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 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-α autocrine and induce RIP1/RIP3/MLKL-dependent necroptosis, thus leading to inhibition of cell proliferation.
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IC50分别约为10 μg/mL、15 μg/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。
Keywords: Nec-1; cisplatin; human head and neck squamous cell carcinoma; necroptosis; z-VAD-fmk.