INHIBITION OF PGP ACTIVITY AND CELL CYCLE-DEPENDENT CHEMOSENSITIVITY TO DOXORUBICIN IN THE MULTIDRUG-RESISTANT LOVO HUMAN COLON-CANCER CELL-LINE

To determine whether the cell cycle affects multidrug resistance (MDR) and its reversal, doxorubicin (DOX) cytotoxicity and the effect of in hibition of P-glycoprotein (Pgp) activity by verapamil (VER) were inve stigated in MDR LoVo cell lines (LoVo-R) in different phases of the ce ll cycle. Synchronised cells were obtained by exposing cells for 24 h to non-toxic concentrations (40 nmol/l) of methotrexate (MTX), which i nduced a reversible blockade in the S phase. DOX cytotoxicity was high er if cells were exposed to DOX shortly after the pretreatment with MT X, when most cells were in the S phase of the cell cycle. At that time , the DOX concentration inhibiting cell growth by 50% (IC50) was decre ased by approximately 4-fold compared to non-synchronised cycling cell s. DOX cytotoxicity remained high during the transition from the S to the G(2)M phase, but was reduced when the cells had shifted to the G(2 )M phase. Inhibition of Pgp activity by VER (6 mu mol/l) enhanced DOX uptake and resulted in an intracellular nuclear compartmentalisation o f DOX in LoVo-R cells. These effects were not significantly different (P = NS) in the different phases of the cell cycle. However, similar i ncreases in intracellular DOX uptake due to the inhibitory effect of V ER on Pgp greatly potentiated DOX cytotoxicity in LoVo-R cells synchro nised in the S or G(2)M phase compared with non-synchronised cycling c ells. The ratio between DOX IC50 in the absence and presence of VER in LoVo-R cells synchronised in the S phase and in cycling cells was 11. 1 and 4.1, respectively (P < 0.01). This greater potentiation could be explained by the increased chemosensitivity of the S- and G(2)M-phase cells to intracellular DOX concentration compared with the non-synchr onised cells. Finally, the combination of synchronisation by MTX and o f inhibition of Pgp activity by VER produced a considerable reduction in DOX IC50 (approximately 50-fold) in LoVo-R cells compared with the cells not treated with MTX and VER. In conclusion, this study demonstr ates that, in LoVo-R cells, the effect of Pgp inhibition on DOX cytoto xicity is dependent on cell cycle phase. DOX cytotoxicity is maximal w hen inhibition of Pgp activity occurs during the S/G(2)M phases. Copyr ight (C) 1996 Elsevier Science Ltd