通过ROS和线粒体凋亡途径抑制HO-1增强多西他赛诱导PC-3细胞凋亡
发布时间：2018-08-30 17:16
【摘要】：目的：探索HO-1在激素非依赖性前列腺癌化疗抵抗中的作用及其分子机制。 在西方发达国家，前列腺癌是最常见的肿瘤，居癌死因的第二位。几乎所有的前列腺癌患者接受雄激素去势治疗后18-24个月均会发展为激素非依赖性前列腺癌，此时转移率很高。虽然使用了各种抗癌药物以缓解疼痛，但总体生存率没有得到改善，并且对标准的放化疗产生了耐药性。化疗抵抗是影响晚期前列腺癌患者生存和生活质量的主要障碍。因此，探索激素非依赖性前列腺癌化疗抵抗中分子靶点和发展全新的治疗策略是至关重要的。 HO-1是一种细胞保护性酶类，是体内抵抗氧化应激主要的防御机制。其代谢产物有二价铁，一氧化碳，胆红素。多种刺激均可诱导HO-1生成，如低氧，重金属，化疗药物以及氧化应激。HO-1具有抗炎，抗氧化，抗凋亡等生理作用。最近，一些研究结果证实相比周围正常组织，肿瘤组织中的内源性HO-1表达增多，如鳞癌，黑色素瘤，恶性胶质瘤，淋巴瘤等。同时，用siRNA敲低HO-1后可抑制膀胱癌的生长。上述结果均说明HO-1是促肿瘤因子。更为重要的是，HO-1可抑制化疗药物诱导的凋亡，，特异性抑制HO-1基因或抑制HO-1活性可增强肿瘤细胞对化疗药物的敏感性。在化疗药物引发的氧化应激环境下，肿瘤细胞本身可能上调抗氧化酶系统，增强抗凋亡能力以抵御抗癌药物引起的氧化应激，这可能是产生化疗抵抗的主要原因之一，但尚缺乏有力的证据。作为强大的抗氧化和抗凋亡酶，抑制HO-1自理论上可以增强化疗药物敏感性。然而，抑制HO-1增强化疗药物的敏感性的确切机制尚不完全明了。 因此本研究在以往实验基础之上，以人前列腺癌标本和PC-3细胞为实验对象，通过观察人前列腺癌标本探索HO-1是否与临床前列腺癌病理特征间存在统计学关系及其与凋亡相关蛋白BCL-2和Bax间的关系;通过观察HO-1是否影响BCL-2、Bax、caspase-3、caspase-9及细胞色素C的表达，并分析其表达与细胞凋亡的关系，阐明HO-1在激素非依赖性前列腺癌化疗抵抗中的作用及其分子机制，为临床选择有效治疗方法提供实验依据。 方法：常规培养人激素非依赖性前列腺癌细胞（PC-3细胞），分为正常组、Znpp组、Hemin组、多西他赛组、多西他赛+Znpp组、多西他赛+Hemin组。 1免疫组化染色分析HO-1、BCL-2、Bax蛋白的表达。 2MTT法分析各实验组细胞存活率。 3DCFH-DA法检测活性氧物质（ROS）含量。 4TUNEL法检测细胞凋亡率。 5Western blot法检测HO-1、Bcl-2、Bax、caspase-3、caspase-9及细胞色素C蛋白含量。 结果： 1HO-1免疫组化表达与前列腺癌临床病理特点间的关系。 HO-1表达量与临床分期，术前PSA，精囊侵犯，Gleason分级呈正相关。与患者年龄，淋巴结转移，尿道侵犯无关。 2在人前列腺癌标本中HO-1与凋亡相关蛋白Bcl-2和Bax免疫组化表达中的关系。 Bcl-2表达随HO-1表达增高而增高，Bax则呈相反的趋势。 3MTT法分析Hemin和Znpp对多西他赛诱导的PC-3细胞毒性的作用。 4抑制HO-1增强多西他赛诱导的PC-3细胞凋亡。 5多西他赛诱导ROS生成以及抑制HO-1可增强多西他赛诱导ROS生成。 6多西他赛诱导的ROS上调Bax，下调Bcl-2表达，而HO-1则可逆转。 7抑制HO-1增强多西他赛诱导的线粒体凋亡途径。 结论：HO-1在前列腺癌中呈高表达趋势，其可能是通过上调Bcl-2表达和下调Bax表达,抑制细胞凋亡而促进前列腺癌的进展。抑制HO-1可增强多西他赛诱导的凋亡，其中潜在的分子机制为依赖于ROS诱导的线粒体凋亡途径。
[Abstract]:Objective: To explore the role and molecular mechanism of HO-1 in the chemoresistance of hormone independent prostate cancer.
Prostate cancer is the most common cancer in western developed countries, ranking second in cancer deaths. Almost all prostate cancer patients develop hormone-independent prostate cancer 18-24 months after androgen castration, with a high metastasis rate. Although various anticancer drugs are used to relieve pain, the overall survival rate is not achieved. Chemotherapy resistance is a major obstacle to the survival and quality of life of patients with advanced prostate cancer. Therefore, it is essential to explore molecular targets and develop novel therapeutic strategies for hormone-independent prostate cancer chemotherapy resistance.
HO-1 is a kind of cell-protective enzymes, which is the main defense mechanism against oxidative stress in vivo. Its metabolites include divalent iron, carbon monoxide, bilirubin. A variety of stimuli can induce HO-1 production, such as hypoxia, heavy metals, chemotherapy drugs and oxidative stress. HO-1 has anti-inflammatory, anti-oxidative, anti-apoptotic and other physiological effects. Recently, some research results. It was confirmed that HO-1 expression was increased in tumor tissues, such as squamous cell carcinoma, melanoma, malignant glioma, lymphoma, and so on. Meanwhile, HO-1 knockdown by siRNA inhibited the growth of bladder cancer. Producing HO-1 gene or inhibiting the activity of HO-1 can enhance the sensitivity of tumor cells to chemotherapeutics. Under the oxidative stress environment induced by chemotherapeutics, tumor cells themselves may up-regulate the antioxidant enzyme system and enhance their anti-apoptosis ability to resist the oxidative stress induced by anticancer drugs. This may be one of the main reasons for chemotherapeutics resistance. Inhibiting HO-1, as a powerful antioxidant and antiapoptotic enzyme, can theoretically enhance chemosensitivity. However, the exact mechanism of inhibiting HO-1 to enhance chemosensitivity is not fully understood.
Therefore, based on previous experiments, this study explored whether HO-1 had statistical relationship with clinicopathological features of prostate cancer and apoptosis-related proteins BCL-2 and Bax by observing human prostate cancer specimens and PC-3 cells, and whether HO-1 affected BCL-2, Bax, caspase-Bax. 3. The expression of caspase-9 and cytochrome C, and the relationship between the expression of HO-1 and apoptosis were analyzed to elucidate the role of HO-1 in hormone-independent prostate cancer chemotherapeutic resistance and its molecular mechanism.
Methods: Human hormone-independent prostate cancer cells (PC-3) were cultured and divided into normal group, Znpp group, hemin group, docetaxel group, docetaxel + Znpp group and docetaxel + hemin group.
1 immunohistochemical staining was used to analyze the expression of HO-1, BCL-2 and Bax protein.
Cell viability was analyzed by 2MTT.
The content of reactive oxygen species (ROS) was detected by 3DCFH-DA.
Apoptosis rate was detected by 4TUNEL.
The contents of HO-1, Bcl-2, Bax, Caspase-3, caspase-9 and cytochrome C protein were detected by 5Western blot.
Result:
The relationship between the expression of 1HO-1 and clinicopathological features of prostate cancer.
HO-1 expression was positively correlated with clinical stage, preoperative PSA, seminal vesicle invasion and Gleason grade, but not with age, lymph node metastasis and urethral invasion.
2 the relationship between HO-1 and apoptosis related protein Bcl-2 and Bax expression in human prostate cancer specimens.
The expression of Bcl-2 increased with the increase of HO-1 expression, while Bax showed an opposite trend.
3MTT assay was used to analyze the effects of Hemin and Znpp on docetaxel induced PC-3 cell toxicity.
4 inhibition of HO-1 enhances docetaxel induced apoptosis in PC-3 cells.
5 docetaxel induced ROS production and inhibition of HO-1 could enhance docetaxel induced ROS production.
6 docetaxel induced ROS up-regulated Bax, down regulated Bcl-2 expression, while HO-1 reversed.
7 inhibition of HO-1 enhances docetaxel induced mitochondrial apoptosis pathway.
CONCLUSION: HO-1 is highly expressed in prostate cancer, which may promote the progression of prostate cancer by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax. Inhibition of HO-1 may enhance the apoptosis induced by docetaxel. The underlying molecular mechanism is ROS-dependent mitochondrial apoptosis pathway.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
[Abstract]:Objective: To explore the role and molecular mechanism of HO-1 in the chemoresistance of hormone independent prostate cancer.
Prostate cancer is the most common cancer in western developed countries, ranking second in cancer deaths. Almost all prostate cancer patients develop hormone-independent prostate cancer 18-24 months after androgen castration, with a high metastasis rate. Although various anticancer drugs are used to relieve pain, the overall survival rate is not achieved. Chemotherapy resistance is a major obstacle to the survival and quality of life of patients with advanced prostate cancer. Therefore, it is essential to explore molecular targets and develop novel therapeutic strategies for hormone-independent prostate cancer chemotherapy resistance.
HO-1 is a kind of cell-protective enzymes, which is the main defense mechanism against oxidative stress in vivo. Its metabolites include divalent iron, carbon monoxide, bilirubin. A variety of stimuli can induce HO-1 production, such as hypoxia, heavy metals, chemotherapy drugs and oxidative stress. HO-1 has anti-inflammatory, anti-oxidative, anti-apoptotic and other physiological effects. Recently, some research results. It was confirmed that HO-1 expression was increased in tumor tissues, such as squamous cell carcinoma, melanoma, malignant glioma, lymphoma, and so on. Meanwhile, HO-1 knockdown by siRNA inhibited the growth of bladder cancer. Producing HO-1 gene or inhibiting the activity of HO-1 can enhance the sensitivity of tumor cells to chemotherapeutics. Under the oxidative stress environment induced by chemotherapeutics, tumor cells themselves may up-regulate the antioxidant enzyme system and enhance their anti-apoptosis ability to resist the oxidative stress induced by anticancer drugs. This may be one of the main reasons for chemotherapeutics resistance. Inhibiting HO-1, as a powerful antioxidant and antiapoptotic enzyme, can theoretically enhance chemosensitivity. However, the exact mechanism of inhibiting HO-1 to enhance chemosensitivity is not fully understood.
Therefore, based on previous experiments, this study explored whether HO-1 had statistical relationship with clinicopathological features of prostate cancer and apoptosis-related proteins BCL-2 and Bax by observing human prostate cancer specimens and PC-3 cells, and whether HO-1 affected BCL-2, Bax, caspase-Bax. 3. The expression of caspase-9 and cytochrome C, and the relationship between the expression of HO-1 and apoptosis were analyzed to elucidate the role of HO-1 in hormone-independent prostate cancer chemotherapeutic resistance and its molecular mechanism.
Methods: Human hormone-independent prostate cancer cells (PC-3) were cultured and divided into normal group, Znpp group, hemin group, docetaxel group, docetaxel + Znpp group and docetaxel + hemin group.
1 immunohistochemical staining was used to analyze the expression of HO-1, BCL-2 and Bax protein.
Cell viability was analyzed by 2MTT.
The content of reactive oxygen species (ROS) was detected by 3DCFH-DA.
Apoptosis rate was detected by 4TUNEL.
The contents of HO-1, Bcl-2, Bax, Caspase-3, caspase-9 and cytochrome C protein were detected by 5Western blot.
Result:
The relationship between the expression of 1HO-1 and clinicopathological features of prostate cancer.
HO-1 expression was positively correlated with clinical stage, preoperative PSA, seminal vesicle invasion and Gleason grade, but not with age, lymph node metastasis and urethral invasion.
2 the relationship between HO-1 and apoptosis related protein Bcl-2 and Bax expression in human prostate cancer specimens.
The expression of Bcl-2 increased with the increase of HO-1 expression, while Bax showed an opposite trend.
3MTT assay was used to analyze the effects of Hemin and Znpp on docetaxel induced PC-3 cell toxicity.
4 inhibition of HO-1 enhances docetaxel induced apoptosis in PC-3 cells.
5 docetaxel induced ROS production and inhibition of HO-1 could enhance docetaxel induced ROS production.
6 docetaxel induced ROS up-regulated Bax, down regulated Bcl-2 expression, while HO-1 reversed.
7 inhibition of HO-1 enhances docetaxel induced mitochondrial apoptosis pathway.
CONCLUSION: HO-1 is highly expressed in prostate cancer, which may promote the progression of prostate cancer by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax. Inhibition of HO-1 may enhance the apoptosis induced by docetaxel. The underlying molecular mechanism is ROS-dependent mitochondrial apoptosis pathway.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
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