Yang Ying, Yan Wei, Liu Zhuogang, Wei Minjie
Department of Hematology, Shengjing Hospital of China Medical University, Shenyang, Liaoning, China.
Department of Pharmacology, School of Pharmacy, China Medical University, Shenyang, Liaoning, China.
Braz J Med Biol Res. 2019;52(5):e8412. doi: 10.1590/1414-431X20198412. Epub 2019 Apr 25.
Multiple myeloma (MM) is a malignant neoplasm of plasma, and exhibits several harmful effects including osteolytic injuries, hypercalcemia, and immune dysfunction. Many patients with MM succumb to the underlying malignancy. An S-phase kinase-related protein 2 (Skp2) inhibitor, designated SKPin C1, has been developed and confirmed to have an inhibitory effect on metastatic melanoma cells. This study aimed to determine the effect of SKPin C1 on MM. Normal B lymphocytes, THP-1 cells, and MM U266 and RPMI 8226 cells were exposed to various dosages of SKPin C1 for 48 h. Cell proliferation was determined by MTT, EdU staining, and cell cycle assays. Western blot assays were performed to assess intracellular protein levels of Skp2, p27, and cleaved caspase-3. The amount of ubiquitin attached to p27 was determined using an immunoprecipitation assay. The viability of U266 and RPMI 8226 cells was significantly inhibited by 10 μM SKPin C1 and the inhibitory effect was enhanced with increasing doses of SKPin C1. In contrast, 50 μM SKPin C1 only marginally decreased viability of normal B lymphocytes in 12 h. Skp2 and p27 expression in U266 and RPMI 8226 cells was higher and lower, respectively, than that in the normal B lymphocytes. Treatment with SKPin C1 or Skp2 knockdown increased p27 protein levels in U266 and RPMI 8226 cells by preventing p27 from being ubiquitinated, which slowed the cell cycle, inhibited cell proliferation, and triggered apoptosis. Therefore, this study suggested SKPin C1 as a potent inhibitor against aberrant proliferation and immortalization of MM.
多发性骨髓瘤（MM）是一种浆细胞恶性肿瘤，具有多种有害影响，包括溶骨性损伤、高钙血症和免疫功能障碍。许多MM患者死于潜在的恶性肿瘤。一种名为SKPin C1的S期激酶相关蛋白2（Skp2）抑制剂已被开发出来，并证实对转移性黑色素瘤细胞有抑制作用。本研究旨在确定SKPin C1对MM的影响。将正常B淋巴细胞、THP-1细胞以及MM U266和RPMI 8226细胞暴露于不同剂量的SKPin C1中48小时。通过MTT、EdU染色和细胞周期分析来测定细胞增殖。进行蛋白质免疫印迹分析以评估Skp2、p27和裂解的半胱天冬酶-3的细胞内蛋白水平。使用免疫沉淀测定法确定附着在p27上的泛素量。10μM的SKPin C1显著抑制了U266和RPMI 8226细胞的活力，并且随着SKPin C1剂量的增加，抑制作用增强。相比之下，50μM的SKPin C1在12小时内仅略微降低了正常B淋巴细胞的活力。U266和RPMI 8226细胞中Skp2和p27的表达分别高于和低于正常B淋巴细胞。用SKPin C1处理或敲低Skp2可通过阻止p27被泛素化来增加U266和RPMI 8226细胞中p27的蛋白水平，这减缓了细胞周期，抑制了细胞增殖并引发了细胞凋亡。因此，本研究表明SKPin C1是一种针对MM异常增殖和永生化的有效抑制剂。
