ABSTRACT
We have recently reported that Ginsenoside Rh2 (G-Rh2) induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. However, the molecular mechanism of its death-inducing function remains unclear. Here we show that G-Rh2 stimulated the activation of both caspase-8 and caspase-9 simultaneously in HeLa cells. Under G-Rh2 treatment, membrane death receptors Fas and TNFR1 are remarkably upregulated. However, the induced expression of Fas but not TNFR1 was contributed to the apoptosis process. Moreover, significant increases in Fas expression and caspase-8 activity temporally coincided with an increase in p53 expression in p53-non-mutated HeLa and SK-HEP-1 cells upon G-Rh2 treatment. In contrast, Fas expression and caspase-8 activity remained constant with G-Rh2 treatment in p53-mutated SW480 and PC-3 cells. In addition, siRNA-mediated knockdown of p53 diminished G-Rh2-induced Fas expression and caspase-8 activation. These results indicated that G-Rh2-triggered extrinsic apoptosis relies on p53-mediated Fas over-expression. In the intrinsic apoptotic pathway, G-Rh2 induced strong and immediate translocation of cytosolic BAK and BAX to the mitochondria, mitochondrial cytochrome c release, and subsequent caspase-9 activation both in HeLa and in SW480 cells. p53-mediated Fas expression and subsequent downstream caspase-8 activation as well as p53-independent caspase-9 activation all contribute to the activation of the downstream effector caspase-3/-7, leading to tumor cell death. Taken together, we suggest that G-Rh2 induces cancer cell apoptosis in a multi-path manner and is therefore a promising candidate for anti-tumor drug development.
Subject(s)
Humans , Apoptosis , Caspase 3 , Metabolism , Caspase 8 , Metabolism , Caspase 9 , Metabolism , Cell Line, Tumor , Cell Proliferation , Cytochromes c , Metabolism , Enzyme Activation , Ginsenosides , Chemistry , Pharmacology , HeLa Cells , Inhibitory Concentration 50 , Mitochondria , Metabolism , Protein Transport , Receptors, Death Domain , Metabolism , Receptors, Tumor Necrosis Factor, Type I , Metabolism , Signal Transduction , Tumor Suppressor Protein p53 , Metabolism , Up-Regulation , bcl-2 Homologous Antagonist-Killer Protein , Metabolism , bcl-2-Associated X Protein , Metabolism , fas Receptor , MetabolismABSTRACT
A high activity isoflavone-glucosidase, which hydrolysis glycosides, was obtainde using liquid fermentation from Absidia sp. R strain. The isoflavone-glucosidase was purified 11 folds with yielding rate of 10.9% after ammonium sulfate precipitation and DEAE-Cellocuse (DE-52) ion exchange chromatography. SDS-PAGE results showed that the molecular weight is 53kD. And the optimum temperature, the optimum pH, Km and pI of the enzyme are 50 deegrees C, 5.0, 1.3 x 10(-2) mol/L and 3.2, respectively. The isoflavone-glucosidase is also rather stable under 60 degrees C and in pH range from 5.0 to 7.0. The enzyme can be activated by Co2+ and Ca2+, and be inhibited by Ag+ and Cu2+.
Subject(s)
Absidia , Glucosidases , Metabolism , Hydrogen-Ion Concentration , Isoflavones , Metabolism , TemperatureABSTRACT
A high active soybean isoflavone glucoside hydrolase-producing mould strain was isolated from spirit qu. Its optimal hydrolase-producing conditions were as follows: 2.5% wheat bran as carbon source, 1% NaNO3 as nitrogen source, initial pH7. 0, culture medium volume 40mL/250mL, inoculating quantity 8% , culture temperature 30℃, revolutions 160r/min and culture time 84h. The enzyme activity reached 82 U/mL. Cu2+ can inhibit Absidia sp. R strain from producing the hydrolase, the influence of other metal ions was not remarkable on it.