背景:中药配方解毒消症饮(JXY)在胃肠道肿瘤中的临床应用,尤其是结直肠癌(CRC),已经很成熟了,然而,其在CRC治疗中的疗效背后的确切生物学机制仍然难以捉摸.
目的:本研究试图揭示JXY调节结直肠癌干细胞的复杂机制,从而阐明了它发挥其有效抗肿瘤作用的途径。
方法:在本研究中,通过网络药理学分析了JXY对CRC细胞信号通路和功能的调节作用。使用HPLC检测JXY的乙酸乙酯提取物的主要化合物,然后处理HCT-116细胞用于RNA-测序(RNA-Seq)。使用蛋白质印迹分析和基质胶球体测定评估JXY提取物处理后HCT-15和HCT-116细胞的蛋白质表达和干性。此外,在有或没有氯化锂(LiCl)刺激的情况下,使用TOPflash报告基因测定评估β-连环蛋白的转录活性。使用干性维持培养基培养患者来源的CRC类器官(CRCPDO),并在JXY提取物处理后使用ATP测定法测量它们的活力。此外,使用源自HCT-15细胞的异种移植小鼠模型评估JXY提取物的抗肿瘤功效。
结果:网络药理学强调了JXY对肿瘤干细胞和Wnt信号通路的影响。HPLC分析证实,在中国药典(迷迭香酸,槲皮素,和山奈酚)。RNA-Seq结果进一步阐明了JXY提取物的作用,特别是靶向癌症干细胞和Wnt信号通路。此外,JXY提取物抑制CRC细胞中的球状体形成并下调CRC-CSC标志物(CD133、DCLK1和C-MYC)。此外,JXY提取物抑制β-catenin的表达和转录活性以及Wnt通路靶蛋白,包括C-MYC和细胞周期蛋白D1。与细胞系的发现一致,JXY提取物抑制了表现出干性特征的CRCPDO的生长。JXY提取物对肿瘤生长具有显著的抑制作用,C-MYC,和β-连环蛋白在异种移植肿瘤中的水平。
结论:这些结果突出了JXY提取物通过调节Wnt信号通路靶向CRC-CSC的新功能,强调其作为治疗CRC的治疗剂的潜力。
BACKGROUND: The clinical application of the traditional Chinese medicinal formula Jiedu Xiaozheng Yin (JXY) for gastrointestinal tumors, particularly colorectal cancer (CRC), is well-established, yet the precise biological mechanism underlying its efficacy in CRC treatment remains elusive.
OBJECTIVE: This study endeavors to unravel the intricate mechanism through which JXY modulates colorectal cancer stem cells, thus elucidating the pathways by which it exerts its potent anti-tumor effects.
METHODS: In this study, the regulatory impact of JXY on the signaling pathway and function of CRC cells was analyzed through Network pharmacology. The ethyl acetate extract of JXY was detected the major compounds using HPLC and then treated the HCT-116 cells for RNA-Sequencing (RNA-Seq). Protein expression and stemness of HCT-15 and HCT-116 cells following JXY extract treatment were assessed using Western blot analysis and matrigel spheroid assays. Additionally, the β-catenin transcriptional activity was evaluated using a TOPflash reporter assay with or without Lithium chloride (LiCl) stimulation. Patient-derived organoids of CRC (CRC PDOs) were cultured using a stemness maintenance medium, and their viability was measured using ATP assays after treatment of JXY extract. Furthermore, the anti-tumor efficacy of JXY extract was assessed using a xenograft mice model derived from HCT-15 cells.
RESULTS: Network pharmacology emphasized the influence of JXY on cancer stem cells and the Wnt signaling pathway. HPLC analysis confirmed that the JXY extract contained the three most prevalent pharmaceutical compounds among the four herbs documented in the Chinese Pharmacopoeia (rosmarinic acid, quercetin, and kaempferol). RNA-Seq results further elucidated the effect of JXY extract, particularly targeting cancer stem cells and the Wnt signaling pathway. Furthermore, JXY extract inhibited spheroid formation in CRC cells and downregulated CRC CSC markers (CD133, DCLK1, and C-MYC). Additionally, JXY extract suppressed the β-catenin expression and transcriptional activity as well as the Wnt pathway target proteins, including C-MYC and Cyclin D1. Consistent with findings from cell lines, JXY extract suppressed the growth of CRC PDOs exhibiting stemness characteristics. And JXY extract demonstrated a significant inhibitory effect on tumor growth, C-MYC, and β-catenin protein levels in xenograft tumors.
CONCLUSIONS: These results highlight the novel function of JXY extract in targeting CRC CSCs by regulating Wnt signaling pathway, underscoring its potential as a therapeutic agent for treating CRC.