摘要
目的研究姜黄素类似物对17β-HSD3活性的抑制率及其诱导前列腺癌LNCaP细胞凋亡的作用。方法用放射性酶标仪检测实验室前期合成的9个化合物对17β-HSD3活性的抑制率,每个化合物设计6个浓度梯度,计算半数抑制浓度(IC50),把最小IC50值化合物作为最佳的17β-HSD3的抑制剂,用其作为主要药物做后续实验研究。培养LNCaP前列腺癌细胞,实验分成4组:空白对照组、姜黄素组(150μmol·L-1)、雄激素受体(AR)抑制剂组(5μmol·L-1)、姜黄素类似物H7组(H7 150μmol·L-1)。噻唑蓝(MTT)法检测LNCaP细胞活性;Q-PCR检测各组LNCaP细胞中凋亡相关基因p53,Caspase-9,Bcl-2,Bax mRNA表达情况;Western blotting方法检测各组LNCaP细胞中凋亡相关蛋白p53,Caspase-9,Bcl-2,Bax蛋白表达情况。酶联免疫吸附测定(ELISA)法检测各组细胞中睾酮含量。结果 H1-H9对大鼠17β-HSD3活性抑制的IC50值为:(20.9±0.02)^(1120.4±124.6)μmol·L-1,对人17β-HSD3活性抑制的IC50值为:(4.9±0.02)^(3 140.7±165.7)μmol·L-1,其中H7的IC50值最小。故选H7为实验药物做细胞实验。与空白对照组比较,姜黄素、AR抑制剂和H7均不同程度抑制了LNCaP细胞活性(P<0.05或P<0.01),并且H7作用比姜黄素明显(P<0.05)。与对照组比较,姜黄素、AR抑制剂和H7均不同程度增加LNCaP细胞中p53,Caspase-9,Bax/Bcl-2的基因和蛋白表达(P<0.05或P<0.01),诱导LNCaP细胞凋亡,并且发现H7诱导LNCaP细胞凋亡的作用更明显。与对照组比较,姜黄素、AR抑制剂和H7均不同程度降低睾酮含量(P<0.01),并且H7作用比姜黄素明显(P<0.01)。结论姜黄素类似物H7可通过抑制LNCaP细胞17β-HSD3的活性,降低睾酮含量从而促进LNCaP细胞凋亡,并有望成为治疗前列腺癌的新靶点候选药物。
Objective To investigate the inhibitory rate of curcumin analogues on 17β-HSD3 activity and the effect on inducing apoptosis of prostate cancer cells LNCaP.Methods Curcumin was used as the lead compound,and 9 compounds were designed and synthesized.The inhibition of 17β-HSD3 activity by the nine compounds was determined by a radioactive microplate assay.Six concentration gradients were designed for each compound,and IC 50 were calculated.The compound with minimum IC 50 value was used as the best inhibitor of 17β-HSD3,and it was used as the main drug for the subsequent study.Prostate cancer cells LNCaP were cultured and divided into 4 groups:blank control group,curcumin group(150μmol·L-1),AR inhibitor group(5μmol·L-1),and curcumin analogue H7 group(150μmol·L-1).The LNCa cell viability was determined by MTT.The expressions of p53,Caspase-9,and Bax/bcl-2 mRNA were measured by Q-PCR.The content of testosterone in cells was determined by ELISA.The apoptosis related proteins p53,Bcl2,and Bax protein in LNCaP cells were detected by western blotting.Results The inhibitory IC 50 of H1-H9 on 17β-HSD3 activity in rats were from(20.9±0.02)to(1120.4±124.6)μmol·L-1,and that in human were from(4.9±0.02)to(3140.7±165.7)μmol·L-1,among which the IC 50 of H7 was the lowest.Therefore,H7 was selected as the experimental drug for the cell study.Compare to the control group,curcumin,AR inhibitor,and H7 inhibited LNCaP cell proliferation in various degrees(P<0.05 or P<0.01),and the effect of H7 was more significant than curcumin(P<0.05).Compared with the control group,curcumin,AR inhibitor,and H7 increased p53,caspase-9,Bax/Bcl-2(P<0.05 or P<0.01)expressions in LNCaP cells in various degrees,and induced LNCaP cell apoptosis.It was found that H7 had more significant effect on LNCaP cell apoptosis.Compare to the control group,testosterone in curcumin,AR inhibitor,and H7 group were reduced in various degrees(P<0.01),and H7 group was more significant than Cur(P<0.01).Conclusion LNCaP cells and testosterone contents are inhibited by curcumin analogue H7.H7 can induce apoptosis of LNCaP cells and is a potential candidate for a new targeted drug in the treatment of prostate cancer.
作者
韩杨
孙文慧
仲崇琦
王猛
包海花
张羽飞
吴丹
袁晓环
HAN Yang;SUN Wenhui;ZHONG Chongqi;WANG Meng;BAO Haihua;ZHANG Yufei;WU Dan;YUAN Xiaohuan(Medical Research Center,Mudanjiang Medical University,Mudanjiang 157011,China)
出处
《医药导报》
CAS
北大核心
2020年第6期740-746,共7页
Herald of Medicine
基金
牡丹江医学院研究生创新基金(2018YJSCX-07MY)
牡丹江市科学技术计划项目(Z2018s055)
黑龙江省高校创新人才培养计划(UNPYSCT-2015110)。
