Killing Effect Of Thermochemotherapy With 5-FC On Human Colon Cancer Cell Line SW480 Transfected Cytosine Deaminase Gene

AIMS: To investigate the killing effect of thermochemotherapy with 5-fluorocytosine ( 5-FC ) on human colorectal carcinoma cell lines SW480 transfected carcinoembryonic antigen (CEA) tissue-specific cytosine deaminase (CD) in vitro, and study the mechanism of thermochemotherapy. METHODS: Recombinant retroviral vector GlCEACDNa, in which the CD gene was controlled under the CEA promoter, was introduced through liposome technique to the human colorectal carcinoma cell line SW480, and then the cells were selectively cultured in G418. The proliferated colonies were treated with 5-FC combined hyperthermia at a temperature of 43 degrees C for 30 min. PCR was used to detect the effect of transfected cytosine deaminase and RT-PCR was performed to analyze the expression of CD gene in target cells after heated. The experiment were performed under different temperature and different heated time and heated times to study the optimum time and temperature of thermochemotherapy with 5-fluorocytosine. Flow cytometry was performed to analyze the cellar cycle and electron microscope was used to observe the change of structure of SW480 cells following thermochemotherapy with 5-fluorocytosine. RESULTS: The expression of CD genes in target cells were detected afterheated. After transfection, SW480 cells were more sensitive to 5-fluorocytosine than their parental cells (P<0.01, t =3.498, n =9 ). The cellular survival rates were 79.5 % (PO.01, t =16.994, n =9 ) and 15.4% (P<0.05, t =2.403, n =9 ) in SW480 cells not transfected CD gene and in SW480 cells transfected CD gene respectively after heated under 43 degree C for 30 min and treated with 5-fluorocytosine for 8d. When the proportion of cells transfected CD gene was 30% in control groups and 50% in heated groups, the killing effect of 5-fluorocytosine on cells were similar to that cells transfected CD gene all. Furthermore, the killing effect of thermochemotherapy with 5-FC at 43 degrees C for 30 min was greater compare to that at other heated temperature and other heated time (P<0.05, ITEM =2.260, n =9; P<0.05, trim =2.179, n =9 ). The proportion of S stage cell was 26.4% in group treated with 5-flurocytosine (P<0.005, t =3.418, n =6) and 41.6% in group (P<0.001, t =7.158, n =6) under the analysis of flow cytometry. Apoptosis were observed after thermochemotherapy with 5-flurocytosine by electron microscope.CONCLUSIONS: 1. Thermochemotherapy with 5-flurocytosine will improve the outcome of the targeting anti-tumor effect of CD/5-FC system on human colorectal carcinoma cell lines SW480 transfected with the CEA tissue-specific CD genes; 2. The killing effect of thermochemotherapy with 5-FC at 43 degrees C for 30 min is optimum. 3. SW480 cells transfected CD gene were blocked in S stage of cellular cycle and apoptosis were induced following treated by 5-FC combined with hyperthermia.