Study On Apoptosis Of SMMC-7721 Cells Induced By Over-expressed Smac Gene Coupling With Cisplatin Or Cadmium Chloride And Related Mechanisms

Smac is a newly-found promoting-apoptosis protein existing in mitochondria, and takes part in each apoptotic pathway, which would supply a new tool for tumor therapy. The research is aimed to investigate the effect of over-expressed Smac gene combined with cisplatin or cadmium chloride on cellular proliferation and apoptosis in SMMC-7721 cells and explore its mechanisms. Firstly, the Smac gene was cloned. Then the recombinant plasmid pcDNA3.1+-FL hSmac which could over-express Smac successfully was constructed. Meanwhile, the hepatoma cell line named SMMC-7721/Smac which could over-express stably Smac was established. The research would provide an experimental basis for the Smac-based tumor gene therapy and the improvement of clinical therapeutic efficacy on hepatoma.1 Construction of recombinant plasmid pcDNA3.1+-FL hSmac1.1 Cloning and identification of human full-length Smac geneAccording to the cDNA full-length sequence of human Smac gene published on Genbank (AF262240), the specific primers for cloning Smac gene were designed and synthesized with the following primers:forward primer——5'-gctctagaatggcggctctgaagagttggctgt-3', including XbaⅠenzyme digestion sites;reverse primer——5'-gcggatcctcaatcctcacgcaggt-3', including BamHⅠenzyme digestion sites.The total mRNA extracted from the Hela cells, a kind of human cervical carcinoma cell line, was acted as the template for reverse transcription. The CDs sequence of Smac gene was amplified by RT-PCR. Then the Smac gene was inserted into the pMD19T vector, and identified by cleavage of endonucleases and sequencing process. The sequence analysis confirmed that the cloned Smac gene was in coincidence with that in Genbank completely, which established a foundation for the following procedures.1.2 Construction and identification of recombinant plasmid pcDNA3.1+-FL hSmacThe Smac gene fragment on pMD19T-FL hSmac was ligated to the eukaryotic expression vector pcDNA3.1+ to construct the recombinant plasmid pcDNA3.1+-FL hSmac through taking the advantage of the related technique in gene engineering, such as the cleavage of endocleases, ligation and so on. The construction of recombinant plasmid pcDNA3.1+-FL hSmac was confirmed correctly through the identification of PCR and endonucleases digestion.2 Expression of recombinant plasmid pcDNA3.1+-FL hSmac in SMMC-7721 cells2.1 Optimization of cell transfection condition and detection of its efficacyLiposome-mediated transfection method was used to transfect the plamids in the experiments. Firstly, the condition for cell transfection was optimized by detecting the EGFP expression in transfected cells with the mixture of pcDNA3.1+-EGFP and liposome in different ratios, which reflected the cell transfection efficacy simultaneously. The results showed that the optimized ratio between plasmids and liposome for the SMMC-7721 cell transfection was 1 to 2.5, and at this point, the transfection efficacy was about 48%. Meanwhile, bright green fluorescence could be seen in cells after being transfected with pcDNA3.1+-EGFP under fluorescence microscopy, which illustrated that the transfection method was practical, so all the following cell transfection was done in this way.2.2 Expression of recombinant plamid pcDNA3.1+-FL hSmac in SMMC-7721 cellsBoth the total RNA and protein were extracted from the SMMC-7721 cells which were transiently transfected with pcDNA3.1+-FL hSmac. The former was detected by RT-PCR, and the later by Western blot and flow cytometry respectively. The results showed that whether on the mRNA level or the protein level, the expression of Smac in the pcDNA3.1+-FL hSmac group was the highest. As compared with that in the control and null-vector transfectant groups respectively, the expression of Smac in the pcDNA3.1+-FL hSmac group increased significantly, which indicated that the recombinant plasmid pcDNA3.1+-FL hSmac could express the gene of interest, Smac, correctly and effectively.3 Effects of over-expressed Smac gene coupling with cisplatin or cadmium chloride on hepatocarcinoma cells3.1 Effects of transiently over-expressed Smac gene coupling with cisplatin or cadmium chloride on hepatocarcinoma cells3.1.1 Iinhibitory effects of transiently over-expressed Smac gene coupling with cisplatin or cadmium chloride on proliferation of hepatocarcinoma cellsMTT assay was used to detect the toxic effects of transiently over-expressed Smac gene coupling with cisplatin or cadmium chloride on hepatocarcinoma cells. The results showed that as compared with those in the control group, except for those in the null-vector transfectant group without significant difference, the inhibitory rates in the rest groups increased significantly (P < 0.001). The inhibitory rates in the only cisplatin or cadmium chloride treated groups increased with the increasing treatment doses. Furthermore, the inhibitory rate in each of the CDDP or cadmium chloride-treated plus Smac groups was significantly higher than that in the corresponding CDDP or cadmium chloride-treated group (P < 0.001). The results indicate that the transiently over-expression of Smac gene could inhibit the cellular proliferation, and enhance the inhibitory effects of cisplatin and cadmium chloride on hepatocarcinoma cells.3.1.2 Effects of transiently over-expressed Smac gene coupling with cisplatin or cadmium chloride on apoptosis of hepatocarcinoma cellsThe change of cell apoptosis was detected by acridine orange-ethidium bromide fluorescent staining (AO/EB) and flow cytometry with Annexin V-PI double-fluoresence staining methods respectively. It showed that the transient over-expression of Smac gene could increase cell apoptosis (P < 0.001). Cisplatin or cadmium chloride alone could induce apoptosis of SMMC-7721 cells, which could significantly induce the increase of apoptosis when coupling with transiently over-expression of Smac gene (P < 0.001). The results acquired by FCM and AO/EB respectively were coincident, which indicate that the transient over-expression of Smac gene could induce tumor cell apoptosis, and promote the apoptosis induced by cisplatin and cadmium chloride. Moreover, from the data of early and late apoptosis acquired by FCM and photographs took by AO/EB detection, it could be seen that the apoptosis induced by cisplatin or cadmium chloride alone was centred by early apoptosis, but after transiently over-expressed Smac gene coupling with cisplatin or cadmium chloride, the total apoptotic rates increased, especially the late apoptotic rates, which indicate that Smac gene transfection could not only improve the apoptosis of SMMC-7721 cells induced by cisplatin or cadmium chloride, but also accelerate the proceeding from early aopotosis to late apoptosis.3.1.3 Effects of transiently over-expressed Smac gene coupling with cisplatin or cadmium chloride on apoptosis-regulatory factors of hepatocarcinoma cellThe effects of transiently over-expressed Smac gene coupling with cisplatin or cadmium chloride on the expressions of caspase-3, -9 and Cyt c in hepatocarcinoma cells were detected by Western blot. The results showed that the expressions of caspase-3, -9 and Cyt c in SMMC-7721 cells increased after transiently transfecting Smac gene, and all the expressions of the three proteins increased significantly after coupling with cisplatin or cadmium chloride, which were higher than those in the only transiently over-expression of Smac gene and the pure treatment of cisplatin or cadmium chloride groups respectively. This indicates that the transient over-expression of Smac gene could increase the expressions of caspase-3, -9 and Cyt c, meanwhile, combining with cisplatin or cadmium chloride could cooperate each other and increase their expression to promote apoptosis. 3.2 Effects of stably over-expressed Smac gene coupling with cisplatin or cadmium chloride on hepatocarcinoma cells3.2.1 Establishment of human hepatoma cell line stably over-expressing SmacpcDNA3.1+-FL hSmac was packed with liposome to transfect the human hepatoma SMMC-7721 cells. Forty eight– seventy two h later, the cells were passaged by 1 : 3, and steadily cultured until the cell density up to 50%– 70%, then selected in medium containing G418. After selection for 10– 20 days, the antibiotics clone could be observed. Through the amplification of cell culture, the cells were gained and named SMMC-7721/Smac, then identified through detecting the expressions of both Smac mRNA and its protein by RT-PCR and Western blot respectively. The results showed that as compared to that in SMMC-7721 cells, the expressions of both the Smac mRNA and its protein in SMMC-7721/Smac cells increased significantly, which indicate that the cell line which could over-express Smac stably was established by liposome-mediated gene transfection. This settles the foundation for the further research.3.2.2 Growth characteristics of hepatoma cell line stably expressing SmacThe morphous of established SMMC-7721/Smac cells was regularly, and most of them were fusiform and polygonal, which had no difference from the SMMC-7721 cells. The passage interval of SMMC-7721 cells was 2– 3 days, otherwise that of SMMC-7721/Smac cells was 5– 6 days during regular cell culture. In order to determine this growth characteristics, the A490 values of the SMMC-7721/Smac and SMMC-7721 cells with the same quantity in different times (12, 24, 36, 48, 72, 96 and 120 h) were detected by microplate reader, which could reflect the cell survival. Then the growth curves of each cell line were drawn accordingly. The results showed that the A490 value of SMMC-7721/Smac cells was significantly lower than that of SMMC-7721 cells in each time-point (P < 0.05, P < 0.001), which indicate that the proliferation speed of SMMC-7721/Smac cells was slower than that of SMMC-7721 cells.3.2.3 Effects of stably over-expressed Smac gene coupling with cisplatin or cadmium chloride on apoptosis-regulatory factorsThe expressions of caspase-3, -9 and Cyt c in the established SMMC-7721/Smac cells were detected by Western blot and immunocytochemistry respectively. The results determined with the two methods were coincident, which showed that the expressions of caspase-3, -9 and Cyt c increased in SMMC-7721/Smac cells when compared with that of SMMC-7721 cells. After the treatment of cisplatin or cadmium chloride, the expressions of caspase-3, -9 and Cyt c increased in both SMMC-7721/Smac and SMMC-7721 cells, but that in former was higher.