Many saponins are characterized as exhibiting a wide spectrum of antitumor activities at low concentrations. Most of the previous studies that aimed to understand the mechanisms underlying anticancer saponins have focused on numerous classical signaling pathways. However, at the oncogene level, little is known about the action of saponins, especially asterosaponin. In this study, CN-3, a new asterosaponin isolated from the starfish Culcita novaeguineae, decreased the proliferation of U87 and U251 cells at low doses in a dose- and time-dependent manner. Microarray analysis revealed CN-3 significantly induced the differential expression of 661 genes that are related to its antiglioma effect in U251. Nine downregulated genes (SCUBE3, PSD4, PGM2L1, ACSL3, PRICKLE1, ABI3BP, STON1, EDIL3, and KCTD12) were selected, for further verification of their low expression. Then, shRNA transfection and high-content screening were performed and significantly decreased U251 cell proliferation rate was only observed for the SCUBE3 knockdown. qPCR confirmed SCUBE3 was highly expressed in U251 and U87 cells, and had medium expression levels in U373 cells. Real-time cellular analysis using iCELLigence demonstrated that SCUBE3 is an oncogene in U251 and U87 cells, with knockdown of SCUBE3 inhibiting U251 and U87 cell proliferation while, conversely, SCUBE3 overexpression promoted their proliferation. Afterward, SCUBE3 protein was found to have high expression in primary glioma specimens from patients examined by immunohistochemistry but low expression in normal brain. PathScan ELISA analysis in conjunction with TEM observation demonstrated that the effect of SCUBE3 knockdown in U251 does not appear to be related to the induction of apoptosis. Employing CCK-8, iCELLigence, flow cytometry, western blotting, and shRNA transfection (knockdown and overexpression) experiments, we reveal that the reduction of SCUBE3 expression, induced by CN-3, mediated both inhibition and G1/S arrest of U251 via the Akt/p-Akt/p53/p21/p27/E2F1 pathway.

许多皂苷的特征在于在低浓度下表现出广泛的抗肿瘤活性。旨在了解抗癌皂苷潜在机制的大多数先前研究都集中在众多经典信号通路上。然而，在致癌基因水平上，关于皂苷，特别是星皂苷的作用了解甚少。在这项研究中，CN-3是一种从海星新星Cucuita novaeguineae分离出的新的星形皂苷元，它在低剂量时以剂量和时间依赖性方式降低了U87和U251细胞的增殖。基因芯片分析显示，CN-3在U251中显着诱导661个与其抗神经胶质瘤作用有关的基因的差异表达。九种下调的基因（SCUBE3，PSD4，PGM2L1，ACSL3，PRICKLE1，ABI3BP，STON1，EDIL3，和KCTD12选择），以进一步验证其低表达。然后，进行了shRNA转染和高内涵筛选，并且仅在SCUBE3敲低的情况下观察到U251细胞增殖速率显着降低。qPCR证实SCUBE3在U251和U87细胞中高度表达，并在U373细胞中具有中等表达水平。使用iCELLigence进行的实时细胞分析表明，SCUBE3是U251和U87细胞中的致癌基因，敲除SCUBE3可抑制U251和U87细胞增殖，相反，SCUBE3的过表达可促进其增殖。之后，发现通过免疫组织化学检查的患者原发性神经胶质瘤标本中SCUBE3蛋白表达高，而正常脑组织中SCUBE3蛋白表达低。PathScan ELISA分析结合TEM观察表明，U251中SCUBE3敲低的作用似乎与诱导细胞凋亡无关。利用CCK-8，iCELLigence，流式细胞仪，western印迹和shRNA转染（敲低和过表达）实验，我们揭示了CN-3诱导的SCUBE3表达的减少介导了U251的抑制和G1 / S阻滞。 Akt / p-Akt / p53 / p21 / p27 / E2F1途径。