Skp2 inhibitor SKPin C1 decreased viability and proliferation of multiple myeloma cells and induced apoptosis

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（Multiple myeloma, MM）是一种浆细胞恶性肿瘤，可引发溶骨性损伤、高钙血症及免疫功能异常等多种有害病理表现，多数MM患者最终死于该恶性肿瘤。一种被命名为SKPin C1的S期激酶相关蛋白2（S-phase kinase-related protein 2, Skp2）抑制剂已被开发，且被证实对转移性黑色素瘤细胞具有抑制活性。本研究旨在探究SKPin C1对MM的作用效果。将正常B淋巴细胞、THP-1细胞、U266及RPMI 8226多发性骨髓瘤细胞置于不同剂量的SKPin C1中处理48小时。采用MTT法、EdU染色法及细胞周期实验检测细胞增殖情况；通过蛋白质印迹实验检测细胞内Skp2、p27及裂解型半胱氨酸天冬氨酸蛋白酶-3（cleaved caspase-3）的蛋白水平；利用免疫沉淀实验检测p27的泛素结合量。实验结果显示，10 μM SKPin C1即可显著抑制U266与RPMI 8226细胞的存活率，且抑制效果随药物剂量升高而增强。与之相反，50 μM SKPin C1仅在12小时内轻度降低正常B淋巴细胞的存活率。相较于正常B淋巴细胞，U266与RPMI 8226细胞中Skp2的表达水平更高，p27的表达水平更低。经SKPin C1处理或Skp2基因敲低后，U266与RPMI 8226细胞内的p27蛋白水平得以升高，其机制为阻断p27的泛素化过程，进而减慢细胞周期进程、抑制细胞增殖并诱导细胞凋亡。因此，本研究表明SKPin C1可作为强效抑制剂，靶向抑制多发性骨髓瘤的异常增殖与永生化。