Antitumor Effects Of Conditionally Replicating Adenovirus Carrying Suicide Gene Dm-dNK And CD40L On Breast Cancer

Objectives:Breast cancer is the most common malignant tumor in women.The mortality rate ranks the second among women with malignant tumor after lung cancer.According to the latest statistics from the American Cancer Society,the death rate dropped by 39%between 1989 and 2015,and the fate of 322600 women with breast cancer in the United States has been reversed due to improvements in therapies?eg,adjuvant chemotherapy,endocrine therapy,targeted therapy?and early screening of breast disease.Over the past20 years,gene therapy has been carried out in many fields such as hereditary diseases,malignant tumors and chronic infectious diseases.Some phase I and phase II clinical trials report significant efficacy of gene therapy in severe blood,immune,and nerve system diseases.The Drosophila melanogaster multidrug deoxyribonucleotide kinase?Dm-dNK?has a wide range ofsubstrate specificity and an amazing catalytic rate 10-100 times higher than nucleic acid kinase.it has been confirmed that suicide gene combined with prodrugs could not only directly kill tumor cells,but also by the bystander effect.In addition,it can effectively increase the sensitivity of certain chemotherapy drugs.CD40 is highly expressed in several type cancer cells such as breast cancer,intestinal cancer and liver cancer.It has been demonstrated that the interaction of CD40-CD40 ligand?CD40L?can enhance the immune response and induce the activation of antigen-presenting cells.It can also directly inhibit tumor growth by inducing cancer cell apoptosis.Oncolytic virus therapy is also one of the strategies for gene therapy of malignant tumors.Conditionally replicating adenovirus not only acts as a vector for gene transfer,but also can kill tumor cells by multiple mechanisms such as direct cleavage by virus replication and induction of anti-tumor immune response in vivo.Therefore,this study aims to construct a conditionally replicating adenovirus carrying suicide gene Dm-dNK and CD40L and assess the combined killing effect in vitro and vivo,and provide a new adea for the treatment of breast cancer.Methods:1.Cell cultureBreast cancer cell lines MDA-MB-231,MCF7,human normal embryonic lung fibroblast cell MRC5 and human renal epithelial cell line HEK293 cells are provided by the Lab 1,Cancer Institute of China Medical University.Cell lines were cultured in High Glucose DMEM.All the medium were supplemented with 10%heat-inactivated fetal bovine serum,100 units/ml penicillin and 100 ug/ml streptomycin and cultured under a5%CO₂ atmosphere at 37?.2.Construction and Validation of conditionally replicating adenovirus and determination of the infection rateP74-dNK,P74-CD40L,P74-dNK-CD40L and Ad-GFP?adenovirus containing green fluorescent protein?were constructed under the guidance of Baize company,Shanghai.Reverse transcriptionpolymerase chain reaction?RT-PCR?was used to verify the transcription of target gene,while western blot was used to test the protein expression.Ad-GFP was used to test the infection rate of different cell lines under the same condition.3.Enzyme AssaysMDA-MB-231,MCF7 and normal cell MRC5 were infected with wild-type adenovirus?Ad-WT?,P74-dNK,P74-dNK-CD40L,nucleotide drug labeled with radioactive[³H]were needed.Radioactivity was measured by a scintillator.4.Cytopathic effect?CPE?MDA-MB-231 and MRC5 were infected with Ad-WT,P74-dNK,P74-CD40L,P74-dNK-CD40L?MOI=0 and 1?at logarithmic growth phase.The medium containing virus was discarded after 4h and replaced with DMEM containing 10%FBS.48h later,oncolysis was observed under an inverted microscope.All cells and supernatants were harvested 24 hours after infection,and cell lysates were obtained after three cycles of freezing and thawing.Adenovirus titers were evaluated in HEK293 cells using TCID50?tissue culture infectious dose?.MTT assay was used to quantitatively detect the cytopathic effect of different cells transfected with different viruses.5.Evaluation of cytotoxicity of oncolytic adenovirus carrying Dm-dNK or CD40LMTT assay was used to detect the cytotoxicity of oncolytic adenovirus carrying P74-dNK combined with BVDU or CD40L alone in different cell lines.6.The cytotoxicity and mechanism of oncolytic adenovirus co-expressing Dm-dNK and CD40LThe MTT assay was used to detect the cytotoxicity of different cell lines infected with P74-dNK-CD40L with/without BVDU and with the changes of MOI of virus and concentration of BVDU.Flow cytometry was used to detect the apoptosis and bystander effect was also explored in MDA-MB-231 cells.7.Assessment of viral replication of oncolytic adenovirus co-expressing Dm-dNK and CD40L combined with BVDUViruses titers were detected before and after addition of BVDU for different cells transfected with Ad-WT,P74-dNK,P74-CD40L,P74-dNK-CD40L.8.The cytotoxicity of oncolytic adenovirus co-expressing Dm-dNK and CD40L in vivoMDA-MB-231 cells were inoculated into the right armpit of 36 BALB/C nude mice and divided into 4 groups:PBS,BVDU,P74-CD40L+BVDU,P74-dNK-CD40L+BVDU.All groups were blinded to the investigators during the experiment.When tumors grew to about 90 mm³ in size,all the mice in the adenovirus treatment groups were intratumoraly injected with a total dose of 10⁹pfu adenovirus,3 times at 48 h intervals.Also BVDU was injected at 5mg/kg twice,on days 2 and 8 after virus injection?for BVDU group,BVDU was injected at the same time with virus injection groups?.The length and width of tumors with vernier caliper every 5 days.The tumor volume?mm³?=1/2*length?mm?*width?mm?²Results:1.The recombinant adenoviral plasmid P74-dNK-CD40L was successfully constructedE1B region was knocked out and hTERT promoter was inserted before Dm-dNK gene.E3 region was knocked out and inserted with CD40L gene to generate P74-dNK,P74-CD40L,P74-dNK-CD40L.The infectivity of the cell lines were determined by the wild-type 5 adenovirus with expression of Green fluorescence?Ad-GFP?.The cancer cell lines MDA-MB-23 1,MCF7 and the normal cell line MRC5exhibited nearly the same infectivity.2.Expression of target genes and measurements of enzymatic activityRT-PCR and western blot verified the mRNA and protein expression of Dm-dNK and CD40L.The expression of E1A protein in MRC5 infected with oncolytic adenovirus was significantly lower than that in Ad-WT,indicating that oncolytic adenovirus replication defect in normal cell lines.The kinase activity of MDA-MB-231 and MCF7infected with recombinant adenovirus P74-dNK and P74-dNK-CD40L was 100-200times than that of Ad-WT,and the activity of all viruses in MRC5 was low.3.Oncolysis in breast cancer cellsMDA-MB-231 and MCF7 showed obvious tumor lysis in all virus groups,while MRC5 transfected with oncolytic adenovirus groups showed no significant tumor lysis except Ad-WT,which was consistent with the viruses titer in all groups.The oncolysis difference was related with the TERT expression in cells and the hTERT promoter in oncolytic adenovirus.4.BVDU enhanced the cell killing effect of Dm-dNK,CD40L have inhibitory effect on CD40 posotive cells.Dm-dNK combined with BVDU showed obvious cell killing effect on breast cancer cell lines,but not on normal cells.Oncolytic adenovirus carrying CD40L showed significant inhibitory effect on CD40 positive breast cancer cell line MDA-MB-231.5.The killing effect of oncolytic adenovirus co-expressing Dm-dNK and CD40L genes combined with BVDU on breast cancer cells was induced by apoptosis and bystander effect.P74-dNK-CD40L combined with BVDU showed most significant cell killing effect on MDA-MB-231,with less virus and lower dosage of BVDU.And with the virus titer and BVDU concentration increased,the killing effect was more obvious.The apoptosis rate of P74-dNK-CD40L combined with BVDU in MDA-MB-231 was significantly better than that of other virus-infected groups,and obvious bystander effect was observed when MOI?1.6.BVDU inhibits oncolytic adenovirus replication carrying Dm-dNK and CD40LAfter BVDU added to MDA-MB-231 transfected with P74-dNK,P74-dNK-CD40L,the virus titer decreased obviously.There were no significant changes in MDA-MB-231other groups and MRC5 cells.7.Inhibitory effects of oncolytic adenovirus co-expressing Dm-dNK and CD40L combined with BVDU in vivoThe volume of nude mice xenografts in PBS group increased significantly,and the tumor growth inhibition of P74-CD40L,especially P74-dNK-CD40L combined with BVDU group was the most obvious,which was consistent with the effect in vitro.Conclusion:Oncolytic adenovirus with E1B region deleted and carrying hTERT promoter co-expressing Dm-dNK and CD40L was successfully constructed.The system was confirmed to replicate only in breast cancer cells not in normal cells,and the kinase activity was more than 100-fold greater than that of normal kinases.oncolytic adenovirus co-expressing Dm-dNK and CD40L kill breat cancer cells significantly through oncolysis,Dm-dNK combined with BVDU induced suicide gene therapy and CD40-CD40L interaction,especially in MDA-MB-231.And confirmed that this inhibitory effect was induced through apoptosis and bystander effect.Meanwhile,the addition of prodrug BVDU to the system not only enhanced the killing effect on cancer cells but also inhibited virus replication and effectively prevents the virus from being overly infected and spread,making the system feasible in terms of safety.This study provides new ideas and strategies for breast cancer gene therapy.