基于AKT/S6K1信号通路探讨白术内酯Ⅰ抑制结直肠癌的作用机制

Investigation of the Mechanism of Atractylodes I Inhibiting Colorectal Cancer via the AKT/S6K1 Signaling Pathway

    摘要
  • 摘要:
    目的 探讨白术内酯Ⅰ抑制结直肠癌的药效及作用机制。
    方法 采用MTT实验从白术的三种主要活性成分中筛选出药效最好的白术内酯Ⅰ并筛选合理给药浓度。克隆形成实验检测白术内酯Ⅰ对LoVo细胞增殖的影响, 流式细胞术检测其对LoVo细胞凋亡和周期的影响, 伤口愈合及Transwell实验验证其对LoVo细胞迁移、侵袭的影响, Western blot检测其对LoVo细胞的增殖、迁移、EMT蛋白的影响。构建CT26小鼠皮下瘤模型, HE染色进行组织病理学分析, Western blot检测小鼠组织EMT蛋白的表达。
    结果 白术内酯Ⅰ能抑制LoVo细胞增殖, 且给药组与空白对照组相比有统计学差异(P < 0.05, P < 0.01)。白术内酯Ⅰ诱导LoVo细胞凋亡, 给药组与空白对照组有差异(P < 0.05, P < 0.01)。白术内酯Ⅰ是通过G2/M期阻滞发挥抗肿瘤作用, 与空白对照组相比, LoVo给药组的G2期细胞数增多, 且有统计学差异(P < 0.05)。细胞划痕和Transwell实验都能表明白术内酯Ⅰ能明显抑制肿瘤细胞的迁移和侵袭, LoVo给药组与对照组相比有统计学差异(P < 0.05, P < 0.01)。Western blot结果证明白术内酯Ⅰ能调控增殖蛋白c-Myc和凋亡蛋白Bcl-2的表达(P < 0.05), 下调周期蛋白CDK1、Cyclin B1、Cyclin D1及血管生成蛋白VEGF、MMP9(P < 0.05), 调控EMT蛋白E-Cadherin、N-Cadherin(P < 0.05), 并下调p-AKT、p-S6K1蛋白(P < 0.05)。
    结论 白术内酯Ⅰ可能通过AKT-S6K1信号通路发挥抗结直肠癌作用。

     

    Abstract:
    OBJECTIVE To investigate the pharmacological efficacy and mechanism of action of Atractylenolide Ⅰ (Atr-Ⅰ) in inhibiting colorectal cancer.
    METHODS Among three active compounds of Atractylodes macrocephala, Atr-Ⅰ exhibited the highest anti-tumor potency by MTT assay. The optimal concentration of Atr-Ⅰ was determined. The effect of Atr-Ⅰ on LoVo cell proliferation was assessed via a clonogenic assay, while its impact on apoptosis and cell cycle progression was evaluated using flow cytometry. The influence of Atr-Ⅰ on the migration and invasion of LoVo cell line was examined through wound healing and Transwell migration assays. Western blot analysis was performed to explore the effects and mechanisms of Atr-Ⅰ on proteins associated with migration, proliferation, and epithelial-mesenchymal transition (EMT) in LoVo cells. The CT26 mouse subcutaneous tumor model was established, and histopathological analysis was conducted using hematoxylin-eosin (HE) staining. Western blot was also used to assess the effects of Atr-Ⅰ on EMT-related proteins in mouse tissues to elucidate underlying mechanisms.
    RESULTS Atr-Ⅰ significantly reduced colorectal cancer cell viability, with statistically significant differences between treatment and control groups (P < 0.05, P < 0.01). Atr-Ⅰ induced apoptosis in LoVo cells, with the treatment group showing significant differences compared to the control (P < 0.05, P < 0.01). Cell cycle analysis revealed that Atr-Ⅰ exerted anti-tumor effects by inducing G2/M phase arrest, with increased G2 phase cell numbers in the LoVo treatment group compared to the control (P < 0.05). Wound healing and Transwell migration assays confirmed that Atr-Ⅰ significantly inhibited tumor cell migration and invasion (P < 0.05, P < 0.01). Western blot analysis demonstrated that Atr-Ⅰ specifically suppressed the expression of c-Myc and Bcl-2 (P < 0.05), as well as cell cycle-related proteins CDK1, Cyclin B1, and Cyclin D1 (P < 0.05), and angiogenesis-related proteins VEGF and MMP9 (P < 0.05). Additionally, Atr-Ⅰ downregulated EMT-related protein N-cadherin and upregulated E-cadherin expression (P < 0.05). It also reduced the expression of p-AKT and p-S6K1 (P < 0.05).
    CONCLUSION Atr-Ⅰ exhibits potent anti-tumor effects against colorectal cancer, potentially through modulation of the AKT/S6K1 signaling pathway.

     

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