| BackgroundLung cancer is a kind of malignant tumor threatening human health seriously, in the United States, the incidence of lung cancer is one of most common malignant neoplasms. A number of patients have lost the opportunity of operation when they were dignosed lung cancer in the middle or late state. Non-surgical treatment methods contain mainly targeted therapy, chemotherapy, radiotherapy and so on, however, the emerging clinical effect and prognosis are poor.The prospect of suicide gene therapy, has been highly concerned by the scholars since it was discovered. The suicide gene systems which are studied deeply include HSVTK I/GCV, NTR/CB1954 and so on. Among these systems, NTR/CB1954 has broader application prospects as its unique characteristics and advantages.In 1967, CB1954, a weak alkylating agents, was synthesized by British Chester Beatty laboratory for the first time, it could kill the tumor cells weakly. The study explored that CB1954 had obvious killing effect to Walker 256 tumor cell in the rat, because the rat Walker 256 cells contain diphosphopyridine nucleotide diaphorase DT-diaphorase, play powerful catabolic effect to the CB954 and the metabolites 4-hydroxylamino derivative can cause the intracellular DNA cross links, induce rat Walker 256 tumor cells apoptosis and necrosis obviously. Further studies show that CB1954 also has strong lethality to Escherichia coli, results in the genomes mutations of Escherichia coli, which is depended on the presence of a metabolic enzymes, which is named nitroreductase (NTR) in the Escherichia coli, the product leads to the death of Escherichia coli. Escherichia coli nitroreductase is encoded by the gene nfsb. Compared with the DT-diaphorase, the reduction activity of NTR to CB1954 is 60 times stronger. Therefore, Escherichia coli nitroreductase shows more research value.CB1954 generates two substances by the action of NTR:4-hydroxy amino 2-nitrobenzene formamide and 2-hydroxy amino 4-nitrophenyl formamide, under the action of a product in intracellular cofactor further metabolism of a cytotoxic substance is, cause tumor cell DNA chain cross-linked, fracture and difficult to be repaired, lead the apoptosis of tumor cells, which is the main form of most of the tumor cell death.The characteristics and advantages of NTR/CB1954 suicide gene system show as follows:experiments confirmed that compared with HSV-TK/GCV, CD/5-FC, NTR/CB1954 suicide gene system not only has cytotoxic effect on the proliferation of the tumor cells, but also dose a killing effect for stationary phase cells; researches show that the destruction process in NTR/CB1954 suicide gene system on breast cancer cells, can specific up-regulate the expression of p53, which is an important tumor suppressor gene,thereby enhances tumor cell apoptosis mediated by p53, and more than 50% of human tumors related the changes of p53 gene; NTR/CB1954 acts the killing effect on tumor cells quickly, W Cui found apoptosis could be observed in the seventh hour in breast cancer cells, and it would reach the peak in the seventeenth hour, while the HSVTK/GCV suicide gene system needs a few days or weeks to achieve a similar effect; CB1954 has strong bystander effect, the reduction product-can pass through cell membranes freely, it can achieve the effect of killing the tumor in the role of a small amount of nitro reductase; CB1954 doesn’t exist cross resistance with other chemotherapeutic agents or prodrug. ObjectiveIn recent years a large number of experiments show that NTR/CB1954 suicide gene system act the killing effect to a variety of malignant tumor cells (breast cancer, cervical cancer, ovarian cancer, etc.). However, in view of the NTR/CB1954 suicide gene system on cell lung cancer related research is rare. The aim of this study is to investigate the killing effect of NTR/CB1954 suicide gene system on Lewis lung cancer cells. First, in vitro, we detect the killing effect of NTR/CB1954 suicide gene system on Lewis lung cancer cell; then, though the establishment of Lewis lung cancer cells in animal models, we detect this effect in vivo. Collect and analyses the experimental results, clear the killing effect of NTR/CB1954 suicide gene system in vitro and vivo environment on Lewis lung cancer cell, preliminary draw a conclusion of the experiment, provide a preliminary experimental evidence for the treatment of lung cancer. MethodsWe chose nfsb as nitro reductase gene, Escherichia coli K12 as the source of genetic strain, and designed the nfsb gene primers. nfsb gene and eukaryotic expression vector were constructed a recombinant vector by gene recombinant technique, eukaryotic expression vector was pcDNA3, named pCDAN3-nfsB. Lewis lung cancer cells were removed from liquid nitrogen, cultured routine, as the cell fusion degree reached 80%, they were subculture. Using liposome transfection method, the empty pcDNA3 vector and the recombinant vector pcDNA3-nfsB transfected Lewis cell and selected by G418 (a kind of aminoglycoside antibiotics) principle and get stable expression of nitroreductase cell line, named for the cells pcDNA3-nfsB-Lewis and empty control vector pcDNA3 transfected cells named cells pcDNA3-Lewis. The experimental cells were divided into three groups:Lewis cells, pcDNA3-Lewis cells and pcDNA3-nfsB-Lewis cells. Relative expression level of pcDNA3-nfsB-Lewis nitro-reductase was detected by the RT-PCR method, the protein expression lever was detected by the application of lauryl sodium sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) technology. The growth of Lewis cells, pcDNA3--Lewis cells and pcDNA3-nfsB-Lewis cells were observed by the method of blue staining, and the growth curves of the cells were drawn, and the effects of the transfection on the growth of the cells were also observed. Cells in each group were added 30umol/L CB1954 and investigated the relationship between the apoptosis rate with the time, and then each group cells were treated with different concentrations of CB1954 (0 μmol/L,12.5μmol/L,25μmol/L,50μmol/L), then detected the death of cells in the 36th hour to explore the difference effect on the cells by the different concentraion CB1954. The apoptosis rate of each group was detected by Hoechest33258/PI fluorescent staining method, and the survival and growth activity of the cells were detected by MTT method. We took C57BL/6 mice as the research object to establish the animal model of transplanted tumor. After 7 days of injection of Lewis cells in C57BL/6 mice cervical, when the tumor could grown to soybean grain size, the mice were sacrificed and the tumors were removed, cut into small pieces, tissue suspension liquid was injected into C57BL/6 mice of the right hind limb subcutaneous injection of 1x106 living cells, the tumor could grow to the size of soybean in a week. The transplanted tumors in experimental animals were randomly divided into three groups:tumor transfected with the recombinant plasmid pcDNA3-nfsB group, empty vector pcDNA3 group and non transfection of any plasmid group and the three groups of rats injected CB1954, then detected tumor body size at different time point and survive time of each experimental animal groups. Study the killing effect of suicide gene system on tumor in vivo.ResultsThe nfsB gene was successfully cloned by primers, and the gene was amplified and stored by coli Escherichia 5. After the eukaryotic expression vector pcDNA3 and objective gene nfsb recombination were connected though gene recombination techniques, recombinant plasmid pcDNA3-nfsB was successfully constructed by enzyme digestion and sequencing. After transfection to Lewis cells, screening by G418 concentration gradient, the ultimate Lewis cells which would stable express the nitroreductase were selected. We amplified nfsb gene partial sequence nfsB1 (about the size of 381bp) by RT-PCR method, and got the 381bp bands through agarose gel electrophoresis, confirmed the Lewis lung cancer cells with the correct expression of target gene nfsb at the RNA level.The results of pcDNA3-nfsB-Lewis cells, pcDNA3-Lewis and Lewis cells proliferation showed that there was no significant difference between groups, P> 0.05. After added 30umol/L CB1954 to cells in each group, the pcDNA3-nfsB-Lewis cells occurred obvious apoptosis at the twelfth hour. Apoptosis rate reached a peak at the 36th hour, showed significantly higher than in the other two groups, with statistical significance (P< 0.01); pcDNA3-Lewis cells and Lewis cell apoptosis rate had no significant difference. After added different concentrations CB1954(0umol/L,12.5 umol/L,25umol/L,50umol/L), the pcDNA3-nfsB-Lewis cells were detected significant apoptosis in 36th hour, and the apoptosis rate showed concentration dependent manner, when the concentration reached 50umol/L, cell apoptosis rate reached the maximum. It showed significant difference compared with the other two groups cells(p<0.05), while the other two groups of cells did not appear obvious apoptosis. The results of NTR/CB1954 suicide gene influenced on cell proliferation through the MTT assay showed that the pcDNA3-nfsB-Lewis cells’od570 was significantly lower than in the other two groups (P< 0.05) cells, the effect showed concentration dependent manner from 0 to 50umol/L.Tumor cells of Lewis lung cancer animal model was established. At the twelfth day and 15th day after intraperitoneal injection of CB1954, the tumor volume growth appears to be relatively slow trend in the pcDNA3-nfsB transfected mice, tumor size were 589.9±19.00mm3,793.6±96.42mm3, and non transfected group were 1106.2±91.83mm3,1649.1±67.79mm3, the pcDNA3 group were 1131.3±60.16mm3,1441.6±224.5mm3, the growth of the former group was significantly slower in the other two groups(P< 0.05). Survival time in the pcDNA3-nfsB intratumoral transfected mice is longer (41.63±7.07 days), and survival time in the transfected with the empty vector pcDNA3 group and non transfection mice were respectively 27.86±4.94 days and 26.75±5.97 days, the former group was significantly longer than the other two groups(P< 0.05). ConclusionNTR/CB1954 suicide gene system can lead apoptosis of Lewis lung cancer cells obviously, which begin in the seventh hour and reach the peak in the 36th hour. The killing effect of CB1954 shows a concentration dependent, the killing effect is growing with the increase of concentration of CB 1954. In vivo, NTR/CB1954 suicide gene system can effectively control the proliferation of Lewis lung cancer cells, and significantly prolong the survival time of experimental animals. This study confirmes that NTR/CB1954 suicide gene system has obvious killing effect on Lewis lung cancer cells in vivo and in vitro conditions, provides a preliminary experimental basis for the therapy for lung cancer by suicide gene system. |
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