地塞米松对前列腺癌PC-3细胞增殖、凋亡、迁移和侵袭的影响及机制研究

Effects and mechanism of dexamethasone on proliferation,apoptosis,migration and invasion of PC-3 cells in prostate cancer

目的:探讨地塞米松(DXMS)通过抑制细胞外信号调节激酶(ERK)/蛋白激酶B(AKT)信号通路对前列腺癌(PCa)PC-3细胞增殖、凋亡、迁移和侵袭的影响。方法:体外培养PCa细胞系PC-3,并分为对照组(二甲基亚砜)、DXMS低剂量组(0.01μmol/L DXMS)、DXMS中剂量组(0.1μmol/L DXMS)、DXMS高剂量组(1μmol/L DXMS)和DXMS高剂量+TPA组(1μmol/L DXMS和50μmol/L ERK/AKT信号通路激活剂TPA),采用qRT-PCR检测糖皮质激素受体(GR)表达;CCK-8检测细胞活性;流式细胞术检测细胞凋亡;Transwell检测细胞迁移和侵袭;Western blot检测GR、细胞凋亡(pro-caspase-3、cleaved-caspase-3、pro-caspase-9、cleaved-caspase-9、Bcl-2、Bax)、转移(Vimentin、N-cadherin、E-cadherin)及ERK、AKT蛋白表达。建立PC-3细胞异种移植裸鼠,当肿瘤直径达到0.5~0.6 cm时开始腹腔注射DXMS,共设置4组:对照组、DXMS低剂量组(1 mg/kg)、DXMS中剂量组(5 mg/kg)、DXMS高剂量组(25 mg/kg)。给药28 d后,测量肿瘤体积和重量,采用TUNEL染色观察组织凋亡,Western blot检测ERK、AKT蛋白表达。结果:随着DXMS剂量的增加,PC-3细胞活力、迁移率和侵袭率逐渐减小,GR mRNA和蛋白水平、凋亡率逐渐增大,cleaved caspase-3、cleaved caspase-9、Bax、E-cadherin表达逐渐升高,Bcl-2、Vimentin、N-cadherin表达逐渐降低,均呈显著的剂量依赖性(P<0.05)。TPA处理后,ERK和AKT磷酸化水平显著升高,能够明显逆转DXMS对PC-3细胞增殖、迁移和侵袭的抑制作用(P<0.05)。体内实验结果显示,DXMS可显著抑制PC-3移植瘤生长,促进其肿瘤组织细胞凋亡,抑制ERK和AKT磷酸化(P<0.05)。结论:DXMS可能通过抑制ERK/AKT信号通路加快PC-3细胞凋亡,阻碍细胞增殖、迁移和侵袭。

Objective:To investigate the effects of dexamethasone(DXMS)on the proliferation,apoptosis,migration and invasion of PC-3 cells in prostate cancer(PCa)cells by inhibiting the extracellular signal-regulated kinase(ERK)/protein kinase B(AKT)signaling pathway.Methods:The PCa cell line PC-3 was cultured in vitro.The cells were divided into control group(dimethyl sulfoxide),DXMS low-dose group(0.01μmol/L DXMS),DXMS medium-dose group(0.1μmol/L DXMS),and DXMS high-dose group(1μmol/L DXMS)and DXMS high-dose+TPA group(1μmol/L DXMS and 50μmol/L ERK/AKT signaling pathway activator TPA).The expression of glucocorticoid receptor(GR)was detected by qRT-PCR.Cell viability was detected by CCK-8 assay.Apoptosis was detected by flow cytometry.Cell migration and invasion were detected by Transwell.The expression of GR,apoptosis(pro-caspase-3,cleaved-caspase-3,pro-caspase-9,cleaved-caspase-9,Bcl-2,Bax),metastasis(Vimentin,N-cadherin,E-cadherin),ERK,and AKT proteins were detected by Western blot.PC-3 cell xenograft nude mice were established.DXMS was injected intraperitoneally when the tumor diameter reached 0.5~0.6 cm.A total of 4 groups were set up:control group,DXMS low-dose group(1 mg/kg),DXMS medium-dose group(5 mg/kg),and DXMS high-dose group(25 mg/kg).After 28 days of administration,the tumor volume and weight were measured.Apoptosis was observed by TUNEL staining,and the expression of ERK and AKT proteins was detected by Western blot.Results:With the increase of DXMS dose,the viability,migration rate and invasion rate of PC-3 cells decreased gradually,the level of GR mRNA and protein,apoptosis rate gradually increased,the expression of cleaved caspase-3,cleaved caspase-9,Bax,E-cadherin gradually increased,the expression of Bcl-2,Vimentin and N-cadherin gradually decreased in a significant dose-dependent manner(P<0.05).After TPA treatment,the phosphorylation levels of ERK and AKT were significantly increased,which could significantly reverse the inhibitory effects of DXMS on proliferation,migration and invasion of PC-3 cells(P<0.05).The in vivo experiment results showed that DXMS significantly inhibited the growth of PC-3 xenograft tumor,promoted the apoptosis of tumor tissues,and inhibited the phosphorylation of ERK and AKT(P<0.05).Conclusion:DXMS may accelerate the apoptosis of PC-3 cells and hinder cell proliferation,migration and invasion by inhibiting ERK/AKT signaling pathway.