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Decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan
J A Moscow1, C A Swanson, K H Cowan
1Medical Breast Cancer Section, National Cancer Institute.
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.
Insights
This study developed a melphalan-resistant breast cancer cell line (Me1R MCF-7) showing reduced melphalan uptake due to altered system L amino acid transporter function, not detoxification mechanisms.
Area of Science:
- Oncology
- Pharmacology
- Cell Biology
Background:
- Acquired resistance to chemotherapy, such as melphalan, is a major challenge in treating human breast cancer.
- Understanding the mechanisms of melphalan resistance is crucial for developing effective therapeutic strategies.
Purpose of the Study:
- To establish and characterize an in vitro model of acquired melphalan resistance in human breast cancer cells.
- To investigate the underlying mechanisms of melphalan resistance, focusing on melphalan transport and cellular detoxification.
Main Methods:
- Serial incubation of MCF-7 cells with increasing melphalan concentrations to develop a resistant cell line (Me1R MCF-7).
- Melphalan uptake studies using inverse-reciprocal plots to determine kinetic parameters (Vmax, Kt).
- Inhibition studies with 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH) to assess transporter involvement.
- Evaluation of glutathione content, glutathione-S-transferase activity, and pi class glutathione S-transferase RNA expression.
Main Results:
- The Me1R MCF-7 cell line exhibited 30-fold resistance to melphalan compared to wild-type (WT) MCF-7 cells.
- Resistant cells showed significantly decreased melphalan accumulation, with a 4-fold reduction in Vmax and decreased Kt.
- Competition studies with BCH implicated an alteration in the system L amino acid transporter in the observed transport defect.
- No significant differences were found in glutathione content, glutathione-S-transferase activity, or RNA expression between resistant and WT cells, and buthionine sulfoximine did not reverse resistance.
Conclusions:
- The Me1R MCF-7 cell line serves as a valuable in vitro model for studying transport-mediated melphalan resistance in human breast cancer.
- Melphalan resistance in this model is primarily mediated by defects in melphalan transport, specifically involving the system L transporter.
- Glutathione-mediated detoxification pathways do not appear to play a significant role in the development of this acquired melphalan resistance.
