Antitumor Effects Of Dm-dNK Combined With Gemcitabine For Primary Breast Cancer

Objective: Breast cancer is one of the most common malignancies in modern women.In China,the incidence of breast cancer has gradually increased in recent decades,and the mortality rate remains high and tends to be younger.In recent decades,gene therapy has been extensively studied in the fields of cancer,hereditary diseases,blood diseases,and infectious diseases,some have achieved remarkable effects in the treatment of the diseases.In tumor treatment,extensive attention has paid to gene therapy because it may eradicate the primary tumor and metastases of malignant tumors without damaging the characteristics of normal tissues.The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster(Dmd NK)is a highly potent nucleotide kinase with a catalytic efficiency 10 to 100 times higher than that of common nucleotide kinases.With extensive substrate specificity,d NK has the capable of catalyzing the phosphorylation of all deoxyribonucleic acids in nature.At present,as a suicide gene,Dm-d NK is used in a variety of malignant cells,in addition to directly killing tumor cells,it can not only kill tumor cells directly,not also by the bystander effect.We have demonstrated in previous studies that the expression of Dm-d NK in tumor cell lines can significantly increase its sensitivity to a variety of cytotoxic nucleoside analog drugs.However,whether the suicide gene system can kill primary cultured tumor cells has not been reported.To this end,this study constructed a selective replication-type adenovirus carrying the suicide gene Dmd NK,and explored the killing effect of the system on primary breast cancer cells,providing new ideas for the treatment of breast cancer.Methods: During the period from 2017-06-01 to 2018-06-01,fresh tumor tissues of 63 surgically resected primary breast cancer patients in the second ward of the Department of Breast Surgery,the First Affiliated Hospital of China Medical University were selected.Primary tumor cells were cultured by three-dimensional culture technique of tumor cells(n=63).Recombinant adenovirus GFP with different multiplicity of infection(MOI)was added,and the percentage of green fluorescent protein(GFP)positive cells was counted under a fluorescence microscope to verify the virus transfection efficiency.The oncolytic adenovirus vector SG500-Dm-d NK was constructed and the expression of related proteins was verified by western blot.SG500 and SG500-d NK with MOI=1,MOI=10,MOI=20 were infected with tumor cells for 48 hours,respectively.DFDC at a concentration of 1 μM,10 μM and 100 μM was added to the tumor cells for 72 h;MOI=10 SG500 and SG500-d NK infected tumor cells and cultured for 48 hours,and then added to the above concentrations of DFDC for 72 hours.The cell survival rates of the above three groups of different treatments were measured using a Primage image analysis system.Results: 1.After GFP-selective replication-type adenovirus infects human breast cancer primary cells,fluorescent cells are observed under a fluorescence microscope,and the infection efficiency increases with the increasing of MOI.Western blot analysis of control group,SG500-d NK and SG500 found that Dm-d NK carried by SG500 can express related protein products in primary breast cancer cells.3.At MOI=1 and MOI=10,there was no significant difference in the cell killing effect of SG500-d NK alone in primary breast cancer cells and the cell killing effect of empty vector alone(p>0.05);when MOI=20,The cell killing effect of SG500-d NK was higher than that of SG500(p<0.05).The killing effect of SG500-d NK combined with DFDC on primary breast cancer cells was significantly higher than that of SG500 combined with DFDC and DFDC alone,and it was more obvious with the gradual increase of DFDC concentration(p<0.05).Conclusion : First,the exogenous gene carried by the replication-type adenovirus SG500 can be transfected into the primary breast cancer cells in the droplet and express the relevant protein product.Second,in the primary breast cancer cells,the combination of Dm-d NK and DFDC can kill tumor cells more effectively and exert their anti-tumor effect.It is a further treatment of breast cancer with Dm-d NK/DFDC suicide gene therapy.Clinical research and applications provide strong evidence.