Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.
BACKGROUND
Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong's Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture.


OBJECTIVE
This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells.


METHODS
Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0 µmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot.


RESULTS
Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC₅₀) values were 0.186, 0.158 and 0.123 µmol/ml at 24, 48 and 72 h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 μmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with down-regulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of pro-apoptotic Bax and p53 proteins.


CONCLUSIONS
Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.
The College of Preclinical Sciences, Shaanxi University of Chinese Medicine , Xianyang , P.R. China .
Xu
X
Chao
Xiaojun
X
Zhou
Gang
G
Zheng
Changhu
C
Dong
Wei
W
Zhang
Xiaomei
X
Song
Tianbo
T
Jin
D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D015153:Blotting, Western; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D031048:Cnidium / Q000737:chemistry; D003374:Coumarins / Q000302:isolation & purification / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000302:isolation & purification / Q000494:pharmacology*; D005638:Fruit / Q000737:chemistry; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D056945:Hep G2 Cells; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints / Q000187:drug effects*; D015394:Molecular Structure; D013997:Time Factors
D000972:Antineoplastic Agents, Phytogenic; D051017:Apoptosis Regulatory Proteins; D003374:Coumarins; D004365:Drugs, Chinese Herbal; C046627:osthol