放射对鼻咽癌细胞凋亡率和相关基因表达水平的影响

目的 探讨放射对人鼻咽高分化鳞癌细胞系(CNE-1)凋亡率和7个凋亡相关基因表达水平影响。方法 体外培养CNE-1,应用流式细胞术及RT-PCR方法检测0、2、4、6、8 Gy下CNE-1凋亡率及Bcl-2、Bcl-xl、Bcl-w、Bax、Bak、Bad、Bid的表达水平。用Pearson法进行基因表达与凋亡率、照射剂量,以及凋亡率与存活分数相关分析。结果 0、2、4、6 Gy下CNE-1早期凋亡率随照射剂量增加而逐渐增加,8 Gy时早期凋亡率不再增加反而下降;晚期凋亡率随照射剂量增加而增加。CNE-1照射后Bax表达上调,与早、晚期凋亡率及照射剂量呈正相关(所有P=0.000);Bcl-xl表达下调,与早、晚期凋亡率呈负相关(P=0.005、0.039),与照射剂量无相关性(P=0.369);Bcl-2表达上调,在4 Gy时到达峰值后开始下降;Bcl-w、Bcl-2、Bad、Bid表达水平与早、晚期凋亡率无相关性(P=0.058~0.894)。凋亡率与存活分数无相关性(P=0.064)。结论 Bax、Bcl-xl与CNE-1细胞凋亡率有一定相关性,但凋亡率与细胞存活分数无相关性。     

肝癌细胞凋亡中TIP30对Bcl-2蛋白家族的表达的调节(英文)

目的 TIP30为一新发现的可引起细胞代谢抑制和凋亡的因子。最新研究表明:TIP30可引起 Bcl-2家族的两个促凋亡的成员 Bad和 Bax 的表达升高并抑制小鼠肿瘤生长。本实验的目的在于研究在肝癌细胞系 HepG2、Hep3B 和 Hu-7的凋亡中,TIP30所参与的信号转导途径。方法采用 MTT、原位 DNA 末端标记、免疫印迹法研究 TIP30引起的细胞凋亡及其对 Bcl-2蛋白家族的调节。结果 TIP30可促使肝癌细胞的凋亡,同时上调 Bax、Bad 的蛋白水平而下调 Bcl-xl 的蛋白水平,但对 Bak 蛋白的水平没有影响。结论 TIP30参与的凋亡调节是通过调节 Bcl-2蛋白家族的水平实现的,阐明 TIP30的凋亡信号转导途径可望为肝癌的治疗开辟新的途径。     

肝癌细胞凋亡中TIP30对Bcl-2蛋白家族的表达的调节

目的TIP30为一新发现的可引起细胞代谢抑制和凋亡的因子。最新研究表明：TIP30可引起Bcl-2家族的两个促凋亡的成员Bad和Bax的表达升高并抑制小鼠肿瘤生长。本实验的目的在于研究在肝癌细胞系HepG2、Hep3B和Hu-7的凋亡中,TIP30所参与的信号转导途径。方法采用MTT、原位DNA末端标记、免疫印迹法研究TIP30引起的细胞凋亡及其对Bcl-2蛋白家族的调节。结果TIP30可促使肝癌细胞的凋亡,同时上调Bax、Bad的蛋白水平而下调Bcl-xl的蛋白水平,但对Bak蛋白的水平没有影响。结论TIP30参与的凋亡调节是通过调节Bcl-2蛋白家族的水平实现的,阐明TIP30的凋亡信号转导途径可望为肝癌的治疗开辟新的途径。     

DNA-PKcs 表达与鼻咽癌细胞株放射敏感性的关系

 目的 探讨不同放射敏感性鼻咽癌细胞株CNE-1（鼻咽高分化鳞癌细胞株）和CNE-2（鼻咽低分化鳞癌细胞株）中DNA依赖蛋白激酶（DNA-PK）的催化亚基DNA-PKCS基因的表达与鼻咽癌细胞放射敏感性的关系。方法 通过克隆形成实验测定CNE-1、CNE-2不同剂量的存活分数，并用线性二次模型拟合剂量存活曲线求出放射生物学参数α、β,SF2、MID值，以及四氮唑蓝比色分析法（MTT法）检测60Co-γ线4Gy照射后12h细胞的存活率，以评价两株细胞的放射敏感性。逆转录实时荧光定量PCR技术（RTrFQPCR）检测照射前、后不同时间及不同剂量CNE-1、CNE-2细胞mRNA水平DNA-PKCS基因的定量表达。结果CNE-1在各个剂量点的存活分数均比CNE-2高，MID值分别为2．78、1．61，SF2值分别为0．627、0．341；4Gy照射后12h的存活率分别为88．2％、72．3％；RT-FQPCR显示两株细胞中均有DNA-PKCS基因的表达，其相对表达量之比为7．54±2．71（t=4．17，P=0．014），表达差异有统计学意义，DNA-PKCS基因在CNE-2细胞中存在时间、剂量依赖关系。结论实验验证了CNB2比CNE-1对射线更敏感，DNA-PKCS基因的表达与鼻咽癌细胞的放射敏感性有关。     

斯钙素1的表达对肺癌细胞A549细胞周期及凋亡的影响

背景与目的：斯钙素1(stanniocalcin l,STC1)在多种癌组织中表达上调,且与癌组织的恶性程度相关,但STC1在肺癌细胞中的分子作用机制尚不明确。本研究旨在探讨STC1的表达对肺癌细胞A549细胞周期及凋亡的影响。方法：构建STC1基因RNA干扰的肺癌细胞株A549-STC1-siRNA和对照细胞株A549-Vector,用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测A549-Vector及A549-STC1-siRNA细胞株的细胞周期蛋白基因CyclinA、CyclinB1、CyclinD1、CyclinE、CDK2、CDK4,凋亡抑制基因Bcl-2、Bcl-xl及凋亡诱导基因Caspase-3、Bax、Bak、Bid的表达水平,用流式细胞术检测STC1基因对A549细胞周期的影响,用原位末端标记(terminal deoxynucleotidyl transferase-mediated nick-end labeling,TUNEL)检测STC1基因对A549细胞凋亡的影响。结果：与A549-Vector细胞相比,A549-STC1-siRNA的细胞周期蛋白基因CyclinA、CyclinB1、CyclinD1、CyclinE、CDK2和CDK4在转录和蛋白表达水平上均显著减少(P<0.05),G0/G1期细胞比例明显增加,S期及G2/M期细胞比例降低(P<0.05),细胞周期受阻;A549-STC1-siRNA的凋亡抑制基因Bcl-2和Bcl-xl表达下调(P<0.05),而凋亡诱导基因Caspase-3、Bax、Bak及Bid显著上调(P<0.05);TUNEL实验表明,A549-STC1-siRNA细胞的凋亡率明显增加。结论：STC1基因的低表达可阻滞肺癌细胞A549的细胞周期,抑制细胞增殖,同时促进细胞凋亡。     

XIAP抑制剂Embelin对人T淋巴瘤细胞Jurkat增殖抑制作用

  目的   研究XIAP抑制剂Embelin体外对人T淋巴瘤细胞Jurkat增殖的影响,并探讨其作用机制。   方法  应用MTS法分析不同浓度Embelin对人T淋巴瘤细胞增殖的影响;光学显微镜下观察经Embelin处理后细胞形态学的改变;经Annexin V/PI双染后用流式细胞术检测Embelin对Jurkat细胞凋亡的影响;Western blot方法检测Embelin作用后细胞XIAP、PARP、Caspase-3、Caspase-8、Caspase-9、促凋亡蛋白Bax和抗凋亡蛋白Bcl-xl、Bcl-2表达的变化。   结果  Emebeli对人白血病细胞具有显著增殖抑制作用（P < 0.05）;Caspase-3抑制剂z-DEVD-fmk,Caspase-9抑制剂Ac-LEHD-CHO能显著下调这种增殖抑制作用。经不同浓度Emebelin处理24 h后,Jurkat细胞凋亡率明显增高,与未处理组比较有显著性差异（P < 0.01）;经Emebelin处理24 h后,Jurkat细胞出现PARP、Caspase3、9裂解片段,且Bax蛋白表达上调,Bcl-2和Bcl-xL蛋白表达水平下调。   结论  Embelin在体外可明显抑制人T淋巴瘤细胞Jurkat的增殖,诱导细胞凋亡,其机制可能是通过拮抗XIAP的作用,激活Caspase依赖性的细胞凋亡内源途径而诱导Jurkat细胞发生凋亡。      

模拟调强模式下CNE-2细胞学中ATM和Ku80表达的研究

目的:研究延长时间的IMRT模式对鼻咽低分化鳞癌细胞株(CNE-2)与DNA损伤修复相关的Ku80、ATM转录水平表达的影响及临床意义.方法:选取鼻咽低分化鳞癌细胞株CNE-2为实验对象,分为常规照射、模拟IMRT两组,分别给予6MV-X线2、4、6、8Gy4个剂量点的照射;应用RT-PCR,检测CNE-2在不同照射模式下,与DNA损伤修复相关的Ku80、ATM转录水平表达.结果:常规照射与模拟IMRT组不同剂量点之间Ku80的转录水平表达差异有统计学意义(P均=0.000);在相同剂量点,常规照射组与模拟IMRT组LSD法两两比较,4个剂量点Ku80的转录水平表达差异均有统计学意义(P均<0.05);接受照射后CNE-2细胞中Ku80表达上调,模拟IMRT组Ku80的表达更高(P均=0.000),在4Gy剂量点时两组细胞Ku80的表达到达峰值,随后表达均下降.接受照射后,与空白对照比较,常规照射与模拟IMRT组CNE-2细胞中ATM转录水平表达均上调(P均<0.005);常规照射与模拟IMRT组不同剂量点之间,ATM的转录水平表达差异无统计学意义(P均>0.05);在相同剂量点,常规照射组与模拟IMRT组LSD法两两比较,ATM的转录水平表达差异均无统计学意义(P均>0.05).结论:由于亚致死性损伤修复增加,大幅延长照射时间的调强放疗的生物效应下降,Ku80的相对高表达可能是鼻咽低分化鳞癌细胞株模拟IMRT的放射生物学效应的下降的原因之一.     

EBV-miR-BART5-3p靶向p53对鼻咽癌细胞放射敏感性的影响

目的:研究爱泼斯坦-巴尔病毒（EBV）-miR-BART5-3p对鼻咽癌细胞放射敏感性的影响,并探讨其作用机制。方法:体外培养人EBV阳性鼻咽癌细胞（C666-1）和人鼻咽癌细胞（CNE-2Z）,将CNE-2Z组细胞设置为正常组,C666-1细胞随机分为对照组、EBV-miR-BART5-3p NC组、EBV-miR-BART5-3p mimics组和EBV-miR-BART5-3p inhibitor组。用实时荧光定量PCR（RT-qPCR）法检测各组细胞及EBV阴性鼻咽癌患者和EBV阳性鼻咽癌患者癌组织中EBV-miR-BART5-3p表达情况,噻唑蓝（MTT）法检测各组细胞存活率,平板克隆实验评估各组放疗敏感性变化情况,膜联蛋白V-异硫氰酸荧光素/碘化丙啶（Annexin V-FITC/PI）法检测各组细胞凋亡敏感性变化,蛋白免疫印迹分析法检测转染后各组细胞p53、Bcl-2相关X蛋白（Bax）、半胱天冬氨酸酶-3（caspase-3）和Bcl-2蛋白表达情况,双荧光素酶报告实验验证EBV-miR-BART5-3p与p53的靶向关系。结果:与EBV阴性组相比,EBV阳性组鼻咽癌组织中EBV-miR-BART5-3p表达水平显著升高（P＜0.05）。与正常组相比,对照组EBV-miR-BART5-3p表达、存活率、克隆数量和Bcl-2蛋白表达水平显著升高（P＜0.05）,凋亡率、p53、Bax和caspase-3蛋白表达水平显著降低（P＜0.05）。与对照组和EBV-miR-BART5-3p NC组相比,EBV-miR-BART5-3p mimics组EBV-miR-BART5-3p表达水平、存活率、克隆数量和Bcl-2蛋白表达水平显著升高（P＜0.05）,凋亡率、p53、Bax和caspase-3蛋白表达水平显著降低（P＜0.05）;与对照组和EBV-miR-BART5-3p NC组相比,EBV-miR-BART5-3p inhibitor组EBV-miR-BART5-3p表达水平、存活率、克隆数量和Bcl-2蛋白表达水平显著降低（P＜0.05）,凋亡率、p53、Bax和caspase-3蛋白表达水平显著升高（P＜0.05）。双荧光素酶报告实验结果显示,与TP53-3' UTR-WT+EBV-miR-BART5-3p NC组比较,TP53-3' UTR-WT+EBV-miR-BART5-3p inhibitor组荧光素酶活性降低（P＜0.05）。结论:下调EBV-miR-BART5-3p可能通过靶向促进p53蛋白表达,提高人EBV阳性鼻咽癌细胞的放射敏感性。     

组蛋白去乙酰化酶抑制剂SAHA体外抑制人卵巢癌SKOV3细胞的实验研究

  目的   目的: 观察组蛋白去乙酰化酶抑制剂辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid, SAHA) 体外对人卵巢癌SKOV3细胞增殖和凋亡的作用, 并探讨其可能机制。   方法  体外应用SAHA作用于人卵巢癌SKOV3细胞, MTT法检测细胞增殖, 流式细胞术检测细胞凋亡率。Western blot法检测细胞内组蛋白H4乙酰化水平, 实时定量PCR方法检测p21、Bcl-2和Bax基因mRNA表达。   结果  经SAHA作用后的人卵巢癌SKOV3细胞生长受到抑制, 细胞凋亡增加(各实验组与对照组比较, 均P < 0.05), 呈剂量依赖性; 同时细胞内组蛋白H4乙酰化水平增加, p21基因和Bax基因mRNA表达增加(各实验组与对照组比较, 均P < 0.05), 呈剂量依赖性, Bcl-2基因表达无明显变化(各实验组与对照组比较, 均P > 0.05)。   结论  SAHA体外可抑制人卵巢癌SKOV3细胞增殖和诱导SKOV3细胞凋亡, 提高组蛋白乙酰化水平, 增加p21和Bax基因表达可能是其作用机制之一。      

姜黄素对人鼻咽癌CNE-2Z细胞增殖及 凋亡的影响

 目的探讨姜黄素对人鼻咽癌CNE-2Z细胞增殖及凋亡的影响及其相关的分子机制。方法体外培养的 CNE-2Z细胞经不同剂量姜黄素处理后,用MTT法观察对细胞增殖的影响,流式细胞术检测细胞周期和凋亡 率的改变,Western blot检测姜黄素处理后相关蛋白Bcl-2、Bax表达水平的变化。结果姜黄素对CNE-2Z细 胞的生长和增殖都具有一定程度的抑制作用,阻断细胞于S期和G2/M期。姜黄素处理后CNE-2Z细胞凋亡抑 制基因Bcl-2蛋白表达减少,同时促凋亡基因Bax蛋白表达增加。 结论姜黄素可抑制CNE-2Z细胞增殖并促进 其凋亡。     

Immunohistochemical detection of apoptotic markers in gastric cancer

Apoptosis proteins may play a role in prognosis and therapy response; however, they have not been fully investigated in gastric cancer. We aimed to assess the expression of proteins in the Bcl-2 family. Immunohistochemistry was employed to examine the expression of the antiapoptotic proteins Bcl-2 and Bcl-XL and the proapoptotic proteins Bad, Bak, Bax, Bid, Bim, and p53 in 21 cases of gastric cancer. Immunopositivity was observed in 12/21 (57%) cases for p53, 16/21 (76%) cases for Bcl-XL, and 5/21 (23%) cases for Bcl-2. For the proapoptotic members of the Bcl family, loss of protein expression was observed: Bid (14/21 cases; 66%), Bad (13/21 cases; 61%), Bax (12/21 cases; 57%), Bak (9/21 cases; 42%), and Bim (4/21 cases; 19%). This study identified apoptosis proteins that exhibit heterogeneous expression between primary gastric carcinomas.     

Expression of bcl-2 family members in malignant pleural mesothelioma

Little is known about the Bcl-2 family members in mesothelioma. These proteins are involved in the control of apoptosis, carrying out both pro- and anti- apoptotic functions. Immunohistochemistry was used to examine the expression of p53 and Bcl-2 family members in 54 archival mesothelioma samples (39 epithelial, 15 sarcomatoid tumours). Overexpression of p53 was observed in 81% (44/54). For anti-apoptotic proteins, overexpression was recorded as follows: Bcl-2 40% (22/54), Bcl-XL 24% (13/54), Mcl-1 92% (50/54). For pro-apoptotic proteins, loss of expression was recorded as follows: Bad 25% (14/54), Bak 24% (13/54), Bax 42% (23/54), Bid 37% (20/54), Bim 18% (10/54). Statistically significant differences between epithelial and sarcomatoid tumours were observed for Bid (p < 0.001), Bad (p = 0.012) and Bcl-XL (p = 0.03). Significant differences in abnormal expression of apoptosis proteins were found between epithelial and sarcomatoid subtypes but histological subtype was the only factor with significant association to patient prognosis.     

肝癌细胞凋亡中TIP30对Bcl-2蛋白家族的表达的调节

Objective: To investigate the role of TIP30 in the apoptotic signal pathway in HepG2, and Hep3B and Hu-7 hepatoblastoma cell lines. Methods: In order to confirm whether TIP30 conducted Bcl-2 family was involved in apoptosis signal pathway, MTT assay, in situ 3 end labelling of DNA assay and Western blot were carried out to detect the diverse apoptotic function of TIP30 and the regulation of Bcl-2 family. Results: TIP30 induced apoptosis as evidenced by morphological changes in hepatoblastoma cells, which was accompanied by up-regulating Bax and Bad proteins and stimulating them from cytoplasm to mitochondria, and down-regulating Bcl-xl, while it had no effect on the level of Bak protein. Conclusion: TIP30 induced apoptosis partly by modulating the protein levels of members of Bcl-2 family in hepatoblastoma cells. Elucidating the mechanism by which TIP30 induces cell death might establish it as an anticancer factor.     

Involvement of proapoptotic molecules Bax and Bak in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced mitochondrial disruption and apoptosis: differential regulation of cytochrome c and Smac/DIABLO release

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L induces apoptosis in a wide variety of cancer and transformed cells. Activation of BID, a "BH3-domain-only" Bcl-2 family member, triggers the oligomerization of proapoptotic family members Bak or Bax, resulting in the release of mitochondrial proteins to cytosol. In this study, we have shown the importance of Bax and Bak in TRAIL-induced apoptosis by studying in murine embryonic fibroblasts (MEFs) from Bax(-/-) and Bak(-/-) animals. TRAIL induced cytochrome c release and apoptosis in wild-type, Bid(-/-), Bax(-/-), or Bak(-/-) MEFs, but not in Bax(-/-) Bak(-/-) double knockout (DKO) MEFs. Bid, which functions upstream of cytochrome c release, was cleaved in all of the knockout cells except in Bid(-/-) MEFs. The release of cytochrome c was correlated with caspase-9 activity. TRAIL increased caspase-3 activity in all of the cells except in DKO cells. TRAIL-induced drop in mitochondrial membrane potential was not observed in DKO MEFs. Unlike cytochrome c release, TRAIL-induced Smac/DIABLO release was blocked in Bid(-/-), Bax(-/-), Bak(-/-), or DKO MEFs, suggesting the differential regulation of these mitochondrial proteins during apoptosis. The apoptotic events downstream of mitochondria were intact in DKO MEFs, because microinjection of cytochrome c, or ectopic expression of mature Smac/DIABLO or pretreatment of Smac N7 peptide completely restored TRAIL sensitivity. In conclusion, the data suggest that Bax and Bak differentially regulate the release of cytochrome c and Smac/DIABLO from mitochondria, and Smac/DIABLO can be used to sensitize cells that are deficient in Bax and Bak genes, or resistant to TRAIL.     

Determination of the in vivo effects of prednisone on Bcl-2 family protein expression in childhood acute lymphoblastic leukemia

Glucocorticoid resistance is often associated with treatment failure in children with acute lymphoblastic leukaemia (ALL) but the underlying molecular mechanisms are still unclear. In 30 consecutive children with ALL treated with prednisone we determined changes in the expression of Bcl-2, Bax and Bcl-xl proteins in leukemic lymphoblasts and related these to clinical features and rate of prednisone-induced apoptosis. The apoptotic index increased after prednisone therapy in 24 of the 30 patients. At diagnosis, we detected expression of Bcl-2 and Bcl-xl protein in 28 samples, while Bax expression protein was detected in 21 of the 30 patients. Prednisone treatment induced a decrease in Bcl-2 and Bcl-xl levels in 17 and 16 of the 28 patients, respectively, while Bax protein increased in 14 of the 21 patients. Twenty of the 30 patients studied were considered to be good prednisone responders, whereas 10 were poor responders. We observed a statistically significant decrease only for Bcl-xl protein expression in T phenotype ALL, in the poor responder group and in patients with >20000/mm(3) white cell count (WBC) at diagnosis. These data suggest a role of Bcl-xl in the mechanisms of protection of leukemic cells from apoptosis induced by glucocorticoids (GCs).     

Activation of mitochondrial voltage-dependent anion channel by apro-apoptotic BH3-only protein Bim

Bcl-2 family of proteins regulates apoptosis by controlling mitochondrial membrane permeability. We have previously shown that the voltage-dependent anion channel (VDAC) plays a crucial role in apoptotic changes of the mitochondria and its activity is directly regulated by some Bcl-2 family members, including Bcl-2/Bcl-x(L) and Bax/Bak but not Bid. Here, we showed that in isolated mitochondria, Bim induced loss of membrane potential and cytochrome c release like Bax/Bak, with these changes being inhibited by an anti-VDAC antibody. In addition, microinjection of the anti-VDAC antibody significantly reduced Bim-induced apoptosis. Study using purified proteins indicated that Bim directly interacts with the VDAC. Immunoprecipitation analysis revealed that Bim interacts with the VDAC and the interaction is remarkably enhanced during apoptosis. An experiment using liposomes indicated that Bim enhanced VDAC activity, as did Bax/Bak. Furthermore, Bim (but not tBid) was able to induce apoptotic changes of yeast mitochondria in a VDAC-dependent manner, and also induced the lysis of red blood cells, with this effect being inhibited by the anti-VDAC antibody. These results indicate that Bim has an ability to activate directly the VDAC, which plays an important role in apoptosis of mammalian cells.