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.

为了帮助阐明顺铂在黑色素瘤治疗中的耐药机制，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色素小体超微结构的变化。测量也被用来确定元素铂的亚细胞摄取。采用高压冷冻、低温丙酮冷冻取代、低温包埋于uv聚合的Lowicryl HM20树脂或LR White树脂中制备样品。切成300 nm厚度的切片，用醋酸铀酰和柠檬酸铅染色，在120 kV加速电压下，安装能量过滤器，用透射电子显微镜成像。我们还分析了未染色的切片，根据黑色素的强度来确定黑素小体的分期，因为黑素小体在这些制剂中很容易看到。为了确定黑素体的数量，采用蒙太奇法获得代表性全细胞的横截面，并对黑素体进行计数。