Involvement of reactive oxygen species and caspase-dependent pathway in berberine-induced cell cycle arrest and apoptosis in C6 rat glioma cells.
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.
Chen
Ting-Ching
TC
Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan, ROC.
Lai
Kuang-Chi
KC
Yang
Jai-Sing
JS
Liao
Ching-Lung
CL
Hsia
Te-Chun
TC
Chen
Guang-Wei
GW
Lin
Jen-Jyh
JJ
Lin
Hui-Ju
HJ
Chiu
Tsan-Hung
TH
Tang
Yih-Jing
YJ
Chung
Jing-Gung
JG
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001599:Berberine / Q000494:pharmacology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects*; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects*; D005910:Glioma / Q000378:metabolism / Q000473:pathology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D001599:Berberine; D045304:Cytochromes c; D020169:Caspases; D002118:Calcium