摘要
背景及目的:肿瘤细胞的一个基本特征是细胞增殖与细胞凋亡的不平衡,而化疗药物杀伤肿瘤细胞的主要机制就是诱导细胞凋亡。已知多个细胞器参与及调控凋亡信号的转导。而内质网在细胞凋亡中发挥的作用日益受到关注。在药物等某些因素作用下,实体瘤中某些细胞的内质网会发生应激反应。内质网可通过特异性信号途径清除未折叠或错误折叠蛋白质对外界应激适应,此种保护性反应被称为未折叠蛋白反应(UPR,unfolded protein response)。近年来发现长时间或过强的UPR可能会诱导线粒体依赖和线粒体非依赖途径的凋亡,但不同细胞系敏感性不一。因此我们检验内质网应激诱导剂衣霉素通过UPR体外诱导胃腺癌细胞凋亡。而肿瘤细胞对化疗不敏感的主要原因之一是其中许多促进细胞增殖存活的信号转导通路被激活,其中MEK/ERK是主要的生存信号途径之一。本研究以胃腺癌细胞为研究对象,了解衣霉素体外诱导胃腺癌细胞凋亡途径,探讨MEK/ERK途径与UPR介导的凋亡相互联系及其作用机制,以寻找克服肿瘤细胞对凋亡抵抗的有效途径。方法:常规体外培养的BGC-823、SGC-7901和人成纤维细胞,细胞传代24h后加入药物处理;流式细胞仪PI单染法检测细胞周期DNA含量分析一系列浓度(1.5～12μM)TM诱导人成纤维细胞和胃腺癌细胞凋亡率,使用MEK特异性抑制剂U0126阻断MEK/ERK信号途径后TM诱导以及加入广谱caspase抑制剂后两者联用诱导胃腺癌细胞的凋亡水平;DAPI染色后荧光显微镜观察药物处理后死亡细胞形态特征及各组差别;Western blot检测TM诱导不同时间点GRP78的表达,并检测TM单独处理前后及U0126与TM共同诱导细胞前后p-ERK、caspase-3、PARP及Bcl-2家族成员表达量的变化;JC-1染色流式细胞仪测各组药物作用前后线粒体膜电位(MMP)的变化:免疫沉淀法检测TM诱导前后蛋白质分子间的相互作用。
结果:胃腺癌细胞相对于人成纤维细胞对TM诱导的内质网应激性凋亡不敏感,但同U0126共同作用后其凋亡水平显著增高,荧光显微镜下可观察到典型的细胞凋亡形态学表现,凋亡细胞数量比TM单独作用明显增多。TM诱导ERK活化形式p-ERK表达增加,表明它在诱发未折叠蛋白反应的同时也活化MEK/ERK信号通路。U0126预作用后,TM诱发胃腺癌细胞caspase-3、PARP分子剪切、活化,而广谱caspase抑制剂Z-VAD-fmk能有效抑制两者联合诱导的胃腺癌细胞凋亡,因而TM诱导的的胃腺癌细胞凋亡极大程度上依赖于caspase家族。U0126与TM共同作用后两株细胞的MMP随着时间的延长下降,抑制凋亡的Mcl-1蛋白量有减少趋势,并且SGC-7901中PUMA分子蛋白表达水平增高,其余Bcl-2家族成员无明显变化。TM诱导两株细胞24h时Mcl-1表达增强,Puma在SGC-7901中随时间推移表达减少特别是36h,而BGC-823中无显著变化。Mcl-1能同PUMA在体外共沉淀,而TM作用24h后SGC-7901细胞的Mcl-1与PUMA形成复合物的量显著增加,在BGC-823细胞中也有增加但不明显。
结论:胃腺癌细胞对TM诱发的凋亡相对不敏感,可能通过活化UPR能够保护自身对长时间的内质网应激适应而免于凋亡。但通过抑制MEK/ERK通路能消除胃腺癌细胞对TM介导内质网应激性凋亡的耐受。TM诱导胃腺癌细胞内质网应激性的凋亡依赖于caspase途径,而线粒体在此凋亡信号转导中起重要作用。MEK/ERK途径可能通过作用于Bcl-2家族中的Mcl-1和PUMA分子:激活Mcl-1可同PUMA分子结合成复合物抑制凋亡,而直接增强PUMA分子活性能促进胃腺癌细胞对TM诱导凋亡的敏感性。
Background & Objective:A fundamental characteristic of cancer cells is to survive induction of apoptosis,which is a major killing mechanism of cancer cells by therapeutic agents.It is known that the transduction of cell apoptosis signaling involves the participation and regulation of many organdies.The role of ER in cell apoptosis is being payed more attention to.A number of cellular adverse conditions,such as drug,may lead to activation of ER stress reaction of some cells in solid tumor.ER can eliminate these unfolded or mis-folded proteins by specific signaling pathway and adapt to ER stress,the cytoprotective response is called UPR.Recent years it is found that prolonged UPR can result in apoptosis through both mitochondria-dependent and -independent pathways, but the sensitivity of different cell lines is varying.For this reason we examine TM-induced apoptosis of gastric adenocarcinoma cells.A number of survival signaling pathways enhancing tumor cell proliferation are known to be constitutively activated in many tumor cells.Among them,MEK/ERK pathway is the main survival signaling pathways.We take gastric adenocarcinoma cells as our research object and explore the apoptotic signaling pathway of TM-induced gastric adenocarcinoma cells.Correlation between MEK/ERK pathway and UPR-mediated apoptosis will be assessed.We try to find an effective methods overcoming the resistance of tumor cells.
Methods:Human gastric adenocarcinoma cell lines SGC-7901、BGC-823 and human fibroblasts were normally cultured and drug was added 24h after cell lines were transferred.Apoptosis of TM-induced gastric adenocarcinoma cell and human fibroblasts with a series of concentration(1.5～12μM) was analyzed by detecting the DNA content of cell cycle with PI staining in flow cytometry.Levels of TM-induced apoptosis in gastric adenocarcinoma cell by blocking MEK/ERK pathway with specific suppressor of MEK,and U0126 and TM co-treatment after adding the pancaspase inhibitor, Z-VAD-fmk,were both studied using the same methods.Morphological characteristics of cell death and apoptotic contrast among the drug treatment groups were examined with DAPI staining using a fluorescence microscope.The expression level of p-ERK,caspase-3,PARP,and Bcl-2 family members before and after TM,U0126 and the two co-treatment were assayed by immunoblotting.We can detect the protein level of GRP78 after TM inducing at varying times with Western blot assay.Changes of MMP among the drug-treated groups were measured with JC-1 staining in flow cytometry.The interaction of different protein moleculars in gastric adenocarcinoma cells was evidenced by carrying out immunoprecipitation before and after TM treatment.
Results:Human gastric adenocarcinoma cell lines compared with human fibroblasts are relatively resistant to TM-induced apoptosis,which were markedly enhanced when U0126 and TM co-treatment.There were typical apoptotic morphology in death cells,and apoptotic cells with U0126 and TM co-treated can be more frequently observed than with TM alone under fluorescent microscope.The activated expression of p-ERK protein was increased after exposure to TM,which suggested that TM induces activation of UPR and at the same time activates MEK/ERK pathway.The cleavage and activation of caspase-3、PARP were induced by TM when cells pretreated with U0126.Induction of apoptosis was largely dependent on caspases in that the pan caspase inhibitor Z-VAD-fmk can effectively inhibited apoptosis by the two in gastric adenocarcinoma cell lines.Treatment with TM and U0126 resulted in a notable decrease changes in the levels of MMP,and up-regulation of anti-apoptotic Mcl-1 protein in both cell lines.Whereas this was followed by an increase in the expression levels of Puma in SGC-7901 cells.The expression levels of other Bcl-2 family members remained,however,unaltered.Whereas Mcl-1 was increased in both cells at 24 hours with the elevated levels of Puma being decreased by 36 hours in SGC-7901 cells,a change in the Puma protein levels in BGC-823 cells had not been observed.PUMA can co-precipitated with Mcl-1 in vitro,After exposure to TM or TG,substantial increases in PUMA were found in Mcl-1 complexes from SGC-7901 cells at 24 hours,but no significant up-regulation were detected in those from BGC-823 cells.
Conclusions:Human gastric adenocarcinoma cell lines are relatively resistant to TM-induced apoptosis,which probably caused by activation of UPR.It protects cells against apoptosis and resulted in the adaptation to prolonged ER stress.We can overcome the resistance to ER stress-induced apoptosis by TM after inhibiting MEK/ERK pathway.Induction of ER stress-induced apoptosis by TM in gastric adenocarcinoma cells is caspase-dependent,and mitochondria plays important roles in this signaling transduction.Mcl-1 and Pmua of Bcl-2 family members are the possible target of MEK/ERK pathway in ER stress-induced apoptosis:activation of Mcl-1 associated with PUMA protects against apoptosis,or promoting the sensitivity of TM-induced apoptosis by directly activating Puma in gastric adenocarcinoma cells.
引文
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