Korean J Phys Anthropol.  2013 Sep;26(3):105-114. 10.11637/kjpa.2013.26.3.105.

Gambogic Acid Induced Apoptosis through Activation of Caspase-dependent Pathway in Aortic Smooth Muscle Cells

• Kim DK
• Lee TJ
• Kim EA
• Kang JH
• Kim KG
• Kim JY

• KMID: 1488821
• DOI: http://doi.org/10.11637/kjpa.2013.26.3.105

Abstract

Gambogic acid (GA) has powerful apoptotic actions. The authors investigated whether GA has apoptotic effects on aortic smooth muscle cells, and compared its potency with that of simvastatin. Smooth muscle cells were isolated from the aortas of Sprague-Dawley rats (4-6 week). Cell purities were confirmed by IF staining using alpha-smooth muscle actin antibody. The IC50 values for cell death by GA and simvastatin were determined using a MTT assay, and the apoptotic effects of 1 microM GA or 30 microM simvastatin (concentrations correspond to IC50 values) were determined after 24 h of treatment using live cell images and by FITC annexin-V and propidium iodide double-staining. In addition, western blotting was used to evaluate apoptosis by quantifying reductions in the expression levels of the PARP and procaspase-3 as well as cleavages of PARP and procaspase-3 after treatment with 1 microM GA or 30 microM simvastatin. The IC50 of GA (1 microM) was lower than that of simvastatin (30 microM). Cell numbers were markedly reduced by both drugs in live cell images. GA (1 microM) produced a higher level of apoptosis than 30 microM simvastatin (26.4+/-2.37% vs. 8.3+/-1.54%, respectively; P<0.05, n=3) by FITC annexin-V & PI double-staining. In addition, 1 microM GA reduced the expressions of PARP, procaspase-3, and Mcl-1 in cells, whereas 30 microM simvastatin did not. Pretreatment with z-VAD-fmk attenuated GA-induced apoptosis and the cleavages of PARP and procaspase-3. The decreased level of Mcl-1 protein induced by GA treatment was recovered by z-VAD-fmk. These results indicate that GA-induced apoptosis was mediated by a caspase-dependent pathway.

Keyword

Gambogic acid; Apoptosis; Smooth muscle cells; Simvastatin

MeSH Terms

Actins
Amino Acid Chloromethyl Ketones
Aorta
Apoptosis
Blotting, Western
Caspase 3
Cell Count
Cell Death
Fluorescein-5-isothiocyanate
Inhibitory Concentration 50
Muscle, Smooth
Muscles
Myocytes, Smooth Muscle
Propidium
Rats, Sprague-Dawley
Simvastatin
Xanthones
Actins
Amino Acid Chloromethyl Ketones
Caspase 3
Fluorescein-5-isothiocyanate
Propidium
Simvastatin
Xanthones