Cisplatin promotes TNF‑α autocrine to trigger RIP1/RIP3/MLKL-dependent necroptosis of human head and neck squamous cell carcinoma cells

doi:10.12122/j.issn.1673-4254.2024.10.13

Abstract

Abstract:

Objective To investigate whether cisplatin induces tumor necrosis factor‑α (TNF‑α) secretion in human head and neck squamous cell carcinoma (HNSCC) cells to trigger RIP1/RIP3/MLKL-dependent necroptosis of the cells. Methods HNSCC cell lines HN4 and SCC4 treated with cisplatin (CDDP) or the combined treatment with CDDP and z-VAD-fmk (a caspase inhibitor) or Nec-1 (a necroptosis inhibitor) for 24 h were examined for changes in cell viability using CCK8 assay and expressions of caspase-8 and necroptosis pathway proteins (RIP1/RIP3/MLKL) using Western blotting. The changes in migration of the cells were assessed with cell scratch assay, and the expressions of epithelial-mesenchymal transition (EMT) marker proteins N-cadherin, vimentin, and E-cadherin as well as the expressions of NF‑κB (p65) and TNF‑α were detected with Western blotting. Results The IC₅₀ of cisplatin was 10 μg/mL in HN4 cells and 15 μg/mL in SCC4 cells. Cisplatin treatment significantly decreased the expressions of caspase-8, N-cadherin and vimentin and increased the expressions of E-cadherin, the necroptosis pathway proteins (RIP1/RIP3/MLKL), TNF‑α, and NF‑κB (p65), and these changes were obviously inhibited by treatment with Nec-1. Cisplatin stimulation also significantly lowered migration of the cells, and this inhibitory effect was strongly attenuated by Nec-1 treatment. Conclusion Cisplatin activates nuclear factor‑κB signaling in HNSCCs to promote TNF‑α autocrine and induce RIP1/RIP3/MLKL-dependent necroptosis, thus leading to inhibition of cell proliferation.

Key words: human head and neck squamous cell carcinoma, cisplatin, z-VAD-fmk, necroptosis, Nec-1

Hongxiao WANG, Detao TAO, Junjie MA, Donglin ZHANG, Zuoyuan SHEN, Chao DENG, Jingping ZHOU. Cisplatin promotes TNF‑α autocrine to trigger RIP1/RIP3/MLKL-dependent necroptosis of human head and neck squamous cell carcinoma cells[J]. Journal of Southern Medical University, 2024, 44(10): 1947-1954.

Figures/Tables 6

Fig.1 CCK8 assay for assessing viability of HN4 and SCC4 cells after stimulation with cisplatin for 24 h.

Fig.2 Western blotting for detecting expression of caspase-8 protein in HN4 (A) and SCC4 cells (B) with different treatments. ##P<0.01, #P<0.05 vs CDDP group.

Fig.3 Western blotting for detecting protein expression levels of RIP1, RIP3, and MLKL in HN4 (A) and SCC4 cells (B) with cisplatin-induced necroptosis. *P<0.05, **P<0.01, ****P<0.0001 vs control group.

Fig.4 Western blotting for detecting protein expressions of epithelial-mesenchymal transition (EMT) marker proteins in HN4 (A) and SCC4 cells (B) with cisplatin-induced necroptosis. *P<0.05, **P<0.01, ***P<0.001 vs control group. ##P<0.01 vs CDDP group.

Fig.5 Cell scratch assay for assessing the impact of cisplatin-induced necroptosis on migratory ability of HN4 (A) and SCC4 cells (B). ****P<0.0001.

Fig.6 Western blotting for detecting expressions of TNF-α and NF-κB (p65) proteins in HN4 (A) and SCC4 cells (B) with cisplatin-induced necroptosis. *P<0.05, **P<0.01, ****P<0.0001 vs control group.

References 32

1	Mohapatra P, Mohanty S, Ansari SA, et al. CMTM6 attenuates cisplatin-induced cell death in OSCC by regulating AKT/c-Myc-driven ribosome biogenesis[J]. FASEB J, 2022, 36(10): e22566.
2	Feng YY, Cao XD, Zhao B, et al. Nitrate increases cisplatin chemosensitivity of oral squamous cell carcinoma via REDD1/AKT signaling pathway[J]. Sci China Life Sci, 2021, 64(11): 1814-28.
3	Sasaya T, Kubo T, Murata K, et al. Cisplatin-induced HSF1-HSP90 axis enhances the expression of functional PD-L1 in oral squamous cell carcinoma[J]. Cancer Med, 2023, 12(4): 4605-15.
4	Choi HS, Kim YK, Yun PY. Cisplatin plus cetuximab inhibits cisplatin-resistant human oral squamous cell carcinoma cell migration and proliferation but does not enhance apoptosis[J]. Int J Mol Sci, 2021, 22(15): 8167.
5	Tang DL, Kang R, Berghe TV, et al. The molecular machinery of regulated cell death[J]. Cell Res, 2019, 29(5): 347-64.
6	Miao YD, Quan WX, Dong X, et al. A bibliometric analysis of ferroptosis, necroptosis, pyroptosis, and cuproptosis in cancer from 2012 to 2022[J]. Cell Death Discov, 2023, 9(1): 129.
7	Liu ZG, Jiao DL. Necroptosis, tumor necrosis and tumorigenesis[J]. Cell Stress, 2019, 4(1): 1-8.
8	Gong YT, Fan ZY, Luo GP, et al. The role of necroptosis in cancer biology and therapy[J]. Mol Cancer, 2019, 18(1): 100.
9	王芳, 李开颖, 蔡振宇. 程序性细胞死亡与肿瘤[J]. 中国细胞生物学学报, 2022, 44(4): 539-50.
10	Zhu XD, Li SL. Ferroptosis, necroptosis, and pyroptosis in gastrointestinal cancers: the chief culprits of tumor progression and drug resistance[J]. Adv Sci, 2023, 10(26): e2300824.
11	Liu L, Huang L, Chen WZ, et al. Comprehensive analysis of necroptosis-related long noncoding RNA immune infiltration and prediction of prognosis in patients with colon cancer[J]. Front Mol Biosci, 2022, 9: 811269.
12	Wang L, Hu CH, Zhao Y, et al. Novel smac mimetic ASTX660 (Tolinapant) and TNF‑α synergistically induce necroptosis in bladder cancer cells in vitro upon apoptosis inhibition[J]. Biochem Biophys Res Commun, 2022, 602: 8-14.
13	Xu Y, Lin ZW, Zhao N, et al. Receptor interactive protein kinase 3 promotes Cisplatin-triggered necrosis in apoptosis-resistant esophageal squamous cell carcinoma cells[J]. PLoS One, 2014, 9(6): e100127.
14	Chen J, Shao B, Wang J, et al. Chlorpyrifos caused necroptosis via MAPK/NF‑κB/TNF‑α pathway in common carp (Cyprinus carpio L.) gills[J]. Comp Biochem Physiol C Toxicol Pharmacol, 2021, 249: 109126.
15	Chen DS, Tong JS, Yang LH, et al. PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors[J]. Proc Natl Acad Sci U S A, 2018, 115(15): 3930-5.
16	张一鸣. LncRNAWSF27/miRNA: 1696调控GPX3参与细胞程序性坏死介导的鸡缺硒性肠炎的研究[D]. 哈尔滨: 东北农业大学, 2023.
17	Ofengeim D, Yuan JY. Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death[J]. Nat Rev Mol Cell Biol, 2013, 14(11): 727-36.
18	Cai HT, Lv MM, Wang TT. PANoptosis in cancer, the triangle of cell death[J]. Cancer Med, 2023, 12(24): 22206-23.
19	Krishnan RP, Pandiar D, Ramani P, et al. Necroptosis in human cancers with special emphasis on oral squamous cell carcinoma[J]. J Stomatol Oral Maxillofac Surg, 2023, 124(6S): 101565.
20	Wu XQ, Nagy LE, Gautheron J. Mediators of necroptosis: from cell death to metabolic regulation[J]. EMBO Mol Med, 2024, 16(2): 219-37.
21	Duan YW, Li QY, Zhou YH, et al. Activation of the TNF‑α-necroptosis pathway in parvalbumin-expressing interneurons of the anterior cingulate cortex contributes to neuropathic pain[J]. Int J Mol Sci, 2023, 24(20): 15454.
22	Lee CS, Hwang G, Nam YW, et al. IKK-mediated TRAF6 and RIPK1 interaction stifles cell death complex assembly leading to the suppression of TNF‑α‑induced cell death[J]. Cell Death Differ, 2023, 30(6): 1575-84.
23	Luo R, Onyshchenko K, Wang LQ, et al. Necroptosis-dependent immunogenicity of cisplatin: implications for enhancing the radiation-induced abscopal effect[J]. Clin Cancer Res, 2023, 29(3): 667-83.
24	Wang S, Liu XY, Liu Y. Hydrogen sulfide protects from acute kidney injury via attenuating inflammation activated by necroptosis in dogs[J]. J Vet Sci, 2022, 23(5): e72.
25	Sulkshane P, Teni T. BH3 mimetic Obatoclax (GX15-070) mediates mitochondrial stress predominantly via MCL-1 inhibition and induces autophagy-dependent necroptosis in human oral cancer cells[J]. Oncotarget, 2017, 8(36): 60060-79.
26	Basit F, Cristofanon S, Fulda S. Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes[J]. Cell Death Differ, 2013, 20(9): 1161-73.
27	Yuan TM, Liang RY, Chueh PJ, et al. Role of ribophorin II in the response to anticancer drugs in gastric cancer cell lines[J]. Oncol Lett, 2015, 9(4): 1861-8.
28	Huang YC, Yuan TM, Liu BH, et al. Capsaicin potentiates anticancer drug efficacy through autophagy-mediated ribophorin II downregulation and necroptosis in oral squamous cell carcinoma cells[J]. Front Pharmacol, 2021, 12: 676813.
29	Uzunparmak B, Gao M, Lindemann A, et al. Caspase-8 loss radiosensitizes head and neck squamous cell carcinoma to SMAC mimetic-induced necroptosis[J]. JCI Insight, 2020, 5(23): e139837.
30	Huang K, Gu XT, Xu HM, et al. Prognostic value of necroptosis-related genes signature in oral squamous cell carcinoma[J]. Cancers, 2023, 15(18): 4539.
31	Seo J, Nam YW, Kim S, et al. Necroptosis molecular mechanisms: recent findings regarding novel necroptosis regulators[J]. Exp Mol Med, 2021, 53(6): 1007-17.
32	Yun HM, Park JE, Lee JY, et al. Latifolin, a natural flavonoid, isolated from the heartwood of Dalbergia odorifera induces bioactivities through apoptosis, autophagy, and necroptosis in human oral squamous cell carcinoma[J]. Int J Mol Sci, 2022, 23(21): 13629.

[1]	Xiaofan CONG, Teng CHEN, Shuo LI, Yuanyuan WANG, Longyun ZHOU, Xiaolong LI, Pei ZHANG, Xiaojin SUN, Surong ZHAO. Dihydroartemisinin enhances sensitivity of nasopharyngeal carcinoma HNE1/DDP cells to cisplatin-induced apoptosis by promoting ROS production [J]. Journal of Southern Medical University, 2024, 44(8): 1553-1560.
[2]	Yuanyuan WANG, Teng CHEN, Xiaofan CONG, Yiran LI, Rui CHEN, Pei ZHANG, Xiaojin SUN, Surong ZHAO. Pristimerin enhances cisplatin-induced apoptosis in nasopharyngeal carcinoma cells via ROS-mediated deactivation of the PI3K/AKT signaling pathway [J]. Journal of Southern Medical University, 2024, 44(5): 904-912.
[3]	ZHU Quan, HUANG Baisheng, WEI Leiyan, LUO Qizhi. Overexpression of LncRNA MEG3 promotes ferroptosis and enhances chemotherapy sensitivity of hepatocellular carcinoma cells to cisplatin [J]. Journal of Southern Medical University, 2024, 44(1): 17-24.
[4]	ZHANG Meng, ZHANG Yuanyuan, NIU Mengzhu, ZHU Yue, TONG Shiyi, KOU Xianjuan. Dihydromyricetin alleviates pyroptosis and necroptosis in mice with MPTP-induced chronic Parkinson's disease by inducing autophagy [J]. Journal of Southern Medical University, 2023, 43(8): 1268-1278.
[5]	LIU Fang, PENG Lanzhu, XI Jingle. High expression of MYH9 inhibits apoptosis of non-small cell lung cancer cells through activating the AKT/c-Myc pathway [J]. Journal of Southern Medical University, 2023, 43(4): 527-536.
[6]	YAO Wanyu, WANG Xiaorui, YANG Yu, YOU Junxiong, JIN Junguo, ZENG Ping, HAN Qinrui, YAO Xueqing, SUN Xuegang, ZHOU Jin. Liposome nanoparticles for targeted delivery of parthenolide induce colorectal cancer necroptosis to ameliorate tumor-infiltrating T cell exhaustion in mice [J]. Journal of Southern Medical University, 2023, 43(10): 1674-1681.
[7]	LIU Xiaofeng, ZHANG Shuang, YU Ranghui, LIN Xiaoping, FAN Wenhao, WANG Yujing, LIANG Zhili, XIE Weidang, LIU Yanan, CHEN Hui. Hydroxysafflor yellow A attenuates heat stroke-induced acute lung injury in mice by inhibiting necroptosis [J]. Journal of Southern Medical University, 2022, 42(12): 1875-1881.
[8]	. miR-483-5p aggravates cisplatin-induced premature ovarian insufficiency in rats by targeting FKBP4 [J]. Journal of Southern Medical University, 2021, 41(6): 801-810.
[9]	. Inhibiting autophagy by silencing ATG5 and ATG7 enhances inhibitory effect of DDP on DDP-resistant I-10 testicular cancer cells [J]. Journal of Southern Medical University, 2021, 41(5): 657-663.
[10]	. IL-17A/lL-17RA reduces cisplatin sensitivity of ovarian cancer SKOV3 cells by regulating autophagy [J]. Journal of Southern Medical University, 2020, 40(11): 1550-1556.
[11]	. Ponatinib inhibits growth of patient-derived xenograft of cholangiocarcinoma expressing FGFR2-CCDC6 fusion protein in nude mice [J]. Journal of Southern Medical University, 2020, 40(10): 1448-1456.
[12]	. Down-regulation of pannexin 2 channel enhances cisplatin-induced apoptosis in testicular cancer I-10 cells [J]. Journal of Southern Medical University, 2020, 40(08): 1090-1096.
[13]	. Chloroquine enhances cisplatin-induced apoptosis of nasopharyngeal carcinoma cells by inhibiting autophagy via upregulating miR129 [J]. Journal of Southern Medical University, 2020, 40(03): 361-369.
[14]	. Overexpression of leukemia inhibitory factor enhances chemotherapy tolerance of endometrial cancer cells in vitro [J]. Journal of Southern Medical University, 2020, 40(01): 20-26.
[15]	. Interaction between necroptosis and apoptosis in MC3T3-E1 cell death induced by dexamethasone [J]. Journal of Southern Medical University, 2019, 39(09): 1030-.