| At present,cancer is one of the biggest diseases threatening human health.It has become the"first killer"threatening human life in developed countries,and it has become the second biggest risk factor in developing countries[1-3].Prostate cancer is currently the most common male malignant disease with high morbidity and mortality.In the United States,prostate cancer has become the top ten new cancers,and the number of male patients in China has been increasing in recent years[4,5].Traditional treatments for cancer include surgery,chemotherapy,radiation therapy,but it is effective in a few patients who have early detection of the disease.It is not ideal in some patients who start in the middle and late stages,and often with side effects,the complete cure rate is low,and it is easy to relapse.In recent years,with the development of biomedical technology and genetic engineering technology,many new treatment programs have been developed one after another,among which gene-targeted therapy and viral vector therapy have received much attention.With the outstanding performance of gene therapy,especially the development and clinical manifestations of adenoviral vector drugs,many researchers have paid attention to it.Therefore,the strategy of targeting tumors with adenovirus as a carrier is expected to replace traditional therapy to treat cancer.Adenovirus as a kind of oncolytic virus itself has a certain killing effect on tumor cells,and the harm of adenovirus to normal human body is very small compared with other viruses.The adenovirus genome is small and can be easily modified.For example,the modification of adenoviral fiber and hexon protein can make adenovirus specifically target certain tumor cells,and after inserting foreign genes into adenoviral vectors,very efficient expression of foreign antigens in large quantities,so adenovirus has great potential in the field of cancer therapy.VP3 is a 13.6 kDa serine-threonine-rich protein containing 121 amino acids with the ability to induce programmed cell death（PCD）in chicken thymocytes.Since the VP3 gene product has the ability to induce death and apoptosis,the gene product was renamed apoptin.Apoptin is a protein that specifically induces apoptosis in tumor cells and does not induce apoptosis in most normal cells.Since this protein is not mediated by p53 protein and is not inhibited by overexpression of Bcl-2,it is considered a novel anti-tumor biological protein.The HTERTp is a promoter that can specifically initiate the transcription of downstream genes in tumor cells.In this study,it can specifically activate the adenovirus promoter gene E1a gene in tumor cells,so that adenovirus can only replicate and express Apoptin protein in tumor cells,which endow recombinant adenovirus with the ability to kill tumors with double specificity.When the anti-tumor effect of anti-tumor drugs on tumor cells is monitored in vivo,immunodeficient animals are generally used for the production of tumor models,and generally nude mice are used.However,traditional animal models cannot dynamically study the growth and movement of tumors,and the killing effect of drugs on tumors,so they are gradually limited.The luciferase gene can be used as a reporter gene to recombine into the tumor cell genome,making the tumor model a step up in accuracy.The use of in vivo imaging technology allows for more continuous,controlled observation of tumor growth and metastasis,which provides a very off-campus tumor model for the field of tumor research.In this study,the human prostate cancer PC-3 and mouse prostate cancer RM-1were performed using the bispecific oncolytic adenovirus Ad-VT and its control viruses Ad-T,Ad-vp3 and Ad-mock for cell inhibition studies.Ad-VT contains a cytomegalovirus（CMV）promoter to activate the apoptin gene（Apoptin）and the tumor-specific promoter（hTERTp,human telomerase reverse transcriptase）to initiate the E1A gene（the essential gene for viral replication）.The virus has the ability to specifically replicate tumors and specifically induce apoptosis of tumor cells.Purposes:This study demonstrated that bispecific oncolytic adenovirus Ad-VT has significant inhibitory effects on human prostate cancer PC-3 and murine prostate cancer RM-1 cells by various experiments in vitro and in vivo.Methods:1.Transfect cells with pGL4.51 carrying the luciferase gene,and screen for human prostate cancer PC-3-luc and mouse prostate cancer RM-1-luc cells stably expressing luciferase by G418 pressure,and both cells were tested for biological properties.2.The killing effect of Ad-VT on human prostate cancer PC-3-luc and mouse prostate cancer RM-1-luc cells was analyzed by crystal violet staining and MTS assay.Hoechst staining and AnnexinV-FITC/PI flow analysis were performed.The main pathways of Ad-VT inhibition of human prostate cancer PC-3-luc and murine prostate cancer RM-1-luc cells were analyzed by methods such as caspase activity assay and JC-1 staining.3.The effects of Ad-VT on the migration and invasion of human prostate cancer PC-3-luc and mouse prostate cancer RM-1-luc cells were analyzed by cell scratch assay,Transwell chamber migration and invasion assay.4.A nude mouse tumor model expressing luciferase was established using human prostate cancer PC-3-luc and murine prostate cancer RM-1-luc cells,and the tumor luminescence intensity was monitored weekly,the tumor size was measured weekly,and the mice was observed every day.Survival conditions were used to analyze the inhibitory effect of Ad-VT on human prostate cancer PC-3-luc and murine prostate cancer RM-1-luc cells in vivo.Result:1.The human prostate cancer PC-3-luc and mouse prostate cancer RM-1-luc cells expressing the luciferase gene were successfully constructed,and the cell line stably expressing the luciferase gene was screened,and there was no significant biological difference from the original cells.2.Inhibition of human prostate cancer PC-3-luc and mouse prostate cancer RM-1-luc cells by crystal violet staining and MTS assay;Hochest staining,Annexin V-FITC/PI flow analysis the detection of caspase activity and JC-1 staining showed that Ad-VT mainly inhibited the growth of human prostate cancer PC-3-luc and mouse prostate cancer RM-1-luc cells by endogenous apoptosis pathway.3.Cell-scratch test,Transwell chamber migration and invasion assay showed that Ad-VT significantly inhibited the migration and invasion of human prostate cancer PC-3-luc and mouse prostate cancer RM-1-luc cells.4.By constructing a nude mouse tumor model expressing human luciferase-expressing human prostate cancer PC-3-luc and murine prostate cancer RM-1-luc cells,it was shown that Ad-VT can also significantly inhibit human prostate cancer PC-3-luc and murine prostate cancer RM-1-luc cell tumor growth in vivo and prolong survival of nude mice.Conclusion:This study successfully constructed human prostate cancer PC-3-luc and mouse prostate cancer RM-1-luc cells,and confirmed Ad-VT against human prostate cancer PC-3-luc and mouse prostate cancer RM-1-luc by various in vitro and in vivo experiments.The Ad-VT has a significant killing effect,indicating that Ad-VT has the potential to become a tumor therapeutic drug. |
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