Biochemical and Biophysical Research Communications
Relevance of the OCT1 transporter to the antineoplastic effect of biguanides
Highlights
► siRNA knockdown of OCT1 reduced sensitivity of EOC cells to metformin, but not to another biguanide, phenformin. ► Suppression of OCT1 also affects the activation of AMP kinase in response to metformin, but not to phenformin. ► Direct actions of metformin may be limited by low OCT1 expression in EOC tumors. ► Phenformin could be used as an alternative biguanide.
Introduction
Metformin is an orally active biguanide that lowers systemic glucose and insulin and is commonly used for the treatment of type II diabetes. Retrospective studies suggest that metformin may have a protective role against cancer, possibly by reducing elevated systemic insulin levels and/or by directly inhibiting cellular proliferation via AMPK pathway activation within neoplastic cells [1]. Studies have demonstrated induction of apoptosis by metformin in pancreatic [2], prostate, and colon cancer [3]. Several publications reported a potential therapeutic effect of metformin in epithelial ovarian cancer (EOC) [4], [5], [6]. We previously demonstrated that metformin enhances ovarian cancer cell cytotoxicity in a dose- and time-dependent manner, an effect potentiated by cisplatin [5]. Metformin induces apoptosis through the modulation of the Bcl-2 family of proteins in some experimental systems [7], but other mechanisms including AMPK-dependent inhibition of mTOR may also play a role [8], [9].
Phenformin is another biguanide with anti-diabetic activity [10]. It was withdrawn from the market in the late 1970s due to a small risk of lactic acidosis in patients treated for diabetes [11]. Phenformin has been shown to have anti-neoplastic activity, including p21 cell-cycle inhibition leading to apoptosis. Moreover, phenformin was shown to reduce tumor growth in several animal models [12], [13], [14], [15], [16]. While risk/benefit considerations clearly favor use of metformin over phenformin for treatment of diabetes, the risk of phenformin-associated lactic acidosis is low enough that this agent certainly would not be contraindicated for cancer treatment if it had demonstrated superior antineoplastic activity.
The organic cation transporter 1 (OCT1) is responsible for organic cation uptake into hepatic cells via facilitated diffusion as well as active transport [17]. It is well known that OCT1 is highly expressed particularly in hepatic cells, where many drugs such as metformin and phenformin act [17], [18]. OCT1 has previously been reported to have several polymorphisms that can influence the cellular uptake of metformin [19]. The most common mutation, OCT1-420del, occurs with an allelic frequency of 16% [20] and was found to be present in 20% of Caucasian Americans displaying a reduced response to metformin [19]. While germ-line OCT1 polymorphisms may have modest but detectable effects on metformin efficacy in diabetes, we conducted in vitro studies to investigate whether differences in OCT1 expression amongst neoplastic tissues may be a more important consideration for potential applications in oncology.
Section snippets
Cell culture
The ovarian cancer cell lines OVCAR-3 and SKOV-3 (American Tissue Culture Collection, Manassas, VA, USA) were grown in RPMI-1640 medium (Wisent Bioproducts, Saint-Bruno, QC, Canada) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 10 μg/ml gentamicin. Each cell line was passaged every 5–7 days and maintained at 37 °C in a 5% CO2, 95% air atmosphere incubator.
Chemicals and antibodies
Metformin (catalog# D150959), Phenformin (catalog# P7045) and Anti-OCT1 (catalog# AV41516) were purchased from
OCT1 is expressed in epithelial ovarian cancer cell lines
OCT1 has been reported to be mainly expressed in liver, kidney and small intestine [23]. We investigated the expression of OCT1 in the human EOC cell lines OVCAR-3 and SKOV-3. We found the presence of detectable levels of OCT1 protein expression (Fig. 1A) in both human EOC cell lines.
OCT1 is crucial for the metformin effect in OVCAR-3 and SKOV-3 cell lines
In order to evaluate the role of OCT1 on the effect of metformin on human EOC, we first exposed the cell lines to OCT1 siRNA and demonstrated a dose-dependent inhibition of OCT1 protein expression in both cell
Discussion
The relevance of germ-line polymorphisms of OCT1 to efficacy of metformin has been studied in diabetes. Much of metformin’s activity in diabetes treatment takes place in the liver, where metformin is thought to initially reduce oxidative phosphorylation [25]. The resulting AMPK activation with secondary suppression of gluconeogenesis [26] leads to decreases in circulating glucose and insulin levels. Germ-line polymorphisms in the OCT1 gene have been correlated with metformin activity, although
Acknowledgments
This work was made possible in part by grants from the Montreal-Israel Cancer Research Fund, the Gloria Shapiro Endowment Fund for Ovarian Cancer Research, the Turqwise Fund for Ovarian Cancer, the Greenberg Foundation, and the Friends for Life charity.
ES was supported by the CIHR/FRSQ training grant FRN53888 in cancer research of the McGill Integrated Cancer Research Training Program.
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