Adenovirus-mediated transfer of RA538 gene and its antitumor effect

The RA538 cDNA was transferred into human ovarian cancer cell line SK-OV-3 and human melanoma cell line WM-983A by its recombinant adenoviral vector constructed through homologous recombination. It was demonstrated that the recombinant adenovirus could transfer RA538 gene with high efficiency, and could obviously inhibit tumor growth, with the inhibiting rates of 85% and 73% respectively, at the same time greatly repress the colony forming ability of the cells. The therapeutic experiments on transplanted subcutaneous tumor model in nude mice demonstrated that RA538 could significantly inhibit tumor growth. Flow cytometry and DNA fragmentation analysis indicated that RA538 could induce the cell cycle G1 arrest/apoptosis of the tumor cells. The expression of c-myc gene was found pronouncedly reduced by Western blot analysis. These results suggest that the RA538 recombinant adenovirus could be a promising drug in cancer gene therapy.     

重组RA—538及反义c—myc腺病毒对人胃癌,食管癌和bcl—2高表达细胞系的作用

本课题研究ＲＡ５３８、反义c-ymc重组腺病毒对人胃癌（ＳＧＣ７９０１）、食管癌（Ｅ Ｃ１０９、ＥＣ８７１２）、正常人胚肺２ＢＳ（２ＢＳ）及ｂｃｌ－２高表达细胞第的体仙外生物学作用及其分子机制。结果显示Ａｄ－ＲＡ５３８及Ａｄ－ＡＳｃ－ｍｙｃ对ＳＧＣ７９０１细胞体内外均具有明显的生长抑制及凋亡诱导作用,并能抑制其ｃ－ｍｙｃ、ｂｃｌ－２、ｃｙｃｌｉｎＤ１基因的表达及刺激ｂａｘ基因的表达。对ＥＣ１０９、ＥＣ８     

ω-6脂肪酸脱氢酶基因在乳腺癌细胞内的表达和作用

为探讨ω- 6脂肪酸脱氢酶基因fat -1在人类乳腺癌细胞MCF- 7中表达和对其生长的作用,将fat -1基因插入到腺病毒载体中,构建腺病毒重组载体(Ad·GFP·fat1) .通过包装细胞系(2 93)产生重组腺病毒,感染MCF 7细胞.用核糖核酸酶保护性分析技术,检测fat -1基因在MCF- 7细胞内的表达,细胞增殖试剂盒(MTT)和凋亡染色试剂盒染色分析fat 1基因对MCF- 7细胞增殖和凋亡的影响,用酶联免疫分析花生酸类(eicosanoids)前列腺素E2 (prostaglandinE2 )的含量.结果显示,腺病毒介导的fat- 1基因能在MCF- 7细胞内有效异源表达,抑制MCF -7细胞的增殖且导致凋亡,前列腺素的含量也明显地减少.结果说明,fat- 1基因在乳腺癌的基因治疗中具有良好利用价值.     

ODC, AdoMetDC双反义腺病毒对肺癌增殖和侵袭抑制作用的体外和体内研究

鸟氨酸脱羧酶(ODC)和S-甲硫氨酸脱羧酶(AdoMetDC)是多胺体内合成的2个关键酶.研究腺病毒Ad-ODC-AdoMetDCas介导的ODC和AdoMetDC反义RNA对肺癌多胺合成,细胞增殖以及侵袭的抑制作用.用活细胞计数和流式细胞术分别检测Ad-ODCas和Ad-ODC-AdoMetDCas对肺癌A-549细胞增殖的影响,蛋白质印迹和HPLC方法分别检测腺病毒对肺癌A-549细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量的抑制作用,TUNEL标记检测法观察Ad-ODC-AdoMetDCas对肺癌细胞凋亡的影响,Matrigel侵袭实验分析腺病毒对肺癌A-549细胞侵袭活性的改变,裸鼠皮下移植瘤模型研究Ad-ODC-AdoMetDCas对体内肺癌生长的抑制作用.实验结果显示,Ad-ODC-AdoMetDCas明显抑制肺癌A-549细胞的增殖,导致细胞凋亡,显著降低肺癌A-549细胞的体外侵袭能力,肺癌A-549细胞感染Ad-ODC-AdoMetDCas后细胞内3种多胺含量都明显降低,Ad-ODC-AdoMetDCas对已形成的裸鼠皮下移植瘤具有明显的抑制作用.实验表明,ODC和AdoMetDC双反义腺病毒具有显著抑制肺癌增殖和侵袭的作用,对于肺癌的防治研究具有一定的前景.     

ODC,AdoMetDC双反义腺病毒对肺癌增殖和侵袭制作用的体外和体内研究

鸟氨酸脱羧酶(ODC)和S-甲硫氨酸脱羧酶(AdoMetDC)是多胺体内合成的2个关键酶.研究腺病毒Ad-ODC-AdoMetDCas介导的ODC和AdoMetDC反义RNA对肺癌多胺合成,细胞增殖以及侵袭的抑制作用.用活细胞计数和流式细胞术分别检测Ad-ODCas和Ad-ODC-AdoMetDCas对肺癌A-549细胞增殖的影响,蛋白质印迹和HPLC方法分别检测腺病毒对肺癌A-549细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量的抑制作用,TUNEL标记检测法观察Ad-ODC-AdoMetDCas对肺癌细胞凋亡的影响,Matrigel侵袭实验分析腺病毒对肺癌A-549细胞侵袭活性的改变,裸鼠皮下移植瘤模型研究Ad-ODC-AdoMetDCas对体内肺癌生长的抑制作用.实验结果显示,Ad-ODC-AdoMetDCas明显抑制肺癌A-549细胞的增殖,导致细胞凋亡,显著降低肺癌A-549细胞的体外侵袭能力,肺癌A-549细胞感染Ad-ODC-AdoMetDCas后细胞内3种多胺含量都明显降低,Ad-ODC-AdoMetDCas对已形成的裸鼠皮下移植瘤具有明显的抑制作用.实验表明,ODC和AdoMetDC双反义腺病毒具有显著抑制肺癌增殖和侵袭的作用,对于肺癌的防治研究具有一定的前景.     

重组p16腺病毒的构建及其对人白血病细胞的抑制作用

为了探讨腺病毒载体用于基因治疗的可行性及野生型 p1 6基因的抗肿瘤特性 ,构建了复制缺陷型重组 p1 6腺病毒 .首先将 p1 6全长 c DNA插入穿梭质粒 p Ad CMV产生重组质粒 p Ad-CMV- p1 6,然后通过脂质体介导与 p JM1 7共转染 2 93细胞 ,经同源重组产生 E1区缺失的重组腺病毒空斑 .用纯化后的腺病毒感染人白血病细胞株 HL- 60后 ,PCR及 Western blot分析显示在感染细胞中有外源性 p1 6 c DNA存在和 p1 6蛋白表达 ;被感染的 HL- 60细胞的生长受到明显抑制 ,而未感染细胞及对照腺病毒感染的细胞没有受到抑制 .结果表明 ,腺病毒作为一种新型基因转移载体 ,可有效地介导肿瘤抑制基因 p1 6的表达 ,在肿瘤基因治疗方面具有很大的应用前景 .     

瘤体内注射凋亡素基因诱导人骨肉瘤S180细胞株凋亡研究

观察瘤体内注射凋亡素基因诱导人骨肉瘤S180细胞株凋亡的效果。利用脂体介导将重组质粒pCDNAVP3转染至荷瘤裸鼠肿瘤结节内。PCR和RT PCR检测结果表明 ,质粒可存在和持续表达 14d以上。抑瘤试验结果证实 ,凋亡素可抑制S180细胞的生长 ,抑瘤率达 2 7.2 %。TUNFL染色法检测结果表明 ,试验组的TUNEL阳性细胞率明显高于对照组。上述结果显示 ,所注射的凋亡素基因可诱导人骨肉瘤S180细胞株凋亡。     

改变细胞膜的脂肪酸组成可促进乳腺癌细胞凋亡

目的: 研究n-6脂肪酸脱氢酶 fat-1基因在人乳腺癌细胞内的表达,改变细胞膜脂肪酸组成,对乳腺癌细胞的凋亡作用。方法: 构建含有fat-1 基因的重组腺病毒载体 (Ad.GFP.fat-1),通过包装细胞系（293）产生的腺病毒,感染人乳腺癌细胞MCF-7。提取细胞的总RNA,以fat-1的反义mRNA 作探针,用Northern Blot检测fat-1 基因在MCF-7细胞内的表达。MTT法分析fat-1 基因对MCF-7细胞增殖的影响,凋亡染色试剂盒检测细胞的凋亡。气相色谱仪分析对MCF-7细胞的n-6 PUFAs/n-3 PUFAs含量影响。结果: 通过基因重组技术,得到预期的重组病毒;fat-1 基因在人乳腺癌细胞MCF-7 中能有效异源表达,2天后,可检测到fat-1 mRNA的条带。与对照细胞相比,fat-1基因有效地抑制了MCF-7细胞的增殖（23%,p<0.05）,促进了凋亡（增加35%）;同时降低了人乳腺癌细胞MCF-7细胞膜n-6 PUFAs/n-3 PUFAs的比率。结论: 腺病毒介导的fat-1 基因能在人乳腺癌细胞MCF-7内有效异源表达,且抑制了MCF-7细胞的增殖。机理为降低了细胞膜的n-6 PUFAs/n-3 PUFAs的比率。     

Adenovirus-mediated expression of both antisense ornithine decarboxylase and S-adenosylmethionine decarboxylase inhibits lung cancer cell growth

Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC). Antisense sequences of ODC and AdoMetDC genes were cloned into an adenoviral vector (named Ad-ODC-AdoMetDCas). To evaluate the effects of recombinant adenovirus Ad-ODC-AdoMetDCas that can simultaneously express both antisense ODC and AdoMetDC, the human lung cancer cell line A-549 was infected with Ad-ODC-AdoMetDCas or the control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were carried out to assess the properties of tumor growth and invasiveness. Our study showed that adenovirus-mediated antisense ODC and AdoMetDC expression inhibits tumor cell growth through blocking the polyamine synthesis pathway. Tumor cells were arrested at the G_1 phase after gene transfer and the invasiveness was reduced. It suggested that the recombinant adenovirus Ad-ODC-AdoMetDCas might be a new anticancer reagent in the treatment of lung cancers.     

裂解型腺病毒Ad-E1A对人脐静脉内皮细胞的影响

以QBI-293A细胞基因组DNA为模板,PCR扩增E1A基因,酶切连接到pAdTrack-CMV转移质粒上,pAdTrack-CMV-E1A经PmeI线性化后,与pAdEasy-1共转化大肠杆菌BJ5183,筛选重组腺病毒质粒pAdEasy-1-pAdTrack-CMV-E1A,经PacI线性化,脂质体转染QBI-293A细胞,获得裂解型腺病毒Ad-E1A。裂解型腺病毒Ad-E1A在ECV304细胞内复制裂解,抑制细胞的生长,并可以降低VEGF的表达,探讨了Ad-E1A可能通过抑制ECV304细胞NF-κB的激活而引起细胞生长抑制的机制,说明Ad-E1A具有抑制肿瘤转移的功能。     

人神经元素3基因重组逆转录病毒表达载体的构建及其包装细胞株的建立

为构建表达人神经元素3基因(neurogenin 3,ngn3)的重组逆转录病毒载体,建立稳定表达ngn3的包装细胞株,本研究以流产人胎儿胰腺组织为材料,通过RT-PCR方法克隆出人ngn3基因,将其连接到pMD18-T载体上并测序,结果表明,测序得到的基因序列与发表的人ngn3基因序列(GenBank Accession No.BC126468)完全一致。将EcoRI和HpaI双酶切后的基因片段构建到pMSCV-neo逆转录病毒载体中,酶切鉴定结果表明,pMSCV-ngn3重组逆转录病毒载体构建成功。脂质体法将pMSCV-ngn3重组载体导入PT67包装细胞,G418筛选后,对得到的细胞株进行RT-PCR和免疫组化检测,结果显示,该细胞株在mRNA水平和蛋白水平均稳定表达Ngn3;收集该细胞株的培养上清液,进行RT-PCR检测及电镜观察,结果表明,该细胞株将导入的重组逆转录病毒载体pMSCV-ngn3包装成了具有感染性的病毒颗粒,并将其释放到了培养上清液中。以上结果表明PT67-ngn3包装细胞株建立成功。该细胞株的成功建立,为下一步将ngn3基因应用于提高人胎儿胰腺祖细胞诱导分化效率方面的研究奠定了基础。     

Enhanced TRAIL sensitivity by E1A expression in human cancer and normal cell lines: inhibition by adenovirus E1B19K and E3 proteins

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines that induces apoptosis in a variety of cancer cells, but not in normal cells. However, more and more tumor cells remain resistant to TRAIL, which limited its application for cancer therapy. Expression of the adenovirus serotype 5 (Ad5) E1A sensitizes tumor cells to apoptosis by TNF-alpha, Fas-ligand, and TRAIL. Here we asked whether E1A overcomes this resistance and enhances TRAIL-induced apoptosis in the tumor cells. Our results revealed that the tumor cell lines, HeLa and HepG2, with infection by Ad-E1A, were highly sensitive to TRAIL-induced apoptosis. Importantly, we found that in normal primary human lung fibroblast cells (HLF) TRAIL is capable of inducing apoptosis in combination with E1A as efficiently as in some tumor cell lines. The adenovirus type 5 encoding proteins, E1B19K and E3 gene products, have been shown to inhibit E1A and TRAIL-induced apoptosis of HLF cells by using the recombinant adenovirus AdDeltaE1B55K, with mutation of E1B55K, containing E1B19K and complete E3 region. Further results demonstrated that the expression of DR5 and TRAIL was down-regulated in the AdDeltaE1B55K co-infected HLF cells. These findings suggest that TRAIL may play an important role in limiting virus infections and the ability of adenovirus to inhibit killing may prolong acute and persistent infections. The results from this study have also suggested the possibility that the combination of E1A with TRAIL could be used in the treatment of human malignancy, or in the selection of the optimal adenovirus mutant as effective delivering vector for cancer therapy.     

p21基因的克隆及其对肺癌细胞生长的抑制

用反转录PCR从正常人胚胎肺细胞中获得了p21基因cDNA,将其插入真核表达载体pMSCVneo,构建成重组质粒pMS21,并将其转染至肺癌细胞株A549。通过集落形成观察到p21对肺癌细胞具有明显的抑制作用,经RNA狭缝杂交、Western印迹分析和免疫细胞化学实验证实这是p21表达的结果。荷瘤裸鼠实验也进一步证实了p21对肺癌细胞具有明显的抑制作用。     

p21基因的克隆及其对肺癌细胞生长的抑制

用反转录ＰＣＲ从正常人胚胎肺细胞中获得了ｐ２１基因ｃＤＮＡ,将其插入真核表达载体ｐＭＳＣＶｎｅｏ,构建成重组质粒,ｐＭＳ２１,并将其转染至肺癌细胞株Ａ５４９．通过集落形成观察到ｐ２１对肺癌细胞具有明显的抑制作用,经ＲＮＡ狭缝杂交、Ｗｅｓｔ－ｅｒｎｂｌｏｔ分析和免疫细胞化学实验证实这是ｐ２１表达的结果．荷瘤裸鼠实验也进一步证实了ｐ２１对肺癌细胞具有明显的抑制作用．为ｐ２１的深入研究提供了基础．     

DLC-1基因表达对结肠癌细胞侵袭转移能力的影响

目的:研究DLC-1基因对结肠癌细胞侵袭迁移能力的影响.方法:将DLC-1 shRNA(短发夹状RNA,short hairpin RNA)序列克隆到质粒pGCsi-U6/Neo载体,采用脂质体介导的转染方法将构建的DLC-1 shRNA表达质粒转入结肠癌细胞系LoVo细胞.采用RT-PCR技术和Western Blot技术分别检测LoVo细胞中DLC-1mRNA和蛋白表达水平的变化.Transwell小室人工重组基底膜侵袭转移实验观察LoVo细胞侵袭迁移能力的改变.结果:结肠癌细胞系LoVo细胞表达DLC-1分子.所构建质粒表达载体能有效地干扰LoVo细胞DLC-1 mRNA和蛋白质表达水平;Transwell小室人工重组基底膜侵袭转移实验结果显示,转染后LoVo细胞侵袭转移能力明显增强(p＜0.05).结论:结肠癌细胞系LoVo细胞表达DLC-1基因,应用RNAi技术可特异性降低其表达.DLC-1的表达水平与结肠癌细胞侵袭转移相关.     

Inhibition of endothelial cell proliferation and tumor angiogenesis by up-regulating NDRG2 expression in breast cancer cells

The N-myc downstream-regulated gene 2 (NDRG2) is involved in tumor cell differentiation and apoptosis, but its function in tumor angiogenesis remains to be established. Here, we employed adenovirus overexpressing NDRG2 (Ad-NDRG2) to efficiently up-regulate target gene expression in the NDRG2-low-expressing, breast cancer cell line MCF-7. Moreover, VEGF secretion was decreased in MCF-7 cells infected by Ad-NDRG2, and medium conditioned by these infected cells could significantly inhibit the proliferation, tube formation and invasion of human umbilical vein endothelial cells (HUVECs). Further study indicated that the angiogenesis promoting factors VEGF and HIF-1α were down-regulated, whereas the angiogenesis suppressing factors p53 and VHL were up-regulated in MCF-7 cells infected by Ad-NDRG2. Finally, in a nude mouse model, intratumoral injections of Ad-NDRG2 every 3 days for 20 days significantly inhibited the growth and angiogenesis of xenografted MCF-7 tumors. In summary, these data indicate that NDRG2 may be involved in angiogenesis by impacting the expression of angiogenesis related factors. Thus, specific overexpression of NDRG2 by adenovirus represents a promising approach for the treatment of tumor angiogenesis.     

Effects of garlic oil on tumoragenecity and intercellular communication in human gastric cancer cell line

Previous studies have demonstrated that garlic oil (GO) and its anti-tumor compound could inhibit DNA and RNA synthesis in human cancer cells.In order to explore the effects of garlic oil on carcinoma cells,a gastric carcinoma cell line,BGC-823 was studied at cellular and molecular levels after garlic oil treatment.Data showed that the cell differentiation and suppression of tumorigenicity were significantly induced in tumor cells after garlic oil treatment.There was a correlation between the cell-cell communication recovery and the increase of p53 and waf1/p21 gene expression in garlic oil-treated cells.This result suggested that tumor suppressor gene waf1/p21 and wt p53 might play an important role in this effect.     

Adenovirus vector-mediated FAM176A overexpression induces cell death in human H1299 non-small cell lung cancer cells

FAM176A (family with sequence similarity 176 member A) is a novel molecule related to programmed cell death. A decreased expression of FAM176A has been found in several types of human tumors in including lung cancers. In the present study, we investigated the biological activities of FAM176A on the human non–small cell lung cancer cell line H1299 cells. We constructed a recombinant adenovirus 5-FAM176A vector (Ad5-FAM176A) and evaluated the expression and anti-tumor activities in vitro. Cell viability analysis revealed that the adenovirus-mediated increase of FAM176A inhibited the growth of the tumor cells in a dose- and time-dependent manner. This inhibitory effect was mediated by both autophagy and apoptosis that involved caspase activation. In addition, cell cycle analysis suggested that Ad5-FAM176A could induce cell cycle arrest at the G2/M phase, all of which suggested that adenovirus-mediated FAM176A gene transfer might present a new therapeutic approach for lung cancer treatment. [BMB Reports 2014; 47(2): 104-109]     

Adenovirus-mediated suicide gene therapy using the human telomerase catalytic subunit (hTERT) gene promoter induced apoptosis of ovarian cancer cell line

Telomerase is a ribonucleoprotein complex the function of which is to add telomeric repeats (TTAGGG)(n) to chromosomal ends, and it is known to play an important role in cellular immortalization. Telomerase is highly active in most tumor cells, yet not in normal cells. As such, it may have possible applications in cancer gene therapy. Telomerase consists of two essential components, telomerase RNA template (hTR) and catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. We here tested the possibility of the utilization of the hTERT promoter in targeted cancer gene therapy. We cloned the hTERT promoter in the replace of the CMV promoter and sub-cloned HSV-TK gene to be controlled by hTERT gene promoter in adenovirus shuttle plasmid. Then we constructed recombinant adenovirus Ad-hT-TK, and infected them into normal and human gynecological cancer cell lines. Through these experiments, we identified the selective tumor specific cell death by Ad-hT-TK. Furthermore, FACS analysis and TUNEL assay suggests that the reduced viability is mediated through the induction of apoptosis, indicating that this approach may be a useful method for suppressing cancer growth in targeted cancer gene therapy. These results show that Ad-hT-TK could be used for gynecological cancer gene therapy.