Platinum Accumulation in Pigmented Granules of Cisplatin-Treated Melanoma Cells

顺铂处理的黑色素瘤细胞色素颗粒中铂的积累

• 批准号: 7967892
• 负责人: Richard Leapman
• 金额: $ 2.38万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967892
• 关键词: Acetone; Antineoplastic Agents; Cells; Cessation of life; Chemicals; Cisplatin; Citrates; Cytoplasmic Granules; Cytotoxic agent; Detection; Drug resistance; Electron Microscope; Electrons; Freeze Substitution; Freezing; Goals; Image; Laboratories; Lead; Measurement; Measures; Mediating; Melanins; Melanoma Cell; Melanosomes; Optics; Outcome; Patients; Pharmaceutical Preparations; Photons; Pigments; Plant Resins; Platinum; Preparation; Property; Radiation; Refractory; Resistance; Resolution; Scanning; Source; Specimen; Staging; Staining method; Stains; Structure; Synchrotrons; Testing; Therapeutic; Thick; United States; base; beamline; chemotherapy; cold temperature; cytotoxicity; improved; melanoma; novel therapeutic intervention; pressure; prevent; research study; resistance mechanism; transmission process; uptake; uranyl acetate; voltage

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.

为了帮助阐明黑色素瘤治疗中顺铂耐药的机制，在培养的 MTN-1 黑色素瘤细胞上进行了实验，以确定顺铂治疗时黑素体超微结构的变化。 还进行了测量以确定元素铂的亚细胞摄取。通过高压冷冻、丙酮中低温冷冻置换以及低温包埋在紫外线聚合的 Lowicryl HM20 树脂或 LR White 树脂中来制备样本。 切成 300 nm 厚度的切片用乙酸双氧铀和柠檬酸铅染色，并用配备能量过滤器、在 120 kV 加速电压下运行的透射电子显微镜成像。 还分析了未染色的切片，根据黑色素的强度确定黑素体阶段，因为黑素体在这些制剂中很容易可见。 为了确定黑素体的数量，通过拼接获得代表性全细胞的横截面，并对黑素体进行计数。 未染色的超薄切片通过 TEM 成像，并在配备场发射源的扫描透射电子显微镜中使用电子探针 X 射线微量分析进行分析。 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 射线微探针对切片进行了分析。 X 射线微探针由于其高亮度同步加速器源而提高了铂检测的灵敏度，但由于波带板 X 射线光学器件的限制而提供了较低的空间分辨率。 这些补充方法的结果表明，长期使用顺铂治疗会增加细胞内色素颗粒（黑素体）的数量，并且重要的是揭示了铂在黑素体中的积累。这些发现提供了证据，证明黑素体通过隔离细胞毒性药物和增加黑素体介导的药物输出来促进黑色素瘤细胞的耐药特性。 通过抑制黑素体的功能或破坏黑素体结构来防止细胞毒性药物的黑素体隔离可能为增强黑色素瘤细胞的化学敏感性提供潜在的方法。 正在进行实验来测试这些治疗方法，方法是在电子显微镜下研究超微结构的变化，并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。