Uticaj metformina na apoptozu ćelija glioma i melanoma in vitro i na rast melanoma in vivo

Abstract

U ovoj disertaciji ispitivan je efekat antidijabetskog leka metformina na apoptozu celijskih linija melanoma i glioma in vitro, u odsustvu i prisustvu hemoterapeutika cisplatina. Takode smo bili zainteresovani za uticaj metformina na rast melanoma in vivo. U odsustvu cisplatina po prvi put je pokazan dvojan antiglioma efekat metformina na celijskoj liniji glioma pacova C6. U celijskim kulturama glioma pacova C6 male gustine metformin je zaustavio progresiju celijskog ciklusa u G0/G1 fazi, bez znacajnog povecanja celijske smrti. Sa druge strane, u konfluentnim kulturama C6 celija metformin je izazvao izuzetno povecanje apoptoze koja je zavisna od kaspaza, depolarizacije mitohondrija, oksidativnog stresa i povezana sa aktivacijom JNK (engl. c-Jun N-terminal kinase). Apoptoza indukovana metforminom je u potpunosti sprecena supstancama koje blokiraju promenu propustljivosti membrane mitohondrija (ciklosporin A) i produkciju reaktivnih kiseonicnih vrsta (N-acetilcistein), dok su je inhibitori aktivacije JNK (SP600125) ili glikolize (natrijumfluorid, jodoacetat) delimicno sprecili. Supstanca C, koja je inhibitor adenozin monofosfatom aktivirane protein kinaze AMPK (engl. AMPactivated protein kinase), je smanjila antiglioma efekat metformina dok je AMPK agonista AICAR (engl. 5-Aminoimidazole-4- carboxamide ribonucleotide) imitirao pomenuti efekat. Primarni astrociti pacova su bili u potpunosti otporni na antiproliferativno i proapoptotsko dejstvo metformina. Dalje je ispitivan in vitro uticaj metformina na antikancersku aktivnost dobro poznatog hemoterapeutika cisplatina. Iako je sam metformin smanjio broj tumorskih celija, iznenadujuce antagonizovao je citotoksicnost cisplatina u U251 celijama glioma. Metformin je smanjio apoptotsku celijsku smrt indukovanu cisplatinom u U251 celijama glioma, tako što je smanjio oksidativni stres i aktivaciju kaspaza. Zapažena citoprotekcija je ocigledno nezavisna od AMPK, jer metformin nije dalje povecao aktivaciju AMPK indukovanu cisplatinom i drugi farmakološki aktivatori AMPK nisu uspeli da inhibiraju apoptozu indukovanu cisplatinom. Sa druge strane, metformin je indukovao aktivaciju Akt u celijama tretiranim cisplatinom, a inhibitor Akt (10-DEBC hidrohlorid) ili fosfoinozitid 3-kinaze/Akt inhibitor (LY294002) su poništili antioksidantna i antiapoptotska dejstva posredovana metforminom. U odsustvu cisplatina in vitro i in vivo antimelanoma efekat metformina je ispitivan, koristeci B16 celijsku liniju melanoma miša. Metformin je izazvao zaustavljanje celijskog ciklusa u G2/M fazi, koje je povezano sa apoptotskom smrcu celija melanoma. Indukcija oksidativnog stresa i depolarizacija membrane mitohondrija su prethodili apoptozi posredovanoj metforminom za koju je pokazano, da je zavisna od kaspaza. Metformin je povecao ekspresiju tumor supresornog proteina p53, dok je smanjio nivo iRNK antiapoptotskog proteina Bcl-2.Tretman metforminom nije stimulisao ekspresiju inhibitora celijskog ciklusa p21, dajuci indikaciju da p21 nije neophodan za zapaženi blok celijskog ciklusa. Za antimelanoma delovanje metformina izgleda da nije potrebna aktivacija AMPK, jer supstanca C nije uspela da povrati vijabilitet B16 celija tretiranih metforminom. Metformin je indukovao autofagiju u B16 celijama, dok su inhibitori autofagije amonijum hlorid i vortmanin delimicno povratili vijabilitet celija melanoma tretiranih metforminom, što ukazuje da je autofagija indukovana metforminom citotoksicna. Konacno, oralna upotreba metformina dovela je do znacajnog smanjenja velicine tumora u mišijem B16 melanoma modelu. Rezultati dobijeni u odsustvu cisplatina na celijskim linijama melanoma i glioma in vitro, kao i na mišijem modelu in vivo, ukazuju na potencijalnu primenu metformina kao antikancerskog agensa u navedenim tumorima. Sa druge strane, u prisustvu cisplatina pokazalo se da treba veoma pažljivo uzeti u razmatranje upotrebu metformina kod pacijenata koji boluju od dijabetesa i kancera i primaju cisplatin, ili kao potencijalne aduvantne terapije u hemoterapeutskim protokolima baziranim na cisplatinu.

We investigated the effect of the well known antidiabetic drug metformin on the viability of melanoma and glioma cell lines in vitro, in the absence and presence of cisplatin. Also, we were interested in the effect of metformin on melanoma growth in vivo. In the absence of cisplatin on rat glioma cell line C6, we have shown for the first time a dual antiglioma effect of metformin. In low-density cultures of the C6 rat glioma cell line, metformin blocked the cell cycle progression in G0/G1 phase without inducing significant cell death. In confluent C6 cultures, on the otherhand, metformin caused massive induction of caspase dependent apoptosis associated with c-JunNterminalkinase (JNK) activation, mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine), while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride, iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C, an inhibitor of AMP activated protein kinase (AMPK), and was mimicked by the AMPK agonist AICAR. Rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. Further, it was investigated the influence of metformin on the in vitro anticancer activity of the well-known chemotherapeutic agent cisplatin. Although metformin reduced the number of tumour cells when applied alone, it surprisingly antagonized the cytotoxicity of cisplatin towards U251 human glioma. In U251 glioma cells metformin suppressed cisplatin-induced apoptotic cell death through inhibition of oxidative stress and caspase activation. The observed cytoprotection was apparently AMPKindependent, as metformin did not further increase cisplatin-induced AMPK activation in U251 cells and other pharmacological AMPK activators failed to block cisplatinmediated apoptosis. On the other hand, metformin induced Akt activation in cisplatintreated cells and Akt inhibitor 10-DEBC hydrochloride or phosphoinositide 3- kinase/Akt inhibitor LY294002 abolished metformin-mediated antioxidant and antiapoptotic effects. In the absence of cisplatin, in vitro and in vivo anti-melanoma effect of antidiabetic drug metformin was investigated using B16 mouse melanoma cell line. Metformin caused a G2/M cell cycle arrest associated with apoptotic death of melanoma cells. Metformin-mediated apoptosis of melanoma cells was preceded by the induction of oxidative stress and mitochondrial membrane depolarization. Metformin caused an increase in the expression of tumor suppressorprotein p53 with concomitant decrease of anti-apoptotic Bcl-2 mRNA levels. However, the treatment with metformin did not stimulate expression of the cycle blocker p21, indicating that p21 was dispensable for the observed cell cycle arrest. The activation of AMP-activated protein kinase (AMPK) was not required for the anti-melanoma action of metformin, as AMPK inhibitor compound C completely failed to restore viability of metformin-treated B16 cells. Metformin induced autophagy in B16 cells, autophagy inhibitors ammonium chloride and wortmannin partly restored the viability of metformin-treated melanoma cells, indicating that metformin-induced autophagy is cytotoxic. Finally, oral administration of metformin led to a significant reduction in tumor size in a B16 mouse melanoma model. Results obtained in cisplatin absence on melanoma and glioma cell lines in vitro and mouse melanoma model in vivo, indicate the potential metformin application as anticancer therapeutic in melanoma and glioma tumor. On the other hand, data in the cisplatin presence warrant caution when considering metformin for treatment of diabetic cancer patients receiving cisplatin, or as a potential adjuvant in cisplatin-based chemotherapeutic regimens.