Summary effect of the trans gene. Method used
Conventional radiation therapy is not effective treating prostate cancer because the required higher dose of radiation to be ‘effective’ causes adverse side effects. Gene therapy could be used as the affected area is easily accessible for the therapeutic gene and gene products of prostate glands can be used for vector targeting however it has low transduction rate, low specificity for the target and high basal activity. Combination of radio-genetic therapy could be used by construction of a promoter that is sensitive to radiation to control the therapeutic gene expression and reduce anti cancer effects of radiation. Recreation of promoter library using different cis acting elements of transcription factors responsive to radiation in prostate cancer cells clone 880 showed the highest response to X-irradiation in LNCap cells, the activity of the constructed promoter was increased by random mutations and named 880-8. Recombinant retroviral vector clone 880-8 and luciferase gene transfected LNCap cells designated LNCap-880-8-luc. Activity in LNCap cells increased up to 9.12+ 0.36 fold at 12h after X-irradiation at 10 Gy. In vivo, the activity of luciferase expression was observed 4.27+1.36 fold at 12h after exposure at 10 Gy. Clone 880-8 responded to radiation in different cancer cell lines which indicates same promoter could be used for their treatments. Another recombinant retrovirus expressing the fcy::fur gene was constructed and transfected LNCap cells named LNCap-880-8-fcy::fur, which demonstrated high enhancement rate. Activation of 880-8 promoter induced slower promoter enhancement in vivo than in vitro. LNCap-880-8-fcy::fur cells showed cell killing in the presence of 5-FC even without X-irradiation, but LNCap880-8luc did not, which indicates it has high basal activity hence the therapeutic cytotoxicity can not be restricted to the targeted area. The promoter in LNCap cells is dose dependent thus can be used as it requires high dose of radiation and the side effects of the radiation could be reduced by the effect of the trans gene. Method used to create radiation sensitive promoter was effective and such promoter can be used in dose dependent gene therapies. Using appropriate transcription factors radiation sensitive promoters could be reconstructed according to cell type and may be used for clinical application in the future.
In the next study activation of artificially constructed promoter in prostate cancer responsive to ultrasound stimulation was examined in LNCap cells using same methods as described in previous study. The difference is when the promoter is exposed to radiation without the fcy::fur gene it indicates high activity without enhancement however no activity observed in the promoter without fcy::fur which suggest that there is no high basal activity hence the promoter could be controlled to the restricted area by ultrasound stimulation and optional concentration of 5FC. Constructed promoter responsive to radiation activation by anti cancer drug called doxorubicin showed similar response in cell killing in fcy::fur gene however the combination of both gene therapy and chemotherapy could be helpful decreasing the side effects and could be used in the future.