基于AR/mTOR信号通路探讨西黄丸对前列腺癌LNCaP细胞增殖、凋亡影响的实验研究

Effects of Xihuang Pills on proliferation and apoptosis of prostate cancer LNCaP cells based on AR/m TOR signaling pathway

基于雄激素受体(androgen receptor,AR)/雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路,研究西黄丸含药血清对前列腺癌LNCaP细胞增殖、凋亡的影响。西黄丸悬液灌胃制备SD大鼠含药血清。细胞增殖-毒性检测试剂盒(cell counting kit-8,CCK-8)法检测低、中、高剂量西黄丸含药血清对前列腺癌LNCaP细胞体外增殖的影响。流式细胞术检测不同浓度西黄丸干预后LNCaP细胞的凋亡水平;蛋白免疫印迹法(Western blot)检测裂解的天冬氨酸特异性半胱氨酸蛋白酶-3(cleaved cysteinyl aspartate specific proteinase caspase-3,cleaved caspase-3)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、AR的蛋白表达以及mTOR蛋白的磷酸化水平。结果显示,与空白血清组相比,各给药组均能使LNCaP细胞的活力显著降低;低、中、高剂量西黄丸含药血清均能使细胞凋亡率显著增加;低、中、高剂量西黄丸含药血清组细胞中cleaved caspase-3蛋白表达均显著升高,Bcl-2蛋白表达显著降低,细胞中AR蛋白表达显著降低,mTOR的磷酸化蛋白p-mTOR水平显著降低。为了研究西黄丸对LNCaP细胞体内生长的影响,采用不同剂量的西黄丸干预接种了LNCaP细胞的皮下移植瘤模型裸鼠,Western blot检测AR、mTOR、p-mTOR、Bcl-2、cleaved caspase-3蛋白的表达水平。结果显示低、中、高剂量西黄丸组皮下移植瘤的体积较模型组显著降低;各给药组皮下移植瘤的质量较模型组显著降低。与模型组相比,低、中、高剂量西黄丸组皮下移植瘤组织内cleaved caspase-3蛋白表达均显著升高,Bcl-2蛋白、AR蛋白表达显著降低;p-mTOR蛋白表达显著降低。进一步实验结果显示,AR激动剂R1881能够减弱西黄丸的抗增殖及促凋亡效应。西黄丸抗前列腺癌的作用机制与抑制AR/mTOR信号通路,抑制LNCaP细胞的增殖、诱导癌细胞凋亡有关。

Based on the androgen receptor(AR)/mammalian target of rapamycin(mTOR)signaling pathway,the effects of Xihuang Pills-medicated serum on the proliferation and apoptosis of prostate cancer LNCaP cells were investigated.The drug-containing serum of SD rats was prepared by intragastric administration of Xihuang Pills suspension.The effects of low-,medium-,and high-dose Xihuang Pills-containing serum on the in vitro proliferation of LNCaP cells were detected by cell counting kit-8(CCK-8).Flow cytometry was used to detect the apoptosis level of LNCaP cells after intervention with different concentrations of Xihuang Pills.Protein expression of cleaved cysteinyl aspartate-specific proteinase caspase-3(cleaved caspase-3),B-cell lymphoma-2(Bcl-2),and AR as well as the phosphorylation level of mTOR protein were detected by Western blot.The results showed that compared with the blank serum,the drug-medicated serum could blunt the activity of LNCaP cells.Low-,medium-,and high-dose Xihuang Pills-containing serum could significantly increase the cell apoptosis rate,increase the expression of cleaved caspase-3 protein,decrease the expression of Bcl-2 protein,reduce the expression of AR protein,and down-regulate the level of phosphorylated mTOR(p-mTOR).To study the effect of Xihuang Pills on the growth of LNCaP cells in vivo,different doses of Xihuang Pills were used to intervene in the subcutaneous graft model in nude mice inoculated with LNCaP cells.The expression levels of AR,mTOR,p-mTOR,Bcl-2,and cleaved caspase-3 were detected by Western blot.The results showed that the volumes of subcutaneous graft tumor in the low-dose,medium-dose,and high-dose Xihuang Pills groups significantly decreased compared with that in the model group.The weight of subcutaneous transplanted tumor in each group with drug intervention was significantly lower than that in the model group.Compared with the model group,the low-dose,medium-dose,and high-dose Xihuang Pills groups showed increased cleaved caspase-3 protein expression,decreased Bcl-2 and AR protein expression,and reduced p-mTOR protein expression.Further experiments showed that AR agonist R1881 could block the anti-proliferation and pro-apoptotic effects of Xihuang Pills.The mechanism of Xihuang Pills against prostate cancer is related to the inhibition of the AR/mTOR signaling pathway,inhibition of LNCaP cell proliferation,and induction of apoptosis in cancer cells.