Tumor-specific Killing Effect Of An Oncolytic Adenovirus Controlled By The HTERT Promoter On Colon Cancer Cells

[Background]: Cancer remains one of the top causes of death. Improvement has been made in the overall survival of cancer patients after the emergence of advanced imaging and diagnostic techniques. However, it is still an obstacle for patients with lung or colorectal cancers. New approaches are urgently needed for better cancer therapy. Currently, tumor biotherapy is one of hot spots in cancer research. The use of viruses that specifically kill tumor cells while sparing normal cells is known as oncolytic virotherapy. Several oncolytic adenoviruses have been created by mutation of some viral genes or under the control of the tumor-specific promoters. One major limitation with the use of these tumor-activated promoters to regulate adenoviruses replication is that they are tissue specific agents. The use of these viruses are limited in certain type of cancers. Telomerase is a type of enzyme that needed for terminal end of a chromosome replication, which is elevated in 85 % of tumor cells, but be deactivated in most of somatic cells. Based on the increased activity of telomerase in tumors, we constructed a recombinant adenovector-Ad/hTERT-GFP-E1 for the purpose of tumor-specific replication of viruses and tumor-targeting therapy, in which El gene and GFP gene were both under control of the human telomerase reverse transcriptase (hTERT) promoter. In this study we will determine the anti-tumoractivity of Ad/hTERT-GFP-E1 in vitro by using several colon cancer cell lines.[Methods]: The vectors were amplified in 293 cells, purified by CsCl banding; Use TCED50 to detect replication titer in vitro.The GFP expression was observed by fluorescence microscope. MTT assay and Cell clonogenic assay were performed to determine the killing effect of the virus in vitro; In Situ Cell Death Detection Kit was used to detect apoptosis after treatment; differences between treatment groups were assessed by using paired Student's t test.[Results]: 1). Replication titer of adenoviruses in vitro: DLD1 cells were treated with Ad/hTERT-GFP-E1 and Ad/CMV-GFP at MOI of 500MOI and 2000MOI for 48 h, then the CPE (cytopathic effect) and expression of GFP were observed. Results showed that CPE and expression of GFP could be seen in DLD1 cells which infected with Ad/hTERT-GFP-E1.However no CPE could be seen and only thimbleful expression of GFP could be detected in NHFB which were infected with Ad/hTERT-GFP-E1. Using above cell lysates, we could see obvious CPE and expression of GFP in DLD1 cells infected with the lysate from DLD1/ Ad/hTERT-GFP-E1 after 48h, but no similar phenomenon for other lysates.. TCID50 assay results showed that the titer of cell lysate from DLD1/ Ad/hTERT-GFP-E1 was obviously higher than the titer of other cell lysates. 2). Killing effect of the oncolytic adenoviruses on tumor cells: Tumor cells (DLD1, HCT8, Lovo, SW116, HCT116) and normal cells(NHFB) were treated with two vectors at different MOI from 100MOI to 2000MOI for 5 days, then cell viability was determined by using MTT assay. Our data demonstrated that Ad/hTERT-GFP-E1 had dramatic cell- killing effects in cancer cells. It was in a dose-dependent manner. Then LOVO and NHFB cells were treated with two vectors at 250 MOI, the cell viability was determined by using the MTT assay on 3, 5, 7, and 9 days after treatment. The data demonstrated that cell killing effects of Ad/hTERT-GFP-E1 was also in a time-dependent manner in LOVO cells, but not for NHFB cells. Cell clonogenic assay demonstrated that treatment with Ad/hTERT-GFP-E1 could dramatically suppress the colony formation. Colony quantification showed that inhibition of colony formation was also in adose-dependent manner. 3). Apoptosis detection: DLD1 cells were treated with two vectors at 500 MOI respectively. PBS alone was used as a mock control. TUNEL assay was used to detect apoptosis 48h after treatment. The percent of apoptotic cells was accounted. Result demonstrated that the percent of apoptotic cells in Ad/hTERT-GFP-E1 group was obviously higher than other control groups.[Conclusion]: 1). The oncolytic adenoviruses Ad/hTERT-GFP-E1 have the ability of tumor-specific replication in cancer cells, the titer of the viruses can maintain high level for a long time. 2). Ad/hTERT-GFP-E1 has tumor-specific cell killing effects in colon cancer cells, but no cytolytic effect in normal human fibroblast. 3). The mechanisms of virus killing effect in cancer cells might involve the apoptotic pathway.