Enhanced anti-proliferative action of busulphan by quercetin on the human leukaemia cell line K562

The alkylating agent busulphan is commonly used as a single agent for the treatment of chronic myeloid leukaemia (CML). Patients receiving busulphan treatment for CML ultimately relapse since the malignant clone is not eradicated. Alternative treatments are clearly needed (Koller & Miller, 1986). Combining two active drugs with different modes of action can sometimes result in enhanced therapeutic efficacy (Chang et al., 1979). Analysis of data obtained from experiments to study this effect has been greatly facilitated with the computer program developed by Chou & Chou (1987), which is based on the median effect plot and multiple drug equation derived by Chou & Talalay (1984). A recent report showed that quercetin enhanced the antiproliferative activity of the alkylating agent nitrogen mustard (Hofmann et al., 1988). Quercetin is a naturally occurring flavonoid which inhibits a wide range of enzymes including protein kinase C (PKC) (Gschwendt et al., 1983), the tyrosine kinase encoded by the src oncogene (Graziani et al., 1983), and other tyrosine kinases (Srivastava & Chiasson, 1986). PKC and several tyrosine kinases are located in or near the cell membrane and are involved in the transduction of growth factor signals to the nucleus. These enzymes therefore represent novel therapeutic targets for anti-cancer agents. Cells from patients with CML express an abnormal tyrosine kinase (Maxwell et al., 1987) and the cells also contain high levels of PKC (Helfman et al., 1983). We have therefore investigated the effect of combining the protein kinase inhibitor quercetin with busulphan on the proliferation of the CML-derived cell line K562 (Lozzio & Lozzio, 1975). K562 cells were grown at 37°C in RPMI 1640 medium containing fetal calf serum (10%), glutamine (2 mM), penicillin (100 units ml) and streptomycin (100l g ml) under an atmosphere of 5% CO2 in air. Cells were incubated with various concentrations of busulphan, quercetin or the two drugs in combination. Drugs were dissolved in DMSO and sterilised by filtration before addition to the tissue culture medium (the final concentration of DMSO was not more than 0.5%). Drugs were freshly made up for each experiment. After 5 days the cells were harvested and the viable cell number was determined by trypan blue exclusion. The ID50, ID70 and ID90 values for quercetin alone and busulphan alone were 59, 106 and 265 pM and 13, 50 and 404 pM respectively, as determined from dose-response curves generated by the dose-effect analysis computer program (Figure 1). The effects of mixtures of quercetin and busulphan on K562 cell proliferation were determined from median effect plots of different concentrations of the two drugs singly and combined in fixed ratios. These effects were quantitated in terms of the 'combination index' (CI) (Chou & Chou, 1987). CI= 1 indicates summation, CI<1 indicates synergism, and CI> 1 indicates antagonism. In order to compensate for variability between experiments, dose-response curves for