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 progression of some patients to fatal and incurable metastatic metastatic castration resistant prostate cancer (castrate-resistant prostate cancer, CRPC), and prostate cancer (Prostate Cancer, PCa) remains the main cause of cancer related deaths in men. The ubiquitin proteasome system (Ubiquitin-Proteasome System, UPS) is fine. Deubiquitinases (DUBs) has become a new target for anti-cancer therapy, which can regulate a variety of cellular processes, including cell cycle control. DNA damage reaction and repair, apoptosis, chromatin modification and various signal transduction pathways. Borteg Zomi (bortezomib/Velcade) is an inhibitor of 20S proteasome. It has been approved by American FDA for the treatment of clinical multiple myeloma and lymphoma, and has achieved good therapeutic effect. It has been proved that the ubiquitin proteasome system is a tumor treatment. The important target is that the UPS component is a new type of effective anti-tumor molecular target, which attracts researchers to study and explore the proteasome system more deeply, so as to seek a more promising antitumor drug. The Androgen Receptor (AR) is closely related to the development of prostate cancer. It is a kind of prostatic cancer. Androgen dependent transcription factors, belonging to the nuclear receptor superfamily. In prostate cancer, the amplification and mutation of the AR gene are usually observed. The AR signaling pathway dominates the survival, proliferation, and growth of the prostate cancer. Our previous study found that the deubiquitinase USP14 could stabilize and regulate the node AR, and the inhibition of USP14 could promote AR ubiquitination degradation from the prostate cancer. The inhibition of AR dependence on the proliferation of prostate cancer cells has no effect on AR non dependent prostate cancer cells. A new small molecule b-AP15 has been identified as an inhibitor of 19S proteasome USP14/UC HL5 (DUBs), which can induce proliferation inhibition and cell apoptosis in various human tumor cell lines. This study intends to further explore the simultaneous inhibition of USP14. Whether or not UCHL5 has a synergistic effect on AR and the antitumor effect on AR dependent and non dependent prostate cancer. According to our experimental results, it is found that b-AP15 can promote the ubiquitination of AR, but this effect is mainly through the function inhibition of USP14 and not related to the function control of UCHL5, and it is found that b-AP15 can inhibit UPS by inducing the inhibition of UPS, Caspases activation, endoplasmic reticulum stress and oxidative stress induced proliferation inhibition and apoptosis of AR dependent and non dependent prostate cancer cells. Experimental methods 1, cell viability detection: the effects of b-AP15 on the activity of prostate cancer and normal prostate epithelial cells by MTS and CCK-8 methods,.2, cloning and formation experiments: cloning and formation experiments The method of treatment was same. The number of clones more than 10 cells was counted by the optical microscope.3, the apoptosis was detected. The apoptotic cells were labeled with Annexin V-FITC/Propidium iodide (PI) and the cell apoptosis was analyzed by flow cytometry and the morphological changes of.4 were recorded by the fluorescence microscope and Si RNA transfected: USP14 and UCHL5 were knocked out. Detection of AR expression.5, Co-IP combined with Western bolt: to detect the expression of related protein.6, mitochondrial membrane potential integrity detection: using Luo Danming 123 staining method to analyze.7 with flow cytometer, reactive oxygen species detection: 10 u M DCFH-DA to stain samples, and then flow cytometry analysis.8, real time fluorescent quantitative PCR: according to Takara kit said After the operation, the Cq value of each detection gene was obtained after the end of amplification? 9, the carrier experiment in nude mice: PC-3 cells were inoculated subcutaneously in the right axilla of the SPF nude mice and b-AP15 was injected intraperitoneally to observe the changes of body weight and tumor volume of nude mice.10, immunohistochemistry: detection of the protein table in the tumor tissue. Experimental results were 1, b-AP15 proliferation of prostate cancer cells. The effect of b-AP15 on AR dependent prostate cancer cells LNCa P, 22Rv1, AR is not dependent on the prostate cancer cell PC-3, DU145 and the WPMY-1 24,48,72 h of the prostatic stromal cells, AR dependent on the inhibition of the proliferation of prostate cancer cells and normal cells. It shows that there is no obvious cell selection for the inhibition of prostate cancer cells and normal cells. In addition, the results of clone formation showed that b-AP15 inhibited the formation of LNCa P and PC-3 cell colony formation.2, and b-AP15 induced cell cycle Stagnation with different concentrations of b-AP15 (0,0.5,1,2 u M) acting on the cell cycle analysis after LNCa P and PC-3 cells. The results showed that the cell cycle of the two cell lines stagnated with the increase of concentration. The results of G0/G1 phase.Western blot show that b-AP15 can significantly reduce the expression of cell cycle protein cyclin D1, CDK6, CDK4, C DK2 and p-Rb (G1 phase to the marker protein), and increase the expression of the tumor suppressor gene that inhibits the function of the cell. .Western blot results showed that the activation of Caspase-3, -8, -9 in two cell lines increased significantly, and PARP appeared in the cleavage band.4. B-AP15 induced apoptosis related to mitochondrial dysfunction mainly regulated by endogenous and exogenous pathways, while endogenous pathways were regulated by endogenous and exogenous pathways. It was also called the mitochondrial pathway. Through the Luo Danming 123 staining method, the flow cytometry detected that b-AP15 could cause the mitochondrial transmembrane potential decline. The concentration and time dependence of the LNCa P and PC-3 cell lines were treated with concentration and time dependence. The results showed that the b-AP15 induced the down regulation of the anti apoptotic protein (Bcl-2), while the apoptotic protein (Bim, Bax, Noxa) was significant. In addition, with the change of membrane permeability, the level of apoptosis inducing factor (cytochrome C) and apoptosis inducible factor (AIF) released into cytoplasm also increased.5, and ROS produced after b-AP15 action. The antioxidant N- acetylcysteine (NAC) could reverse b-AP15 induced apoptosis.6. B-AP15 could obviously inhibit LNCa P and eggs. The expression of white enzyme body function and androgen receptor (AR) produces endoplasmic reticulum stress. Low concentration of b-AP15 can cause the aggregation of ubiquitin protein (Ub-prs). The inhibition of proteasome function is earlier than the production of cell apoptosis. It shows that b-AP15 can induce apoptosis by inhibiting the function of UPS. The study found that b-AP15 can produce endoplasmic reticulum stress and inhibit male. The expression of hormone receptor (AR) was detected by RT-PCR and immunoprecipitation assay. The inhibition of AR was mediated by ubiquitination and not gene transcription. In addition, LNCa P cells were transfected with UCHL5 Si RNA. Western blot was used to detect AR expression. The effect of the system was mainly by inhibiting the effect of USP14 on.7. The effect of b-AP15 on xenotransplantation in BALB/c nude mice was compared with that of the control group. The results showed that the tumor volume and tumor weight of the b-AP15 group decreased obviously, and there was no statistical difference between the two groups of nude mice. The proteasome was ubiquitous in the b-AP15 treated tumor tissues after immunohistochemical staining. The aggregation of vegetated substrate protein, p27 and activated caspase-3 expression increased. The indexes of creatine kinase, alanine transaminase and creatinine in two groups of nude mice were detected. The results showed that b-AP15 had no effect on the heart, liver and kidney function, indicating that the toxic effect of b-AP15 was controllable and has a broad treatment for prostate cancer. Conclusion. 1, b-AP15 can induce androgen receptor dependent and independent prostate cancer cell apoptosis in vivo and in vitro.
【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

【相似文献】