| BackgroundBreast cancer is one of the major disease that threatens the health of women, with the incidence increasing in all of the world.The conventional cancer therapies such as operation,radiation therapy,chemistry therapy,incretion therapy are accompanied by their intrinsic limitations,therefore newly satisfied treatment methods are urgently needed.With the development of oncobiology and molecular technology,gene therapy represents a novel treatment model in cancer therapy,and suicide therapy is a promising strategy of gene therapy by its high effective and clinical useful potentiality.Suicide gene therapy is one of cancer gene therapy which transfers prodrug-activating enzyme genes that are found only in viruses and bacteria but not in mammalian cells into cancer cells by some gene engineering methods to express some kinds of enzymes which could catalyze nontoxic prodrugs into cytotoxic substance so as to kill the cancer cells.The specific expression of suicide genes in the tumor cells so as to selectively damage tumor cells could be realized by taking advantage of some target technology which could deliver drugs to the tumour cells accurately.The tumor specific transcription modulating elements were used to control suicide gene expression in some studies.For example,erb2 promoter was introduced for breast cancer and AFP promoter was commonly applied in the suicide gene therapy for hepatic cancer.However,most of the common promoters used nowadays are specific for some kind of cancer and their usage are relatively limited.With the role of angiogenesis in tumor growth and progression was recognized, antiangiogenic therapy has been a new modality to treat human cancers. Antiangiogenesis limits the oxygen and nutrient supply to the surrounding tumor cells, inhibits the growth and metastasis of tumor cells.Recently,most of the studies demonstrated that the growth velocity of tumor neovascularization was 50-100 times as fast as that of normal tissue,and reproductive activity of tumor neogenetic vascular endothelial cells was the keystone of tumor growth.However,Vascular endotheial growth factor(VEGF),among a number of angiogenic mediators,played an important role in the process of tumor neovascularization,the angiogenic action of VEGF were mediated via endothelial-specific VEGF receptor KDR.It has been revealed that KDR over-expresses in the majority of the solid cancer cells and neogenetic vascular endothelial cells of neoplasma but not in normal cells.So,if the suicide gene was driven by KDR promoter,it could make genes of interest over-express exclusively in tumor cells and its neogenetic vascular endothelial cells and target kill cancer cells, vascular endothelial cells.Not only did this gene therapy system enlarge the effect of therapy,but also was suitable for many kinds of cancer.Modem molecular biology studies demonstrated that neoplasm was a complicated disease involved of multiple factor,mutiple stage,polygene.Because of many different tumour cell clones existing in tumour tissue and having different sensitivity to different gene therapy systems,the single gene therapy can't get better anti-tumour effect.The combination of gene therapy which means two or more kinds of gene therapy are used at a same time can take advantage of the virtue of each different gene,improve anti-tumour effect and decrease side-effect to normal tissue.It is well-knowed that tumour is not only due to the disorder of cell proliferation and differentiation but also owing to the abnormality of cell apoptosis.The disorder of regulation in apoptosis may well prolong the survival of cells aberrantly,being subject to accumulation of mutation which leads to transformation and assisting cells by resisting monitoring from immune system,and thereby involves in carcinogenesis. Following understanding of the importance of aberrant regulation of apoptosis in carcinogenesis,transferring apoptosis-activating gene or apoptosis-inactivating suppressor gene into tumor cells has become one of most intriguing therapeutic strategies.Survivin is a newly discovered member of inhibitor of apoptosis proteins family and play an imporyant role in the regulation of apoptosis.It expresses in human embryonic tissue and takes part in the histological differentiation,organ formation, but almost not in normal differentiated human tissues.Meanwhile,survivin low-expresses in non-proliferation vascular endothelial cells and over-expresses in neogenetic vascular endothelial cells.Therefore,survivin has been paid significant attention as a new target for anti-tumor and anti-vessel therapy.To make up for the shortages of single gene and exert the predominance of combination of gene therapy,in my research work,the combination of recombinant adenoviruses containing CD/TK double suicide gene driven by KDR and survivin antisense oligonucleotide will be used at the same time and the anti-tumor effect of the double suicide gene or/and survivin antisense oligonucleotide on MCF-7 tumor cells and ECV304 vascular endothelial cells can be examined in vitro and in vivo respectively.Note:the study was supported by science and technology foundation of Guangdong province(2005B31201009).ObjectivesTo explore the transfection and expression of double suicide gene driven by KDR in MCF-7 and ECV304 cells respectively.The killing effects were measured after transfected cells were cultured in culture mediums containing different concentration ofprodrugs.To observe survivin antisense oligonucleotides' blocking effect on the expression of survivin for MCF-7,ECV304 cells and inhibitory effect on the growth of cell line respectively.To study the anti-tumour effect of combination of recombinant adenoviruses containing CD/TK double suicide gene driven by KDR and survivin antisense oligonucleotide on MCF-7,ECV304 cells and the expression of two kinds of gene.Further,tumors,derived from MCF-7 human breast cancer cells,were established in nude mice,and the anti-tumor effect of combination of recombinant adenoviruses containing CD/TK double suicide gene driven by KDR and survivin antisense oligonucleotide in vivo was studied.Methods1.Recombinant adenovirus constructionThe recombinant plasmid pAdEsayKDRP-CDglyTK composed of CD/TK confusion gene driven by KDR promoter was transfected into 293 cells to generate recombinant adenoviruses AdKDRP-CDglyTK.The viral production was conveniently followed with the aid of green fluorescent protein and the viruses were amplified by repeatedly infecting 293 cells with recombinant adenoviruses supernatant and then purified by CsCl banding procedure,which were then verified by PCR.2.Selective killing effect of adenoviral vectors containing CDglyTK fusion gene on MCF-7 cells and ECV304 cells in vitro.To determine whether recombinant adenoviruses containing the KDR promoter upstream of the CDglyTK fusion gene would specifically render KDR-producing cells sensitive to prodrugs(5-FC and GCV)-mediated cell killing.MCF-7 cells, ECV304 cells and LS174T ceils were infected with the recombinant adenoviruses AdKDR-CDglyTK.Then,the expression of CDglyTK gene was detected by RT-PCR and the infection rate was observed.Moreover,cell viability and bystander effect were measured by MTT cell proliferation assay after treatment with prodrugs.3.Inhibitory effect of survivinASODN on the growth of MCF-7,ECV304 cellsSurvivinASODN target to the promotor region of Survivin mRNA was designed and synthesized.SurvivinASODN was transfected into MCF-7,ECV304 cell lines at variety concentration using liposomal transfection reagent.The expression of survivin protein after transfection was assessed by western blot.RT-PCR method was used to detect the expression of survivin mRNA.The change of inhibitory rate of cell growth were studied and the apoptosis rate of cells was measured by flow cytometry.4.Killing effect of combination of double suicide gene driven by KDR and survivinASODN on MCF-7,ECV304 cellsThe KDR expressive cells MCF-7,ECV304 were infected with recombinant adenoviruses AdKDR-CDglyTK and SurvivinASODN was transfected into the same cells meanwhile.The infection rate of recombinant adenoviruses and transfection efficiency of survivinASODN were observed.The expression of CDglyTK mRNA was detected by RT-PCR and the expression of survivin protein was assessed by western blot after transfection.The killing effect and bystander effect on MCF-7, ECV304 cells were measured by MTT cell proliferation assay.Transmission electron microscope(TEM)was used to observe the change of cells' ultrastructure after treatment with prodrugs.5.Inhibitory effect of combination of recombinant adenoviruses containing KDR-CDglyTK fusion gene system and survivinASODN on human breast carcinoma transplanted subcutaneously in nude mice.To establish human breast carcinoma transplanted subcutaneously in nude mice, 4-to 6-week-old female Balb/c nude mice were used as hosts xenografts,MCF-7 cells were injected subcutaneously into the left axillary fat pad.All tumors were 0.5cm mean diameter and mice were grouped randomly according to treatment regimen(ie survivinASODN,AdKDR-CDglyTK,AdKDR-CDglyTK+survivinASODN,negative control).For survivinASODN group,survivinASODN was injected into or around the tumour.For AdKDR-CDglyTK group,the recombinant adenoviruses was injected into or around the tumour,followed by GCV and 5-FC treatment.For AdKDR-CDglyTK+survivinASODN group,the recombinant adenoviruses and survivinASODN was injected into or around the tumour,followed by GCV and 5-FC treatment.For negative control group,the liposomal transfection reagent LipolifectmainTM2000was injected into or around the tumour,followed by GCV and 5-FC treatment.The perpencicular tumor diameter was measured before and after treatment.The weight of tumors was studied and the growth inhibition rate was calculated after treatment.Moreover,Transmission electron microscope(TEM)was used to observe the change of tunour cells' ultrastructure and immunohistochemistry method was used to detect microvessel density(MVD)of tumor tissues.Meanwhile, CDglyTK gene expression of tumor tissues was detected by RT-PCR method and survivin protein expression of tumor tissues was assessed by western blot.Results1.Construction and identification of the recombinant adenovirusAfter the pAdtrack-KDR-CDglyTK was linearized by Enzymatic digestion, (Pac I)the recombinant adenoviral plasmid was transfected into 293 cells.About 24 hours after transfecting,the expression of green fluorescent protein could be seen and became intensified at 3-5 days or so.As lots of 293 cells turned round and plaques could form at 7-10 days,the 293 cells were gathered.Recombinant adenoviruses whose titer was as high as 2×1012pfu/ml have been achieved by repeatedly infecting 293 cells with recombinant adenoviruses supernatant,followed by CsCl banding procedure.The recombinant adenoviruses were further confirmed by PCR which could produce 580bp DNA fragment with the same size as KDR promoter and 2.4kb DNA fragment with the same size as CD and TK fusion gene fragment.2.Specific killing effect of adenoviral vectors containing CDglyTK fusion gene on MCF-7,ECV304 and LS174T cells in vitro2.1 MCF-7,ECV304 and LS 174T cells infected by the recombinant adenovirusesThe infection rate of all cells were no difference,and it gradully increased with the increasing of the MOI of AdKDR-CDglyTK(given by Zhujiang hospital). Almost all of them were infected as MOI of the recombinant adenoviruses was 200, and there was CDglyTK expression in all the transfeced cells except for the LS 174T cells infected with AdKDR-CDglyTK.2.2 Inhibitory effect of adenoviral vector containing CDglyTK gene on MCF-7 and ECV304 cellsThree kinds of cells infected with AdKDR-CDglyTK at MOI of 100 were maintained in culture medium of different concentration of GCV and 5-FC,it was revealed that the infected cells exhibited different sensibility to the two prodrugs: MCF-7 and ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs.Moreover,the survival ratio of cells decreased gradually with the increasing of the concentration of prodrugs.Compared with them,LS174T cells infected with Ad-KDR-CDglyTK appeared to be unsensitive to the two prodrugs.The survival ratio of cells has no change with the increasing of the concentration of prodrugs(P=0.816).While the concentration of GCV,5-FC was high than 40μg/ml and 250μg/ml respectively,the survival ratio of MCF-7 and ECV304 cells was significant lower than that of LS174T cells at the condition of the same concentration of prodrugs was given(P＜0.001).2.3 Bystander effect of CDglyTK geneThe nontransfected cells were cocultured with different ratios of the transfected cells,bystander effect of CDglyTK gene was observed in MCF-7,ECV304 cells and no killing effect by recombinant adenovimses was observed in LS174T cells.While the transfected cells ratio was the same,the survival ratio of MCF-7 and ECV304 cells was significant lower than that of LS 174T cells(P＜0.001).3.Inhibitory effect of survivin antisense oligodeoxynucleotides on MCF-7,ECV304 cells3.1 Examining the rate of inhibition of MCF-7,ECV304 cells growth by MTT cell proliferation assayThe comparison of cell inhibition rates among blank control group,liposomes control group,and nonsense oligodeoxynucleotides control group showed that there was no significant difference observed.(P＞0.05).Campared with three control groups, the inhibition rate of cells for antisense oligodeoxynucleotides group was significant higher(P＜0.001).All cells treated with survivinASODN in 3 different kinds of concentration were inhibited,and with the increase of ASODN,the effect was enhanced showing dose-dependent correlation.There was significant difference among different ASODN groups(P＜0.05).3.2 Expression of survivin mRNA in MCF-7,ECV304 cellsSurvivin bands were found near 191bp in three control groups and survivinASODN group.There was no significant difference among blank control group,liposomes control group,and nonsense oligodeoxynucleotides group(P＞0.05). After 200ng/ml,400ng/ml,600ng/ml ASODN were transferred into cells,the expression is significantly different from three control groups(P＜0.01).With the increase of ASODN,the expression of survivinmRNA decreased gradually and significant difference appeared among different ASODN groups(P＜0.05).3.3 Expression of survivin protein in MCF-7,ECV304 cellsSurvivin protein highly expressed in MCF-7,ECV304 cells,there was no significant difference among among blank control group,liposomes control group, and nonsense oligodeoxynucleotides group(P＞0.05).After 200ng/ml,400ng/ml, 600ng/ml ASODN were transferred into cells,the expression is significantly different from three control groups(P＜0.01).With the increase of ASODN,the expression of survivin protein decreased gradually and significant difference appeared among different ASODN groups(P＜0.05).3.4 Influence of survivinASODN on the apoptosis rate of MCF-7,ECV304 cellsThe apparent apoptosis morphology with aneuploid peak was found before G1 stage in survivin ASODN groups after 48h by comparison with blank group, liposomes group,and nonsense oligodeoxynucleotides group and the rate of cell apoptosis was statistically significant(P＜0.05).With the increase of ASODN,the apoptosis rate increased gradually and significant difference appeared among different ASODN groups(P＜0.05).4.Killing effect of combination of double suicide gene driven by KDR and survivinASODN on MCF-7,ECV304 cells4.1 Efficiency of survivinASODN transfection and recombinant adenovirus infectionSurvivinASODN at a concentration of 400ng/ml was transfected into cells,brown fluorescence was visible in 56%of MCF-7 cells and 51%of ECV304 cells 48 hours later.After cells were infected by adenovirus at a MOI of 100,GFP expression was visible in 84%of MCF-7 cells and 78%of ECV304 cells 48 hours later.After recombinant adenovirus at a MOI of 100 and survivinASODN at a concentration of 400ng/ml were added to cells together,brown fluorescence was visible in 52%of MCF-7 cells and 49%of ECV304 cells and GFP expression in 86%of MCF-7 cells and 72%of ECV304 cells.The comparison between survivinASODN group and combination of survivinASODN and recombinant adenovirus group showed that there was no significant difference observed(P＞0.05).Similary results appeared between recombinant adenovirus group and combination of survivinASODN and recombinant adenovirus(P＞0.05).4.2 Expression of survivin protein in MCF-7,ECV304 cellsWestern blot showed survivin protein highly expressed in MCF-7,ECV304 cells. There was no significant difference between survivinASODN group and survivinASODN+AdKDR-CDglyTK group in the expression of survivin protein(P＞0.05),but significant difference compared with AdKDR-CDglyTK group and negative control group(P＜0.05).The difference between AdKDR-CDglyTK group and negative control group was not significant(P＞0.05).4.3 Expression of CDglyTKmRNA in MCF-7,ECV304 cellsRT-PCR analysis indicated that CDglyTKmRNA expressed in both MCF-7 and ECV304 cells infected with CDglyTK at a MOI of 100 and no CDglyTKmRNA expressed in negative control group and survivinASODN group cells.4.4 Inhibitory effect of combined gene therapy on MCF-7,ECV304 cellsMCF-7,ECV304 cells for negative control group appeared to be unsensitive to the prodrugs.While the concentration of GCV,5-FC was high than 40μg/ml, 500μg/ml respectively,The survival rate of MCF-7,ECV304 cells for negative control group was significant different with other groups at the condition of the same concentration of prodrug was given(P＜0.001).The survival ratio of MCF-7,ECV304 cells treated with combined gene(survivinASODN and AdKDR-CDglyTK)decreased gradually with the increasing of the concentration ofprodrugs(P＜0.001).While the concentration of GCV,5-FC was high than 40μg/ml,500μg/ml respectively,The survival rate of MCF-7,ECV304 cells treated with combined gene(survivinASODN and AdKDR-CDglyTK)was significant less than that treated with AdKDR-CDglyTK or survivinASODN group at the condition of the same concentration of prodrug was given(P＜0.001).But the survival rate of MCF-7,ECV304 cells treated with combined gene(survivinASODN and AdKDR-CDglyTK)was less than that treated with AdKDR-CDglyTK and the difference between them was not significant(P＞0.05), while the concemtration of GCV was 100μg/ml and 5-FC 2000μg/ml.The synergistic effects were significant with CDI(coefficient of drug interaction)＜1 for combined gene therapy(SurvivinASODN and AdKDR-CDglyTK),while the concentration of GCV,5-FC was less than 100μg/ml,2000μg/ml.The synergistic effects were very significant with CDI＜0.7,while the concentration of GCV reached 60μg/ml and 5-FC 500μg/ml.4.5 Bystander effect of combined gene therapyThe cellular survival of the mixed cell population for survivinASODN+AdKDR-CDglyTK group was less than that for AdKDR-CDglyTK and survivinASODN group,when the transfected cells were mixed with untransfected cells at the same ratio and the same concentration ofprodrug was given.4.6 MCF-7 cells and ECV304 cells were observed by transmission electron microscopy(TEM)after treatmentCell shrinkage,chromatin gathered along the nuclear membrane.Chromatin condensation,fragmentation with intensive electron density and apoptotic body were observed by TEM in some cells.5.Anti-tumor effect of combination of recombinant adenoviruses containing KDR-CDglyTK fusion gene system and survivinASODN on human breast carcinoma transplanted subcutaneously in nude mice.5.1 Anti-tumor effect of the combined gene therapy in vivoThe volume of four group tumours has no significant difference before treatment start(P＞0.05).The volume and weight of four group tumours was measured when treatment ended.The volume of tumours for negative control group increased significantly when treatment ended(P＜0.001),and the volume of turnouts for experimental group decreased significantly(P＜0.05).The volume and weight of tumours for experimental group was significant less than that for negative control group(P＜0.001).The volume and weight of tumours for combined gene therapy group was significant less comparing with the single gene therapy group(P＜0.05). The ratio of tumor growth inhibition for combined gene therapy was significant higher than that for survivinASODN or AdKDR-CDglyTK group(P＜0.05).5.2 Measurement of microvessel density in tumourThe microvessel density of tumour for SurvivinASODN,AdKDR-CDglyTK, survivinASODN+AdKDR-CDglyTK group was significant lower than that for negative control group(P＜0.05).The microvessel density of tumour for combined gene therapy group was significant low comparing with the single gene therapy group(P＜0.05).5.3 Expression of survivin protein in tumoursSurvivin protein highly expressed in transplant tumour cells.The difference between AdKDR-CDglyTK group and negative control group was not significant(P＞0.05).There was no significant difference between survivinASODN group and survivinASODN+AdKDR-CDglyTK group in the expression of survivin protein(P＞0.05),but significant difference compared with AdKDR-CDglyTK group and negative control group(P＜0.05).5.4 Expression of CDglyTKmRNA in tumonrsRT-PCR analysis showed that CDglyTKmRNA expressed in transplant tumours treated with AdKDR-CDglyTK and no CDglyTKmRNA expressed in negative control group and survivinASODN group.5.5 Tumour cells were observed by transmission electron microscopy(TEM)after treatmentCell shrinkage,chromatin gathered along the nuclear membrane.Chromatin condensation,fragmentation with intensive electron density and apoptotic body were observed by TEM in some cells.Conclusions1.The recombinant adenoviral plasmids containing CD/TK fusion suicide gene under the control of KDR have been constructed.Recombinant virus was shown high-performance transfectious ratio in MCF-7 cells,LS174T cells and ECV304 cells,and the tranfectious ratio of the three cells wasn't different.2.Cytosine deaminase and thymidine kinase could be effectively expressed in the recombinant adenoviruses-infeeted MCF-7 cells and ECV304 cells,so recombinant adenoviral plasmids containing CD/TK fusion suicide gene under the control of KDR exerted target killing effect on MCF-7 cells and ECV304 cells and showed stronger killing effect following concentration of progrugs rised in vitro.3.The in vitro antitumor mechanism of this therapeutic system included directly toxic effect on suicide gene modified cells and bystander killing effect on neighboring nonmodified cells.4.Survivin antisense oligodeoxynucleotides mediated by liposomes could be used to reduce survivin mRNA and protein levels in MCF-7,ECV304 cells.5.The proliferation of MCF-7 and ECV304 cells treated with survivinASODN at different concentration was inhibited,and with the increase of ASODN,the effect was enhanced showing dose-dependent correlation.The antitumour mechanism of survivinASODN was inducing gene modified cell apoptosis.6.The combination of adenovirus mediated double suicide gene driven by KDR and survivinASODN gene therapy showed the MCF-7,ECV304 cells infected by recombinant adenoviruses meadiated double suicide gene could be transfected with the survivinASODN and the infection rate was not affected as well as the transfection rate.7.The combination of adenovirus mediated double suicide gene driven by KDR and survivinASODN gene therapy showed Cytosine deaminase and thymidine kinase could be effectively expressed in MCF-7,ECV304 cells and had no influence on the expression of survivinASODN.8.The combination of survivinASODN and AdKDR-CDglyTK gene therapy showed stronger killing effect on MCF-7,ECV304 cells comparing with the separate use of each treatment alone and synergistic killing efficacy in low concentration of GCV and 5-FC.9.The combination of survivinASODN and AdKDR-CDglyTK gene therapy showed stronger bystander effect on MCF-7,ECV304 cells comparing with the separate use of each treatment alone. 10.The survivinASODN or AdKDR-CDglyTK gene therapy displayed evidently tumor growth inhibition and reduction of microvessel density in nude mice.The combined gene therapy group showed stronger antitumour efficacy and lower microvessel density in vivo than single gene.11.Survivin protein highly expressed in transplant tumour cells.SurvivinASODN gene therapy decreased the survivin protein level effectively.Cytosine deaminase and thymidine kinase could be effectively expressed in the recombinant adenoviruses-infected transplant tumour cells.12.The combination of adenovirus mediated double suicide gene driven by KDR and survivinASODN gene therapy showed CD and TK gene could be effectively expressed in transplant tumour cells and had no influence on the expression of survivinASODN.
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