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.