摘要
目的通过蛋白激酶抑制剂敏感性分析揭示伴PTEN突变前列腺癌细胞株PC-3细胞增殖的分子机制。方法选择9种关键激酶抑制剂作用于PC-3细胞,CCK8法检测蛋白激酶抑制剂对PC-3细胞增殖的影响;PTEN基因经RT-PCR扩增后测序检测PC-3细胞中PTEN的突变;Western blot分析PI3K和mTOR抑制剂对PC-3细胞信号通路中关键激酶表达水平的影响;细胞经蛋白激酶抑制剂处理后,采用Annexin V-FITC和溴化丙啶染色,荧光显微镜观察PC-3细胞的凋亡情况。结果GSK2126458、AZD6482、Rapamycin和OSI-027可明显抑制PC-3细胞增殖,且AZD6482和Rapamycin对PC-3细胞增殖具有协同抑制作用;测序结果显示PC-3细胞存在PTEN第78位氨基酸错义突变;Western blot结果表明,与对照组和AZD6482单药组相比,Rapamycin单药以及AZD6482联合Rapamycin作用于PC-3细胞后AKT蛋白磷酸化水平明显下降,差异有统计学意义(P<0.05)。药物作用于细胞48和72 h后,AZD6482单药组、Rapamycin单药组和AZD6482+Rapamycin联合组的细胞凋亡比例较对照组明显升高,差异有统计学意义(P<0.05)。结论PI3K/AKT信号通路在伴PTEN突变的PC-3细胞增殖和凋亡过程中起关键作用,从而参与前列腺癌的发展过程。
Objective To study the molecular mechanism of prostate cancer cell line PC-3 proliferation by sensitivity analysis of protein kinases inhibitors(PKIs).Methods PC-3 cells were treated with nine PKIs,and the inhibition of cell viability was detected by CCK8 assay.RT-PCR and sequencing were used to detect the mutation of PTEN in PC-3 cell line.PTEN gene was amplified by RT-PCR,and sequencing to find out the mutation in PTEN.Western blot was used to analyze the expression level of protein kinases in cell signaling pathway of PC-3 cells;cells were stained by Annexin V-FITC and propane bromide(PI)after treated with PKIs,and fluorescence microscopy was used to observe the apoptosis rate of PC-3 cells induced by PKIs.Results PKIs inhibited experiments showed that GSK2126458,AZD6482,Rapamycin and OSI-027 potently inhibit the proliferation of PC-3 cells,and AZD6482 and Rapamycin had a synergistic effect on the inhibition PC-3 cells proliferation.It is revealed by sequencing result that there is a missense mutation of amino acid at position 78 of PTEN in PC-3 cells.Western blot result showed that compared with the control group and AZD6482 group,the AKT protein phosphorylation level of PC-3 cells treated with rapamycin alone and AZD6482 combined with rapamycin decreased significantly(P<0.05).After 48 and 72 hours,the apoptotic rate of AZD6482,Rapamycin and AZD6482+Rapamycin group was significantly higher than that of control group(P<0.05).Conclusion PC-3 cell line with PTEN mutation mainly promote cell proliferation through activating PI3K/AKT signaling pathway,and thus participating in the development of prostate cancer.
作者
徐凝馨
曹琼
李小文
李文龙
孙心旖
林晓雨
刘铭
覃艳红
李莉
XU Ning-xin;CAO Qiong;LI Xiao-wen;LI Wen-long;SUN Xin-yi;LIN Xiao-yu;LIU Ming;QIN Yan-hong;LI Li(School of Basic Medicine,Shanxi Medical University,Taiyuan Shanxi 030001,China;Department of Hematology,the Second Hospital of Shanxi Medical University,Taiyuan Shanxi 030001,China)
出处
《毒理学杂志》
CAS
CSCD
2020年第4期285-290,共6页
Journal of Toxicology
基金
国家自然科学青年基金(81400139)
山西省自然科学基金(2014011039-2)。
