联合应用MS-275和MDV3100对前列腺肿瘤干细胞样微球的作用  

Experimental study on the combination of MDV3100 and MS-275 for the treatment of prostate tumorosphere cells

作　　者：汤思洁 江佳佳[2] 程慧颖 郭佳倩 黄灿 李晓华 顾洛[1] Tang Sijie;Jiang Jiajia;Cheng Huiyin;Guo Jiaqian;Huang Can;Li Xiaohua;Gu Luo(Department of Physiology, NMU, Nanjing 211166;The Center for Pathology & Laboratory Medicine, the Affiliated Aoyang Hospital of Jiangsu University, Suzhou 215617;National Center for Gene Testing Technology Application & Demonstration, Center for Cancer Diagnostics & Clinical Genomics, Hefei King Med Diagnostics Co., Ltd, Hefei 230088, China)

机构地区：[1]南京医科大学生理学系,江苏南京211166 [2]江苏大学附属澳洋医院病理和检验医学中心,江苏苏州215600 [3]国家基因检测技术应用示范中心合肥金域医学检验所有限公司,安徽合肥230088

出　　处：《南京医科大学学报（自然科学版）》2019年第4期465-471,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81572741;81372319);吴阶平医学基金会临床科研专项资助基金(320.6750.14120);苏州市科技发展计划(SYSD2014009)

摘　　要：目的:探讨雄激素受体(androgen receptor,AR)拮抗剂和组蛋白去乙酰化酶(histone deacetylase,HDAC)抑制剂对前列腺肿瘤干细胞样微球的作用,并探讨其可能的分子机制。方法:采用无血清悬浮培养前列腺LNCaP和22RV1肿瘤细胞以获取干细胞样肿瘤微球细胞,然后单独和联合使用AR拮抗剂MDV3100和HDACⅠ类抑制剂MS-275分别处理2种微球细胞,镜下观察细胞形态,评估其克隆形成能力。应用实时荧光定量聚合酶链反应检测微球细胞中CD133的转录水平,Western blot检测DNA损伤标志物磷酸化H2A.X(p-H2A.X)的表达和细胞凋亡标志蛋白聚ADP-核糖聚合酶(PARP)特异性裂解片段的水平,同时检测Wnt信号通路关键分子β-catenin以及原癌基因c-Myc、cyclin D1蛋白表达情况。结果:MDV3100单独处理能明显降低肿瘤干细胞标志物CD133的表达;而MS-275单独或联合MDV3100处理均能明显抑制肿瘤干细胞样肿瘤微球的形成、降低肿瘤微球细胞CD133的表达,并促进肿瘤微球细胞产生PARP的特异性裂解,使p-H2A.X表达水平升高,同时可以明显降低β-catenin、c-Myc及cyclin D1的表达。结论:联合应用MS-275和MDV3100可以显著增强MDV3100的抗肿瘤活性,具体表现在抑制肿瘤干细胞样微球细胞生成及克隆形成、损伤细胞的DNA、诱导细胞凋亡等。该结果为临床联合应用HDACⅠ类抑制剂MS-275和AR拮抗剂MDV3100治疗前列腺肿瘤提供了一定的实验依据。Objective:To study the treatment effects of androgen receptor(AR)antagonist combined with class I histone deacetylase(HDAC)inhibitors on prostate tumorosphere,and to investigate the possible molecular mechanisms involved in the process. Methods:Prostate cancer LNCaP and 22 RV1 cells were cultured in serum-free suspension condition,and the obtained two tumorosphere stem-like cells were treated with AR antagonist MDV3100 with or without the presence of HDAC class I inhibitor MS-275 to study the morphology change and the ability of cell clone formation in monolayer culture condition. Then,quantitative real-time fluorescence polymerase chain reaction(qRT-PCR)was utilized to analyze cancer stem cell marker CD133 expression,and Western blot was used to detect the levels of DNA damage marker H2 A.X(p-H2 A.X),the cleavage of poly ADP-ribose polymerase(PARP),β-catenin,proto-oncogene c - Myc and cyclin D1. Results:Treatment with MDV3100 alone inhibited CD133 expression in tumorosphere cells.Combination treatment of MDV3100 with MS-275 reduced the number of tumorosphere,inhibited CD133 transcription,enhanced the level of both PARP cleavage and p-H2 A.X,decreased expression of β-catenin,c-Myc and cyclin D1. Conclusion:Compared with the treatment of MDV3100 alone,combination treatment of MDV3100 with MS-275 significantly inhibited cancer stem-like tumorosphere cell formation and promoted apoptotic cell death. The drugs-reduced over activation of Wnt/β-catenin/c-Myc/cyclin D1 pathway was possibly involved in the antitumor process. These results provide guidance for clinical application of MDV3100 and MS-275 in prostate cancer management of personal medicine.

关 键 词：前列腺肿瘤干细胞样微球 MDV3100 MS-275 

分 类 号：R737.25[医药卫生—肿瘤]