雄激素受体介导多西他赛抵抗性前列腺癌形成的机制研究

发布时间：2018-07-21 15:53

【摘要】：前列腺癌(Prostate Cancer,PCa)作为世界上男性第二大发病的肿瘤性疾病,已严重影响全球男性的身心健康与生活质量。前列腺癌转移发生率高,许多患者诊断时已伴有骨转移等。对于转移性激素敏感性前列腺癌(metastatic hormone-sensitive prostate cancer,mHSPC),治疗首选雄激素剥夺治疗(androgen deprivation therapy,ADT),其次有化疗、免疫治疗等。尽管ADT治疗有效率高达80%,但绝大多数患者在治疗后的一到两年内疾病会继续恶化,进展为转移性去势抵抗性前列腺癌(metastatic castration-resistant prostate cancer,mCRPC)。多西他赛(docetaxel)作为mCRPC的一线化疗药,因可显著延长患者的生存率,而被广泛应用。然而,几乎全部mCRPC患者在接受多西他赛化疗后均会出现抵抗性,从而形成多西他赛抵抗性前列腺癌(docetaxel resistant prostate cancer,DRPC)。深入研究DRPC的形成机制,对于患者的治疗至关重要。本实验室其他研究小组发现在DRPC细胞中,AR的表达明显增强,其AR信号通路相关基因的表达也发生了相应改变。选取本实验室已有的高表达AR的PC3-AR9细胞与AR阴性的PC3细胞,在不同梯度浓度的多西他赛作用下,PC3-AR9细胞的死亡率显著低于PC3细胞(P0.05)。从而证明,AR信号通路的激活参与DRPC的形成。PC3-AR9细胞与PC3细胞在不同浓度的多西他赛处理后,应用碘化丙啶(Propidium iodide,PI)与赫斯特荧光染料33258(Hoechst33258)行荧光染色,共聚焦显微镜下发现PC3细胞会发生坏死性凋亡(Necroptosis),且坏死性凋亡率随药物处理浓度增加而升高;而PC3-AR9细胞的坏死性凋亡率均显著低于PC3细胞(P0.0001)。PC3细胞内坏死复合体(Necrosome)核心蛋白混合谱系激酶结构区域样蛋白(Mixed Lineage Kinase Domain-like Protein,MLKL)的总表达量与磷酸化表达量,会随多西他赛处理浓度的增加而增加;而PC3-AR9细胞的MLKL的总表达量与磷酸化表达量则不随多西他赛处理浓度变化,且表达量均低于PC3细胞。总结数据得出,多西他赛可诱导前列腺癌细胞系发生坏死性凋亡,且AR可抑制多西他赛对坏死性凋亡的诱导作用。在TNF-α+Smac+Z-VAD-fmk(TSZ)联合诱导下建立坏死性凋亡细胞模型;在ShAR质粒转染下,建立敲减AR的LNCaP与C4-2细胞模型。在TSZ诱导下,PC3、C4-2-Sh AR与LNCaP-ShAR细胞的死亡率分别高于PC3-AR9(P0.01)、C4-2-Scramble(P0.01)与LNCa P-Scramble(P0.05)细胞。在TSZ诱导后应用PI与Hoechst33258行荧光染色,可见PC3细胞发生了坏死性凋亡,且其坏死率显著高于PC3-AR9细胞。同理验证,发现C4-2-ShAR与LNCaP-ShAR细胞也发生坏死性凋亡,且坏死率均分别高于C4-2-Scramble与LNCaP-Scramble细胞。表明AR的激活可抑制坏死性凋亡的发生,且随AR表达量的升高其对坏死性凋亡的抑制作用也在增强。后提取TSZ处理后的PC3与PC3-AR9细胞蛋白,Western Blot检测后证实在AR存在的PC3-AR9细胞中MLKL的总表达量与磷酸化表达量均明显低于PC3细胞。可得知,AR通过抑制MLKL的表达及磷酸化而抑制坏死性凋亡。后应用MLKL第86位半胱氨酸共价调节剂Necrosulfonamide(NSA)与TSZ共同处理,可逆转C4-2-ShAR与LNCaP-ShAR细胞的坏死性凋亡发生。TSZ联合NSA共同处理与TSZ单独处理相比,C4-2-Scramble与LNCaP-Scramble细胞的坏死性凋亡率无明显变化。同样处理条件下,荧光染色观察C42-shAR与C42-Scramble细胞的差异,发现NSA可抑制细胞坏死性凋亡的发生,且AR的抑制作用与NSA相似。这也间接证实,AR对坏死性凋亡的抑制作用是通过抑制MLKL的表达与磷酸化而实现的。选取42例原发性前列腺癌组织标本行免疫组化分析,基于AR表达含量的不同,分为低表达组(Low)与高表达组(High),发现随AR表达的增强,RIP3(R=-0.919,P0.01)与MLKL(R=-0.909,P0.01)的表达均减弱。从而体内证实,PCa中AR的激活抑制了坏死性凋亡的发生,且随AR表达量的升高其对坏死性凋亡的抑制作用也在增强。上述结果显示,多西他赛通过促进PCa细胞发生坏死性凋亡而对mCRPC起到治疗作用,但在治疗过程中,由于AR信号通路的重新建立,AR表达水平逆转性升高,抑制了MLKL的表达与磷酸化,转而抑制坏死性凋亡,且随AR表达量的增加其对坏死性凋亡的抑制作用也在增强,最终导致mCRPC对多西他赛出现抵抗性,发展为DRPC。对于mCRPC的患者,早期多西他赛化疗结合ADT治疗方案可能是新的治疗策略。
[Abstract]:Prostate Cancer (PCa), as the second most common oncology disease in the world, has seriously affected the physical and mental health and quality of life of men worldwide. The incidence of metastatic prostate cancer is high, and many patients have been diagnosed with bone metastases at the time of diagnosis. For metastatic hormone sensitive prostate cancer (metastatic hormone-sensitive prostat) E cancer, mHSPC), the first choice for androgen deprivation therapy (androgen deprivation therapy, ADT), followed by chemotherapy and immunotherapy. Although the effective rate of ADT therapy is up to 80%, the overwhelming majority of patients will continue to deteriorate within one to two years after treatment, progressing to metastatic castration resistant prostate cancer (metastatic castration-resistan). T prostate cancer, mCRPC). As a first-line chemotherapeutic agent for mCRPC, docetaxel is widely used because it can significantly prolong the survival rate of patients. However, almost all patients with mCRPC are resistant to chemotherapy after docetaxel, thus forming a poly (docetaxel resistant prostate cancer, D), D (cancer, D). RPC). In-depth study of the formation mechanism of DRPC is very important for the treatment of patients. Other research teams in this laboratory have found that the expression of AR is obviously enhanced in DRPC cells, and the expression of the AR signaling pathway related genes also changes. The selection of PC3-AR9 cells with high expression of AR in our laboratory and AR negative PC3 cells are different. With the gradient concentration of docetaxel, the death rate of PC3-AR9 cells was significantly lower than that of PC3 cells (P0.05). Thus, the activation of AR signaling pathway was involved in DRPC formation of.PC3-AR9 cells and PC3 cells at different concentrations of docetaxel, and the use of propidium iodide (Propidium iodide, PI) and Hearst fluorescent dye 33258 (Hoechst33258) The necrotizing apoptosis (Necroptosis) was found in PC3 cells under confocal microscopy, and the necrotic apoptosis rate increased with the increase of drug concentration, while the necrotic apoptosis rate of PC3-AR9 cells was significantly lower than that of the PC3 cell (P0.0001).PC3 cell necrosis complex (Necrosome) core protein mixed pedigree kinase structure. The total expression and phosphorylation of protein (Mixed Lineage Kinase Domain-like Protein, MLKL) increased with the concentration of docetaxel, while the total expression and phosphorylation of MLKL in PC3-AR9 cells did not change with the concentration of docetaxel, and the expression was lower than that of PC3 cells. The cell line can induce necrotic apoptosis in the prostate cancer cell line, and AR can inhibit the inducing effect of docetaxel on necrotic apoptosis. The necrotizing apoptotic cell model is established under the joint induction of TNF- alpha +Smac+Z-VAD-fmk (TSZ); the LNCaP and C4-2 cell model of the knockout AR is established under the ShAR plasmid. PC3, C4-2-Sh AR and PC3 under the induction of TSZ The mortality of AR cells was higher than that of PC3-AR9 (P0.01), C4-2-Scramble (P0.01) and LNCa P-Scramble (P0.05) cells. The necrotizing apoptosis was observed in PI and Hoechst33258 line after TSZ induction, and the necrosis rate of the PC3 cells was significantly higher than that of the cells. Apoptosis and necrosis rate were higher than that of C4-2-Scramble and LNCaP-Scramble cells respectively. The results showed that the activation of AR inhibited the occurrence of necrotic apoptosis, and the inhibitory effect on necrotic apoptosis was also enhanced with the increase of AR expression. Then, PC3 and PC3-AR9 cell protein after TSZ treatment were extracted, and Western Blot detected PC3-AR9 thin AR after Western Blot detection. The total expression and phosphorylation of MLKL in the cell were significantly lower than that of PC3 cells. It was found that AR inhibited the necrotic apoptosis by inhibiting the expression of MLKL and phosphorylation. Then the MLKL eighty-sixth cysteine covalent modifier Necrosulfonamide (NSA) was treated with TSZ, which could reverse the necrotic apoptosis of C4-2-ShAR and LNCaP-ShAR cells. Compared with the combined treatment of NSA and TSZ alone, the necrotic apoptosis rate of C4-2-Scramble and LNCaP-Scramble cells was not significantly changed. Under the same treatment conditions, the difference between C42-shAR and C42-Scramble cells was observed by fluorescence staining. It was found that NSA inhibited the occurrence of necrotic apoptosis of cells, and the inhibition of AR was similar to NSA, which was also indirectly confirmed. The inhibitory effect of AR on necrotic apoptosis was achieved by inhibiting the expression and phosphorylation of MLKL. 42 cases of primary prostate cancer specimens were selected by immunohistochemical analysis. Based on the difference of AR expression, they were divided into low expression group (Low) and high expression group (High). The enhancement of AR expression, RIP3 (R=-0.919, P0.01) and MLKL (R=-0.909, P0.01) were found. The expression of AR was weakened in vivo. In vivo, the activation of PCa inhibited the occurrence of necrotic apoptosis and increased the inhibitory effect on necrotic apoptosis with the increase of AR expression. The results showed that docetaxel could play a therapeutic role in mCRPC by promoting necrotic apoptosis in PCa cells, but in the course of treatment, because AR The reestablishment of the signal pathway, the elevation of AR expression level, the inhibition of the expression and phosphorylation of MLKL, and the inhibition of necrotic apoptosis, and the increase of the inhibitory effect on necrotic apoptosis with the increase of AR expression, eventually lead to the resistance of mCRPC to docetaxel, and the development of DRPC. to mCRPC patients and early docetaxel. Chemotherapy combined with ADT regimen may be a new therapeutic strategy.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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