黄芩素对多西他赛体外抗前列腺癌活性的影响

doi:10.3969/j.issn.1673-8640.2018.06.019

摘要/Abstract

摘要：

目的探讨黄芩素对多西他赛体外抗前列腺癌活性的影响和机制。方法将人前列腺癌细胞系LNCaP按不同处理方式分为对照组（空质粒转染处理）、黄芩素组（单用10 mmol/L黄芩素处理）、多西他赛组（单用1 nmol/L多西他赛处理）、多西他赛+黄芩素组（用1 nmol/L多西他赛和10 mmol/L黄芩素处理）和多西他赛+黄芩素+鼠双微体2（MDM2）质粒组（用MDM2质粒转染后再用1 nmol/L多西他赛和10 mmol/L黄芩素处理）。采用噻唑蓝（MTT）实验检测LNCaP细胞的活力,流式细胞术检测LNCaP细胞的凋亡情况,免疫印迹法检测LNCaP细胞中MDM2、蛋白53（p53）、佛波醇-12-肉豆酸酯-13-乙酰基诱导蛋白1（PMAIP1,又称Noxa）、受p53基因上调表达的凋亡调控基因（Puma）蛋白的表达水平以及细胞色素c的释放水平、半胱氨酸天冬氨酸特异性蛋白酶（Caspase）-9和Caspase-3的活化水平。结果多西他赛+黄芩素组对LNCaP细胞活力的抑制率和LNCaP细胞凋亡率均明显高于多西他赛+黄芩素+MDM2质粒组、多西他赛组、黄芩素组和对照组（P<0.05）。黄芩素组和多西他赛+黄芩素组LNCaP细胞MDM2 mRNA和MDM2蛋白水平均明显低于多西他赛+黄芩素+MDM2质粒组、多西他赛组和对照组（P<0.05）。多西他赛+黄芩素组p53、Puma和Noxa蛋白的表达水平、细胞色素c释放水平及Caspase-9、Caspase-3活化水平均明显高于多西他赛+黄芩素+MDM2质粒组、多西他赛组、黄芩素组和对照组（P<0.05）,而相对线粒体膜电位则明显低于其他各组（P<0.05）。结论黄芩素能通过MDM2/p53途径提高多西他赛的体外抗前列腺癌活性。

关键词: 黄芩素, 多西他赛, 前列腺癌, 鼠双微体2, 蛋白53, 凋亡

Abstract:

Objective To investigate the influence and mechanism of baicalein on in vitro anti-tumor activity of docetaxel for prostate cancer. Methods According to different processing modes,human prostate cancer LNCaP cells were classified into control group（empty plasmid transfection）,baicalein group（10 mmol/L baicalein）,docetaxel group（1 nmol/L docetaxel）,docetaxel+baicalein group（1 nmol/L docetaxel and 10 mmol/L baicalein） and docetaxel+baicalein+murine double minute 2（MDM2） plasmid group （1 nmol/L docetaxel and 10 mmol/L baicalein after MDM2 plasmid transfection）. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay was performed to determine the activity of LNCaP cells. Flow cytometry was used to determine the apoptosis of LNCaP cells. Western blotting analysis was performed to determine the expression levels of MDM2,protein 53（p53）,phorbol-12-myriistate-13-acetate-induced protein 1（PMAIP1,also known as Noxa） and p53 upregulated modulator of apoptosis（Puma） and the activation levels of cysteine-containing aspartate-specific protease（Caspase）-9 and Caspase-3 in LNCaP cells. Results The activity inhibitory rate and apoptotic rate of LNCaP cells in docetaxel+baicalein group were higher than those in docetaxel+baicalein+MDM2 plasmid group,docetaxel group,baicalein group and control group（P<0.05）. The levels of MDM2 mRNA and MDM2 protein in LNCaP cells of baicalein group and docetaxel+baicalein group were lower than those in docetaxel+baicalein+MDM2 plasmid group,docetaxel group and control group（P<0.05）. The p53,Puma and Noxa expression levels,the releasing levels of cytochrome c and the activation levels of Caspase-9 and Caspase-3 in docetaxel+baicalein group were higher than those in docetaxel+baicalein+MDM2 plasmid group,docetaxel group,baicalein group and control group（P<0.05）,while relatively mitochondrial membrane potential was lower than those in the other groups（P<0.05）. Conclusions Baicalein increases the in vitro anti-tumor activity of docetaxel for prostate cancer through MDM2/p53.

Key words: Baicalein, Docetaxel, Prostate cancer, Murine double minute 2, Protein 53, Apoptosis

中图分类号:

R446.1

应晓, 王育瑛, 王再红, 王振华. 黄芩素对多西他赛体外抗前列腺癌活性的影响[J]. 检验医学, 2018, 33(6): 556-562.

YING Xiao, WANG Yuying, WANG Zaihong, WANG Zhenhua. Influence of baicalein on in vitro anti-tumor activity of docetaxel for prostate cancer[J]. Laboratory Medicine, 2018, 33(6): 556-562.

图/表 7

表1 各组LNCaP细胞的活力抑制率和凋亡率比较（%,x±s ）

组别	细胞活力抑制率	凋亡率
对照组	0	1.5±0.2
黄芩素组	8.8±0.7	4.5±0.5
多西他赛组	19.8±1.6^*#△	10.6±0.8^*#△
多西他赛+黄芩素组	68.6±6.3^*#	38.1±3.3^*#
多西他赛+黄芩素+ MDM2质粒组	27.9±2.4^*#△	14.6±1.3^*#△

图1 各组LNCaP细胞的凋亡率比较注：（a）对照组;（b）黄芩素组;（c）多西他赛组;（d）多西他赛+黄芩素组;（e）多西他赛+黄芩素+MDM2质粒组

图2 黄芩素和多西他赛对LNCaP细胞MDM2 mRNA表达的影响注：与对照组比较,*P<0.05;与多西他赛组比较,#P<0.05;与多西他赛+黄芩素组比较,△P<0.05

图3 黄芩素和多西他赛对LNCaP细胞MDM2蛋白表达的影响注：（a）免疫印迹法结果;（b）各组MDM2蛋白相对表达量（灰度值）;与对照组比较,*P<0.05;与多西他赛组比较,#P<0.05;与多西他赛+黄芩素组比较,△P<0.05

图4 黄芩素和多西他赛对p53依赖的凋亡相关蛋白表达的影响

图5 多西他赛和黄芩素对LNCaP细胞线粒体膜电位的影响注：（a）各组红色荧光强度;（b）各组相对线粒体膜电位;与对照组比较,*P<0.05;与多西他赛组比较,#P<0.05;与多西他赛+黄芩素组比较,△P<0.05

图6 黄芩素和多西他赛对LNCaP细胞Caspase-9、Caspase-3活化及细胞色素c释放的影响

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