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. 2016 Jan;63(1):173-84.
doi: 10.1002/hep.28251. Epub 2015 Nov 26.

V体育ios版 - Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells

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"VSports注册入口" Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells

Xiaofang Sun et al. Hepatology. 2016 Jan.

Abstract (V体育官网)

Ferroptosis is a recently recognized form of regulated cell death caused by an iron-dependent accumulation of lipid reactive oxygen species. However, the molecular mechanisms regulating ferroptosis remain obscure. Here, we report that nuclear factor erythroid 2-related factor 2 (NRF2) plays a central role in protecting hepatocellular carcinoma (HCC) cells against ferroptosis. Upon exposure to ferroptosis-inducing compounds (e VSports手机版. g. , erastin, sorafenib, and buthionine sulfoximine), p62 expression prevented NRF2 degradation and enhanced subsequent NRF2 nuclear accumulation through inactivation of Kelch-like ECH-associated protein 1. Additionally, nuclear NRF2 interacted with transcriptional coactivator small v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog proteins such as MafG and then activated transcription of quinone oxidoreductase-1, heme oxygenase-1, and ferritin heavy chain-1. Knockdown of p62, quinone oxidoreductase-1, heme oxygenase-1, and ferritin heavy chain-1 by RNA interference in HCC cells promoted ferroptosis in response to erastin and sorafenib. Furthermore, genetic or pharmacologic inhibition of NRF2 expression/activity in HCC cells increased the anticancer activity of erastin and sorafenib in vitro and in tumor xenograft models. .

Conclusion: These findings demonstrate novel molecular mechanisms and signaling pathways of ferroptosis; the status of NRF2 is a key factor that determines the therapeutic response to ferroptosis-targeted therapies in HCC cells V体育安卓版. .

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Figures

Figure 1
Figure 1. Increased NRF2 expression levels during ferroptosis
(A, B) Indicated HCC cells were treated with erastin (10 μM), BSO (200 μM), and sorafenib (5 μM) with or without indicated inhibitors (ZVAD-FMK,10 μM; ferrostatin-1, 1 μM; necrosulfonamide, 0.5 μM) for 24 hours and cell viability (A) and MDA levels (B) were assayed (n=3, *p < 0.05). (C, D) Indicated HCC cells were treated with erastin (10 μM), BSO (200 μM), and sorafenib (5 μM) for 24 hours and NRF2 protein (C) and mRNA (D) levels were assayed (n=3, *p < 0.05). (E) HepG2 cells were treated with erastin (10 μM) with or without cycloheximide (“CHX”, 20μg/ml) or MG-132 (5 μM) for 24 hours and NRF2 protein level was assayed. (F) HepG2 cells were treated with 20 μg/ml CHX over a 1h time period (left) or treated with 10 μM erastin for 4h followed by 20 μg/ml CHX over a 1h time period. Cells were lysed at the indicated time points. Cell lysates were subjected to western blot analysis with anti-NRF2 and anti-actin antibodies.
Figure 2
Figure 2. Interaction between p62 and Keap1 regulates NRF2 expression levels during ferroptosis
(A, B) Indicated HCC cells were treated with erastin (10 μM) and sorafenib (5 μM) for 24 hours. The protein levels of Keap1 and p62 (A), as well as interaction between Keap1 and p62 (B), were assayed using western blot and immunoprecipitation (IP) (n=3, *p < 0.05). (C, D) Indicated p62 knockdown HCC cells were treated with erastin (10 μM) and sorafenib (5 μM) for 24 hours. The protein levels of NRF2, Keap1, and p62 (C), as well as the interaction between NRF2 and Keap1 (D), were assayed with western blot and IP. (E) Knockdown of Keap1 by shRNA reversed loss of p62-inceased degradation of NRF2 following erastin (10 μM) treatment in HepG2 cells.
Figure 3
Figure 3. NRF2 expression contributes to ferroptosis resistance
(A) Western blot analysis of NRF2 expression in indicated NRF2 knockdown HCC cells. (B) Indicated knockdown HCC cells were treated with erastin (1.25–10 μM) and sorafenib (1.25–10 μM) for 24 hours and cell viabilities were assayed (n=3, *p < 0.05). (C) Indicated knockdown HCC cells were treated with erastin (10 μM) and sorafenib (5 μM) for 24 hours and GSH, MDA, and iron levels were assayed (n=3, *p < 0.05). (D) Indicated NRF2 knockdown HCC cells were treated with erastin (10 μM) and sorafenib (5 μM) with or without indicated inhibitors (ferrostatin-1, 1 μM; liproxstatin-1, 100nM; ZVAD-FMK,10 μM; necrostatin-1, 10 μM; necrosulfonamide, 0.5 μM) for 24 hours and cell viability was assayed (n=3, *p < 0.05).
Figure 4
Figure 4. NRF2 activation contributes to ferroptosis resistance
(A–C) Indicated HCC cells were treated with erastin (10 μM) and sorafenib (5 μM) for 24 hours. The expression of NRF2 in nuclear extracts (A), transcription activity of NRF2 (B), and interaction between NRF2 and MafG (C) were assayed (n=3, *p < 0.05). (D) Indicated NRF2 knockdown HCC cells were treated with erastin (10 μM) and sorafenib (5 μM) for 24 hours. The mRNA expression of NQO1, HO1, and FTH1 were assayed by Q-PCR (n=3, *p < 0.05). (E) Knockdown of NQO1, HO1, and FTH1 by specific shRNA enhanced growth inhibition in indicated HCC cells following treatment with erastin (10 μM) and sorafenib (5 μM) for 24 hours (n=3, *p < 0.05). (F) The NRF2 inhibitor alkaloid trigonelline (0.5 μM) inhibited NQO1, HO1, and FTH1 mRNA expression and enhanced growth inhibition in indicated HCC cells following treatment with erastin (10 μM) and sorafenib (5 μM) for 24 hours (n=3, *p < 0.05).
Figure 5
Figure 5. Suppression of NRF2 enhances ferroptosis in vivo
(A, B) NRF2 knockdown Hepa1–6 cells were more sensitive to erastin and sorafenib in vivo. (A) C57BL/6 mice were injected subcutaneously with indicated Hepa1–6 cells (1 × 106 cells/mouse) and treated with erastin (30 mg/kg i.p., twice every other day) and sorafenib (10 mg/kg i.p., once every other day) at day seven for two weeks. Tumor volume was calculated weekly. Data represents mean ± SE (n=5–8 mice/group, * p < 0.05). (B) Q-PCR analysis of the indicated gene expression in isolated tumor at day 28. (C, D) The alkaloid trigonelline increased the anticancer activity of erastin and sorafenib in vivo. (C) C57BL/6 mice were injected subcutaneously with indicated Hepa1–6 cells (1 × 106 cells/mouse) and treated with erastin (30 mg/kg i.p., twice every other day) and sorafenib (10 mg/kg i.p., once every other day) with or without the alkaloid trigonelline (1 mg/kg i.p., once every other day) at day seven for two weeks. Tumor volume was calculated weekly. Data represent mean ± SE (n=5–8 mice/group, * p < 0.05). (D) Q-PCR analysis of the indicated gene expression in isolated tumor at day 28.
Figure 6
Figure 6
Activation of NRF2 confers resistance to ferroptosis in hepatocellular carcinoma cells.

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