UNLABELLED: Pivaloyloxymethyl butyrate (AN-9), a butyric acid (BA) prodrug, exhibited low toxicity and significant anticancer activity in vitro and in vivo. The purpose of this study was to elucidate the basis for AN-9 increased anticancer activity compared to BA, by studying the uptake of BA and AN-9 into the cells. METHODS: The uptake rate and level of [14C]-AN-9 and [14C]-BA, labeled on the carboxylic moiety of BA, into HL-60 and MEL leukemic cell lines was measured. The cells were filtered and the retained radioactivity was determined. The dependence of the uptake on the activity of cellular esterases and membrane fluidity was investigated. RESULTS: The uptake level in cells incubated with [14C]-AN-9 increased rapidly, peaked after 30 min in MEL and 1 h in HL-60 cells, and declined thereafter. This decline could be attributed to the hydrolysis of AN-9 by cellular esterases and catabolism of the released BA to CO2. In cells pretreated with an esterase inhibitor and incubated with [14C]-AN-9, the reduction of radioactivity was less precipitous. In cells exposed to [14C]-BA, the intracellular radioactivity level was low and unaffected by treatment with an esterase inhibitor. The uptake of [14C]-AN-9 decreased significantly at 4 degrees C compared to that at 37 degrees C. CONCLUSION: The higher potency of AN-9 compared to BA could be at least partially attributed to the more rapid uptake of the lipophilic AN-9 and the release of BA in the cells.
UNLABELLED: Pivaloyloxymethyl butyrate (AN-9), a butyric acid (BA) prodrug, exhibited low toxicity and significant anticancer activity in vitro and in vivo. The purpose of this study was to elucidate the basis for AN-9 increased anticancer activity compared to BA, by studying the uptake of BA and AN-9 into the cells. METHODS: The uptake rate and level of [14C]-AN-9 and [14C]-BA, labeled on the carboxylic moiety of BA, into HL-60 and MEL leukemic cell lines was measured. The cells were filtered and the retained radioactivity was determined. The dependence of the uptake on the activity of cellular esterases and membrane fluidity was investigated. RESULTS: The uptake level in cells incubated with [14C]-AN-9 increased rapidly, peaked after 30 min in MEL and 1 h in HL-60 cells, and declined thereafter. This decline could be attributed to the hydrolysis of AN-9 by cellular esterases and catabolism of the released BA to CO2. In cells pretreated with an esterase inhibitor and incubated with [14C]-AN-9, the reduction of radioactivity was less precipitous. In cells exposed to [14C]-BA, the intracellular radioactivity level was low and unaffected by treatment with an esterase inhibitor. The uptake of [14C]-AN-9 decreased significantly at 4 degrees C compared to that at 37 degrees C. CONCLUSION: The higher potency of AN-9 compared to BA could be at least partially attributed to the more rapid uptake of the lipophilic AN-9 and the release of BA in the cells.