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...

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...