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.

nasopharyngeal carcinoma  /  gamabufotalin  /  PFKFB4  /  autophagy  /  GSK-3β/mTOR signaling pathway