Verapamil-induced augmentation of etoposide accumulation in L1210 cells in vitro

The effects of the calcium antagonist verapamil on the intracellular disposition of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (etoposide) (VP-16) as well as on subsequent DNA damage and cytotoxicity were studied in L1210 cells in vitro. At extracellular VP-16 concentrations of 1 to 5 microM, verapamil (10 microM) addition resulted in an increase of DNA single-strand break frequency comparable to that found when VP-16 was present alone at a 3-fold higher concentration. In addition, the elevation of cellular VP-16 levels in the presence of verapamil was linearly correlated with the enhancement of DNA damage and increased cell kill. Verapamil-mediated increase in net VP-16 transport was rapid (1 to 2 min), and allowed for the same elevation of steady-state VP-16 concentration, whether verapamil was added simultaneously with VP-16 or was added after a steady-state level of VP-16 was achieved. Verapamil-mediated elevation of VP-16 levels was not seen at reduced temperature (0 degrees C). Studies of bidirectional VP-16 transport revealed that verapamil (40 microM) did not alter influx of VP-16 (15 microM), but lowered the unidirectional rate constant for efflux by 93%, resulting in the observed increase of steady-state level of the epipodophyllotoxin. Removal of verapamil resulted in a rapid return of VP-16 to levels comparable to that seen with VP-16 alone. When VP-16 was allowed to flow out of the cell in the presence of verapamil, less than 5% of cellular epipodophyllotoxin was retained, suggesting that the calcium antagonist is not acting by enhancing intracellular binding of VP-16. These results indicate that verapamil potentiates VP-16 activity by elevation of intracellular exchangeable epipodophyllotoxin; an activity which seems to be due to inhibition of the efflux mechanism for VP-16. The low intracellular retention of this epipodophyllotoxin and the good correlation between intracellular VP-16 and subsequent DNA damage and cytotoxicity suggest that the epipodophyllotoxin class of anticancer agents may be more useful for probing calcium antagonist effects on drug transport in sensitive cells and in cells exhibiting pleiotropic drug resistance than the vinca alkaloids and anthracyclines which have large tight binding intracellular components.