To help elucidate the mechanisms for cisplatin drug resistance in the treatment of melanoma, experiments have been performed on cultured MTN-1 melanoma cells to determine changes in melanosome ultrastructure that occur with cisplatin treatment. Measurements have also been made to determine subcellular uptake of elemental platinum. Specimens were prepared by high-pressure freezing, low-temperature freeze-substitution in acetone and low-temperature embedding in UV-polymerized Lowicryl resin. Unstained ultramicrotomed sections of thickness approximately 100 nanometers were imaged by TEM and analyzed using electron probe x-ray microanalysis in a scanning transmission electron microscope, equipped with a field-emission source. Sections of thickness approximately 200 nanometers were also analyzed using the x-ray microprobe at the Advanced Photon Source in Argonne National Laboratory. The x-ray microprobe provides increased sensitivity for platinum detection due to its high brightness synchrotron source but gives lower spatial resolution due to limits of the zone-plate x-ray optics. Results from these complementary approaches showed that prolonged treatment with cisplatin increased the number of intracellular pigmented granules (melanosomes), and importantly revealed accumulation of platinum in the melanosomes. The findings provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome mediated drug export. Preventing melanosomal sequestration of cytotoxic drugs by inhibiting the functions of melanosomes or disrupting melanosomal structures might offer a potential approach for enhancing the chemosensitivity of melanoma cells. Experiments are being conducted to test these therapeutic approaches by studying ultrastructure changes in the electron microscope and by measuring differences in cisplatin uptake using the synchrotron-based x-ray microprobe.
| Chen, Kevin G; Valencia, Julio C; Lai, Barry et al. (2006) Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas. Proc Natl Acad Sci U S A 103:9903-7 |
