凋亡调节蛋白在手术切除的原发性非小细胞肺癌中的表达

目的 检测凋亡调节蛋白抑癌基因p53和Bcl-2家族在非小细胞肺癌(NSCLC)标本中的相关性和预后意义。方法 采用免疫组化法用特异性针对人体的p53和Bcl-2家族的抗体检测这些凋亡调节基因在50例存档的、手术彻底切除的NSCLC患者标本中的表达情况。分析它们之间的相互关系，并追踪患者的生存情况，分析其表达的预后意义。结果 (1)31例(62％)的NSCLC表达抑癌基因p53，典型的p53染色免疫阳性是细胞核染色。典型的Bcl-2家族蛋白呈胞浆染色．少数也可以呈胞核染色阳性。(2)p53的表达与患者的吸烟史呈正相关(P＜0．05)，p53与Bcl-2的表达呈显著的正相关(P＜0．05)，p53与Bax的表达之间呈显著的负相关(P＜0．05)。(3)p53的表达阴性的肿瘤患者没有发生远处转移，p53的表达与远处转移的发生呈正相关(P＜0．05)。Bcl-2的表达与原发性NSCLC手术切除患者的生存时间(P＜0．05)呈负相关。结论 抑癌基因p53和Bcl-2家族蛋白在NSCLCK中的表达等都很常见。p53突变可能使Bcl-2表达增加，Bax表达减少而增加远处转移的风险。     

非小细胞肺癌凋亡及相关基因bcl-2和bax的表达

目的:探讨非小细胞肺癌(NSCLC)细胞凋亡与相关基因bcl-2、bax表达产物的关系及其临床意义。方法:应用流式细胞术和间接免疫荧光标记法进行检测。结果:凋亡率,Bcl-2、Bax蛋白标记率在NSCLC组与对照组中的差别有统计学意义(P<0.05)。NSCLCI期凋亡率明显高于II、III期组(P<0.01)。NSCLCI期组Bcl-2蛋白标记率明显低于II、III期组(P<0.01)。NSCLC中Bcl-2蛋白标记率与凋亡率呈负相关(r=-0.667,P<0.01)。Bax标记率与凋亡率无直线相关性(r=0.230,P>0.05)。Bcl-2/Bax>1组凋亡率明显低于Bcl-2/Bax≤1组(P<0.05)。结论:随着NSCLC临床分期的进展凋亡率下降,Bcl-2通过抑制凋亡影响NSCLC的进展。NSCLC细胞内存在一种对死亡讯号的应答模式:bcl-2/bax。     

非小细胞肺癌中神经内分泌分化与bcl-2、Bax表达的相关性研究

随机收集 5 1例手术切除的 NSCLC石蜡标本 ,采用免疫组化 S-P法检测了神经元特异性烯醇化酶 (NSE)、嗜铬颗粒蛋白 A(Cg A)及 bcl-2、Bax基因蛋白的表达。结果显示 ,NSCLC中相当一部分伴有 NE分化 ,NSCLC-NE具有与其它 NSCL C不同的生物学特性。NSCLC-NE中可以检测到高表达的 bcl-2蛋白 ,bcl-2的表达与 Bax的表达有密切关系 ,bcl-2、Bax的失衡在 NSCL C-NE的发生和发展中可能起重要的作用     

实验性肺癌癌变过程中肿瘤细胞凋亡以及基因Fas、Bcl-2蛋白表达的动态研究

为探讨实验性肺癌癌变过程中细胞凋亡情况及 Fas、Bcl-2蛋白的表达、相互关系及与肺癌的早期诊断、预后判断的关系 ,用化学致癌物 3-甲基胆蒽 ( MCA)及二乙基亚硝胺 ( DEN)诱发大鼠肺鳞状细胞癌 ,应用免疫组化 S-P法检测癌变过程中 Fas、Bcl-2蛋白的表达 ,同时应用原位 TUNEL法检测细胞凋亡情况。结果显示 ,凋亡率及 Bcl-2蛋白表达情况是肺癌启动、发展及预后判断的有效指标 ,Fas是肺癌进展及预后判断的重要指标。 Bcl-2与凋亡协同存在 ,Bcl-2表达率及细胞凋亡率高 ,是肺组织癌变的重要标志 ;Bcl-2表达率及细胞凋亡率低 ,同时 Fas表达率高 ,是肺癌预后不良的标志。     

过表达microRNA-144通过靶向调节泛素样PHD和环指结构域1基因诱导非小细胞肺癌凋亡

目的探讨microRNA-144 (miR-144)对非小细胞肺癌(Non-small-cell lung cancer,NSCLC)细胞增殖以及预后的影响。方法通过癌症基因组图谱(The cancer genome atlas,TCGA)检测NSCLC患者组织中miR-144以及泛素样PHD和环指结构域包含1(Ubiquitin like PHD and ring finger domain 1,UHRF1)的表达预后以及两者表达的相关性。采用CCK-8、AO/EB以及γH2A检测不同表达miR-144对NSCLC细胞活性、细胞凋亡、增殖以及DNA损伤的影响,Western blot检测PCNA、Bax、Bcl-2及UHRF1的表达。荧光素酶报告证明miR-144以及UHRF1的靶向关系。结果在NSCLC患者组织中,miR-144表达水平较低,低表达miR-144 NSCLC患者生存时间明显低于高表达miR-144者。UHRF1在NSCLC患者组织中明显升高,且在不同年龄、性别、TNM分期和种族有显著差异。低表达UHRF1非小细胞肺癌患者生存率明显高于高表达UHRF1的NSCLC患者。过表达miR-144能够明显降低A549细胞活性,诱导A549细胞凋亡,增加γH2ax表达;抑制PCNA以及Bax蛋白表达,上调Bcl-2表达。荧光素酶报告证明UHRF1是miR-144的靶基因。同时,低表达miR-144能够使UHRF1表达增加,过表达miR-144能够抑制UHRF1的表达。结论过表达miR-144通过靶向UHRF1诱导A549细胞凋亡以及DNA损伤,参与NSCLC的病理生理过程,进而影响预后。     

环磷酰胺和紫杉醇联合化疗诱导小鼠Lewis肺癌细胞的凋亡

目的观察小剂量环磷酰胺(Ctx)和紫杉醇(Px)联合化疗对小鼠Lewis肺癌细胞凋亡的影响并探讨其可能的机制。方法建立小鼠Lewis肺癌模型,将38只C57BL/6♂小鼠随机分为4组:生理盐水对照组(A)、Ctx单药组(B)、Px单药组(C)、Ctx Px组(D)。采用免疫组化技术检测各组肿瘤组织中Bcl-2、Bax及Caspase-9的蛋白表达水平。结果D组Bcl-2蛋白表达水平明显低于A、B、C组,D组Bax、Caspase-9蛋白表达水平及Bax/Bcl-2则明显高于A、B、C组。结论小剂量Ctx和Px联合化疗比单用具有更强的促进小鼠Lewis肺癌细胞凋亡的作用,其机制可能与增加Bax/Bcl-2比值和激活Caspase-9等有关。     

Mcl-1蛋白的结构、功能及其抑制剂的研究进展

细胞凋亡与许多人类疾病的发生密切相关,过度凋亡引发神经退行性疾病,而抑制凋亡则与肿瘤等疾病的产生有关.研究表明,Bcl-2蛋白家族在细胞凋亡中发挥重要作用,特别是近几年对Bcl-2蛋白家族成员Mcl-1及其抑制剂研究越来越多.本文介绍了Mcl-1蛋白的结构与功能特点,综述了近几年作为抗肿瘤药物的Mcl-1抑制剂的研究进展.     

Bax、Livin和Survivin在非小细胞肺癌组织中的表达

目的:研究Bax、Livin和Survivin蛋白在非小细胞肺癌(NSCLC)组织中的表达情况及其与NSCLC病理因素的关系,探讨Bax、Livin和Survivin蛋白表达的相关性.方法:随机抽取NSCLC切除组织石蜡标本40例为NSCLC组,正常肺组织石蜡标本10例作为对照组.采用免疫组化非生物素二步法(Pv)检测2组中Bax、Livin和Survivin蛋白的表达情况,根据肿瘤组织细胞的染色率和染色强度进行判定.结果:对照组及NSCLC组中的阳性表达率Bax分别为80.00％和42.50％,Livin分别为10.00％和65.00％,Survivin分别为0和50.00％;有和无淋巴结转移患者的表达率Bax分别为23.81％、63.16％,Livin分别为85.71％、42.11％;Ⅰ、Ⅱ期和Ⅲ、Ⅳ期患者的表达率Bax分别为55.56％、15.38％,Livin分别为51.85％、92.31％;中高分化与低分化患者的Survivin表达率分别为39.29％、75.00％,差异均有统计学意义(P＜ 0.05);NSCLC组织中Bax和Livin的表达呈念相关性(rn=-0.4294,P＜0.05);Bax和Survivin,Livin和Survivin的表达均无相关性(P＞0.05).结论:检测Bax、Livin和Survivin的表达对NSCLC的诊断和预后评估有一定价值.     

Caspase抑制剂对大鼠缺血再灌注心肌细胞凋亡及Bcl-2、Bax蛋白表达的影响

目的 探讨Caspase抑制剂z-VAD-fmk对缺血-再灌注(I-R)心肌细胞凋亡及Bcl-2、Bax蛋白表达的影响.方法 24只大鼠随机分为假手术(C)组、I-R对照(B)组和z-VAD-fmk治疗(A)组,以穿线结扎或松扎左冠状动脉制备大鼠心肌I-R模型,TUNEL法检测心肌细胞凋亡指数(AI),免疫组化法检测Bcl-2、Pax蛋白表达,并分析心肌组织病理学损害程度.结果 B组和A组Bcl-2、Bax基因蛋白表达水平均明显高于C组(P<0.05).A组凋亡指数显著低于B组;与B组比较,A组Bax蛋白表达水平有所下降,而Bcl-2蛋白表达水平显著升高(P<0.05),Bcl-2/Bax比值较对照组明显增加.结论 Caspase抑制剂可抑制I-R后心肌细胞凋亡,其机制可能与上调Bcl-2和下调Bax蛋白表达有关.     

欧前胡素对顺铂的骨肉瘤细胞抗肿瘤作用的增效作用

目的 研究欧前胡素是否能提高人骨肉瘤细胞(human osteosarcoma cells,HOS细胞)对顺铂的敏感性。方法 HOS细胞用欧前胡素和顺铂处理,MTT法检测体外细胞活力,Western blot检测Bcl-2 蛋白家族成员(Mcl-1、Bcl-2、Bcl-xl、Bad和Bax)的表达水平,流式细胞术检测细胞凋亡水平和线粒体膜电位的变化情况。构建Mcl-1真核表达载体,MTT法检测Mcl-1表达载体转染对欧前胡素联合顺铂治疗骨肉瘤疗效的影响。结果 欧前胡素在体外可显著提高顺铂对骨肉瘤细胞系HOS的杀伤活性。欧前胡素可显著降低HOS细胞Mcl-1的表达,而顺铂对Mcl-1的表达水平无影响。相比于欧前胡素或顺铂单治疗组,两者联合可显著诱导HOS细胞发生凋亡并降低其线粒体膜电位。体外转染Mcl-1真核表达载体显著降低顺铂联合欧前胡素对HOS细胞的杀伤活性。结论 欧前胡素通过靶向于Mcl-1增强顺铂对骨肉瘤细胞的杀伤活性。     

Apoptotic markers p53, Bcl-2 and Bax in primary lung cancer

BACKGROUND: Apoptosis is the fundamental process necessary for eliminating damaged or unwanted cells. Alterations in the apoptotic pathway appear to be key events in cancer development and progression. The aim of the study was to determine the p53, Bcl-2 and Bax expressions in lung cancer, taking into account histological heterogeneity and the adjacent bronchial resection margin. MATERIALS AND METHODS: Tissue specimens from 60 histopathologically verified lung cancer specimens and 12 bronchial stumps were evaluated. The presence of the studied markers was revealed by immunocytochemistry on paraffin-embedded tissue. RESULTS: The percentage of p53- and Bax-positive lung cancers was comparable (51.6% for both proteins), while Bcl-2 immunoreactivity was observed in fewer (31.6%) cases. There was no p53 accumulation in bronchial stumps, while Bcl-2 and Bax staining formed a repeatable specific pattern in bronchial epithelium. The differences in apoptotic marker expression between non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) were revealed, especially regarding p53 and Bax expression (60% vs. 10%, p = 0.005 and 58% vs. 20%, p = 0.04, respectively). Taking into account the histological structure of NSCLC, Bax expression appeared to be more frequent in adenocarcinoma than in squamous cell lung cancers (88% vs. 42%, p = 0.004). No interrelationship between the studied proteins in lung cancer tissue was revealed. CONCLUSION: The expression of p53, Bcl-2 and Bax was altered in lung cancer tissue compared to histologically normal bronchial epithelium. The difference between apoptotic marker expression in NSCLC and SCLC could reflect the different pathogenesis of these two lung pathologies.     

Different contribution of BH3-only proteins and caspases to doxorubicin-induced apoptosis in p53-deficient leukemia cells

Bcl-2 family proteins are key regulators of the intrinsic apoptotic pathway, either facilitating (Bax, Bak, BH3-only) or inhibiting (Bcl-2, Bcl-x_L, Mcl-1, A1) mitochondrial release of apoptogenic factors. The role of caspases in this process is a matter of controversy. We have analyzed the relative contribution of caspases and Bcl-2 family of proteins in the induction phase of apoptosis triggered by doxorubicin in two p53-deficient leukemia cell lines, Jurkat and U937. First, we have found that caspases are dispensable for the induction phase of doxorubicin-induced apoptosis in both cell lines but they are needed to speed up the execution phase in Jurkat cells, not expressing Bax. Thus, down-regulation of Bak expression by siRNA significantly prevented doxorubicin-induced apoptosis in Jurkat but not in U937 cells. Reduction of Mcl-1 protein levels with siRNA increased sensitivity to apoptosis in both cell lines. Moreover, our results indicate that the contribution of BH3-only proteins to apoptosis is cell line specific. In Jurkat cells simultaneous silencing of Bim and PUMA was necessary to reduce doxorubicin-induced apoptosis. In U937 cells silencing of Bim or Noxa reduced sensitivity to doxorubicin. Immunoprecipitation experiments discarded an interaction between Mcl-1 and Bak in both cell lines and underscored the role of Bim and PUMA as mediators of Bax/Bak activation.     

Monosaccharide digitoxin derivative sensitize human non-small cell lung cancer cells to anoikis through Mcl-1 proteasomal degradation

Advanced stage cancers acquire anoikis resistance which provides metastatic potential to invade and form tumors at distant sites. Suppression of anoikis resistance by novel molecular therapies would greatly benefit treatment strategies for metastatic cancers. Recently, digitoxin and several of its novel synthetic derivatives, such as α-l-rhamnose monosaccharide derivative (D6-MA), have been synthesized and studied for their profound anticancer activity in various cancer cell lines. In this study, we investigated the anoikis sensitizing effect of D6-MA compared with digitoxin to identify their anti-metastatic mechanism of action. D6-MA sensitized NSCLC H460 cells to detachment-induced apoptosis with significantly greater cytotoxicity (IC50 = 11.9 nM) than digitoxin (IC50 = 90.7 nM) by activating caspase-9. Screening of the Bcl-2 protein family revealed that degradation of anti-apoptotic Mcl-1 protein is a favorable target. Mcl-1 over-expression and knockdown studies in D6-MA and digitoxin exposed cells resulted in rescue and enhancement, respectively, indicating a facilitative role for decreased Mcl-1 expression in NSCLC anoikis. Transfection with mutant Mcl-1S159 attenuated detachment-induced cell death and correlated with a remaining of Mcl-1 level. Furthermore, D6-MA suppressed Mcl-1 expression via ubiquitin proteasomal degradation that is dependent on activation of glycogen synthase kinase (GSK)-3β signaling. In addition, D6-MA also targeted Mcl-1 degradation causing an increased anoikis in A549 lung cancer cells. Anoikis sensitizing effect on normal small airway epithelial cells was not observed indicating the specificity of D6-MA and digitoxin for NSCLC. These results identify a novel cardiac glycoside (CG) sensitizing anoikis mechanism and provide a promising anti-metastatic target for lung cancer therapy.     

氯美昔布诱导肺癌细胞凋亡的作用机制

目的:探讨氯美昔布诱导肺癌细胞凋亡的可能作用机制。方法:人肺癌细胞株A549和NCI-H460细胞先加入终浓度IL-1β11μg/L刺激24h,再加入不同浓度氯美昔布继续培养24h,采用Western blot法检测ERK2、p-ERK及Bcl-2表达水平。氯美昔布体外处理A549、NCI-H460后,采用Western blot法检测Bcl-2、Bax表达水平。结果:氯美昔布作用后肺癌细胞中ERK2及p-ERK的磷酸化水平均下降,并伴有Bcl-2水平的下降,差异有统计学意义(P<0.01)。并且随着氯美昔布浓度的增大,Bcl-2蛋白表达量逐渐下降,而Bax蛋白表达在A549细胞中增强,在NCI-H460细胞中不变,Bcl-2/Bax的比值下降。结论:氯美昔布诱导肺癌细胞凋亡的机制可能与通过ERK信号转导途径调节肺癌细胞产生Bcl-2,进而诱导Bcl-2/Bax的比值下降有关。     

Small-molecule inhibitors of Bcl-2 family proteins as therapeutic agents in cancer

This review focuses on the recent patents and use of small-molecule inhibitors (SMIs) of Bcl-2 family proteins as therapeutic agents against cancer. Bcl-2 members are crucial regulators of apoptotic cell death. Apoptosis is an evolutionarily conserved process of programmed cell death that plays an essential role in organism development and tissue homeostasis. Several mechanisms exist allowing cells to escape programmed cell death among them is the overexpression of the antiapoptotic proteins. Cancer cells are often found to overexpress many of these members such as Bcl-2, Bcl-X(L), Mcl-1, Bcl-w and A1/Bfl1 and are usually resistant to a wide range of anti-cancer drugs and treatments. Many groups have been working to develop anti-cancer drugs that block the function of anti-apoptotic Bcl-2 members, thus favoring cell death. Methods include the downregulation of Bcl-2 expression or the use of peptides or small organic molecules to the Bcl-2 binding pocket, preventing its sequestration of proapoptotic proteins such as Bid and Bim. One of the most promising aspects of SMIs in treating cancer is that their targets and mechanisms of action are different from those of cytotoxic drugs and radiation. This makes it feasible to combine SMIs with other treatments, creating a synergistic therapy, without likely development of cross-resistance or increased toxicity. A broad-spectrum or "pan" SMI which targets multiple Bcl-2 family proteins is the goal.     

−(−)Gossypol promotes the apoptosis of bladder cancer cells in vitro

Dysregulation of Bcl2 family member proteins has been associated with poor chemotherapeutic response in bladder cancer, suggesting that agents targeting these crucial proteins may provide an interventional strategy to slow or halt bladder cancer progression and metastasis. In this study, we investigated whether the cottonseed polyphenol, -(-)gossypol, a BH3 mimetic, can reduce the expression of pro-survival, or increase the expression of pro-apoptotic, Bcl2 family proteins and thereby effectively sensitize otherwise resistant bladder cancer cells to the standard chemotherapeutic drugs gemcitabine, paclitaxel and carboplatin. These studies show that gossypol induced apoptosis in both chemosensitive UM-UC2 and chemoresistant resistant UM-UC9 bladder cancer cells in vitro in a dose and time dependent manner via a caspase mediated death signaling pathway. Moreover, in combined treatments, gossypol synergized with gemcitabine and carboplatin to induce apoptosis in chemoresistant bladder cancer cells. This effect was associated with the down-regulation the Bcl-xl and Mcl-1 pro-survival Bcl2 family proteins and up-regulation of the Bim and Puma BH3-only Bcl2 family proteins. Overall, these studies show that gossypol sensitizes bladder cancer cells to standard chemotherapeutic drugs and may provide a promising new strategy for bladder cancer treatment.     

Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway

We aimed to investigate the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human breast cancer cells. The molecular mechanisms involved in the apoptotic activity exerted by nimbolide were studied on the estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. The growth inhibitory effect of nimbolide was assessed by MTT assay. Apoptosis induction by nimbolide treatment was determined by JC-1 mitochondrial membrane potential staining, cytochrome c release, caspase activation, cleavage of PARP and AO/EtBr dual staining. The modulation of apoptotic proteins (intrinsic pathway: Bax, bad, Bcl-2, Bcl-xL, Mcl-1, XIAP-1 and caspase-3, 9; extrinsic pathway: TRAIL, FasL, FADDR and Caspase-8) were studied by western blot and real time PCR analysis. Treatment with nimbolide resulted in dose and time-dependent inhibition of growth of MCF-7 and MDA-MB-231 cells. The occurrence of apoptosis in these cells was indicated by JC-1 staining, modulation of both intrinsic and extrinsic apoptotic signaling molecules expression and further apoptosis was confirmed by AO/EtBr dual staining. These events were associated with: increased levels of proapoptotic proteins Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c and reduced levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP-1. Nimbolide induces the cleavage of pro-caspase-8, pro-caspase-3 and PARP. The above data suggest that nimbolide induces apoptosis by both the intrinsic and extrinsic pathways. With evidence of above data it is suggested that nimbolide exhibit anticancer effect through its apoptosis-inducing property. Thus, nimbolide raises new hope for its use in anticancer therapy.     

非小细胞肺癌中survivin表达及其与bcl-2表达的关系

目的：探讨非小细胞肺癌（non-small-cell lung cancer，NSCLC）组织中survivin蛋白表达及其与bcl-2蛋白表达的相关性。方法：应用免疫组织化学方法检测60例非小细胞肺癌组织及15例正常肺组织中survivin蛋白的表达，分析其与NSCLC临床病理参数之间的关系，并探讨其与bcl-2蛋白的表达的相互关系。结果：60例NSCLC组织中，survivin蛋白的阳性表达率为61．7％（37／60），15例正常肺组织中无survivin蛋白表达（P〈0．05）。survivin蛋白的表达与肺癌TNM分期、淋巴结转移和细胞分化程度有密切关系（P〈0．05），但与患者年龄、性别、吸烟与否及肿瘤组织学类型无关（p〉0．051。此外。survivin与bcl-2蛋白的表达具有相关性（P〈0．05）。结论：survivin蛋白在肺癌组织中高表达并与肺癌TNM分期、淋巴结转移和细胞分化程度有密切关系，提示该基因对NSCLC的发生发展起重要作用。survivin基因有望成为肺癌基因治疗的新靶点并可作为判断病情和评价预后的指标。survivin蛋白表达与bcl-2蛋白表达呈正相关．二者在非小细胞肺癌的发生中可能起协同作用。