Multidrug‐resistant human kb carcinoma cells are highly resistant to the protein phosphatase inhibitors okadaic acid and calyculin A. Analysis of potential mechanisms involved in toxin resistance

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Abstract
In this study we show that multidrug-resistant (MDR) human KB-VI cells are highly resistant to the cytotoxicity of okadaic acid and calyculin A, 2 toxins from marine sponges that are potent inhibitors of type-I and type-2A protein phosphatases (PP1 and PP2A). Cytotoxicity and colony-forming assays indicated that, relative to parental drug-sensitive KB-3 cells, KB-VI cells are 35-fold more resistant to okadaic acid and 70-fold more resistant to calyculin A. Cytotoxicity of the toxins was associated with mitotic arrest characterized by chromosome scattering and over-condensation, with KB-3 cells being more sensitive than KB-VI cells and calyculin A being more potent than okadaic acid. The resistance of KB-VI cells to both okadaic acid and calyculin A was completely reversed by verapamil, suggesting that the toxins may be transported by P-glycoprotein (P-gp). To further assess the possibility of an interaction with P-gp, the toxins were employed as potential modulators of the photoaffinity labeling of P-gp by [3H]azidopine. Relative to vinblastine, which effectively competed with [3H]azidopine for P-gp photolabeling, calyculin A was 100-fold less potent and okadaic acid did not inhibit photolabeling at concentrations up to 50 μM. To determine whether the resistance mechanism involved differences in toxin-sensitive phosphatase activity, the activity was assayed in extracts from both cell lines and found to be slightly higher (1.6-fold) in KB-VI than in KB-3 cells. Our results demonstrate a novel, marked resistance of MDR KB-VI cells to these phosphatase inhibitors and suggest that a major mechanism of resistance may involve toxin transport by P-gp at sites apparently different from those which bind azidopine.