Uspostavljanje rezistentnih tumorskih ćelijskih linija kao modela za testiranje novih hemioterapeutika: molekularna karakterizacija rezistencije nastale dugotrajnim izlaganjem paklitakselu

Abstract

Glavni uzrok neuspeha hemioterapije u lečenju kancera je pojava višestruke(engl. „multi-drug“) rezistencije (MDR). Efikasnost paklitaksela (PTX) je čestoograničena pojavom rezistencije. Cilj ove doktorske disertacije je bio ispitivanjemolekularnih i fenotipskih promena u toku razvoja MDR-a indukovanih PTX-om kodćelijskih linija humanog karcinoma debelog creva (DLD1) i glioblastoma (U87).Takođe je testirana upotrebljivost dobijenih MDR modela u evaluaciji četiri anti-kanceragensa.Kontinuirani tretman PTX-om doveo je do razvoja MDR-a kod obe ispitivanećelijske linije koje su postale rezistentne na strukturno i funkcionalno različitehemioterapeutike. Nakon potvrde prisustva ukrštene rezistencije kod novouspostavljenihćelijskih linija DLD1-TxR i U87-TxR, analizirana je ekspresijamembranskih trasportera uključenih u razvoj MDR-a na nivou iRNK. Ćelije su imalepovišen nivo ekspresije mdr1 gena i smanjen nivo ekspresije mrp1 gena. Prekomernaekspresija P-glikoproteina (P-gp), koji kodira mdr1 gen, je uočena kod obe MDRćelijske linije. Analiza na protočnom citofluorimetru je pokazala da je akumulacija Pgpsupstrata (rodamina 123 i doksorubicina) kod DLD1-TxR i U87-TxR ćelija značajnomanja u poređenju sa odgovarajućim parentalnim ćelijama, DLD1 i U87. Značajnosmanjenje ekspresije gst-π gena i koncentracije glutationa (GSH) je uočeno kod U87-TxR ćelija. Sekrecija vaskularnog endotelijalnog faktora rasta (VEGF) je inhibirana ujednokratnom tretmanu kod ćelijskih linija karcinoma debelog creva i u kontinuiranomtretmanu kod ćelijske linije glioblastoma. Analiza ćelijskog ciklusa je pokazala da jejednokratni tretman PTX-om kod ćelijskih linija humanog karcinoma debelog crevapraćen porastom subG0 faze, odnosno povećanjem procenta mrtvih ćelija, dok je kodćelija glioblastoma došlo do umiranja tokom interfaze (G1, S ili G2). MDR tumorskećelijske linije su stekle nove strukturne i numerićke hromozomske aberacije. SticanjeMDR fenotipa kod U87-TxR ćelija je praćeno smanjenem jednog nivoa ploidije.Takođe je uočen gubitak hromozomskog regiona 6q kod obe rezistentne ćelijske linije,kao i inaktivacija p53 tumor spresor gena kod U87-TxR ćelija i PTEN tumor supresorgena kod DLD1-TxR ćelija.

Multi-drug resistance (MDR) is a major obstacle to successful cancer treatment.The efficacy of paclitaxel (PTX) is often limited by appearance of drug resistance. The aim ofthis study was to explore molecular and phenotypic alterations during development of MDRinduced by PTX in human colon carcinoma (DLD1) and glioblastoma (U87) cell lines. Wealso tested the usefulness of developed MDR models in the evaluation of four anti-canceragents.Continuous treatment with PTX led to the development of MDR in both testedcancer cell lines that became resistant to structurally and functionally unrelatedchemotherapeutics. After confirmation of the cross-resistance in newly established DLD1-TxRand U87-TxR, we analyzed the mRNA expression of membrane transporters involved inMDR. The cells had increased levels of mdr1 gene expression, while mrp1 was decreased.Over-expression of P-glycoprotein (P-gp), coded by mdr1, was observed in both MDR cancercell lines. Flow cytometry analyzes showed that the accumulation of P-gp substrates(rhodamine 123 and doxorubicin) in DLD1-TxR and U87-TxR was significantly lowercompared to DLD1 and U87, respectively. The significant depletion of gst-π gene expressionand glutathione (GSH) concentration was observed in U87-TxR. Vascular Endothelial GrowthFactor (VEGF) secretion was inhibited by single PTX treatment of colon cancer and incontinuous treatment of glioblastoma cell lines. The analysis of cell cycle kinetics revealedextensive cell death in colon cancer cells that were accumulated in subG0 phase after PTXtreatment, while glioblastoma cells died through interphase (G1, S or G2). The MDR cancercell lines acquired novel structural or numerical chromosomal aberrations. Polyploidyreduction was observed after development of MDR in U87-TxR. Losses of 6q in both resistantcancer cell lines and inactivation of p53 in U87-TxR and PTEN in DLD1-TxR were alsorevealed.We evaluated the anti-cancer activities and MDR reversal potential of the Aktinhibitor (GSK690693), the Ras inhibitor (Tipifarnib) and two P-gp inhibitors (jatrophanediterpenoids Euphodendrophane H-Euph H and Euphodendrophane S -Euph S). Their effectsvary due to the cell-type differences, existence of MDR phenotype or tumor suppressors’alterations. Tipifarnib, Euph H and S, significantly sensitized MDR cancer cells to PTX.