Li Xiaoliang, Zhou Mingyan, Zhu Zhe, Wang Zhe, Zhang Xuguang, Lu Lu, Xie Zhenrui, Wang Bingshu, Pan Yipeng, Zhang Junqing, Xu Jian
Hepatobiliary and Liver Transplantation Department of Hainan Digestive Disease Center, Institute of Clinical Medicine, The Second Affiliated Hospital of Hainan Medical University, Haikou, Hainan, 570311, China; Engineering Research Center of Tropical Medicine Innovation and Transformation of Ministry of Education & International Joint Research Center of Human-machine Intelligent Collaborative for Tumor Precision Diagnosis and Treatment of Hainan Province & Hainan Provincial Key Laboratory of Research and Development on Tropical Herbs, School of Pharmacy, Hainan Medical University, Haikou, Hainan, 571199, China.
Hepatobiliary and Liver Transplantation Department of Hainan Digestive Disease Center, Institute of Clinical Medicine, The Second Affiliated Hospital of Hainan Medical University, Haikou, Hainan, 570311, China.
J Ethnopharmacol. 2024 Oct 28;333:118430. doi: 10.1016/j.jep.2024.118430. Epub 2024 Jun 8.
Alpinia officinarum Hance (A. officinarum), a perennial herb known for its medicinal properties, has been used to treat various ailments, such as stomach pain, abdominal pain, emesis, and digestive system cancers. A. officinarum is extensively cultivated in the Qiongzhong and Baisha regions of Hainan, and it holds substantial therapeutic value for the local Li people of Hainan. Kaempferol, a flavonoid derived from A. officinarum, has demonstrated anticancer properties in various experimental and biological studies. Nevertheless, the precise mechanisms through which it exerts its anti-hepatocellular carcinoma (HCC) effects remain to be comprehensively delineated.
This investigation aims to elucidate the anti-HCC effects of kaempferol derived from A. officinarum and to delve into its underlying mechanistic pathways.
Using ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS) to identify active compounds in A. officinarum. HCCLM3 and Huh7 cells were used to study the anti-HCC effect of kaempferol from A. officinarum. The cytotoxicity and proliferation of kaempferol and A. officinarum were measured using CCK-8 and EDU staining. Wound-healing assays and three-dimensional tumor spheroid models were further used to evaluate migration and the anti-HCC activity of kaempferol. The cell cycle and apoptosis were evaluated by flow cytometry. Western blot and qRT-PCR were used to detect the expression of proteins and genes associated with the cell cycle checkpoints. Finally, bioinformatics was used to analyze the relationship between the differential expression of core targets in the ATM/CHEK2/KNL1 pathway and a poor prognosis in clinical HCC samples.
UPLC-MS/MS was employed to detect five active compounds in A. officinarum, such as kaempferol. The CCK-8 and EDU assays showed that kaempferol and A. officinarum significantly inhibited the proliferation of HCC cells. A wound-healing assay revealed that kaempferol remarkably inhibited the migration of HCC cells. Kaempferol significantly suppressed the growth of tumor spheroids. In addition, kaempferol markedly induced G2/M arrest and promoted apoptosis of HCC cells. Mechanically, kaempferol significantly reduced the protein and mRNA expression levels of ATM, CHEK2, CDC25C, CDK1, CCNB1, MPS1, KNL1, and Bub1. Additionally, the combination of kaempferol and the ATM inhibitor KU55933 had a more significant anti-HCC effect. The results of bioinformatics showed that ATM, CHEK2, CDC25C, CDK1, and KNL1 were highly expressed in patients with HCC and cancer tissues, indicating that these genes have certain value in the clinical diagnosis of HCC.
Collectively, our results revealed that kaempferol from A. officinarum inhibits the cell cycle by regulating the ATM/CHEK2/KNL1 pathway in HCC cells. In summary, our research presents an innovative supplementary strategy for HCC treatment.
高良姜是一种以药用特性闻名的多年生草本植物，已被用于治疗各种疾病，如胃痛、腹痛、呕吐和消化系统癌症。高良姜在海南琼中和白沙地区广泛种植，对海南当地黎族具有重要的治疗价值。从高良姜中提取的黄酮类化合物山奈酚在各种实验和生物学研究中已显示出抗癌特性。然而，其发挥抗肝细胞癌（HCC）作用的确切机制仍有待全面阐明。
本研究旨在阐明从高良姜中提取的山奈酚的抗HCC作用，并深入探究其潜在的作用机制途径。
采用超高效液相色谱 - 质谱联用/质谱（UPLC - MS/MS）鉴定高良姜中的活性成分。使用HCCLM3和Huh7细胞研究高良姜中山奈酚的抗HCC作用。采用CCK - 8和EDU染色检测山奈酚和高良姜的细胞毒性及增殖情况。进一步通过伤口愈合实验和三维肿瘤球体模型评估山奈酚的迁移和抗HCC活性。通过流式细胞术评估细胞周期和凋亡情况。采用蛋白质印迹法和qRT - PCR检测与细胞周期检查点相关的蛋白质和基因表达。最后，利用生物信息学分析ATM/CHEK2/KNL1通路中核心靶点的差异表达与临床HCC样本预后不良之间的关系。
采用UPLC - MS/MS检测到高良姜中的五种活性成分，如山奈酚。CCK - 8和EDU实验表明，山奈酚和高良姜显著抑制HCC细胞的增殖。伤口愈合实验显示，山奈酚显著抑制HCC细胞的迁移。山奈酚显著抑制肿瘤球体的生长。此外，山奈酚显著诱导HCC细胞的G2/M期阻滞并促进其凋亡。机制上，山奈酚显著降低了ATM、CHEK2、CDC25C、CDK1、CCNB1、MPS1、KNL1和Bub1的蛋白质和mRNA表达水平。此外，山奈酚与ATM抑制剂KU55933联合使用具有更显著的抗HCC作用。生物信息学结果表明，ATM、CHEK2、CDC25C、CDK1和KNL1在HCC患者和癌组织中高表达，表明这些基因在HCC临床诊断中具有一定价值。
总体而言，我们的结果表明，高良姜中的山奈酚通过调节HCC细胞中的ATM/CHEK2/KNL1通路抑制细胞周期。综上所述，我们的研究为HCC治疗提供了一种创新的辅助策略。
