Apoptotic effects of extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines

doi:10.1016/j.canlet.2004.08.012

Cancer Letters

Volume 221, Issue 1, 18 April 2005, Pages 77-89

https://doi.org/10.1016/j.canlet.2004.08.012 Get rights and content

Abstract

The fruiting body of Antrodia camphorata is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. camphorata (EAC) fruiting bodies in two human liver cancer cell lines, Hep G2 and PLC/PRF/5. Treatment with EAC decreased the cell growth of Hep G2 and PLC/PRF/5 cells in a dose dependent manner. In Fas/APO-1 positive-Hep G2 cells, EAC increased the expression level of Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), in a p53-indenpendent manner. In addition, EAC also initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 both in Hep G2 and PLC/PRF/5 cells. Furthermore, EAC also inhibited the cell survival signaling by enhancing the amount of IκBα in cytoplasm and reducing the level and activity of NF-κB in the nucleus, and subsequently attenuated the expression of Bcl-X_L in Hep G2 and PLC/PRF/5 cells. EAC therefore decreased the cell growth and induced apoptosis both in Hep G2 and PLC/PRF/5 cells.

Introduction

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, and is also one of the four most prevalent malignant diseases of adults in China, Taiwan, Korea, and sub-Africa [1], [2]. Several etiologic factors have been classified as high-risk factor in association with HCC, including exposure to aflatoxin B1, and infection with hepatitis B virus and hepatitis C virus [1], [2], [3].

Antrodia camphorata is one of the most important traditional Chinese crude drugs for treating diarrhea, hypertension, and liver cancer. Previous studies have shown that it possesses a wide range of biological functions, such as antioxidative, anti-inflammatory, hepatoprotective, vasorelaxative and anti-hepatitis B virus effects [4], [5], [6], [7], [8]. A recent study reported that the mycelium of A. camphorata showed appreciable cytotoxic activity against tumor cells [9]. In this study, we determined the apoptotic activity of ethylacetate extract from A. camphorata (EAC) in two human liver cancer cell lines, Hep G2 and PLC/PRF/5. Furthermore, to establish the anticancer mechanism of EAC, we assayed the death receptor and mitochondrial apoptotic pathway-related molecules, including Fas/APO-1, Fas ligand, caspase-8, cytochrome c, caspase-9, Bcl-2 family proteins, and NF-κB signaling molecules, which are strongly associated with the signal transduction pathway of apoptosis and affect the chemosensitivity of tumor cells to anticancer agents.

Section snippets

Materials

Fetal calf serum (FCS), Dulbecco's modified Eagle's medium (DMEM), penicillin G, streptomycin and amphotericin B were obtained from GIBCO BRL (Gaithersburg, MD). Dimethyl sulfoxide (DMSO), ribonuclease (RNase), and propidium iodide (PI) were purchased from Sigma Chemical (St Louis, MO). XTT and p53 pan ELISA kits were obtained from Roche Diagnostics GmbH (Germany). Nucleosome ELISA, WAF1 ELISA, Fas Ligand, and Fas/APO-1 ELISA kits were purchased from Calbiochem (Cambridge, MA). Bax, Bak, Bcl-X_L

Cell growth inhibition effect of EAC in Hep G2 and PLC/PRF/5

Cell growth inhibition was determined by XTT assay. As shown in Fig. 1, EAC treatment induced proliferation inhibition in Hep G2 and PLC/PRF/5. At 48 h, the maximal effect on proliferation inhibition was observed with 100 μg/ml EAC, which inhibited cell proliferation in 92.82% of Hep G2 cells, and the IC₅₀ value was 42.57 μg/ml (Fig. 1A). Similarly, the maximum effect on cell proliferation inhibition was observed with 100 μg/ml EAC that inhibited in 89.23% of PLC/PRF/5 cells, with an IC₅₀ value of

Discussion

A. camphorata, a traditional Chinese herb, is locally found in Taiwan. It has been widely used to treat cancer and inflammation. Previous studies revealed that an activated steroid acid of A. camphorata fruiting bodies, zhankuic acid, exhibited a significant cytotoxic effect against P338 murine leukemia [13]. Hseu et al. [14] have reported that aqueous extract of A. camphorata mycelia possess cytotoxic effect against HL-60 leukemia cells. The maleic and succinic acid derivatives of mycelium of

Acknowledgements

This study was supported by a research grant from the National Science Council of Taiwan (NSC92-2321-B-037-001).

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Apoptotic effects of extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines

Abstract

Introduction

Section snippets

Materials

Cell growth inhibition effect of EAC in Hep G2 and PLC/PRF/5

Discussion

Acknowledgements

Adv. Cancer Res.

Life Sci.

Life Sci.

Life Sci.

Hum. Pathol.

Cell

Hepatology

Cell

Blood

Hepatocellular carcinoma: from bedside to proteomics

Proteomics

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Hepatology

Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata

Planta Med.

Antioxidative and hepatoprotective effects of Antrodia camphorata extract

J. Agric. Food Chem.

Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects

Fed. Eur. Microbiol. Soc. Microbiol. Lett.