Gilteritinib Suppresses Hepatocellular Carcinoma Cells in Association with Reduced Calpain-1 Expression and Inhibition of STAT3/AKT Signaling

Background/Objectives: Hepatocellular carcinoma (HCC) is a leading cause of cancer-associated mortality worldwide, and current targeted therapeutic options remain inadequate. Gilteritinib, a clinically approved tyrosine kinase inhibitor, has shown antitumor activity in hematologic malignancies; however, its therapeutic role and underlying mechanisms in HCC remain unclear. Methods: The effects of gilteritinib on HCC were evaluated in Hepa1-6 and Huh-7 cells. Cell proliferation, migration, invasion, apoptosis, and cell cycle distribution were assessed by CCK-8, wound healing, Transwell, and flow cytometry assays. Protein expression and total calpain activity were assessed by Western blotting and enzymatic activity assays. Surface plasmon resonance (SPR) analysis was used to examine the cellular association of gilteritinib with HCC cells. Antitumor efficacy and safety were further evaluated in a Hepa1-6 allograft mouse model. Results: Gilteritinib significantly inhibited the proliferation, migration, and invasion of HCC cells and induced G2/M-phase arrest and apoptosis. Gilteritinib treatment was associated with reduced calpain-1 expression and total calpain activity. These changes were accompanied by decreased phosphorylation of STAT3 and AKT, downregulation of Bcl-2, and upregulation of Bax. In vivo, gilteritinib markedly suppressed tumor growth in Hepa1-6 tumor-bearing mice. Conclusions: Collectively, gilteritinib exerts antitumor effects against HCC both in vitro and in vivo, and these effects are closely associated with reduced calpain-1 expression and inhibition of STAT3/AKT signaling. These findings suggest that gilteritinib has therapeutic potential in HCC and highlight calpain-1 as a candidate downstream correlate worthy of further mechanistic study.