周期蛋白依赖性激酶5对去势抵抗性前列腺癌细胞生长及侵袭转移的影响

The effect of cyclin dependent kinase 5 on the growth,invasion,and metastasis of castrated-resistant prostate cancer cells

目的研究周期蛋白依赖性激酶5(CDK5)对去势抵抗性前列腺癌(CRPC)细胞DU145生长及侵袭转移的影响。方法将CDK5小分子抑制剂20-223添加到细胞中,根据其浓度分为对照组(20-223浓度为0.00μmol/L)、2.00μmol/L组(20-223浓度为2.00μmol/L)、4.00μmol/L组(20-223浓度为4.00μmol/L)以及8.00μmol/L组(20-223浓度为8.00μmol/L),每组100株。采用CDK5小干扰RNA(siRNA)干扰CDK5在DU145细胞内的表达,CDK5抑制剂20-223抑制CDK5在DU145细胞内的活性;采用噻唑蓝(MTT)检测细胞生长活力,单克隆形成实验测定细胞增殖能力,采用膜联蛋白V-异硫氰酸荧光素(AnnexinV-FITC)和碘化丙啶(PI)双染法测定细胞凋亡,划痕实验和Transwell实验测定细胞的迁移能力,Western Blot测定Myc促癌基因(c-Myc)、SRY-box转录因子4(SOX4)及侵袭转移相关蛋白的表达情况。对比分析CDK5抑制剂20-223对CRPC细胞DU145生长、凋亡、侵袭转移能力,以及四组细胞c-Myc、SOX4、上皮间质转化(EMT)相关蛋白的表达量。结果在用药后24、48、72 h的细胞生长抑制率方面,2.00μmol/L组、4.00μmol/L组、8.00μmol/L组均高于对照组,差异有统计学意义(P<0.01)。CRPC细胞DU145生长活力随着CDK5抑制剂的浓度升高而降低,其生长受到显著抑制。2.00μmol/L组、4.00μmol/L组、8.00μmol/L组用药后48 h Q1象限、Q2象限、Q3象限、Q4象限的细胞凋亡率均高于对照组,差异有统计学意义(P<0.01)。4.00μmol/L组在用药后48 h的迁移率(2.28%)低于对照组(2.37%),差异有统计学意义(P<0.01)。细胞经过2 d的处理后,2.00μmol/L组、4.00μmol/L组、8.00μmol/L组c-Myc、SOX4、Vimentin、Zeb1蛋白的表达量均低于对照组,而E-cadherin的表达量高于对照组。CDK5基因沉默组CDK5、c-Myc、SOX4蛋白的表达明显低于对照组和NC组。结论CDK5在CRPC中的表达及活性下降,在一定程度上抑制了DU145细胞的恶性增殖及浸润,并且对其凋亡起到了促进作用。CDK5的潜力很强,属于去势抵抗药物的一种,当对其的活性进行抑制后,c-Myc、SOX4等蛋白水平明显下降,进而上调E-cadherin、Vimentin、Zeb1等蛋白水平,从而减弱了去势抵抗药物敏感性。

Objective To study the effect of cyclin dependent kinase 5(CDK5)on the growth,invasion,and metastasis of castrated-resistant prostate cancer(CRPC)cells.Methods CDK5 small molecule inhibitor 20-223 was added to cells at four levels of concentration,with 0.00μmol/L for control group,2.00μmol/L for 2.00μmol/L group,4.00μmol/L for 4.00μmol/L group and 8.00μmol/L for 8.00μmol/L group,with 100 stains in each group.CDK5 small interfering RNA(siRNA)was used to interfere with the expression of CDK5 in DU145 cells,and CDK5 inhibitor 20-223 inhibited the activity of CDK5 in DU145 cells;Cell growth activity was measured using methyl-thiazole-tetrazolium(MTT),and cell proliferation ability was measured using monoclonal formation assay;Cell apoptosis was measured using Annexin V-fluorescein isothio-cyanate(Annexin V-FITC)and propidium iodide(PI)double staining methods;The scratch test and Transwell test were used to determine the migration ability of cells;Western Blot was used to determine the expression of Myc oncogenic gene(c-Myc),SRY-box transcription factor 4(SOX4),and invasion and metastasis related proteins.Comparatively analysis of CDK5 inhibitor 20-223 on CRPC cell DU145 growth,apoptosis,invasive metastasis ability,as well as the expression of c-Myc,SOX4,and epithelial mesenchymal transition(EMT)-related proteins in the four groups of cells.Results The cell growth inhibition rates of the 2.00µmol/L group,4.00μmol/L group,and 8.00µmol/L group were higher than those of the control group at 24,48,and 72 h after medication,with a statistically significant difference(P<0.01).CRPC cell DU145 growth viability decreased with increasing concentrations of CDK5 inhibitors and their growth was significantly inhibited.The incidence of cell apoptosis of Q1 quadrant,Q2 quadrant,Q3 quadrant,and Q4 quadrant in the 2.00µmol/L group,4.00μmol/L group,and 8.00µmol/L group were higher than those in the control group at 48 h after medication,with a statistically significant difference(P<0.01).The migration rate in the 4.00μmol/L group was 2.28%at 48 h after medication,which was lower than 2.37%in the control group,with a statistically significant difference(P<0.01).After 2 days of treatment,the expression levels of c-Myc,SOX4,Vimentin,and Zeb1 proteins in the 2.00µmol/L group,4.00μmol/L group,and 8.00µmol/L group were lower than those of the control group,while the expression of E-cadherin was higher than that of the control group.The expression of CDK5,c-Myc,and SOX4 proteins in CDK5 gene silenced tissues was significantly lower than that in the control group and NC group(P<0.05).Conclusion The decreased expression and activity of CDK5 in CRPC can inhibit the malignant proliferation and infiltration of DU145 cells to a certain extent,and promote their apoptosis.CDK5 has a strong potential and belongs to a castration-resistant drug.When its activity is inhibited,the levels of c-Myc,SOX4 and other proteins are significantly decreased,and then the levels of E-cadherin,Vimentin,Zeb1 and other proteins are up-regulated,thus weakening the sensitivity of castration-resistant drugs.