Metformin suppresses Nrf2-mediated chemoresistance in hepatocellular carcinoma cells by increasing glycolysis

Results

Nrf2 conferred cisplatin resistance in HepG2 hepatocellular carcinoma cells

Hyperactivation of the Nrf2 pathway has been reported to contribute to multiple drug resistance in some cancers [22, 23, 46, 47]. We tested if this was the case for hepatocellular carcinoma. To this end, we collected tissue samples from 16 patients (8 have undergone cisplatin treatment) with hepatocellular carcinoma and carried out immunohistochemistry (IHC) assay by using a specific Nrf2 antibody. The results showed that Nrf2 protein levels were increased in cisplatin-treated tumor tissues (Figure 1A and 1B). Second, we asked if Nrf2 was activated in cisplatin-resistant HepG2 cells (HepG2/DDP) as compared to parental HepG2 cells. The cisplatin-resistant HepG2/DDP cells were obtained by continuously culturing HepG2 cells in the presence of cisplatin, a process mimicking the clinical development of cisplatin resistance. Consistently, Western blot showed that HepG2/DDP cells expressed much higher levels of Nrf2 protein (Figure 1C). To further confirm the role of Nrf2, we carried out RT-qPCR experiment and found that HepG2/DDP cells had higher expression of Nrf2 target genes (NQO1, GCLC, GCLM and HO-1), compared with HepG2 (Figure 1D). Upon knocking down with Nrf2 siRNA, the expression of Nrf2 target genes was significantly reduced in both HepG2 and HepG2/DDP cells (Figure 1E and 1F). Last, we examined the cell viability through CellTiter-Glo, which detected intracellular ATP concentration proportional to the number of live cells. We found that siRNA knockdown of Nrf2 preferentially sensitized the HepG2/DDP cells to chemotherapy drug cisplatin (Figure 1G and 1H) at multiple concentrations, confirming the role of Nrf2 in the drug resistance of HepG2/DDP liver cancer cells. Consistently, activation of Nrf2 through knocking down the negative regulator KEAP1, preferentially increased cisplatin resistance in HepG2 cells compared to HepG/DDP cells (Supplementary Figure 1).

Figure 1. Nrf2 activation contributes to cisplatin-resistance of HepG2/DDP cells. (A) Nrf2 protein levels were increased in hepatocellular carcinoma tissues from patients undergone cisplatin treatment. Sections of paraffin-embedded hepatocellular carcinoma tissues were stained with a Nrf2-specific antibody in immunochemistry (IHC) experiments. Representative images were shown. Scale bars are 20 μm. (B) Quantifying Nrf2 expression in tumor tissues from 16 patients (8 undergone cisplatin treatment). Scores were obtained by pathologists according to hospital protocols. Statistical analysis by student’s t-test showed significant difference (*, P<0.05). (C) Nrf2 protein levels were elevated in cisplatin-resistant HepG2/DDP cells. Cell lysates of HepG2 and cisplatin-resistant HepG2/DDP from 2 different cultures were subjected to Western blot analysis with Nrf2 and Tubulin antibodies separately. Shown are samples from two different cultures. Representative images of were shown. Quantification of N= 2 biological repeats were shown in bar graph. (D) Nrf2 target gene expression was upregulated in HepG2/DDP cells. mRNA was isolated from HepG2 and HepG2/DDP and the relative mRNA levels of Nrf2 target genes (NQO1, GCLC, GCLM and HO-1) was compared by RT-qPCR. Relative expression of each gene as compared to that in HepG2 cells in fold change. Significance was tested by student’s t-test (** P<0.001, *** P<0.0001). (E, F) Nrf2 knockdown repressed target genes expression in HepG2 and HepG2/DDP cells. Nrf2 was knocked down by transfecting cells with siRNA pools specific to Nrf2 gene for 48 hours. mRNA was isolated and RT-qPCR was conducted with specific primers for both Nrf2 gene and its target genes (HO-1 and GCLM). For each gene, data were normalized to non-transfected controls (Ctrl). Significance was tested by student’s t-test (* P<0.05, ** P<0.001). (G) Nrf2 knockdown sensitized HepG2/DDP cells to cisplatin. HepG2/DDP and HepG2 cells were transfected with siRNAs specific to Nrf2 gene for 48 hours and cells were treated with cisplatin at indicated concentrations for 24 hours. Cell survival was measured with Cell Titer-Glo reagent. Data from 3 independent experiments was normalized to the average of non-treated controls. Significance was tested by student’s t-test (* P<0.05, ** P<0.001). (H) Survival fold change by Nrf2 knockdown. Data in (G) were used to calculate the fold change caused by Nrf2 siRNA knockdown at each cisplatin concentration. Significance was tested by student’s t-test (* P<0.05, ns, not significant).

Metformin sensitized HepG2/DDP cells to cisplatin through inhibiting Nrf2-dependent transcription

There are several studies and clinical trials showing the benefits of metformin on reducing cancer incidence [28–32]. Some studies suggest that metformin could benefit patients through improving their response to chemotherapy. We aimed to study the mechanisms at the cellular and molecular levels. First, we tested if metformin would increase cisplatin toxicity on HepG2/DDP cells. After 48 hours of 10 ug/ml of cisplatin treatment, ~ 60% of HepG2/DDP cells remained alive. However, adding metformin from 0.5-4 mM significantly enhanced the killing effect of cisplatin (Figure 2A). Although metformin also enhanced cisplatin toxicity in HepG2 cells, its effect on HepG2/DDP cells were much stronger (Figure 2B).

Figure 2. Metformin increased cisplatin sensitivity of HepG2/DDP through down-regulation of Nrf2-dependent transcription. (A) Metformin sensitized HepG2/DDP cells to cisplatin. HepG2/DDP and HepG2 cells were treated with metformin at various concentrations and cell survival was measured by Cell Titer-Glo. Relative percentage of survival after 10 ug/mL cisplatin treatment for 24 hours was plotted. Difference between cisplatin-treated HepG2/DDP and HepG2 was tested by student’s t-test (** P<0.001). (B) Data from (A) were normalized to cisplatin non-treated controls and shown in bar plot. Metformin’s effect on HepG2/DDP and HepG2 was tested by student’s t-test (** P<0.001). (C) Metformin treatment did not affect Nrf2 protein levels. HepG2/DDP cells were treated with or without 1mM metformin for 24 hours and total cell lysates were subjected to Western blotting. Culture #1 and #2 were different cell clones. Representative images of were shown. Quantification of N= 4 biological repeats were shown in bar graph. (D) Metformin repressed Nrf2 target genes expression. HepG2/DDP cells were treated with or without 1mM metformin for 24 hours and mRNA was isolated and reversed transcribed. RT-qPCR were carried out with HO-1 and GCLM specific primers. For each gene, data of cisplatin-treated samples were normalized to that of non-treated controls. Significance was tested by student’s t-test (* P<0.05, ** P<0.001). (E) Metformin and Nrf2(siRNA) had no additive effect on increasing cisplatin toxicity. HepG2/DDP and HepG2 cells were transfected with Nrf2-specific siRNAs for 24 hours then treated metformin for 24 hours. 10ug/ml cisplatin were added for another 24 hours and relative cell viability was measured with Cell Titer-Glo. Data from 2 independent experiments were normalized to the average of non-treated controls. Significance was tested by student’s t-test (ns, not significant, * P<0.01, ** P<0.001). (F) Nrf2 activation prevented metformin from increasing cisplatin toxicity. HepG2/DDP cells were treated as in (E) except KEAP1-specific siRNA was transfected. Data from 4 independent experiments were normalized to the average of metformin non-treated control. Significance was tested by student’s t-test (ns, not significant, ** P<0.001).

We then asked if metformin would sensitize the HepG2/DDP cells to cisplatin through down-regulating Nrf2. To this end, we first tested if metformin would change Nrf2 protein levels. We treated HepG2/DDP cells with 1 mM metformin. However, no change was observed by Western blotting Nrf2 in the cell lysates (Figure 2C). Second, we examined the change of Nrf2 target gene expression by RT-qPCR. Interestingly, although the protein levels of Nrf2 were not affected, the expression of Nrf2 target genes HO-1 and GCLM were robustly reduced (Figure 2D). Third, we asked if metformin’s effect on cisplatin toxicity was additive to Nrf2 knockdown. If metformin improves the killing of cisplatin through Nrf2 pathway, Nrf2 knockdown and metformin should not be additive. By knocking down Nrf2 and treat cancer cells with 1mM metformin, we showed that there was no additive effect on cisplatin killing of HepG2/DDP cells (Figure 2E). Last, we tested if constitutively activating Nrf2 would block metformin’s effect on enhancing cisplatin toxicity. Interestingly, KEAP1 knockdown promoted the survival of HepG2/DDP cells and prevented the beneficial effect of metformin (Figure 2F). Consistently, KEAP1 knockdown also blocked the effect of metformin on reducing Nrf2 target gene expression induced by cisplatin (Supplementary Figure 2). Together, our data suggest that metformin sensitizes HepG2/DDP cells to cisplatin through inhibiting Nrf2-dependent transcription.

Metformin increased glucose uptake and glycolysis in HepG2/DDP cells

Metformin is a diabetes drug functioning to decrease plasma glucose levels. One of the possible mechanisms of such effect is through increasing glucose uptake in peripheral tissues such as fat tissues and muscles [27]. Also, several recent reports suggest that enhanced glucose metabolisms could down-regulate Nrf2 target genes expression [17, 19, 48]. We wondered if metformin would regulate Nrf2 transcriptional activity through modulating intracellular glucose metabolism. To this end, we first examined glucose absorption in HepG2/DDP cells after metformin treatment. Consistent with previous reports in muscle and fat cells [49–51], metformin increased glucose uptake in HepG2 cells, as determined by Glucose Uptake-Glo assay (Figure 3A). This was further confirmed by the upregulation of glucose transporter Glut1 and Glut4 as demonstrated by western blotting (Figure 3B). Knocking down GLUT1 gene preferentially reduced glucose uptake in HepG2/DDP cells compared to HepG2 (Supplementary Figure 3). Next, we asked if metformin could increase intracellular glucose levels in HepG2/DDP and HepG2 cells by using the Glucose-Glo Assay. Consistently, enhanced glucose uptake by metformin resulted in elevated intracellular glucose levels (Figure 3C). Third, we examined the protein levels of key enzymes in the glycolysis pathway, including the Hexokinase 2 (HK2) and Lactate dehydrogenase A (LDHA). Our results showed that these enzymes were elevated ~3 to 4 folds upon 1mM metformin treatment (Figure 3D). Last, we examined if increased glucose levels and glycolysis enzymes could result in elevated production of glycolytic metabolites such as lactate, NAD/NADH and NADP/NADPH. HepG2/DDP and HepG2 cells treated with and without metformin for 24 hours were trypsinized from plate and equal number of cells were used in the assay. As shown in Figure 3E–3G, lactate concentration was increased in metformin-treated cells, so were NAD/NADH and NADP/ NADPH levels. Metformin also increased the metabolite concentrations in a similar manner in HepG2 cells. Our results suggest that metformin increased glucose concentration and enhanced glycolytic activity in HepG2/DDP cells.

Figure 3. Metformin increased glucose uptake and glycolysis in HepG2/DDP cells. (A) Metformin increased glucose uptake. Indicated cells were treated with or without metformin (1mM) for 24 hours and glucose uptake assay were conducted with Glucose Uptake-Glo. Cell Titer-Glo was also carried out to measure the relative viability, which was used to normalize the data in glucose uptake assay. Data from 3 independent biological samples of 3 replicates were statistically analyzed by student’s t-test (*** P<0.0001). (B) Metformin increased the expression of glucose transporter Glut1 and Glut4. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours and total cell lysates were separated by SDS-PAGE. Glut1 and Glut4 protein levels were detected by Western blot using specific antibodies to Glut1 and Glut4. Tubulin was used as internal control. Representative images of were shown. Quantification of N= 2 biological repeats were shown in bar graph. (C) Metformin increased intracellular glucose concentration in HepG2/DDP cells. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours, washed extensively and intracellular glucose concentration was measured by using Glucose-Glo kit. Data from 2 independent biological samples of 3 replicates were plotted and statistically analyzed by student’s t-test (** P<0.001). (D) Metformin increased the protein levels of glycolytic enzymes HK2 and LDHA. Experiment was conducted as in (B) except HK2 and LDHA antibodies were used. Representative images of were shown. Quantification of N= 3 biological repeats were shown in bar graph. (E) Metformin increased intracellular lactate production. Indicated cells were treated with or without metformin (1mM) for 24 hours, washed extensively then intracellular lactate concentration was measured by using lactate-Glo kit. Data from 2 independent biological samples of 3 replicates were plotted and statistically analyzed by student’s t-test (** P<0.001, *** P<0.0001). (F) Metformin increased intracellular NAD/NADH production. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours and lactate concentration was measured by using NAD/NADH -Glo kit. Data from 2 independent biological samples of 3 replicates plotted and statistically analyzed by student’s t-test (* P<0.05, **P<0.001). (G) Metformin increased intracellular NADP/NADPH production. HepG2/DDP cells were treated with or without metformin (1mM) for 24 hours and lactate concentration was measured by using NADP/NADPH -Glo kit. Data from 2 independent biological samples of 3 replicates were plotted and statistically analyzed by student’s t-test (*** P<0.0001).

High glucose suppressed Nrf2-mediated transcription and sensitized HepG2/DDP cells to cisplatin

If increased glycolysis by metformin suppresses Nrf2-dependent transcription and improves cisplatin killing of HepG2/DDP cells, adding extra glucose in the medium might also show the same benefits. To test this possibility, HepG2/DDP cultured overnight in standard RPMI medium (containing 2g/L glucose) were shifted to RPMI medium with 2g/L, 4g/L, 8g/L or 12g/L glucose overnight. We first examined if Nrf2-dependent transcription of HO-1 gene would be suppressed using RT-qPCR. As shown in Figure 4A, glucose significantly suppressed HO-1 gene expression in a dose-dependent manner. Similarly, at the concentration of 8g/L glucose, expression of other Nrf2 target genes NQO1, GCLC and GCLM were also significantly reduced (Figure 4B). Next, we tested if high glucose medium could sensitize HepG2/DDP cells to cisplatin as did the metformin. Indeed, cells cultured in high glucose medium (8g/L) became more vulnerable to killing by cisplatin (Figure 4C).

Figure 4. High glucose medium suppressed Nrf2-mediated transcription in HepG2/DDP cells. (A) High glucose decreased Nrf2 target gene HO-1 expression in a dose-dependent manner. HepG2/DDP cells were cultured RPMI-1640 with indicated concentrations of glucose medium for 24 hours and total mRNA was extracted and reverse transcribed. RT-qPCR was carried out to compare the relative expression of HO-1. Significance was tested by student’s t-test (*P<0.05, **P<0.001). (B) Glucose at 8g/L suppressed other Nrf2 target genes. Experiments were conducted as in (A) by using qPCR primers specific to NQO1, GCLC and GCLM. Data were normalized to 2g/L glucose (normal RPMI glucose concentration) for each individual gene. Significance was tested by student’s t-test (*P<0.05, ***P<0.0001). (C) Glucose at 8g/L increased cisplatin killing of HepG2/DDP cells. HepG2/DDP and HepG2 cells cultured in normal RPMI-1640) were shifted to normal (2g/L) or high glucose (8g/L) RPMI-1640 for 24 hours then cisplatin at indicated concentrations was added. After 24 hours, cell viability was measured with Cell Titer-Glo. Data from 2 independent experiments were normalized to the average of non-treated controls. Significance was tested by student’s t-test (** P<0.001, *** P<0.0001). (D) KEAP1 knockdown increased Nrf2 protein levels in a glucose-independent manner. HepG2/DDP cells were transfected with KEAP1-specific siRNAs for 24 hours and shifted to normal or high glucose medium for 24 hours. Total cell lysates were analyzed by SDS-PAGE and Western blotting. Representative images were shown. Quantification of N= 2 biological repeats were shown in bar graph. (E–H) KEAP1 knockdown prevented glucose from suppressing Nrf2 target genes. siRNA knockdown and glucose conditioning conducted as in (D). Relative expression of indicated genes was quantified by RT-qPCR. Comparison of fold change was shown in (G) and (H). Significance was tested by student’s t-test ((* P<0.05, ** P<0.001, ns, not significant). (I) KEAP1 knockdown preventing glucose from enhancing cisplatin toxicity. siRNA knockdown and glucose conditioning conducted as in (D). HepG2/DDP cells then treated with cisplatin (10ug/ml) for 24 hours. Relative cell viability was measured by Cell Titer-Glo. Data from 2 independent of 3 replicates were normalized to the control and analyzed with student’s t-test (** P<0.001, ns, not significant).

To ask if glucose regulation of cisplatin sensitivity in HepG2/DDP cells was dependent on Nrf2 function, we siRNA knocked down the expression of the Nrf2 inhibitor KEAP1. KEAP1 was successfully inhibited as Nrf2 proteins were elevated in Western blot assay (Figure 4D). Interestingly, although high glucose medium suppressed the Nrf2 target genes (Figure 4A and 4B), Nrf2 was not affected at the protein levels (Figure 4D), similar to the results in metformin treatment (Figure 2C). Upon KEAP1 knockdown, glucose was no longer able to suppress Nrf2 target genes (Figure 4E-H). Consistently, upon KEAP1 knockdown, glucose was no longer able to increase cisplatin toxicity to HepG2/DDP cells (Figure 4I). Our data suggest that high glucose, similar to metformin, sensitizes HepG2/DDP cells to cisplatin through KEAP1-Nrf2 pathway.

Metformin decreased Nrf2-dependent transcription and increased cisplatin sensitivity through glycolysis

Metformin and high glucose both sensitize HepG2/DDP liver cancer cells to cisplatin (Figure 2A and Figure 4C). Whether they act through the same pathway in the upstream of Nrf2 remained unknown. To test this, we first asked if metformin and high glucose would act additively in suppressing Nrf2 target gene expression and cisplatin resistance of HepG2/DDP cells. Cell were cultured under normal and high glucose conditions and then treated with and without 1mM metformin. RT-qPCR showed that the target genes expression of NQO1 and GCLC were reduced by glucose or metformin treatment alone, but not further reduced by combining glucose and metformin, suggesting a non-additive effect (Figure 5A and 5B). Second, we treated HepG2/DDP cells cultured under above conditions with 10 ug/ml cisplatin and examined the viability. As shown in Figure 5C, glucose or metformin alone sensitized the HepG2/DDP cells to cisplatin toxicity, however, combination of metformin and glucose did not further increase cisplatin toxicity. These results suggest that high glucose and metformin target the same metabolic process (glycolysis) and pathway (Nrf2-dependent oxidative stress response pathway) to regulate drug response of HepG2/DDP cells.

Figure 5. Metformin regulated Nrf2-dependent transcription and cisplatin sensitivity through glucose metabolism and glycolysis in HepG2/DDP cells. (A, B) Metformin and glucose had no additive effect on Nrf2-dependent transcription. HepG2/DDP cells in normal RPMI (2g/L) were shifted to normal or high glucose (8g/L) medium supplemented with or without 1mM metformin for 24 hours. Total mRNA was extracted and relative expression of indicated gene was quantified by RT-qPCR. Data from 2 independent experiments (3 replicates) were normalized to non-treated controls and statistically analyzed by student’s t-test (ns, not significant, *P<0.05, **P<0.001). (C) Metformin and glucose had no additive effect on cisplatin toxicity. HepG2/DDP cells cultured and treated with glucose and metformin as in (B). After cisplatin (10ug/ml) treatment for another 24 hours, relative cell viability was measured by Cell Titer-Glo. Data from 2 experiments with 3 replicates were plotted and analyzed by student’s t-test (** P<0.001, ns, not significant). (D–E) Inhibition of glycolysis with 2-DG (20mM) attenuated Metformin’s effect on repressing Nrf2-dependent target gene expression. HepG2/DDP cells under normal glucose condition were treated with or without 1mM metformin and 20mM 2-DG for 24 hours. Relative expression of indicated genes was quantified by RT-qPCR for 2 independent times (3 replicates each time). Right panels are normalized data to compare the effect of 2-DG. Student’s t-test was used to test statistical significance (*P<0.05, **P<0.001, ***P<0.0001). (F) Glycolysis inhibition by 2-DG attenuated metformin’s effect on increasing cisplatin toxicity. HepG2/DDP cells were cultured and treated with metformin as in (E) then cisplatin (10ug/ml) was added for 24 hours. Relative cell viability was measured by Cell Titer-Glo. Data from 2 experiments of 3 replicates were plotted and analyzed by student’s t-test (*P<0.05, ***P<0.0001, ns, not significant).

Next, we chemically inhibited the cellular glycolysis by using 2-Deoxy-d-glucose (2-DG), a non-non-metabolizable glucose analog. 2-DG is a potent inhibitor of glycolysis via its action on hexokinase, the rate limiting step of glycolysis [52]. As shown in Figure 5D and 5E, metformin repressed Nrf2 target gene (NQO1 and GCLC) expression in both 2-DG treated and non-treated conditions. However, such repression was blunted by 2-DG (Figure 5D and 5E, right panels). Interestingly, 2-DG alone increased the expression of NQO1 and GCLC, consistent with a negative role of glycolysis in Nrf2 regulation. In addition, we tested the effect of 2-DG and metformin on cisplatin toxicity in HepG2/DDP cells. We found that the beneficial effect of metformin on enhancing cisplatin killing of HepG2/DDP cells was also significantly blunted by 2-DG (Figure 5F). Together, these data suggest that glycolysis is an important biological process mediating the effect of metformin and glucose on repressing Nrf2-dependent transcription and sensitizing HepG2/DDP cells to cisplatin.

Author Contributions

LC, XJ, and JZ designed the experiments. LC, XJ and LL performed the experiments and collected the data. LC, XJ, and JZ interpreted the data and wrote the manuscript.

Conflicts of Interest

These authors declare no conflicts of interest.

Funding

The present study was supported by the National Natural Science Foundation of China (grant nos. 81272193, 81302075 and 81402001) and the Key Research and Development Program of Hunan Province (Grant No: 2019DK2011).

References

• 1. Massarweh NN, El-Serag HB. Epidemiology of hepatocellular carcinoma and intrahepatic cholangiocarcinoma. Cancer Control. 2017; 24:1073274817729245. https://doi.org/10.1177/1073274817729245 [PubMed]
• 2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016; 66:7–30. https://doi.org/10.3322/caac.21332 [PubMed]
• 3. McGlynn KA, Petrick JL, London WT. Global epidemiology of hepatocellular carcinoma: an emphasis on demographic and regional variability. Clin Liver Dis. 2015; 19:223–38. https://doi.org/10.1016/j.cld.2015.01.001 [PubMed]
• 4. Raghunath A, Sundarraj K, Arfuso F, Sethi G, Perumal E. Dysregulation of Nrf2 in hepatocellular carcinoma: role in cancer progression and chemoresistance. Cancers (Basel). 2018; 10:481. https://doi.org/10.3390/cancers10120481 [PubMed]
• 5. Costantini S, Di Bernardo G, Cammarota M, Castello G, Colonna G. Gene expression signature of human HepG2 cell line. Gene. 2013; 518:335–45. https://doi.org/10.1016/j.gene.2012.12.106 [PubMed]
• 6. Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980; 209:497–99. https://doi.org/10.1126/science.6248960 [PubMed]
• 7. Ji C, Ren F, Ma H, Xu M. The roles of p38MAPK and caspase-3 in DADS-induced apoptosis in human HepG2 cells. J Exp Clin Cancer Res. 2010; 29:50. https://doi.org/10.1186/1756-9966-29-50 [PubMed]
• 8. Fujita H, Hirose K, Sato M, Fujioka I, Fujita T, Aoki M, Takai Y. Metformin attenuates hypoxia-induced resistance to cisplatin in the HepG2 cell line. Oncol Lett. 2019; 17:2431–40. https://doi.org/10.3892/ol.2018.9869 [PubMed]
• 9. Li J, Zhou P, Li L, Zhang Y, Shao Y, Tang L, Tian S. Effects of cationic microbubble carrying CD/TK double suicide gene and αVβ3 integrin antibody in human hepatocellular carcinoma HepG2 cells. PLoS One. 2016; 11:e0158592. https://doi.org/10.1371/journal.pone.0158592 [PubMed]
• 10. Wu S, Zhang T, Du J. Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway. Drug Des Devel Ther. 2016; 10:3471–81. https://doi.org/10.2147/DDDT.S110505 [PubMed]
• 11. Galluzzi L, Vitale I, Michels J, Brenner C, Szabadkai G, Harel-Bellan A, Castedo M, Kroemer G. Systems biology of cisplatin resistance: past, present and future. Cell Death Dis. 2014; 5:e1257. https://doi.org/10.1038/cddis.2013.428 [PubMed]
• 12. Wen J, Zeng M, Shu Y, Guo D, Sun Y, Guo Z, Wang Y, Liu Z, Zhou H, Zhang W. Aging increases the susceptibility of cisplatin-induced nephrotoxicity. Age (Dordr). 2015; 37:112. https://doi.org/10.1007/s11357-015-9844-3 [PubMed]
• 13. Bayat Mokhtari R, Homayouni TS, Baluch N, Morgatskaya E, Kumar S, Das B, Yeger H. Combination therapy in combating cancer. Oncotarget. 2017; 8:38022–43. https://doi.org/10.18632/oncotarget.16723 [PubMed]
• 14. Rangasamy T, Cho CY, Thimmulappa RK, Zhen L, Srisuma SS, Kensler TW, Yamamoto M, Petrache I, Tuder RM, Biswal S. Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest. 2004; 114:1248–59. https://doi.org/10.1172/JCI21146 [PubMed]
• 15. Thimmulappa RK, Mai KH, Srisuma S, Kensler TW, Yamamoto M, Biswal S. Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide microarray. Cancer Res. 2002; 62:5196–203. [PubMed]
• 16. Tang W, Jiang YF, Ponnusamy M, Diallo M. Role of Nrf2 in chronic liver disease. World J Gastroenterol. 2014; 20:13079–87. https://doi.org/10.3748/wjg.v20.i36.13079 [PubMed]
• 17. Wang C, Zhang Y, Li F, Wei Y. Conserved roles of glucose in suppressing reactive oxygen species-induced cell death and animal survival. Aging (Albany NY). 2019; 11:5726–43. https://doi.org/10.18632/aging.102155 [PubMed]
• 18. Gào X, Schöttker B. Reduction-oxidation pathways involved in cancer development: a systematic review of literature reviews. Oncotarget. 2017; 8:51888–51906. https://doi.org/10.18632/oncotarget.17128 [PubMed]
• 19. Li L, Chen Y, Chenzhao C, Fu S, Xu Q, Zhao J. Glucose negatively affects Nrf2/SKN-1-mediated innate immunity in C. Elegans. Aging (Albany NY). 2018; 10:3089–103. https://doi.org/10.18632/aging.101610 [PubMed]
• 20. Lewis KN, Mele J, Hayes JD, Buffenstein R. Nrf2, a guardian of healthspan and gatekeeper of species longevity. Integr Comp Biol. 2010; 50:829–43. https://doi.org/10.1093/icb/icq034 [PubMed]
• 21. So HS, Kim HJ, Lee JH, Lee JH, Park SY, Park C, Kim YH, Kim JK, Lee KM, Kim KS, Chung SY, Jang WC, Moon SK, et al. Flunarizine induces Nrf2-mediated transcriptional activation of heme oxygenase-1 in protection of auditory cells from cisplatin. Cell Death Differ. 2006; 13:1763–75. https://doi.org/10.1038/sj.cdd.4401863 [PubMed]
• 22. Wu J, Bao L, Zhang Z, Yi X. Nrf2 induces cisplatin resistance via suppressing the iron export related gene SLC40A1 in ovarian cancer cells. Oncotarget. 2017; 8:93502–15. https://doi.org/10.18632/oncotarget.19548 [PubMed]
• 23. Syu JP, Chi JT, Kung HN. Nrf2 is the key to chemotherapy resistance in MCF7 breast cancer cells under hypoxia. Oncotarget. 2016; 7:14659–72. https://doi.org/10.18632/oncotarget.7406 [PubMed]
• 24. Zhang M, Zhang C, Zhang L, Yang Q, Zhou S, Wen Q, Wang J. Nrf2 is a potential prognostic marker and promotes proliferation and invasion in human hepatocellular carcinoma. BMC Cancer. 2015; 15:531. https://doi.org/10.1186/s12885-015-1541-1 [PubMed]
• 25. Hayashi A, Suzuki H, Itoh K, Yamamoto M, Sugiyama Y. Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts. Biochem Biophys Res Commun. 2003; 310:824–29. https://doi.org/10.1016/j.bbrc.2003.09.086 [PubMed]
• 26. Vollrath V, Wielandt AM, Iruretagoyena M, Chianale J. Role of Nrf2 in the regulation of the Mrp2 (ABCC2) gene. Biochem J. 2006; 395:599–609. https://doi.org/10.1042/BJ20051518 [PubMed]
• 27. Rena G, Hardie DG, Pearson ER. The mechanisms of action of metformin. Diabetologia. 2017; 60:1577–85. https://doi.org/10.1007/s00125-017-4342-z [PubMed]
• 28. Heckman-Stoddard BM, DeCensi A, Sahasrabuddhe VV, Ford LG. Repurposing metformin for the prevention of cancer and cancer recurrence. Diabetologia. 2017; 60:1639–47. https://doi.org/10.1007/s00125-017-4372-6 [PubMed]
• 29. Yu X, Mao W, Zhai Y, Tong C, Liu M, Ma L, Yu X, Li S. Anti-tumor activity of metformin: from metabolic and epigenetic perspectives. Oncotarget. 2017; 8:5619–28. https://doi.org/10.18632/oncotarget.13639 [PubMed]
• 30. Zhou PT, Li B, Liu FR, Zhang MC, Wang Q, Li YY, Xu C, Liu YH, Yao Y, Li D. Metformin is associated with survival benefit in pancreatic cancer patients with diabetes: a systematic review and meta-analysis. Oncotarget. 2017; 8:25242–50. https://doi.org/10.18632/oncotarget.15692 [PubMed]
• 31. Whitburn J, Edwards CM, Sooriakumaran P. Metformin and prostate cancer: a new role for an old drug. Curr Urol Rep. 2017; 18:46. https://doi.org/10.1007/s11934-017-0693-8 [PubMed]
• 32. Cao X, Wen ZS, Wang XD, Li Y, Liu KY, Wang X. The clinical effect of metformin on the survival of lung cancer patients with diabetes: a comprehensive systematic review and meta-analysis of retrospective studies. J Cancer. 2017; 8:2532–41. https://doi.org/10.7150/jca.19750 [PubMed]
• 33. Qu Z, Zhang Y, Liao M, Chen Y, Zhao J, Pan Y. In vitro and in vivo antitumoral action of metformin on hepatocellular carcinoma. Hepatol Res. 2012; 42:922–33. https://doi.org/10.1111/j.1872-034X.2012.01007.x [PubMed]
• 34. Do MT, Kim HG, Choi JH, Jeong HG. Metformin induces microRNA-34a to downregulate the Sirt1/Pgc-1α/Nrf2 pathway, leading to increased susceptibility of wild-type p53 cancer cells to oxidative stress and therapeutic agents. Free Radic Biol Med. 2014; 74:21–34. https://doi.org/10.1016/j.freeradbiomed.2014.06.010 [PubMed]
• 35. Bai M, Yang L, Liao H, Liang X, Xie B, Xiong J, Tao X, Chen X, Cheng Y, Chen X, Feng Y, Zhang Z, Zheng W. Metformin sensitizes endometrial cancer cells to chemotherapy through IDH1-induced Nrf2 expression via an epigenetic mechanism. Oncogene. 2018; 37:5666–81. https://doi.org/10.1038/s41388-018-0360-7 [PubMed]
• 36. Riaz MA, Sak A, Erol YB, Groneberg M, Thomale J, Stuschke M. Metformin enhances the radiosensitizing effect of cisplatin in non-small cell lung cancer cell lines with different cisplatin sensitivities. Sci Rep. 2019; 9:1282. https://doi.org/10.1038/s41598-018-38004-5 [PubMed]
• 37. Zhang J, Jiao K, Liu J, Xia Y. Metformin reverses the resistance mechanism of lung adenocarcinoma cells that knocks down the Nrf2 gene. Oncol Lett. 2018; 16:6071–80. https://doi.org/10.3892/ol.2018.9382 [PubMed]
• 38. Han G, Gong H, Wang Y, Guo S, Liu K. AMPK/mTOR-mediated inhibition of survivin partly contributes to metformin-induced apoptosis in human gastric cancer cell. Cancer Biol Ther. 2015; 16:77–87. https://doi.org/10.4161/15384047.2014.987021 [PubMed]
• 39. Zhao D, Long XD, Lu TF, Wang T, Zhang WW, Liu YX, Cui XL, Dai HJ, Xue F, Xia Q. Metformin decreases IL-22 secretion to suppress tumor growth in an orthotopic mouse model of hepatocellular carcinoma. Int J Cancer. 2015; 136:2556–65. https://doi.org/10.1002/ijc.29305 [PubMed]
• 40. Wang Y, Wang Y, Zhang Z, Park JY, Guo D, Liao H, Yi X, Zheng Y, Zhang D, Chambers SK, Zheng W. Mechanism of progestin resistance in endometrial precancer/cancer through Nrf2-AKR1C1 pathway. Oncotarget. 2016; 7:10363–72. https://doi.org/10.18632/oncotarget.7004 [PubMed]
• 41. Fan R, Wang Y, Wang Y, Wei L, Zheng W. Mechanism of progestin resistance in endometrial precancer/cancer through Nrf2-survivin pathway. Am J Transl Res. 2017; 9:1483–91. [PubMed]
• 42. Yu C, Jiao Y, Xue J, Zhang Q, Yang H, Xing L, Chen G, Wu J, Zhang S, Zhu W, Cao J. Metformin sensitizes non-small cell lung cancer cells to an epigallocatechin-3-gallate (EGCG) treatment by suppressing the Nrf2/HO-1 signaling pathway. Int J Biol Sci. 2017; 13:1560–69. https://doi.org/10.7150/ijbs.18830 [PubMed]
• 43. Do MT, Kim HG, Khanal T, Choi JH, Kim DH, Jeong TC, Jeong HG. Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of raf-ERK-Nrf2 signaling and AMPK-independent pathways. Toxicol Appl Pharmacol. 2013; 271:229–38. https://doi.org/10.1016/j.taap.2013.05.010 [PubMed]
• 44. Ikeda M, Morizane C, Ueno M, Okusaka T, Ishii H, Furuse J. Chemotherapy for hepatocellular carcinoma: current status and future perspectives. Jpn J Clin Oncol. 2018; 48:103–14. https://doi.org/10.1093/jjco/hyx180 [PubMed]
• 45. Dickson MA, Carvajal RD, Merrill AH Jr, Gonen M, Cane LM, Schwartz GK. A phase I clinical trial of safingol in combination with cisplatin in advanced solid tumors. Clin Cancer Res. 2011; 17:2484–92. https://doi.org/10.1158/1078-0432.CCR-10-2323 [PubMed]
• 46. Wang XJ, Sun Z, Villeneuve NF, Zhang S, Zhao F, Li Y, Chen W, Yi X, Zheng W, Wondrak GT, Wong PK, Zhang DD. Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2. Carcinogenesis. 2008; 29:1235–43. https://doi.org/10.1093/carcin/bgn095 [PubMed]
• 47. Zhang HX, Chen Y, Xu R, He QY. Nrf2 mediates the resistance of human A549 and HepG2 cancer cells to boningmycin, a new antitumor antibiotic, in vitro through regulation of glutathione levels. Acta Pharmacol Sin. 2018; 39:1661–69. https://doi.org/10.1038/aps.2018.21 [PubMed]
• 48. Alcántar-Fernández J, González-Maciel A, Reynoso-Robles R, Pérez Andrade ME, Hernández-Vázquez AJ, Velázquez-Arellano A, Miranda-Ríos J. High-glucose diets induce mitochondrial dysfunction in caenorhabditis elegans. PLoS One. 2019; 14:e0226652. https://doi.org/10.1371/journal.pone.0226652 [PubMed]
• 49. Galuska D, Zierath J, Thörne A, Sonnenfeld T, Wallberg-Henriksson H. Metformin increases insulin-stimulated glucose transport in insulin-resistant human skeletal muscle. Diabete Metab. 1991; 17:159–63. [PubMed]
• 50. Galuska D, Nolte LA, Zierath JR, Wallberg-Henriksson H. Effect of metformin on insulin-stimulated glucose transport in isolated skeletal muscle obtained from patients with NIDDM. Diabetologia. 1994; 37:826–32. https://doi.org/10.1007/BF00404340 [PubMed]
• 51. Fischer M, Timper K, Radimerski T, Dembinski K, Frey DM, Zulewski H, Keller U, Müller B, Christ-Crain M, Grisouard J. Metformin induces glucose uptake in human preadipocyte-derived adipocytes from various fat depots. Diabetes Obes Metab. 2010; 12:356–59. https://doi.org/10.1111/j.1463-1326.2009.01169.x [PubMed]
• 52. Chen W, Guéron M. The inhibition of bovine heart hexokinase by 2-deoxy-D-glucose-6-phosphate: characterization by 31P NMR and metabolic implications. Biochimie. 1992; 74:867–73. https://doi.org/10.1016/0300-9084(92)90070-u [PubMed]
• 53. Elgogary A, Xu Q, Poore B, Alt J, Zimmermann SC, Zhao L, Fu J, Chen B, Xia S, Liu Y, Neisser M, Nguyen C, Lee R, et al. Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer. Proc Natl Acad Sci USA. 2016; 113:E5328–36. https://doi.org/10.1073/pnas.1611406113 [PubMed]
• 54. Blackwell TK, Steinbaugh MJ, Hourihan JM, Ewald CY, Isik M. SKN-1/nrf, stress responses, and aging in caenorhabditis elegans. Free Radic Biol Med. 2015; 88:290–301. https://doi.org/10.1016/j.freeradbiomed.2015.06.008 [PubMed]
• 55. Goldstein LD, Lee J, Gnad F, Klijn C, Schaub A, Reeder J, Daemen A, Bakalarski CE, Holcomb T, Shames DS, Hartmaier RJ, Chmielecki J, Seshagiri S, et al. Recurrent loss of NFE2L2 exon 2 is a mechanism for Nrf2 pathway activation in human cancers. Cell Rep. 2016; 16:2605–17. https://doi.org/10.1016/j.celrep.2016.08.010 [PubMed]
• 56. Liberti MV, Locasale JW. The warburg effect: how does it benefit cancer cells? Trends Biochem Sci. 2016; 41:211–18. https://doi.org/10.1016/j.tibs.2015.12.001 [PubMed]
• 57. Kowalik MA, Columbano A, Perra A. Emerging role of the pentose phosphate pathway in hepatocellular carcinoma. Front Oncol. 2017; 7:87. https://doi.org/10.3389/fonc.2017.00087 [PubMed]
• 58. Hu ZY, Xiao L, Bode AM, Dong Z, Cao Y. Glycolytic genes in cancer cells are more than glucose metabolic regulators. J Mol Med (Berl). 2014; 92:837–45. https://doi.org/10.1007/s00109-014-1174-x [PubMed]
• 59. Gerards MC, van der Velden DL, Baars JW, Brandjes DP, Hoekstra JB, Vriesendorp TM, Gerdes VE. Impact of hyperglycemia on the efficacy of chemotherapy-a systematic review of preclinical studies. Crit Rev Oncol Hematol. 2017; 113:235–41. https://doi.org/10.1016/j.critrevonc.2017.03.007 [PubMed]
• 60. Bhattacharya B, Low SH, Soh C, Kamal Mustapa N, Beloueche-Babari M, Koh KX, Loh J, Soong R. Increased drug resistance is associated with reduced glucose levels and an enhanced glycolysis phenotype. Br J Pharmacol. 2014; 171:3255–67. https://doi.org/10.1111/bph.12668 [PubMed]
• 61. Pandey V, Chaube B, Bhat MK. Hyperglycemia regulates MDR-1, drug accumulation and ROS levels causing increased toxicity of carboplatin and 5-fluorouracil in MCF-7 cells. J Cell Biochem. 2011; 112:2942–52. https://doi.org/10.1002/jcb.23210 [PubMed]
• 62. Menegon S, Columbano A, Giordano S. The dual roles of NRF2 in cancer. Trends Mol Med. 2016; 22:578–93. https://doi.org/10.1016/j.molmed.2016.05.002 [PubMed]
• 63. Sporn MB, Liby KT. NRF2 and cancer: the good, the bad and the importance of context. Nat Rev Cancer. 2012; 12:564–71. https://doi.org/10.1038/nrc3278 [PubMed]
• 64. Allard JS, Perez EJ, Fukui K, Carpenter P, Ingram DK, de Cabo R. Prolonged metformin treatment leads to reduced transcription of Nrf2 and neurotrophic factors without cognitive impairment in older C57BL/6J mice. Behav Brain Res. 2016; 301:1–9. https://doi.org/10.1016/j.bbr.2015.12.012 [PubMed]
• 65. Prasad S, Sajja RK, Kaisar MA, Park JH, Villalba H, Liles T, Abbruscato T, Cucullo L. Role of Nrf2 and protective effects of metformin against tobacco smoke-induced cerebrovascular toxicity. Redox Biol. 2017; 12:58–69. https://doi.org/10.1016/j.redox.2017.02.007 [PubMed]
• 66. Ashabi G, Khalaj L, Khodagholi F, Goudarzvand M, Sarkaki A. Pre-treatment with metformin activates Nrf2 antioxidant pathways and inhibits inflammatory responses through induction of AMPK after transient global cerebral ischemia. Metab Brain Dis. 2015; 30:747–54. https://doi.org/10.1007/s11011-014-9632-2 [PubMed]
• 67. Fourtounis J, Wang IM, Mathieu MC, Claveau D, Loo T, Jackson AL, Peters MA, Therien AG, Boie Y, Crackower MA. Gene expression profiling following NRF2 and KEAP1 siRNA knockdown in human lung fibroblasts identifies CCL11/Eotaxin-1 as a novel NRF2 regulated gene. Respir Res. 2012; 13:92. https://doi.org/10.1186/1465-9921-13-92 [PubMed]