携带人TRAIL基因的双靶向溶瘤腺病毒与X线联合抑瘤效应

目的探讨携带人TRAIL基因的溶瘤腺病毒CNHK500-hTRAIL,联合X线照射对A549细胞生长的影响。方法将质粒pPE3-hTRAIL与pSG500用Lipofectamine2000共转染至293细胞内同源重组,包装出的溶瘤腺病毒CNHK500-hTRAIL,采用PCR法进行鉴定,并在293细胞中大量扩增病毒,采用50%组织培养感染计量法测定病毒滴度,以MOI为5的溶瘤腺病毒CNHK500-hTRAIL感染A549细胞24 h,用RT-PCR法检测hTRAIL基因在肿瘤细胞中的表达。用不同MOI值(0.01～40)的CNHK500-hTRAIL感染A549细胞,感染5 d,通过四甲基偶氮唑蓝(MTT)比色法检测其对A549细胞的细胞存活率,以便确定适宜的感染滴度。用CNHK500-hTRAIL感染A549细胞,感染后48 h,用不同剂量的X线(1和2 Gy)照射,照射后3 d,通过MTT比色法检测CNHK500-hTRAIL联合X线照射下A549细胞的细胞存活率。结果 PCR法鉴定表明溶瘤腺病毒CNHK500内携带hTRAIL基因,制备的CNHK500-hTRAIL病毒滴度为3.25×109PFU/ml,且CNHK500-hTRAIL携带的hTRAIL基因在A549细胞中有效表达。经不同MOI值CNHK500-hTRAIL感染A549细胞,细胞存活率为64.26%～96.02%。CNHK500-hTRAIL感染联合不同剂量X线照射组细胞存活率较单纯X线照射(1和2 Gy)和单纯CNHK500-hTRAIL感染组明显降低(P<0.01),细胞存活率分别为50.77%和45.56%,增敏效应比值(E/O)为1.5和1.6,均大于1.4。结论成功构建了携带hTRAIL基因的溶瘤腺病毒CNHK500-hTRAIL,CNHK500-hTRAIL感染与X射线联合照射对A549细胞生长有协同抑制作用。     

TRAIL与环磷酰胺诱导LN215细胞凋亡的作用

目的探讨人恶性胶质瘤细胞株LN215细胞对TRAIL的敏感性及TRAIL与环磷酰胺诱导LN215细胞凋亡的作用。方法培养LN215细胞,在倒置显微镜下观察细胞的形态及生长特性,应用酸性磷酸酶法检测LN215细胞对TRAIL单独及联合环磷酰胺作用诱导凋亡的程度。结果 LN215细胞在TRAIL低浓度时无明显的凋亡发生,甚至出现轻微的增殖现象;TRAIL浓度达到1 ng/ml时,细胞开始出现凋亡;随TRAIL浓度升高,凋亡水平逐渐升高,在300 ng/ml时,凋亡率可达到12%。TRAIL与环磷酰胺联合作用组(0.11±0.01)与对照组(0.29±0.02)相比具有显著性差异(P﹤0.01)。结论 LN215是TRAIL耐药细胞株,与环磷酰胺联合后可发挥协同作用,引起细胞发生明显的凋亡反应。     

XAF1增加TRAIL诱导的肝癌细胞凋亡的敏感性

背景:肿瘤坏死因子(TNF)相关凋亡诱导配体(TRAIL)可特异性诱导肿瘤细胞凋亡,但部分肿瘤细胞对TRAIL不敏感甚至耐药。目的:探讨X连锁凋亡抑制蛋白(XIAP)相关因子1(XAF1)是否可增加TRAIL诱导的肝癌细胞株凋亡的敏感性。方法:重组人TRAIL(rhTRAIL)因子分别加入3株肝癌细胞株SMMC7721、HepG2和Bel-7404中培养,联合或不联合相同感染复数(MOI)的重组腺病毒Ad5/F35-XAF1和对照空病毒Ad5/F35-Null。作用48h后,以MTT法检测细胞活力,以Annexin V-FITC/PI法检测细胞凋亡率。结果:随着TRAIL浓度的增加,3株肝癌细胞株的细胞活力均不同程度降低。TRAIL与Ad5/F35-XAF1联合作用后,其细胞活力显著低于单独TRAIL组,而Ad5/F35-Null组细胞活力与单独TRAIL组无明显差异。与空白对照组和Ad5/F35-Null组相比,TRAIL组和Ad5/F35-XAF1组3株肝癌细胞株的凋亡率均显著增加。TRAIL与Ad5/F35-XAF1联合作用后,细胞凋亡率显著高于Ad5/F35-XAF1组或TRAIL组。结论:XAF1可明显增加TRAIL诱导的肝癌细胞凋亡的敏感性,两者联合应用对诱导不同肝癌细胞凋亡具有协同作用。     

TRAIL 基因转染对人宫颈癌 Hela 细胞凋亡的影响

目的：探讨外源性肿瘤坏死因子相关凋亡配体（ TRAIL）基因对人宫颈癌Hela细胞凋亡的影响。方法将构建的真核表达质粒pEGFP-TRAIL用Lipofectamine2000转染试剂盒转染到Hela细胞中,共聚焦显微镜下观察质粒的表达,分别用DAPI细胞核染色法和四甲基偶氮唑蓝（ MTT）比色法细胞染色法检测Hela细胞凋亡率。结果真核表达质粒pEGFP-TRAIL构建成功且能在Hela细胞中持续表达;转染真核表达质粒pEGFP-TRAIL的Hela细胞、转染空载体pEGFP-C1的Hela细胞凋亡率分别为12．1％、61．2％,两组比较,P＜0．05。结论外源性TRAIL基因可诱导Hela细胞凋亡。     

人端粒酶逆转录酶干扰对肿瘤坏死因子相关凋亡诱导配体诱导肝癌细胞凋亡的影响

目的研究人端粒酶逆转录酶(hTERT)干扰对肝癌．HepG2、SMMC-7221细胞生物学形为的影响和对肿瘤坏死因子相关的凋亡诱导配体(TRAIL)诱导凋亡的影响。方法将HepG2细胞和SMMC-7721细胞分为转染组 (转染重组质粒真核表达载体)、对照组(转染空载体质粒)和未转染组。采用聚合酶链反应方法检测hTERT干扰序列, 逆转录聚合酶链反应方法检测hTERT表达,HE染色、生长曲线和流式细胞术方法分别检测细胞形态、增殖情况和细胞周期,β-半乳糖苷酶染色方法检测细胞状态,Armexin V／PI染色流式细胞术检测细胞凋亡。结果转染组细胞内均存在hTERT干扰序列,HepG2和SMMC 7221细胞hTERT干扰率分别为100％和43．3％;与未转染组细胞相比, 转染细胞核质比明显缩小,增殖率下降差异有统计学意义(P<0．05),老化细胞和G2-M期细胞明显增加(P<0．05)。细胞老化率分别由未转染组的0增加到转染组的20．4％,由3．60%,增加到10．O％;G2-M期分别由未转染组的7．1％、6．9％增加到转染组的10．6％、7．9％。hTERT干扰显著增加肝癌细胞凋亡和TRAIL诱导凋亡敏感性(P<0．05)。两株肝癌细胞凋亡率分别由未转染组的3．5％、4．8％增至转染组的5．2%、7．9％;100 ng／ml TRAIL作用24 h后两株肝癌细胞凋亡率分别由未转染组的5．3％、13．9％增加到转染组的10．4％、77．2％,而对照组细胞各指标均无显著变化。结论 hTERT干扰明显影响肝癌细胞的生物学行为,显著增加细胞凋亡和TRAIL诱导凋亡的敏感性。     

肿瘤坏死因子相关凋亡诱导配体与肝细胞凋亡的相关性

肿瘤坏死因子相关的调亡诱导配体是TNF家族成员之一,通过其死亡受体诱导凋亡.研究显示TRAIL/死亡受体途径参与多种肝脏病理过程,本文就TRAIL/死亡受体途径的特性、致凋亡的机制及与肝细胞凋亡关系的最新进展作一综述.     

TRAIL联合顺铂诱导胶质瘤细胞凋亡的机制

作为新型的抗肿瘤药物,肿瘤坏死因子相关凋亡诱导配体(TRAIL)已进入临床Ⅱ期试验阶段[1].研究发现,TRAIL可以诱导包括胶质瘤在内的多种肿瘤细胞发生凋亡,但大多恶性胶质瘤细胞对TRAIL耐药[2].前期研究的实验发现,原代培养的胶质瘤细胞对TRAIL诱导凋亡的敏感性不同,检测发现死亡受体(DR)5的表达量不同,并当TRAIL与化疗药物联合作用后可发挥协同作用[3～5].因此,本文进一步对TRAIL与化疗药物联合作用诱导胶质瘤细胞凋亡的机制进行研究,为TRAIL作为肿瘤治疗新药提供新的实验依据.     

紫杉醇增强TRAIL诱导的胃癌BGC823细胞凋亡的机制

目的 探讨紫杉醇增强TRAIL诱导的胃癌BGC823细胞凋亡的机制。方法胃癌BGC823细胞传代堵养后,取对数生长期细胞用于实验。采用MTT法测定细胞活力,流式细胞仪检测细胞凋亡,Westernblot检测Akt、p-Akt蛋白表达。结果在胃癌BGC823细胞中,100ng／ml的TRAIL可致少量的细胞凋亡,同时检测到Akt的磷酸化。紫杉醇作用胃癌BGC823细胞24h,IC50剂量为8．97μg／ml。与单药TRAIL和紫杉醇相比,TRAIL（100ng／m1）联合紫杉醇（8．97μg／ml,24h的IC50剂量）对细胞的诱导凋亡作用明显增强（P〈0．05）。免疫印迹结果显示,TRAIL（100ng／ml）作用BGC823细胞24h,活化了Akt蛋白,而8．97μg／ml的紫杉醇抑制了Akt的磷酸化。TRAII（100ng／ml）联合紫杉醇（8．97μg/ml）作用后,TRAIL引起的Akt磷酸化被抑制。结论紫杉醇通过抑制TRAIL引起的Akt磷酸化,从而增强了TRAIL诱导的胃癌BGC823细胞凋亡。     

重组腺病毒对Hela、MCF-7的转染效率及其介导的hTRAIL基因体外抑瘤作用

目的 研究重组腺病毒感染宫颈癌细胞Hela和乳腺癌细胞MCF-7的转染效率,并观察重组腺病毒介导的人肿瘤坏死因子(TNF)相关诱导凋亡配体(hTRAIL)基因对Hela、MCF-7生长的影响.方法 将Hela和MCF-7细胞接种至12孔板,培育24 h后加入不同感染滴度的重组腺病毒Ad-CMV-EGFP,用流式细胞仪检测其转染效率;将Hela细胞和MCF-7细胞接种至96孔板,24 h后转染重组腺病毒Ad-CMV-hTRAIL,72 h用四甲基偶氮唑盐(MTT)比色法检测肿瘤细胞的存活率.结果 Ad-CMV-EGFP对Hela和MCF-7细胞的转染效率在一定范围内随着感染复数(MOI)的增加而增高,转染 Ad-CMV-hTRAIL的Hela细胞的存活率明显低于转染Ad-CMV-EGFP的病毒对照组(P＜0.05).结论 腺病毒对Hela和MCF-7细胞均有较高的转染效率,且腺病毒介导的hTRAIL基因对Hela细胞具有明显的体外抗瘤作用.     

TRAIL体外诱导人肝星形细胞LX-2凋亡作用及调控机制

目的研究肿瘤坏死因子相关凋亡诱导配体（TRAIL）诱导肝星形细胞凋亡情况及调控机制。方法用RT-PCR检测LX-2中α-SMAmRNA和DR5mRNA的表达;MTT比色法、流式细胞术检测外源性TRAIL对LX-2细胞增殖和诱导细胞凋亡的影响;采用Westernblot检测Bax、Caspase3表达。结果培养的LX-2表达α-SMAmRNA和DR5mRNA逐渐增加,TRAIL可以抑制其细胞增殖,与剂量相关（P〈0.05）。与对照组比较,TRAIL诱导活化的LX-2细胞凋亡明显增加（P〈0.05）,Western blot分析显示TRAIL作用下,LX-2线粒体Bax、细胞质Caspase3表达上调。结论外源性TRAIL可以诱导活化的LX-2凋亡,其机制与DR5及线粒体Bax表达上调有关。     

Tumor necrosis factor-related apoptosis-inducing ligand gene on human colorectal cancer cell line HT29

AIM: To evaluate the therapeutic efficiency of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) gene on human colorectal cancer cell line HT29. METHODS: Human embryonal kidney cells transformed by introducing sheared fragments of Ad5 DNA (293 cell) were used for amplification of adenoviral vectors: Ad/GT-TRAIL,Ad/GT-Bax, Ad/GT-LacZ and Ad/PGK-GV16. Human colorectal cancer cell line HT29 was transfected with binary adenovirus-mediated TRAIL gene. Bax gene was used as positive control, LacZ gene was used as the vector control,and cells treated with PBS only were used as a mock control. The morphological changes, cell growth and apoptosis were measured by reversmicroscope, MTT method and flow cytometry. RESULTS: All adenoviral vectors titer determined by optical absorbency at A260nm were 1X10(10) viral particle/ml(vp/ml).Obviously morphological changes of HT29 cells were observed when infected with Ad/GT-TRAIL, and these changes were much more obviously when Ad/PGK-GV16 was coinfected. The cell suppression percentage and the percentage of apoptotic cells were 52.5 % and 16.5 % respectively when infected with Ad/GT-TRAIL alone, while combining with Ad/PGK-GV16, the growth of HT29 was suppressed by 85.2 % and the percentage of apoptotic cells was 35.9 %. It showed a significantly enhanced therapeutic efficiency with binary system (P<0.05). CONCLUSION: A binary adenoviral vector system provides an effective approach to amplify viral vectors that express potentially toxic gene, TRAIL. Ad/GT-TRAIL showed a significantly enhanced therapeutic efficiency for HT29 when coinfected with Ad/PGK-GV16. Ad/GT-TRAIL could induce apoptosis of HT29 and inhibit its growth.     

蜂毒素基因重组腺病毒诱导肝癌细胞凋亡的作用

目的 观察蜂毒素基因重组腺病毒诱导肝癌细胞凋亡的作用。 方法 通过形态学观察、DNA电泳、原位缺口末端标记(TUNEL)、流式细胞仪等方法观察携蜂毒素基因重组腺病毒对肝癌细胞凋亡的影响。 结果 光镜下见部分肝癌细胞出现凋亡形态学变化,DNA电泳可见梯形条带,TUNEL染色结果显示,Ad-rAFP-Mel组、Ad-rAFP组和对照组的凋亡率分别为(21.5±2.4)％、(10.5±4.4)％和(3.0±1.4)％,各组间差异有显著性(F=3 8.0,P<0.0 5)。流式细胞仪定量分析结果显示,3组凋亡率分别为(7.3±0.5)％、(3.9±0.1)％和(0.8±0.1)％,各组间差异有显著性(F=415.1,P<0.05)。 结论 诱导细胞凋亡可能是蜂毒素抗肿瘤的作用机制之一。     

Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.     

Synergistic suppression effect on tumor growth of hepatocellular carcinoma by combining oncolytic adenovirus carrying XAF1 with cisplatin

Purpose

The potent anticancer efficacy of oncolytic viruses has been verified in Clinic in recent years. Cisplatin (DDP) is one of most common chemotherapeutic drugs, but is accompanied by side effects and drug resistance. Our previous studies have shown the strategy of cancer -targeting gene-viro-therapy (CTGVT) mediated by the oncolytic virus ZD55 containing the XAF1 cDNA (ZD55-XAF1), which exhibited potent antitumor effects in various tumor cells and no apparent toxicities on normal cells. In the study, the CTGVT strategy is broadened by combining DDP with ZD55-XAF1 for growth inhibition of hepatocellular carcinoma (HCC) cells.

Methods

The transgenic expression was evaluated by both in vitro and in vivo experiments, and the enhanced inhibitory effect of ZD55-XAF1 combined with cisplatin was assessed in HCC cells. The cytotoxicity on normal liver cells was evaluated by MTT assay and apoptotic cell staining. Activation of caspase-9 and PARP for apoptosis was further detected by Western blot analysis. The in vivo antitumor efficacy of combination treatment with cisplatin and ZD55-XAF1 was estimated in an HCC xenograft mouse model.

Results

We found that the combination of ZD55-XAF1 and cisplatin showed enhanced inhibitory effects on the proliferation of HCC cells in vitro and tumor growth in mice. Furthermore, the combined treatment of ZD55-XAF1 and DDP decreases the chemotherapy dose needed to achieve the same inhibitory effect without overlapping toxicities on normal liver cells and induces tumor cell apoptosis via the activation of caspase-9/PARP pathway.

Conclusion

Thus, these data suggest that the chemo-gene-viro-therapeutic strategy by combining ZD55-XAF1 and DDP reveals a novel therapeutic strategy for hepatocellular carcinoma.

     

Increased hepatotoxicity of tumor necrosis factor-related apoptosis-inducing ligand in diseased human liver

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor cells but not in most normal cells and has therefore been proposed as a promising antitumor agent. Recent experiments suggested that isolated primary human hepatocytes but not monkey liver cells are susceptible to certain TRAIL agonists, raising concerns about the use of TRAIL in cancer treatment. Whether TRAIL indeed exerts hepatotoxicity in vivo and how this is influenced by chemotherapeutic drugs or liver disease are completely unknown. Employing different forms of recombinant TRAIL, we found that the cytokine can induce proapoptotic caspase activity in isolated human hepatocytes. However in marked contrast, these different TRAIL preparations induced little or no cytotoxicity when incubated with tissue explants of fresh healthy liver, an experimental model that may more faithfully mimic the in vivo situation. In healthy liver, TRAIL induced apoptosis only when combined with histone deacetylase inhibitors. Strikingly, however, TRAIL alone triggered massive apoptosis accompanied by caspase activation in tissue explants from patients with liver steatosis or hepatitis C viral infection. This enhanced sensitivity of diseased liver was associated with an increased expression of TRAIL receptors and up-regulation of proapoptotic Bcl-2 proteins. CONCLUSION: These results suggest that clinical trials should be performed with great caution when TRAIL is combined with chemotherapy or administered to patients with inflammatory liver diseases.     

Proteasome inhibitors synergize with tumor necrosis factor-related apoptosis-induced ligand to induce anaplastic thyroid carcinoma cell death

CONTEXT: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive types of cancer characterized by complete refractoriness to multimodal treatment approaches. Therapeutic strategies based on the simultaneous use of proteasome inhibitors and death receptor ligands have been shown to induce apoptosis in several tumor types but have not yet been explored in ATC. OBJECTIVE AND METHODS: The aim of this study was to investigate the ability of the proteasome inhibitor Bortezomib to induce apoptosis in ATC cell lines. Bortezomib was used as a single agent or in combination with TNF-related apoptosis-induced ligand (TRAIL), a member of the TNF family that selectively induces tumor cell apoptosis. The molecular effects of Bortezomib were investigated by analyzing the expression of key regulators of cell cycle and apoptosis and the activation of different apoptotic pathways. RESULTS: Bortezomib induced apoptosis in ATC cells at doses achieved in the clinical setting, differently from conventional chemotherapeutic agents. Simultaneous treatment with low doses of Bortezomib and TRAIL had a synergistic effect in inducing massive ATC cell apoptosis. Bortezomib increased the expression of cytotoxic TRAIL receptors, p21 (WAF/CIP1) and proapoptotic second mitochondria-derived activator of caspases/direct inhibitor of apoptosis binding protein with low pI, and reduced the expression of antiapoptotic mediators such as cellular Fas-associated death domain-like IL-1beta converting enzyme inhibitory protein, Bcl-2, Bcl-X(L), and inhibitor of apoptosis-1, thus resulting in cell death induction through the mitochondrial apoptotic pathway. CONCLUSIONS: The combination of proteasome inhibitors and TRAIL synergizes to induce the destruction of chemoresistant neoplastic thyrocytes and could represent a promising therapeutic strategy for the treatment of anaplastic thyroid carcinoma.     

Antitumor activity of an hTERT promoter-regulated tumor-selective oncolytic adenovirus in human hepatocellular carcinoma

INTRODUCTIONHepatocellular carcinoma (HCC) is one of the malignant diseases with a high incidence and mortality in China. Today, malignant tumors have already become the first death cause in Chinese city residents, and the second in country residents. Amo…     

XAF1基因诱导胰腺癌细胞株BxPC3凋亡的实验研究

目的 探讨Ad5/F35腺病毒介导的XAF1基因诱导胰腺癌细胞BxPC3凋亡及其可能的作用机制.方法 将前期构建的重组腺病毒Ad5/F35-XAF1感染胰腺癌细胞BxPc3.采用半定量RTPCR、蛋白质印迹检测法检测感染前后BxPC3细胞XAF1 mRNA和蛋白表达的变化;运用Annexin-V/PI和TUNEL法检测细胞的凋亡率;免疫蛋白印迹法检测细胞Caspase-3、PARP、Caspase-8和Bcl-2蛋白的表达.结果 Ad5/F35-XAF1感染后,BxPC3细胞的XAF1 mRNA和蛋白表达明显增高,与空白组和Ad5/F35-Null对照组比较,差异显著(P＜0.05);两种方法 检测的细胞凋亡率分别为(19.90±3.09)%、(9.29±2.13)%,较Ad5/F35-Null组的(6.72±0.76)%、(2.73±0.51)%和空白组的(7.22±1.53)%、(1.56±0.47)%均有显著差异(P＜0.01);Caspase-3、PARP、Caspase-8蛋白表达显著增强,Bcl-2表达降低.结论 Ad5F35-XAF1重组腺病毒感染胰腺癌细胞BxPC-3能明显诱导细胞的凋亡,其机制可能是激活死亡受体途径和线粒体途径.