Molecular basis for the synergistic interaction of adriamycin with the formaldehyde-releasing prodrug pivaloyloxymethyl butyrate (AN-9)

The interaction of Adriamycin and pivaloyloxymethyl butyrate (AN-9) was inv estigated in IMR-32 neuroblastoma and MCF-7 breast adenocarcinoma cells. Ad riamycin is a widely used anticancer drug, whereas AN-9 is an anticancer ag ent presently undergoing Phase II clinical trials. The anticancer activity of AN-9 has been attributed to its ability to act as a butyric acid prodrug , although it also releases formaldehyde and pivalic acid. Adriamycin and A N-9 in combination display synergy when exposed simultaneously to cells or when AN-9 treatment is up to 18 h after Adriamycin administration. However, the reverse order of addition results in antagonism. These interactions ha ve been established using cell viability assays and classical isobologram a nalysis. To understand the molecular basis of this synergy, the relative le vels of Adriamycin-DNA adducts were determined using various treatment comb inations. Levels of Adriamycin-DNA adducts were enhanced when treatment com binations known to be synergistic were used and were diminished using those treatments known to be antagonistic. The relative timing of the addition o f Adriamycin and AN-9 was critical, with a 20-fold enhancement of Adriamyci n-DNA adducts occurring when AN-9 was administered 2 h after the exposure o f cells to Adriamycin. The enhanced levels of these adducts and the accompa nying decreased cell viability were directly related to the esterase-depend ent release of formaldehyde from AN-9, providing evidence for the formaldeh yde-mediated activation of Adriamycin.