背景:结直肠癌(CRC)是癌症相关死亡的最常见原因之一,并显着损害生活质量。黄芪郁金(AC)在中医治疗CRC中被广泛使用,但确切的机制尚不清楚。
目的:本研究旨在阐明AC抑制CRC进展的机制。
方法:使用UPLC-MS/MS分析鉴定AC的活性组分。使用CT26-Lucifer细胞系在BALB/c小鼠中建立原位移植结直肠肿瘤模型以评估AC的作用。使用IVIS成像技术监测肿瘤体积。用苏木精和伊红(H&E)染色进行肿瘤形态的组织学检查。进行小鼠肿瘤样品的转录组测序以鉴定关键途径和分子靶标。使用CCK-8和伤口愈合试验评估AC对细胞活力和迁移的影响。分别。为了研究AC对CRC细胞的影响,使用氯化钴(CoCl2)建立了体外低氧模型,缺氧诱导剂.通过构建稳定的慢病毒载体实现HIF-2α过表达。从RNA-seq中确定的关键靶标,比如c-Myc,Ki-67,β-连环蛋白,使用蛋白质印迹法评估切割的胱天蛋白酶3,CD133和CD44,qRT-PCR,和免疫荧光分析。采用上皮-间充质转化(EMT)和球体克隆测定来评估癌症干细胞的表型变化。
结果:确定了12种AC成分。AC在体内有效抑制CRC进展。转录组分析强调低氧信号是一个显著富集的途径,暗示其在AC抑制CRC进展中的作用。在低氧模型中,AC在体外抑制CRC细胞的增殖和迁移。此外,AC通过下调干性标志物来降低癌症干性,抑制EMT,减少肿瘤球的形成。低氧反应的下调和AC引起的干性改变涉及HIF-2α和WNT/β-catenin信号传导的减弱。
结论:这项研究提供了第一个证据,表明AC降低CRC的干性,AC抑制CRC向干细胞样细胞的转变与HIF-2α/β-catenin通路的下调密切相关,尤其是在缺氧条件下。
BACKGROUND: Colorectal cancer (CRC) is one of the most common causes of cancer-related mortality and significantly impairs quality of life. Astragali Radix-Curcumae Rhizoma (AC) is widely employed in the treatment of CRC in Chinese medicine, but the precise mechanisms remain unclear.
OBJECTIVE: This study aimed to elucidate the mechanisms by which AC inhibits CRC progression.
METHODS: The active components of AC were identified using UPLC-MS/MS analysis. An orthotopic transplantation colorectal tumor model was established in BALB/c mice using the CT26-Lucifer cell line to evaluate the effects of AC. Tumor volumes were monitored using IVIS imaging technology. Histological examination of tumor morphology was performed with hematoxylin and eosin (H&E) staining. Transcriptomic sequencing of mouse tumor samples was conducted to identify critical pathways and molecular targets. The impact of AC on cell viability and migration was assessed using CCK-8 and wound healing assays, respectively. To investigate the effects of AC on CRC cells, an in vitro hypoxic model was established using cobalt chloride (CoCl2), a hypoxia inducer. HIF-2α overexpression was achieved by constructing stable lentiviral vectors. Key targets identified from RNA-seq, such as c-Myc, Ki-67, β-catenin, cleaved caspase 3, CD133, and CD44, were evaluated using western blotting, qRT-PCR, and immunofluorescence assays. Epithelial-Mesenchymal Transition (EMT) and spheroid cloning assays were employed to evaluate phenotypic changes in cancer stem cells.
RESULTS: Twelve components of AC were identified. AC effectively inhibited CRC progression in vivo. Transcriptomic analysis highlighted hypoxic signaling as a significantly enriched pathway, implicating its role in suppressing CRC progression by AC. In the hypoxic model, AC inhibited the proliferation and migration of CRC cells in vitro. Furthermore, AC reduced cancer stemness by downregulating stemness markers, inhibiting EMT, and decreasing tumor sphere formation. The downregulation of hypoxic responses and the shift in stemness by AC involved attenuation of HIF-2α and WNT/β-catenin signaling.
CONCLUSIONS: This study provides the first evidence that AC reduces the stemness of CRC and the inhibition of the transition of CRC to stem-like cells by AC is closely related to the downregulation of the HIF-2α/β-catenin pathway, especially under hypoxic conditions.