帕比司他逆转前列腺癌细胞hepaCAM基因表达机制研究

目的:研究帕比司他(Panobinostat)逆转抑癌基因肝细胞粘附分子(hepatocyte cell adhesion molecule,hepaCAM)表达,协同hepaCAM抑制前列腺癌(prostate cancer,PCa)细胞生长。方法:不同浓度帕比司他作用于体外培养的PC3细胞,首先采用MTT法检测帕比司他对细胞增殖的影响,RT-PCR和Western blot法检测hepaCAM、组蛋白去乙酰化酶(histone deacetylases, HDACs)以及乙酰化组蛋白H3 赖氨酸9(Ac-H3K9)的表达变化。接着用不同因素处理细胞,MTT法检测细胞增殖活性,流式细胞术检测细胞周期改变,RT-PCR和Western blot法检测细胞周期调节因子cyclinD1 和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的基因表达。结果:帕比司他抑制PC3细胞生长与作用浓度增加和作用时间呈正相关, hepaCAM mRNA、蛋白以及细胞核中Ac-H3K9表达随帕比司他浓度升高而增高,HDAC1、 HDAC3、 HDAC4 mRNA和蛋白的表达随浓度升高而降低;单独过表达hepaCAM腺病毒和单独使用帕比司他组PC3细胞生长明显受到抑制且随作用时间延长抑制率增加,两者联用更为明显,差异有统计学意义(P < 0.05);与单独hepaCAM 腺病毒组和帕比司他组相比,两者联用S期细胞比例明显增高,差异有统计学意义(P < 0.05),且可进一步下调cyclinD1、 PCNA mRNA和蛋白的表达,差异有统计学意义(P < 0.05)。结论:帕比司他可通过抑制HDACs活性,加强PCa PC3细胞组蛋白H3 N-端的赖氨酸残基乙酰化,逆转hepaCAM表达;hepaCAM腺病毒和帕比司他联用可通过阻滞细胞周期于S期协同抑制PC3 细胞生长,作用机制可能与下调cyclinD1 和PCNA 的表达有关。这对揭示抑癌基因hepaCAM在肿瘤缺失的原因及为将帕比司他应用于临床治疗肿瘤提供了科学支持。

The Mechanisms of Panobinostat Reversing HepaCAM Gene Expression in Prostate Cancer

Objective: To study the reversing effect of Panobinostat on the expression of tumor suppressor gene hepatocyte cell adhesion molecule (hepaCAM) and its synergistic inhibitory effect with hepaCAM on the growth of prostate cancer (PCa) cells. Methods: PC3 cells cultured in vitro were treated with Panobinostat at different concentrations and the effect of Panobinostat on cell proliferation was detected by MTT assay, followed by the expression changes of hepaCAM, histone deacetylase (HDACs), and the acetylation of lys9 of histone H3 (Ac-H3K9) detected by RT-PCR and Western blot. Then after the cells were treated with different factors, cell proliferation activity was detected by MTT assay, cell cycle change was detected by flow cytometry, and gene expressions of cell cycle regulating factors CyclinD1 and proliferating cell nuclear antigen (PCNA) were detected by RT-PCR and Western blot. Results: The inhibitory effect of Panobinostat on the growth of PC3 cells was positively correlated to its concentration and duration. The expression levels of hepaCAM mRNA, its protein, and intranuclear Ac-H3K9 protein were increased, while that of both mRNA and protein of HDAC1, HDAC3, and HDAC4 were decreased when the concentration of Panobinostat increased; Cell growth was significantly inhibited in both the adenovirus-hepaCAM adenovirus alone group and the Panobinostat alone group, with an inhibition rate increased along with the prolonged action time. This inhibition became even particularly noticeable when the two were combined, with a difference of statistical significance (P < 0.05); the percentage of cells in S phase was significantly higher by the combined treatment compared with ad-hepaCAM alone and Panobinostat alone, respectively, with statistically significant differences (P < 0.05). This combination could further downregulate the mRNA and protein expressions of cyclinD1 and PCNA, and the difference was statistically significant (P < 0.05).Conclusion: Panobinostat may enhance the acetylation of lys9 of histone 3 and reverse the hepaCAM expression through its inhibitory effect on HDACs activity in PCa PC3 cells; hepaCAM-expressed adenovirus combined with Panobinostat may synergistically inhibit the growth of PC3 cells, via a potential mechanism associated with the down-regulation of the expression of CyclinD1 and PCNA. This reveals the reasons of hepaCAM deficiency in tumors and provides scientific support for the application of Panobinostat in clinical therapy.