Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3.
OBJECTIVE
To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22.


METHODS
Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry.


RESULTS
MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G(1) phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner.


CONCLUSIONS
ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.
Dai
Zhi-Jun
ZJ
Department of Oncology, the Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China. dzj0911@126.com
Wang
Xi-Jing
XJ
Li
Zong-Fang
ZF
Ji
Zong-Zheng
ZZ
Ren
Hong-Tao
HT
Tang
Wei
W
Liu
Xiao-Xu
XX
Kang
Hua-Feng
HF
Guan
Hai-Tao
HT
Song
Ling-Qin
LQ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D053148:Caspase 3 / Q000187:drug effects / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008930:Mitochondria, Liver / Q000378:metabolism*; D010936:Plant Extracts / Q000494:pharmacology*; D031363:Scutellaria
D010936:Plant Extracts; C524947:Scutellaria barbata extract; D045304:Cytochromes c; D053148:Caspase 3