Experimental Studies Of Therapeutic Effect Of The Gene Encoding Drosophila Melanogaster Multisubstrate Deoxyribonucleoside Kinase On Breast Cancer By Retrovirus-Mediated; Expression Of CD133, PAX2, ESA, And GPR30 In Invasive Ductal Breast Carcinomas

Part 1:The study of the therapeutic effect and bystander effect of Dm-dNK mediated by retroviral vector on breast cancer cells in vitroObjective:To amplify and harvest the recombinant retrovirus with Dm-dNK gene, and establish the new gene recombinant breast cancer cell lines(MCF7,ER+ and MDA-MB-231,ER-) expressing Dm-dNK stably for Dm-dNK/AraT(AraC) gene therapy study.Then to study the therapeutic effect of Dm-dNK/nucleoside analog gene therapy system on breast cancer cells in vitro.Mehtods:Recombinant retrovirus plasmids with Dm-dNK gene and without Dm-dNK gene was amplifed with Bacillus coliαand QIAGEN midi-abstract kit.Then these plasmids transfected packing cells PT67.Supernatant of the cloned packing cells was harvested.Breast cancer cells were infected by the recombinant retroviruses with Dm-dNK or without Dm-dNK.The cells were selected by neomycin(G418) at the screening concentration.During the process,hexadime bromide(also named polybrene) was added to 8μg/ml concentration into the infecting system to assist the recombinant retrovirus infection.After 6 hrs,the culture fluid was refreshed.Another 24 hrs culture was going and the infecting process was repeated.The cells were cultured for 48 hrs and then G418 was added into culture fluid to the final screening concentration.The cell clones of anti-G418 were selected under G418 for 21 days.These breast cancer cell lines containing Dm-dNK or pLXSN genes were named MCF7-dNK,MCF7-pLXSN, MDA-MB-231-dNK,and MDA-MB-231-pLXSN,respectively.The cells of MCF7-dNK/MDA-MB-231-dNK,MCF7-pLXSN/MDA-MB-231-pLXSN,and MCF7 /MDA-MB-231 were cultured respectively and then AraT/AraC was administered into their culture fluids.Breast cancer cells infected with Dm-dNK and their pro-generation cells were mixed at different proportions.The AraT/AraC cytotoxicity efficacies to these cells were evaluated by MTT methods.Then the growth rates were counted.Result:It was comfirmed that packing cell PT67 was transfected with GFP.Then these plasmids transfected packing cells PT67.Supernatant of the cloned packing cells infected by the recombinant retroviruses was harvested.These breast cancer cell lines MCF7-dNK,MCF7-pLXSN,MDA-MB-231-dNK,and MDA-MB-231-pLXSN were established.The suicide gene Dm-dNK combined with AraT/AraC could significantly kill MCF7-dNK and MDA-MB-231-dNK.Their growth rates were remarkable decrease comparing with control cells,and more remarkable,more concentrations of AraT/AraC.The growth inhibition rates of MCF7-dNK and MDA-MB-231-dNK were 77.0%and 75.4%at the concentrations of 10^-1 mmol/L AraT,respectively,73.4%and 78.2%,10^-3 mmol/L AraC.Bystander effect existed in the mixed cells of MCF7-DNK/ MDA-MB-231-DNK and pro-generation cells.Conclusion:The pLXSN-dNK/AraT(AraC) suicide/prodrug system can significantly inhibit the growth of breast cancer cells in vitro and existed bystander effect on breast cancer cells. Part 2:The therapeutic effect of Dm-dNK on breast in femine nude mice in vivoObjective:To study the therapeutic effect of Dm-dNK/nucleoside analog gene therapy system on breast cancer cells in vivo.Mehtods:Femine nude mice were xenografted by MDA-MB-231 cells.After the cells were implanted subcutaneously for 7 days and the xenografted tumors free grew up a little bit,viral supernatant and AraC was injected into tumors and intraperitoneally, respectively and continuously.The tumors'volumes were measured routinely and survival days of the nude mice counted in following days.Result:In vivo,after AraC administration the growth of xenogrfted tumor in experimental groups was sifnificantly suppressed,the tumors'volumes were much smaller and the life spans of experimental xenografted tumor nude mice were prolonged distinctly comparing with control groups.Conclusion:The pLXSN-dNK/AraC suicide/prodrug system can significantly inhibit the growth of breast cancer in femine nude mice and prolongs the survival of femine nude mice with breast cancer. Part 3:Expression of CD133,PAX2,ESA,and GPR30 in invasive ductal breast carcinomasObjective Biomarkers in breast neoplasms provide invaluable information regarding prognosis and help determining the optimal treatment.Methods In 74 invasive ductal breast carcinomas,we investigated the protein expression of these molecular markers by immunohistochemistry,and their associations with known Clinicopathological parameters,tumor recurrence,and expression of some known biomarkers.We studied the interrelationship between the expressions of these proteins.Results CD133,a putative CSC marker,was positively related to tumor size, tumor stage,and lymph node metastasis.PAX2 was negatively correlated with tumor recurrence.ESA,one of the breast CSC markers,was an indicator of tumor recurrence.GPR30 was associated with hormone receptors.Despite the correlation between GPR30 and the nuclear estrogen receptor,the expression was dependent. Positive staining of GPR30 in tumors displayed a significant association with high C-erbB2 expression and a tendency for tumor recurrence.A positive relationship between GPR30 and CD133 existed.Conclusion Detecting the expression of CD133,PAX2,ESA,and GPR30 in invasive ductal breast carcinomas may be of help in more accurately predicting the aggressive properties of breast cancer and determining the optimal treatment.