Study On The Targeting Antitumor Effect Of CD/TK Fusion Suicide Gene Under The Control Of KDRP On Breast Cancer

BackgroundBreast cancer is the most frequent carcinoma that threatens health and life of women worldwide, with the incidence increasing in many countries. However, conventional cancer therapies 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 suicide genes, namely 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 confers drug sensitivity to the cancer cells. It has been found that not only suicide gene-transfected cancer cells but also non-transfected cells will be killed during the suicide gene therapy by means of the direct killing effect and so-called bystander effect, so the anti-tumor effect is quite satisfied with mild side-effects.Herpes simplex virus thymidine kinase (HSV-TK)/ ganciclovir (GCV) and E. coli cytosine deaminase (CD)/5-fluorocytosine (5-FC) are the two most mature suicide gene therapies so far and several clinical test protocols have been approved by FDA in U.S.A.. With regards to the fact that different types of tumor cells have different sensitivity to different suicide gene therapies and drug resistance is easy to occur at the presence of a single suicide gene therapy system, combined use of different suicide gene systems is recommended and CD/TK double suicide gene is the most interesting. There are several advantages of using CD/5-FC and TK/GCV at the same time, such as achieving synergetic effect, improving anti-tumor effect and avoiding drug resistance, etc.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 tumor specific transcription modulating elements, such as promoter and enhancer. For example, AFP promoter was commonly applied in the suicide gene therapy for hepatic cancer and erb2 promoter was introduced for breast 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 firmly established, considerable efforts have been made to antiangiogenic therapy as a new modality to treat human cancers. Antiangiogenesis limit the oxygen and nutrient supply to the surrounding tumor cells, inhibit to growth and metastasis of tumor cells ,so antiangiogenesis therapy is a particularly attractive antitumor modality for the past few years. 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,high-affinity receptor tyrosine kinase flk-1/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, and many research results have demonstrated that KDR promoter could make genes of interest over-express exclusively in tumor cells and its neogenetic vascular endothelial cells.Most of the vectors for suicide gene therapy are viral vectors. At present, adenoviruses, adeno-associated viruses, herpes simplex viruses and retroviruses are frequently used. Owing to some special biological characteristics, such as high viral titer, large capacity, being able to infect both dividing and non-dividing tumor cells, not integrating into the host genome and being applied in vivo experiment, etc, adenoviruses are widely used in every kind of gene therapy, especially in the suicide gene therapy. Before the homologous recombination in bacteria was found, recombinant adenoviruses could be constructed only in a few large research centers. With the development of molecular biology and gene engineering, constructing recombinant adenoviruses have become much simpler, especially after AdEasier-1 system was used.To make up for the shortages of single suicide and common promoter, in my our research work, a new kind of recombinant adenoviruses containing CD/TK fusion suicide gene under the control of KDR promoter will be constructed by a simplified and high performance AdEasier-1 system which is kindly given by professor Belt Vogelstein and the anti-tumor effect of the recombinant adenoviruses containing CD/TK double suicide gene driven by KDRP on MCF-7 tumor cells and vascular endothelial cells can be examined too in vitro and in vivo respectively.Note: the study was supported by biological and modern agriculture technologyresearch foundation of "863 project" of China (2001AA217171) and natural science foundation of Guangdong province (013072) respectively.ObjectivesTo examine the in vitro and in vivo anti-tumor effect of the recombinant adenoviruses containing CD/TK double suicide gene driven by KDRP on MCF-7 tumor cells and vascular endothelial cells respectively. The recombinant adenoviral plasmid pAdKDRP-CDglyTK was constructed by modified AdEsay system, namely 'Two-step transformation protocol", and the newly constructed plasmids were transfected to 293 packaging cells to grow adenoviruses. MCF-7 tumor cells and ECV304 cells were infected with the resultant recombinant adenoviruses, the infection rate were measured with the aid of GFP expression. Infected cells were cultured in culture mediums containing different concentration of GCV and 5-FC, and the killing effects were measured. Further, tumors, derived from MCF-7 human breast cancer cells, were established in nude mice, and the anti-tumor effect of the recombinant adenoviruses containing CD/TK double suicide gene driven by KDRP in vivo was studied by injecting the recombinant adenoviruses and prodrug(5-Fc and GCV).Methods1. Recombinant adenovirus constructionFirst, KDR promoter sequence, CD gene sequence and TK gene sequence were generated by PCR protocol to construct pcDNA3-KDRP-CDglyTK. Second, pcDNA3-KDRP-CDglyTK was digested to harvest KDRP-CDglyTK-pA which was recombined into pAdtrack to construct pAdtrackKDRP-CDglyTK. Third, pAdEsayKDRP-CDglyTK was generated with Adeasy-1 system in a "two steptransformation protocol. Finally, pAdEsayKDRP-CDglyTK was transfected into 293 cells to generate recombinant adencviruses 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 to 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 breast cancer cells, ECV304 cells and LS174T cells were infected with the recombinant adenoviruses AdKDRP-CDglyTK and AdCMV-CDglyTK. Then, CDglyTK gene expression was detected. Moreover, cell viability and bystander effect was measured using an MTT cell proliferation assay, and flow cytometry, transmission electron microscope(TEM) and acridine orange(OA) fluorescein stain were used to observe change of cell cycle and apoptotic cells after treatment by using prodrugs.3. In vivo toxicity studied with injections of the recombinant adenoviruses and prodrugsTo examine the therapeutic effect of the recombinant adenoviruses in vivo, 4-to 6-week-old female Balb/c nu/nu ethymic mice were used as hosts for MCF-7 cellxenografts, a total of 0.5xlO7 MCF-7 cells in 0.2ml PBS were injected subcutaneously into the mammary fat pad using an 18-guage needle, and tumors were allowed to grow for 2 weeks before randomizing mice by size. All tumors were at least 0.5cm and mice were grouped according to treatment regimen (ie viruses+ 5-Fc + GCV, viruses only, 5-Fc + GCV only, blank). The perpencicular tumordiameter was measured with sliding caliper at 3-day intervals, and tumor weight(W) was calculated by the formula: W=A><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.2. Specific killing effect of adenoviral vectors containing CDglyTK fusion gene to MCF-7 cells and ECV304 cells in vitro2.1 MCF-7, ECV304 and LS174T cells were infected with the recombinant adenovirusesThe infection rate of all cells were no difference, and it gradully increased with the increasing of the MOI of AdKDRP-CDglyTK and AdCMV-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 LS174T cell infected by AdKDRP-CDglyTK.2.2 specific cytotoxicity of adenoviral vector containing CDglyTK gene on MCF-7 and CEV304 cellsThree kinds of cells infected with AdKDR-CDglyTK and AdCMV-CDglyTK at MOI of 100 were maintained in culture medium of different concentration of GCV and/or 5-FC for 96 hours, it was revealed that the infected cells exhibited different sensibility to the two prodrugs: all the cells infected Ad-CMV-CDglyTK and the MCF-7 and ECV304 cells infected Ad-KDR-CDglyTK were highly sensitive to the prodrugs, and there were no significant differences among them (P=0.984) . Compared with them ,the LS174T cells infected with Ad-KDR-CDglyTK appeared to be unsensitive to the two prodrugs (F=9201.903, P=0.000) , Moreover, survival ratioof the sensitive cells decreased gradually with the increasing of the concentration of prodrugs. It was verifed that specific and high-performance killing effect of Ad-KDR-CDglyTK on MCF-7 and CEV304 cells. To make a comparison between single suicide gene and double suicide gene, we exerted different kinds of prodrugs on MCF-7 and CEV304 cells, as a result, the effect of double suicide gene was much stranger than that of each single suicide gene.2.3 The study of bystander effect of CDglyTK geneThe nontransfected cells were cocultured with different ratios of the transfected cells, bystander effect of CDglyTK gene on cells increased gradually with the increasing of rate of transfected cells, as the ratio was 40%, survival rates of MCF-7 and ECV304 cells were (11.42+2.66)% and (10.50 ±3.05)%respectively, but no killing effect by recombinant adenoviruses was observed in LS174T cells (F=5421.483,^=0.000) .2.4 The effect of progugs on cell cycle of transfected MCF-7 and ECV304 cellsAt concentrations of GCV and 5-Fc were 40mg/L and 250mg/L respectively, by 12h after treatment, cell cycle of tranfected cells was monitored. DNA content distribution indicates that the locations of different cells in the cell cycle, in nonprodrug-treated and prodrug-treated MCF-7 cells, the ratios of G2 phase cells were (11.58±0.15)% and (0.43 ±0.10)% respectively (*=125.315, P=0.000) , but that of S phase were (33.90+0.08)% and (57.20±0.09)% respectively (￡=403.568, P=0.000). In nonprodrug-treated and prodrug-treated ECV304 cells, the ratios of G2 phase cells were( 16.48±0.10)% and (0.20±0.08)% respectively (/=258.681,P= 0.000) , and that of S phase were (34.83 ±0.17)% and (62.60 + 0.14)% respectively (t=250.542, P=0.000) . It was revealed that the cell cycle of MCF7 and ECV304 cells was arrested at S phase.2.5 MCF-7 cells and ECV304 cells were observed by TEM after treatmentCell shrinkage, chromatin gathered along the nuclear membrane, cell budding. Chromatin condensation, fragmentation with intensive electron density and apoptotic body were observed by TEM in some cells. And the others exhibited necrosis.2.6 AO fluorescein stain of transfected MCF-7 and ECV304 cells after treatment withprodrugsThe characteristic morphology of MCF-7 and CEV304 cells by fluorescencestaining was showed under microscope, included cell shrinkage, nuclear condensationand fragmentation with flavovirens fluorescence and flavovirens apoptotic body.3. In vivo anti-tumor effect of the recombinant adenoviruses3.1 Anti-tumor effect of the recombinant adenoviruses containing fusion suicide gene in vivoHuman breast tumor cells MCF-7 were injected into the corresponding syngeneic mice and allowed to established tumors of 50mm mean diameter, we injected the recombinant adenoviruses (l><1010pfu) by introtumor, followed by GCV and 5-FC treatment(intraperitoneal injection of 5omg/kg/d and 500 mg/kg/d for 14 days respectively). After prodrugs treatment ended, ratio of tumor growth inhibition were 95.03% in I group, tumor weights hadn't difference between II group, III group and IV group.3.2 Histology of tumor and CDglyTK expression in tumorTumors were excised and analyzed histological after treatment, tumors from each group (control, treatment) were analyzed by hematoxylin-eosin (H-E) staining and immunohistochemical staining. H-E sections showed lamellar necrosis of tumor cell , leukocyte infiltration tumors treated. Control tumors and tumors treated with the recombinant adenoviruses were stained by immunohistochemical method, however, treatment with the recombinant adenoviruses and prodrugs reduced theMVD, but not in control groups(i=64.126,^<0.001). Further, the CDglyTK gene expression was confirmed by RT-PCR in tumor in nude mice treated by AdKDRP-CDglyTK.3.3 Histology of heart, liver, lung, kidney and small intestineTo observe whether the recombinant adenoviruses and prodrugs contained systemic toxicity, liver , heart ,lung, kidney and small intestine of trentment group nude mice were detected by H-E staining, it was revealed that the histology of heart, liver, lung, kidney and small intestine hadn't abnormal.3.4 Visible nude mice tumor modelTo establish visible nude mice tumor model, transfected MCF-7 cells were injected subcutaneously into the mammary fat pad, and size and MVD of tumor in mice bearing could be measured by LTSYSMIC, the advantage of the animal model included monitoring tumor formation, infiltration and metastasis.Conclusions1. It is the first time that modified AdEsay system has been used to construct recombinant adenoviruses in China, and recombinant adenoviral plasmids containing CD/TK fusion suicide gene under the control of KDRP have been constructed by the system at a frequency of 90%.The titer of recombinant adenoviruses is as high as 2xl012pfu/ml.2. Recombinant virus was shown high-performance transfectious ratio in MCF-7 tumor cells, LS174T tumor cells and ECV304 cells, and the tranfectious ratio of the three cells wasn't different.3. Cytosine deaminase and thymidine kinase could be effectively expressed in the recombinant adenoviruses-transfected MCF-7 tumor cells and ECV304 cells, so recombinant adenoviral plasmids containing CD/TK fusion suicide gene underthe control of KDRP exerted specific killing effect on MCF-7 tumor cells and ECV304 cells and could be killed more effectivelt following concentration of progrugs rised in vitro. Moreover, a double approach (SHV/TK and CD/5-FC) is superior to a single suicide gene/prodrug approach.4. 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, and treated cell presented cell cycle arrest, cellular apoptosis and necrosis.5. Breast tumor common mice model and visible model derived from human breast cancer MCF-7 cells can be established stably, they can facilitate monitoring tumor formation, infiltration and metastasis.6. CDglyTK fusion gene could be effectively expressed in the recombinant adenoviruses-transfected tumors in nude mice, as a result, the suicide gene/produgs system displayed satisfactory antitumor efficacy in vivo, and in vivo antitumor efficacy of the therapeutic system involved tumor growth inhibition, reduction of microvessel density and tumor necrosis.7. The histology of heart, liver, lung, kidney and small intestine in treatment group hadn't abnormal, it was revealed that the recombinant adenoviruses and prodrugs didn't exert systemic toxicity to nude mice.