Platinum Accumulation in Pigmented Granules of Cisplatin-Treated Melanoma Cells

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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 HM20 resin or in LR White resin. Sections cut to a thickness of 300 nm were stained with uranyl acetate and lead citrate, and imaged with a transmission electron microscope operating at 120 kV accelerating voltage, equipped with an energy filter. Unstained sections were also analyzed to determine the melanosomes stages according to the intensity of melanin pigments, because the melanosomes are easily visible in these preparations. To determine numbers of melanosomes, cross sections of representative whole cells were obtained by montaging, and melanosomes were counted. Unstained ultramicrotomed sections 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 were also analyzed using the x-ray microprobe situated on beamline 2-ID-D of the Advanced Photon Source at 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.