姜黄素联合白藜芦醇诱导人喉癌细胞Hep-2凋亡的作用机制

The mechanism of curcumin combined with resveratrol inducing the apoptosis of human laryngeal cancer cells Hep-2

目的利用姜黄素及其联合白藜芦醇在体外作用于喉癌细胞Hep-2,观察其对人喉癌细胞的影响,并探讨可能的抗肿瘤作用机制。方法采用MTT法检测各组药物对喉癌细胞增殖的抑制作用;Hoechst 33258染色法观察各组药物对喉癌细胞核的形态影响;流式细胞术检测细胞凋亡率;RT-PCR检测p65和caspase-3的基因表达情况;Western blot法检测各组药物对喉癌细胞p65蛋白和caspase-3蛋白表达情况的影响。结果 MTT结果显示姜黄素联合白藜芦醇组抑制喉癌细胞增殖的作用要明显高于单独姜黄素组和单独白藜芦醇组(P<0.05);Hoechst 33258染色法的结果显示,经各组药物处理后的细胞核形态与对照组相比呈凋亡特征性改变;流式细胞术分析结果显示姜黄素联合白藜芦醇组的细胞凋亡率明显高于单独姜黄素组和单独白藜芦醇组(P<0.05);RT-PCR结果显示姜黄素联合白藜芦醇组的p65基因表达量与二者单独使用时相比明显降低(P<0.05),而姜黄素联合白藜芦醇组的caspase-3基因表达量与二者单独使用时相比明显升高(P<0.05);Western blot结果显示与对照组比较,单独姜黄素组和单独白藜芦醇组的p65蛋白表达明显下调,caspase-3蛋白表达则明显上调,而姜黄素联合白藜芦醇组对p65和caspase-3表达调控的影响与单独姜黄素组和单独白藜芦醇组相比差异有统计学意义(P<0.05)。结论姜黄素联合白藜芦醇在诱导人喉癌细胞Hep-2的凋亡时具有协同效应,其作用机制可能是通过阻断转录因子NF-κB(p65)信号通路的活化,上调caspase-3蛋白的表达来实现的。

Objective To observe the effect of curcumin and its combination with resveratrol on the proliferation and apoptosis of laryngeal cancer cells in vitro, and to investigate the possible mechanism of anti-tumor action on Hep-2. Methods Cell growth inhibition rate was detected by MTT method; The morphological changes of the nucleus were observed by Hoechst 33258 staining method;Apoptosis rate was detected by flow cytometry;The expression of p65 and caspase-3 gene was detected by RT-PCR;Western blot method was uesd to detect the effect of drugs on the expression of p65 protein and caspase-3 protein in laryngeal carcinoma cells. Results The result of MTT showed that the effect of curcumin combined with resveratrol on the proliferation of laryngeal cancer cells was significantly higher than that of curcumin and resveratrol used alone （ P 〈 0.05 ） ; The results of Hoechst 33258 staining showed that the morphology of nuclei in each treatment group showed a characteristic change of apoptosis compared with the control group ; The results of flow cytometry analysis showed that the apoptosis rate of curcumin combined with resveratrol group was significantly higher than that of curcumin and resveratrol alone group （ P 〈 0.05 ） ; RT-PCR results showed that the expression of p65 gene in curcumin combined with resveratrol group was significantly lower than that in the control group （ P 〈 0.05 ）, and the expression of caspase-3 gene in curcumin combined with resveratrol group was significantly higher than in the control group（ P 〈 0.05 ） ;Western blot analysis showed that expression of p65 protein was down-regulated and the expression of caspase-3 protein was significantly up-regulated in curcumin group alone and resveratrol alone group compared with the control group, while the expression of p65 and caspase-3 in curcumin combined with resveratrol group had significant difference compared with single curcumin and resveratrol alone group （ P 〈 0. 05 ）. Conclusions Curcumin and resveratrol has synergistic effect on inducing apoptosis of human laryngeal carcinoma cell Hep-2, and the mechanism is probably by blocking transcription factor kappa B NF- （ p65 ） signal pathway activation and up- regulating expression of caspase-3 protein.