FoxO proteins' nuclear retention and BH3-only protein Bim induction evoke mitochondrial dysfunction-mediated apoptosis in berberine-treated HepG2 cells

Mammalian forkhead-box family members belonging to the 'O' category (FoxO) manipulate a plethora of genes modulating a wide array of cellular functions including cell cycle regulation, apoptosis, DNA damage repair, and energy metabolism. FoxO overexpression and nuclear accumulation have been reported to show correlation with hindered tumor growth in vitro and size in vivo, while FoxO's downregulation via phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway has been linked with tumor promotion. In this study, we have explored for the first time intervention of berberine, a plant-derived isoquinoline alkaloid, with FoxO family proteins in hepatoma cells. We observed that berberine significantly upregulated the mRNA expression of both FoxO1 and FoxO3a. Their phosphorylation-mediated cytoplasmic sequestration followed by degradation was prevented by berberine-induced downmodulation of the PI3K/Akt/mTOR pathway which promoted FoxO nuclear retention. PTEN, a tumor suppressor gene and negative regulator of the PI3K/Akt axis, was upregulated while phosphorylation of its Ser380 residue (possible mechanism of PTEN degradation) was significantly decreased in treated HepG2 cells. Exposure to berberine induced a significant increase in transcriptional activity of FoxO, as shown by GFP reporter assay. FoxO transcription factors effectively heightened BH3-only protein Bim expression, which in turn, being a direct activator of proapoptotic protein Bax, altered Bax/Bcl-2 ratio, culminating into mitochondrial dysfunction, caspases activation, and DNA fragmentation. The pivotal role of Bim in berberine-mediated cytotoxicity was further corroborated by knockdown experiments where Bim-silencing partially restored HepG2 cell viability during berberine exposure. In addition, a correlation between oxidative overload and FoxO's nuclear accumulation via JNK activation was evident as berberine treatment led to a pronounced increase in JNK phosphorylation together with enhanced ROS generation, lipid peroxidation, decreased activities of superoxide dismutase and catalase, and diminished glutathione levels. Thus, our findings suggest that the antiproliferative effect of berberine may in part be due to mitochondria-mediated apoptosis with Bim acting as a pivotal downstream factor of FoxO-induced transcriptional activation.