联合应用MS-275和MDV3100对前列腺肿瘤干细胞样微球的作用
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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.
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.
引文
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[12]江佳佳,印金徐,郭佳倩,等.曲古抑菌素A和多西他赛联合应用治疗前列腺肿瘤的实验研究[J].中华临床实验室管理电子杂志,2018,6(3):145-152
[13]Bernardo MM,Kaplun A,Dzinic SH,et al. Maspin expres-sion in prostate tumor cells averts stemness and stratifies drug sensitivity[J]. Cancer Res,2015,75(18):3970-3979
[14]Li X,Kaplun A,Lonardo F,et al. HDAC1 inhibition by maspin abrogates epigenetic silencing of glutathione S-transferase pi in prostate carcinoma cells[J]. Mol Cancer Res,2011,9(6):733-745
[15] Bai P. Biology of poly(ADP-ribose)polymerases:The fac-totums of cell maintenance[J]. Mol Cell,2015,58(6):947-958
[16] Sharma A,Singh K,Almasan A,et al. Histone H2AX phosphorylation:a marker for DNA damage[J]. Methods Mol Biol,2012,920:613-626
[17]Liu T,Chi H,Chen J,et al. Curcumin suppresses prolifer-ation and in vitro invasion of human prostate cancer stem cells by ceRNA effect of miR-145 and lncRNA-ROR[J].Gene,2017,631:29-38
[18]da Silva-Diz V,Lorenzo-Sanz L,Bernat-Peguera A,et al.Cancer cell plasticity:Impact on tumor progression and therapy response[J]. Semin Cancer Biol,2018,53:48-58
[2] Loriot Y,Zoubeidi A,Gleave ME. Targeted therapies in metastatic castration-resistant prostate cancer:beyond the androgen receptor[J]. Urol Clin North Am,2012,39(4):517-531
[3] Siegel RL,Miller KD,Jemal A. Cancer Statistics,2017[J]. CA Cancer J Clin,2017,67(1):7-30
[4] Korpal M,Korn JM,Gao X,et al. An F876L mutation in androgen receptor confers genetic and phenotypic resis-tance to MDV3100(enzalutamide)[J]. Cancer Discov,2013,3(9):1030-1043
[5] Roos WP,Krumm A. The multifaceted influence of his-tone deacetylases on DNA damage signalling and DNA re-pair[J]. Nucleic Acids Res,2016,44(21):10017-10030
[6] Greer CB,Tanaka Y,Kim YJ,et al. Histone deacetylases positively regulate transcription through the elongation machinery[J]. Cell Rep,2015,13(7):1444-1455
[7] Kim YJ,Greer CB,Cecchini KR,et al. HDAC inhibitors induce transcriptional repression of high copy number genes in breast cancer through elongation blockade[J].Oncogene,2013,32(23):2828-2835
[8] Bose P,Dai Y,Grant S. Histone deacetylase inhibitor(HDACI)mechanisms of action:emerging insights[J].Pharmacol Ther,2014,143(3):323-336
[9] Kurz EU,Wilson SE,Leader KB,et al. The histone deacet-ylase inhibitor sodium butyrate induces DNA topoisomer-ase II alpha expression and confers hypersensitivity to eto-poside in human leukemic cell lines[J]. Mol Cancer Ther,2001,1(2):121-131
[10] Nishioka C,Ikezoe T,Yang J,et al. Blockade of mTOR signaling potentiates the ability of histone deacetylase in-hibitor to induce growth arrest and differentiation of acute myelogenous leukemia cells[J]. Leukemia,2008,22(12):2159-2168
[11] Larsson C. Epigenetic aspects on therapy development for gastroenteropancreatic neuroendocrine tumors[J]. Neuro-endocrinology,2013,97(1):19-25
[12]江佳佳,印金徐,郭佳倩,等.曲古抑菌素A和多西他赛联合应用治疗前列腺肿瘤的实验研究[J].中华临床实验室管理电子杂志,2018,6(3):145-152
[13]Bernardo MM,Kaplun A,Dzinic SH,et al. Maspin expres-sion in prostate tumor cells averts stemness and stratifies drug sensitivity[J]. Cancer Res,2015,75(18):3970-3979
[14]Li X,Kaplun A,Lonardo F,et al. HDAC1 inhibition by maspin abrogates epigenetic silencing of glutathione S-transferase pi in prostate carcinoma cells[J]. Mol Cancer Res,2011,9(6):733-745
[15] Bai P. Biology of poly(ADP-ribose)polymerases:The fac-totums of cell maintenance[J]. Mol Cell,2015,58(6):947-958
[16] Sharma A,Singh K,Almasan A,et al. Histone H2AX phosphorylation:a marker for DNA damage[J]. Methods Mol Biol,2012,920:613-626
[17]Liu T,Chi H,Chen J,et al. Curcumin suppresses prolifer-ation and in vitro invasion of human prostate cancer stem cells by ceRNA effect of miR-145 and lncRNA-ROR[J].Gene,2017,631:29-38
[18]da Silva-Diz V,Lorenzo-Sanz L,Bernat-Peguera A,et al.Cancer cell plasticity:Impact on tumor progression and therapy response[J]. Semin Cancer Biol,2018,53:48-58