Kaempferol from Alpinia officinarum hance induces G2/M cell cycle arrest in hepatocellular carcinoma cells by regulating the ATM/CHEK2/KNL1 pathway

Ethnopharmacological relevance: 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.

Aim of the study: This investigation aims to elucidate the anti-HCC effects of kaempferol derived from A. officinarum and to delve into its underlying mechanistic pathways.

Materials and methods: 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.

Results: 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.

Conclusions: 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.

ATM/CHEK2/KNL1 pathway; Alpinia officinarum hance; Cell cycle; Hepatocellular carcinoma; Kaempferol.