Pharmacological and biological evidence for differing mechanisms of doxorubicin resistance in two human tumor cell lines

The cellular pharmacology of doxorubicin resistance (DOX^R) has most commonly been associated with decreased drug uptake, enhanced drug efflux, cross-resistance to multiple anticancer agents, and the overproduction of a M_r 170,000 cell surface glycoprotein (termed P-glycoprotein). In this study, the pharmacological and genetic characteristics of two newly derived human DOX^R sublines were examined. These DOX^R sublines were established following continuously increasing DOX exposure until a 222-fold resistant fibrosarcoma subline (HT1080/DR4) and a 285-fold resistant colon adenocarcinoma subline (LoVo/DR5) were developed. However, three major lines of evidence suggest that despite the similar selection strategy, the mechanism of DOX^R differs significantly between these two cell lines. First, Western blotting using the C219 antibody specific to P-glycoprotein revealed the overexpression of the M_r 170,000 cell surface glycoprotein in LoVo DOX^R cells but not in HT1080 DOX^R cells. Second, LoVo DOX^R cells are cross-resistant to vincristine, actinomycin D, colchicine, etoposide, and gramicidin D, but not to 1 -β-D-arabinofuranosylcytosine. In contrast, HT1080 DOX^R cells display cross-resistance to vincristine, acdnomycin D, vinblastine, and etoposide; however, they are not cross-resistant to gramicidin D, and show an increased (~18-fold) cross-resistance to 1-β-D-arabinofurano-sylcytosine. Third, intracellular DOX accumulation (as measured by [¹⁴C]DOX at 1-h and high-performance liquid chromatography analysis) was decreased ~2.7-fold in LoVo DOX^R cells and ~2.0-fold in HT1080/DR4 cells. However, while net accumulation studies in the presence of 5 μg/ml verapamil reversed DOX^R to parental values in LoVo colon adenocarcinoma cells, it only minimally decreased DOX resistance (12.6%) in HT1080/DR4 cells. Efflux patterns of [¹⁴C]DOX were similar for the DOX^R sublines with an ~50% decrease in DOX retention after 1 h when compared to their respective parental cell lines. Our results suggest that DOX^R in LoVo/DR5 cells may result from overexpression of P-glycoprotein. In contrast, DOX^R in HT1080/DR4 appears to be non-P-glycoprotein mediated and may be related to an alternative mechanism capable of altering drug efflux or differential drug binding.