| Colon cancer is one of the common malignant tumor, according to the data of the International Agency for Research on Cancer shows that the global incidence of new cases of colorectal cancer in men in the third malignant tumor, while female patients are second only to breast cancer. The incidence of colon cancer in our country is located in the fifth place after lung cancer, gastric cancer, liver cancer and breast cancer.Despite many advances achieved in cancer prevention and treatment strategies such as surgery, radiotherapy and chemotherapy, these could not get a radical cureare in the late stage of cancer therapy,but limited to a more satisfactory effect in the treatment of early stage tumors. The current realities have highlighted the need for more novel cancer therapies to induce effective responses in clinical trials. Gene therapy is a promising strategy for patients resistant totraditional therapies because they target defects inmalignant cells, selectively correcting or eradicatingdefective tissues. However, efficacy and specificityremain major challenges for existing cancer gene therapy. Oncolytic virotherapy is an attractive drug platform of cancer gene therapy consistent with the bothgoals. The oncolytic viruses exhibit an ability toreplicate selectively in tumor tissues to the exclusion ofnormal cells. Furthermore, they can be genetically manipulated to express multiple cancer cell-specifictherapeutic elements. This antitumor treatment combined virus therapy with gene therapy using selective oncolytic viruses in tumor cell proliferation and replication, increased oncolytic virus carrying anticancer gene copy number and improved the expression level, thereby enhancing the killing effect on tumor cells.These characteristics demonstrate the utility of oncolytic virotherapy in the clinicsand provide the basis for potent and specific selective killing to cancer gene therapy.Adenovirus-based vectors are the most widely usedplatforms in gene delivery. However, non-replicatingadenoviruses are seldom effective in eradicating tumorcells. Therefore, very high concentrations and multiple administrations are generally needed to producesignificant anti-tumor responses; such regimens, however, often induce anti-viral immune responses thatresult in the neutralization of the viral vectors in subsequent immunizations and toxicity to the tissues.To circumvent these limitations, conditional replication-competent adenoviruses(CRCA) have been developedand are being extensively evaluated; these viruses replicate specifically in tumor cells with subsequent oncolysisand release of virus progeny to further infect anddestroy neighboring cancer cells. Furthermore, ithas been recently suggested that antibodies which neutralize replication-incompetent adenoviruses have limited effects on the replication-competent adenoviruses. It is therefore reasonable to anticipate that replication-selective tumor-specific adenoviruses would havepotent effects in Cancer gene therapy.In this study, we constructed an oncolytic adenovirususing a cancer-specific promoter(human telomerasereverse transcriptase promoter) and a cancer cell selective apoptosis-inducing gene(Apoptin) that demonstrated significant anti-tumor activity toward solidand metastatic tumors such as gastric cancer and prostate cancer.Telomerase activity isstrikingly higher in about 90% of cancers than in normalcells, and it has been widely used as a tumor marker.One of the three subunits of telomerase, human telomerase reverse transcriptase(h TERT), is the determinant of the telomerase activity and is highly active inimmortalized cell lines and over 85% of human cancers.Therefore, its promoter has been used for tumor specific expression of transgenes. Apoptin, the product of thechicken anemia virus VP3 gene, shows specificity and efficiency toward a wide range of transformed andmalignant cells of human origin, including hepatomas,lymphomas, cholangiocarcinomas, melanomas, andbreast, lung, and colon carcinomas, while sparing non-transformed primary cells such as fibroblasts, keratinocytes, or smooth muscle cells. At present, there is no study on the inhibitory effect of Ad-Apoptin-h TERT-E1 a on colon cancer. This study was to detect the target effect of Ad-EGFP-h TERT-E1 a, Ad-Apoptin, Ad-Apoptin-h TERT-E1 a, Ad-EGFP and other recombinant adenovirus on tumor tissue by using series of in vitro and in vivo experiments. At the same time, to investigate the effect of Ad-Apoptin-h TERT-E1 a on tumor growth and apoptosis of tumor cells.Objective:This study intends to investigate the specific killing effect of Ad-Apoptin-h TERT-E1 a on colon cancer cells, and to investigate the inhibitory effect and inhibition mode of Ad-Apoptin-h TERT-E1 a on tumor cells and its mechanism.Method: In in vitro experiments,recombinant adenovirus Ad-Apoptin-h TERT-E1 a, Ad-EGFP-h TERT-E1 a, Ad-Apoptin, and Ad-EGFP were infected with human colon cancer cells SW1116.On the other had,the normal cell line GES was used as control group. MTT staining, AO/EB staining, DAPI staining and flow cytometry were clear for the targeted effect of recombinant adenovirus and its inhibitory effect. At the same time, Ad-EGFP-h TERT-E1 a, Ad-Apoptin and Ad-EGFP different recombinant adenovirus were as control group,the inhibitory effect of Ad-Apoptin-h TERT-E1 a on colon cancer cells was clearly defined by MTT staining, AO/EB staining, DAPI staining and flow cytometry. Thereafter, blot Western and flow cytometry were used to detect the levels of Caspase, reactive oxygen species(ROS) and mitochondrial membrane potential(MMP) in order to detect related mechanisms of inhibitory effects furtherly and to study on tumor suppressor pathway of recombinant adenovirus from the molecular.In vivo experiments, BALB/c mice bearing tumor model and lung metastasis tumor model were constructed by using CT26 cells. The recombinant adenovirus was inoculated by subcutaneous injection and tail vein injection. To investigate the effect of Ad-Apoptin-h TERT-E1 a on tumor inhibition in vivo by observing the growth trend of tumor, the tumor inhibition rate at different point of time and survival time.Results: MTT staining, light microscopy, AO/EB staining and V Annexin staining showed that the recombinant adenovirus can induce apoptosis in different degree, and Ad-Apoptin-h TERT-E1 a induced apoptosis in human colon cancer cell line SW1116 was the strongest, while the recombinant adenovirus had no obvious inhibition effect on the normal cell line GES. MTT staining also showed that the inhibitory effect of Ad-Apoptin-h TERT-E1 a on SW1116 in vitro was dose-dependent and time-dependent. The expression levels of caspase 3, caspase 6 and caspase 7 were detected by blot Western assay and flow cytometry, and the changes of mitochondrial membrane potential, reactive oxygen species and cytochrome C were detected at the same time. The results showed that the activities of Caspase-3, Caspase-6 and Caspase-7 in SW1116 cells were significantly enhanced after Ad-Apoptin-h TERT-E1 a infection, which accompanied by the changes of Cyto C, MMP and ROS.The results suggest that the mechanism of Ad-Apoptin-h TERT-E1 a induced apoptosis in human colon cancer cells is the activation of the mitochondrial membrane potential and the activation of the apoptotic pathway, but the normal gastric mucosal cell GES was not affected. The experimental results of two kinds of tumor bearing models showed that inoculation Ad-Apoptin-h TERT-E1 a, Ad-Apoptin and recombinant adenovirus by the injection or blood vessel could both significantly inhibited the proliferation of tumor in mice and significantly prolonged the survival time of the model mice, and no obvious toxic and side effects.The inhibition effect affected by Ad-Apoptin-h TERT-E1 a T was the strongest. To further verify the specificity of recombinant oncolytic adenovirus on tumor cell killing effect.Conclusions: HTERT promoter of oncolytic adenovirus expressing apoptin gene, namely Ad-Apoptin-h TERT-E1 a can can make the majority of SW1116 tumor cells showed apoptotic morphologyis which is characterized by a typical dose- dependent and time dependent manner. In SW1116 cells infected with Ad-Apoptin-h TERT-E1 a, the activities of apoptosis related proteins Caspase-3/6/7 were significantly enhanced, and the mitochondrial membrane potential was down regulated, and the levels of reactive oxygen species and C Cyto were up-regulated, suggesting that Ad-Apoptin-h TERT-E1 a may induce apoptosis and inhibit proliferation of human colon cancer cells through mitochondrial pathway. The tumor suppressor effect of Ad-Apoptin-h TERT-E1 a induced apoptosis in tumor cells has a specific targeting of tumor cells, which can not induce apoptosis in normal cells. Ad-Apoptin-h TERT-E1 a significantly inhibited the proliferation of tumor in mice, and prolonged the survival time of CT26 tumor bearing mice. In addition, no obvious toxic side effect was found.To sum up, the Ad- Apoptin- h TERT- E1 a presents potential applied in anti-tumor treatment, can also used in the treatment of primary and metastatic colon cancer. |
PDF Full Text Request