Ruth S. Everett

Organic Cation Transporter 1 (OCT1/mOct1) Is Localized in the Apical Membrane of Caco-2 Cell Monolayers and Enterocytes

Organic cation transporters (OCTs) are members of the solute carrier 22 family of transporter proteins that are involved in absorption, distribution, and excretion of organic cations. OCT3 is localized in the apical (AP) membrane of enterocytes, but the literature is ambiguous about OCT1 (mOct1) localization, with some evidence suggesting a basolateral (BL) localization in human and mouse enterocytes. This is contrary to our preliminary findings showing AP localization of OCT1 in Caco-2 cell monolayers, an established model of human intestinal epithelium. Therefore, this study aims at determining the localization of OCT1 (mOct1) in Caco-2 cells, and human and mouse enterocytes. Functional studies using OCT1-specific substrate pentamidine showed transporter-mediated AP but not BL uptake in Caco-2 cells and human and mouse intestinal tissues. OCT1 inhibition decreased AP uptake of pentamidine by ∼50% in all three systems with no effect on BL uptake. A short hairpin RNA-mediated OCT1 knockdown in Caco-2 cells decreased AP uptake of pentamidine by ∼50% but did not alter BL uptake. Immunostaining and confocal microscopy in all three systems confirmed AP localization of OCT1 (mOct1). Our studies unequivocally show AP membrane localization of OCT1 (mOct1) in Caco-2 cells and human and mouse intestine. These results are highly significant as they will require reinterpretation of previous drug disposition and drug-drug interaction studies where conclusions were drawn assuming BL localization of OCT1 in enterocytes. Most importantly, these results will require revision of the regulatory guidance for industry in the United States and elsewhere because it has stated that OCT1 is basolaterally localized in enterocytes.

Four Cation-Selective Transporters Contribute to Apical Uptake and Accumulation of Metformin in Caco-2 Cell Monolayers

Metformin is the frontline therapy for type II diabetes mellitus. The oral bioavailability of metformin is unexpectedly high, between 40 and 60%, given its hydrophilicity and positive charge at all physiologic pH values. Previous studies in Caco-2 cell monolayers, a cellular model of the human intestinal epithelium, showed that during absorptive transport metformin is taken up into the cells via transporters in the apical (AP) membrane; however, predominant transport to the basolateral (BL) side occurs via the paracellular route because intracellular metformin cannot egress across the BL membrane. Furthermore, these studies have suggested that the AP transporters can contribute to intestinal accumulation and absorption of metformin. Transporter-specific inhibitors as well as a novel approach involving a cocktail of transporter inhibitors with overlapping selectivity were used to identify the AP transporters that mediate metformin uptake in Caco-2 cell monolayers; furthermore, the relative contributions of these transporters in metformin AP uptake were also determined. The organic cation transporter 1, plasma membrane monoamine transporter (PMAT), serotonin reuptake transporter, and choline high-affinity transporter contributed to approximately 25%, 20%, 20%, and 15%, respectively, of the AP uptake of metformin. PMAT-knockdown Caco-2 cells were constructed to confirm the contribution of PMAT in metformin AP uptake because a PMAT-selective inhibitor is not available. The identification of four intestinal transporters that contribute to AP uptake and potentially intestinal absorption of metformin is a significant novel finding that can influence our understanding of metformin pharmacology and intestinal drug-drug interactions involving this highly prescribed drug.

Why Does the Intestine Lack Basolateral Efflux Transporters for Cationic Compounds? A Provocative Hypothesis

Transport proteins in intestinal epithelial cells facilitate absorption of nutrients/compounds that are organic anions, cations, and zwitterions. For two decades, we have studied intestinal absorption and transport of hydrophilic ionic compounds, with specific focus on transport properties of organic cations and their interactions with intestinal transporters and tight junction proteins. Our data reveal how complex interactions between a compound and transporters in intestinal apical/basolateral (BL) membranes and tight junction proteins define oral absorption, and that the BL membrane lacks an efflux transporter that can transport positively charged compounds. Based on our investigations of transport mechanisms of zwitterionic, anionic, and cationic compounds, we postulate that physicochemical properties of these ionic species, in relation to the intestinal micro pH environment, have exerted evolutionary pressure for development of transporters that can handle apical uptake/efflux of all 3 ionic species and BL efflux of anions and zwitterions, but such evolutionary pressure is lacking for development of a BL efflux transporter for cationic compounds. This review provides an overview of intestinal uptake/efflux transporters and describes our studies on intestinal transport of cationic, anionic, and zwitterionic drugs that led to hypothesize that there are no cation-selective BL efflux transporters in the intestine.

Cation-selective transporters are critical to the AMPK-mediated antiproliferative effects of metformin in human breast cancer cells

The antidiabetic drug metformin exerts antineoplastic effects against breast cancer and other cancers. One mechanism by which metformin is believed to exert its anticancer effect involves activation of its intracellular target, adenosine monophosphate‐activated protein kinase (AMPK), which is also implicated in the antidiabetic effect of metformin. It is proposed that in cancer cells, AMPK activation leads to inhibition of the mammalian target of rapamycin (mTOR) and the downstream pS6K that regulates cell proliferation. Due to its hydrophilic and cationic nature, metformin requires cation‐selective transporters to enter cells and activate AMPK. This study demonstrates that expression levels of cation‐selective transporters correlate with the antiproliferative and antitumor efficacy of metformin in breast cancer. Metformin uptake and antiproliferative activity were compared between a cation‐selective transporter‐deficient human breast cancer cell line, BT‐20, and a BT‐20 cell line that was engineered to overexpress organic cation transporter 3 (OCT3), a representative of cation‐selective transporters and a predominant transporter in human breast tumors. Metformin uptake was minimal in BT‐20 cells, but increased by >13‐fold in OCT3‐BT20 cells, and its antiproliferative potency was >4‐fold in OCT3‐BT20 versus BT‐20 cells. This increase in antiproliferative activity was associated with greater AMPK phosphorylation and decreased pS6K phosphorylation in OCT3‐BT20 cells. In vitro data were corroborated by in vivo observations of significantly greater antitumor efficacy of metformin in xenograft mice bearing OCT3‐overexpressing tumors versus low transporter‐expressing wildtype tumors. Collectively, these findings establish a clear relationship between cation‐selective transporter expression, the AMPK‐mTOR‐pS6K signaling cascade, and the antiproliferative activity of metformin in breast cancer.

Abstract 4639: Do cation-selective transporters help or hurt the antitumor efficacy of metformin in breast cancer

Introduction Metformin is effective against many cancers including breast cancer. Activation of intracellular adenosine monophosphate-activated protein kinase (AMPK) is implied in metformin anticancer efficacy. Due to its hydrophilicity and positive charge, metformin relies on cation-selective transporters for cellular uptake. Accordingly, metformin cellular uptake, AMPK activation and anti-proliferative effects increased when organic cation transporter 3 (OCT3) was expressed in BT20 cells (BT20-OCT3) that have very low levels of cation-selective transporters (Cai et al., AAPS Abstract W4368, 2013). Our previous studies showed that human breast cancer cell lines and tumors exhibit wide variability in expression of metformin transporters, e.g. OCT1-3, plasma monoamine transporter (PMAT), and multidrug and toxin extrusion (MATE) transporters 1-2 (Zhang et al., AACR Abstract 9800, 2012). This study aims to assess the pivotal role of transporters in metformin antitumor effects using xenograft mice with tumors from BT20 and OCT3-BT20 cells. Methods BT20 cells stably transfected with OCT3 were produced, and OCT3 activity verified by [14C]metformin (50µM) uptake in presence/absence of 50µM famotidine (OCT3 and MATE1 inhibitor) or 500 µM quinidine (pan transporter inhibitor). Optimum metformin dose for tumor study was assessed by a dose-range pharmacokinetic study in mice dosed intraperitoneally (IP) with 15, 30, 50, 100, and 150mg/kg [14C]metformin. Xenograft mice were produced using 2×106 BT20/OCT3-BT20 cells. Doxorubicin (DOX; 4mg/kg) or DOX + 100mg/kg metformin was given IP every 5 days, and tumor volumes measured over 25 days. Results Metformin plasma AUC0-24hr was linear up to 100 mg/kg (AUC0-24hr of 95, 160, 366, 770 and 510 µmol*hr/L at 15, 30, 50, 100 and 150 mg/kg, respectively); hence, this dose was used for antitumor efficacy study. Surprisingly, tumors were larger in DOX + metformin group compared to DOX (45.3 vs. 23.5 mm3) in mice with OCT3-BT20 tumors. In mice with BT20 tumors, tumors were smaller in DOX + metformin group compared to DOX (20.5 vs. 61.8 mm3). Since BT20 tumors grew slowly, tumor measurements were possible in only 2 mice/group. Hence, the more aggressive MCF-7 human breast cancer cell was used to develop an OCT3-expressing (OCT3-MCF7) clone in which metformin uptake was 4.5-fold higher than in MCF-7 cells (65.66 vs. 15.17 pmol/mg protein/min). Metformin antitumor effect in xenograft mice with MCF-7 and OCT3-MCF7 tumors is under evaluation. Conclusion In vitro data imply that OCT3 expression in human breast cancer cells enhances metformin cellular uptake and anti-proliferative effect. In vivo xenograft mice studies show that BT20 tumors over-expressing OCT3 are less responsive to metformin + DOX than native BT20 tumors; however, further studies are needed to show statistical significance. Citation Format: Hao Cai, Muhammad Wahajuddin, Ruth Everett, Dhiren R. Thakker. Do cation-selective transporters help or hurt the antitumor efficacy of metformin in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4639. doi:10.1158/1538-7445.AM2014-4639

Abstract 4427: Multiple cation-selective transporters contribute to the anti-proliferative effects of metformin in ovarian cancer cell lines.

PURPOSE: Recent studies show that the anti-diabetic drug metformin exhibits anticancer effects against a broad spectrum of cancers including ovarian cancer. Because metformin is highly hydrophilic with a net positive charge at all physiologic pH values, it requires cation-selective transporters to enter into cells and interact with its intracellular target 5’ adenosine monophosphate-activated protein kinase. The expression pattern and level of these transporters in cancer cells would determine the intracellular concentrations and subsequently the anti-proliferative activity of metformin. This study aims to investigate metformin transporter expression and the impact of these transporters on metformin uptake and anti-proliferative activity in human ovarian cancer cell lines that are routinely used to assess potential anti-cancer agents against this cancer type. METHODS: Total RNA from human ovarian cancer cell lines (IG, SKOV3 and HEY) was subjected to real-time PCR to determine expression levels of the common cation transporters organic cation transporter (OCT) 1-3, plasma membrane monoamine transporter (PMAT), multidrug and toxin extrusion transporters (MATE) 1 and 2. Uptake of [14C]metformin (50μM) in the presence or absence of the cation-selective transporter inhibitors famotidine and 1-methyl-4-phenylpyridinium (MPP+) was assessed to determine the contributions of transporters to metformin uptake in these cell lines. RESULTS: OCT3 mRNA was highly expressed in IG cells, with low levels of OCT2, PMAT and MATE1. Metformin uptake into IG cells was significantly (P CONCLUSIONS: The ovarian cancer cell lines used in this study are utilized as models to test anticancer agents against ovarian cancer. In order to use these cell lines to investigate the anti-proliferative activity of metformin so as to predict its anticancer effects in ovarian cancer, the expression of metformin transporters and metformin uptake in relation to the expression of these transporters needs to be elucidated. Studies on determining the IC50 values of metformin in ovarian cancer cell lines, as well as cation-selective transporter expression studies in human ovarian tumor tissue samples are currently in progress. Citation Format: Ruth S. Everett, Yunhui Zhang, Innocent M. Ononiwu, Victoria L. Bae-Jump, Dhiren R. Thakker. Multiple cation-selective transporters contribute to the anti-proliferative effects of metformin in ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4427. doi:10.1158/1538-7445.AM2013-4427

Abstract LB-345: Differences in metformin transporter expression between breast tumor and breast cancer cell lines: selecting a relevant breast cancer cell model for metformin therapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: Recent studies show that the anti-diabetic drug metformin induces significant anticancer effects in several cancers including breast cancer via activation of intracellular AMPK. Because of its hydrophilic nature and net positive charge at physiologic pHs, metformin requires cation-selective transporters such as OCT1-3, PMAT and MATE1-2 to enter cells and activate AMPK. Expression of these transporters in cancer cells would determine the intracellular concentrations of metformin that can be achieved for anticancer effects. Therefore, elucidating metformin transport in breast cancer is central to understanding optimum dosing and transporter-associated inter-subject variability in its anticancer efficacy. However, there is limited information on metformin transport in breast cancer. Studies in our laboratory suggest that cation-selective transporters are expressed at negligible levels in MCF-7 cells, a widely used in vitro breast cancer model, which is not consistent with the robust anticancer effect of metformin in xenograft mouse models and clinical studies. Therefore, this study aims at determining a) metformin transporter expression in human breast tumor, b) if metformin transporter expression in breast cancer cell lines is relevant to that in breast tumor tissue and c) there is differential metformin transporter expression in tumor and non-malignant breast tissue. Methods: Total RNA isolated from breast cancer cell lines MCF-7, SK-BR-3, MDA-MB-231, ZR-75-1, BT-474, MDA-MB-435S, MDA-MB-468, BT-20 and BT-549 and from 14 pairs of human breast tumor and adjacent non-malignant breast tissues was subjected to.real-time PCR to determine relative expression levels of OCT1-3, PMAT, MATE1 and 2. Results: Most cell lines expressed low levels of candidate cation-selective transporters. MDA-MB-468 and BT-549 showed high expression of MATE1 and MDA-MB-231 expressed high levels of OCT3 and MATE1. In tumor and non-malignant breast tissue, the predominant transporters were OCT3 and PMAT. Nine of the total 14 tumor samples showed downregulation of OCT3 by 85% and PMAT by 66% compared to non-malignant tissue. Conclusions: The expression profile of cation-selective transporters is different between tumor and normal breast tissue and between breast tumor tissue and cell lines. Therefore, one needs to use caution in choosing a relevant in vitro cell model for investigating anticancer efficacy of metformin whose uptake is transporter-mediated. Expression data suggest that OCT3 and PMAT may play a major role in metformin intracellular uptake in breast cancer. MDA-MB-231 may be a useful model to assess the metformin anticancer effects in breast cancer. Clearly, genetic variability in metformin transporters may lead to differential responses to metformin anticancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-345. doi:1538-7445.AM2012-LB-345