过表达PRRX1 通过p53 介导的线粒体凋亡途径诱导肝癌SMMC7721 细胞凋亡

[摘要] 目的：探讨配对相关同源框1 蛋白（PRRX1）过表达对肝癌SMMC7721 细胞凋亡的影响及其分子机制。方法：分别用慢病毒介导PRRX1 过表达载体（pGMLV-PRRX1）、空载质粒（Vector）感染人肝癌SMMC7721 细胞,用qPCR和WB实验检测慢病毒感染后细胞中PRRX1 mRNA和蛋白的表达变化,用CCK-8 法、Annexin-V FITC/PI 染色流式细胞术分别检测PRRX1 过表达对SMMC7721 细胞增殖、凋亡的影响,用线粒体膜电位检测试剂盒（JC-10 染色法）检测细胞线粒体膜电位变化,用caspase 活性检测试剂盒（分光光度法）测定细胞中caspase-8 和caspase-9 酶活性,用WB实验检测细胞中p53、Bcl-2、Bax、Fas、Cleaved-caspase-3以及线粒体和细胞质中细胞色素C（Cty C）蛋白的表达。结果：成功构建PRRX1 过表达的SMMC7721 细胞株,感染细胞中PRRX1 mRNA和蛋白的表达水平显著升高（均P<0.01）。与对照组和空载组比较,PRRX1 过表达组SMMC7721 细胞的增殖能力显著下降、细胞凋亡率显著增高、Cleaved-caspase-3 剪切水平显著升高、线粒体膜电位显著下降、线粒体中Cty C蛋白表达下调、胞质中Cty C蛋白表达上调以及caspase-9 酶活性升高（P<0.05 或P<0.01）,同时p53 和Bax 蛋白表达增加而Bcl-2 蛋白表达降低（均P<0.05）,但Fas 蛋白表达及caspase-8 酶活性无显著变化（均P>0.05）。结论: PRRX1 过表达可诱导肝癌SMMC7721 细胞凋亡,其机制可能与p53介导的线粒体凋亡途径被激活有关。     

胆管癌p53-bax线粒体凋亡通路的甲基化研究

目的探讨胆管癌p53-bax线粒体凋亡通路中多个基因的甲基化状态及其在胆管癌发生过程中的意义。方法采用甲基化特异性聚合酶链反应（MSP），对胆管癌组织和癌旁组织中的p14^ARF、DAPK和TMS1／ASC基因启动子的甲基化状态进行检测，并对胆管癌组织中p53基因外显子5～8进行DNA序列分析。结果36例胆管癌组织标本中，有24例（66．7％）至少存在1个抑癌基因的甲基化，其中p14^ARF、DAPK和TMS1／ASC基因甲基化的比率分别为25．0％、30．6％和36．1％；癌旁组织中，有5例（13．9％）存在抑癌基因的甲基化，其中TMS1／ASC3例（8．3％），DAPK2例（5．6％）。36例胆管癌组织标本巾，有22例（61．1％）存在p53基因的突变。p53突变伴1个以上抑癌基因甲基化者共14例，占38．9％，其发生率与胆管癌的病理类型、分化程度和浸润深度有关（P〈0．05）。结论p53-bax线粒体凋亡通路中，DNA甲基化是胆管癌中常见的分子事件。癌旁组织中，DAPK和TMS1／ASC基因的甲基化率虽然较低，但可能有早期诊断意义。p53突变伴抑癌基因的甲基化与胆管癌的病理生物学行为有关，并趋向于较高的恶性程度。     

腺病毒介导野生型p53基因诱导膀胱癌细胞凋亡

p53基因是一种抑癌基因,它在肿瘤发生过程中有重要作用.p53基因的突变可引起细胞增殖,导致悼瘤的发生.迄今已在许多恶性肿瘤细胞中发现p53基因的突变.将外源野生型p53基因导入p53基因突变的肿瘤细胞,能抑制其恶性增殖,逆转其恶性表型.这些研究为肿瘤的基因治疗奠定了基础;值得注意的是,至今大多数研究均用有p53基因缺失或突变的肿瘤细胞,但并非所有的肿瘤细胞都发生p53基因突变;本文将外源野生型p53基因导入表达内源野生型p53基因的膀胱癌细胞株EJ,观察其抑制效应及凋亡的诱导作用.     

TGF-β1诱导视网膜母细胞瘤细胞凋亡及对p53的影响

肺癌与其它实体瘤一样,其生长、转移和预后均依 赖血管生成,以新生血管为靶点,抑制肿瘤血管生成, 阻断肿瘤的营养来源和迁移通道,已成为癌症治疗的 新策略。有研究发现,血管内皮生长因子(Vascular en- dothelial growth factor,VEGF)为一高度特异的血管内 皮细胞有丝分裂素,可直接诱导肿瘤血管生成。我 们利用近年兴起的反义技术,设计VEGF反义寡核苷 酸片断,使其特异性封闭肿瘤细胞中的VEGF mRNA, 通过研究它对肿瘤细胞合成VEGF及对肿瘤生长的影 响,探讨其对肺癌治疗新途径的可能性。     

神经酰胺通过非p53途径诱导鼻咽癌细胞中p21WAF1的表达

[目的]探讨第二信使神经酰胺信号传导诱导鼻咽癌细胞p21^WAF1表达的机理。[方法]Western blot法检测p53,p21,Jun,NFкB基因表达,试剂盒检测SAPK/JNK的活性。[结果]在鼻咽癌CNE2细胞中阿霉素能刺激p53表达,不能上调p21^WAF1的表达,可见在CNE2细胞中p53为功能异常型,C2-神经酰胺处理CNE2细胞后,可见p53蛋白呈现下降趋势;而p21^WAF1蛋白在处理4、16小时时,p21^WAF1表达明显增高,但36小时后p21^WAF1恢复到基础水平,在6.25μmmol/L,12.5μmmol/L,25μmmol/L的C2-神经酰胺处理24小时后p21^WAF1上升,而50μmmol/L的C2-神经酰胺处理24小时后,p21^WAF1开始下降,C2-神经酰胺处理CNE2细胞2小时后,JNK的活性开始增加,6小时时保持激活状态,12小时时下降,c-Jun/AP1蛋白在神经酰胺处理后3小时开始明显增高,持续到12小时,24小时下降至基础水平;C2-神经酰胺对CNE2细胞中NFкB蛋白表达没有明显的变化。[结论]C2-神经酰胺在鼻咽癌细胞CNE2中,通过p53非依赖性途径诱导p21^WAF1的表达,神经酰胺激活JNK,从而激活AP-1因子,可能是其诱导p21^WAF1表达的机理。     

胆管癌p53-Bax线粒体凋亡通路DNA甲基化机制的研究

Objective: To study the methylation status of several genes on p53-Bax mitochondrial apoptosis pathway and clinical significance in cholangiocarcinoma. Methods: Promoter hypermethylation of DAPK, p14 and ASC genes were de-tected by methylation-specific PCR. p53 gene status (exon 5-8) were examined by automated sequencing, combined with the clinical documents of patients by statistics analysis. Results: (1) We found 66.7% of 36 cases cholangiocarcinoma had meth-ylation of at least one tumor suppressor gene. The frequency of tumor suppressor gene methylation in cholangiocarcinoma was: p14 (24%), DAPK (30.6%), TMSI/ASC (36.1%). The frequency of tumor suppressor gene methylation in tissues near cancer was: DAPK (5.6%), TMSI/ASC (8.3%). (2) p53 gene mutations were found in 22 of 36 patients (61.1%). (3) There were no statistically relationship among the methylation of DAPK, p14 and ASC genes. There were negative relationship differences between the methylation of p14 and p53 gene mutation (P<0.05). (4) p53 gene mutation combined with the methylation of tumor suppressor were 14 cases (38.9%). There were statistically differences on extent of pathologic biology, differentiation and invasion (P<0.05). Conclusion: Our study indicated that methylation of p53-Bax mitochondrial apoptosis pathway in cholangiocarcinoma was a common epigenetic event. Although the methylation of ASC, DAPK genes was low, it might be significance for early diagnosis, p53 gene mutation combined with the methylation of tumor suppressor might be relationship with pathologic biology, it trended to more malignancy.     

食管癌变过程中p53表达及其与细胞凋亡水平的关系

[目的]探讨食管癌变过程中食管鳞状上皮p53表达情况及其与凋亡水平的关系。[方法]824例食管黏膜活检组织（包括正常黏膜、异型增生和鳞癌）,行DNA末端标记（TUNEL法）检测及p53免疫组化检查。[结果]①轻—中度不典型增生组p53表达阳性率高于正常及炎症组和良性增生组（P=0.003）,低于重度不典型增生/鳞癌组（P=0.036）;良性增生组与轻—中度不典型增生组间无明显差异（P=0.192）;②正常及炎症组和良性增生组随年龄增长p53表达呈上升趋势（P〈0.01）;轻中—度不典型增生组及重度不典型增生组各年龄组间差异不明显;③随着p53表达的增加,上皮颗粒层细胞凋亡指数（AI）逐步下降（χ2=87.266,P=0.000）,基底层AI呈轻度增加趋势（χ2=34.253,P=0.000）。[结论]食管癌变过程中伴有p53基因的突变和颗粒层细胞凋亡水平的下降,且两者关系密切。     

Livin蛋白在食管癌中的表达及与p53蛋白相关性研究

目的：探讨凋亡抑制蛋白Livin在食管癌组织中的表达及其与p53蛋白之间的关系。方法：采用免疫组织化学S-P法检测Livin蛋白和p53蛋白在食管癌组织和癌旁正常食管组织中的表达情况。结果：Livin在食管癌组织中的表达明显高于癌旁正常食管组织（P〈0.01）。Livin表达与癌组织浸润深度和淋巴结转移呈正相关（P〈0.05）,而与癌组织分化程度无关（P〉0.05）。p53在食管癌组织中表达明显高于癌旁正常食管组织（P〈0.01）。在食管癌组织中Livin与p53表达无关（P〉0.05）。结论：Livin在食管癌组织中异常表达,有望成为食管癌诊断和基因治疗的新靶点。抑癌基因p53的失活与凋亡抑制基因Livin在食管癌的发生方面无相互促进作用。     

冬凌草甲素诱导食管癌细胞凋亡过程中线粒体结构与膜电位的改变

目的:研究冬凌草甲素(Oridonin,ORI)诱导食管癌细胞凋亡的过程中线粒体超微结构和功能的变化。方法:采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法和透射电镜法检测细胞凋亡和超微结构改变;罗丹明123(Rhodamine 123,Rho123)荧光探针标记流式细胞仪检测和分析线粒体跨膜电位(MTP,△Ψm)的改变。结果:32μg/ml ORI作用2h后电镜下SHEEC细胞线粒体增多,4h后线粒体肿胀空泡化、内部结构消失,8h后细胞核染色质成块状边集,细胞凋亡。ORI作用24h后,代表线粒体膜电位的Rho123荧光强度降低。结论:在ORI诱导下,SHEEC细胞线粒体有明显的形态和功能改变伴随线粒体△Ψm降低,线粒体改变可能是ORI诱导食管癌细胞凋亡过程的重要环节。     

滋肾固髓汤通过调控p38MAPK/p53信号通路诱导结直肠癌HCT-116细胞凋亡

目的:探讨滋肾固髓汤对结直肠癌HCT-116细胞凋亡的影响及其分子机制。方法:制备滋肾固髓汤含药血清,首先采用不同体积分数滋肾固髓汤含药血清处理HCT-116细胞,噻唑蓝(MTT)检测细胞增殖活性;再将HCT-116细胞分为空白组、滋肾固髓汤低、中、高剂量组、SB203580(p38MAPK抑制剂)组和高剂量滋肾固髓汤+SB203580组,分别加入相应药物进行干预后,Hoechst 33258染色观察细胞凋亡的形态学变化;Annexin V-FITC流式细胞术检测细胞的凋亡水平;Western blot检测细胞中p38MAPK、磷酸化(p)-p38MAPK(Thr180/Tyr182)、鼠双微染色体2(MDM2)、p53、Cleaved caspase-3、Bax和Bcl-2等蛋白表达水平。结果:滋肾固髓汤可抑制HCT-116细胞增殖活性,并呈时间和浓度依赖性。不同浓度滋肾固髓汤处理可诱导HCT-116细胞呈现核聚集、皱缩或碎裂等凋亡形态变化,促进细胞凋亡,上调p-p38MAPK、p53、Bax和Cleaved caspase-3等蛋白表达水平,下调MDM2和Bcl-2蛋白表达水平。S...     

Chloroquine activates the p53 pathway and induces apoptosis in human glioma cells

Glioblastoma is the most common malignant brain tumor in adults. The currently available treatments offer only a palliative survival advantage and the need for effective treatments remains an urgent priority. Activation of the p53 growth suppression/apoptotic pathway is one of the promising strategies in targeting glioma cells. We show that the quinoline derivative chloroquine activates the p53 pathway and suppresses growth of glioma cells in vitro and in vivo in an orthotopic (U87MG) human glioblastoma mouse model. Induction of apoptosis is one of the mechanisms underlying the effects of chloroquine on suppressing glioma cell growth and viability. siRNA-mediated downregulation of p53 in wild-type but not mutant p53 glioblastoma cells substantially impaired chloroquine-induced apoptosis. In addition to its p53-activating effects, chloroquine may also inhibit glioma cell growth via p53-independent mechanisms. Our results clarify the mechanistic basis underlying the antineoplastic effect of chloroquine and reveal its therapeutic potential as an adjunct to glioma chemotherapy.     

Overexpression of Sp1 leads to p53‐dependent apoptosis in cancer cells

Numerous studies have documented that Sp1 expression level were elevated in various human cancers. However, the promoters of many pro‐apoptotic genes have been found to contain the Sp1 binding elements and are activated by Sp1 overexpression. To better understand the role and the mechanism of increased Sp1 levels on apoptosis, we used adenovirus to ectopically express GFP‐Sp1 protein in various cancer cell lines. First, in HeLa and A549 cells, we found that Sp1 overexpression suppressed the cell growth and increased the detection of sub‐G1 fraction, caspase‐3 cleavage, and annexin‐V signal revealed that apoptosis occurred. Furthermore, when cells entered the mitotic stage, the cell apoptosis was induced by Sp1 overexpression through affecting mitotic chromatin packaging. We also verified that p53 protein was accumulated and activated the p53‐dependent apoptotic pathways in the wild‐type p53 cells but not in the p53‐mutated or p53‐deleted cell lines when these cells were infected with adeno‐GFP‐Sp1 virus. In addition, A549 (p53^+/+) cells could be protected from apoptosis under Sp1 overexpression when p53 was knockdown by p53 shRNA. Finally, H1299 (p53^?/?) cell viability was significantly inhibited by adeno‐GFP‐Sp1 virus infection in the expression of p53. In conclusion, p53 was an essential factor for Sp1 overexpression‐induced apoptotic cell death in transforming cells. © 2009 UICC     

Dulxanthone A induces cell cycle arrest and apoptosis via up-regulation of p53 through mitochondrial pathway in HepG2 cells

Natural products derived from plants provide a rich source for development of new anticancer drugs. Dulxanthone A was found to be an active cytotoxic component in Garcinia cowa by bioactivity-directed isolation. Studies to elucidate the cytotoxic mechanisms of dulxanthone A showed that dulxanthone A consistently induced S phase arrest and apoptosis in the most sensitive cell line HepG2. Furthermore, p53 was dramatically up-regulated, leading to altered expression of downstream proteins upon dulxanthone A treatment. Cell cycle related proteins, such as cyclin A, cyclin B, cyclin E, cdc-2, p21 and p27 were down-regulated. Some apoptosis correlated proteins were also altered following the drug treatment. Bcl-2 family members PUMA was up-regulated while Bcl-2 and Bax were down-regulated. However, the expression ratio of Bax/Bcl-2 was increased. This resulted in the release of cytochrome C from the mitochondria to the cytosol. Concurrently, Apaf-1 was stimulated with p53 by dulxanthone A. In result, cytochrome C, Apaf-1 and procaspase-9 form an apoptosome, which in turn triggered the activation of caspase-9, caspase-3 and downstream caspase substrates. Lamin A/C and PARP were down-regulated or cleaved, respectively. Moreover, cell cycle arrest and apoptosis in HepG2 cells induced by dulxanthone A were markedly inhibited by siRNA knockdown of p53. In summary, dulxanthone A is an active cytotoxic component of G. cowa. It induces cell cycle arrest at lower concentrations and triggers apoptosis at higher concentrations via up-regulation of p53 through the intrinsic mitochondrial pathway in HepG2 cells. Dulxanthone A is therefore likely a promising preventive and/or therapeutic agent against Hepatoma.     

Simvastatin induces apoptosis in human breast cancer cells: p53 and estrogen receptor independent pathway requiring signalling through JNK

The effect of simvastatin, a widely used statin for the treatment of hypercholesterolemia, was investigated in the estrogen receptor (ER)-positive MCF-7, and the ER-negative MDA-MB 231 human breast cancer cell lines. Simvastatin induced cell cycle arrest and apoptosis in both cells. These effects of simvastatin were not altered by 17-β-estradiol treatment.

MCF-7 cells express wild-type tumor suppressor protein p53, whereas MDA-MB 231 cells carry a p53 mutation. However, no alteration in the level or localisation of p53 was observed with simvastatin treatment in either cell line. On the other hand, simvastatin strongly stimulated phosphorylation of c-jun which was completely abolished by the c-jun NH₂-terminal kinase (JNK) inhibitor SP600125, which also significantly reduced the antiproliferative and apoptotic effects of simvastatin in these cells.

In conclusion, we describe here that simvastatin induces apoptosis via involvement of JNK in breast cancer cells independent of their ER or p53 expression status. These findings indicate a great potential for statins for the treatment of cancers resistant to currently used drugs, and target the JNK signalling pathway for a novel approach of breast cancer treatment.     

Id-1 promotes proliferation of p53-deficient esophageal cancer cells

The helix-loop-helix protein inhibitor of differentiation and DNA binding (Id-1) is known to promote cellular proliferation in several types of human cancer. Although it has been reported that Id-1 is over-expressed in esophageal squamous cell carcinoma (ESCC), its function and signaling pathways in esophageal cancer are unknown. In our study, we investigated the direct effects of Id-1 on esophageal cancer cell growth by transfecting an Id-1 expression vector into an ESCC cell line (HKESC-3), which showed serum-dependent Id-1 expression. Ectopic Id-1 expression resulted in increased serum-independent cell growth and G1-S phase transition, as well as up-regulation of mouse double minute 2 (MDM2) and down-regulation of p21Waf1/Cip1 protein expressions in the transfectant clones in a p53-independent manner. However, overexpression of Id-1 had no effect on the pRB, CDK4 and p16INK4A expressions. Stable transfection of Id-1 antisense expression vector to inhibit the expression of endogenous Id-1 in another ESCC cell line (HKESC-1) reversed the effects on MDM2 and p21Waf1/Cip1. In addition, Id-1 expression protected ESCC cells from Tumor Necrosis Factor (TNF)-alpha-induced apoptosis by up-regulating and activating Bcl-2. In conclusion, our study provides evidence for the first time that Id-1 plays a role in both proliferation and survival of esophageal cancer cells. Our findings also suggest that unlike prostate, hepatocellular and nasopharyngeal carcinomas in which Id-1 induces cell proliferation through inactivation of p16INK4A/RB pathway, the increased cell proliferation observed in ESCC cells may be mediated through a different mechanism.