Figure 4.

Baicalein modulated expression of a subset of anti-apoptotic Bcl-2 family proteins and induced cytochrome c release from mitochondria. A, endogenous expression of anti-apoptotic Bcl-2 family proteins in BxPC-3 (B), HPAF-II (H), MIA PaCa-2 (M), Panc-1 (P), Capan-2 (C), and AsPc-1 (A) cells. B, cells were treated with baicalein for 24 h and changes in anti-apoptotic Bcl-2 family protein expression were measured. C, cells were treated with 5 µM (BxPC-3) or 15 µM (MIA PaCa-2) baicalein for the indicated times and the expression of Mcl-1 and Bcl-2 were measured. D, cells were treated with baicalein for 2 h, after which total RNA was isolated and Mcl-1 mRNA was quantified by real time RT-PCR. Values represent the means ± SD for three independent experiments performed in triplicate and are expressed as a fold increase relative to the non-treated cells. *p<0.01. E, cells were pre-treated with the proteasome inhibitor MG132 (0.2 µM) for 1 h, followed by 5 µM (BxPC-3) or 15 µM (MIA PaCa-2) baicalein for another 6 h. Mcl-1 protein expression was measured by Western blot (lower panel) and the signal density measured by Image J software and was expressed as Mcl-1/GAPDH (%) (upper panel). F, cells were treated with the indicated concentrations of baicalein for 12 h, after which mitochondrial and cytosolic fraction were separated as described in materials and methods. Levels of cytochrome c in each subfraction were measured by Western Blot. Cox IV and GAPDH served as loading controls for mitochondrial and cytosolic fraction, respectively.