Abstract
A combination of 5-fluorouracil (5-FU) and cisplatin (cis-Diamminedichloroplatinum (II) ; DDP) was administered to the human pharyngeal carcinoma KB cell line in vitro in order to examine the effects of 5-FU pretreatment on DDP cytotoxicity and binding of platinum (Pt) to DNA (Pt-DNA) or its removal from DNA. DDP cytotoxicity was determined by cell survival in the presence (0.5, 1.0 μg/ml) or absence of 5-FU pretreatment. Cultures were exposed to 5-FU for 24 h in Eagle's minimum essential medium (MEM) with 10 % fetal bovine serum, and treated with DDP for 2 h after a 24 h drug-free interval in arginine-deficient MEM (ADM) containing 2.5 % dialyzed fetal bovine serum (DFBS), and then incubated for the required time interval in ADM with DFBS. After 0, 6, 12, 24 h postincubation, DNA was isolated from the cells, and Pt-DNA was measured by atomic absorption spectrophotometry.
By comparing 50 % inhibitory concentration in survival, the pretreatment with 0.5 and 1.0 μg 5-FU/ml increased DDP cytotoxicity 1.4- and 1.7-fold in KB cells, respectively.
The amount of Pt-DNA in KB cells, which was measured immediately after treatment with DDP, increased with doses up to 2.0 μg/ml of 5-FU, and the cells treated with 10.0 μg/ml of DDP had approximately 1.7-fold higher Pt-DNA than the cells treated with 5.0 μg/ml of DDP at each 5-FU dose tested.
In the time-course study, the cells exposed to 0.5 μg 5-FU/ml lost 19.5 and 42.7 % of their total Pt-DNA at 12 and 24 h after 5.0 μg DDP/ml treatment, respectively, compared with 46.4 and 47.6 % for the cells without 5-FU exposure. The cells exposed to 1.0 μg 5-FU/ml lost 24.4 and 31.7 % of their total Pt-DNA at the same respective times. The cells exposed to 0.5 μg 5-FU/ml also lost 16.4 and 31.1 % of their total Pt-DNA at 12 and 24 h after 10.0 μg DDP/ml treatment in comparison with 34.7 and 57.9 % for the cells without 5-FU exposure, and the cells exposed to 1.0 μg 5-FU/ml did 16.4 and 25.6 % of their total Pt-DNA at those times.
These results indicate that 5-FU pretreatment is associated with both increasing effects on Pt binding to DNA and retarding effects on the removal of Pt-DNA in a KB cell line, thereby potentiating the cytotoxicity of DDP.
