Regression of Established Tumors Expressing P-glycoprotein by Combinations of Adriamycin, Cyclosporin Derivatives, and MRK-16 Antibodies

Abstract

Overexpression of P-glycoprotein, a transmembrane protein capable of transporting a broad spectrum of anticancer drugs out of cells, likely contributes to tumor drug resistance. Strategies for overcoming this resistance include the use of specific compounds, such as cyclosporin derivatives, that modulate P-glycoprotein function and antibodies that bind to the protein, thereby altering its activity. We examined the antitumor activity of combination treatment with the anti-P-glycoprotein monoclonal antibody MRK-16, a cyclosporin derivative (either cyclosporin A [CsA] or PSC 833), and the anticancer drug Adriamycin (ADM) against human colorectal carcinoma cells in vitro and established xenografts of these cells in vivo. The human colorectal carcinoma cell line HCT-15 and its ADM-resistant subline HCT-15/ADM2-2 were used in this study. Cellular staining with a tetrazolium dye was used to assess the antitumor (i.e., antiproliferative) effects of treatment in vitro. Caliper measurement of tumor volumes was used to assess the antitumor effects of treatment in vivo. Cell surface binding of MRK-16 was measured by means of an immunofluorescence assay. Differences in the patterns of tumor cell growth in vitro and tumor growth rates in vivo were evaluated by means of repeated measure analysis of variance. Synergy in the combined effects of treatment was evaluated by means of the fractional product method. HCT-15 cells were found to express P-glycoprotein intrinsically; HCT-15/ADM2-2 cells expressed approximately five times more P-glycoprotein than the parental cells. HCT-15/ADM2-2 cells were also found to be about eight times more resistant to ADM in vitro than the parental cells. Incubation of both cell types in vitro with either MRK-16 and ADM or one of the cyclosporin derivatives and ADM inhibited cell growth minimally; however, ternary treatment with MRK-16, one of the cyclosporin derivatives, and ADM dramatically reduced the growth of both cell types. An analysis of treatment effects indicated that synergistic effects were obtained with ternary treatment. When athymic mice bearing established tumors (either HCT-15 or HCT-15/ADM2-2) were treated similarly with various combinations of the tested agents, the most pronounced antitumor effects were observed with ternary treatment. In some mice bearing HCT-15/ADM2-2 xenografts, ternary treatment led to complete tumor regression. Finally, CsA and PSC 833 were both shown to enhance MRK-16 binding to HCT-15 cells and HCT-15/ADM2-2 cells in vitro. Combination treatment with a cyclosporin derivative and an anti-P-glycoprotein antibody can be effective in circumventing P-glycoprotein-mediated drug resistance.