Summary Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes a, /31, ,B2, y, 6 and e in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDRI-transfected HeLa clone, 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 PKCa has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-71 I)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up to 1 gm final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKCa by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl>dexverapamil-HCl>GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular model systems and the selective, albeit general, PKC inhibitor GF 109203X.

Multidrug resistance (MDR) is frequently associated with overexpression of a 170-kDa P-glycoprotein (Pgp). Data suggest altered protein kinase C (PKC) activity in cells expressing the multidrug-resistant phenotype. The staurosporine derivative CGP 41251, an experimental anticancer drug, has been shown to exert selectivity for inhibition of protein kinase C activity and to exhibit antitumor activity in vitro and in vivo. Here we show that CGP 41251 is also able to reverse MDR. After treatment of the multidrug-resistant human lymphoblastoid cell line CCRF-VCR1000 with 500 nM Adriamycin, cell proliferation was reduced to 81% of untreated controls. A combination of 500 nM Adriamycin with a non-toxic concentration of 150 nM CGP 41251 (IC50 for inhibition of cell proliferation 420 nM CGP 41251) inhibits cell proliferation of CCRF-VCR1000 cells to 29% of untreated controls. In sensitive CCRF-CEM cells no enhancement of Adriamycin-induced cytotoxicity was observed upon addition of 150 nM CGP 41251. Strong synergism of the inhibition of cell proliferation was also observed after concomitant treatment of KB-8511 cells with CGP 41251 and Vinblastine or Adriamycin. Drug-sensitive KB-31 cells could not be further sensitized to Adriamycin or Vinblastine with CGP 41251 doses above 100 nM. Pretreatment with 50-1000 nM CGP 41251 for 30 min led to a dose-dependent increase in the intracellular accumulation of rhodamine 123, a substrate of P-glycoprotein. Treatment of multidrug-resistant CCRF-VCR1000 cells with CGP 41251 for 10 min was sufficient to inhibit the efflux of rhodamine 123. Preincubation with CGP 41251 for 12 or 24 hr did not alter multidrug resistance gene (mdrI)-mRNA levels. CGP 41251, a drug with antitumor efficacy in experimental systems, might offer an attractive combination partner for the treatment of tumors expressing the MDR phenotype.

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