Role of differential drug uptake, efflux, and binding of etoposide in sensitive and resistant human tumor cell lines: implications for the mechanisms of drug resistance.
In order to study the mechanism of etoposide (VP-16) resistance in human tumor cells and to assess the role of P-170 glycoprotein in VP-16 accumulation, we have examined the uptake and efflux of VP-16 in both sensitive and multidrug-resistant MCF-7 human breast and HL60 human promyelocytic leukemia cells. The drug-resistant cells, MCF-7/ADR and HL60/ADR, were selected for resistance to adriamycin and were 200- to 250-fold resistant to VP-16. Whereas MCF-7/ADR cells overexpress the P-170 glycoprotein and show the multidrug-resistant phenotype, HL60/ADR cells do not overexpress the P-170 glycoprotein. Although there was a 2-fold decrease in accumulation of VP-16 in MCF-7/ADR cells, this decrease did not correlate with a 250-fold resistance to the drug. VP-16 efflux was rapid and almost complete from MCF-7 cell lines and it was decreased at 4 degrees. Further, there was a significant increase in VP-16 accumulation in the MCF-7/ADR cells in the presence of glucose-free medium supplemented with sodium azide. However, no change in the pattern of VP-16 efflux was observed. Under these conditions, addition of glucose caused release of VP-16 from MCF-7/ADR cells, suggesting energy-dependent modifications in the drug binding. Coincubation of vincristine with VP-16 also increased the drug accumulation and decreased the rate of efflux of VP-16 in both sensitive and resistant MCF-7 cells, suggesting that vincristine and VP-16 may compete for similar binding and efflux mechanisms in these cell lines. In contrast, daunorubicin increased VP-16 accumulation only in the sensitive MCF-7 cell line, whereas the efflux rate of VP-16 was not significantly changed in either cell line. HL60 sensitive cells accumulated 4- to 5-fold more VP-16 than the resistant subline. Both sensitive and resistant cells showed an important noneffluxable pool of the drug, 3-fold larger for sensitive cells (79 +/- 12 versus 25 +/- 2 pmol of VP-16/mg of protein, for sensitive and resistant cells, respectively). The efflux of VP-16 was temperature dependent only in sensitive cells. VP-16 accumulation in HL60/ADR cells was increased in glucose-free medium supplemented with sodium azide; however, the noneffluxable pool of VP-16 was not significantly changed. In contrast, although these conditions had no effect on the drug accumulation in the parental line, they caused a decrease in the noneffluxable pool of VP-16, suggesting an energy-dependent binding and retention of VP-16.(ABSTRACT TRUNCATED AT 400 WORDS)
Politi
P M
PM
Clinical Pharmacology Branch, National Cancer Institute, Bethesda, Maryland 20892.
Sinha
B K
BK
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000255:Adenosine Triphosphate / Q000032:analysis; D001386:Azides / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005047:Etoposide / Q000493:pharmacokinetics* / Q000494:pharmacology; D005260:Female; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000502:physiology; D019810:Sodium Azide; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014750:Vincristine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D008562:Membrane Glycoproteins; D014750:Vincristine; D005047:Etoposide; D000255:Adenosine Triphosphate; D019810:Sodium Azide