Molecular mechanisms of echinocystic acid-induced apoptosis in HepG2 cells

https://doi.org/10.1016/j.bbrc.2004.07.004Get rights and content

Abstract

Echinocystic acid (EA), a natural triterpone enriched in various herbs, has been showed to have cytotoxic activity in some cancer cells, and is used for medicinal purpose in many Asian countries. In the present study, we found that EA could induce apoptosis in human HepG2 cells, as characterized by DNA fragmentation, activation of caspase-3, -8, and -9, and PARP cleavage. The efficacious induction of apoptosis was observed at 45 μM for 24 h. Molecular data showed that EA induced the truncation of Bid protein and reduction of Bcl-2 protein. EA also caused the loss of mitochondrial membrane potentialΨm) and cytochrome c release from mitochondria to cytosol. Moreover, EA could activate c-Jun NH2-terminal kinase (JNK) and p38 kinase, and JNK-specific inhibitor SP600125 and p38 kinase-specific inhibitor SB200235 could block serial molecular events of EA-induced apoptosis such as Bid truncation, Bcl-2 reduction, cytochrome c release, caspase activation, and DNA fragmentation in HepG2 cells. These findings indicate that JNK- and p38 kinase-mediated mitochondrial pathways might be involved in EA-induced apoptosis and enhance our understanding of the anticancer function of EA in herbal medicine.

Section snippets

Materials and methods

Materials and cell culture. Fetal bovine serum (FBS) was from Equitech-Bio. 3,3′-Dihexyloxacarbocyanine iodide (DiOC6(3)) was from Molecular Probes. The antibodies against poly(ADP)ribose polymerase (PARP), Bid, caspase-3, -8, and -9, JNK, p38, Phos-SAPK/JNK, Phos-p38, Phos-c-Jun, and α-tubulin were from Cell Signaling Technology. Cytochrome c and Bcl-2 antibodies were obtained from Santa Cruz. Caspase-8 (IETD-CHO) and caspase-9 (LEHD-CHO) inhibitors were from Calbiochem. Caspase-3

EA inhibits the proliferation of HepG2 cells through the induction of apoptosis

To test the effect of EA on the proliferation of HepG2 cells, the cells were treated with different concentrations of EA. After 24 h culture, the survival rate of HepG2 cells was investigated by a MTT assay. As shown in Fig. 1, EA inhibited cell proliferation in a dose-dependent manner with an IC50 (50% inhibitory concentration) value of 45.4 μM at 24 h treatment. To elucidate whether EA inhibits the proliferation of HepG2 cells by inducing apoptosis, we confirmed the apoptotic characterizations

Discussion

Apoptosis is a tightly regulated progress under the control of several signaling pathways, such as caspase and mitochondrial pathways [2], [3], [32]. In the present study, EA treatment caused activation of caspase-3, -8, and -9 in a time–response manner that is consistent with the results of PARP inactivation and DNA fragmentation. Moreover, a time-dependent release in mitochondrial cytochrome c and a concomitant increase in cytosolic cytochrome c were also observed in EA-treated HepG2 cells.

Acknowledgment

This work was partly supported by Cooperation of Innovative Technology and Advanced Research in Evolutional Area (CITY AREA).

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