Platinum Accumulation in Pigmented Granules of Cisplatin-Treated Melanoma Cells

为了帮助阐明顺铂耐药治疗黑色素瘤的机制，我们在培养的MTN-1黑色素瘤细胞上进行了实验，以确定顺铂治疗后黑素小体超微结构的变化。还进行了测量，以确定亚细胞对元素铂的摄取。样品经高压冷冻、丙酮低温冷冻取代和低温包埋于UV聚合的Lowicryl HM20树脂或LR White树脂中制备。切片厚度为300 nm，用醋酸铀酰和柠檬酸铅染色，在配有能量滤光片的120kV加速电压下工作的透射电子显微镜下成像。未染色的切片也被分析以根据黑色素的强度来确定黑素小体的阶段，因为黑素小体在这些制剂中很容易被看到。为了确定黑素小体的数量，通过蒙太奇方法获得具有代表性的整个细胞的横截面，并对黑素小体进行计数。 未染色的超微切片用透射电子显微镜成像，并在配有场发射源的扫描电子显微镜上用电子探针X射线显微分析进行分析。还使用位于阿贡国家实验室先进光子源的光束线2-ID-D上的X射线微探头对切片进行了分析。X射线微探头由于其高亮度同步加速器源而提高了铂检测的灵敏度，但由于波带片X射线光学的限制而提供了较低的空间分辨率。这些互补方法的结果显示，顺铂的长期治疗增加了细胞内色素颗粒(黑素体)的数量，并重要地显示了铂在黑素体中的积聚。这些发现提供了证据，证明黑素小体通过隔离细胞毒药物和增加黑素小体介导的药物输出而导致黑色素瘤细胞的难治性。通过抑制黑素小体的功能或破坏黑素小体的结构来阻止黑素小体对细胞毒药物的隔离，可能为提高黑色素瘤细胞的化疗敏感性提供了一种潜在的途径。目前正在进行实验，通过研究电子显微镜中的超微结构变化，以及使用基于同步加速器的X射线微探针测量顺铂摄取的差异，来测试这些治疗方法。

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.