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天花粉蛋白对人宫颈癌细胞增殖及凋亡的影响及其机制研究

2019-09-07王凡邓心燕姚铭辉田甜刘影王慧王佳董小耘

中国医药导报 2019年17期
关键词:细胞凋亡细胞周期

王凡 邓心燕 姚铭辉 田甜 刘影 王慧 王佳 董小耘

[摘要] 目的 研究天花粉蛋白(TCS)對人宫颈癌细胞(HeLa细胞)诱导凋亡的作用及机制。 方法 低剂量(7 μmol/L)、中剂量(14 μmol/L)和高剂量(28 μmol/L)TCS处理HeLa细胞10~82 h后,观察细胞形态及细胞生长情况。同样梯度剂量的TCS处理HeLa细胞24 h后,检测细胞周期,凋亡变化,应用蛋白免疫印迹法检测细胞凋亡相关蛋白表达。 结果 TSC处理后HeLa细胞形态皱缩,贴壁变差,增殖减少,呈剂量依赖性,低、中剂量TCS组随时间延长,细胞数逐步减少(r = -0.9727、-0.9757,P < 0.05)。处理24 h后中、高剂量TCS组G0/G1期比例较空白对照组增加(P < 0.05),低、中、高剂量TCS组S期比例较空白对照组降低,差异有统计学意义(P < 0.05)。低、中、高剂量TCS组HeLa细胞凋亡率较空白对照组增加(P < 0.05)。与空白对照组比较,低、中、高剂量TCS组Caspase-3、Caspase-9、聚腺苷二磷酸核糖聚合酶(PARP)和磷酸化细胞外调节蛋白激酶(p-ERK)蛋白表达水平逐渐减少,与TCS呈现浓度依赖关系(r = -0.969,P < 0.05)。 结论 TCS通过活化Caspase家族蛋白、诱导细胞凋亡,进而抑制宫颈癌HeLa细胞增殖,从而发挥抗肿瘤作用。

[关键词] 天花粉蛋白;细胞凋亡;细胞周期;半胱天冬酶;宫颈癌细胞

[中图分类号] R737.33          [文献标识码] A          [文章编号] 1673-7210(2019)06(b)-0013-05

Effect of trichosanthin on proliferation and apoptosis of human cervical cancer cells and research on its mechanism

WANG Fan1,2   DENG Xinyan1,2   YAO Minghui1   TIAN Tian1   LIU Ying1   WANG Hui1   WANG Jia1   DONG Xiaoyun1,2

1.Medical College of Yangzhou University, Jiangsu Province, Yangzhou   225000, China; 2.Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Jiangsu Province, Yangzhou   225000, China

[Abstract] Objective To study the effect and mechanism of trichosanthin (TCS) on inducing apoptosis of human cervical cancer HeLa cells. Methods HeLa cells were treated with low (7 μmol/L), medium (14 μmol/L) and high (28 μmol/L) doses of TCS for 10-82 h, the cell morphology and inhibition of cell growth was observed. The cell cycle and apoptosis was detected and Western blot was performed to detect the protein expression of apoptosis proteins of cervical cancer cells HeLa after treated with TCS for 24 h. Results HeLa cells of cervical cancer treated by TSC showed wrinkles and deformation, the growth of HeLa cells was inhibited. The number of cells in low and medium dose of TCS group decreased with time (r = -0.9727, -0.9757, P < 0.05). Compared with the blank control group, the G0/G1 phase of HeLa cells in medium, high dose of TCS group were significantly increased (P < 0.05), and the S phase in low, medium, high dose of TCS group were decreased after TCS treated for 24 h, the differences were statistically significant (P < 0.05). The apoptosis rate of HeLa cells in the low, medium and high dose of TCS group was increased compared with that in the blank control group (P < 0.05). Compared with the blank control group, the protein expressions levels of Caspase-9, Caspase-3, polyadenosine diphosphate ribosome polymerase (PARP) and p-extracellular regulated protein kinase (ERK) in low, medium, high dose of TCS group were gradually decreased in a dose-dependent manner after of TCS treatment (r = -0.969, P < 0.05). Conclusion TCS can induce apoptosis by activating the Caspase family of proteins to inhibit the proliferation of cervical cancer HeLa cells to exert anti-tumor effects.

与空白对照组比较,低、中、高剂量TCS组Caspase-9、Caspase-3、PARP、p-ERK及ERK蛋白表达均降低(P < 0.05)。与低剂量TCS组比较,中剂量TCS组Caspase-9、Caspase-3、PARP及p-ERK蛋白表达均明显降低(P < 0.05)。高剂量TCS组的Caspase-9、Caspase-3、PARP及p-ERK蛋白表达较低、中剂量TCS组降低(P < 0.05),而ERK表达量与低、中剂量TCS组比较差异无统计学意义(P > 0.05)。Caspase-9、Caspase-3、PARP和p-ERK蛋白表达水平与TCS浓度呈负相关关系(r = -0.969,P < 0.05)。见图5、表3。

3 讨论

细胞凋亡在治疗癌症中有重要地位[7]。研究表明,TCS可诱导多种肿瘤细胞凋亡[8-9]。本研究结果显示,经TCS作用后的HeLa细胞出现了皱缩、间隙增大、细胞数减少等细胞凋亡的形态学改变。经过TCS处理后的HeLa细胞凋亡率随着浓度和时间的增加而逐渐升高。同时,TCS也使HeLa细胞出现G0/G1期的比例增高,S期比例降低,提示G1期阻滞。S期为DNA合成期,TCS使细胞阻滞于G1期,细胞无法进行DNA合成,继而引起细胞凋亡。

蛋白酶降解细胞内蛋白质从而参与细胞凋亡过程[10]。Caspase家族如Caspase-3和Caspase-9在细胞凋亡中起着关键作用[11-13]。正常情况下,Caspase-9以及Caspase-3以无活性的酶原形式存在于细胞内,细胞发生凋亡时线粒体可释放促凋亡因子,进入细胞质促进启动子Caspase-9的激活,后者反过来激活效应Caspase,如Caspase-3[14]。Caspase-3也是多种凋亡途径的共同下游效应部分[15]。活化裂解后的Caspase-3可切割PARP,PARP为DNA修复酶,从而使PARP失去对DNA的修复功能[6]。本研究结果显示,TCS使Caspase-9及Caspase-3不再以无活性的酶原形式存在于细胞内,Caspase-9以及Caspase-3活化后裂解PARP,DNA修复受阻,从而使发生细胞凋亡。

ERK信号通路激活与细胞生长、增殖有关[17-18],如早在1996年便发现可通过Ras/Raf/ERK途径促凋亡功能[19]。本研究发现随着TCS浓度的升高,p-ERK表达依次降低,提示TCS可抑制细胞ERK磷酸化。黄益玲等[20]发现TCS抑制PC3细胞增殖的机制可能为TCS能抑制ERK磷酸化,与本研究结果类似,ERK磷酸化减少可能与细胞凋亡有关,其分子机制有待进一步研究。

综上所述,TCS通过活化Caspase家族蛋白,诱导细胞凋亡,进而抑制宫颈癌细胞增殖,从而发挥抗肿瘤作用,但其具体分子机制仍需进一步的研究。

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