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:330.
https://hdl.handle.net/21.15107/rcub_ibiss_5394 .
Jovanović Stojanov, Sofija, Stepanović, Ana, Ljujić, Mila, Lupšić, Ema, Dragoj, Miodrag, Jovanović, Mirna, Dinić, Jelena, Pešić, Milica, "Inhibicija  autofagije  senzitizuje  ćelije  glioblastoma  na inhibitore  Src  tirozin-kinaze,  derivate  pirazolo[3,4-d]pirimidina Si306 i pro-Si306" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):330,
https://hdl.handle.net/21.15107/rcub_ibiss_5394 .

Multidrug-resistant cancer cells are sensitive to abietane diterpenoids from Plectranthus species

Jovanović, Mirna; Bangay, Gabrielle; Jovanović Stojanov, Sofija; Dragoj, Miodrag; Stepanović, Ana; Lupšić, Ema; Podolski-Renić, Ana; Dinić, Jelena; Rijo, Patricia; Pešić, Milica

(Belgrade: Serbian Plant Physiology Society, 2022)

TY  - CONF
AU  - Jovanović, Mirna
AU  - Bangay, Gabrielle
AU  - Jovanović Stojanov, Sofija
AU  - Dragoj, Miodrag
AU  - Stepanović, Ana
AU  - Lupšić, Ema
AU  - Podolski-Renić, Ana
AU  - Dinić, Jelena
AU  - Rijo, Patricia
AU  - Pešić, Milica
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5369
AB  - Plants of the genus Plectranthus (Lamiaceae) are used in traditional medicine. Here, the anti-cancer effects of the three abietane diterpenoid derivatives from Plectranthus species are described. Compounds’ effects (comp. 1 = VI31.1.1, comp. 2 = VI20.1.1, comp. 3 = RoyBz) were tested
in human lung cancer cells, in sensitive NCI-H460 and chemoresistant NCI-H460/R, as well as in colon cancer cells, sensitive DLD1, and hemoresistant DLD1-TxR. The resistant cells were more sensitive than corresponding parental cells to 1 and 2 in MTT assay, with IC50 values ranging from 3 to 10 μM. For the most potent 3 (IC50 as low as 1 μM), resistant cells had up to 2 times higher IC50 values than sensitive cells. The growth inhibition effect by all three compounds was more ronounced in cancer cells, compared to normal human fibroblasts (MRC-5). Only 2 induced a significant cell death effect showing 70% non-viable cells in NCI-H460. The effect the compoundsmight have on the P-gp extrusion pump was also tested using doxorubicin and rhodamine 123 accumulation assays. Compounds 1 and 2 caused a significant increase in the accumulation of both P-gp substrates, doxorubicin, and rhodamine 123. The compounds isolated from Plectranthus showed anticancer potential in lung and colon cancer cells. Importantly, they displayed colateral sensitivity - a phenomenon when the chemoresistant cells are more sensitive to the compounds than corresponding sensitive cells. The compounds inhibited the P-gp activity implying MDR modulating potential.
PB  - Belgrade: Serbian Plant Physiology Society
C3  - 4th International Conference on Plant Biology [and] 23rd SPPS Meeting; 2022 Oct 6-8; Belgrade, Serbia
T1  - Multidrug-resistant cancer cells are sensitive to abietane diterpenoids from Plectranthus species
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5369
ER  - 
@conference{
author = "Jovanović, Mirna and Bangay, Gabrielle and Jovanović Stojanov, Sofija and Dragoj, Miodrag and Stepanović, Ana and Lupšić, Ema and Podolski-Renić, Ana and Dinić, Jelena and Rijo, Patricia and Pešić, Milica",
year = "2022",
abstract = "Plants of the genus Plectranthus (Lamiaceae) are used in traditional medicine. Here, the anti-cancer effects of the three abietane diterpenoid derivatives from Plectranthus species are described. Compounds’ effects (comp. 1 = VI31.1.1, comp. 2 = VI20.1.1, comp. 3 = RoyBz) were tested
in human lung cancer cells, in sensitive NCI-H460 and chemoresistant NCI-H460/R, as well as in colon cancer cells, sensitive DLD1, and hemoresistant DLD1-TxR. The resistant cells were more sensitive than corresponding parental cells to 1 and 2 in MTT assay, with IC50 values ranging from 3 to 10 μM. For the most potent 3 (IC50 as low as 1 μM), resistant cells had up to 2 times higher IC50 values than sensitive cells. The growth inhibition effect by all three compounds was more ronounced in cancer cells, compared to normal human fibroblasts (MRC-5). Only 2 induced a significant cell death effect showing 70% non-viable cells in NCI-H460. The effect the compoundsmight have on the P-gp extrusion pump was also tested using doxorubicin and rhodamine 123 accumulation assays. Compounds 1 and 2 caused a significant increase in the accumulation of both P-gp substrates, doxorubicin, and rhodamine 123. The compounds isolated from Plectranthus showed anticancer potential in lung and colon cancer cells. Importantly, they displayed colateral sensitivity - a phenomenon when the chemoresistant cells are more sensitive to the compounds than corresponding sensitive cells. The compounds inhibited the P-gp activity implying MDR modulating potential.",
publisher = "Belgrade: Serbian Plant Physiology Society",
journal = "4th International Conference on Plant Biology [and] 23rd SPPS Meeting; 2022 Oct 6-8; Belgrade, Serbia",
title = "Multidrug-resistant cancer cells are sensitive to abietane diterpenoids from Plectranthus species",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5369"
}
Jovanović, M., Bangay, G., Jovanović Stojanov, S., Dragoj, M., Stepanović, A., Lupšić, E., Podolski-Renić, A., Dinić, J., Rijo, P.,& Pešić, M.. (2022). Multidrug-resistant cancer cells are sensitive to abietane diterpenoids from Plectranthus species. in 4th International Conference on Plant Biology [and] 23rd SPPS Meeting; 2022 Oct 6-8; Belgrade, Serbia
Belgrade: Serbian Plant Physiology Society..
https://hdl.handle.net/21.15107/rcub_ibiss_5369
Jovanović M, Bangay G, Jovanović Stojanov S, Dragoj M, Stepanović A, Lupšić E, Podolski-Renić A, Dinić J, Rijo P, Pešić M. Multidrug-resistant cancer cells are sensitive to abietane diterpenoids from Plectranthus species. in 4th International Conference on Plant Biology [and] 23rd SPPS Meeting; 2022 Oct 6-8; Belgrade, Serbia. 2022;.
https://hdl.handle.net/21.15107/rcub_ibiss_5369 .
Jovanović, Mirna, Bangay, Gabrielle, Jovanović Stojanov, Sofija, Dragoj, Miodrag, Stepanović, Ana, Lupšić, Ema, Podolski-Renić, Ana, Dinić, Jelena, Rijo, Patricia, Pešić, Milica, "Multidrug-resistant cancer cells are sensitive to abietane diterpenoids from Plectranthus species" in 4th International Conference on Plant Biology [and] 23rd SPPS Meeting; 2022 Oct 6-8; Belgrade, Serbia (2022),
https://hdl.handle.net/21.15107/rcub_ibiss_5369 .

Ispitivanje matriksnih metaloproteinaza i fokalne adhezione kinaze kao farmakoloških meta kod nesitnoćelijskog karcinoma pluća

Jovanović Stojanov, Sofija; Podolski-Renić, Ana; Jovanović, Mirna; Pešić, Milica; Stanković, Tijana; Dragoj, Miodrag

(Belgrade: Serbian Biological Society, 2022)

TY  - CONF
AU  - Jovanović Stojanov, Sofija
AU  - Podolski-Renić, Ana
AU  - Jovanović, Mirna
AU  - Pešić, Milica
AU  - Stanković, Tijana
AU  - Dragoj, Miodrag
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5393
AB  - Неситноћелијски  карцином  плућа  је  најчешћи  облик  рака  плућа.  У  време  постављања  дијагнозе,  велики  проценат  пацијената  већ  има  развијене  метастазе  одговорне за изузетно високу стопу смртности. Фокална адхезиона киназа (FAK) и  матриксне  металопротеиназе  (MMP2  и  ММР9)  су  међу  кључним  молекулима  укљученим  у  процес  метастазирања.  Прекомерно  су  експримирани  у  већини  тумора, повећавајући инвазивни и метастатски капацитет  туморских ћелија. FAK  своју проинвазивну улогу делом остварује регулисањем производње и ослобађања 
ММР2 и ММР9. У овој студији је анализирана експресија ММР2 и ММР9 у односу  на  повећану  експресију  FAK-а  код  пацијената  са  неситноћелијским  карциномом  плућа.  Показано  је  да  истовремено  повећана  експресија  FAK  и  макар  једне  од  испитиваних  матриксних  металопротеиназа  негативно  утичу  на  преживљавање.  Стога  је  in  vitro  испитиван  антиинвазивни  и  антитуморски  ефекат  истовремене  циљане  инхибиције  FAK  и  ММР2/MMP9  одговарајућим  инхибиторима  малих  молекула.  Добијени  резултати  су  показали  да  истовремена  инхибиција  FAK  и  ММР2/MMP9  не  утиче  значајно  на  смањење  инвазивних  способности  туморских  ћелија  у  односу  на  појединачне  третмане  инхибиторима.  Насупрот  томе,  истовремена инхибиција FAK и ММР2/MMP9 има изражен антитуморски ефекат,  смањује вијабилност ћелија доводећи до апоптозе. На основу добијених података  може  се  закључити  да  би  истовремена  циљана  инхибиција  FAK  и  ММР2/MMP9  могла бити обећавајући терапијски приступ за неситноћелијски карцином плућа.
AB  - Nesitnoćelijski karcinom pluća je najčešći oblik raka pluća. U vreme postavljanja dijagnoze, veliki procenat pacijenata već ima razvijene metastaze odgovorne za izuzetno visoku stopu smrtnosti. Fokalna adheziona kinaza (FAK) i matriksne metaloproteinaze (MMP2 i MMR9) su među ključnim molekulima uključenim u proces metastaziranja. Prekomerno su eksprimirani u većini tumora, povećavajući invazivni i metastatski kapacitet tumorskih ćelija. FAK svoju proinvazivnu ulogu delom ostvaruje regulisanjem proizvodnje i oslobađanja MMR2 i MMR9. U ovoj studiji je analizirana ekspresija MMR2 i MMR9 u odnosu na povećanu ekspresiju FAK-a kod pacijenata sa nesitnoćelijskim karcinomom pluća. Pokazano je da istovremeno povećana ekspresija FAK i makar jedne od ispitivanih matriksnih metaloproteinaza negativno utiču na preživljavanje. Stoga je in vitro ispitivan antiinvazivni i antitumorski efekat istovremene ciljane inhibicije FAK i MMR2/MMP9 odgovarajućim inhibitorima malih molekula. Dobijeni rezultati su pokazali da istovremena inhibicija FAK i MMR2/MMP9 ne utiče značajno na smanjenje invazivnih sposobnosti tumorskih ćelija u odnosu na pojedinačne tretmane inhibitorima. Nasuprot tome, istovremena inhibicija FAK i MMR2/MMP9 ima izražen antitumorski efekat, smanjuje vijabilnost ćelija dovodeći do apoptoze. Na osnovu dobijenih podataka može se zaključiti da bi istovremena ciljana inhibicija FAK i MMR2/MMP9 mogla biti obećavajući terapijski pristup za nesitnoćelijski karcinom pluća.
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - Ispitivanje  matriksnih  metaloproteinaza  i  fokalne  adhezione kinaze kao farmakoloških meta kod nesitnoćelijskog karcinoma pluća
T1  - Испитивање матриксних металопротеиназа и фокалне адхезионе киназе као фармаколошких мета код неситноћелијског карцинома плућа
SP  - 331
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5393
ER  - 
@conference{
author = "Jovanović Stojanov, Sofija and Podolski-Renić, Ana and Jovanović, Mirna and Pešić, Milica and Stanković, Tijana and Dragoj, Miodrag",
year = "2022",
abstract = "Неситноћелијски  карцином  плућа  је  најчешћи  облик  рака  плућа.  У  време  постављања  дијагнозе,  велики  проценат  пацијената  већ  има  развијене  метастазе  одговорне за изузетно високу стопу смртности. Фокална адхезиона киназа (FAK) и  матриксне  металопротеиназе  (MMP2  и  ММР9)  су  међу  кључним  молекулима  укљученим  у  процес  метастазирања.  Прекомерно  су  експримирани  у  већини  тумора, повећавајући инвазивни и метастатски капацитет  туморских ћелија. FAK  своју проинвазивну улогу делом остварује регулисањем производње и ослобађања 
ММР2 и ММР9. У овој студији је анализирана експресија ММР2 и ММР9 у односу  на  повећану  експресију  FAK-а  код  пацијената  са  неситноћелијским  карциномом  плућа.  Показано  је  да  истовремено  повећана  експресија  FAK  и  макар  једне  од  испитиваних  матриксних  металопротеиназа  негативно  утичу  на  преживљавање.  Стога  је  in  vitro  испитиван  антиинвазивни  и  антитуморски  ефекат  истовремене  циљане  инхибиције  FAK  и  ММР2/MMP9  одговарајућим  инхибиторима  малих  молекула.  Добијени  резултати  су  показали  да  истовремена  инхибиција  FAK  и  ММР2/MMP9  не  утиче  значајно  на  смањење  инвазивних  способности  туморских  ћелија  у  односу  на  појединачне  третмане  инхибиторима.  Насупрот  томе,  истовремена инхибиција FAK и ММР2/MMP9 има изражен антитуморски ефекат,  смањује вијабилност ћелија доводећи до апоптозе. На основу добијених података  може  се  закључити  да  би  истовремена  циљана  инхибиција  FAK  и  ММР2/MMP9  могла бити обећавајући терапијски приступ за неситноћелијски карцином плућа., Nesitnoćelijski karcinom pluća je najčešći oblik raka pluća. U vreme postavljanja dijagnoze, veliki procenat pacijenata već ima razvijene metastaze odgovorne za izuzetno visoku stopu smrtnosti. Fokalna adheziona kinaza (FAK) i matriksne metaloproteinaze (MMP2 i MMR9) su među ključnim molekulima uključenim u proces metastaziranja. Prekomerno su eksprimirani u većini tumora, povećavajući invazivni i metastatski kapacitet tumorskih ćelija. FAK svoju proinvazivnu ulogu delom ostvaruje regulisanjem proizvodnje i oslobađanja MMR2 i MMR9. U ovoj studiji je analizirana ekspresija MMR2 i MMR9 u odnosu na povećanu ekspresiju FAK-a kod pacijenata sa nesitnoćelijskim karcinomom pluća. Pokazano je da istovremeno povećana ekspresija FAK i makar jedne od ispitivanih matriksnih metaloproteinaza negativno utiču na preživljavanje. Stoga je in vitro ispitivan antiinvazivni i antitumorski efekat istovremene ciljane inhibicije FAK i MMR2/MMP9 odgovarajućim inhibitorima malih molekula. Dobijeni rezultati su pokazali da istovremena inhibicija FAK i MMR2/MMP9 ne utiče značajno na smanjenje invazivnih sposobnosti tumorskih ćelija u odnosu na pojedinačne tretmane inhibitorima. Nasuprot tome, istovremena inhibicija FAK i MMR2/MMP9 ima izražen antitumorski efekat, smanjuje vijabilnost ćelija dovodeći do apoptoze. Na osnovu dobijenih podataka može se zaključiti da bi istovremena ciljana inhibicija FAK i MMR2/MMP9 mogla biti obećavajući terapijski pristup za nesitnoćelijski karcinom pluća.",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "Ispitivanje  matriksnih  metaloproteinaza  i  fokalne  adhezione kinaze kao farmakoloških meta kod nesitnoćelijskog karcinoma pluća, Испитивање матриксних металопротеиназа и фокалне адхезионе киназе као фармаколошких мета код неситноћелијског карцинома плућа",
pages = "331",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5393"
}
Jovanović Stojanov, S., Podolski-Renić, A., Jovanović, M., Pešić, M., Stanković, T.,& Dragoj, M.. (2022). Ispitivanje  matriksnih  metaloproteinaza  i  fokalne  adhezione kinaze kao farmakoloških meta kod nesitnoćelijskog karcinoma pluća. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 331.
https://hdl.handle.net/21.15107/rcub_ibiss_5393
Jovanović Stojanov S, Podolski-Renić A, Jovanović M, Pešić M, Stanković T, Dragoj M. Ispitivanje  matriksnih  metaloproteinaza  i  fokalne  adhezione kinaze kao farmakoloških meta kod nesitnoćelijskog karcinoma pluća. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:331.
https://hdl.handle.net/21.15107/rcub_ibiss_5393 .
Jovanović Stojanov, Sofija, Podolski-Renić, Ana, Jovanović, Mirna, Pešić, Milica, Stanković, Tijana, Dragoj, Miodrag, "Ispitivanje  matriksnih  metaloproteinaza  i  fokalne  adhezione kinaze kao farmakoloških meta kod nesitnoćelijskog karcinoma pluća" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):331,
https://hdl.handle.net/21.15107/rcub_ibiss_5393 .

On optimal temozolomide scheduling for slowly growing glioblastomas

Segura-Collar, Berta; Jiménez-Sánchez, Juan; Gargini, Ricardo; Dragoj, Miodrag; Sepúlveda-Sánchez, Juan M; Pešić, Milica; Ramírez, María A; Ayala-Hernández, Luis E; Sánchez-Gómez, Pilar; Pérez-García, Víctor M

(Oxford: Oxford University Press, 2022)

TY  - JOUR
AU  - Segura-Collar, Berta
AU  - Jiménez-Sánchez, Juan
AU  - Gargini, Ricardo
AU  - Dragoj, Miodrag
AU  - Sepúlveda-Sánchez, Juan M
AU  - Pešić, Milica
AU  - Ramírez, María A
AU  - Ayala-Hernández, Luis E
AU  - Sánchez-Gómez, Pilar
AU  - Pérez-García, Víctor M
PY  - 2022
UR  - http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC9616068
UR  - https://academic.oup.com/noa/article/doi/10.1093/noajnl/vdac155/6722624
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5182
AB  - Background Temozolomide (TMZ) is an oral alkylating agent active against gliomas with a favorable toxicity profile. It is part of the standard of care in the management of glioblastoma (GBM), and is commonly used in low-grade gliomas (LGG). In-silico mathematical models can potentially be used to personalize treatments and to accelerate the discovery of optimal drug delivery schemes. Methods Agent-based mathematical models fed with either mouse or patient data were developed for the in-silico studies. The experimental test beds used to confirm the results were: mouse glioma models obtained by retroviral expression of EGFR-wt/EGFR-vIII in primary progenitors from p16/p19 ko mice and grown in-vitro and in-vivo in orthotopic allografts, and human GBM U251 cells immobilized in alginate microfibers. The patient data used to parametrize the model were obtained from the TCGA/TCIA databases and the TOG clinical study. Results Slow-growth "virtual" murine GBMs benefited from increasing TMZ dose separation in-silico. In line with the simulation results, improved survival, reduced toxicity, lower expression of resistance factors, and reduction of the tumor mesenchymal component were observed in experimental models subject to long-cycle treatment, particularly in slowly growing tumors. Tissue analysis after long-cycle TMZ treatments revealed epigenetically driven changes in tumor phenotype, which could explain the reduction in GBM growth speed. In-silico trials provided support for implementation methods in human patients. Conclusions In-silico simulations, in-vitro and in-vivo studies show that TMZ administration schedules with increased time between doses may reduce toxicity, delay the appearance of resistances and lead to survival benefits mediated by changes in the tumor phenotype in slowly-growing GBMs.
PB  - Oxford: Oxford University Press
T2  - Neuro-Oncology Advances
T1  - On optimal temozolomide scheduling for slowly growing glioblastomas
IS  - 1
VL  - 4
DO  - 10.1093/noajnl/vdac155
SP  - vdac155
ER  - 
@article{
author = "Segura-Collar, Berta and Jiménez-Sánchez, Juan and Gargini, Ricardo and Dragoj, Miodrag and Sepúlveda-Sánchez, Juan M and Pešić, Milica and Ramírez, María A and Ayala-Hernández, Luis E and Sánchez-Gómez, Pilar and Pérez-García, Víctor M",
year = "2022",
abstract = "Background Temozolomide (TMZ) is an oral alkylating agent active against gliomas with a favorable toxicity profile. It is part of the standard of care in the management of glioblastoma (GBM), and is commonly used in low-grade gliomas (LGG). In-silico mathematical models can potentially be used to personalize treatments and to accelerate the discovery of optimal drug delivery schemes. Methods Agent-based mathematical models fed with either mouse or patient data were developed for the in-silico studies. The experimental test beds used to confirm the results were: mouse glioma models obtained by retroviral expression of EGFR-wt/EGFR-vIII in primary progenitors from p16/p19 ko mice and grown in-vitro and in-vivo in orthotopic allografts, and human GBM U251 cells immobilized in alginate microfibers. The patient data used to parametrize the model were obtained from the TCGA/TCIA databases and the TOG clinical study. Results Slow-growth "virtual" murine GBMs benefited from increasing TMZ dose separation in-silico. In line with the simulation results, improved survival, reduced toxicity, lower expression of resistance factors, and reduction of the tumor mesenchymal component were observed in experimental models subject to long-cycle treatment, particularly in slowly growing tumors. Tissue analysis after long-cycle TMZ treatments revealed epigenetically driven changes in tumor phenotype, which could explain the reduction in GBM growth speed. In-silico trials provided support for implementation methods in human patients. Conclusions In-silico simulations, in-vitro and in-vivo studies show that TMZ administration schedules with increased time between doses may reduce toxicity, delay the appearance of resistances and lead to survival benefits mediated by changes in the tumor phenotype in slowly-growing GBMs.",
publisher = "Oxford: Oxford University Press",
journal = "Neuro-Oncology Advances",
title = "On optimal temozolomide scheduling for slowly growing glioblastomas",
number = "1",
volume = "4",
doi = "10.1093/noajnl/vdac155",
pages = "vdac155"
}
Segura-Collar, B., Jiménez-Sánchez, J., Gargini, R., Dragoj, M., Sepúlveda-Sánchez, J. M., Pešić, M., Ramírez, M. A., Ayala-Hernández, L. E., Sánchez-Gómez, P.,& Pérez-García, V. M.. (2022). On optimal temozolomide scheduling for slowly growing glioblastomas. in Neuro-Oncology Advances
Oxford: Oxford University Press., 4(1), vdac155.
https://doi.org/10.1093/noajnl/vdac155
Segura-Collar B, Jiménez-Sánchez J, Gargini R, Dragoj M, Sepúlveda-Sánchez JM, Pešić M, Ramírez MA, Ayala-Hernández LE, Sánchez-Gómez P, Pérez-García VM. On optimal temozolomide scheduling for slowly growing glioblastomas. in Neuro-Oncology Advances. 2022;4(1):vdac155.
doi:10.1093/noajnl/vdac155 .
Segura-Collar, Berta, Jiménez-Sánchez, Juan, Gargini, Ricardo, Dragoj, Miodrag, Sepúlveda-Sánchez, Juan M, Pešić, Milica, Ramírez, María A, Ayala-Hernández, Luis E, Sánchez-Gómez, Pilar, Pérez-García, Víctor M, "On optimal temozolomide scheduling for slowly growing glioblastomas" in Neuro-Oncology Advances, 4, no. 1 (2022):vdac155,
https://doi.org/10.1093/noajnl/vdac155 . .
8
2
2

A 3D Biomimetic System for Testing Anticancer Drug Sensitivity.

Dragoj, Miodrag; Stojkovska, Jasmina; Jovanović Stojanov, Sofija; Obradović, Bojana; Pešić, Milica; Baiocchi, Marta

(Humana Press Inc., 2022)

TY  - CHAP
AU  - Dragoj, Miodrag
AU  - Stojkovska, Jasmina
AU  - Jovanović Stojanov, Sofija
AU  - Obradović, Bojana
AU  - Pešić, Milica
AU  - Baiocchi, Marta
PY  - 2022
UR  - https://link.springer.com/10.1007/978-1-0716-2513-2_1
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5093
AB  - 3D cultures of cancer cells enable better mimicking of physiological conditions compared to traditional monolayer 2D cultures. Here we describe alginate scaffold-based model that can be used in both static and biomimetic conditions for studying drug sensitivity in cancer cells and multidrug resistance (MDR) mechanisms. This 3D culture model resembles in vivo conditions and provides relevant and reproducible results. It is easy to set up and allows for facile manipulation for downstream analyses. All these remarkable features make this 3D culture model a promising tool in drug discovery and cancer cell biology research.
PB  - Humana Press Inc.
PB  - New York: Humana Press Inc.
T2  - Cancer Drug Resistance: Methods and Protocols
T1  - A 3D Biomimetic System for Testing Anticancer Drug Sensitivity.
VL  - 2535
DO  - 10.1007/978-1-0716-2513-2_1
SP  - 1
EP  - 9
ER  - 
@inbook{
author = "Dragoj, Miodrag and Stojkovska, Jasmina and Jovanović Stojanov, Sofija and Obradović, Bojana and Pešić, Milica and Baiocchi, Marta",
year = "2022",
abstract = "3D cultures of cancer cells enable better mimicking of physiological conditions compared to traditional monolayer 2D cultures. Here we describe alginate scaffold-based model that can be used in both static and biomimetic conditions for studying drug sensitivity in cancer cells and multidrug resistance (MDR) mechanisms. This 3D culture model resembles in vivo conditions and provides relevant and reproducible results. It is easy to set up and allows for facile manipulation for downstream analyses. All these remarkable features make this 3D culture model a promising tool in drug discovery and cancer cell biology research.",
publisher = "Humana Press Inc., New York: Humana Press Inc.",
journal = "Cancer Drug Resistance: Methods and Protocols",
booktitle = "A 3D Biomimetic System for Testing Anticancer Drug Sensitivity.",
volume = "2535",
doi = "10.1007/978-1-0716-2513-2_1",
pages = "1-9"
}
Dragoj, M., Stojkovska, J., Jovanović Stojanov, S., Obradović, B., Pešić, M.,& Baiocchi, M.. (2022). A 3D Biomimetic System for Testing Anticancer Drug Sensitivity.. in Cancer Drug Resistance: Methods and Protocols
Humana Press Inc.., 2535, 1-9.
https://doi.org/10.1007/978-1-0716-2513-2_1
Dragoj M, Stojkovska J, Jovanović Stojanov S, Obradović B, Pešić M, Baiocchi M. A 3D Biomimetic System for Testing Anticancer Drug Sensitivity.. in Cancer Drug Resistance: Methods and Protocols. 2022;2535:1-9.
doi:10.1007/978-1-0716-2513-2_1 .
Dragoj, Miodrag, Stojkovska, Jasmina, Jovanović Stojanov, Sofija, Obradović, Bojana, Pešić, Milica, Baiocchi, Marta, "A 3D Biomimetic System for Testing Anticancer Drug Sensitivity." in Cancer Drug Resistance: Methods and Protocols, 2535 (2022):1-9,
https://doi.org/10.1007/978-1-0716-2513-2_1 . .

Sclareol, a fragrant natural compound, suppresses P-glycoprotein activity and sensitizes resistant cancer cells to doxorubicin

Lupšić, Ema; Stepanović, Ana; Nikolić, Andrea M.; Dragoj, Miodrag; Jovanović Stojanov, Sofija; Novaković, Miroslav; Opsenica, Igor M.; Pešić, Milica

(STRATAGEM, 2021)

TY  - CONF
AU  - Lupšić, Ema
AU  - Stepanović, Ana
AU  - Nikolić, Andrea M.
AU  - Dragoj, Miodrag
AU  - Jovanović Stojanov, Sofija
AU  - Novaković, Miroslav
AU  - Opsenica, Igor M.
AU  - Pešić, Milica
PY  - 2021
UR  - https://stratagem-cost.eu/2021/09/stratagems-4th-annual-conference-in-prague-czechia-to-take-place-on/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5957
AB  - Multidrug resistance (MDR) is one of the major obstacles to successful cancer treatment. How to
overcome cancer MDR is still an unsolved issue in clinical practice although several generations of MDR
transporters’ inhibitors have been developed and widely investigated so far. Nature is an important source
of potential anticancer agents capable to suppress the activity of membrane transporters implicated in MDR
such as P-glycoprotein (P-gp). In this study, we evaluated the effects of sclareol (SC), a naturally occurring
labdane type diterpene, on the P-gp activity and its potential to sensitize different human cancer cell lines
to doxorubicin (DOX). To that end, we used several human cancer cell lines (colorectal carcinoma, DLD1,
and its MDR variant DLD1-TxR, non-small cell lung carcinoma NCI-H460, and its MDR variant NCIH460/R, glioblastoma U251, U87, and its MDR variant U87-TxR) and normal human embryonic lung fibroblasts (MRC-5). The effects of SC alone and in combination with DOX on cell viability were assessed by
MTT, while the effects on DOX and rhodamine 123 (Rho 123) accumulation as determinants of P-gp activity
were assessed by flow cytometry. The efficient concentrations of SC that significantly decreased cell viability
(IC50 values) ranged between 20 µM for DLD1 and 60 µM for MRC-5. The presence of MDR phenotype did
not diminish the SC effect on cell viability, even more, SC was more potent in U87-TxR than in U87 cells.
The effects of 72 h simultaneous treatment of SC (10 and 20 µM) with DOX (20, 50, 100, 200 and 500 nM)
demonstrated the considerable potential of SC to sensitize DLD1, DLD1-TxR, NCI-H460/R, U87-TxR and
U251 cells to DOX. However, the observed sensitization was not due to the P-gp inhibition in all MDR cancer
cell lines. Only in NCI-H460/R the obvious suppression of P-gp was observed due to the significant increase
in the accumulation of both P-gp substrates (DOX and Rho 123). SC did not affect the P-gp activity in DLD1
and DLD1-TxR cells. On the contrary, DOX and Rho123 accumulation increased in U87 and U87-TxR albeit
the fact that U87 cells do not express P-gp. Results obtained in this study showed a considerable potential
of SC to sensitize cancer cells to DOX. However, the effects of SC are cancer type-specific and not solely
dependent on the suppression of P-gp activity. Further investigations are envisioned to determine molecular
mechanisms of SC in different cancer cell types.
PB  - STRATAGEM
C3  - 4th Annual STRATAGEM Conference: New Diagnostic and therapeutic tools against multidrug resistant tumours; 2021 Sep 6-8; Prague; Czechia
T1  - Sclareol, a fragrant natural compound, suppresses P-glycoprotein activity and sensitizes resistant cancer cells to doxorubicin
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5957
ER  - 
@conference{
author = "Lupšić, Ema and Stepanović, Ana and Nikolić, Andrea M. and Dragoj, Miodrag and Jovanović Stojanov, Sofija and Novaković, Miroslav and Opsenica, Igor M. and Pešić, Milica",
year = "2021",
abstract = "Multidrug resistance (MDR) is one of the major obstacles to successful cancer treatment. How to
overcome cancer MDR is still an unsolved issue in clinical practice although several generations of MDR
transporters’ inhibitors have been developed and widely investigated so far. Nature is an important source
of potential anticancer agents capable to suppress the activity of membrane transporters implicated in MDR
such as P-glycoprotein (P-gp). In this study, we evaluated the effects of sclareol (SC), a naturally occurring
labdane type diterpene, on the P-gp activity and its potential to sensitize different human cancer cell lines
to doxorubicin (DOX). To that end, we used several human cancer cell lines (colorectal carcinoma, DLD1,
and its MDR variant DLD1-TxR, non-small cell lung carcinoma NCI-H460, and its MDR variant NCIH460/R, glioblastoma U251, U87, and its MDR variant U87-TxR) and normal human embryonic lung fibroblasts (MRC-5). The effects of SC alone and in combination with DOX on cell viability were assessed by
MTT, while the effects on DOX and rhodamine 123 (Rho 123) accumulation as determinants of P-gp activity
were assessed by flow cytometry. The efficient concentrations of SC that significantly decreased cell viability
(IC50 values) ranged between 20 µM for DLD1 and 60 µM for MRC-5. The presence of MDR phenotype did
not diminish the SC effect on cell viability, even more, SC was more potent in U87-TxR than in U87 cells.
The effects of 72 h simultaneous treatment of SC (10 and 20 µM) with DOX (20, 50, 100, 200 and 500 nM)
demonstrated the considerable potential of SC to sensitize DLD1, DLD1-TxR, NCI-H460/R, U87-TxR and
U251 cells to DOX. However, the observed sensitization was not due to the P-gp inhibition in all MDR cancer
cell lines. Only in NCI-H460/R the obvious suppression of P-gp was observed due to the significant increase
in the accumulation of both P-gp substrates (DOX and Rho 123). SC did not affect the P-gp activity in DLD1
and DLD1-TxR cells. On the contrary, DOX and Rho123 accumulation increased in U87 and U87-TxR albeit
the fact that U87 cells do not express P-gp. Results obtained in this study showed a considerable potential
of SC to sensitize cancer cells to DOX. However, the effects of SC are cancer type-specific and not solely
dependent on the suppression of P-gp activity. Further investigations are envisioned to determine molecular
mechanisms of SC in different cancer cell types.",
publisher = "STRATAGEM",
journal = "4th Annual STRATAGEM Conference: New Diagnostic and therapeutic tools against multidrug resistant tumours; 2021 Sep 6-8; Prague; Czechia",
title = "Sclareol, a fragrant natural compound, suppresses P-glycoprotein activity and sensitizes resistant cancer cells to doxorubicin",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5957"
}
Lupšić, E., Stepanović, A., Nikolić, A. M., Dragoj, M., Jovanović Stojanov, S., Novaković, M., Opsenica, I. M.,& Pešić, M.. (2021). Sclareol, a fragrant natural compound, suppresses P-glycoprotein activity and sensitizes resistant cancer cells to doxorubicin. in 4th Annual STRATAGEM Conference: New Diagnostic and therapeutic tools against multidrug resistant tumours; 2021 Sep 6-8; Prague; Czechia
STRATAGEM..
https://hdl.handle.net/21.15107/rcub_ibiss_5957
Lupšić E, Stepanović A, Nikolić AM, Dragoj M, Jovanović Stojanov S, Novaković M, Opsenica IM, Pešić M. Sclareol, a fragrant natural compound, suppresses P-glycoprotein activity and sensitizes resistant cancer cells to doxorubicin. in 4th Annual STRATAGEM Conference: New Diagnostic and therapeutic tools against multidrug resistant tumours; 2021 Sep 6-8; Prague; Czechia. 2021;.
https://hdl.handle.net/21.15107/rcub_ibiss_5957 .
Lupšić, Ema, Stepanović, Ana, Nikolić, Andrea M., Dragoj, Miodrag, Jovanović Stojanov, Sofija, Novaković, Miroslav, Opsenica, Igor M., Pešić, Milica, "Sclareol, a fragrant natural compound, suppresses P-glycoprotein activity and sensitizes resistant cancer cells to doxorubicin" in 4th Annual STRATAGEM Conference: New Diagnostic and therapeutic tools against multidrug resistant tumours; 2021 Sep 6-8; Prague; Czechia (2021),
https://hdl.handle.net/21.15107/rcub_ibiss_5957 .

Anticancer effects of sclareol and its derivatives in glioblastoma cells

Stepanović, Ana; Lupšić, Ema; Stojković, Pavle; Dragoj, Miodrag; Jovanović Stojanov, Sofija; Terzić- Jovanović, Nataša; Novaković, Miroslav; Opsenica, Igor M.; Pešić, Milica

(Belgrade: Serbian Association for Cancer Research, 2021)

TY  - CONF
AU  - Stepanović, Ana
AU  - Lupšić, Ema
AU  - Stojković, Pavle
AU  - Dragoj, Miodrag
AU  - Jovanović Stojanov, Sofija
AU  - Terzić- Jovanović, Nataša
AU  - Novaković, Miroslav
AU  - Opsenica, Igor M.
AU  - Pešić, Milica
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4995
AB  - Background: Glioblastoma is the most common, aggressive and lethal brain tumor in adults with high proliferation rate, infiltrating nature and presence of multidrug resistance (MDR). Sclareol (SC) is a naturally occurring labdane type diterpene, derived from Salvia sclarea. We examined cell growth inhibition effect of SC and its derivatives (PAS and TNT groups of compounds) - hybrid (chimeric) molecules. Sclareol was covalently bonded to [1,2,4]triazolo[1,5-a]pyrimidin-7-amine scaffold, and different diamines were used as linkers. We also studied SC potential to reverse DOX resistance and its accumulation. The combination of SC with DOX has been earlier described to potentiate DOX cytotoxicity if simultaneously delivered in nanoparticles. Material and Methods: SC in combination with DOX as well as SC derivatives were tested on human glioma cell line U87, and its MDR counterpart - U87-TxR. MTT assay was used to examine inhibition of cell growth. Accumulation of DOX was measured by flow cytometry. Results: Thirteen out of nineteen TNT derivatives and three out of six PAS derivatives showed stronger anti-glioma effect than SC. Simultaneous treatment of SC with DOX demonstrated potential of SC to reverse DOX resistance. Even more, SC significantly increased DOX accumulation in both glioblastoma cell lines. Conclusion: Results obtained in this study showed a considerable synergy of SC and DOX in glioma cells. Better results observed with SC derivatives make them good candidates for further testing.
PB  - Belgrade: Serbian Association for Cancer Research
C3  - Abstract book: The fifth congress of the Serbian association for cancer research:“Translational potential of cancer research in Serbia“; 2021 Dec 3; Virtual.
T1  - Anticancer effects of sclareol and its derivatives in glioblastoma cells
SP  - 72
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4995
ER  - 
@conference{
author = "Stepanović, Ana and Lupšić, Ema and Stojković, Pavle and Dragoj, Miodrag and Jovanović Stojanov, Sofija and Terzić- Jovanović, Nataša and Novaković, Miroslav and Opsenica, Igor M. and Pešić, Milica",
year = "2021",
abstract = "Background: Glioblastoma is the most common, aggressive and lethal brain tumor in adults with high proliferation rate, infiltrating nature and presence of multidrug resistance (MDR). Sclareol (SC) is a naturally occurring labdane type diterpene, derived from Salvia sclarea. We examined cell growth inhibition effect of SC and its derivatives (PAS and TNT groups of compounds) - hybrid (chimeric) molecules. Sclareol was covalently bonded to [1,2,4]triazolo[1,5-a]pyrimidin-7-amine scaffold, and different diamines were used as linkers. We also studied SC potential to reverse DOX resistance and its accumulation. The combination of SC with DOX has been earlier described to potentiate DOX cytotoxicity if simultaneously delivered in nanoparticles. Material and Methods: SC in combination with DOX as well as SC derivatives were tested on human glioma cell line U87, and its MDR counterpart - U87-TxR. MTT assay was used to examine inhibition of cell growth. Accumulation of DOX was measured by flow cytometry. Results: Thirteen out of nineteen TNT derivatives and three out of six PAS derivatives showed stronger anti-glioma effect than SC. Simultaneous treatment of SC with DOX demonstrated potential of SC to reverse DOX resistance. Even more, SC significantly increased DOX accumulation in both glioblastoma cell lines. Conclusion: Results obtained in this study showed a considerable synergy of SC and DOX in glioma cells. Better results observed with SC derivatives make them good candidates for further testing.",
publisher = "Belgrade: Serbian Association for Cancer Research",
journal = "Abstract book: The fifth congress of the Serbian association for cancer research:“Translational potential of cancer research in Serbia“; 2021 Dec 3; Virtual.",
title = "Anticancer effects of sclareol and its derivatives in glioblastoma cells",
pages = "72",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4995"
}
Stepanović, A., Lupšić, E., Stojković, P., Dragoj, M., Jovanović Stojanov, S., Terzić- Jovanović, N., Novaković, M., Opsenica, I. M.,& Pešić, M.. (2021). Anticancer effects of sclareol and its derivatives in glioblastoma cells. in Abstract book: The fifth congress of the Serbian association for cancer research:“Translational potential of cancer research in Serbia“; 2021 Dec 3; Virtual.
Belgrade: Serbian Association for Cancer Research., 72.
https://hdl.handle.net/21.15107/rcub_ibiss_4995
Stepanović A, Lupšić E, Stojković P, Dragoj M, Jovanović Stojanov S, Terzić- Jovanović N, Novaković M, Opsenica IM, Pešić M. Anticancer effects of sclareol and its derivatives in glioblastoma cells. in Abstract book: The fifth congress of the Serbian association for cancer research:“Translational potential of cancer research in Serbia“; 2021 Dec 3; Virtual.. 2021;:72.
https://hdl.handle.net/21.15107/rcub_ibiss_4995 .
Stepanović, Ana, Lupšić, Ema, Stojković, Pavle, Dragoj, Miodrag, Jovanović Stojanov, Sofija, Terzić- Jovanović, Nataša, Novaković, Miroslav, Opsenica, Igor M., Pešić, Milica, "Anticancer effects of sclareol and its derivatives in glioblastoma cells" in Abstract book: The fifth congress of the Serbian association for cancer research:“Translational potential of cancer research in Serbia“; 2021 Dec 3; Virtual. (2021):72,
https://hdl.handle.net/21.15107/rcub_ibiss_4995 .

Sclareol, a natural compound, inhibits P-glycoprotein activity in cancer cells

Stepanović, Ana; Lupšić, Ema; Nikolić, Andrea M.; Dragoj, Miodrag; Jovanović Stojanov, Sofija; Novaković, Miroslav; Opsenica, Igor M.; Pešić, Milica

(Belgrade: Faculty of Chemistry: Serbian Biochemical Society, 2021, 2021)

TY  - CONF
AU  - Stepanović, Ana
AU  - Lupšić, Ema
AU  - Nikolić, Andrea M.
AU  - Dragoj, Miodrag
AU  - Jovanović Stojanov, Sofija
AU  - Novaković, Miroslav
AU  - Opsenica, Igor M.
AU  - Pešić, Milica
PY  - 2021
UR  - http://www.bds.org.rs/download/SBS_Conference_10_2021.pdf
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5607
AB  - P-glycoprotein (P-gp) is often expressed at the cellular membrane of cancer cells where it plays a significant role in protecting cancer cells from extracellular assault. It works as an export transporter for many substrates - xenobiotics including chemotherapeutics. Several generations of P-gp inhibitors have been developed and studied but they have not yet been introduced into clinics. The most promising fourth-generation comprises natural compounds. In this study, we evaluated the potential of sclareol, a naturally occurring labdane diterpene, to inhibit P-gp activity in human glioblastoma (U87, and its resistant variant U87-TxR with P-gp overexpression) and non-small cell lung carcinoma (NCI-H460, and its resistant variant NCI-H460/R with P-gp overexpression) cell lines. To that end, we used the accumulation assays of fluorescent P-gp substrates (rhodamine 123 and doxorubicin) that were analyzed by flow cytometry. An increase in the accumulation of the P-gp substrate corresponds to the level of P-gp activity suppression. Our results showed that simultaneous application of sclareol (20 μM and 50 μM) with either rhodamine 123 (5 μM) or doxorubicin (20 μM) significantly increased their accumulation in resistant cells (U87-TxR and NCI-H460/R) than in their corresponding sensitive cells (U87 and NCI-H460). The doxorubicin accumulation was also considerably increased in sensitive U87 cells implying that sclareol may interact with doxorubicin through other mechanisms in glioblastoma cells (not only by P-gp inhibition). Further investigations are envisioned to reveal the mechanisms behind sclareol and doxorubicin interaction in glioblastoma cells.
PB  - Belgrade: Faculty of Chemistry: Serbian Biochemical Society, 2021
C3  - Serbian Biochemical Society Tenth Conference: with international participation: Biochemical Insights into Molecular Mechanisms; 2021 Sep 24; Kragujevac, Serbia
T1  - Sclareol, a natural compound, inhibits P-glycoprotein activity in cancer cells
SP  - 77
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5607
ER  - 
@conference{
author = "Stepanović, Ana and Lupšić, Ema and Nikolić, Andrea M. and Dragoj, Miodrag and Jovanović Stojanov, Sofija and Novaković, Miroslav and Opsenica, Igor M. and Pešić, Milica",
year = "2021",
abstract = "P-glycoprotein (P-gp) is often expressed at the cellular membrane of cancer cells where it plays a significant role in protecting cancer cells from extracellular assault. It works as an export transporter for many substrates - xenobiotics including chemotherapeutics. Several generations of P-gp inhibitors have been developed and studied but they have not yet been introduced into clinics. The most promising fourth-generation comprises natural compounds. In this study, we evaluated the potential of sclareol, a naturally occurring labdane diterpene, to inhibit P-gp activity in human glioblastoma (U87, and its resistant variant U87-TxR with P-gp overexpression) and non-small cell lung carcinoma (NCI-H460, and its resistant variant NCI-H460/R with P-gp overexpression) cell lines. To that end, we used the accumulation assays of fluorescent P-gp substrates (rhodamine 123 and doxorubicin) that were analyzed by flow cytometry. An increase in the accumulation of the P-gp substrate corresponds to the level of P-gp activity suppression. Our results showed that simultaneous application of sclareol (20 μM and 50 μM) with either rhodamine 123 (5 μM) or doxorubicin (20 μM) significantly increased their accumulation in resistant cells (U87-TxR and NCI-H460/R) than in their corresponding sensitive cells (U87 and NCI-H460). The doxorubicin accumulation was also considerably increased in sensitive U87 cells implying that sclareol may interact with doxorubicin through other mechanisms in glioblastoma cells (not only by P-gp inhibition). Further investigations are envisioned to reveal the mechanisms behind sclareol and doxorubicin interaction in glioblastoma cells.",
publisher = "Belgrade: Faculty of Chemistry: Serbian Biochemical Society, 2021",
journal = "Serbian Biochemical Society Tenth Conference: with international participation: Biochemical Insights into Molecular Mechanisms; 2021 Sep 24; Kragujevac, Serbia",
title = "Sclareol, a natural compound, inhibits P-glycoprotein activity in cancer cells",
pages = "77",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5607"
}
Stepanović, A., Lupšić, E., Nikolić, A. M., Dragoj, M., Jovanović Stojanov, S., Novaković, M., Opsenica, I. M.,& Pešić, M.. (2021). Sclareol, a natural compound, inhibits P-glycoprotein activity in cancer cells. in Serbian Biochemical Society Tenth Conference: with international participation: Biochemical Insights into Molecular Mechanisms; 2021 Sep 24; Kragujevac, Serbia
Belgrade: Faculty of Chemistry: Serbian Biochemical Society, 2021., 77.
https://hdl.handle.net/21.15107/rcub_ibiss_5607
Stepanović A, Lupšić E, Nikolić AM, Dragoj M, Jovanović Stojanov S, Novaković M, Opsenica IM, Pešić M. Sclareol, a natural compound, inhibits P-glycoprotein activity in cancer cells. in Serbian Biochemical Society Tenth Conference: with international participation: Biochemical Insights into Molecular Mechanisms; 2021 Sep 24; Kragujevac, Serbia. 2021;:77.
https://hdl.handle.net/21.15107/rcub_ibiss_5607 .
Stepanović, Ana, Lupšić, Ema, Nikolić, Andrea M., Dragoj, Miodrag, Jovanović Stojanov, Sofija, Novaković, Miroslav, Opsenica, Igor M., Pešić, Milica, "Sclareol, a natural compound, inhibits P-glycoprotein activity in cancer cells" in Serbian Biochemical Society Tenth Conference: with international participation: Biochemical Insights into Molecular Mechanisms; 2021 Sep 24; Kragujevac, Serbia (2021):77,
https://hdl.handle.net/21.15107/rcub_ibiss_5607 .

Evaluation of different temozolomide treatment modalities in a novel long-term 3D glioblastoma cell culture

Jovanović Stojanov, Sofija; Dragoj, Miodrag; Jovanović, Mirna; Stojkovska, Jasmina; Stankovic, Tijana; Dinić, Jelena; Podolski-Renić, Ana; Stepanović, Ana; Obradović, Bojana; Pešić, Milica

(The European Association for Cancer Research, 2021)

TY  - CONF
AU  - Jovanović Stojanov, Sofija
AU  - Dragoj, Miodrag
AU  - Jovanović, Mirna
AU  - Stojkovska, Jasmina
AU  - Stankovic, Tijana
AU  - Dinić, Jelena
AU  - Podolski-Renić, Ana
AU  - Stepanović, Ana
AU  - Obradović, Bojana
AU  - Pešić, Milica
PY  - 2021
UR  - https://www.eacr.org/login.php?referrer=%2Fconference%2FGoodbyeflatbiology2021virtual%2Fdigital-abstracts%2Fdetail%2F4208
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4904
AB  - Glioblastoma (GMB) is the most common and aggressive primary malignant brain tumor. Median life expectancy with the only clinically approved treatment option, Stupp protocol, is 11-15 months. This protocol involves surgical resection followed by 6 weeks of radiation and chemotherapy and then 6 cycles of 5-day chemotherapy treatment and 23 days of recovery. The only approved chemotherapeutic drug currently used is temozolomide (TMZ) that improves patients' survival for 2.5 months compared to radiotherapy alone. Such limited therapeutic options could be partly due to the lack of appropriate glioblastoma models for testing novel drugs and new treatment modalities.  
Therefore we established a novel long-term 3D glioblastoma biomimicking model system that would enable optimal drug testing in clinically more relevant duration. Glioblastoma U87 cells were immobilized in alginate microtubes and cultivated for 28 days, initially under static conditions. Cell viability, morphology and aggregate formation were monitored under fluorescent and confocal microscopes upon double staining with calcein-AM/propidium iodide. Most importantly, we investigated the effects of two different TMZ treatment modalities on cell viability and expression of resistance-related genes, MGMT and ABCB1. All treatments with 100 μm TMZ started on day 7 (X=7). We compared 3-day subsequent treatment modality (day by day treatments, X+1) with protractive treatment modality (every 7th day, X+7). 
In established 3D model system, cells managed to grow for up to 28 days without propagation, formed aggregates and constantly increased their number. Both treatment modalities had the same effects on cell viability. However, three day treatment in a row led to a tremendous increase in the expression of resistance markers, which was significantly higher compared to protractive treatment modality.
	The results showed that our 3D model system is suitable for drug testing and revealed that protractive treatment modality could be more beneficial for GBM patients.
PB  - The European Association for Cancer Research
C3  - Goodbye Flat Biology: Next Generation Cancer Models; 2021 Oct 5-6; Virtual event, Worldwide
T1  - Evaluation of different temozolomide treatment modalities in a novel long-term 3D glioblastoma cell culture
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4904
ER  - 
@conference{
author = "Jovanović Stojanov, Sofija and Dragoj, Miodrag and Jovanović, Mirna and Stojkovska, Jasmina and Stankovic, Tijana and Dinić, Jelena and Podolski-Renić, Ana and Stepanović, Ana and Obradović, Bojana and Pešić, Milica",
year = "2021",
abstract = "Glioblastoma (GMB) is the most common and aggressive primary malignant brain tumor. Median life expectancy with the only clinically approved treatment option, Stupp protocol, is 11-15 months. This protocol involves surgical resection followed by 6 weeks of radiation and chemotherapy and then 6 cycles of 5-day chemotherapy treatment and 23 days of recovery. The only approved chemotherapeutic drug currently used is temozolomide (TMZ) that improves patients' survival for 2.5 months compared to radiotherapy alone. Such limited therapeutic options could be partly due to the lack of appropriate glioblastoma models for testing novel drugs and new treatment modalities.  
Therefore we established a novel long-term 3D glioblastoma biomimicking model system that would enable optimal drug testing in clinically more relevant duration. Glioblastoma U87 cells were immobilized in alginate microtubes and cultivated for 28 days, initially under static conditions. Cell viability, morphology and aggregate formation were monitored under fluorescent and confocal microscopes upon double staining with calcein-AM/propidium iodide. Most importantly, we investigated the effects of two different TMZ treatment modalities on cell viability and expression of resistance-related genes, MGMT and ABCB1. All treatments with 100 μm TMZ started on day 7 (X=7). We compared 3-day subsequent treatment modality (day by day treatments, X+1) with protractive treatment modality (every 7th day, X+7). 
In established 3D model system, cells managed to grow for up to 28 days without propagation, formed aggregates and constantly increased their number. Both treatment modalities had the same effects on cell viability. However, three day treatment in a row led to a tremendous increase in the expression of resistance markers, which was significantly higher compared to protractive treatment modality.
	The results showed that our 3D model system is suitable for drug testing and revealed that protractive treatment modality could be more beneficial for GBM patients.",
publisher = "The European Association for Cancer Research",
journal = "Goodbye Flat Biology: Next Generation Cancer Models; 2021 Oct 5-6; Virtual event, Worldwide",
title = "Evaluation of different temozolomide treatment modalities in a novel long-term 3D glioblastoma cell culture",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4904"
}
Jovanović Stojanov, S., Dragoj, M., Jovanović, M., Stojkovska, J., Stankovic, T., Dinić, J., Podolski-Renić, A., Stepanović, A., Obradović, B.,& Pešić, M.. (2021). Evaluation of different temozolomide treatment modalities in a novel long-term 3D glioblastoma cell culture. in Goodbye Flat Biology: Next Generation Cancer Models; 2021 Oct 5-6; Virtual event, Worldwide
The European Association for Cancer Research..
https://hdl.handle.net/21.15107/rcub_ibiss_4904
Jovanović Stojanov S, Dragoj M, Jovanović M, Stojkovska J, Stankovic T, Dinić J, Podolski-Renić A, Stepanović A, Obradović B, Pešić M. Evaluation of different temozolomide treatment modalities in a novel long-term 3D glioblastoma cell culture. in Goodbye Flat Biology: Next Generation Cancer Models; 2021 Oct 5-6; Virtual event, Worldwide. 2021;.
https://hdl.handle.net/21.15107/rcub_ibiss_4904 .
Jovanović Stojanov, Sofija, Dragoj, Miodrag, Jovanović, Mirna, Stojkovska, Jasmina, Stankovic, Tijana, Dinić, Jelena, Podolski-Renić, Ana, Stepanović, Ana, Obradović, Bojana, Pešić, Milica, "Evaluation of different temozolomide treatment modalities in a novel long-term 3D glioblastoma cell culture" in Goodbye Flat Biology: Next Generation Cancer Models; 2021 Oct 5-6; Virtual event, Worldwide (2021),
https://hdl.handle.net/21.15107/rcub_ibiss_4904 .

Novel anti-cancer compound – inibitor of TrxR distress GSH system in glioma cells

Jovanović, Mirna; Dragoj, Miodrag; Jovanović Stojanov, Sofija; Stepanović, Ana; Dinić, Jelena; Nešović, Marija; Pešić, Milica; Podolski-Renić, Ana

(Elsevier Inc., 2021)

TY  - CONF
AU  - Jovanović, Mirna
AU  - Dragoj, Miodrag
AU  - Jovanović Stojanov, Sofija
AU  - Stepanović, Ana
AU  - Dinić, Jelena
AU  - Nešović, Marija
AU  - Pešić, Milica
AU  - Podolski-Renić, Ana
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4867
AB  - Glioblastoma is the most frequent malignant brain tumor, with limited therapy options due to aggressive invasiveness and resistance to therapy . Elevated expression of redox system components, thioredoxin (Trx) and thioredoxin reductase (TrxR), is a common feature of cancer cells and correlates with cancer progression and poor prognosis. Therefore, Trx and TrxR are attractive targets for chemotherapy development. Inhibition of Trx system may also affect another important redox system – the glutathione (GSH) system. Here, we investigated weather GSH system can compensate Trx system inhibition by novel TrxR1 inhibitor (DVD-444) in human and rat drug resistant glioma cell lines and their sensitive counterparts. RT-qPCR analysis showed that DVD-444 decreased GSH peroxidase 1 (GPX1) and 4 (GPX4) mRNA expression in all glioma cell lines. Decrease in GPX expression indicates suppression of another antioxidant defense system, besides Trx system, leaving cells vulnerable to oxidative stress. Tested compound caused an increase in expression of GSH reductase (GR) and GSH-S transferase π (GSTπ). The observed increase in GR could be the consequence of oxidative stress imposed by treatment with TrxR1inhibitor, while elevated GSTπ implies that GSTπ is highly involved in detoxification of the applied compound. Furthermore, colorimetric GSH assay showed that DVD-444 increased GSH cell content in glioma cell lines. AV/PI staining following treatment with TrxR1 inhibitor demonstrated significant level of cell death in rat glioma cell lines. Based on CFSE staining DVD-444 showed antiproliferative effect in human glioma cells. In conclusion, TrxR1 inhibitor caused changes in components of GSH system. However, the changes in GSH system did not prevent inhibition of cell proliferation and cell death evasion after TrxR1 inhibition, making DVD-444 perspective candidate for glioblastoma treatment strategy
PB  - Elsevier Inc.
C3  - Free Radical Research Europe (SFRR-E) Annual Meeting Abstracts “Redox biology in the 21st century: a new scientific discipline” 15-18 June 2021, Belgrade, Serbia
T1  - Novel anti-cancer compound – inibitor of TrxR distress GSH system in glioma cells
DO  - 10.1016/j.freeradbiomed.2021.08.137
SP  - S97
ER  - 
@conference{
author = "Jovanović, Mirna and Dragoj, Miodrag and Jovanović Stojanov, Sofija and Stepanović, Ana and Dinić, Jelena and Nešović, Marija and Pešić, Milica and Podolski-Renić, Ana",
year = "2021",
abstract = "Glioblastoma is the most frequent malignant brain tumor, with limited therapy options due to aggressive invasiveness and resistance to therapy . Elevated expression of redox system components, thioredoxin (Trx) and thioredoxin reductase (TrxR), is a common feature of cancer cells and correlates with cancer progression and poor prognosis. Therefore, Trx and TrxR are attractive targets for chemotherapy development. Inhibition of Trx system may also affect another important redox system – the glutathione (GSH) system. Here, we investigated weather GSH system can compensate Trx system inhibition by novel TrxR1 inhibitor (DVD-444) in human and rat drug resistant glioma cell lines and their sensitive counterparts. RT-qPCR analysis showed that DVD-444 decreased GSH peroxidase 1 (GPX1) and 4 (GPX4) mRNA expression in all glioma cell lines. Decrease in GPX expression indicates suppression of another antioxidant defense system, besides Trx system, leaving cells vulnerable to oxidative stress. Tested compound caused an increase in expression of GSH reductase (GR) and GSH-S transferase π (GSTπ). The observed increase in GR could be the consequence of oxidative stress imposed by treatment with TrxR1inhibitor, while elevated GSTπ implies that GSTπ is highly involved in detoxification of the applied compound. Furthermore, colorimetric GSH assay showed that DVD-444 increased GSH cell content in glioma cell lines. AV/PI staining following treatment with TrxR1 inhibitor demonstrated significant level of cell death in rat glioma cell lines. Based on CFSE staining DVD-444 showed antiproliferative effect in human glioma cells. In conclusion, TrxR1 inhibitor caused changes in components of GSH system. However, the changes in GSH system did not prevent inhibition of cell proliferation and cell death evasion after TrxR1 inhibition, making DVD-444 perspective candidate for glioblastoma treatment strategy",
publisher = "Elsevier Inc.",
journal = "Free Radical Research Europe (SFRR-E) Annual Meeting Abstracts “Redox biology in the 21st century: a new scientific discipline” 15-18 June 2021, Belgrade, Serbia",
title = "Novel anti-cancer compound – inibitor of TrxR distress GSH system in glioma cells",
doi = "10.1016/j.freeradbiomed.2021.08.137",
pages = "S97"
}
Jovanović, M., Dragoj, M., Jovanović Stojanov, S., Stepanović, A., Dinić, J., Nešović, M., Pešić, M.,& Podolski-Renić, A.. (2021). Novel anti-cancer compound – inibitor of TrxR distress GSH system in glioma cells. in Free Radical Research Europe (SFRR-E) Annual Meeting Abstracts “Redox biology in the 21st century: a new scientific discipline” 15-18 June 2021, Belgrade, Serbia
Elsevier Inc.., S97.
https://doi.org/10.1016/j.freeradbiomed.2021.08.137
Jovanović M, Dragoj M, Jovanović Stojanov S, Stepanović A, Dinić J, Nešović M, Pešić M, Podolski-Renić A. Novel anti-cancer compound – inibitor of TrxR distress GSH system in glioma cells. in Free Radical Research Europe (SFRR-E) Annual Meeting Abstracts “Redox biology in the 21st century: a new scientific discipline” 15-18 June 2021, Belgrade, Serbia. 2021;:S97.
doi:10.1016/j.freeradbiomed.2021.08.137 .
Jovanović, Mirna, Dragoj, Miodrag, Jovanović Stojanov, Sofija, Stepanović, Ana, Dinić, Jelena, Nešović, Marija, Pešić, Milica, Podolski-Renić, Ana, "Novel anti-cancer compound – inibitor of TrxR distress GSH system in glioma cells" in Free Radical Research Europe (SFRR-E) Annual Meeting Abstracts “Redox biology in the 21st century: a new scientific discipline” 15-18 June 2021, Belgrade, Serbia (2021):S97,
https://doi.org/10.1016/j.freeradbiomed.2021.08.137 . .

Pyrazolo[3,4-d]pyrimidine derivatives, Si306 and pro-Si306, induce oxidative stress and cell death in patient-derived glioblastoma cells

Stepanović, Ana; Podolski-Renić, Ana; Jovanović Stojanov, Sofija; Nešović, Marija; Dragoj, Miodrag; Jovanović, Mirna; Nikolić, Igor; Tasić, Goran; Schenone, Silvia; Pešić, Milica; Dinić, Jelena

(Elsevier Inc., 2021)

TY  - CONF
AU  - Stepanović, Ana
AU  - Podolski-Renić, Ana
AU  - Jovanović Stojanov, Sofija
AU  - Nešović, Marija
AU  - Dragoj, Miodrag
AU  - Jovanović, Mirna
AU  - Nikolić, Igor
AU  - Tasić, Goran
AU  - Schenone, Silvia
AU  - Pešić, Milica
AU  - Dinić, Jelena
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4801
AB  - Glioblastoma is the most frequent and aggressive brain tumor in adults. The main characteristics of glioblastoma include high proliferation rate, infiltrating nature, and resistance to chemotherapy and radiation. Glioblastoma cells highly express Src tyrosine kinase which has a key role in regulating survival, proliferation, angiogenesis and invasiveness of tumor cells. This tyrosine kinase is also an important regulator of reactive oxygen species production and cellular homeostasis. Anticancer properties of two pyrazolo[3,4-d]pyrimidine derivatives and Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306, were investigated in human glioblastoma cell line U87, its multidrug-resistant (MDR) counterpart U87-TxR, and patient-derived glioblastoma cell culture. Si306 and pro-Si306 triggered reactive oxygen species and reactive nitrogen species production in sensitive and MDR glioblastoma cell lines and primary glioblastoma cells as evidenced by elevated levels of superoxide anion, hydrogen peroxide and peroxynitrite anion. Additionally, western blot analysis revealed elevated expression of superoxide dismutase 1, superoxide dismutase 2, and thioredoxin reductase 1 in glioblastoma cells after Si306 and pro-Si306 treatments. The levels of phosphorylated histone H2A.X increased in all glioblastoma cells after treatments with these inhibitors, demonstrating DNA damage. Both compounds also induced significant cell death in primary glioblastoma culture. In addition, the Src tyrosine kinase inhibitors prompted primary glioblastoma cells to enter senescence. The presence of the MDR phenotype did not reduce the activity of the compounds. Overall, the investigated pyrazolo[3,4-d]pyrimidines displayed significant anti-glioblastoma effects making them good candidates for further development as anticancer agents
PB  - Elsevier Inc.
C3  - Free Radical Research Europe (SFRR-E) Annual Meeting Abstracts “Redox biology in the 21st century: a new scientific discipline” 15-18 June 2021, Belgrade, Serbia
T1  - Pyrazolo[3,4-d]pyrimidine derivatives, Si306 and pro-Si306, induce oxidative stress and cell death in patient-derived glioblastoma cells
DO  - 10.1016/j.freeradbiomed.2021.08.082
SP  - S75
ER  - 
@conference{
author = "Stepanović, Ana and Podolski-Renić, Ana and Jovanović Stojanov, Sofija and Nešović, Marija and Dragoj, Miodrag and Jovanović, Mirna and Nikolić, Igor and Tasić, Goran and Schenone, Silvia and Pešić, Milica and Dinić, Jelena",
year = "2021",
abstract = "Glioblastoma is the most frequent and aggressive brain tumor in adults. The main characteristics of glioblastoma include high proliferation rate, infiltrating nature, and resistance to chemotherapy and radiation. Glioblastoma cells highly express Src tyrosine kinase which has a key role in regulating survival, proliferation, angiogenesis and invasiveness of tumor cells. This tyrosine kinase is also an important regulator of reactive oxygen species production and cellular homeostasis. Anticancer properties of two pyrazolo[3,4-d]pyrimidine derivatives and Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306, were investigated in human glioblastoma cell line U87, its multidrug-resistant (MDR) counterpart U87-TxR, and patient-derived glioblastoma cell culture. Si306 and pro-Si306 triggered reactive oxygen species and reactive nitrogen species production in sensitive and MDR glioblastoma cell lines and primary glioblastoma cells as evidenced by elevated levels of superoxide anion, hydrogen peroxide and peroxynitrite anion. Additionally, western blot analysis revealed elevated expression of superoxide dismutase 1, superoxide dismutase 2, and thioredoxin reductase 1 in glioblastoma cells after Si306 and pro-Si306 treatments. The levels of phosphorylated histone H2A.X increased in all glioblastoma cells after treatments with these inhibitors, demonstrating DNA damage. Both compounds also induced significant cell death in primary glioblastoma culture. In addition, the Src tyrosine kinase inhibitors prompted primary glioblastoma cells to enter senescence. The presence of the MDR phenotype did not reduce the activity of the compounds. Overall, the investigated pyrazolo[3,4-d]pyrimidines displayed significant anti-glioblastoma effects making them good candidates for further development as anticancer agents",
publisher = "Elsevier Inc.",
journal = "Free Radical Research Europe (SFRR-E) Annual Meeting Abstracts “Redox biology in the 21st century: a new scientific discipline” 15-18 June 2021, Belgrade, Serbia",
title = "Pyrazolo[3,4-d]pyrimidine derivatives, Si306 and pro-Si306, induce oxidative stress and cell death in patient-derived glioblastoma cells",
doi = "10.1016/j.freeradbiomed.2021.08.082",
pages = "S75"
}
Stepanović, A., Podolski-Renić, A., Jovanović Stojanov, S., Nešović, M., Dragoj, M., Jovanović, M., Nikolić, I., Tasić, G., Schenone, S., Pešić, M.,& Dinić, J.. (2021). Pyrazolo[3,4-d]pyrimidine derivatives, Si306 and pro-Si306, induce oxidative stress and cell death in patient-derived glioblastoma cells. in Free Radical Research Europe (SFRR-E) Annual Meeting Abstracts “Redox biology in the 21st century: a new scientific discipline” 15-18 June 2021, Belgrade, Serbia
Elsevier Inc.., S75.
https://doi.org/10.1016/j.freeradbiomed.2021.08.082
Stepanović A, Podolski-Renić A, Jovanović Stojanov S, Nešović M, Dragoj M, Jovanović M, Nikolić I, Tasić G, Schenone S, Pešić M, Dinić J. Pyrazolo[3,4-d]pyrimidine derivatives, Si306 and pro-Si306, induce oxidative stress and cell death in patient-derived glioblastoma cells. in Free Radical Research Europe (SFRR-E) Annual Meeting Abstracts “Redox biology in the 21st century: a new scientific discipline” 15-18 June 2021, Belgrade, Serbia. 2021;:S75.
doi:10.1016/j.freeradbiomed.2021.08.082 .
Stepanović, Ana, Podolski-Renić, Ana, Jovanović Stojanov, Sofija, Nešović, Marija, Dragoj, Miodrag, Jovanović, Mirna, Nikolić, Igor, Tasić, Goran, Schenone, Silvia, Pešić, Milica, Dinić, Jelena, "Pyrazolo[3,4-d]pyrimidine derivatives, Si306 and pro-Si306, induce oxidative stress and cell death in patient-derived glioblastoma cells" in Free Radical Research Europe (SFRR-E) Annual Meeting Abstracts “Redox biology in the 21st century: a new scientific discipline” 15-18 June 2021, Belgrade, Serbia (2021):S75,
https://doi.org/10.1016/j.freeradbiomed.2021.08.082 . .

Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors Induce Oxidative Stress in Patient-Derived Glioblastoma Cells

Stepanović, Ana; Jovanović Stojanov, Sofija; Podolski-Renić, Ana; Nešović, Marija; Dragoj, Miodrag; Nikolić, Igor; Tasić, Goran; Schenone, Silvia; Pešić, Milica; Dinić, Jelena

(Basel : MDPI, 2021)

TY  - JOUR
AU  - Stepanović, Ana
AU  - Jovanović Stojanov, Sofija
AU  - Podolski-Renić, Ana
AU  - Nešović, Marija
AU  - Dragoj, Miodrag
AU  - Nikolić, Igor
AU  - Tasić, Goran
AU  - Schenone, Silvia
AU  - Pešić, Milica
AU  - Dinić, Jelena
PY  - 2021
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/4258
AB  - Background: Glioblastoma (GBM) highly expresses Src tyrosine kinase involved in survival, proliferation, angiogenesis and invasiveness of tumor cells. Src activation also reduces reactive oxygen species (ROS) generation, whereas Src inhibitors are able to increase cellular ROS levels. Methods: Pro-oxidative effects of two pyrazolo[3,4-d]pyrimidine derivatives—Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306—were investigated in human GBM cells U87 and patient-derived GBM-6. ROS production and changes in mitochondrial membrane potential were assessed by flow cytometry. The expression levels of superoxide dismutase 1 (SOD1) and 2 (SOD2) were studied by Western blot. DNA damage, cell death induction and senescence were also examined in GBM-6 cells. Results: Si306 and pro-Si306 more prominently triggered ROS production and expression of antioxidant enzymes in primary GBM cells. These effects were followed by mitochondrial membrane potential disruption, double-strand DNA breaks and senescence that eventually led to necrosis. Conclusion: Src kinase inhibitors, Si306 and pro-Si306, showed significant pro-oxidative potential in patient-derived GBM cells. This feature contributes to the already demonstrated anti-glioblastoma properties of these compounds in vitro and in vivo and encourages clinical investigations.
PB  - Basel : MDPI
T2  - Brain Sciences
T1  - Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors Induce Oxidative Stress in Patient-Derived Glioblastoma Cells
IS  - 7
VL  - 11
DO  - 10.3390/brainsci11070884
SP  - 884
ER  - 
@article{
author = "Stepanović, Ana and Jovanović Stojanov, Sofija and Podolski-Renić, Ana and Nešović, Marija and Dragoj, Miodrag and Nikolić, Igor and Tasić, Goran and Schenone, Silvia and Pešić, Milica and Dinić, Jelena",
year = "2021",
abstract = "Background: Glioblastoma (GBM) highly expresses Src tyrosine kinase involved in survival, proliferation, angiogenesis and invasiveness of tumor cells. Src activation also reduces reactive oxygen species (ROS) generation, whereas Src inhibitors are able to increase cellular ROS levels. Methods: Pro-oxidative effects of two pyrazolo[3,4-d]pyrimidine derivatives—Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306—were investigated in human GBM cells U87 and patient-derived GBM-6. ROS production and changes in mitochondrial membrane potential were assessed by flow cytometry. The expression levels of superoxide dismutase 1 (SOD1) and 2 (SOD2) were studied by Western blot. DNA damage, cell death induction and senescence were also examined in GBM-6 cells. Results: Si306 and pro-Si306 more prominently triggered ROS production and expression of antioxidant enzymes in primary GBM cells. These effects were followed by mitochondrial membrane potential disruption, double-strand DNA breaks and senescence that eventually led to necrosis. Conclusion: Src kinase inhibitors, Si306 and pro-Si306, showed significant pro-oxidative potential in patient-derived GBM cells. This feature contributes to the already demonstrated anti-glioblastoma properties of these compounds in vitro and in vivo and encourages clinical investigations.",
publisher = "Basel : MDPI",
journal = "Brain Sciences",
title = "Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors Induce Oxidative Stress in Patient-Derived Glioblastoma Cells",
number = "7",
volume = "11",
doi = "10.3390/brainsci11070884",
pages = "884"
}
Stepanović, A., Jovanović Stojanov, S., Podolski-Renić, A., Nešović, M., Dragoj, M., Nikolić, I., Tasić, G., Schenone, S., Pešić, M.,& Dinić, J.. (2021). Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors Induce Oxidative Stress in Patient-Derived Glioblastoma Cells. in Brain Sciences
Basel : MDPI., 11(7), 884.
https://doi.org/10.3390/brainsci11070884
Stepanović A, Jovanović Stojanov S, Podolski-Renić A, Nešović M, Dragoj M, Nikolić I, Tasić G, Schenone S, Pešić M, Dinić J. Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors Induce Oxidative Stress in Patient-Derived Glioblastoma Cells. in Brain Sciences. 2021;11(7):884.
doi:10.3390/brainsci11070884 .
Stepanović, Ana, Jovanović Stojanov, Sofija, Podolski-Renić, Ana, Nešović, Marija, Dragoj, Miodrag, Nikolić, Igor, Tasić, Goran, Schenone, Silvia, Pešić, Milica, Dinić, Jelena, "Pyrazolo[3,4-d]pyrimidine Tyrosine Kinase Inhibitors Induce Oxidative Stress in Patient-Derived Glioblastoma Cells" in Brain Sciences, 11, no. 7 (2021):884,
https://doi.org/10.3390/brainsci11070884 . .
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