Purpose Metformin, a first-line drug for treatment of Type 2 diabetes, is effective against multiple type of cancers including endometrial cancer. The anticancer effects of metformin, in part, are mediated via activation of its intracellular target adenosine monophosphate-activated protein kinase (AMPK) and subsequent modulation of the downstream signaling of Mammalian Target of Rapamycin (mTOR) and P70S6K. Since metformin is positively charged (pKa 12.4) and hydrophilic (logD -6.13 at pH 6.0) it requires organic cation transporters (OCT) 1-3, plasma membrane monoamine transporter (PMAT) and multidrug and toxin extrusion transporters (MATE) 1 and 2 to mediate its cellular uptake/egress. The goal of this study is to assess the expression of these transporters in the endometrial cancer (EC) cell lines ECC-1, Ishikawa and SPEC-2. Methods EC cells (ECC-1, Ishikawa and SPEC-2) were treated with varying concentrations (0.1 mM-100 mM) of metformin for 72 hours, cell proliferation was assessed by MTT assay. Cellular uptake (5 min) of [14C] metformin at 50μM concentration was measured in the presence or absence of cation-selective transporter inhibitors, 1-methyl-4-phenylpyridinium (MPP+) (500 μM), desipiramine (200 μM) and pyrimethamine (2.4 μM). Expression of 6 cation-selective transporters (OCT 1-3, MATE1, MATE2 and PMAT) and phosphorylation of AMPK and P70S6K was evaluated by Western blot analysis. Results Metformin exhibited dose-dependent anti-proliferative activity against ECC-1, Ishikawa and SPEC-2 cell lines with an IC50 of 4.3, 2.2 and 8.8mM respectively. Protein expression analysis showed that OCT 1-3 were highly expressed in ECC-1 and Ishikawa cells whereas MATE1 and 2 were the predominant in SPEC-2 cells. [14C] metformin uptake into these 3 cell lines was mediated by cation-selective transporters as evidenced by the observation that >80% uptake was inhibited by the pan cation inhibitors MPP+ (500 μM). In the ECC-1 and Ishikawa cell lines, ≈50% of [14C]metformin uptake was inhibited by OCT1-3 and PMAT inhibitor desipiramine (200 μM) and ≈50% of uptake was inhibited by MATE1 and 2 inhibitor pyrimethamine (2.4 μM). In contrast, pyramithamine inhibited 72% of [14C]metformin uptake in SPEC-2 cells. Western blot analysis revealed that 5 µM pyrimethamine reversed metformin-mediated phosphorylation of AMPK and inhibition of pP70S6K in all the EC cell lines. Discussion Our results demonstrate that the OCT 1-3 and MATE 1-2 transporters play an approximately equal role in the uptake of metformin in ECC-1 and Ishikawa cells, whereas MATE 1-2 play a major role in metformin uptake into the SPEC-2 cells. As expected, metformin promoted phosphorylation of AMPK (activation) and attenuated phosphorylation of P70S6K (inhibition) in all of the EC cell lines. This work highlights the importance of transporters in the anti-proliferative activity of metformin in EC cells.

Citation Format: Aruljothi Muralidharan, Victoria L. Bae-Jump, Dhiren R. Thakker. OCT and MATE transporters mediate uptake and antiproliferative activity of the anticancer drug metformin in endometrial cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4916.