EFFECTS OF THE SELECTIVE BISINDOLYLMALEIMIDE PROTEIN-KINASE-C INHIBITOR GF 109203X ON P-GLYCOPROTEIN-MEDIATED MULTIDRUG-RESISTANCE

Inhibition of protein kinase C (PKC) is discussed as a new approach fo r overcoming multidrug resistance (MDR) in cancer chemotherapy. For ev aluation of this concept we applied the bisindolylmaleimide GF 109203X , which shows a highly selective inhibition of PKC isozymes alpha, bet a 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this c ompound in modulation of MDR was examined using several P-glycoprotein (P-gp)overexpressing cell lines including a MDR1-transfected HeLa clo ne, and was compared with the activities of dexniguldipine-HCl (DNIG) and dexverapamil-HCl (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp -mediated MDR, cell lines exhibiting different expression levels of th is PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a c-fos(-711)CAT-transfected NIH 3T3 clone, the inhibitor y qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up to 1 mu M final concentrations of the PKC in hibitor GF 109203X a concentration at which many PKC isozymes should b e blocked substantially, no cytotoxic or MDR-reversing effects whatsoe ver were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, deplet ion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no in fluence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal a ctivity of GF 109203X was seen at higher final drug concentrations, ho wever. Remarkably, [H-3]vinblastine-sulphate binding competition exper iments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modu lators, i.e. decreasing efficacy in the series dexniguldipine-HCl>dexv erapamil-HCl>GF 109203X. Similar interaction with the P-gp in the micr omolar concentration range was revealed by competition of GF 109203X w ith photoincorporation of [H-3]azidopine into P-gp-containing crude me mbrane preparations. No significant effect of the PKC inhibitor on MDR 1 expression was seen, which was examined by cDNA-PCR. Thus, the bisin dolylmaleimide GF 109203X probably influences MDR mostly via direct bi nding to P-Ep. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not sup port the concept of a major contribution of PKC to a P-gp-associated M DR, al least using the particular cellular model systems and the selec tive, albeit general, PKC inhibitor GF 109203X.