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

doi:10.21873/anticanres.16256

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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.
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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.
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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.
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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.
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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.