葫芦素B对喉癌细胞增殖和凋亡的影响及其机制研究

目的:体内外观察葫芦素B对人喉癌细胞系Hep-2的抑制增殖和诱导凋亡作用并探讨其作用机制,为临床应用提供实验依据.方法:用0.1、1.0、10.0和100.0 μmol/L的葫芦素B作用于Hep-2细胞24、48和72 h, MTT法检测细胞增殖.用0.1、1.0和10.0 μmol/L的葫芦素B作用于Hep-2细胞24 h或10 μmol/L葫芦素B作用8、12和24 h,流式细胞仪检测细胞周期分布和细胞凋亡率,荧光显微镜观察细胞凋亡.Western blot检测p-STAT3、cyclin B1和Bcl-2的蛋白表达.建立喉癌裸鼠移植瘤模型,观察葫芦素B的体内抑瘤作用.结果:MTT结果显示葫芦素B对Hep-2 细胞的增殖抑制作用具有明显的剂量和时间依赖性;流式细胞仪检测发现随着葫芦素B浓度增高或作用时间延长,处于G2/M期细胞的比例逐渐升高,同时伴有G0/G1期细胞减少,细胞凋亡率逐渐升高,经统计学分析,各实验组之间及其与对照组之间的均差异有统计学意义(P<0.05或P<0.01);荧光显微镜观察可见典型细胞凋亡;Western blot检测显示葫芦素B可剂量依赖性抑制p-STAT3、cyclin B1和Bcl-2的蛋白表达.体内实验发现低、中、高剂量组的抑瘤率分别是32.43%、43.24%和70.27%.结论:葫芦素B通过抑制STAT3活化,抑制cyclin B1和Bcl-2的蛋白表达,引起喉癌细胞G2/M期阻滞、增殖抑制和凋亡,发挥对喉癌的抗癌效应.

Effects of cucurbitacin B on cell proliferation and apoptosis in Hep-2 cells

Objective:To investigate the mechanism underlying the anticancer activity of cucurbitacin B on human laryngeal cancer.Method:Hep-2 cells were treated with different concentrations of cucurbitacin B for different time.MTT assay was used to evaluate cell proliferation.Flow cytometry with PI staining and fluorescent microscopy with Hoechst 33258 staining were used to estimate cell cycle distribution and cell apoptosis.Expression of p-STAT3,cyclin B1 and Bcl-2 proteins was evaluated by Western blot assay.In vivo inhibitory effects of cucurbitacin B on tumor growth was evaluated in a nude mouse xenograft model.Result:Cucurbitacin B inhibited cellular proliferation in a dose and time dependent manner(P<0.05 or 0.01).Flow cytometry analysis showed that treatment with cucurbitacin B resulted in accumulation of cells at the G2/M phase of the cell cycle and cell apoptosis in a dose and time dependent manner(P<0.05 or P<0.01).Marked morphological changes of cell apoptosis including condensation of chromatin,nuclear fragmentation and apoptotic bodies were observed clearly by Hoechst 33258 staining.Western blot analysis demonstrated that the expression of p-STAT3,cyclin B1 and Bcl-2 proteins was suppressed significantly.In vivo studies showed that the inhibitory rates on laryngeal squamous carcinoma xenograft model were 32.43%,43.24% and 70.27% for lower,moderate and higher dosage group,respectively.Conclusion:Cucurbitacin B inhibited cell proliferation and induced apoptosis of Hep-2 cells by suppressing STAT3 signal pathway,down regulating the expression of cyclin B1 and Bcl-2 proteins.