Experimental Studies Of Gene-therapy Effect On Ovarian Epithelium Cancer By Recombinant Adenovirus-mediated Canstatin Gene Driven By HTERT Core Promoter

Now the prognosis of patients with ovarian cancer in advanced stage is poor. There are approximately 26,600 new cases of ovarian cancer,resulting in about 14,500 deaths each year in USA.It is reported that there are approximately 191,000 new cases of ovarian cancer in the world yearly and less than 23%of the cases can be diagnosed early,being of extremely delitescence.The total 5-year survival rate is no more than 30%among the diagnosed patients,although combination therapy has been adopted.Consequently ovarian cancer has been usually known as silent killer. Therefore,it is urgent to develop new treatment strategy for this malignant disease.Angiogenesis,the formation of new capillary blood vessels,plays an essential role in pathological processes,such as solid tumor growth and metastases.Solid tumors cannot grow beyond a few millimeters in diameter without generation of tumor vasculature.Furthermore,the dense the blood vessel formation,the higher the rate of tumor growth,the greater the metastatic potential.Typeâ…£collagen plays an essential role in angiogenesis.Typeâ…£collagen is one of essential components to constitute basilar membrane of blood vessels and can promote cell adhesion,migration, differentiation and growth.Typeâ…£collagen is a potential therapeutic target to regulate neovascularization in cancerous diseases.Canstatin is a novel discovered endogenous inhibitor of angiogenesis following endostatin and comes from noncollagenous domain of collagen typeⅣα2-chains (non-collagenousl,NC1).NC1 area participate the assembly ofα-chains and plays a functional role in the formation of trimer of typeâ…£collagen and affect the formation of basilar membrane,and the basilar membrane plays all-important functional role in the formation of neovascularization.Previous studies have elucidated that canstatin can inhibits vascular endothelial cell growth and migration,and induces tumor cell apoptosis.It is demonstrated that canstatin can inhibits xenografted tumors growth by inhibitting neovascularization.It is reported that systemic injection of recombinant canstatin gene intratumorally can efficiently inhibits the growth of xenografted tumors such as pancreatic cancer,prostate cancer and breast cabcer compared with placebo-treated mice.However,it is still unknown whether canstatin gene is effective in the treatment of ovarian epithelium cancer xenograft.Telomerase is a ribonucleoprotein enzyme,which is positively expressed in most malignant tumor cells,while negatively expressed in normal somatic ones.The hTERT promoter plays a significant role in the development of tumor and has become a new target for the gene therapy of cancer.There is now a body of evidence that hTERT-mediated driver can regulate a wide of solid tumor,including colon cancer, lung cancer,hepatocellular carcinoma prostate cancer,ovarian cancer,and et al.It is reported that hTERT-mediated virus replication is in tumor cells mainly,but not normal cells.In this study,in order to investigate the effect of canstatin gene on ovarian epithelium cancer and improve the target properties of canstatin,we constructed a recombinant adenovirus plasmid encoding canstatin gene driven by human telomerase reverse transcriptase(hTERT) core promoter(Adxsi-GFP-hTERT-Canstatin, AdhTERT-Can),and investigated its anticancer properties on ovarian epithelium cancer in vitro and vivo.In vitro,the effect of HO8910PM cells growth is studied after transfected by recombinant plasmid AdhTERT-Can.Finally,the models of human ovarian epithelium cancer xenografts in immunodeficiency BALB/C nude mouse are set up,and then both antitumor activity and mechnism are in study.This study is divided into the following two parts. Partâ… Construction of recombinant adenovirus plasmid carring human canstatin gene and hTERT core promoterMethods1.The ovarian cancer tissue was kindly provided by a woman with ovarian epithelium cancer whom underwent operation in our hospital.The total RNA and the genomic DNA were extracted from the tissue.The fragment of canstatin was obtained by RT-PCR and the fragment ofhTERT was obtained by PCR.2.Both gene fragment of canstatin and hTERT were cloned into pMD18T vector to construct cloning vector pMD-hTERT-Canstatin.Then transform competent E.coli DH5α.The plasmid were sieved by antibiotics,and then incubated with vigorous shaking.Finally,plasmids were isolated,restriction enzymes analysis and sequencing were used to confirm the recombinant plasmid.The nucleotide sequence was compared with that in GeneBank database.3.The fragment of hTERT-Canstatin were conjugated to pShuttle-EGFP-BGHPolyA from pMDI8T vector and then transform competent E.coli DH5α.The plasmid were sieved by antibiotics,and then incubated with vigorous shaking.Finally, plasmids were isolated,restriction enzymes analysis was used to confirm the recombinant plasmid.4.The fragment of hTERT-Canstatin were conjugated to pAdxsi from pShuttle-EGFP-BGHPolyA vector and then transform competent E.coli DH5α.The plasmid were sieved by antibiotics,and then incubated with vigorous shaking.Finally, plasmids were isolated,restriction enzymes analysis was used to confirm the recombinant plasmid.5.Human embryonic kidney cell line HEK 293 cells was recoveried.Cell culture and transfer of culture were in progress.The recombinant adenovirus vector pAdhTERT-Can were digest with incision enzyme pacâ… ,then were transfected into HEK 293 cells by means of Lipofectamin.Freeze-thawing were in progress for times to obtain recombinant adenovirus plasmid AdhTERT-Can.The recombinant adenovirus was confirmed by GFP expression in HEK 293 cell.Repeated infection-collection-freeze-thawing procedure to amplify recombinant adenovirus plasmid AdhTERT-Can.The titer of virus was measured by plaque assay.Results1.The human canstatin gene showed one clear bands after 2%agarose gel electrophoresis.The length of the target gene is coincidence with the canstatin cDNA(684bp) in Gene Bank.The hTERT gene showed one clear bands after 2% agarose gel electrophoresis.The length of the target gene is coincidence with the hTERT DNA(332bp) in Gene Bank2.Restriction enzymes analysis of small-scale preparations of the recombinant plasmids pMD-hTERT-Canstatin showed the plasmid in one colony were separated into two bands near the locations of primary plasmid and the joining gene fragment -canstatin(684bp) and hTERT(332bp).DNA sequencing demonstrated the sequence of this coloned was completely homologous to the canstatin and hTERT gene sequence represented in Genbank.3.Restriction endonucleases analysis of small-scale preparations of plasmids pShuttle-hTERT-Canstatin showed the plasmid in one colony were separated into two bands of 1kb/4.5kb,one of which was the target gene fragment.It is demonstrated pShuttle-hTERT-Canstatin vector was constructed successfully.4.After digested by XhoI,restriction endonucleases analysis of small-scale preparations of plasmids pAdhTERT-Can showed the plasmid in one colony were separated into seven bands of 14Kbp/11.8Kbp/5.1Kbp/2.47Kbp/ 1.45Kbp/0.6Kbp/0.49Kbp.Linearize pAdhTERT-Can and transfect to HEK293 cell by Lipofectamine and the HEK 293 erupt green fluorescence by the expression of reporter genes green fluorescent protein(GFP) under fluorescence microscope.It is indicated that the recombinant adenovirus vector containing canstatin gene and hTERT gene had constructed successfully.The titer of the recombinant adenovirus vector was 1.6×10¹¹ pfu/ml enough for the requirement of the study in vitro. Partâ…¡Experimental studies of recombinant plasmid AdhTERT-Can on ovarian epithelium cancer in vitro and vivoMethods1.Recombinant adenovirus plasmid AdhTERT-Can infect HO8901PM cells in vitro mediated with lipofectamine.After infected,the recombinant adenovirus plasmid was confirmed by green fluorescence(GFP) expression under fluorescence microscope.2.After infected by plasmid AdhTERT-Can,Canstatin gene in the infected ovarian cancer were detected by RT-PCR,which the primer and method is the same as the anterior.3.HO8910PM cells were harvested and then the cells growth cycle were determined by flow cytometry(FCM)4.Establish models of human ovarian epithelium cancer transplantation tumor in nude mouse.When the animal models were set up and tumor diameter exceeded 5mm×5mm,all bearing cancer nude mouse were randomized into three groups: AdhTERT-Can,Ad-Can and PBS groups,6 mouse for each group.And all bearing cancer nude mouse accepted gene therapy:0.1ml/each injection of 4.0×10¹⁰ PFU/ ml viral supernatant solution or isovolumic PBS buffer by caudal vein respectively, for a total of two injections separated by a 72-hour interval.5.During the treatment period,the size(length and width diameter) of tumors were measured and the tumor volume was calculated.The anti-tumor efficacy was evaluated by tumor volume.6.All the animals were sacrificed in 30 days after injection and tumors were resected for both HE and immunohistochemical examinations(Flk-1,caspase-3,CD105).Flk-1 and caspase-3 was used to explore the mechanism of inhibition of tumor growths.。7.CD105 was used to detecte the microvessel density(MVD) in tumors.8.Statistical treatment:Statistics analysis was performed by SPSS13.0 software, adopt standard deviation,chi-square test and ANOVA,test standardα=0.05. Results1.Recombinant plasmid AdhTERT-Can infect HO8901PM cells mediated with lipofectamine.Premodinantly ovarian cancer cells infected by AdhTERT-Can erupt green fluorescence under fluorescence microscope.It is indicated that HO8910PM cells were infected by AdhTERT-Can successfully.2.Canstatin gene in the infected ovarian cancer was detected,and the fragment of canstatin can be expressed by RT-PCR.It showed one clear bands near 684bp after agarose gel electrophoresis of which length of the target gene is coincidence with the canstatin cDNA(684bp) in Gene Bank.It is indicated that there are canstatin gene expression in the transfected ovarian cancer.3.There are no marked change of the cell growth cycle among AdhTERT-Can,Ad-Can and PBS group by flow cytometry.It is indicated that the growth of ovarian cancer cells were not influenced by canstatin gene in vitro.4.Sclerosises like granule size were observed under the location of vaccination in all mouse subcutaneous injections 14d later.It is indicated that the models of human ovarian epithelium cancer xenografts in BALB/C mouse were set up.5.In the AdhTERT-Can group,the tumor volume was inhibited significantly compared with control groups from 8th day to the 30 days end point of experiment(P＜0.01).It is indicated that AdhTERT-Can plasmid inhibits significantly the growth of human ovarian cancer xenografted tumors in mouse by caudal vein and the inhibition have target.6.The expression of Flk-1 in tumor cell membrane decreased in mouse treated with AdhTERT-Can compared with control groups,however caspase-3 increased (P＜0.05).It is indicated that the mechanism of inhibition tumor growth partly results from increased the expression of caspase-3 and decreased Flk-1 in tumor cell membrane of which induces tumor cell apoptosis and inhibits angiogenesis of tumor.7.CD105 particle main located in membrane or cytoplasm of vascular endothelial cell and were stained buffy.The number of MVD were 9.86±3.21/mm², 15.54±4.67/mm²,18.43±6.55/mm² respectively in AdhTERT-Can,Ad-Can and PBS groups(P＜0.05).CD105 expression and the MVD in AdhTERT-Can group was lower than that in control group.Blood vessels were main located in marginal part of tumor.It is indicated that recombinant plasmid AdhTERT-Can can inhibits the growth of ovarian epithelium cancer transplanted tumor by restraining the angiogenesis of tumor.8.The HE staining slices all showed necrotic regions of tumors in each group, especially in the AdhTERT-Can group and the other organs showed normal.9.The study in vivo showed that the inhibition perstetured until the 30 days end point of experiment.Conclusion1.The recombinant adenovirus plasmid AdhTERT-Can has been constructed successfully.2.The recombinant adenovirus plasmid AdhTERT-Can has significant infection ability for ovarian epithelium cancer cells HO-8910PM.It provides a useful transduction vector for gene therapy of ovarian cancer.3.The growth of ovarian epithelium cancer cells were not infected by AdhTERT-Can vector in vitro.4.It is indicated that AdhTERT-Can plasmid inhibits significantly the growth of human ovarian cancer xenografted tumors in mouse by caudal vein and the inhibition have target.5.The recombinant adenovirus plasmid AdhTERT-Can inhibits the growth of ovarian epithelium cancer transplanted tumor by restraining the angiogenesis of tumor.6.The mechanism of inhibition tumor growth partly results from increased the expression of caspase-3 and decreased Flk-1 in tumor cell of which induced tumor cell apoptosis and inhibitted angiogenesis of tumor.7.The recombinant adenovirus plasmid AdhTERT-Can showed no toxic and side-effect for the other organs. Innovations1.The hTERT core promoter was linked to canstatin for the first time to construct recombinant adenovirus plasmid carting human canstatin gene and hTERT gene core promoter.The topic use ovarian epithelium cancer as empirical study object of canstatin gene for the first time.2.AdhTERT-Can plasmid inhibits significantly the growth of human ovarian cancer xenografted tumors in mouse by caudal vein and the inhibition have target.3.The mechanism of inhibition tumor growth partly results from increased the expression of caspase-3 and decreased Flk-1 in tumor cell of which induced tumor cell apoptosis and inhibitted angiogenesis of tumor.