Bufalin treats hepatocellular carcinoma through inhibiting PI3K/AKT signaling pathway

Aim To investigate the therapeutic effects of bufalin, a single-component compound from traditional Chinese medicine, on hepatocellular carcinoma and the underlying mechanisms. Methods CCK-8 and colony formation assays were used to examine the effects of different concentrations of bufalin on the proliferation of human hepatocellular carcinoma cell lines HepG2 and SK-HEP-1. The Hoechst staining assay and flow cytometry were used to detect cell apoptosis. A wound healing assay was performed to assess cell migration. RNA-seq and Western blot were used to investigate the effects of bufalin on the expression and phosphorylation levels of key proteins in the phosphatidylinositol-3 kinase(PI3K)/protein kinase B (AKT) pathway. An in vivo tumor growth assay using KM mice was conducted to evaluate the antitumor efficacy and safety of bufalin. Results Bufalin significantly inhibited the proliferation of hepatocellular carcinoma cells in a concentration- and time-dependent manner, and reduced their colony-forming ability. Bufalin significantly increased cell apoptosis. The wound healing assay showed that bufalin inhibited tumor cell migration. Mechanistic studies revealed that bufalin downregulated PI3K expression and inhibited AKT phosphorylation at Ser473, thereby blocking PI3K/AKT pathway activation. RNA-seq analysis indicated that bufalin regulated autophagy and protein phosphorylation pathways, upregulating the autophagy-related gene Beclin-1 and downregulating SQSTM1 expression. In vivo, the high-dose group of bufalin(1. 5 mg· kg-1)significantly inhibited tumor growth(with tumorinhibiting effects comparable to those of 5-Fu), and showed no toxicity to the heart, liver, spleen, lungs, or kidneys of mice(as evidenced by organ coefficients and HE staining). Conclusions Bufalin exerts significant antitumor activity both in vitro and in vivo by inhibiting the phosphorylation of the PI3K/AKT pathway, inducing apoptosis, arresting the cell cycle, and suppressing the migration of hepatocellular carcinoma cells. Additionally, bufalin demonstrates good safety profiles, making it a promising candidate for the treatment of hepatocellular carcinoma.