| Background and Objective:Ovarian cancer is malignant tumor of ovary, one of the most common malignant tumors of female genital system. Due to the lack of early diagnose methods, it often be diagnosed too late. Its mobility is tending to become the leading course of gynecologic malignant tumors. Though the prognosis of patients suffered from ovarian cancer is much better than before, but the 5-year survival rate is only 25- 30%. With the rapid development of molecular biology, gene therapy for cancer is come into clinic use. Gene therapy becomes the lively research field of neoplasm. Because of its characteristic of localization in the peritoneum cavity, ovarian cancer is often used for animal and clinic studies. But the development is not perfectly, the leading obstacle is the traditional gene therapy often use replication-deficient adenovirus or physical chemistry methods for gene transfer, and these cause lower gene transfection and expression, vectors lack of target, so the therapeutic purpose can not achieve.Adenovirus vector has being widely used for gene therapy and clinic studies. But most adenoviruses exist disadvantages, such as short time expression of exotic gene, strong immunity, and obvious cytotoxicity in high titer. The emergence of tumor-selective replication-competent virus has provided a new way for the tumor gene therapy, which makes up some disadvantages of replication-deficient adenovirus vector in tumor gene therapy. The virus can selectively replicate in tumor cells and lead to the lysis of the cells and not the normal cells, thus to form high concentration of virus in the primary and metastasized tumor tissue. This method has no severe toxic and side effects compared with traditional ways. ONYX-015 is a adenovirus chimera of type 2 and type 5 with the large part of Elb 55Kda gene deleted and it can selectively replicate in tumor cells with abnormal p53 gene but spare the normal cells. P53 gene is one of the most easily mutant tumor suppress gene in solid tumors of mankind. It was showed that about 55-80%primary ovarianl tumor cells are with the p53 abnormality. ONYX-015 has been used for ovarian cancer experimental trial.An important characteristic of solid tumor is that to satisfy its abundant blood supply in the course of tumor development by angiogenesis, and angiogenesis plays indispensable role in the tumor's growth and metastasis. Ovarian cancer has plentiful amount of blood vessels, and is apt to cause peritoneum cavity metastasis. Antiangiogenesis has become the new strategy for tumor therapy. In the found antiangiogenesis factors, endostatin is the most potent one and there are no report for toxicity and side effects either in animal experiments or in the latest finished Phase I clinical test. Endostatin can effectively inhibit the migration and vessel formation of the endothelia induced by the vascular endothelial growth factor (VEGF) and inhibit the growth and metastasis of solid tumor in animal experiments. Endostatin can be administrated for long time without the fear of immunogenesis and drug resistance. There were data showing that the expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) in ovarian cancer tissue are closing related with the biological behavior of ovarian cancer, such as recurrence and metastasis.The huge amount of therapeutic dosage, when directly administrated with pure hEndo protein, is far beyond the achievement of traditional bioengineering method, and its bioactivity is difficult to maintain in normal condition outside, which hampered its direct clinical application. The problem is possible to be solved by the method of gene therapy. There had been successful reports on the replication-deficient adenovirus vector mediated endostatin gene therapy, which led to continuous high concentration of expressed endostatin. However, for the safety consideration, the large part of El gene region were deleted in the early reformation for replication-deficient adenovirus vector and at the same time this also restricted its replication ability in tumor cells and the efficient expression of therapeutic gene. How to make the therapeutic gene continuous high concentration of expression in the tumor cells but spare the normal cells is a critic problem. At the same time the target and safety of vector, that is are also needed to be concerned in tumor gene therapy.Tumor-selective replication-competent virus has provided a new way for the tumor gene therapy. If the virus armed with anti-cancer gene, the gene can be efficiently expressed in the tumor tissue with the viruses' replication. The combinedtherapeutic effects of both virus and gene will improve the results of tumor therapy.Methods and results:1. Construction and preparation of replication-deficient and replication-competent adenovirus vector carrying human endostatin geneFirstly, by the method of overlap PCR, inserted a synthetic oligonucleotide coding for the signal peptide of M-oncogenous protein to upstream of the endostatin gene and an EcoRI and Xbal endoneuclease site was respectively introduced into either side of the full-length human endostatin cDNA for subcloning into pBluescriptSK. After sequence confirm, then an EcoR I -Xba I fragment from pBlue-endo containing the fused signal sequence and endostatin gene was inserted into the replication-deficient adenovirus shuttle vector pCA13, between the two Bglll endoneuclease site of which is the exotic gene expression cassette (containing CMV promotor + fused signal sequence and endostatin gene +SV40 polyA termination signal). Then the cassette from plasmid pCA13-Endo after digested with Bglll was cloned into the replication-competent adenovirus Ad A Elb-hE,CNHK200-hE (CK-hE) shuttle vector pXC7c.Then plasmid pCA13-hEndo, pXC7c-hEndo, pCA13-GFP and pXC7c-GFP were each co-transfected with pBHGE3 (an El-deleted backbone plasmid of type 5 adenoviral right arm) into 293 cells using the Lipofectamine2000 Effectence Transfection Reagent to construct the recombinant adenoviruses, and the obtained adenoviruses were each designed as CK-hE, Ad-hE, CK-GFP and Ad-GFP. After plague purification for three times, the adenovirus carrying human endostatin gene were identified by PCR, and the adenovirus carrying GFP gene were directly cloned under the fluorescent microscope.Finally, the adenoviruses were amplified in 293 cells to the wanted quantity;high titer virus was purified by ultra-centrifugation on cesium chloride (CsCl) gradients. The titers of all adenoviruses were determined with the Tissue Culture Infectious Dose 50 (TCID50) method and showed as plaque-forming units per milliliter (pfu/ml). The final titer of CK-hE> Ad-I^ CK-GFP or Ad-GFP is 4X 1010pfu/ml, 1X 10npfu/ml, 3.8X 1010piVml or 7.5 X 1010pfu/ml respectively.2. The therapeutic effects of replication-competent adenovirus carrying human endostatin gene on ovarian cancer in vitroThe killing effects difference between CK-hE. Ad-hE and ONYX-015 werefirstly observed by the method of cytopathic effect (CPE) assays after L02, OV-90 cells were infected with these three virus at different MOI respectively. It was showed that CK-hE, even though with the insertion of about 1.2kb-long human endostatin gene, similar to ONYX-015, could still selectively replicate in and kill the tumor cells but did not influence the growth of normal cells, and its ability of replicating in tumor cells is far superior to Ad-hE. The CPEs of Ad-hE to L02 were similar to that of CK-hE and ONYX-015, but to tumor cells was quite another situation, the number of MOI at which Ad-hE could kill all the tumor cells was three exponential times more than that of CK-hE and ONYX-015. The killing effect difference between each virus were determined by the method of MTT, and it was showed that the killing rates of CK-hE and ONYX-015 to OV-90 cells were much larger than that of Ad-hE 3 days after infection (P<0.01) and there was no difference between those of CK-hE and ONYX-015(P>0.05).Virus replication experiments elucidated the mechanism of the different CPEs and killing effects of the three viruses to these three different cell lines. None of the viruses replicated in the normal liver cell line L02, the largest replication fold was 8.47 at 96 hours after infection. However, in the ovarian cancer cell line OV-90, the replication ability of CK-hE and ONYX-015 were superior to that of Ad-hE with the replication fold being more than 2000 at 96 hours after infection, whereas the replication fold of Ad-hE in OV-90 cell was only 3.42 folds at 96 hours after infection.By Western Blot, it showed that the 20kDa endostatin protein could be detected in the culture supernatant of OV-90 cells after infected either with CK-hE or Ad-hE, which suggested that either of these two adenovirus vector could express secreted human endostatin protein. Then we determined the concentrations of endostatin protein quantitatively in the culture supernatant of OV-90 cell after infected either with CK-hE or Ad-hE by Enzyme-Linked Immunoabsordent Assay (ELISA). With the time going, the tendency of endostatin expression was according to that of CK-hE's replicating in OV-90 cells. The concentration of endostatin expressed by CK-hE was increased , was 8 times more than that expressed by Ad-hE at the 5th day after infection.By the use of embryonic chorioallantoic membrane (CAM) assay, which confirmed that the biological antiangiogenesis activity of endostatin expressed in the ovarian cancer cells by CK-hE was similar to that of the standard sample. At thesame time, we also observed that the large quantity ot aaenovirus couia aiso uu some harm to the normal vessels.It could be showed easily and more directly that the differences of vector target, therapeutic gene expression and CPEs to tumor cells between the replication-deficient and replication-competent adenovirus by L02 and OV-90 cells were infected with either virus carrying GFP report gene. The fluorescence in the two kinds of cells infected with Ad-GFP or CK-GFP and in L02 infected with CK-hE remained unchanged, whereas the fluorescence region continually enlarged and at last the central tumor cells went dead with the fluorescent protein decomposed in OV-90 infected with CK-hE.3. The therapeutic effects on ovarian cancer of replication-competent adenovirus carrying human endostatin gene in vivoTo establish the animal model bearing human ovarian cancer, the BALB/c nude mice were inoculated subcutaneous with OV-90 cells. Then these nude mice were cured by the method of directly intratumoral injection with virus. The tumors in the group treated with CK-hE grew at a more slowly speed and the tumor sizes were markedly smaller than those of the group treated with Ad-hE ( P<0.05) and the control group treated with PBS ( P<0.01 ) . No therapeutic effect of tumor disappears or growth halting was observed. Following experiments explained the mechanism of anti-tumor synergy of endostatin and replication-competent adenovirus.ELISA results showed that the endostatin could be detected in the blood serum of nude mice bearing human ovarian cancer after single intratumoral injection with adenovirus carrying human endostatin gene with the time going and the concentration of expressed endostatin by CK-hE was increased much higher. The endostatin levels of nude mice in CK-hE treated group were much higher and increased faster than those of mice in Ad-hE treated group all along. At the 7th day after injection the endostatin concentration of nude mice in CK-hE treated group(1897±196ng/ml) was significantly higher than that of nude mice in Ad-hE treated group(637 ± 121ng/ml) ( P<0.05).Immunohistochemical staining for vWF of vessel endothelial cells revealed that a decreased microvessel in the tumor tissues treated with CK-hE compared with ONYX-015.In the tumor tissues treated with CK-hE, immunohistochemical staining for Hexon of adenovirus capsid showed there were large quantities of positive tumor cells Crystal grid in the tumor cell's nucleolus, which was an affirmative proof for the replication of adenovirus in cells, and the super-micro structure change before lysis of the cell were clear seen under the electron microscopy. All of above suggested that the replication-competent adenovirus could selectively replicate in tumor cells, which not only led to the lysis of tumor cells as a therapeutic effects but to the increase of virus vector and gene expression, thus the therapeutic effects were enlarged.Conclusions:Compared the therapeutic differences of ovarian cancer between replication-competent and replication-deficient adenovirus vector carrying human endostatin gene both in vitro and in vivo in this experiment, verified that replication—competent adenovirus vector not only can selectively replicate in and lyse ovarian cancer cells but can lead to a much higher level of endostatin expression with bioactivity than replication-deficient adenovirus do. The therapeutic synergy enhanced the ideal therapeutic effects on ovarian cancer. The results suggested that the combination of viral therapy and gene therapy for tumor could exert the advantages of both simultaneously, and the therapeutic effect was far superior to either gene therapy or viral therapy alone. However, there was no therapeutic effect of tumor disappear or growth halting observed in nude mice bearing human ovarian cancer after repeated intratumoral injection with CK-hE. |
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