Upregulation and activation of p53 by erastin‑induced reactive oxygen species contribute to cytotoxic and cytostatic effects in A549 lung cancer cells

Huang,Chaoli; Yang,Mengchang; Deng,Jia; Li,Peng; Su,Wenjie; Jiang,Rong

doi:10.3892/or.2018.6585

Upregulation and activation of p53 by erastin‑induced reactive oxygen species contribute to cytotoxic and cytostatic effects in A549 lung cancer cells

Authors:
- Chaoli Huang
- Mengchang Yang
- Jia Deng
- Peng Li
- Wenjie Su
- Rong Jiang
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Affiliations: Department of Nephrology, Affiliated Hospital, Chengdu University, Chengdu, Sichuan 610081, P.R. China, Department of Anesthesiology, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, Sichuan 610072, P.R. China
Published online on: July 20, 2018 https://doi.org/10.3892/or.2018.6585
Pages: 2363-2370

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Abstract

The tumour‑suppressor protein p53 is a key regulator of multiple cellular processes and exerts its tumour‑suppressor function by inducing apoptotic cell death. However, emerging evidence indicates that p53 is also involved in inducing ferroptosis, which is a unique iron‑dependent form of non‑apoptotic cell death triggered by the RAS‑selective lethal small molecule erastin. Previous studies have shown that erastin exposure induces increased ROS accumulation and oxidative stress. In the present study, we incubated A549 cells with erastin and detected ROS accumulation. Semi‑quantitative western blotting was performed to analyse the effect of the induced ROS on p53 activity. To determine how ROS activate p53, NAC, an ROS scavenger, and KU‑55933, an ATM kinase inhibitor, were employed to co‑incubate with erastin, followed by western blot analysis. Either p53 or SLC7A11 siRNA was introduced into A549 cells to silence the target‑gene expression, followed by ROS detection to illustrate the regulatory role of ROS‑activated p53 on its target gene SLC7A11. Annexin V‑FITC/PI staining was performed to detect the induction of apoptotic cell death by erastin exposure. To further assess the effects of erastin treatment on cellular proliferation, EdU staining and cell cycle flow cytometric analysis were performed. Erastin exposure upregulated and activated p53 and thus, transcriptionally activated its downstream target genes, including p21 and Bax, in lung cancer A549 cells dependent on erastin‑induced ROS. Subsequently, activated p53 by erastin treatment suppressed SLC7A11 and induced ROS accumulation, indicating the potential feedback loop between p53 and erastin‑induced ROS. By employing the caspase inhibitor Z‑VAD‑FMK, it was revealed that erastin‑induced p53 contributed to both ferroptotic and apoptotic cell death and inhibited cell proliferation via arresting the cell cycle at G1 phase. Collectively, these results indicated that p53 may contribute to the cytotoxic and cytostatic effects associated with establishing a feedback loop with ROS induced by erastin.

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1	Yagoda N, von Rechenberg M, Zaganjor E, Bauer AJ, Yang WS, Fridman D, Wolpaw AJ, Smukste I, Peltier JM, Boniface JJ, et al: RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature. 447:864–868. 2007. View Article : Google Scholar : PubMed/NCBI
2	Dolma S, Lessnick SL, Hahn WC and Stockwell BR: Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer Cell. 3:285–296. 2003. View Article : Google Scholar : PubMed/NCBI
3	Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, et al: Ferroptosis: An iron-dependent form of non-apoptotic cell death. Cell. 149:1060–1072. 2012. View Article : Google Scholar : PubMed/NCBI
4	Huo H, Zhou Z, Qin J, Liu W, Wang B and Gu Y: Erastin Disrupts mitochondrial permeability transition pore (mPTP) and induces apoptotic death of colorectal cancer cells. PLoS One. 11:e01546052016. View Article : Google Scholar : PubMed/NCBI
5	Vogelstein B, Lane D and Levine AJ: Surfing the p53 network. Nature. 408:307–310. 2000. View Article : Google Scholar : PubMed/NCBI
6	Jiang L, Kon N, Li T, Wang SJ, Su T, Hibshoosh H, Baer R and Gu W: Ferroptosis as a p53-mediated activity during tumour suppression. Nature. 520:57–62. 2015. View Article : Google Scholar : PubMed/NCBI
7	Gao M, Monian P, Quadri N, Ramasamy R and Jiang X: Glutaminolysis and transferrin regulate ferroptosis. Mol Cell. 59:298–308. 2015. View Article : Google Scholar : PubMed/NCBI
8	Tsai MH, Liu JF, Chiang YC, Hu SC, Hsu LF, Lin YC, Lin ZC, Lee HC, Chen MC, Huang CL and Lee CW: Artocarpin, an isoprenyl flavonoid, induces p53-dependent or independent apoptosis via ROS-mediated MAPKs and Akt activation in non-small cell lung cancer cells. Oncotarget. 8:28342–28358. 2017. View Article : Google Scholar : PubMed/NCBI
9	Zhou Z, Yin Y, Chang Q, Sun G, Lin J and Dai Y: Downregulation of B-myb promotes senescence via the ROS-mediated p53/p21 pathway, in vascular endothelial cells. Cell Prolif. 50:2017. View Article : Google Scholar
10	Assaily W, Rubinger DA, Wheaton K, Lin Y, Ma W, Xuan W, Brown-Endres L, Tsuchihara K, Mak TW and Benchimol S: ROS-mediated p53 induction of Lpin1 regulates fatty acid oxidation in response to nutritional stress. Mol Cell. 44:491–501. 2011. View Article : Google Scholar : PubMed/NCBI
11	Seo SU, Cho HK, Min KJ, Woo SM, Kim S, Park JW, Kim SH, Choi YH, Keum YS, Hyun JW, et al: Thioridazine enhances sensitivity to carboplatin in human head and neck cancer cells through downregulation of c-FLIP and Mcl-1 expression. Cell Death Dis. 8:e25992017. View Article : Google Scholar : PubMed/NCBI
12	Wang SJ, Li D, Ou Y, Jiang L, Chen Y, Zhao Y and Gu W: Acetylation is crucial for p53-mediated ferroptosis and tumor suppression. Cell Rep. 17:366–373. 2016. View Article : Google Scholar : PubMed/NCBI
13	Shi Y, Nikulenkov F, Zawacka-Pankau J, Li H, Gabdoulline R, Xu J, Eriksson S, Hedström E, Issaeva N, Kel A, et al: ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis. Cell Death Differ. 21:612–623. 2014. View Article : Google Scholar : PubMed/NCBI
14	Yang J, Zhao X, Tang M, Li L, Lei Y, Cheng P, Guo W, Zheng Y, Wang W, Luo N, et al: The role of ROS and subsequent DNA-damage response in PUMA-induced apoptosis of ovarian cancer cells. Oncotarget. 8:23492–23506. 2017.PubMed/NCBI
15	An JJ, Shi KJ, Wei W, Hua FY, Ci YL, Jiang Q, Li F, Wu P, Hui KY, Yang Y and Xu CM: The ROS/JNK/ATF2 pathway mediates selenite-induced leukemia NB4 cell cycle arrest and apoptosis in vitro and in vivo. Cell Death Dis. 4:e9732013. View Article : Google Scholar : PubMed/NCBI
16	Suh DH, Kim MK, Kim HS, Chung HH and Song YS: Mitochondrial permeability transition pore as a selective target for anti-cancer therapy. Front Oncol. 3:412013. View Article : Google Scholar : PubMed/NCBI
17	Yamaguchi H, Hsu JL, Chen CT, Wang YN, Hsu MC, Chang SS, Du Y, Ko HW, Herbst R and Hung MC: Caspase-independent cell death is involved in the negative effect of EGFR inhibitors on cisplatin in non-small cell lung cancer cells. Clin Cancer Res. 19:845–854. 2013. View Article : Google Scholar : PubMed/NCBI
18	Aresvik DM, Pettersen RD, Abrahamsen TG and Wright MS: 5-Fluorouracil-induced death of Jurkat T-cells-A role for caspases and MCL-1. Anticancer Res. 30:3879–3887. 2010.PubMed/NCBI
19	Siskind LJ: Mitochondrial ceramide and the induction of apoptosis. J Bioenerg Biomembr. 37:143–153. 2005. View Article : Google Scholar : PubMed/NCBI
20	Colombini M: Ceramide channels and their role in mitochondria-mediated apoptosis. Biochim Biophys Acta. 1797:1239–1244. 2010. View Article : Google Scholar : PubMed/NCBI
21	Lago CU, Sung HJ, Ma W, Wang PY and Hwang PM: p53, Aerobic metabolism, and cancer. Antioxid Redox Signal. 15:1739–1748. 2011. View Article : Google Scholar : PubMed/NCBI
22	Downs I, Liu J, Aw TY, Adegboyega PA and Ajuebor MN: The ROS scavenger, NAC, regulates hepatic Vα14iNKT cells signaling during Fas mAb-dependent fulminant liver failure. PLoS One. 7:e380512012. View Article : Google Scholar : PubMed/NCBI
23	Jürgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D and Reed JC: Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA. 95:4997–5002. 1998. View Article : Google Scholar : PubMed/NCBI
24	Horn HF and Vousden KH: Coping with stress: Multiple ways to activate p53. Oncogene. 26:1306–1316. 2007. View Article : Google Scholar : PubMed/NCBI
25	Zhang F, Wang W, Tsuji Y, Torti SV and Torti FM: Post-transcriptional modulation of iron homeostasis during p53-dependent growth arrest. J Biol Chem. 283:33911–33918. 2008. View Article : Google Scholar : PubMed/NCBI