Research ArticleExperimental Studies

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

TAKANOBU NADAI, KATSUYA NARUMI, YUTO MUKAI, HINATA UEDA, AYAKO FURUGEN, YOSHITAKA SAITO and MASAKI KOBAYASHI
Anticancer Research March 2023, 43 (3) 1113-1120; DOI: https://doi.org/10.21873/anticanres.16256

Abstract

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.

Key Words:

Breast cancer is the most common cancer in women worldwide, and approximately 70% of breast cancer patients have been diagnosed with the estrogen receptor (ER)-positive type (1, 2). Tamoxifen is an ER antagonist used as the first-choice medicine before and after surgery (3). It is metabolized to 4-hydroxytamoxifen (4-OHT) and N-4-hydroxy-N-desmethyl-tamoxifen by cytochrome P450 2D6 and 3A4, and their metabolites have anticancer activity against ER-positive breast cancer (4). In addition to its anti-estrogen activity, tamoxifen has been suggested to produce reactive oxygen species (ROS) by inhibiting oxidative phosphorylation and reducing cellular ATP levels (5). However, long-term tamoxifen treatment induces self-resistance, and has become a severe problem for ER-positive breast cancer treatment in clinical practice (3). Bhattacharya et al. reported that the Warburg effect can influence drug efficacy and that cancer cells cultured under different glucose conditions are expected to respond differently to different drugs (6). In cancer cells, glycolysis, which is the main process for ATP production from glucose, is known to be different from that in normal cells because of the use of the electron transport chain as a major energy source (Warburg effect) (7). In recent years, the glycolysis byproduct lactate has also been demonstrated to be used as a source of energy in cancer cells (reverse Warburg effect) (8). Monocarboxylate transporters (MCTs) are members of the solute carrier 16A protein family and comprise 14 subtypes (9). Among these, MCT1–4 are proton-dependent lactate transporters; MCT1 and MCT4 have attracted attention as potential therapeutic targets for cancer (10). The expression of MCT1 and MCT4 has been identified in breast cancer tissues and is partly related to poor prognosis in patients with breast cancer (11, 12). Therefore, inhibition of MCTs is expected to increase breast cancer therapy efficiency. However, the subtypes of MCT1 and MCT4 that are related to cancer survival are poorly recognized. We previously showed that high expression of hypoxia-inducible factor-1α (HIF-1α) under high glucose conditions altered 4-OHT-induced cytotoxicity by bindarit, an MCT4 inhibitor (13). However, the effect of MCT1, 4 substrate/inhibitor on 4-OHT-induced cytotoxicity under normal glucose conditions is unclear. The glucose-dependent regulation of HIF-1α protein expression was reported in cancer cells (14). In this study, we evaluated the effect of MCT1, 4 substrate/inhibitor on 4-OHT-induced cytotoxicity under normal glucose conditions.

Materials and Methods

Cell culture. The ER-positive breast cancer cell line MCF-7 was incubated in high (25 mM) or normal glucose (5.5 mM) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. The cells were grown in a humidified atmosphere at 37°C and 5% CO2, and cells within passage 50 were used for the experiments.

Materials. 4-Hydroxytamoxifen was purchased from Sigma-Aldrich (St. Louis, MO, USA, Catalog number T176). Bindarit was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA, Catalog number 11479). 5-Oxoproline (5-OP) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan, Catalog number P0573). All other reagents were commercially available and of guaranteed purity and were used without further purification.

Western blotting. The MCF-7 cells were cultured in 6-well plates under high or normal glucose DMEM conditions. The cells were then collected from the plates, pelleted, and resuspended in radio immunoprecipitation assay (RIPA) buffer. Protein samples were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto poly vinylidene fluoride (PVDF) membranes. Western blotting was performed using antibodies against HIF-1α and β-actin. Anti-rabbit HIF-1α (D2U3T; 1:1,000 dilution) and anti-rabbit β-actin (ab179467; 1:5,000 dilution) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) and Abcam (Cambridge, MA, USA), respectively.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and intracellular ATP assay. The MTT assay was performed as previously described (13). The MCF-7 cells were seeded at a concentration of 4.0×103 cells/well. Experiments were performed by treatment with 4-OHT or 5-OP or both for 48 h, beginning 24 h after seeding. Absorbance was measured at 590 nm. The 50% inhibitory concentration (IC50) values for cell viability were calculated using SigmaPlot 12.5 (Systat Software Inc., San Jose, CA, USA). The “Cell” ATP Assay Reagent (Toyo B-Net, Ltd., Tokyo, Japan) was used for ATP measurement according to the manufacturer’s instructions. Bioluminescence was evaluated using an Infinite 200 PRO Multimode Reader (Tecan, Männedorf, Switzerland).

Real-time PCR. Real-time PCR was performed using a LightCycler 480 II System (Roche Diagnostics GmbH, Mannheim, Germany) as previously described (15). The PCR conditions used were 40 cycles at 95°C for 3 s, 95°C for 10 s, 56°C for 20 s, and 72°C for 1 s. The mRNA levels were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was selected as the housekeeping gene because it showed the least variation compared to other housekeeping genes. The primer sequences are listed in Table I.

View this table:
Table I.

Primer sequences for real-time PCR.

Intracellular ROS assay. Total ROS was measured using 2′,7′-dichlorodihydrofluorescein diacetate (16). The MCF-7 cells were seeded on a 6-well plate at a density of 2.0×105 cells/well. After 24 h of seeding, the medium was aspirated, and the cells were exposed to 4-OHT or 5-OP or both for 48 h.

Statistical analysis. The experiments were performed at least three times. All results are presented as mean±standard error (SE). Two-group comparisons were analyzed by unpaired Student’s t-test. Statistical significance among means of more than two groups was evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett’s test or Tukey-Kramer test. Statistical significance was set at p<0.05.

Results

Changes in HIF-1α, MCTs, and cellular myelocytomatosis oncogene (c-MYC) mRNA levels in media with different glucose concentrations. First, we examined HIF-1α protein expression under high or normal glucose conditions. Normal glucose conditions significantly decreased HIF-1α expression (Figure 1A). In addition, the mRNA levels of MCT1 and c-MYC, a positive transcription factor of MCT1, increased, whereas those of MCT4 decreased under normal glucose conditions (Figure 1B).

Figure 1.
Figure 1.

Effect of glucose condition in the culture medium on HIF-1α protein expression level (A) and MCT1, MCT4, and c-MYC mRNA levels (B) in MCF-7 cells. (A) HIF-1α protein expression levels were analyzed by western blot analysis in MCF-7 cells. *p<0.05, compared to the high glucose condition using the unpaired Student’s t-test. Data are presented as the mean±standard error (SE) of three independent experiments. (B) Gene expression levels were analyzed by real-time PCR. *p<0.05 and **p<0.01, compared to the high glucose condition using the unpaired Student’s t-test. Data are presented as the mean±SE of three independent experiments.

Combination effect with MCT inhibitor and 4-OHT in MCF-7 cells. We next investigated whether the MCT substrate/inhibitor affected the reduction in MCF-7 cell viability by 4-OHT. The IC50 value of 4-OHT was 15.7±0.2 μM in MCF-7 cells (Figure 2). Bindarit, an MCT4 inhibitor, increased the 4-OHT IC50 value to 27.4±1.5 μM. In contrast, 5-OP, an MCT1 substrate/inhibitor, significantly enhanced the sensitivity to 4-OHT in MCF-7 cells with an IC50 value of 12.7±0.2 μM.

Figure 2.
Figure 2.

Effect of 4-OHT combined with MCT inhibitors on MCF-7 cell viability under normal glucose conditions. Experiments were performed by exposing cells to 4-OHT combined with 5-OP or bindarit for 48 h. The sigmoid curve and IC50 values for cell growth inhibition by 4-OHT were determined using the MTT assay. *p<0.05 and **p<0.01, compared to the control using ANOVA followed by Dunnett’s test. Data are presented as the mean±standard error (SE) of three independent experiments. 4-OHT, 4-Hydroxytamoxifen; 5-OP, 5-oxoploline.

Effect of combined 5-OP and 4-OHT treatment in MCF-7 cells. To test the combined effects of 5-OP and 4-OHT, MCF-7 cells were visualized under the microscope. The results showed that 4-OHT alone suppressed the growth of MCF-7 cells, whereas no change was observed in MCF-7 cells treated with 5-OP alone (Figure 3A). The effect of 4-OHT-induced suppression of cell growth was enhanced by 5-OP treatment. Moreover, we investigated the effects of 4-OHT and 5-OP on intracellular ATP levels in MCF-7 cells. As shown in Figure 3B, 4-OHT significantly decreased ATP levels in a dose-dependent manner, whereas 5-OP alone did not affect ATP levels, and combination with 5-OP further decreased ATP levels when compared with 4-OHT treatment alone (Figure 3B).

Figure 3.
Figure 3.

Confocal microscopy of cell morphology (A) and intracellular ATP levels (B) in MCF-7 cells during exposure to 4-OHT and 5-OP under normal glucose conditions. These experiments were performed by exposing cells to 4-OHT or 5-OP for 48 h. (A) Images of cell morphology observed by confocal microscopy. (B) Intracellular ATP was measured by the firefly luciferase luminescence method using an ATP assay kit. *p<0.05 and **p<0.01, compared to the control group; †p<0.01 compared to the 4-OHT 5 μM group; ‡p<0.01, compared to the 4-OHT 10 μM using ANOVA followed by Tukey-Kramer’s test. Data are presented as mean±standard error (SE) of three independent experiments. 4-OHT, 4-Hydroxytamoxifen; 5-OP, 5-oxoploline.

5-Oxoproline enhances 4-OHT-induced superoxide dismutase 2 (SOD2) mRNA and ROS generation in MCF-7 cells. To further examine the combined effects of 4-OHT and 5-OP on ATP levels, we performed real-time PCR, focusing on the expression levels of oxidative stress markers. Levels of SOD1 and SOD3 were not significantly increased by 4-OHT or 5-OP (Figure 4A and C). In contrast, SOD2 mRNA levels were significantly increased by 4-OHT treatment. In addition, combination treatment with 5-OP tended to enhance this effect (Figure 4B). Furthermore, combination of 4-OHT and 5-OP significantly increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor known to regulate SOD2 (Figure 5A). Finally, we examined whether the combination of 4-OHT and 5-OP affected ROS generation in MCF-7 cells. Although 5-OP alone did not affect ROS levels, the combination with 4-OHT significantly increased 4-OHT-induced ROS generation (Figure 5B).

Figure 4.
Figure 4.

Effect of 4-OHT combined with 5-OP on SOD1 (A), SOD2 (B), and SOD3 (C) mRNA levels in MCF-7 cells under normal glucose conditions. SOD mRNA levels were measured by real-time PCR. *p<0.05 and **p<0.01, compared to the control group; †p<0.05 compared to the 5-OP group using ANOVA followed by Tukey-Kramer’s test. Data are presented as the mean±standard error (SE) of three independent experiments. 4-OHT, 4-Hydroxytamoxifen; 5-OP, 5-oxoploline.

Figure 5.
Figure 5.

Effect of 4-OHT combined with 5-OP on Nrf2 mRNA levels (A) and ROS (B) in MCF-7 cells under normal glucose conditions. (A) Nrf2 mRNA levels were measured by real-time PCR. **p<0.01, compared to the control group; †p<0.05, compared to the 5-OP group using ANOVA followed by Tukey-Kramer’s test. Data are presented as the mean±standard error (SE) of three independent experiments. (B) Total ROS levels were evaluated by DCFH in MCF-7 cells. *p<0.05 and **p<0.01, compared to the control group; †p<0.05, ††p<0.01 compared to the 5-OP group; ‡p<0.05 compared to the 4-OHT group using ANOVA followed by Tukey-Kramer’s test. Data are presented as the mean±SE of three independent experiments. 4-OHT, 4-Hydroxytamoxifen; 5-OP, 5-oxoploline.

Discussion

Tamoxifen is mainly used to treat ER-positive breast cancer. However, as long-term dosage induces tamoxifen resistance, a new strategy for breast cancer therapy using tamoxifen and other medicines is required (3). Several MCT inhibitors have been developed to treat cancer in recent years (17). However, there is little evidence to show the effectiveness of MCT inhibitors combined with anticancer effects on cancer cells. Previously, we identified 5-OP as an MCT1 substrate/inhibitor and bindarit as an MCT4 inhibitor (18, 19), and examined the combined effect of these inhibitors on 4-OHT-induced cytotoxicity in ER-positive breast cancer cells. Our previous study under high glucose conditions showed that MCT inhibitors did not synergize with tamoxifen, and protein expression of HIF-1α increased by bindarit reduced tamoxifen sensitivity in MCF-7 cells (13). The expression of HIF-1α protein was reported to be increased in cancer cells in a glucose-dependent manner (14), suggesting that the effect of MCT inhibition may be masked by high glucose conditions. Based on these findings, we hypothesized that MCT inhibitors would affect 4-OHT-induced cytotoxicity under normal glucose conditions. Thus, we investigated whether MCT inhibitors increased 4-OHT-induced cytotoxicity under normal glucose conditions in ER-positive breast cancer cells. First, we examined the changes in HIF-1α protein expression under different glucose conditions. The results indicated lower HIF-1α protein expression under normal glucose conditions than under high glucose conditions, consistent with findings of a previous study (Figure 1A) (14). The mRNA levels of MCT4 under normal glucose conditions were significantly lower than those under high glucose conditions in parallel with HIF-1α protein expression. In addition, MCT1 mRNA levels significantly increased under normal glucose conditions (Figure 1B). We then focused on the relationship between MCT1 and c-MYC, which is a positive transcription factor of MCT1. The mRNA expression of c-MYC was higher under normal glucose conditions than under high-glucose conditions (Figure 1B). c-MYC has been reported to promote MCT1 expression (20, 21). High c-MYC expression is related to oxygen consumption, mitochondrial mass and function, and mitochondrial DNA content (22). Furthermore, a study using MCF-7 cells showed that a decrease in the glucose concentration in the medium decreased the efficiency of the glycolytic system (23). Collectively, our results suggest that a metabolic shift from glycolysis to oxidative phosphorylation occurs under normal glucose conditions. In oxidative cancer cells, MCT1 has been suggested for the metabolic switch from lactate-fueled oxidative phosphorylation to aerobic glycolysis in these cells (24). Moreover, lactate is converted to pyruvate, which is the fuel of the tricarboxylic acid cycle, by lactate dehydrogenase in oxidative cancer cells (25). Thus, MCT1 inhibition may affect the viability of oxidatively phosphorylated cells that use lactate as the energy source. Next, we examined the combined effect of MCT inhibitors on 4-OHT under normal glucose conditions using an MTT assay. Bindarit suppressed 4-OHT-induced growth inhibition, as reported in our previous study (Figure 2) (13). 5-Oxoproline significantly decreased the 4-OHT IC50 value in MCF-7 cells. Therefore, it is conceivable that 5-OP enhances the 4-OHT-induced suppression of cell growth under normal glucose conditions. We further evaluated the cell morphology and intracellular ATP levels with 5-OP, 4-OHT, and both compounds in MCF-7 cells. These results showed that 5-OP increased the suppression of 4-OHT-induced cell growth under normal glucose conditions in MCF-7 cells (Figure 3). 4-Hydroxytamoxifen has also been suggested to reduce oxidative phosphorylation efficiency in rat liver mitochondria by inducing depolarization of the mitochondrial membrane potential (26). Thus, the combination of 4-OHT and 5-OP in the present study may have a combined effect on MCT1 inhibition and inhibition of oxidative phosphorylation.

Tamoxifen is not only an ER antagonist but is also known to induce oxidative stress in cells (27). We investigated the mRNA expression of SOD1–3 as oxidative stress markers. 4-Hydroxytamoxifen significantly increased the mRNA levels of SOD2, a marker of mitochondrial oxidative stress (Figure 4B). These results indicated that 4-OHT induces oxidative stress in breast cancer cells. In addition, compared with each compound alone, the combination of 4-OHT and 5-OP significantly increased SOD2 mRNA levels, but not those of SOD1 and SOD3, in MCF-7 cells (Figure 4A and C). Superoxide dismutase 2 is regulated by Nrf2. No significant increase was observed in Nrf2 mRNA levels when treated with 4-OHT alone, but it increased in combination with 5-OP (Figure 5A). Based on these results, we examined the combined effects of 5-OP and 4-OHT on ROS levels. The results showed that 5-OP significantly increased the 4-OHT-induced ROS levels in MCF-7 cells. In contrast, 5-OP alone had no effect on the intracellular ROS levels in MCF-7 cells. A study has shown that high 5-OP accumulation induces oxidative stress, although the effect is weak (28). Reactive oxygen species are generated in mitochondria, and ROS accumulation is associated with cell survival (29). Tamoxifen also induces cell death via the regulation of mitochondrial nitric oxide synthase (27). These reports suggest that tamoxifen induces cell death by decreasing intracellular ATP levels through the accumulation of ROS in the mitochondria. In addition, lactate metabolizes to pyruvate in the mitochondria, and plays a central role in ATP production related to NAD+/NADH in OXPHOS (30). MCT1 has been reported to be expressed in the mitochondria of MCF-7 cells and has been confirmed to play a role in lactate transport (31). Furthermore, Chiu et al. reported that the high expression of HIF-1α decreases mitochondrial ROS (32). Our results suggest that susceptibility to mitochondrial oxidative stress is high under normal glucose conditions, as HIF-1α expression is reduced compared to that under high glucose conditions. This difference in the effect of oxidative stress owing to the difference in the medium glucose concentration is considered to have a significant effect on cell viability, unlike observations in our previous studies. Collectively, 5-OP may affect energy production by MCT1 inhibition and enhance the cytotoxicity of MCF-7 cells by 4-OHT under normal glucose conditions.

Conclusion

In conclusion, we found that 5-OP enhances 4-OHT-induced cytotoxicity in MCF-7 cells under normal glucose conditions. Moreover, 5-OP increases the 4-OHT-induced ROS levels. Therefore, our results suggest that 5-OP increases mitochondrial oxidative stress induced by 4-OHT. These results indicate that the combination of MCT1 inhibitors and 4-OHT may help to treat breast cancer. In the future, we hope that novel cancer treatment strategies can be established by combining other MCT1 inhibitors with mitochondria-targeted anticancer drugs.

Acknowledgements

This research was supported by JSPS KAKENHI (Grant No. JP 18K14416 to K.N and Grant No. JP 20K07171 to M.K). We thank the Nagai Memorial Research Scholarship awarded by the Pharmaceutical Society of Japan (to T.N).

Footnotes

  • ↵# Present address: Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan

  • Authors’ Contributions

    T.N, K.N and M.K designed the study. T.N, K.N, Y.M and H.U conducted the experiments. T.N, K.N and M.K performed data analysis. T.N, K.N, Y.M, H.U, A.F, Y.S and M.K wrote or contributed to the writing of the manuscript.

  • Conflicts of Interest

    The Authors declare that there are no competing financial interests in relation to this study.

  • Received December 23, 2022.
  • Revision received January 3, 2023.
  • Accepted January 10, 2023.

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5-Oxoproline Enhances 4-Hydroxytamoxifen-induced Cytotoxicity by Increasing Oxidative Stress in MCF-7 Breast Cancer Cells
TAKANOBU NADAI, KATSUYA NARUMI, YUTO MUKAI, HINATA UEDA, AYAKO FURUGEN, YOSHITAKA SAITO, MASAKI KOBAYASHI
Anticancer Research Mar 2023, 43 (3) 1113-1120; DOI: 10.21873/anticanres.16256
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