Bim在黑色素瘤细胞内质网应激状态中的异常调控及作用
发布时间：2018-10-21 17:22
【摘要】：背景及目的:很多实体肿瘤包括黑色素瘤对治疗的耐受或者在反复治疗的情况下引起的获得性耐受成为临床治疗的重大障碍,这很大程度上是由于肿瘤细胞对化疗药物诱导凋亡不敏感。目前研究表明除了死亡受体和线粒体途径外,内质网也在调节化疗药物诱发的细胞凋亡中起着重要的作用。一些化疗药物能够引发内质网应激,尽管内质网应激时的未折叠蛋白反应可通过减缓内质网应激状态而保护细胞,但过强或持续时间过长的应激反应可引起细胞凋亡。近期研究结果表明:黑色素瘤对内质网应激诱导的凋亡并不敏感,这种抗凋亡机制目前尚未完全明确。但已经清楚和线粒体途径密切相关的BH3-only家族蛋白对内质网应激性凋亡起到重要调节作用,其中Bim在很多对内质网应激性凋亡敏感的细胞中发生上调并发挥重要的促凋亡作用,但目前对内质网应激状态下的黑色素瘤细胞中Bim的表达情况了解甚少,本课题旨在探讨Bim在黑色素瘤细胞内质网应激状态中的调控以及其在黑色素瘤细胞对内质网应激性凋亡耐受中的作用,从而更加完善黑色素瘤对化疗诱导凋亡不敏感的分子机制。 方法:采用天然核苷抗生素衣霉素(TM)处理黑色素瘤Mel-RM细胞株、MM200细胞株和对照组HEK293细胞株,建立内质网应激模型。通过流式细胞术(FCM)AnnexinⅤ/ PI双染法及Hoechst染色法检测细胞凋亡;Western blot检测caspase-3,-9及PARP的活化和Bim,GRP78,CHOP及FOXO1等蛋白水平;实时荧光定量PCR检测Bim,CHOP及FOXO1的mRNA水平;小干扰RNA(siRNA)技术特异性“沉默”Bim基因,Western blot检测Bim的“沉默”效率和“沉默”前后caspase-3的活化情况;采用流式细胞术检测“沉默”Bim前后TM诱导HEK293细胞凋亡率的变化。 结果:TM作用黑色素瘤细胞和HEK293细胞后,两株细胞对药物诱导的凋亡均呈剂量和时间依赖性,但黑色素瘤细胞较HEK293细胞相比,对内质网应激性凋亡表现为耐受。对照组HEK293细胞中caspase-3,-9明显活化,但在黑色素瘤细胞中却不明显。在内质网应激状态下的黑色素瘤细胞中Bim的蛋白水平没有上调,mRNA水平下调。检测黑色素瘤细胞中内质网应激状态下调控Bim的转录因子CHOP的表达水平上调,另一转录因子FOXO1的表达水平下调。在TM诱导的HEK293细胞中特异性“沉默”Bim基因后caspase-3的活化程度下降,细胞凋亡率也明显下降。 结论:Bim在内质网应激状态下的黑色素瘤细胞中受到异常调控,这可能和转录因子CHOP及FOXO1有关,这种异常调控可能是导致黑色素瘤细胞对内质网应激性凋亡耐受的重要原因,因此Bim是提高黑色素瘤对内质网应激性凋亡敏感性的一个重要靶点。
[Abstract]:Background and objective: the tolerance of many solid tumors, including melanoma, to treatment or acquired tolerance in the case of repeated treatment has become a major obstacle to clinical treatment. This is largely due to the insensitivity of tumor cells to chemotherapeutic drugs. In addition to death receptors and mitochondrial pathways, endoplasmic reticulum plays an important role in regulating apoptosis induced by chemotherapeutic drugs. Some chemotherapeutic drugs can induce endoplasmic reticulum stress. Although the unfolded protein response to endoplasmic reticulum stress can protect cells by slowing down the endoplasmic reticulum stress state, excessive or prolonged stress response can induce apoptosis. Recent studies have shown that melanoma is not sensitive to endoplasmic reticulum stress-induced apoptosis, and the mechanism of anti-apoptosis is not completely clear. However, it is well known that BH3-only family proteins, which are closely related to mitochondrial pathway, play an important role in regulating endoplasmic reticulum stress-induced apoptosis, in which Bim up-regulates and plays an important role in promoting apoptosis in many cells sensitive to endoplasmic reticulum stress apoptosis. However, little is known about the expression of Bim in melanoma cells under endoplasmic reticulum stress. The aim of this study was to investigate the role of Bim in the regulation of endoplasmic reticulum stress in melanoma cells and its role in the tolerance of melanoma cells to endoplasmic reticulum stress-induced apoptosis. Thus, the molecular mechanism of melanoma insensitivity to chemotherapy-induced apoptosis was improved. Methods: the endoplasmic reticulum (ER) stress model was established by treating melanoma Mel-RM cell line, MM200 cell line and control HEK293 cell line with natural nucleoside antibiotic itamycin (TM). The activation of caspase-3,-9 and PARP and the levels of Bim,GRP78,CHOP and FOXO1 were detected by (FCM) Annexin 鈪，
本文编号：2285817
[Abstract]:Background and objective: the tolerance of many solid tumors, including melanoma, to treatment or acquired tolerance in the case of repeated treatment has become a major obstacle to clinical treatment. This is largely due to the insensitivity of tumor cells to chemotherapeutic drugs. In addition to death receptors and mitochondrial pathways, endoplasmic reticulum plays an important role in regulating apoptosis induced by chemotherapeutic drugs. Some chemotherapeutic drugs can induce endoplasmic reticulum stress. Although the unfolded protein response to endoplasmic reticulum stress can protect cells by slowing down the endoplasmic reticulum stress state, excessive or prolonged stress response can induce apoptosis. Recent studies have shown that melanoma is not sensitive to endoplasmic reticulum stress-induced apoptosis, and the mechanism of anti-apoptosis is not completely clear. However, it is well known that BH3-only family proteins, which are closely related to mitochondrial pathway, play an important role in regulating endoplasmic reticulum stress-induced apoptosis, in which Bim up-regulates and plays an important role in promoting apoptosis in many cells sensitive to endoplasmic reticulum stress apoptosis. However, little is known about the expression of Bim in melanoma cells under endoplasmic reticulum stress. The aim of this study was to investigate the role of Bim in the regulation of endoplasmic reticulum stress in melanoma cells and its role in the tolerance of melanoma cells to endoplasmic reticulum stress-induced apoptosis. Thus, the molecular mechanism of melanoma insensitivity to chemotherapy-induced apoptosis was improved. Methods: the endoplasmic reticulum (ER) stress model was established by treating melanoma Mel-RM cell line, MM200 cell line and control HEK293 cell line with natural nucleoside antibiotic itamycin (TM). The activation of caspase-3,-9 and PARP and the levels of Bim,GRP78,CHOP and FOXO1 were detected by (FCM) Annexin 鈪，
本文编号：2285817
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