Cyclopamine对神经母细胞瘤细胞系SK-N-SH细胞生长周期的影响及其机制

Effect Cyclopamine on the Proliferation and Cell Cycle of Human Neuroblastoma Cell Line SK-N-SH and Its Mechanism

目的探讨Shh信号途径的抑制剂cyclopamine对人神经母细胞瘤细胞系SK-N-SH细胞生长周期的影响及其分子生物学机制。方法以不同浓度的cyclopamine处理SK-N-SH细胞不同时间,在相差倒置显微镜下观察药物作用后的该细胞形态学变化。用四甲基偶氮唑盐(MTT)分析法检测各组SK-N-SH细胞的增殖情况,流式细胞仪检测不同浓度cyclopamine作用后对细胞周期的影响,Westernblot检测药物作用前后细胞周期蛋白D1、P16和P21蛋白表达的变化。结果cyclopamine各处理组SK-N-SH细胞形态学有所不同。MTT法检测结果显示,cyclopamine对SK-N-SH细胞增殖的抑制是浓度依赖性和时间依赖性的(P<0.05)。流式细胞仪检测发现随着药物浓度的增加,G0/G1期细胞百分比明显增加(P<0.05),而S期细胞百分比明显降低(P<0.05)。Westernblot检测结果显示,随着药物浓度的增加,SK-N-SH细胞中细胞周期蛋白D1表达逐渐降低,P16和P21表达逐渐增高(P<0.05)。结论cyclopamine抑制SK-N-SH细胞生长,阻滞细胞从G1期向S期转化,其机制可能与调控细胞周期蛋白表达有关。cyclopamine可能成为未来治疗神经母细胞瘤的药物之一。

Objective To investigate the shh signaling pathway inhibitors, cyclopamine on proliferation and cell cycle of human neuroblastoma cell line SK-N-SH and its mechanism. Methods The SK-N-SH cells were treated for different duration by the different concentrations of cyclopamine. The changes in the cells morphology induced by the drug were observed under the inverted microscope. Methabenzthiazuron（MTT） assay was used to evaluate the proliferation of the cells. The effect of cyclopamine of different concentration on the cell cycle was detected by flow cytometry. The expressions of the cyclin D1, P16 and P21 in SK-N-SH cells were determined by western blot. Results With the different concentrations of cyclopamine and different duration to treat, the morphology of SK-N-SH cells was varied. MTT assay showed that the proliferation rate of SK-N-SH cells was concentration-dependent and time-dependent on cyclopamine （P0.05）. The cyclopamine significantly inhibited SK-N-SH cell poliferation （P0.05）. The percentage of G0/G1 phase cells significantly increased and that of sphase cells significantly decreased with the increase in the drug concentration （P0.05）. The western blot showed that in SK-N-SH cells the expression level of cyclin D1 significantly decreased and, the expressions of P21 and P16 significantly increased with the increase in the dose of cyclopamine （P0.05）. Conclusions The inhibitory effect of cyclopamine on SK-N-SH cell poliferation is the concentration- and time-dependent, and it can block transformation of the cells from G1 phase to S phase. This effect of cyclopamine may be related to the inhibition of the cyclin D1 expression and to the increase in the cyclin P16 and P21 expression. Maybe cyclopamine is a potential drug to treat the neuroblastomas.