新型去泛素化酶抑制剂b-AP15诱导雄激素受体依赖和非依赖的前列腺癌细胞凋亡的研究

发布时间：2018-06-21 09:29

本文选题：b-AP15 + 去泛素化酶抑制剂　；参考：《广州医科大学》2017年硕士论文

【摘要】：背景与目的随着部分患者的病情进展为致命和不能治愈的转移性去势抵抗性前列腺癌(castrate-resistant prostate cancer,CRPC),前列腺癌(Prostate Cancer,PCa)仍然是男性癌症相关死亡的主要原因。泛素-蛋白酶体系统(Ubiquitin-Proteasome System,UPS)是细胞蛋白质高度特异和选择性降解的途径,通过调节底物泛素化和去泛素化之间的动态平衡来掌控大多数蛋白质的命运。近期研究表明去泛素化酶(Deubiquitinases,DUBs)已成为抗癌治疗的新靶点,它可以调节多种细胞过程,包括细胞周期控制,DNA损伤反应和修复,凋亡,染色质修饰以及各种信号转导途径。硼替佐米(bortezomib/Velcade)是20S蛋白酶体的抑制剂,已被美国FDA批准用于治疗临床多发性骨髓瘤和淋巴瘤等疾病并取得良好的疗效,证实了泛素蛋白酶体系统是肿瘤治疗的一个重要靶点,这也证明UPS组份是一种新型的有效抗肿瘤分子靶标,吸引了研究者对蛋白酶体系统进行更深入的研究和探索,从而寻求更有治疗前景的抗肿瘤药物。雄激素受体(Androgen Receptor,AR)与前列腺癌的发生发展密切相关,它是一种雄激素依赖的转录因子,属于核受体超家族。在前列腺癌中通常能观察到AR基因的扩增和突变,AR信号通路支配前列腺癌的存活,增殖和生长。我们课题组前期研究发现去泛素化酶USP14可以稳定并调节AR,抑制USP14可以促进AR泛素化降解从而抑制AR依赖前列腺癌细胞增殖,但对AR非依赖前列腺癌细胞没有影响。一个新型的小分子b-AP15已被鉴定为19S蛋白酶体USP14/UC HL5(DUBs)的抑制剂,能诱导多种人类肿瘤细胞系的增殖抑制和细胞凋亡。本研究我们拟进一步探讨同时抑制USP14和UCHL5是否对AR产生协同调控作用以及对AR依赖与非依赖前列腺癌的抗肿瘤效应。根据我们的实验结果发现b-AP15可以促进AR的泛素化降解,但这种作用主要是通过USP14的功能抑制,而与UCHL5的功能调控无关,并发现b-AP15通过诱导UPS的抑制、caspases活化、内质网应激与氧化应激的产生引起AR依赖和非依赖性前列腺癌细胞的增殖抑制和凋亡。实验方法1、细胞活力检测:采用MTS法和CCK-8法检测b-AP15对前列腺癌和正常前列腺上皮细胞活力的影响。2、克隆形成实验:克隆形成实验的处理方法同上。用光学显微镜计数大于10个细胞的克隆数。3、细胞凋亡情况检测:采用Annexin V-FITC/Propidium iodide(PI)标记死亡细胞用流式细胞仪分析细胞凋亡情况以及荧光显微镜观察并拍照记录细胞形态学变化。4、Si RNA转染:敲除USP14和UCHL5检测AR的表达。5、Co-IP联合Western bolt:检测相关蛋白的表达。6、线粒体膜电位完整性检测:采用罗丹明123染色法用流式细胞仪分析。7、活性氧检测:采用10μM DCFH-DA对样本进行染色,随后用流式细胞仪分析。8、实时荧光定量PCR:根据Takara试剂盒说明书操作,扩增结束后得到各检测基因的Cq值?9、裸鼠载体实验:SPF级裸鼠右侧腋皮下接种PC-3细胞,b-AP15腹腔注射给药,观察裸鼠体重和肿瘤体积变化。10、免疫组化:检测肿瘤组织中的蛋白表达情况。实验结果1、b-AP15对前列腺癌细胞增殖的影响不同处理浓度(0-5μM)的b-AP15作用于AR依赖前列腺癌细胞LNCa P,22Rv1,AR非依赖前列腺癌细胞PC-3,DU145和前列腺基质细胞WPMY-1 24,48,72 h后,都有不同程度抑制增殖作用,表明b-AP15对前列腺癌细胞和正常细胞的抑制作用没有明显的细胞选择性。此外,克隆形成的结果显示b-AP15抑制了LNCa P和PC-3细胞集落的形成。2、b-AP15诱导细胞周期停滞不同浓度的b-AP15(0,0.5,1,2μM)作用于LNCa P和PC-3细胞24h后进行细胞周期分析。结果表明随着b-AP15浓度的增加,两个细胞系的细胞周期都停滞在G0/G1期。Western blot结果显示,b-AP15可显著降低细胞周期蛋白cyclin D1,CDK6,CDK4,C DK2和p-Rb(G1期到S期的标记蛋白)的表达,并使抑制cyclin-CDK功能的抑癌基因p27表达增加。3、b-AP15诱导caspase依赖的细胞凋亡b-AP15作用于LNCa P和PC-3细胞后Annexin-V/PI阳性率显着增加,出现细胞凋亡的形态学变化。Western blot结果显示,两个细胞系活化的caspase-3,-8,-9都显著升高,PARP出现切割带。4、b-AP15诱导的细胞凋亡与线粒体功能障碍有关细胞凋亡主要通过内源性和外源性两种途径调控,而内源性途径又称为线粒体途径。通过罗丹明123染色法,流式细胞仪进行检测发现b-AP15能引起线粒体跨膜电位下降。对LNCa P和PC-3两种细胞系进行浓度和时间依赖处理,结果表明b-AP15引起抗凋亡蛋白(Bcl-2)的表达下调,而促凋亡蛋白(Bim,Bax,Noxa)则显著增加。此外,随着膜通透性改变,释放至细胞质中的促凋亡因子(细胞色素C)和细胞凋亡诱导因子(AIF)水平也显着增加。5、b-AP15作用后能诱导ROS产生,抗氧化剂N-乙酰半胱氨酸(NAC)可逆转b-AP15诱导的凋亡。6、b-AP15可明显抑制LNCa P和PC-3细胞的蛋白酶体功能和雄激素受体(AR)的表达,产生内质网应激。低浓度的b-AP15即能引起泛素化蛋白(Ub-prs)的聚集,蛋白酶体功能抑制早于细胞凋亡的产生,说明b-AP15是通过抑制UPS的功能诱导细胞凋亡。研究发现b-AP15能产生内质网应激以及抑制雄激素受体(AR)的表达,通过RT-PCR和免疫共沉淀实验检测发现AR的抑制是通过泛素化降解而不是基因转录水平。另外,用UCHL5的si RNA转染LNCa P细胞,Western blot检测AR的表达,结果显示敲除UCHL5并不能抑制AR,结合前期实验结果说明b-AP15对AR的抑制主要是通过抑制USP14发挥的作用。7、b-AP15对BALB/c裸鼠异种移植瘤的影响与对照组比较,结果显示b-AP15治疗组的肿瘤体积和肿瘤重量明显降低,两组之间裸鼠体重无统计学差异。免疫组化染色后发现b-AP15处理的肿瘤组织中,蛋白酶体泛素化底物蛋白聚集,p27以及活化的caspase-3表达增加。检测两组裸鼠的心肌,肝脏以及肾脏组织中肌酸激酶,谷丙转氨酶和肌酐的指标,结果表明b-AP15并对心,肝,肾功能没有影响,说明b-AP15的毒性作用是可控的,对前列腺癌具有广阔的治疗前景。结论1、b-AP15可以诱导体内外雄激素受体依赖和非依赖的前列腺癌细胞凋亡。
[Abstract]:Background and objective: with the progress of some patients with deadly and incurable metastatic castration resistant prostate cancer (castrate-resistant prostate, cancer, CRPC), prostate cancer (Prostate Cancer PCa) is still a major cause of cancer-related deaths in men. The ubiquitin proteasome system (Ubiquitin-Proteasome, System, UPS) is fine Methods cell protein highly specific and selective degradation, and go through the dynamic balance between the ubiquitination of most proteins to control the fate of ubiquitin to substrate regulation. Recent studies show that deubiquitinating enzymes (Deubiquitinases, DUBs) has become a new target for cancer therapy, it can regulate a variety of cellular processes, including cell cycle control And in response to DNA damage and repair, apoptosis, chromatin modification and various signal transduction pathways. Boron for Zomi (bortezomib/Velcade) is the inhibitor of the 20S proteasome, has been approved by the FDA for treatment of multiple myeloma and lymphoma and other diseases and has good curative effect, confirmed by the ubiquitin proteasome system in cancer treatment a An important target, it also proves that the UPS component is an effective antitumor new molecular target, attracted a further study and exploration of the proteasome system, so as to seek more promising anticancer drugs. The androgen receptor (Androgen Receptor, AR) is closely related with the occurrence and development of prostate cancer it is a kind of. Androgen dependent transcription factor that belongs to the nuclear receptor superfamily. Usually can be observed in AR gene amplification and mutation in prostate cancer, the survival of AR signal pathway governs prostate cancer, proliferation and growth. We find that the deubiquitinating enzyme USP14 can stabilize and adjust the AR of the previous studies, inhibition of USP14 can promote AR ubiquitination degradation from The inhibition of AR dependent proliferation of prostate cancer cells, but had no effect on AR non dependent prostate cancer cells. A novel small molecule b-AP15 has been identified as 19S USP14/UC HL5 (DUBs) proteasome inhibitor, inhibition of proliferation and apoptosis can be induced by a variety of human tumor cell lines. In this study, we intend to further investigate and restrain USP14 And UCHL5 are synergistic and regulation effects on AR dependent and non dependent prostate cancer antitumor effect of AR. According to the results of our experiments showed that b-AP15 can promote the degradation of AR, but this effect is mainly through inhibition of USP14 function, and functional regulation of UCHL5 control has nothing to do, and found that b-AP15 inhibition induced by UPS, The activation of caspases, resulting in endoplasmic reticulum stress and oxidative stress caused by AR dependent and non dependent prostate cancer cell proliferation inhibition and apoptosis. Methods 1, cell viability was detected by MTS method and CCK-8 method to detect the effect of b-AP15 on prostate cancer and normal prostate epithelial cell viability.2, clone formation experiment: Clone Formation experiment Processing methods above. The number of clones with.3 optical microscope count greater than 10 cells, the cell apoptosis detected by Annexin V-FITC/Propidium iodide (PI) markers of cell death by apoptosis analysis and fluorescence microscopy and flow cytometry and photographed cell morphological changes of.4, Si RNA and UCHL5 USP14 transfection: knockout The expression of.5 AR, to detect the expression of.6 Co-IP protein combined with Western bolt:, the integrity of the mitochondrial membrane potential detection with flow cytometry analysis of.7 123 by Luo Danming staining, ROS detection: using 10 M DCFH-DA staining of samples, followed by flow cytometry analysis of.8, real-time fluorescence quantitative PCR: Takara Kit according to said The book, after the end of each gene amplification detection value of Cq? 9, the carrier in nude mice experiment: SPF grade right flank of nude mice inoculated subcutaneously with PC-3 cells, b-AP15 intraperitoneal injection, observe the mice weight and tumor volume changes of.10, immunohistochemistry, tumor tissue in the detection of protein expression. Results 1. B-AP15 on the proliferation of prostate cancer cells Effect of different concentration (0-5 M) with b-AP15 AR on prostate cancer cell line LNCa P, 22Rv1, AR on prostate cancer cells PC-3, DU145 and WPMY-1 in prostatic stromal cells 24,48,72 after h, there are different degrees of inhibition of proliferation, showed that the inhibitory effect of b-AP15 on prostate cancer cells and normal cells is not obvious cell selection Optional. In addition, the results show that the formation of colony formation.2 b-AP15 inhibited LNCa P and PC-3 cell colony, b-AP15 induced cell cycle arrest in different concentrations of b-AP15 (0,0.5,1,2 M) on LNCa P and PC-3 24h cells after cell cycle analysis. The results show that with the increase of b-AP15 concentration, cell cycle two cell lines have been stuck in G0/G1.Western blot showed that b-AP15 significantly decreased the cell cycle protein cyclin D1, CDK6, CDK4, C DK2 and p-Rb (G1 to S marker protein) expression, and the expression of tumor suppressor gene p27 inhibited the function of cyclin-CDK increased.3, b-AP15 induced caspase dependent apoptosis in LNCa P b-AP15 PC-3 and Annexin-V/PI positive cells Of a significant increase in the percentage of morphological changes of.Western blot results of apoptosis showed that two cell lines and the activation of Caspase-3, -8, -9 increased significantly, PARP cutting with.4, b-AP15 induced apoptosis and mitochondrial dysfunction related to apoptosis mainly through two ways of endogenous and exogenous and endogenous regulation. Way Also known as the mitochondrial pathway. By 123 Luo Danming staining detection showed that b-AP15 can lead to the mitochondrial membrane potential decreased by flow cytometry. LNCa P and PC-3 two cell lines were concentration and time dependent processing, the results showed that b-AP15 induced anti apoptosis protein (Bcl-2) of the expression, and pro apoptotic protein (Bim, Bax, Noxa) significantly Increased. In addition, with the change of membrane permeability, the release of Pro apoptotic factors to the cytoplasm (cytochrome C) and apoptosis inducing factor (AIF) levels were also significantly increased.5, b-AP15 treatment can induce the production of ROS, antioxidant N- acetylcysteine (NAC) on apoptosis of.6 induced by b-AP15 reversal, b-AP15 inhibited LNCa P and PC-3 cells of eggs White body enzyme function and androgen receptor (AR) expression, resulting in endoplasmic reticulum stress. Low concentration of b-AP15 can cause the ubiquitinated protein (Ub-prs) aggregation, inhibition of proteasome function in early apoptosis, indicating that b-AP15 is the induction of apoptosis by inhibiting the function of UPS. It was found that b-AP15 could produce endoplasmic reticulum stress and inhibition of male Hormone receptor (AR) expression assays showed the inhibition of AR through ubiquitin mediated degradation rather than gene transcription by RT-PCR and immunoprecipitation. In addition, with the UCHL5 Si RNA P was transfected into LNCa cells, the expression of Western blot detected by AR, results showed that knockdown of UCHL5 did not inhibit AR, combined with the preliminary experimental results b-AP15 of AR suppression The main business is the role of.7 by inhibiting USP14 play, compared the effect of b-AP15 on BALB/c xenografts in nude mice and the control, the results showed that b-AP15 treatment group, the tumor volume and tumor weight decreased significantly, no significant difference between the two groups of nude mice. The tumor weight of immunohistochemical staining after the b-AP15 treatment, proteasome pan In the substrate protein aggregation, activation of p27 and increase the expression of Caspase-3. Two groups were detected in nude mice myocardium, liver and kidney tissues of creatine kinase, alanine aminotransferase and creatinine index, the results show that the b-AP15 and the heart, liver, kidney function has no effect, that the toxicity effect of b-AP15 is controllable, and has broad the treatment of prostate cancer Future. Conclusion 1 b-AP15 can induce in vivo androgen receptor dependent and non prostate cancer cell apoptosis.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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