The cytotoxicity of
berberine on C6 rat
glioma cells indicated that
berberine induced morphological changes and caused cell death through G2/M arrest and apoptosis. While undergoing apoptosis, there was a remarkable accumulation of G2/M cells with the upregulatoin of Wee1 but it also inhibited
cyclin B, CDK1 and Cdc25c that led to G2/M arrest. Along with cytotoxicity in C6 cells, several apoptotic events including mitochondrial
cytochrome c release, activation of
caspase-9, -3 and -8 and DNA fragmentation were induced.
Berberine increased the levels of GADD153 and GRP 78 in C6 cells based on the examination of Western blotting and this is a major hallmark of endoplasmic reticulum (ER) stress. We also found that
berberine promoted the production of
reactive oxygen species and Ca2+ in C6 cells. Western blotting assay also showed that
berberine inhibited the levels of
anti-apoptotic protein Bcl-2 but increased the levels of
pro-apoptotic protein Bax before leading to a decrease in the levels of mitochondrial membrane potential (DeltaPsim) followed by
cytochrome c release that caused the activations of capase-9 and -3 for apoptotic occurrence. The
caspase-8, -9 and -3 were activated by
berberine in C6 cells based on the substrate
solution (PhiPhiLux-G1D1, CaspaLux 8-L1D2, CaspaLux 9-M1D2 for
caspase-3, -8 and -9, respectively) and analyzed by flow cytometer and each inhibitor of
caspase-8, -9 and -3 led to increase the percentage of viable C6 cells after exposure to
berberine. This finding was also confirmed by Western blot assay which showed that
berberine promoted the active form of
caspase-8, -9 and -3. These results demonstrate that the cytotoxicity of
berberine in C6 rat
glioma cells is attributable to apoptosis mainly through induced G2/M-arrested cells, in an ER-dependent manner, via a mitochondria-dependent
caspase pathway regulated by Bax and Bcl-2.