Enhancement Of Expression Of The Survivin Promoter-driven Double Suicide Genes CD/TK System By The Nuclear Matrix Attachment Region In The Targeting Therapy Of Gastric Cancer

Gastric cancer, the most common malignancy of the digestive tract, remains amajor public health issue because of its high incidence and mortality worldwide. Withthe development of cancer research, the clinical diagnosis and treatment of gastriccancer have progressed greatly. However, the effect of traditional treatments, such aschemotherapy, radiation therapy, and surgical operations, is not satisfactory becauseof poor prognosis, low five-year survival rate, and relatively high level of mortality.Therefore, finding a new, effective treatment to improve the survival rate of patientsis necessary. With the developments in molecular biology and genetic engineering,gene therapy has become an effective and promising method of cancer treatment.Suicide gene therapy, with its unique mechanism, has been given much attentionin research. By introducing a suicide gene, known as a prodrug-converting enzymegene, into tumor cells and inducing the tumor cells to express this gene, the suicidegene enables the conversion of non-toxic compounds into highly toxic metabolites,effectively killing the cancer cells. Therefore, suicide gene therapy can be apromising strategy or safe modality for advanced diseases, including gastric cancer.As suicide gene therapy has been successfully used in many studies in vitro and invivo, its application for the treatment of cancer patients has not yet reached thedesirable clinical significance. For example, the transfection of exogenous gene still lacks effective targeting and specificity; a poor vector-specific receptor results in lowexpression of viral vectors in tumor cells.One way to enhance gene expression specific to tumor cells, not normal cells, isto use tumor tissue-specific promoters to regulate gene expression. Previous studiesshow that survivin, a16.5kDa protein also known as baculoviral IAPrepeat-containing protein5, is significantly overexpressed in cancers. The survivinpromoter has been used for cancer therapy because of this specific expression. Thematrix attachment region (MAR) is a DNA sequence of eukaryotic chromatin thatbinds specifically to the nuclear matrix or nuclear scaffold. Through specificinteraction with MAR-binding proteins, MAR plays an important regulatory role inenhancing transgene expression, decreasing the expression variation amongindividuals of different transformants and serving as the replication origin. Therefore,MAR as a cis-regulatory element has been applied to transgenic manipulation.We hypothesized that the survivin promoter and MAR could be used as atumor-specific targeting approach to drive and enhance the expression of suicidegenes, resulting in the considerable improvement of the targeted ability and sensitivity.In this paper, the survivin promoter-driven double suicide gene system [Escherichiacoli cytosine deaminase (CD)/5-fluorocytosine (5-FC) and virus thymidine kinase(TK)/ganciclovir (GCV)] enhanced by MAR was developed and transfected intohuman gastric cancer SGC-7901cells. The results showed that the expression ofCD/TK double suicide genes could be significantly enhanced by MAR, and thissystem could selectively reduce proliferation and enhance apoptosis in vivo in gastriccancer cells. The findings provide important theoretical basis and clinical value forthe gene therapy of gastric cancer.Our studies in this paper were divided into four parts as follows. Part I: Construction of the recombinant vector pMS-CD/TKAim:To clone CD, TK, MAR and survivin promoter genes, and to construct therecombinant vector pMS-CD/TK containing MAR and the survivin promoter-drivenCD/TK double suicide gene system.Methods:1. The recombinant vector pMS-CD/TK containing MAR and the survivinpromoter-driven CD/TK double suicide gene system were developed as follows. Afragment-encoding CD gene was cloned from E. coli BL21genome DNA bypolymerase chain reaction (PCR). A fragment-encoding TK gene was amplified froma vector pHSV-TK, which was previously developed in our laboratory. GenomicDNA was extracted from human HeLa cells using a DNA extraction kit. A survivinpromoter fragment and a MAR fragment were respectively amplified from humanHeLa cell genome DNA by PCR.2. After the CMV promoter of the pEGFP-C1vector (Clontech) was replaced bythe survivin promoter, the CD and TK gene fragments were in turn subcloned into thedownstream of survivin promoter to generate the plasmid pS-CD/TK. Finally, theamplified MAR fragment was inserted into the upstream of the survivin promoter togenerate the plasmid pMS-CD/TK. Vectors pS-CD/TK and pMS-CD/TK wereconfirmed by restriction enzyme digestion, agarose gel electrophoresis, and DNAsequencing.Results:1. The sequencing results suggested that A948bp survivin promoter fragmentand a787bp MAR fragment were respectively cloned by PCR from human HeLacell genome DNA. 2. A1.3kb fragment encoding CD gene was cloned from E. coli BL21genomeDNA, and a1.2kb fragment encoding TK gene was amplified from the vectorpHSV-TK.3. Vectors pS-CD/TK and pMS-CD/TK were generated as described above. Theresults of identification by restriction enzyme digestion, agarose gel electrophoresis,and DNA sequencing suggested that we successfully established the recombinantscontaining MAR and the survivin promoter-driven CD/TK double suicide genesystem.Part II: Transfection and expression analysis of the recombinantvector pMS-CD/TK in the gastric cancer cellsAim:To transfect the gastric cancer cell SGC-7901using the recombinant vectorpMS-CD/TK or pS-CD/TK. To dectet expression of the double suicide genes CD/TKin the transfected cells.Methods:For all experiments, SGC-7901and GES-1cells grown in the exponential phasewere seeded at1.0×104cells/well in24-well plates containing100μl of medium.The cells were divided into six groups: Group I, GES-1cells untreated with vector;Group II, GES-1cells treated with pS-CD/TK; Group III, GES-1cells treated withpMS-CD-TK; Group IV, SGC-7901cells untreated with vector; Group V, SGC-7901cells treated with pS-CD/TK; and Group VI, SGC-7901cells treated with pMS-CD/TK. Cells were transfected with10μg of recombinant plasmid DNA in the presenceof50μg of Lipofectin Transfection Reagent according to the manufacturer’s manual.The transfected cells were screened by adding500μg/ml of G418. After selectionfor two weeks, G418-resistant cells were isolated and transferred onto individualplates to propagate for the follow-up experiments. Expression of the double suicidegenes CD/TK in the transfected cells was detected by quantitative real-time PCR(qPCR) and Western blot. Results:1. Reverse transcription PCR (RT-PCR) analysis revealed that an expected187bp fragment of the CD/TK fusion gene was detected only in SGC-7901cellstransfected with the plasmid pS-CD/TK or pMS-CD/TK. Gene expression was notdetected in the control GES-1cells.2. The result of qPCR showed that the expression of the CD/TK fusion geneenhanced by the regulation of MAR significantly increased7.7-fold in the SGC-7901cells transfected with the plasmid pMS-CD/TK compared with the SGC-7901cellstransfected with the plasmid pS-CD/TK.3. The result of Western blot analysis indicated that approximately48kDa of aspecific single protein band was detected by an anti-CD antibody in the SGC-7901cells transfected with the plasmid pMS-CD/TK or pS-CD/TK but not in theuntransfected SGC-7901cells.Part IIII: Killing effect of the double suicide gene CD/TK enhancedby MAR on the gastric cancer cell in vitroAim:To investigate the selective killing efficacy of the double suicide genes (CD/TK)regulated by the survivin promoter and enhanced by the MAR geneMethods:To determine the function of the exogenous CD/TK gene in tumor cells, theSGC-7901and GES-1cells transfected with or without pS-CD/TK or pMS-CD/TKwere treated with GCV and5-FC. Cellular survival rates were assayed using the MTTmethod. Cellular apoptosis was analyzed by flow cytometry analysis.Results:1. A high survival rate reached almost100%in cells that did not have theCD/TK gene but were given the prodrugs (5-FC+GCV) and in cells that had theCD/TK gene but were not given the prodrugs (5-FC+GCV). However, treatment with 5-FC+GCV could significantly decrease the cellular survival rate in SGC-7901cellstransfected with pS-CD/TK, especially in SGC-7901cells transfected with pMS-CD/TK.2. Similar to the cellular survival rate results, treatment with5-FC+GCV did notaffect the cellular apoptosis in GES-1cells transfected with or without the pS-CD/TKor pMS-CD/TK. However, treatment with5-FC+GCV markedly induced SGC-7901cell apoptosis after transfection with pS-CD/TK or pMS-CD/TK (P <0.01) comparedwith untreated cells (negative control:5.01%). Furthermore, the results revealed thatthe proportion of positive SGC-7901cells transfected with pMS-CD/TK for AnnexinV and PI (Region II)(26.25%) was significantly higher (P <0.01) than that ofpositive SGC-7901cells transfected with pS-CD/TK (17.65%).Part IV: Anti-tumor effect of the double suicide CD/TK systemenhanced by MAR on the nude mice transplanted gastric cancer invivoAim:To evaluate the anti-tumor effect of the double suicide CD/TK system enhancedby MAR on the nude mice transplanted gastric cancer in vivo.Methods:Tumor-bearing Balb/c mice model was established by subcutaneous injection ofgastric cancer cell SGC-7901. After successful tumor formation,18tumor-bearingmice were randomly divided into three groups: the control group (Group A) treatedwith PBS; the recombinant vector pS-CD/TK add prodrugs group (Group B); the therecombinant vector pMS-CD/TK add prodrugs group (Group C). After treatment, themice were sacrificed by cervical dislocation, tumor tissues were removed, weighed,sectioned, stained by HE method. The changes of tumor volume, weight andtumor-inhibition rate were evaluated. The tumor pathological changes were alsoobserved under microscope. CD/TK expression in the tumor tissue was detected byqPCR. Results:1. Tumor-bearing Balb/c mice model was successfully established bysubcutaneous injection of gastric cancer cell SGC-7901.2. Tumor size (mm3), final tumor weight (mg) in groups B (256.7±6.4,251.7±14.2) and C (187.2±3.5,118.8±10.2) were significantly smaller than those inthe control group A (424.2±5.5,557.1±8.9)(P <0.05). Meanwhile, the tumorinhibition rate in groups B (63.1%) and C (82.6%) were significantly higher than thatin the control group A (P <0.05). The results of HE stained sections showed that nonecrosis observed in the control group C while combination therapy in the group Aand B showed increased differentiation and necrosis.3. Expression of CD/TK gene was detected by qPCR in the treatment groups Aand B.Conclusions1. The recombinant vector pMS-CD/TK containing MAR and the survivinpromoter-driven CD/TK double suicide gene system were successfully established byidentification of restriction enzyme digestion, agarose gel electrophoresis, and DNAsequencing.2. Expression of the CD/TK fusion gene could be detected and enhanced byMAR in the transfected gastric cancer cell SGC-7901.3. The double suicide CD/TK fusion gene system has stronger killing effect onthe gastric cancer cell SGC-7901and also shows obvious bystander effect.4. The killing effect on the transfected gastric cancer cell could be improved byMAR through enhancing the expression of the CD/TK genes.5. The double suicide genes CD/TK system containing MAR could producesignificant tumor inhibition effect on gastric cancer xenograft in nude mice.