Interactions of mitoxantrone with cytokeratin in human and murine tumor cells

Abstract

The prolonged intracellular retention of the anti-cancer drug mitoxantrone has been linked to its cytotoxicity by several investigators. The persistent association of mitoxantrone with intermediate filament cytokeratin proteins was likewise observed, and its relationship to drug cytotoxicity was investigated. To test whether this association was indicative of mitoxantrone damage to cytokeratin, human WiDr/S colon carcinoma cells or mitoxantrone-resistant WiDr/R cells were treated with mitoxantrone and isolated cytokeratins were examined. Although an affinity of mitoxantrone for cytokeratin proteins could be demonstrated, no drug damage was apparent using a two-dimensional gel electrophoretic analysis. Neither did mitoxantrone affect the assembly or disassembly of tetrameric cytokeratin complexes from or to monomers. Chronic exposure of WiDr/R cells to mitoxantrone does not affect cytokeratin structure or dynamics as determined by electrophoretic analyses. To test whether drug binding to cytokeratin might prevent drug damage at other sites, murine fibroblasts transfected with sequences encoding cytokeratins were subjected to drug cytotoxicity assays. Transfectants were not only resistant to mitoxantrone, but also to doxorubicin and the unrelated drugs methotrexate, melphalan, vincristine and colcemid relative to untransfected and mock-transfected fibroblasts. Transfectants were especially resistant to the cytotoxic effects of anti-tubulin drugs, possibly due to stabilizing interactions between the ectopic cytokeratins and the endogenous microtubules. Drug resistance can not be attributed to differences in growth rates, cell cycle distribution, drug accumulation or drug efflux. The intranuclear distribution of doxorubicin is different in transfected than parental cells, which may affect the resistance phenotype. The data suggest that cytokeratins may function to protect cells from drug damage, which could be a novel explanation for intrinsic drug resistance in some tumors.