新生霉素诱导前列腺癌PC-3细胞自噬的研究

The research of autophagy induced by novobiocin in prostate cancer cell line PC-3

目的:通过体外细胞实验,观察新生霉素是否抑制了前列腺癌PC-3细胞的增殖,并在分子水平和形态学上证实新生霉素通过细胞自噬抑制了前列腺癌PC-3细胞的增殖。为新生霉素应用于去势抵抗性前列腺癌(castration-resistant prostate cancer,CRPC)的临床治疗提供理论和实验依据。方法:设置对照组和不同浓度的新生霉素实验组,作用前列腺癌PC-3细胞24小时后,利用MTT法测定对照组和不同浓度实验组的增殖率,并计算出新生霉素的半抑制率。透射电镜观察对照组及实验组细胞内的自噬体。利用免疫荧光技术,在荧光显微镜下观察对照组和不同浓度实验组发生细胞自噬时的自噬标志性蛋白LC-3Ⅱ和自噬底物蛋白p62的不同。利用RT-PCR在mRNA水平上检测对照组和不同浓度实验组的自噬相关基因LC-3Ⅱ、Beclin-1的表达。结果:MTT结果示新生霉素能使PC-3细胞的增殖明显受到抑制(F=569.26,P=0.000),其抑制率随着药物浓度的增加而增加,根据公式计算出其IC50=1.52mmol/L。透射电镜观察可见实验组PC-3细胞质内自噬体和自噬溶酶体数量较对照组多,且随着浓度的增加自噬体数量增加。自噬体在细胞中的分布通过绿色荧光FITC标记的自噬特异性蛋白LC-3Ⅱ在荧光显微镜下清晰显示,在细胞膜附近有绿色荧光呈散点状分布,随着实验组新生霉素浓度的增加荧光强度增加。而绿色荧光FITC标记的自噬底物蛋白p62在荧光显微镜下的强度随着药物浓度的增加而降低。RT-PCR在mRNA水平上检测到随着药物浓度的增加自噬相关基因LC-3Ⅱ、Beclin-1的表达量升高。结论:在体外细胞实验中,新生霉素能够在一定浓度范围内呈浓度依赖性的抑制PC-3细胞增殖。新生霉素能在mRNA水平上增强Beclin-1、LC-3Ⅱ的表达。新生霉素可能是通过诱导细胞自噬性死亡来抑制前列癌PC-3细胞的增殖。

Objective： To study the cell autophagy induced by novobiocin in prostate cancer cell line PC- 3 and explore the possible mechanisms and to provide theoretical and experimental basis for novobiocin treatment of CRPC.Methods： Prostate cancer cell line PC- 3 was incubated with 0 mmol / L,0. 2 mmol / L,0. 4 mmol / L,0. 8mmol / L,1. 2mmol / L,1. 6mmol / L novobiocin in culture medium in vitro for 24 h,and was divided into experimental groups and control group. The proliferation inhibition rate of PC- 3 cells was assayed by MTT among different novobiocin concentration groups. The ultrastructure changes of cells were observed by transmission electron microscope（ TEM）,different amounts of autophagosome were observed in the experimental group and the control group. The level of protein expression of LC- 3Ⅱ and p62 were examined by immunofluorescence. The level of mRNA of autophagy gene LC- 3Ⅱ and Beclin- 1 was examined by RT- PCR among the experimental groups and the control group. Results： Within the concentration of（ 0 - 1. 6） mmol / L,the proliferation rate of PC- 3 in vitro was obviously inhibited by novobiocin. As the novobiocin concentration increasing,the inhibition ratio was stepping up and the half inhibition ratio was 1. 52 mmol /L. The autophagosome with double- membrane structure can be examined by the TEM,and the number was stepping up as the concentration increasing. Immunofluorescence showed the expression of p62 was decreased in PC- 3 cells after treated with novobiocin,and the expression of LC- 3Ⅱ was increased. RT- PCR showed the level of mRNA of Beclin- 1 and LC- 3 Ⅱ was increased by novobiocin with the increase of the concentration of novobiocin. Conclusion： Novobiocin could inhibit the proliferation of PC- 3 cells in vitro by a dose- dependent manner. Within a certain drug concentration,the level of expression mRNA and protein of LC- 3 and beclin 1 in PC- 3 cells could be improved by novobiocin. The proliferation rate of PC- 3 cells could be inhibited by novobiocin by induceing autophagy cell death.