5-Oxoproline Enhances 4-Hydroxytamoxifen-induced Cytotoxicity by Increasing Oxidative Stress in MCF-7 Breast Cancer Cells.

BACKGROUND/AIM: Monocarboxylate transporters (MCTs) transport short-chain monocarboxylates, such as lactate, and have been reported to be related to poor prognosis in breast cancer. Our previous studies showed that a high glucose state altered MCT expression and changed the sensitivity of the tamoxifen active metabolite 4-hydroxytamoxifen (4-OHT) via hypoxia-inducible factor-1α (HIF-1α) protein expression. We hypothesized that MCT inhibitors affect 4-OHT-induced cytotoxicity under normal glucose conditions by decreasing HIF-1α protein expression. To test this hypothesis, we evaluated the combined effect of MCT inhibitor and 4-OHT using the estrogen receptor (ER)-positive breast cancer cell line MCF-7, under normal glucose conditions. MATERIALS AND METHODS: Expression of MCTs and oxidative stress markers was evaluated by real-time PCR. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Reactive oxygen species (ROS) were measured using the cell permeability probe 2',7'-dichlorodihydrofluorescein diacetate. RESULTS: MCT1 expression increased under normal glucose conditions. The MCT1 substrate/inhibitor, 5-oxoproline (5-OP), enhanced 4-OHT-induced cytotoxicity. Bindarit, a selective MCT4 inhibitor, decreased 4-OHT sensitivity, similar to results of our previous study under high glucose conditions. In contrast, the combination of 5-OP and 4-OHT decreased ATP levels compared with that by 4-OHT alone in MCF-7 cells. Furthermore, 5-OP significantly increased the ROS production induced by 4-OHT. CONCLUSION: 5-OP enhances 4-OHT-induced cytotoxicity in ER-positive breast cancer cells under normal glucose conditions.

Involvement of SLC16A1/MCT1 and SLC16A3/MCT4 in l-lactate transport in the hepatocellular carcinoma cell line.

Fourteen isoforms of the monocarboxylate transporter (MCT) have been reported. Among the MCT isoforms, MCT1, MCT2, and MCT4 play a role in l-lactate/proton cotransport and are involved in the balance of intracellular energy and pH. Therefore, MCT1, MCT2, and MCT4 are associated with energy metabolism processes in normal and pathological cells. In the present study, we evaluated the expression of MCT1, MCT2, and MCT4 and the contribution of these three MCT isoforms to l-lactate uptake in hepatocellular carcinoma (HCC) cells. In HepG2 and Huh-7 cells, l-lactate transport was pH-dependent, which is characteristic of MCT1, MCT2, and MCT4. Furthermore, l-lactate uptake was selectively inhibited by MCT1 and MCT4 inhibitors in HepG2 and Huh-7 cells. Kinetic analysis of HepG2 cells demonstrated that l-lactate uptake was biphasic. Although the knockdown of MCT1 and MCT4 in the HepG2 cells decreased the uptake of l-lactate, the knockdown of MCT2 had no effect on the uptake of l-lactate. Consequently, we concluded that both MCT1 and MCT4 were involved in the transport of l-lactate in HepG2 and Huh-7 cells at pH 6.0. In contrast, PXB-cells, freshly isolated hepatocytes from humanized mouse livers, showed lower MCT4 expression and l-lactate uptake at pH 6.0 compared to that in HCC cell lines. In conclusion, MCT4, which contributes to l-lactate transport in HCC cells, is significantly different in HCC compared to normal hepatocytes, and has potential as a target for HCC treatment.

Pharmacological Inhibition of MCT4 Reduces 4-Hydroxytamoxifen Sensitivity by Increasing HIF-1α Protein Expression in ER-Positive MCF-7 Breast Cancer Cells.

The rate of glycolysis in cancer cells is higher than that of normal cells owing to high energy demands, which results in the production of excess lactate. Monocarboxylate transporters (MCTs), especially MCT1 and MCT4, play a critical role in maintaining an appropriate pH environment through lactate transport, and their high expression is associated with poor prognosis in breast cancer. Thus, we hypothesized that inhibition of MCTs is a promising therapeutic target for adjuvant breast cancer treatment. We investigated the effect of MCT inhibition in combination with 4-hydroxytamoxifen (4-OHT), an active metabolite of tamoxifen, using two estrogen receptor (ER)-positive breast cancer cell lines, MCF-7 and T47D. Lactate transport was investigated in cellular uptake studies. The cytotoxicity of 4-OHT was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In both cell lines evaluated, MCT1 and MCT4 were constitutively expressed at the mRNA and protein levels. [14C]-L-lactate uptake by both cells was significantly inhibited by bindarit, a selective MCT4 inhibitor, but weakly affected by 5-oxoploline (5-OP), a selective MCT1 inhibitor. The results of the MTT assay showed that combination with bindarit, but not 5-OP, decreased 4-OHT sensitivity. Bindarit significantly increased the levels of hypoxia-inducible factor-1α (HIF-1α) in MCF-7 cells. Moreover, HIF-1α knockdown significantly increased 4-OHT sensitivity, whereas induction of HIF-1α by hypoxia decreased 4-OHT sensitivity in MCF-7 cells. In conclusion, pharmacological MCT4 inhibition confers resistance to 4-OHT rather than sensitivity, by increasing HIF-1α protein levels. In addition, HIF-1α inhibition represents a potential therapeutic strategy for enhancing 4-OHT sensitivity.

In vitro and in vivo evaluation of organic anion-transporting polypeptide 2B1-mediated pharmacokinetic interactions by apple polyphenols.

Organic anion-transporting polypeptide (OATP) 2B1 plays a critical role in the intestinal absorption of substrate drugs. Apple juice reportedly interacts with OATP2B1 substrate drugs. The purpose of this study was to investigate the effect of two apple polyphenols, phloretin and phloridzin, on OATP2B1-mediated substrate transport in vitro and to evaluate the effect of phloretin on rosuvastatin pharmacokinetics in rats.In vitro studies revealed that both polyphenols inhibited OATP2B1-mediated uptake of estrone-3-sulfate. Despite preincubation with phloretin and subsequent washing, the inhibitory effect was retained. Phloretin markedly decreased OATP2B1-mediated rosuvastatin uptake, with an IC50 value of 3.6 µM.On coadministering rosuvastatin and phloretin in rats, the plasma concentration of rosuvastatin 10 min after oral administration was significantly lower than that in the vehicle group. The area under the plasma concentration-time curve of rosuvastatin was not significant, showing a tendency to decrease in the phloretin group when compared with the vehicle group. The in-situ rat intestinal loop study revealed the inhibitory effect of phloretin on rosuvastatin absorption.Phloretin has potent and long-lasting inhibitory effects on OATP2B1 in vitro. Phloretin may inhibit OATP2B1-mediated intestinal absorption of rosuvastatin; however, it failed to significantly impact the systemic exposure of rosuvastatin in rats.