Abstract
An in vitro model of acquired melphalan resistance was developed by serial incubation of an MCF-7 human breast cancer cell line in increasing concentrations of melphalan. The resulting derivative cell line, Me1R MCF-7, was 30-fold resistant to melphalan. Uptake studies demonstrated decreased initial melphalan accumulation in Me1R MCF-7 cells. Inverse-reciprocal plots of initial melphalan uptake revealed a 4-fold decrease in the apparent Vmax of Me1R MCF-7 compared with WT MCF-7 (516 amol cell-1 min-1 vs 2110 amol cell-1 min-1 respectively) as well as a decrease in the apparent Kt (36 microM vs 70 microM respectively). Two amino acid transporters have previously been identified as melphalan transporters: system L, which is sodium-independent and inhibited by 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH), and system ASC which is sodium dependent and unaffected by BCH. At low concentrations of melphalan (3-30 microM), 1mM BCH competition eliminated the differences between the two cell lines, thus implicating an alteration of the system L transporter in the transport defect in the resistant cells. Me1R MCF-7 cells were also evaluated for glutathione-mediated detoxification mechanisms associated with melphalan resistance. There was no difference between Me1R MCF-7 and WT MCF-7 in glutathione content, glutathione-S-transferase activity and expression of pi class glutathione S-transferase RNA. In addition, buthionine sulfoximine did not reverse melphalan resistance in Me1R MCF-7 cells. Therefore, Me1R MCF-7 cells provide an in vitro model of transport-mediated melphalan resistance in human breast cancer cells.
Highlights
Melphalan (1-phenylalanine mustard, L-PAM, Alkeran) is a rationally designed alkylating agent which incorporates the amino acid phenylalanine as a part of its structure
WT MCF-7 and MelR MCF-7 cells were grown in Improved Minimal Essential Medium (IMEM) with (Gibco) and 5% foetal calf serum (Gibco) as previously described (Batist et al, 1986)
Melphalan resistant MCF-7 cells were developed by serial incubation of MCF-7 cells in increasing concentrations of melphalan as described in Methods
Summary
Melphalan (1-phenylalanine mustard, L-PAM, Alkeran) is a rationally designed alkylating agent which incorporates the amino acid phenylalanine as a part of its structure. Melphalan is active against ovarian cancer, myeloma, breast cancer and rhabdomyosarcoma. Most in vitro models of melphalan resistance have involved glutathione-mediated pathways, a finding observed in a wide variety of rodent cell lines including Chinese hamster ovary (Begleiter et al, 1983) and murine L1210 leukaemia cells (Ahmad et al, 1987a; Ahmad et al, 1987b); and human cell lines, including ovarian (Green et al, 1984), myeloma (Gupta et al, 1989; Bellamy et al, 1991) and prostate (Bailey et al, 1992) cells
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