Mcl-1 调控NSCLC 对EGFR-TKIs敏感性的实验研究

目的:探讨人髓细胞白血病基因-1(myeloid cell leukemia-1,Mcl-1)是否参与调控非小细胞肺癌(non-small cell lung cancer,NSCLC)对EGFR-TKIs的敏感性,为非小细胞肺癌的治疗提供新的思路。方法:通过Western blot方法检测EGFR-TKIs敏感细胞H3255和耐药细胞H1975中Mcl-1蛋白的表达水平。分别给予敏感细胞H3255和耐药细胞H1975 EGFR-TKIs处理后检测细胞凋亡情况和Mcl-1蛋白表达水平。设计并合成特异性si RNA下调耐药细胞H1975中Mcl-1的表达,采用脂质体转染后通过流式细胞技术检测细胞的凋亡情况。结果:H3255细胞Mcl-1表达水平明显低于H1975细胞。一代EGFR-TKIs Gefitinib显著降低H3255细胞Mcl-1表达而不能减少H1975细胞Mcl-1表达。H1975细胞经二代EGFR-TKIs Afatinib和三代EGFR-TKIs AZD9291处理后Mcl-1表达明显减少。特异性si RNA下调H1975细胞Mcl-1表达可以促进细胞凋亡。结论:Mcl-1参与了调节NSCLC对EGFR-TKIs的敏感性,可能成为防止或逆转NSCLC对EGFR-TKIs耐药的潜在靶点。     

Mcl-1在胆盐（GCDA）诱导的肝癌细胞耐药中的作用

目的：探讨抗凋亡蛋白Mcl-1在GCDA诱导的肝癌细胞耐药中的作用及其机制。方法：培养3种肝癌细胞系,用免疫荧光法和Western blot技术检测Mcl-1的表达;GCDA±CYC处理HepG2细胞,采用Western blot技术检测Mcl-1的半衰期变化;用抗癌药物Irinotecan与GCDA对HepG2细胞进行处理,采用MTT法和Western blot技术分别检测细胞增殖抑制率和Mcl-1的表达变化;用RNA干扰技术下调Mcl-1,检测化疗药物对HepG2细胞的敏感性。结果：Mcl-1在肝癌细胞中广泛表达;GCDA能延长Mcl-1的半衰期至6h以上,并明显减弱化疗药物对抗凋亡蛋白Mcl-1的抑制作用,降低癌细胞的药物敏感性;RNA干扰下调Mcl-1能增加癌细胞的药物敏感性。结论：胆盐（GCDA）能诱导HepG2细胞产生耐药性,其作用机制可能是通过延长Mcl-1半衰期增加其蛋白稳定性和抗凋亡作用来促使肝癌细胞抗药的。     

RNA干扰Mcl-1基因表达对淋巴瘤Raji细胞增殖和凋亡的机制研究

目的:探讨抑制Mcl-1基因表达对淋巴瘤Raji细胞增殖和凋亡的影响及机制。方法:NC-siRNA、Mcl-1-siRNA转染Raji细胞,以不作任何处理的细胞作为空白对照组,48h后Western blot检测各组细胞中Mcl-1的蛋白表达;CCK8实验和流式细胞仪分别检测细胞的增殖和凋亡情况;Western blot检测Cleaved caspase3、Notch1、Hes1蛋白表达。结果:转染Mcl-1-siRNA后Mcl-1的蛋白表达显著降低;与对照组及NC-siRNA组比较,Mcl-1-siRNA组细胞存活率显著降低,细胞凋亡率显著升高,Cleaved caspase3蛋白显著上调表达,Notch1和Hes1蛋白显著下调表达。结论:RNA干扰抑制Mcl-1基因表达可显著降低Raji细胞增殖及诱导细胞凋亡,其机制与抑制Notch1信号通路有关。     

抗凋亡蛋白Mcl-1及其抑制剂的研究进展

骨髓细胞白血病蛋白Mcl-1是Bcl-2家族蛋白中重要的抗凋亡蛋白成员,其在多种恶性肿瘤(急性细胞性白血病、多发性骨髓瘤等)中都具有高表达的特点,导致肿瘤细胞对传统化疗药物及Bcl-2抑制剂产生耐药性。Mcl-1作为抗肿瘤药物研发的重要靶点正日益受到相关研究人员的关注,其中Mcl-1新型抑制剂以及联合抑制剂的研究取得了较大进展。本文将对Mcl-1蛋白结构和功能以及相关抑制剂的研究做更深入的分析和总结。     

USP9X低表达通过下调Mcl-1促进肝癌细胞凋亡

研究抑制泛素特异性蛋白酶9X（ubiquitin-specific protease 9X,USP9X）对人肝癌（primary hepatocellular carcinoma,HCC）细胞SMMC7721和HepG2中髓细胞白血病-1（myeloid cell leukemia-1,Mcl-1）蛋白的表达调控及对细胞凋亡和生长活力的影响。实验分为USP9X-siRNA组和阴性对照NC组两组进行分析。通过Western blot技术分别检测USP9X在肝癌细胞SMMC7721、HepG2和正常人肝细胞株L02中的蛋白表达情况;应用化学合成USP9X-siRNA转染肝癌细胞SMMC7721和HepG2,通过Western blot、流式细胞仪和MTT检测转染前后Mcl-1的蛋白表达差异以及细胞凋亡和生长活力变化。结果表明,USP9X在肝癌细胞SMMC7721和HepG2中的蛋白表达水平均高于正常肝细胞L02（t=15.155,P=0.000;t=9.171,P=0.001）;SMMC7721和HepG2细胞中抑制USP9X能明显下调Mcl-1的蛋白表达,并导致细胞凋亡增加和生长活力降低。提示,肝癌细胞SMMC7721和HepG2中USP9X表达上调;USP9X表达降低可能通过下调Mcl-1的蛋白表达进而诱导人肝癌细胞SMMC7721和HepG2的凋亡。     

Wnt/β-catenin信号通路对细胞凋亡和坏死的调控研究进展

Wnt信号通路是一条与细胞增殖分化和机体平衡密切相关且高度保守的信号通路,主要包括Wnt/β-catenin信号通路、Wnt-Ca2+信号通路和平面细胞极性信号通路。其中,以经典Wnt/β-catenin信号炎性反应和细胞命运方面的研究最为深入。现已证实,Wnt/β-catenin信号对细胞命运的调控作用具有两面性,不仅通过调节Survivin、Cyclin、C-myc等基因的表达抑制一些肿瘤细胞凋亡,而且可通过上调促凋亡蛋白BIM、Bax和下调抗凋亡蛋白Mcl-1、Bcl-xl的表达量来促进细胞凋亡。同时,该信号还可以通过抑制某些炎性因子的过度分泌,并下调活性氧(reactive oxygen species,ROS)的含量及坏死相关蛋白PARP-1的表达来抑制细胞坏死。该文对Wnt/β-catenin信号对细胞凋亡和坏死的调控研究进展进行综述。     

Mcl-1参与非小细胞肺癌对吉非替尼耐药的实验研究

目的:探究人髓细胞白血病基因-1(myeloid cell leukemia-1,Mcl-1)是否参与非小细胞肺癌对吉非替尼的耐药。方法:应用Western blot检测Mcl-1在吉非替尼敏感细胞PC-9和耐药细胞H1975表达差异;梯度浓度的吉非替尼作用于PC-9细胞后,Western blot实验检测B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)家族中抗凋亡蛋白的表达变化;应用Western blot实验检测Mcl-1在PC-9和H1975细胞内降解速度差异。结果:Mcl-1在PC-9细胞内的表达明显低于H1975细胞,差异有统计学意义(P0.05),并且随着吉非替尼作用浓度的升高,Mcl-1表达逐渐降低,而Bcl-2和Bcl-x L表达基本不变,并且PC-9细胞内Mcl-1降解更迅速,半衰期明显缩短。结论:非小细胞肺癌对吉非替尼耐药可能与Mcl-1的表达量上调,降解速度减慢,半衰期延长有关。     

Spautin-1通过抑制自噬可增强舒尼替尼诱导的肾癌细胞凋亡

目的:研究spautin-1(一种自噬抑制剂)是否能抑制舒尼替尼诱导的肾癌细胞的自噬以及对舒尼替尼诱导的肾癌细胞凋亡的影响。方法:以肾癌细胞系786-O细胞为模型,Western Blot检测spautin-1对舒尼替尼诱导786-O细胞自噬的影响;Cell Counting Kit-8(CCK-8)检测spautin-1和舒尼替尼对786-O细胞增殖的影响;应用流式细胞术,检测spautin-1对舒尼替尼诱导的786-O细胞凋亡的影响;Western Blot检测spautin-1的促凋亡作用与PI3K/AKT/GSK3β信号通路及抗凋亡蛋白Bcl-2和Mcl-1的关系。结果:与舒尼替尼单独处理组相比,spautin-1能通过降低Beclin-1的表达显著抑制舒尼替尼在786-O细胞中诱导的自噬;Spautin-1和舒尼替尼联合作用明显增强舒尼替尼对786-O细胞增殖的抑制作用;Spautin-1能进一步增强舒尼替尼诱导的786-O细胞的凋亡;Spautin-1和舒尼替尼联合处理786-O细胞时,可以显著降低p-AKTSer473和p-GSK3βSer9的蛋白表达水平,增强GSK3β的活性,进而下调Bcl-2、Mcl-1的表达。结论:Spautin-1能通过抑制AKT活性并活化GSK3β,进一步降低抗凋亡蛋白Bcl-2、Mcl-1的表达,增强舒尼替尼诱导的肾癌细胞凋亡。     

白藜芦醇对胰腺癌细胞增殖和凋亡影响的研究

为了研究白藜芦醇对人胰腺癌细胞增殖和存活的影响,利用不同浓度白藜芦醇处理PANC-1和Bx PC-3细胞,通过MTS和软琼脂集落实验检测白藜芦醇对胰腺癌细胞的停泊依赖和停泊非依赖增殖的抑制。同时,采用免疫印迹的方法,检测不同浓度白藜芦醇对胰腺癌细胞增殖相关信号通路的调控,及不同时间点凋亡激活相关蛋白分子的表达。结果发现白藜芦醇能剂量依赖性抑制PANC-1和Bx PC-3细胞增殖,并且这种增殖抑制作用与表皮生长因子受体(epithelial growth factor receptor,EGFR)及下游信号通路的抑制有关。一定浓度的白藜芦醇能诱导胰腺癌细胞发生凋亡,caspase3和caspase7被剪切活化。通过检测Bcl-2(B-cell lymphoma-2,Bcl-2)促存活家族蛋白,证实白藜芦醇能有效抑制Mcl-1(myeloid cell leukemia-1,Mcl-1)的表达,并不改变Bcl-2和Bcl-XL的表达。实验结果初步表明,白藜芦醇抑制胰腺癌的增殖与存活与EGFR信号通路及促存活蛋白Mcl-1的表达下调有关。     

染色质改构因子BRG1降低UV照射引起的细胞凋亡

生命体的遗传物质基础是DNA分子,多种因素可以作用于细胞内的DNA分子,导致多种类型的DNA损伤。若受损的DNA得不到及时和有效的修复,细胞将走向凋亡或发生变异。染色质改构复合物(chromatin remodeling complex)在基因表达调控和DNA复制等方面扮演着重要角色。依赖ATP的染色质改构复合物SWI/SNF的核心亚基Brahma Related Gene1(BRG1)在染色质结构调整和基因转录调控等多个细胞进程中具有重要作用,仅有有限的文献报道BRG1参与到DNA的损伤修复过程。因此,进一步研究与验证BRG1在调控DNA的损伤修复进而挽救细胞凋亡中的作用十分重要。本文通过利用不同强度的UV照射检测细胞凋亡的情况,初步建立了DNA损伤修复的实验体系。将BRG1表达质粒瞬时转染到SW13(BRG1-/-)细胞系中,并利用30J/m2的UV照射,分别在0h、6h和24h检测细胞早期凋亡程度。结果表明,SW13(BRG1-/-)细胞中瞬时表达BRG1可以明显降低由UV照射引起的细胞凋亡,其中UV照射后24h的细胞表现最明显。我们进一步在HeLa细胞中通过瞬时表达BRG1验证了上述结果。由于BRG1通过染色质改构在基因的转录调控、复制和重组等方面起着重要的作用,我们推测BRG1可能通过染色质改构参与了DNA的损伤修复过程,进而影响了细胞凋亡。     

Simultaneous knock-down of Bcl-xL and Mcl-1 induces apoptosis through Bax activation in pancreatic cancer cells

Anti-apoptotic Bcl-2 family proteins have been reported to play an important role in apoptotic cell death of human malignancies. The aim of this study was to delineate the mechanism of anti-apoptotic Bcl-2 family proteins in pancreatic cancer (PaCa) cell survival. We first analyzed the endogenous expression and subcellular localization of anti-apoptotic Bcl-2 family proteins in six PaCa cell lines by Western blot. To delineate the functional role of Bcl-2 family proteins, siRNA-mediated knock-down of protein expression was used. Apoptosis was measured by Cell Death ELISA and Hoechst 33258 staining. In the results, the expression of anti-apoptotic Bcl-2 family proteins varied between PaCa cell lines. Mcl-1 knock-down resulted in marked cleavage of PARP and induction of apoptosis. Down-regulation of Bcl-2 or Bcl-xL had a much weaker effect. Simultaneous knock-down of Bcl-xL and Mcl-1 strongly induced apoptosis, but simultaneous knock-down of Bcl-xL/Bcl-2 or Mcl-1/Bcl-2 had no additive effect. The apoptosis-inducing effect of simultaneous knock-down of Bcl-xL and Mcl-1 was associated with translocation of Bax from the cytosol to the mitochondrial membrane, cytochrome c release, and caspase activation. These results demonstrated that Bcl-xL and Mcl-1 play an important role in pancreatic cancer cell survival. Targeting both Bcl-xL and Mcl-1 may be an intriguing therapeutic strategy in PaCa.     

Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2-related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)-induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system.     

A small hairpin RNA targeting myeloid cell leukemia-1 enhances apoptosis in host macrophages infected with <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Myeloid cell leukemia-1 (Mcl-1) plays an important role in various cell survival pathways. Some studies indicated that the expression of Mcl-1 was upregulated in host cells during infection with the virulent Mycobacterium tuberculosis strain, H37Rv. The present study was designed to investigate the effect of inhibiting Mcl-1 expression both in vivo and in vitro on apoptosis of host macrophages infected with M. tuberculosis using a small hairpin (sh)RNA. Mcl-1 expression was detected by the real time-polymerase chain reaction, western blotting, and immunohistochemistry. Flow cytometry and transmission electron microscopy were used to measure host macrophage apoptosis. We found elevated Mcl-1 levels in host macrophages infected with M. tuberculosis H37Rv. The expression of Mcl-1 was downregulated efficiently in H37Rv-infected host macrophages using shRNA. Knockdown of Mcl-1 enhanced the extent of apoptosis in H37Rv-infected host macrophages significantly. The increased apoptosis correlated with a decrease in M. tuberculosis colony forming units recovered from H37Rv-infected cells that were treated with Mcl-1-shRNA. Reducing Mcl-1 accumulation by shRNA also reduced accumulation of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax, in H37Rv-infected host macrophages. Our results showed that specific knockdown of Mcl-1 expression increased apoptosis of host macrophages significantly and decreased the intracellular survival of a virulent strain of M. tuberculosis. These data indicate that interference with Mcl-1 expression may provide a new avenue for tuberculosis therapy.     

Sodium salicylate promotes neutrophil apoptosis by stimulating caspase-dependent turnover of Mcl-1

Mcl-1 is an antiapoptotic member of the Bcl-2 family of proteins that plays a central role in cell survival of neutrophils and other cells. The protein is unusual among family members in that it has a very short half-life of 2-3 h. In this report, we show that sodium salicylate (at 10 mM) greatly enhances the rate at which neutrophils undergo apoptosis and, in parallel, greatly accelerates the turnover rate of Mcl-1, decreasing its half-life to only 90 min. Whereas constitutive and GM-CSF-modified Mcl-1 turnover is regulated by the proteasome, the accelerated sodium salicylate-induced Mcl-1 turnover is mediated largely via caspases. Sodium salicylate resulted in rapid activation of caspase-3, -8, -9, and -10, and salicylate-accelerated Mcl-1 turnover was partly blocked by caspase inhibitors. Sodium salicylate also induced dramatic changes in the activities of members of the MAPK family implicated in Mcl-1 turnover and apoptosis. For example, sodium salicylate blocked GM-CSF-stimulated Erk and Akt activation, but resulted in rapid and sustained activation of p38-MAPK, an event mimicked by okadaic acid that also accelerates Mcl-1 turnover and neutrophil apoptosis. These data thus shed important new insights into the dynamic and highly regulated control of neutrophil apoptosis that is effected by modification in the rate of Mcl-1 turnover.     

New face of antiapoptotic proteins. I. Protein Mcl-1

Main regulators of apoptosis belong to Bcl-2 protein family and apoptosis inhibitory proteins--IAPs. In this review the apoptosis inhibitor--Mcl-1 protein is profoundly characterized. It is important that this unique short-living protein--the member of Bcl-2 family may also operate as apoptosis promoting agent, which results of alternative splicing of its pre-mRNA, posttranslational modifications or proteolysis. The review presents also other functions of Mcl-1, i.e. involvement in cell cycle regulation, elongation of telomers. Elevated expression of Mcl-1 accompanies the development of various cancers, neurodegenerative disorders and also infectious diseases. The obtained results indicate that expression level of Mcl-1 may be useful in treatment decisions of large number of diseases. Ablating expression of this protein may be an attractive therapeutic strategy in the treatment of various cancers, and the diseases where Mcl-1 may play a key role in apoptosis supression.     

Cyclooxygenase-2 inducing Mcl-1-dependent survival mechanism in human lung adenocarcinoma CL1.0 cells. Involvement of phosphatidylinositol 3-kinase/Akt pathway

Cyclooxygenase 2 (COX-2) has been reported to be commonly expressed in advanced stages of human lung adenocarcinoma. In this study, the COX-2 constitutive expression vector was transfected into a human lung adenocarcinoma cell line CL1.0 and several clones were obtained which stably expressed COX-2. These COX-2-overexpressed clones demonstrated remarkable resistance to apoptosis induced by Ultraviolet B (UVB) irradiation, vinblastine B (VBL) cell lymphoma-2 (Bcl-2), or other anti-cancer drugs. To understand how COX-2 prevents apoptosis, the investigators examined the expression level of Bcl-2 family members. Mcl-1, but not other Bcl-2 members, was significantly up-regulated by COX-2 transfection or prostaglandin E(2) (PGE(2)) treatment. Treatment of COX-2-overexpressed cells (cox-2/cl.4) with two specific COX-2 inhibitors, NS-398 and celecoxib, caused an effective reduction of the increased level of Mcl-1. These data suggest that the expression level of Mcl-1 is tightly regulated by COX-2. Moreover, transfection of cox-2/cl.4 cells with antisense Mcl-1 enhanced apoptosis induced by UVB irradiation, revealing that Mcl-1 plays a crucial role in cell survival activity mediated by COX-2. Furthermore, COX-2 transfection or PGE(2) treatment evidently activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Inhibition of the PI3K pathway by LY294002 or wortmannin effectively attenuated the increased level of Mcl-1 induced by COX-2 or PGE(2). Blocking the PI3K activity with a dominant-negative vector, DN-p85, also greatly diminished the level of Mcl-1 and enhanced UVB-elicited cell death in cells transfected by COX-2. In a similar way, LY294002 inhibited cell survival and Mcl-1 level in PGE(2)-treated CL1.0 cells. These findings suggest that COX-2 promotes cell survival by up-regulating the level of Mcl-1 by activating the PI3K/Akt-dependent pathway.     

Mcl-1

Mcl-1 is a Bcl-2 family protein which can act as an apical molecule in apoptosis control, promoting cell survival by interfering at an early stage in a cascade of events leading to release of cytochrome c from mitochondria. Mcl-1 has a short half life and is a highly regulated protein, induced by a wide range of survival signals and also rapidly down regulated during apoptosis. Mcl-1 can also readily be cleaved by caspases during apoptosis to produce a cell death promoting molecule. The multiple levels of control of Mcl-1 expression suggest that Mcl-1 plays a critical role in controlling life and death decisions in response to rapidly changing environmental cues and Mcl-1 is required for embryonic development and the function of the immune system. Expression of Mcl-1 may be useful in informing decision making in the treatment of various cancers, and countering Mcl-1 function may be an attractive therapeutic strategy in malignancy, inflammatory conditions and infectious disease where Mcl-1 may play a major role in suppressing apoptosis.     

Mcl-1: a highly regulated cell death and survival controller

Summary Mcl-1 is one member of the Bcl-2 family that has a very short protein half-life. Since its identification in 1993, a great number of studies have implicated that Mcl-1 plays an important role in various cell survival pathways. However, not until recently did the molecular mechanism by which Mcl-1 antagonizes apoptosis have begun to be elucidated. Mcl-1 is rapidly degraded in response to cell death signals and is immediately re-induced by survival stimuli. These results indicate that Mcl-1 plays an apical role in many cell death and survival regulatory programs.