Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin, China.
Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin, China.
Phytomedicine. 2024 Dec;135:156156. doi: 10.1016/j.phymed.2024.156156. Epub 2024 Oct 13.
Targeting Wnt/β-catenin signaling emerges as one of the promising strategies for colorectal cancer (CRC) treatment, as this signaling is highly activated in CRC progression. Despite reports on the cytotoxic effects of hirsutine (HT), an indole alkaloid found in herbal medicines from the genus Uncaria, its therapeutic potential for CRC and the involved mechanisms are poorly understood. This study investigates the anticancer efficacy and the probable mechanisms of HT against CRC.
To evaluate in vitro anticancer activity of HT, cell growth examined by MTT and colony formation assay, and apoptosis examined by flow cytometry were analyzed. To explore the mechanisms, RNA-sequencing, western blotting, dual-luciferase reporter assays, immunofluorescence, and co-immunoprecipitation were performed. Mouse model of azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colon cancer was utilized to assess HT's in vivo anticancer efficacy.
HT significantly inhibited CRC cell proliferation with IC values of 22.25 ± 3.27 μM for SW620 cells and 22.24 ± 2.36 μM for HCT116 cells, and induced apoptosis. HT decreased protein levels of Wnt3a and β-catenin dose- and time-dependently, and inhibited TOP/FOP FLASH reporter activity, nuclear travel of β-catenin, and downstream targets like c-Myc, Cyclin D1, VEGF. HT reduced β-catenin protein half-life, and the reversal of this effect by MG132 indicated that HT facilitated proteasome-dependent degradation of β-catenin in these two cell lines. HT also increased β-catenin ubiquitination without affecting Axin and β-TrCP levels. HT treatment for 24 h induced YAP cytoplasmic retention, enhanced YAP interacting with β-catenin and β-TrCP, triggering destruction complex formation and β-catenin ubiquitination and degradation, while YAP siRNA impaired these effects. Additionally, β-catenin overexpression and LiCl treatment counteracted HT-induced inhibition on cell growth and Wnt/β-catenin cascade. In model of AOM/DSS-induced mouse colon cancer, compared with AOM/DSS treatment group, HT recovered colon length, reduced tumor numbers and radius, and downregulated β-catenin and Ki-67, while upregulated cleaved PARP in the colorectal tissue with tumors.
HT exhibits anticancer activity against CRC probably by inhibiting Wnt/β-catenin signaling, with YAP playing an indispensible role during the process, highlighting HT as a potential novel candidate drug for CRC therapy.
靶向 Wnt/β-连环蛋白信号通路已成为结直肠癌（CRC）治疗的一种有前途的策略之一，因为该信号通路在 CRC 进展中高度激活。尽管有报道称，从钩藤属草药中发现的吲哚生物碱毛钩藤碱（HT）具有细胞毒性作用，但对于 CRC 的治疗潜力及其相关机制知之甚少。本研究旨在探讨 HT 对 CRC 的抗癌作用及其可能的机制。
通过 MTT 和集落形成实验检测 HT 的体外抗癌活性，通过流式细胞术检测细胞凋亡。通过 RNA 测序、western blot、双荧光素酶报告基因检测、免疫荧光和免疫共沉淀实验来探讨相关机制。利用氧化偶氮甲烷/葡聚糖硫酸钠（AOM/DSS）诱导的结肠癌细胞小鼠模型来评估 HT 的体内抗癌疗效。
HT 显著抑制 CRC 细胞增殖，SW620 细胞和 HCT116 细胞的 IC 值分别为 22.25±3.27 μM 和 22.24±2.36 μM，同时诱导细胞凋亡。HT 呈剂量和时间依赖性地下调 Wnt3a 和 β-连环蛋白的蛋白水平，抑制 TOP/FOP FLASH 报告基因活性、β-连环蛋白核转位以及下游靶标如 c-Myc、Cyclin D1 和 VEGF。HT 缩短了 β-连环蛋白的半衰期，MG132 的逆转作用表明 HT 促进了这两种细胞系中β-连环蛋白的蛋白酶体依赖性降解。HT 还增加了 β-连环蛋白的泛素化，而不影响 Axin 和 β-TrCP 的水平。HT 处理 24 小时后诱导 YAP 细胞质保留，增强 YAP 与β-连环蛋白和β-TrCP 的相互作用，触发破坏复合物的形成和β-连环蛋白的泛素化和降解，而 YAP siRNA 则削弱了这些作用。此外，β-连环蛋白过表达和 LiCl 处理可拮抗 HT 诱导的对细胞生长和 Wnt/β-连环蛋白级联的抑制作用。在 AOM/DSS 诱导的小鼠结肠癌模型中，与 AOM/DSS 处理组相比，HT 恢复了结肠长度，减少了肿瘤数量和半径，并下调了结直肠组织中的β-连环蛋白和 Ki-67，同时上调了肿瘤组织中的 cleaved PARP。
HT 对 CRC 具有抗癌活性，可能是通过抑制 Wnt/β-连环蛋白信号通路，YAP 在该过程中发挥不可或缺的作用，提示 HT 可能成为 CRC 治疗的一种有前途的新型候选药物。
