Combination of Metformin and Sorafenib Induces Ferroptosis of Hepatocellular Carcinoma Through p62-Keap1-Nrf2 Pathway

Abstract

Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world. Sorafenib is the first small-molecule multi-kinase inhibitors approved by FDA for treatment of advanced HCC. Metformin has been demonstrated to have benefit for preventing cancer progression. In human recurrent HCCs, NF-E2-related factor 2 (Nrf2) was overexpressed and associated with poor survival. Nrf2 related signaling pathway plays central role to mediate cellular resistance to sorafenib through protecting HCC cells from ferroptosis. The effect of Combination treatment for HCC cells and the intrinsic mechanism have not been reported. In this study, metformin augmented the anti-tumor effect of sorafenib for HCC through ferroptosis induction by inhibiting Nrf2 related pathway. Based on the results of Nrf2 knockdown and p62 knockdown study, the combination of sorafenib and metformin suppressed proliferation of HCC cells through p62-Keap1-Nrf2/HO1 signaling way. Size of xenografts treated with the combination of sorafenib and metformin was smaller than other groups in vivo. Moreover, the combination treatment greatly induced ferroptosis in HCC cells through inhibiting Nrf2 expression. Based on our findings, the combination treatment suppressed proliferation of HCC cells through ferroptosis induction, by p62-Keap1-Nrf2/HO1 signaling way.

Keywords: Hepatocellular carcinoma (HCC); NF-E2-related factor 2 (Nrf2); ferroptosis; metformin; sorafenib.

Figure 1

Nrf2 is activated in HCC population and contributes to poor patient survival. A Tissue microarray consisting of 50 tumor tissues and corresponding nontumor liver tissues was subjected to HE and IHC analysis. Representative HE and IHC images show Nrf2 expression in HCC and its corresponding nontumor counterpart (case 16). Two HCC cases, one with high Nrf2 expression (case 9) and the other with low Nrf2 expression (case 21), are shown. Scale bar represent 20 um. B The overall survival rate of HCC patients after tumor recurrence. Patients with high Nrf2 overexpression were with significantly lower OS compared with those with low Nrf2 expression (p=0.003).

Figure 2

Combination treatment with metformin and sorafenib synergistically suppresses proliferation in HCC cells through inhibiting NRF2 expression. A HepG2 cells were treated with different concentrations of sorafenib (IC50=11.04 uM) and metformin (IC50=16.69 mM). B Huh-7 cells were treated with different concentrations of sorafenib (IC50=9.63 uM) and metformin (IC50=15.34 mM). C HepG2 cells were co-treated with sorafenib (10 uM) and metformin (15 mM). D Huh-7 cells were co-treated with sorafenib (10 uM) and metformin (15 mM). E combination treatment synergistically inhibited P62-Keap1-Nrf2/HO-1 signal way in HepG2 and Huh-7 cells. F Nrf2 knockdown HepG2 cell was significantly inhibited by sorafenib and metformin compared with control group. G Nrf2 knockdown Huh-7 cell was significantly inhibited by sorafenib and metformin compared with control group. H Protein levels of Nrf2 and HO-1 were assayed with western blot for control group and Nrf2 knockdown group. I Protein levels of P62, Keap1, Nrf2 and HO-1 were assayed with western blot for Nrf2 knockdown group treated with sorafenib (10 uM) and metformin (15 mM).

Figure 3

The combination treatment suppresses proliferation in HCC cells through P62-Keap1-Nrf2 signal way. A P62 knockdown HepG2 cell was significantly inhibited by sorafenib and metformin compared with control group. B P62 knockdown Huh-7 cell was significantly inhibited by sorafenib and metformin compared with control group. C Protein levels of Nrf2, HO-1, P62 and Keap1 were assayed with western blot for P62 knockdown group treated with sorafenib (10 uM) and metformin (15 mM). D Protein level of P62 was assayed with western blot for control group and P62 knockdown group. E P62 knockdown HepG2 cell has similar reaction to sorafenib and metformin compared with Nrf2 knockdown group. Both were significantly inhibited by sorafenib and metformin compared with control group. F P62 knockdown Huh-7 cell has similar reaction to sorafenib and metformin compared with Nrf2 knockdown group. Both were significantly inhibited by sorafenib and metformin compared with control group.

Figure 4

A Nrf2 knockdown HCC cells were treated with metformin (15 mM) and sorafenib (10 uM) with or without indicated inhibitors (ferrostatry-1, 1 uM; liproxstatin-1, 100nM; ZVAD-FMK, 10uM; necrostatin, 10uM; necrosulfonamide, 0.5 uM) for 24 hours, and cell viability was assayed (n=3, *p<0.05). B Knockdown HCC cells were treated with metformin (15 mM) and sorafenib (10 uM) for 24 hours and GSH level was assayed (n=3, *p<0.05). C Knockdown HCC cells were treated with metformin (15 mM) and sorafenib (10 uM) for 24 hours and MDA level was assayed (n=3, *p<0.05). D Knockdown HCC cells were treated with metformin (15 mM) and sorafenib (10 uM) for 24 hours and iron level was assayed (n=3, *p<0.05).

Figure 5

The combination treatment suppresses proliferation of HCC cells through inhibiting Nrf2 expression in vivo. A Representative image of xenograft-bearing tumors on day 35. B Balb/c nude mice were subcutaneously grafted with 1*10⁶ HepG2/Nrf2 knockdown HepG2 cells. Treatment with metformin, sorafenib or the combination was initiated on day 7. Tumor volume was calculated weekly. C GSH level analysis of the indicated gene expression in isolated tumor at day 35. D qRT-PCR analysis of the Nrf2 expression in isolated tumor at day 35. E qRT-PCR analysis of the HO-1 expression in isolated tumor at day 35. Data represents mean ± standard error (n=5-8 mice/group, p<0.05).

Figure 6

Combination of metformin and sorafenib induces ferroptosis of hepatocellular carcinoma through p62-Keap1-Nrf2 pathway.