目的 研究日蟾蜍它灵（gamabufotalin，CS-6）对鼻咽癌细胞增殖的影响，从细胞周期阻滞和自噬探讨CS-6抑制鼻咽癌细胞增殖的机制。方法 通过CCK-8实验、克隆形成实验检测CS-6对HK-1和5-8F细胞的增殖抑制作用；使用流式细胞仪检测CS-6对HK-1和5-8F细胞周期分布和凋亡的影响；采用Western blotting检测细胞周期蛋白B1（Cyclin B1）和周期蛋白依赖性激酶1（cyclin dependent protein kinase 1，CDK1）的表达；采用转录组测序技术筛选CS-6作用于细胞后的差异表达基因，IPA软件分析差异表达基因的富集通路；采用透射电镜观察CS-6作用于细胞后自噬体的超微结构；采用Western blotting检测微管相关蛋白轻链3（microtubule-associated protein light chain 3，LC3）、p62、糖原合成酶激酶-3β（glycogen synthase kinase-3β，GSK-3β）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）通路相关蛋白表达；通过瞬时转染mCherry-EGFP-LC3质粒后，使用激光共聚焦显微镜观察CS-6作用于细胞后自噬通量的变化。为了研究PFKFB4在CS-6诱导鼻咽癌细胞增殖、周期阻滞及自噬中的作用，HK-1和5-8F细胞转染PFKFB4质粒后，采用qRT-PCR和Western blotting检测6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶-4（6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4，PFKFB4）的表达，检测细胞存活率、集落形成、自噬通量、细胞周期和自噬相关蛋白表达的变化。体内建立鼻咽癌移植瘤小鼠模型，给予CS-6干预后，测定肿瘤体积和质量，采用苏木素-伊红染色观察肿瘤、肝、肾组织病理变化，采用免疫组化法检测肿瘤组织Ki67表达，采用Western blotting检测肿瘤组织中LC3、p62、Cyclin B1、CDK1、p-mTOR、PFKFB4、p-GSK-3β蛋白表达。结果 CS-6作用于细胞48 h后，能够显著抑制HK-1和5-8F细胞的增殖（P＜0.05、0.001），其半数抑制浓度分别为8.684、14.320 nmol/L；CS-6引起HK-1和5-8F细胞发生明显的G₂/M期阻滞（P＜0.05、0.01），同时增加Cyclin B1和CDK1的蛋白表达（P＜0.05、0.01、0.001）；CS-6在48 h处理条件下未诱导HK-1和5-8F细胞发生凋亡。CS-6显著降低PFKFB4的mRNA及蛋白表达（P＜0.05、0.01、0.001），在细胞内观察到自噬典型的双层膜结构及自噬溶酶体，上调LC3-II/I以及下调p62的蛋白表达（P＜0.05、0.01、0.001），促进自噬体与溶酶体的融合。过表达PFKFB4能部分逆转CS-6对细胞增殖的抑制以及对细胞周期、自噬相关蛋白表达的调控作用（P＜0.05、0.01、0.001），并使自噬小体和溶酶体的融合受阻。CS-6显著增加p-GSK-3β表达并抑制p-mTOR的表达（P＜0.05、0.01、0.001）；过表达PFKFB4可部分逆转CS-6对p-GSK-3β和p-mTOR的调控作用（P＜0.05、0.01）。体内实验中，CS-6显著抑制荷瘤小鼠肿瘤生长（P＜0.001），诱导肿瘤组织发生病理损伤，抑制肿瘤细胞增殖（P＜0.001），上调肿瘤组织中LC3-II/I、p-GSK3β表达并下调p62表达（P＜0.001），下调p-mTOR、PFKFB4、Cyclin B1和CDK1的蛋白表达（P＜0.01、0.001），且对肝肾组织无影响。结论 CS-6通过下调PFKFB4，进而调控GSK-3β/mTOR信号通路，激活自噬及细胞周期过程，从而发挥抗鼻咽癌作用。

Objective To study the effect of gamabufotalin (CS-6) on the proliferation of nasopharyngeal carcinoma cells, and explore the mechanism by which CS-6 inhibits nasopharyngeal carcinoma cell proliferation through cell cycle arrest and autophagy. Methods The inhibitory effect of CS-6 on the proliferation of HK-1 and 5-8F cells was detected through CCK-8 assay and colony formation assay. Flow cytometry was used to detect the effect of CS-6 on the cell cycle distribution and apoptosis of HK-1 and 5-8F cells. Western blotting was used to detect the expressions of Cyclin B1 and cyclin dependent protein kinase 1 (CDK1). Transcriptome sequencing was used to screen for differentially expressed genes after CS-6 treatment, and IPA software was used to analyze the enrichment pathways of differentially expressed genes. The ultrastructure of autophagosomes after CS-6 acts on cells was observed using transmission electron microscopy. Western blotting was used to detect the expressions of microtubule associated protein light chain 3 (LC3), p62, and glycogen synthase kinase-3β (GSK-3β)/mammalian target of rapamycin (mTOR) pathway related proteins. After transient transfection of mCherry-EGFP-LC3 plasmid, the changes in autophagy flux of cells treated with CS-6 were observed using laser confocal microscopy. In order to investigate the effect of PFKFB4 on CS-6-induced proliferation, cell cycle arrest and autophagy of nasopharyngeal carcinoma cells, HK-1 and 5-8F cells were transfected with PFKFB4 plasmid, qRT-PCR and Western blotting were used to detect the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 (PFKFB4), as well as changes in cell survival rate, colony formation, autophagy flux, cell cycle and autophagy related protein expressions. A mouse model of nasopharyngeal carcinoma transplantation in vivo was established, after CS-6 administration, tumor volume and weight were measured, pathological changes in tumor, liver and kidney tissues were observed using hematoxylin-eosin staining, Ki67 expression in tumor tissues was detected using immunohistochemistry, LC3, p62, Cyclin B1, CDK1, p-mTOR, PFKFB4 and p-GSK-3β protein expressions in tumor tissues were detected using Western blotting. Results After 48 h of treatment, CS-6 significantly inhibited the proliferation of HK-1 and 5-8F cells (P < 0.05, 0.001), with half-maximal inhibitory concentrations of 8.684 and 14.320 nmol/L, respectively. CS-6 caused significant G₂/M phase arrest in HK-1 and 5-8F cells (P < 0.05, 0.01), while increasing protein expressions of Cyclin B1 and CDK1 (P < 0.05, 0.01, 0.001). CS-6 did not induce apoptosis in HK-1 and 5-8F cells under treatment conditions of 48 h. CS-6 significantly reduced the mRNA and protein expressions of PFKFB4 (P < 0.05, 0.01, 0.001), typical double-membrane autophagosomes and autolysosomes were observed in the cells, CS-6 upregulated LC3-II/I and downregulated p62 protein expressions (P < 0.05, 0.01, 0.001), promoted the fusion of autophagosomes and lysosomes. Overexpression of PFKFB4 could partially reverse the inhibitory effect of CS-6 on cell proliferation and regulate the expressions of cell cycle and autophagy related proteins (P < 0.05, 0.01, 0.001), and inhibit the fusion of autophagosomes and lysosomes. CS-6 significantly increased the expression of p-GSK-3β and inhibited the expression of p-mTOR (P < 0.05, 0.01, 0.001). Overexpression of PFKFB4 could partially reverse the regulatory effects of CS-6 on p-GSK-3β and p-mTOR (P < 0.05, 0.01). In vivo, CS-6 significantly inhibited tumor growth in tumor bearing mice (P < 0.001), induced pathological damage to tumor tissue, inhibited tumor cell proliferation (P < 0.001), upregulated LC3-II/I, p-GSK-3β expressions and downregulated p62 expression in tumor tissue (P < 0.001), downregulated protein expressions of p-mTOR, PFKFB4, Cyclin B1 and CDK1 (P < 0.01, 0.001), and had no effect on liver and kidney tissues. Conclusion CS-6 exerts an anti-nasopharyngeal carcinoma effect by downregulating PFKFB4, thereby regulating GSK-3β/mTOR signaling pathway and inducing autophagy and cell-cycle arrest.

R285.5

山东省自然科学基金面上项目（ZR2024MH059）；滨州医学院中医药学科融合科技计划专项项目（2024YYKJ12）；国家自然科学基金青年基金项目（82104463）；山东省自然科学基金青年基金项目（ZR202103060169）