In vitro metformin anti-neoplastic activity in epithelial ovarian cancer
Introduction
Ovarian cancer is the fifth most common cause of cancer related mortality in western countries [1]. The most common type is of epithelial origin. Although the majority of patients have a satisfactory initial clinical response, more than 70% will experience recurrences and ultimately die of the disease [2]. New therapeutic modalities are therefore necessary to overcome the high recurrence rate and to change response to treatment into cure.
Increasing basic science evidence and dietary findings suggests that insulin and insulin-like growth factors (IGFs) play a role in carcinogenesis and disease progression in epithelial ovarian cancer [reviewed in [3], [4], [5], [6]]. Therefore downregulation of the IGF signaling pathway attained by “caloric restriction” has been postulated as a therapeutic approach to prevent cancer development [7], [8]. Among other effects, “caloric restriction” leads to the activation of the AMP-activated protein kinase (AMPK) which is the primary activator of cellular response to lowered ATP levels [9], [10]. Activated AMPK has been associated with growth inhibition of human cancer cell lines [11], [12].
Upon activation, the phosphorylated AMPK suppresses the mammalian target of Rapamycin (mTOR) signaling pathway, which, via its downstream molecular effectors 4E-BP1 and p70S6, ultimately affects cellular transcription and translation [13], [14]. Several experimental approaches have demonstrated the therapeutic potential of mTOR inhibition, inducing drastic anti-proliferative and anti-angiogenic effects in pre-clinical models [14], [15], [16], [17].
Metformin, a commonly used oral anti-hyperglycemic agent of the biguanide family, also activates AMPK [18]. Taken together with recent population-based studies that suggested that metformin may reduce cancer risk and improve prognosis [19], [20], we evaluated the in vitro anti-neoplastic activity of metformin in epithelial ovarian cancer, and its relationship to AMPK activation and downstream pathway.
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Cell lines and treatments
The human ovarian cancer cell lines OVCAR-3 (American tissue culture collection, Manassas, VA) and OVCAR-4 were grown in RMPI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 10 μg/ml of garamycin at 37 °C in 5% CO2. The cell cultures were routinely passaged every 3–5 days. Assays were conducted under serum-free conditions as previously described [3]. Metformin was obtained from Sigma Laboratories (Oakville, Ontario). Cisplatin was obtained from Mayne Pharmaceuticals
Metformin inhibits the proliferation of OVCAR-3 and OVCAR-4 cells in a dose- and time-dependent manner
Metformin induced significant proliferation inhibition on both the OVCAR-3 and OVCAR-4 cells lines in a dose- (Fig. 1) and time-dependent manner (Fig. 2).
Metformin potentiates the effect of cisplatin
Cisplatin remains the most active treatment for epithelial ovarian carcinoma, however sensitivity of the tumor cells to cisplatin varies. Here we demonstrate that metformin could increase the cytotoxicity of cisplatin on OVCAR-3 cells (Fig. 3).
Compound C inhibits the anti-proliferative effect induced by metformin
Compound C is known to function as an ATP-competitive inhibitor of AMPK [21]. Here we demonstrate
Discussion
The potential link between Insulin/IGF-I signaling pathways and cancer has attracted substantial attention during the last years [22]. As a result there is a growing interest in targeting this pathway for cancer treatment. AMPK is a conserved serine/threonine protein kinase regulator of cellular metabolism that is activated in response to nutrient deprivation and pathological stresses [23]. Interestingly, several in vitro and in vivo studies show cancer growth inhibition following AMPK
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was in part supported by grants from the Israel Cancer Research Foundation, the Canadian Foundation for Women's Health, the Gloria Shapiro Foundation, Génome Québec, the Fonds de recherche en santé du Québec, the Norych Career Scientist Award, and the Schouella Distinguished Scientist Award.
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