临床肿瘤学杂志

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3,3’-二吲哚甲烷对宫颈癌SiHa细胞增殖抑制的实验研究

李源,朱俊勇,陈祖华,曾川,韦准   

  1. 武汉大学人民医院妇产科
  • 收稿日期:2012-05-07 修回日期:2012-06-17 出版日期:2012-09-29 发布日期:2012-09-29
  • 通讯作者: 朱俊勇

  • Received:2012-05-07 Revised:2012-06-17 Online:2012-09-29 Published:2012-09-29

摘要: 目的 研究吲哚-3-甲醇(I3C)的重要衍生物3,3’-二吲哚甲烷(DIM)对宫颈癌SiHa细胞增殖和凋亡的影响。方法 采用CCK-8法检测不同浓度DIM对SiHa细胞体外生长的抑制作用,流式细胞术检测DIM对SiHa细胞凋亡的影响,荧光显微镜观察细胞形态的变化;Western blotting检测不同浓度DIM对SiHa细胞凋亡相关蛋白表达的影响。结果 DIM对SiHa细胞的抑制作用呈时间-剂量依赖性,DIM对SiHa细胞干预48h的半数抑制浓度(IC50)为44.44μmol/L。分别以25、50、100μmol/L 的DIM处理48h后,SiHa细胞的凋亡率分别为(10.09±1.32)%、(21.11±3.36)%和(55.46±6.33)%,阴性对照组为(4.56±0.52)%,组间差异有统计学意义(P<0.05)。不同浓度DIM处理48h后,荧光显微镜下SiHa细胞呈现皱缩、变圆,并形成明显的凋亡小体。Western blotting显示随着DIM浓度的增加,SiHa细胞中MAPK家族的ERK、p38和pp38蛋白的表达水平受抑制,JNK、p-JNK蛋白的表达水平上调,PI3K家族的Akt、p-Akt、PI3K p110α、PI3K p110β、PI3K class Ⅲ、GSK3β、pPDK1和pcRaf蛋白的表达也受到抑制。结论 DIM能抑制SiHa细胞的生长并诱导细胞凋亡,其作用机制是通过对MAPK家族和PI3K家族凋亡相关蛋白表达的调控实现的,并将有可能成为一种有效的抗宫颈癌药物。

Abstract: Objective To investigate the potential antiproliferative and proapoptotic effects of 3,3diindolylmethane(DIM), important derivatives of Indo3carbinol(I3C) on SiHa cells. Methods Cell proliferation was detected by cell counting Kit8 and cell apoptosis was analyzed by flow cytometry. Morphological changes accompanying cell apoptosis were observed with fluorescence microscope after Hoechst 33258 staining. In addition, changes of proteins expression involved in MPAK and PI3K pathway were determined by Western blotting. Results DIM treatment inhibited the proliferations and induced the apoptosis of SiHa cells significantly in a time and dosedependent manner. After DIM treatment for 48h,the calculated IC50 of DIM for SiHa cells was 4444μmol/L. The apoptotic ratio in SiHa cells were(4.56±0.52)%,(10.09±1.32)%,(21.11±3.36)% and(55.46±6.33)% at 0, 25, 50 and 100μmol/L DIM. Evident morphological changes including membrane shrinking, round shaping and budding to form apoptotic bodies were observed in SiHa cells after DIM treatment for 48h at different concentration. In addition, expressions of ERK, p-ERK, p38 and p-p38, which were involved in MAPK pathway, were downregulated when the dose of DIM increased. Another proteins involved in MAPK pathway, such as JNK and p-JNK were upregulated. Furthermore, DIM treatment significantly suppressed the expressions of Akt, p-Akt, PI3K p110α, PI3K p110β, PI3K class Ⅲ, GSK3-β, p-PDK1 and p-c-Raf which were involved in PI3K pathway. Conclusion These results demonstrated that DIM exerted antitumor effects on SiHa cells through its antiproliferative and proapoptotic roles. The molecular mechanism for these effects may be related to its regulatory effects on MAPK and PI3K pathway and apoptosis proteins. DIM might be a preventive and therapeutic agent against cervical cancer.

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