title: In vitro and in vivo anti-melanoma action of metformin.
abstract: The in vitro and in vivo anti-melanoma effect of antidiabetic drug metformin was investigated using B16 mouse melanoma cell line. Metformin caused a G(2)/M cell cycle arrest associated with apoptotic death of melanoma cells, as confirmed by the flow cytometric analysis of cell cycle/DNA fragmentation, phosphatidylserine exposure and caspase activation. Metformin-mediated apoptosis of melanoma cells was preceded by induction of oxidative stress and mitochondrial membrane depolarization, measured by flow cytometry in cells stained with appropriate fluorescent reporter dyes. The expression of tumor suppressor protein p53 was increased, while the mRNA levels of anti-apoptotic Bcl-2 were reduced by metformin, as revealed by cell-based ELISA and real-time RT-PCR, respectively. 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, as demonstrated by flow cytometry-detected increase in intracellular acidification and immunoblot-confirmed upregulation of autophagosome-associated LC3-II. Autophagy inhibitors ammonium chloride and wortmannin partly restored the viability of metformin-treated melanoma cells. Finally, oral administration of metformin led to a significant reduction in tumor size in a B16 mouse melanoma model. These data suggest that anti-melanoma effects of metformin are mediated through p21- and AMPK-independent cell cycle arrest, apoptosis and autophagy associated with p53/Bcl-2 modulation, mitochondrial damage and oxidative stress.
authors.affiliation: Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade, Serbia.
authors.forename: Kristina
authors.initials: K
authors.lastname: Janjetovic
authors.forename: Ljubica
authors.initials: L
authors.lastname: Harhaji-Trajkovic
authors.forename: Maja
authors.initials: M
authors.lastname: Misirkic-Marjanovic
authors.forename: Ljubica
authors.initials: L
authors.lastname: Vucicevic
authors.forename: Darko
authors.initials: D
authors.lastname: Stevanovic
authors.forename: Nevena
authors.initials: N
authors.lastname: Zogovic
authors.forename: Mirjana
authors.initials: M
authors.lastname: Sumarac-Dumanovic
authors.forename: Dragan
authors.initials: D
authors.lastname: Micic
authors.forename: Vladimir
authors.initials: V
authors.lastname: Trajkovic
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004195:Disease Models, Animal; D004789:Enzyme Activation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D008546:Melanoma, Experimental / Q000188:drug therapy* / Q000473:pathology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008687:Metformin / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use*; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D008687:Metformin; D055372:AMP-Activated Protein Kinases; D020169:Caspases