顺铂诱导TNF-α自分泌引发头颈部鳞状癌细胞RIP1/RIP3/MLKL的坏死性凋亡  

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

作　　者：汪虹晓 陶德韬 马俊杰 张东林 沈左媛 邓超 周静萍 WANG Hongxiao;TAO Detao;MA Junjie;ZHANG Donglin;SHEN Zuoyuan;DENG Chao;ZHOU Jingping(School of Stomatology,Wannan Medical College,Wuhu 241002,China;Department of Oral and Maxillofacial Surgery,First Affiliated Hospital(Yijishan Hospital)of Wannan Medical College,Wuhu 241100,China;Anhui Engineering Research Center for Oral Materials and Application,Hefei 230031,China)

机构地区：[1]皖南医学院口腔医学院,安徽芜湖241002 [2]皖南医学院第一附属医院(皖南医学院弋矶山医院)口腔颌面外科,安徽芜湖241100 [3]安徽省口腔材料与应用转化工程研究中心,安徽合肥230031

出　　处：《南方医科大学学报》2024年第10期1947-1954,共8页Journal of Southern Medical University

基　　金：安徽省高等学校科学研究项目(2024AH040246);皖南医学院弋矶山医院引进人才基金项目(YR202108);芜湖市科技计划项目(2022JC30);安徽省大学生创新创业训练项目计划(S202210368003)。

摘　　要：目的探讨顺铂是否能够诱导头颈部鳞状癌细胞产生TNF-α,进而激活RIP1/RIP3/MLKL依赖性的坏死性凋亡通路,抑制鳞癌细胞的增殖,并探讨其分子机制。方法选取头颈部鳞状癌细胞系HN4和SCC4作为实验对象,分为对照组、顺铂组、caspases抑制组、坏死性凋亡抑制组。利用CCK-8法检测顺铂刺激后24 h的细胞存活率。随后采用Western blotting检测caspase-8以及坏死性凋亡通路蛋白(RIP1/RIP3/MLKL)和NF-κB(p65)、TNF-α表达情况;结合细胞划痕实验和Western blotting检测上皮间充质转化相关蛋白(N-cadherin、Vimentin、E-cadherin)的表达情况,评估坏死性凋亡对头颈部鳞状癌细胞迁移能力的影响。结果顺铂对HN4、SCC4细胞毒性IC_(50)分别约为10μg/mL、15μg/mL。在顺铂刺激下,与坏死性凋亡抑制剂组相比,caspase-8表达降低(P<0.05),N-cadherin、Vimentin表达降低(P<0.05)、E-cadherin表达升高(P<0.05),坏死性凋亡通路蛋白(RIP1/RIP3/MLKL)表达升高(P<0.05),TNF-α和NF-κB(p65)蛋白表达均升高(P<0.05)。在顺铂组中,细胞愈合率显著降低于对照组和坏死性凋亡抑制剂组。结论顺铂作用可激活头颈部鳞状癌细胞NF-κB信号通路,并促进TNF-α自分泌,从而引发鳞癌细胞经由RIP1/RIP3/MLKL通路依赖性的坏死性凋亡反应,并抑制肿瘤细胞性增殖。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-αwere detected with Western blotting.Results The IC_(50) of cisplatin was 10μg/mL in HN4 cells and 15μg/mL in SCC4 cells.Cisplatin treatment significantly decreased the expressions of caspase-8,N-cadherin and vimentin and increased the expressions of Ecadherin,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-αautocrine and induce RIP1/RIP3/MLKL-dependent necroptosis,thus leading to inhibition of cell proliferation.

关 键 词：头颈部鳞状癌细胞 顺铂 Z-VAD-FMK 坏死性凋亡 Nec-1 

分 类 号：R739.91[医药卫生—肿瘤]