Kaempferol activates BNIP3-mediated mitophagy initiation and induces cytotoxic autophagy arrest to inhibit hepatocellular carcinoma progression

Background Kaempferol, a flavonoid isolated from Alpinia officinarum Hance, has demonstrated inhibitory effects against hepatocellular carcinoma (HCC) cell proliferation in numerous experimental models.However,the exact molecular mechanisms underlying these effects remain elusive. This research was designed to comprehensively investigate kaempferol's anti-cancer properties,identify its molecular targets, and elucidate the precise mechanisms underpinning its effects against HCC through experiments conducted both in vitro and in vivo. Materials and methods The human HCC cell lines HepaG2 and HCCLM3 were chosen to assess kaempferol’s cytotoxicity and anti-proliferation capacity. To evaluate the inhibitory effect of kaempferol on cellular growth, assays including Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation were implemented. Apoptosis induction was measured using flow cytometry (FC) along with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. Autophagic activity was assessed employing monodansylcadaverine (MDC)-based detection methods and transmission electron microscopy (TEM). Kaempferol's antitumor efficacy in vivo was explored using a xenograft nude mouse model, where the suppression of tumor growth was verified by serum biochemical marker analyses. To uncover potential molecular mechanisms, high-resolution quantitative proteomic analysis, immunohistochemistry (IHC), and immunofluorescence (IF) staining were conducted. Additionally, the levels of crucial proteins involved in mitophagy were quantitatively measured using Western blot (WB) analysis. Results The CCK-8 and EdU assays confirmed the robust inhibitory effects of kaempferol on the proliferation of HCC cells, and similar growth-suppressive effects were observed in the nude mouse xenograft model. Furthermore, kaempferol treatment induced marked apoptosis in HCC cells. Mechanistic investigations revealed significant upregulation of mitophagy-related proteins, including BNIP3, SQSTM1 (p62), TAX1BP1, and MAP1LC3B2 (LC3B2), indicating the initiation of mitophagy accompanied by the blockade of autophagic flux. Additionally, apoptosis was notably increased in HCC cells treated with kaempferol. Conclusions In summary, our findings suggest that kaempferol activates BNIP3-mediated mitophagy initiation and induces cytotoxic autophagy arrest, thereby inhibiting HCC progression. This study provides novel insights into kaempferol's anti-HCC mechanisms and offers an innovative supplementary strategy for HCC treatment.