目的 探讨蟾蜍它灵对人头颈部鳞状细胞癌（head and neck squamous cell carcinoma，HNSCC）的影响及其潜在的作用机制。方法 采用CCK-8法和克隆形成实验，考察蟾蜍它灵对人舌鳞癌Cal-27细胞和人咽鳞癌FaDu细胞活力及增殖的影响；采用流式细胞术检测蟾蜍它灵对Cal-27和FaDu细胞周期分布的影响；采用Western blotting检测蟾蜍它灵对Cal-27和FaDu细胞内细胞周期蛋白依赖性激酶4（cyclin-dependent kinase 4，CDK4）和细胞周期蛋白D1（cyclin D1）表达的影响。构建裸鼠异种移植瘤模型，进一步验证蟾蜍它灵的体内抗肿瘤作用。通过蛋白质组学检测经蟾蜍它灵处理后的Cal-27细胞内差异表达的蛋白，并分析其发挥抗肿瘤作用的信号通路；采用Western blotting检测蛋白激酶R样内质网激酶（protein kinase R-like endoplasmic reticulum kinase，PERK）/真核翻译起始因子2α亚基（eukaryotic initiation factor 2α，eIF2α）信号通路相关蛋白的表达。结果 体外实验结果显示，蟾蜍它灵可显著降低Cal-27和FaDu细胞的活力（P＜0.01、0.001），并抑制其增殖（P＜0.01、0.001），诱导细胞周期阻滞于G₀/G₁期（P＜0.05、0.01、0.001），下调FaDu细胞内CDK4和cyclin D1的蛋白表达（P＜0.05、0.001），下调Cal-27细胞内CDK4蛋白表达（P＜0.05）。体内实验结果显示，蟾蜍它灵可显著抑制荷瘤小鼠体内肿瘤的生长（P＜0.01、0.001）。蛋白质组学和IPA分析表明蟾蜍它灵能引起内质网应激，上调Cal-27和FaDu细胞内p-PERK和p-eIF2α的表达水平（P＜0.05、0.001）。结论 蟾蜍它灵可通过诱导内质网应激，激活EIF2信号通路，诱导HNSCC细胞阻滞于G₀/G₁期，进而抑制肿瘤细胞的增殖和荷瘤小鼠体内肿瘤的生长。

Objective To investigate the effect and potential mechanism of bufotalin on growth of human head and neck squamous cell carcinoma (HNSCC). Methods The effect of bufotalin on viability and proliferation of Cal-27 and FaDu cells were observed by CCK-8 method and plate clone formation experiment. The effect of bufotalin on cell cycle distribution of Cal-27 and FaDu cells were detected by flow cytometry. Western blotting was used to detect the effect of bufotalin on expressions of cyclin-dependent kinase 4 (CDK4) and cyclin D1 in Cal-27 and FaDu cells. To further explore the anti-tumor effects of bufotalin in vivo, a xenograft tumor model of HNSCC was established in nude mice. Proteomics technology was applied to identify differentially expressed proteins in bufotalin-treated Cal-27 cells, and the associated signaling pathways underlying its anti-tumor effect were analyzed. Western blotting was performed to assess the expressions of protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) signaling pathway related proteins. Results In vitro experimental results showed that bufotalin significantly reduced the viability of Cal-27 and FaDu cells (P < 0.01, 0.001), inhibited their proliferation (P < 0.01, 0.001), induced cell cycle arrest in G₀/G₁ phase (P < 0.05, 0.01, 0.001), down-regulated the protein expressions of CDK4 and cyclin D1 in FaDu cells (P < 0.05, 0.001), and down-regulated the protein expression of CDK4 in Cal-27 cells (P < 0.05). The in vivo experimental results showed that bufotalin could significantly inhibit the growth of tumors in tumor bearing mice (P < 0.01, 0.001). Proteomics and IPA analysis showed that bufotalin could induce endoplasmic reticulum stress and up-regulate the expression levels of p-PERK and p-eIF2α in Cal-27 and FaDu cells (P < 0.05, 0.001). Conclusion Bufotalin could induce endoplasmic reticulum stress, activate EIF2 signaling pathway, and induce the arrest of HNSCC cells in G₀/G₁ phase, thereby inhibiting tumor cell proliferation and tumor growth in tumor-bearing mice.

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国家中医药管理局科技司-山东省卫生健康委员会共建中医药科技项目（GZY-KJS-SD-2023-094）