目的:癌症干细胞(CSC)的标志之一是由于Wnt拮抗剂如分泌型卷曲相关蛋白4(SFRP4)的存在减少而导致的过度活跃的Wntβ-catenin信号传导。富含半胱氨酸的结构域(CRD)和netrin样结构域(NLD)是具有抗肿瘤特性的SFRP4的两个功能结构域。在这项研究中,我们已经探索了短微肽SC-301(来自CRD)和SC-401(来自NLD)对CSC特性的有效性,EMT,从PA-1和SKOV-3细胞系富集的卵巢CSC的凋亡和自噬。
方法:基因表达分析,对卵巢CSC进行Western印迹和免疫细胞化学以评估微肽对各种CSC相关致癌特性的抑制潜力。进行免疫共沉淀以检测CD24与β-连环蛋白复合物的结合。CYTO-ID自噬检测试剂盒2.0用于监测肽处理的CSC中的自噬通量。
结果:可以清楚地看到,来自两个结构域的微肽抑制Wnt途径,启动细胞凋亡,抑制迁移和化学致敏CSC。具体来说,CD24(卵巢CSC的定义标记)被肽处理抑制。值得注意的是,CD24和β-catenin之间的相互作用在肽处理后被破坏。SFRP4肽处理还抑制了对CSC存活至关重要的自噬过程。
结论:研究表明,尽管两种肽都具有抑制作用,SC-401在靶向CSC特性和下调Wntβ-连环蛋白机制方面更有效。
OBJECTIVE: One of the hallmarks of cancer stem cells (CSC) is hyperactive Wnt β-catenin signaling due to the decreased presence of Wnt antagonists such as secreted frizzled related protein 4 (SFRP4). Cysteine-rich domain (CRD) and netrin-like domain (NLD) are the two functional domains of SFRP4 having anti-tumor properties. In this study, we have explored the effectiveness of short micropeptides SC-301 (from CRD) and SC-401 (from NLD) on CSC properties, EMT, apoptosis and autophagy in ovarian CSCs enriched from PA-1 and SKOV-3 cell lines.
METHODS: Gene expression analysis, Western blot and immunocytochemistry were performed on ovarian CSCs to evaluate the inhibitory potential of micropeptides to various CSC associated oncogenic properties. Co-immunoprecipitation was performed to detect the binding of CD24 to β-catenin protein complex. CYTO-ID Autophagy Detection Kit 2.0 was used to monitor autophagic flux in peptide treated CSCs.
RESULTS: It is clearly seen that the micropeptides derived from both the domains inhibit Wnt pathway, initiate apoptosis, inhibit migration and chemosensitize CSCs. Specifically, CD24, a defining marker of ovarian CSC was suppressed by peptide treatment. Notably, interaction between CD24 and β-catenin was disrupted upon peptide treatment. SFRP4 peptide treatment also suppressed the autophagic process which is crucial for CSC survival.
CONCLUSIONS: The study demonstrated that although both peptides have inhibitory effects, SC-401 was emphatically more effective in targeting CSC properties and down regulating the Wnt β-catenin machinery.