Targeted Molecular Therapy Of Oncolytic Adenovirus Harboring Multisubstrate Deoxyribo-nucleoside Kinase Of Arabidopsis Thaliana(At-dNK) In Lung Cancer

Objective:Lung cancer is the highest malignant tumors in China and all the world,but also the world's leading cause of death of malignant tumors.Even after radical surgery,there is still the risk of metastasis,recurrence,poor prognosis of death.Surgery is currently the best treatment for non-small cell lung cancer,but the operation of patients with little less,about 20%-30%of the total number of illness,the vast majority of patients found in the lung lesions have been lost Surgical Opportunity,Chemotherapy has become an important treatment for non-small cell lung cancer,but the traditional chemotherapy drugs lack of targeting,a wide range of anti-effects on the tumor cells,while the killing of normal cells is also very obvious;Toxic side effects,to patients with great pain.Therefore,focusing on lung cancer molecular biology and other fields as the basis of lung cancer gene molecular targeting research to explore its pathophysiology pathogenesis and comprehensive treatment of lung cancer research has become an important issue.This research aimed to select adenovirus-mediated At-dNK gene targeting molecular therapy for lung cancer as the research content.In this study,we observed the dual therapeutic effect of suicide gene combined with prodrug system on lung cancer cells in vitro and in vivo,the aim of this study is to establish a new molecular therapy for effective intervention of lung cancer.Methods:1.Construction and expression of an adenovirus plasmid and detection of its enzyme activity1.1 The construction of adenovirus vector and transfection:The At-dNK was cloned into the plasmid vector pENTER12 by polymerase chain reaction(PCR)according to the cDNA of At-dNK,a primer having EcoRI and XhoI restriction sites was designed at both ends thereof,and the plasmid pENTER12 contains the CMV promoter,the multiple cloning site,and the SV40 poly A tail.PENER12-dNK and plasmid PPE3-RC were recombined with endoglucanase LR and co-transfected with recombinant adenovirus plasmid pZD55 into adenovirus packaging cell HEK293 cells,9-14 days after the virus plaques,the use of DNA extraction kit to extract the recombinant virus DNA,verified by PCR named ZD55-dNK.In addition,the At-dNK termination codon was knocked out and fused with the green fluorescent protein gene(GFP)to form dNK-GFP.At-dNK expression and transfection efficiency were observed by fluorescence.Replication defect Adenovirus Ad-GFP was used to compare the differences in infection rates among different cell lines.1.2 The expression of At-d NK gene and the detection of enzyme activity:A-549,NCI-H520,549/dNK and 520/dNK,the lung cancer cell lines A-549,NCI-H520 after adenovirus ZD55-dNK infection and the normal cell line MRC-5 were isolated and purified by TRIZOL method.The expression of the target gene was detected by using a reverse transcription PCR kit.Enzyme activity was determined by extracting cellular proteins,labelingnucleotides with radioactive[3H]as substrate.The mixture was reacted on Whatman DE-81 filter paper for different times and the radioactivity was measured by a liquid scintillation meter.2.Study on the killing effect of At-dNK gene combined with nucleoside analogues on tumor cells in vitro and in vivo2.1 Nucleotide analogues inhibit the cytotoxicity of ITCH to lung cancer cells:After phosphorylation,nucleoside analogues can inhibit the synthesis of DNA,break the DNA strand,induce p53 dependent apoptosis,and regulate the Wnt/?-catenin pathway.Inhibiting the expression of ITCH-ITCH could inhibit the proliferation of tumor cells and promote the apoptosis of tumor cells.SiRNA and siNC(negative control group)were used to transfect lung cancer cell lines and silence ITCH.After 48 hours of transfection,the proliferation of tumor cells was measured by MTT method.The apoptosis and the proportion of apoptosis were observed by V-FITC double stainingassay kit.The cells were plated in 6-well plates and treated with add nucleoside drug,by Annexin V-FITC and propidium iodide double staining,then using the flow cytometry to analyze the proportion of apoptosis.2.2 The killing effect of At-dNK gene combined with nucleoside analogues on lung cancer cells:Stable transfected cells 549/dNK and 520/dNK and untransfected control cells A-549 and NCI-H520 were plated,added different concentrations of ara-C,ara-T and CdA,the cell proliferation was detected by MTT assay after 5 days co-culture.Lung cancer cell lines A549,NCI-H520 and normal cell line MRC-5 are plated.The medium was replaced with virus solution ZD55-dNK,ZD55,DL1520,and WtAd on day2,respectively.After 2 days of infection,the cells were incubated with different concentration of BVDU And ara-T at concentrations of 0,0.001,0.01,0.1,1,and 10?M.The cell proliferation was measured by MTT assay after 5 days co-culture.Annexin V-FITC double-staining apoptosis detection kit was used to observe the apoptosis and percentage of apoptosis after treatment.The cells were plated in 6-well plates and treated with add nucleoside drug,by Annexin V-FITC and propidium iodide double staining,then using the flow cytometry to analyze the proportion of apoptosis.2.3 Inhibitory effect of At-dNK gene combined with nucleoside analogues on transplanted tumor in nude mice:The tumor models were implanted subcutaneously in nude mice of 100-150 mm~3,and 549/dNK cells and control A-549 cells were subcutaneously injected into BALB/C nude mice at 6-8 weeks of age.The treatment group consisted of 10 animals:5 of A-549 metastases and 5 of 549/dNK metastases;all treated animals were treated with ara-T intraperitoneally 6 days after tumor injection for one month.The control group consisted of 8 nude mice:4 of A-549 metastases,4 of549/dNK metastases,and 6 days after injection into the control group,PBS or ara-T was intraperitoneally injected.After treatment,nude mice were sacrificed and the tumor volume was measured according to the formula(a2×b)/2(a is a minor axis).The expression of At-dNK was observed by fluorescence in vitro imaging in 549/dNK-EGFP metastatic nude mice.3.Research on security mechanism detection3.1 Inhibitory Effect of Recombinant Adenovirus ZD55-dNK Combined with BVDU on Adenovirus:A-549,a lung cancer cell line in logarithmic growth phase and Normal fibroblast MRC-5 were plated overnight in 24-wells plate.The cells were infected with MOI=1 virus ZD55-dNK,ZD55,DL1520 and WtAd5 for 2 days and 1?M of BVDU or ara-T was added to each well of the cells for 3 days.The cells were treated with different virus and the medium were collected,and the virus was released by repeated freezing and thawing.The virus titer was measured by TCID50(tissue culture infectious dose)method.The titer of the virus was measured before and after the drug treatment by renal fibroblast293.In the same way,the cells were treated with virus and drug as above.Cell protein was extracted by cell lysates and the expression of E1A protein was detected by Western blotting.Results:1.The construction of adenovirus vector and transfection:The549/dNKEGFP cells screened by transfection of A-549 cells showed that the fusion protein expression of At-dNK was concentrated in the nucleus.NCI-H520 as well.DNA from adenovirus ZD55-dNK was extracted using PCR to identify whether the virus was correctly constructed.The length of the CMV promoter was 521bp and the At-dNK sequence length was 779bp.The infection rate of the cell line to the virus was determined by the wild type 5 adenovirus(Ad5-GFP)expressing the green fluorescent protein.Results The lung cancer cell line A-549,NCI-H520 and normal cell line MRC-5 showed the same infection rate.2.The expression of At-dNK gene and the detection of enzyme activity:The mRNA of transfected stably 549/dNK and 520/dNK cells and non-transfected A-549 and NCI-H520 cells were detected by RT-PCR and electrophoresis,and the expression of At-dNK was detected only in the transfected cells.The expression of At-dNK in lung cancer cells A-549 and NCI-H520 was significantly increased in cells infected with oncolytic adenovirus ZD55-dNK but the expression of MRC-5 in normal cells were lower,indicating the targeting of adenovirus ZD55-dNK.The protein activity of stably transfected cells549/dNK and 520/dNK(3857.47pmol/mg/min)was significantly higher than that of A-549 and NCI-H520(84.93pmol/mg/min).Similarly,the phosphorylation of ara-C,ara-T and CdA in cells 549/dNK and 520/dNK was much higher than that of A-549 and NCI-H520.These results demonstrate that the At-dNK gene is capable of stably expressing kinase activity in lung cancer cells.The activity of lung cancer cell lines A-549 and NCI-H520 after infection with ZD55-dNK was significantly higher than that of the untransfected cells(25 and 50 times,respectively).The activity of MRC-5 in the normal cell line after infection with ZD55-dNK was 10-fold lower than that of the post-infection cancer cell line.These results demonstrate that adenovirus ZD55-dNK can selectively express and stably express kinase activity in lung cancer cells.3.Cytotoxicity of nucleoside analogues to lung cancer cells:the protein expression of ITCH silenced by siRNA was significantly inhibited in H1975 and Calu3 cell lines:The percentage of apoptotic cells was 2.1%and 1.9%in the H1975 cells and Calu3 cells separately.Meanwhile,the apoptotic rates in the siRNA control groups were less than 3%in both cell lines.However,when the cells were treated with siITCH,the number of apoptotic cells(43.2%in H1975 cells,45.7%in Calu3 cells)were dramatically increased(p=0.001).These results suggested that siITCH effectively promoted the apoptosis of lung cancer cells.4.The killing effect of At-dNK gene combined with nucleoside analogues on lung cancer cells:Two At-dNK-expressing cells(549/dNK and 520/dNK)showed significant drug sensitivity,except 520/dNK cells in combination with CdA.The most sensitive is549/dNK cells in combination with ara-T,with 1-10?M ara-T only 5%or less cell viability.Moreover,the drug concentration of ara-T required to achieve a median lethal dose of the 549/dNK and 520/dNK cells was nearly 800-fold less than that of the control group,and the sensitivity of other nucleoside analogues was slightly weaker than ara-T.The killing effect of At-dNK/nucleoside analogue system on lung cancer cells is to induce cell death through apoptosis.549/dNK and 520/dNK cells under a 1?M dose of ara-T,the percentage of apoptotic cells were 75%and 58%,In contrast,A-549 and NCI-H520 cells were treated with the same dose of ara-T,the percentage of apoptotic cells was 18%and 22%,respectively.The killing effect of At-dNK/nucleoside analogue system on lung cancer cells is to induce cell death through apoptosis.The oncolytic adenovirus ZD55-dNK combined with BVDU can kill lung cancer cells more effectively than the other three viruses that do not carry the suicide gene,even under very low drug concentrations(0.01?M BVDU),whereas in normal cells,ZD55-dNK infected cells with or without drugs,showed lower toxicity than the other three viruses,light microscopic observed cytopathic effect(CPE)also confirmed the same results,And BVDU has a higher substrate specificity for At-dNK than ara-T.5.Inhibitory effect of At-dNK gene combined with nucleoside analogues on transplanted tumor in nude mice:The At-dNK/nucleoside analogue system had a metastatic tumor volume of 430±197 mm~3 and 1068±227 mm~3 after 549/dNK and520/dNK metastases of lung cancer cells treated with At-dNK.The tumor volume of the PBS treated group was 3325±506 mm~3 in the control group(549/dNK).The tumor growth curve showed that there was no significant difference in tumor growth rate between A-549 metastatic tumor ara-T treatment group and A-549 metastatic PBS treatment group and 549/dNK metastatic PBS treatment group(P>0.05 all observation time points).However,the tumor growth was significantly inhibited in the ara-T treatment group(549/dNK)compared with the control group(P<0.05).6.Recombinant adenovirus ZD55-dNK combined with BVDU can inhibit adenovirus proliferation:TCID50 method was used to detect the lung cancer cell line A-549 and normal cell line MRC-5 after infection with ZD55-dNK,ZD55,DL1520 and WtAd.BVDU and ara-T were added to detect the cells and supernatant Virus titer in lung cancer cells,ZD55-dNK combined BVDU treatment of viral titer decreased the most obvious,compared with other viral titers,reduced by nearly 1000-fold.Western Blot method on the virus early transcription unit E1A observation also confirmed this point,due to the selective expression of oncolytic virus,E1A was only expressed in lung cancer cells after infection with ZD55-dNK,ZD55-dNK combined with BVDU significantly reduced the expression of E1A,While the E1D of ZD55 or DL1520 did not change obviously after dosing,but in the normal cells,the E1A bands of oncolytic virus were not obvious except the expression of wild-type virus in MRC-5.Conclusion:The At-dNK kinase gene can be expressed at high levels in lung cancer cell lines A-549 and NCI-H520 and maintain protein kinase activity.A variety of nucleotide analogues,dThd,ara-C,ara-T and CdA,which are continuously phosphorylated as substrates,exhibit extensive substrate specificity and high catalytic activity.We constructed the oncolytic adenovirus ZD55-dNK,which can specifically express At-dNK at high level in lung cancer cell lines A-549 and NCI-H520,and maintain protein kinase activity.At the same time,tumor lysis of oncolytic virus,with the nucleotide analogues of BVDU and ara-T,which phosphorylate At-dNK as a substrate,can cause tumor cell death through apoptosis,and have little effect on normal cells.In addition,adenovirus combined with BVDU in the treatment of lung cancer cells,the toxicity of BVDU-TP not only on lung cancer cell killing effect but also on the proliferation of the virus itself also has inhibitory effect,although the mechanism is not clear,but the synergistic inhibitory effect To prevent the excessive infection and spread of the virus,in order to effectively intervene the occurrence of lung cancer,development and clinical application of adenovirus to provide a strong experimental basis.At-dNK/nucleoside analogue therapy system can be used as a new suicide gene therapy for lung cancer diagnosis and treatment to provide new and efficient treatment.