Study Of Novel Chimeric Gene Promoters In Cancer Therapy

Part I Construction and activity assay of radio-inducible and cancer-specific chimeric promotersObjective We combined the radio-inducible CArG element with cancer-specific human telomerase reverse transcriptase (hTERT) gene promoter, and constructed novel chimeric promoters. We detected the cancer-specificity and radio-induicbility of chimeric promoters to explore their ability of application to cancer therapy.Methods We synthesized different enhacers containing 4,6,8,10 CArG elements, respectively, inserted them into the upstream of hTERT promoter, and denoted the vector containing different number of CArG element as pC4-hTERT-luc, pC6-hTERT-luc, pC8-hTERR-luc, pC10-hTERT-luc. We also made pControl-luc which containing SV40-promoter as positive control. Three cancer cell lines (human epithelial cervical cancer cells HeLa, adenocarcinomic human alveolar basal epithelial cells A549, and human hepatocellμlar carcinoma cells MHCC97) and one nomal cell line (human erythroleukemia cells hEL) were transient transfected with these plasmids, followed by radiation treatment with different dose. The activity of these promoters in cancer and normal cells were detected using luc-reporter gene. We alse detected inducible effect of different types of radiation treatment (a single 6 Gy dose, or fractionated irradiation dose,3×2Gy)Results Synthetic promoter containing 6 repeat CArG units has the best radio-inducibility than any other promoters containing different number of CArG units, even higher than that of positive-control promoter (P<0.05), and nearly maximum levels obtained at 4-6Gy. Very low activities of the chimeric promoters could be detected in normal hEL cells. A similar level of reporter gene expression was seen after 3 doses of 2 Gy compared with a single dose of 6 Gy in cancer cells (P>0.05).Conclusions The cancer-specific chimeric promoter containing 6 CArG elements showed the best radio-response, and the chimeric promoter system has the potential for use in cancer gene therapy.Part II In vitro study of gene therapy mediated by chimeric promoterObjective To detect the specific cell killing effect for combing radiotherapy with horseradish peroxidase (HRP)/indole-3-acetic (IAA) suicide gene therapy controlled by a novel radio-inducible and cancer-specific chimeric gene promoter.Methods We screened out chimeric hTERT promoter containing 6 CArG elements for further study of gene therapy. We constructed the plasmid expressing HRP enzyme under the control of chimeric promoter carrying 6 CArG elements, and denoted as pC6-hTERT-HRP. We also made phTERT-HRP as single promoter control, pControl-HRP as positive control, and pControl-luc as negative control. Cells (HeLa, A549, MHCC97, and hEL) were transient transfected with these plasmids, followed by irradiation treatment (6 Gy) or not. The HRP expression was detected by using Western blotting analysis. The proliferation inhibition and apoptosis effect of this chimeric promoter system were analyzed by using MTS assay and Annexin V-FITC staining after the treatment of prodrug-IAA, and the effect of radiosensitivity for this system in cancer cells were detected by clonogenic assays.Results After transient transfection, HRP expression could be dectected in three cancer cell lines. And after 6-Gy irradiation treatment, higher HRP-expression could be seen by using Western blotting analysis. While in the normal hEL cells, HRP-expression could only be seen in the pControl-HRP transfectead cells. After 6-Gy irradiation treatment, the chimeric promoter system showed stronger proliferation inhibition and apoptosis inducing effect of lung cancer cells compared to the control plasmid, even higher than the positive control plasmid (P<0.05), and lower killing effect of normal hEL cells were detected compared with the positive control (P<0.05). The results of c clonogenic assays showed that radiation sensitizer enhancement ratio (SER) of chimeric promoter was calculated to be 3.35 in HeLa cells,3.45 in A549 cells, and 3.45 in MHCC97 cells, which were higher than the control plasmid.Conclusions The new chimeric promoter system provided novel cancer therapeutic modalities, and has the potential for use in cancer therapy.PartⅢIn vivio study of gene therapy mediated by chimeric promoterObjective Objective To detect the selective inhibitory effects of irradiation plus replicated-deficient adenovirus-mediated HRP/IAA suicide gene system using tumor-specific and radio-inducible chimeric promoters on human hepatocellμlar carcinoma subcutaneous xenograft in nude mouse model.Methods Recombinant replicated-deficient adenovirus vector containing chimeric promoter was constructed, and denoted as Ad-C6-hTERT-HRP. The adenovirus Ad-CMV-GFP was also made as negative control. MHCC97 cells were subcutaneously injected into the left dorsal of the mice to establish a human subcutaneous transplanting hepatocellμlar carcinoma model. The mice were given intratumoral injection of viruses on the 11th,14th, and 17th day post inoculation, and followed by 6Gy-irradiation treatment on the next day or not. All mice were intraperitoneally injected with IAA from the 13th to 27th day. Tumor volumes were estimated every week and the survival time of each mouse was estimated. The tumors and normal tissues of each mouse were taken for H&E staining and terminal deoxynucleotidyl transferase-mediated dUTP labeling (TUNEL) assay at the end of 8th week post inoculation.Results The combination of adenovirus-mediated gene therapy and radiotherapy significantly inhibited tumor volume compared with adenovirus group. Furthermore, the relative tumor volume in Ad-C6-hTERT-HRP plus IR group was significantly smaller than the Ad-CMV-GFP plus IR group since the 5th week post inoculation. The mice in Ad-C6-hTERT-HRP plus IR group showed a significant prolongation of survival compared with any other groups (P<0.05), and the mean survival duration in Ad-C6-hTERT-HRP plus IR was 54.2 days. The TUNEL staining of the tumor showed that the combination of Ad-C6-hTERT-HRP and radiotherapy resulted the highest apoptotic level (P<0.05). The general samples of the tissues had no obvious change under a microscope and no toxicity effect associated with treatment on normal tissues could be detected.Conclusions Adenovirus-mediated suicide gene therapy plus radiotherapy dramatically inhibited tumor growth and prolonged median survival time. It provided a promising therapeutic modality for hepatocellμlar carcinoma therapy.