Partial reversal of doxorubicin resistance by forskolin and 1,9-dideoxyforskolin in murine sarcoma S180 variants

Acquired resistance to chemotherapeutic agents is an important clinical problem. One preclinical model, termed multidrug resistance (MDR), is characterized by a complex phenotype of cross-resistance to biochemically unrelated antineoplastic agents, the presence of a high-molecular-weight membrane glycoprotein, and impaired accumulation of drug. To determine whether MDR is mediated in part by altered cyclic 3',5'-adenosine monophosphate (cAMP) levels, the effect of incubation with the Adenylate Cyclase agonist, forskolin, was investigated in the murine sarcoma S180 cell line and two MDR variants (A5-.8, A5-2.5). Basal cAMP levels in sensitive and MDR lines were not significantly different (range, 0.15 +/- 0.05 to 0.31 +/- 0.09 pmol/mg protein); however, 1-h incubation with forskolin, 10 microM, elevated intracellular cAMP 2-fold in the parent line and 43- and 35-fold in the variants. The Adenylate Cyclase agonists, prostaglandin E2 and cholera toxin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine had no significant effect on cAMP levels. To determine the effect of forskolin on doxorubicin-induced cell lethality, S180 and MDR lines were incubated with doxorubicin plus forskolin for 1 h and cloned in soft agar. Coincubation with forskolin partially reversed doxorubicin resistance in the MDR lines in a dose-dependent fashion. To determine whether this effect was mediated solely by elevation of intracellular cAMP, the inactive 1,9-dideoxy analogue of forskolin (DF) was used. Incubation with DF resulted in no elevation of cAMP levels in the sensitive or resistant cell lines; however, DF also partially reversed doxorubicin resistance in the MDR variants. Furthermore, coincubation of the A5-2.5 cell line with doxorubicin and 8-bromo cAMP, 1 mM, did not result in reversal of resistance to doxorubicin. To determine whether the reversal of resistance by the diterpenes was associated with alteration of doxorubicin transport, uptake and efflux of [14C]doxorubicin were measured. Coincubation with both forskolin and DF, 10 microM, enhanced [14C]doxorubicin uptake in the resistant cells, while drug efflux was significantly affected only in the cell line exhibiting intermediate resistance. Since both forskolin and its inactive analogue are effective in partially reversing resistance to doxorubicin and augmenting anthracycline uptake, a mechanism other than elevation of cAMP is most likely responsible.