蜂毒肽通过线粒体凋亡途径促进皮肤鳞状细胞癌A431细胞株对5-FU的敏感性

Melittin promoting 5-FU sensitivity of skin squamous cell carcinoma A431 cell line through mitochondrial apoptosis pathway

摘要:

目的探究蜂毒肽通过线粒体凋亡途径促进皮肤鳞状细胞癌A431细胞株对5-氟尿嘧啶(5-FU)敏感性机制。方法筛选蜂毒肽作用于A431细胞株的半数抑制浓度(IC₅₀),并用于后续干预浓度。将A431细胞随机分为对照组(不干预)、蜂毒肽组(0.5 μmol/L蜂毒肽)、5-FU组(0.25 μmol/L 5-FU)和联合组(0.5 μmol/L蜂毒肽+0.25 μmol/L 5-FU)。检测各组A431细胞在24、48、72 h细胞活性、72 h细胞周期占比,以及细胞凋亡率及细胞线粒体凋亡蛋白(Smac)、活化半胱氨酸蛋白酶-3(cleaved Caspase-3)、细胞色素C(Cyt C)、促凋亡蛋白(Bax)的蛋白表达量。采用裸鼠荷瘤实验观察各组细胞的肿瘤生长抑制率。结果与对照组相比,不同时间点下蜂毒肽组、5-FU组、联合组的细胞存活率降低(P＜0.05)；与蜂毒肽组和5-FU组相比,不同时间点下联合组的细胞存活率降低(P＜0.05)。与对照组比较,蜂毒肽组、5-FU组、联合组的细胞凋亡率、G0/G1期细胞比例、Smac、cleaved Caspase-3、Cyt C、Bax蛋白表达增加(P＜0.05)；与蜂毒肽组和5-FU组相比,联合组的细胞凋亡率、G0/G1期细胞比例、Smac、cleaved Caspase-3、Cyt C、Bax蛋白表达增加(P＜0.05)。与对照组比较,蜂毒肽组、5-FU组、联合组的S期与G2/M期细胞比例降低(P＜0.05)；与蜂毒肽组和5-FU组比较,联合组的S期与G2/M期细胞比例降低(P＜0.05)。与蜂毒肽组和5-FU组比较,联合组肿瘤生长第30天的肿瘤生长抑制率升高(P＜0.05)。结论蜂毒肽可增强人皮肤鳞状细胞癌A431细胞对5-FU的敏感性,蜂毒肽和5-FU联合使用可将A431细胞周期有效阻滞在G0/G1期,促进细胞凋亡,抑制细胞增殖,其机制可能与激活线粒体凋亡途径有关。

Abstract:

To explore the mechanism of melittin promoting 5-FU sensitivity of skin squamous cell carcinoma A431 cell line (5-fluorouracil) through mitochondrial apoptosis pathway. MethodsThe median inhibitory concentration (IC₅₀) of melittin on A431 cell line was screened and used for subsequent intervention. A431 cells were randomly divided into control group (no intervention), melittin group (0.5 μmol/L melittin), 5-FU group (0.25 μmol/L 5-FU) and combined group (0.5 μmol/L melittin +0.25 μmol/L 5-FU). The cell viability of A431 cells in each group was detected at 24,48 and 72 hours, and the percentage of cell cycle and apoptosis rate of cells, the protein expressions of mitochondrial apoptotic protein (SMAC), cleaved Caspase-3, Cyt C and pro-apoptotic protein (Bax) at 72 hours were detected. The tumor growth inhibition rate of different groups of cells was observed by nude mice bearing tumor experiments. ResultsCompared with the control group, the cell viability in the melittin group, 5-FU group and combined group decreased at different time points (P＜0.05). Compared with melittin group and 5-FU group, the cell viability in the combined group decreased at different time points (P＜0.05). Compared with the control group, the apoptosis rate of cells in the melittin group, 5-FU group and combined group, the cell proportion of G0/G1 phase combined group, the expression of Smac, cleared-caspase-3, cytochrome c and Bax protein increased (P＜0.05). Compared with the melittin group and 5-FU group, the apoptosis rate, G0/G1 phase cell ratio, SMAC, cleaved Caspase-3, Cyt C and Bax protein expression in the combined group increased (P＜0.05). Compared with the control group, the percentage of cells in S phase, G2/M phase melittin group, 5-FU group and combined group decreased (P＜0.05). Compared with the melittin group and 5-FU group, the proportion of cells in S phase and G2/M phase combined group decreased (P＜0.05).Compared with the melittin group and 5-FU group, the tumor growth inhibition rate increased on the 30th day of tumor growth (P＜0.05). ConclusionsMelittin can enhance the sensitivity of human skin squamous cell carcinoma cell line A431 to 5-FU. The combination of melittin and 5-FU can effectively block the cell cycle of A431 in G0/G1 phase, promote cell apoptosis and inhibit cell proliferation. The mechanism may be related to the activation of mitochondrial apoptosis pathway.