Pashikanti Srinath, Afrin Farjana, Meldrum Trevor C, Stegelmeier John L, Pavek Adriene, Habashi Yashar A, Fatema Kaniz, Barrott Jared J
Department of Biomedical and Pharmaceutical Sciences, Idaho State University, Pocatello, ID 83209, USA.
Methods Protoc. 2019 Aug 31;2(3):76. doi: 10.3390/mps2030076.
Sphingolipid metabolism is an important process in sustaining the growth needs of rapidly dividing cancer cells. Enzymes that synthesize sphingolipids have become attractive targets in cancer pharmacology. Ceramide is a precursor for synthesizing sphingolipids such as sphingomyelin, sphingosine-1-phosphate, and glucosylceramide. Sphingomyelin synthase (SMS) is the enzyme that transfers a phosphatidylcholine to ceramide to generate sphingomyelin. To test the inhibition of SMS, scientists assess the buildup of ceramide in the cell, which is cytotoxic. Because ceramide is a small lipid molecule, there are limited tools like antibodies to detect its presence. Alternatively, designated machines for small-molecule separation coupled with mass spectrometry detection can be used; however, these can be cost-prohibitive. We used a commercially available NBD-ceramide to apply to human cancer cell lines in the presence or absence of a known SMS inhibitor, jaspine B. After short incubation times, we were able to collect cell lysates and using solvent extraction methods, run the cellular material on a thin-layer chromatography plate to determine the levels of intact fluorescently labeled ceramide. Brighter fluorescence on the TLC plate correlated to greater SMS inhibition. Small molecules can then be screened quantifiably to determine the biological impact of inhibiting the sphingolipid metabolism pathways involving ceramide.
鞘脂代谢是维持快速分裂的癌细胞生长需求的重要过程。合成鞘脂的酶已成为癌症药理学中具有吸引力的靶点。神经酰胺是合成鞘脂如鞘磷脂、鞘氨醇 -1- 磷酸和葡萄糖神经酰胺的前体。鞘磷脂合酶（SMS）是一种将磷脂酰胆碱转移至神经酰胺以生成鞘磷脂的酶。为了测试对SMS的抑制作用，科学家们评估细胞中神经酰胺的积累情况，神经酰胺具有细胞毒性。由于神经酰胺是一种小的脂质分子，用于检测其存在的工具如抗体有限。或者，可以使用与质谱检测联用的小分子分离专用仪器；然而，这些仪器成本可能过高。我们使用市售的NBD - 神经酰胺，在存在或不存在已知的SMS抑制剂茉莉刺桐碱B的情况下应用于人类癌细胞系。经过短时间孵育后，我们能够收集细胞裂解物，并使用溶剂萃取方法，将细胞物质在薄层色谱板上进行分析，以确定完整的荧光标记神经酰胺水平。薄层色谱板上较亮的荧光与更强的SMS抑制作用相关。然后可以对小分子进行定量筛选，以确定抑制涉及神经酰胺的鞘脂代谢途径的生物学影响。
