Elsevier

Life Sciences

Volume 71, Issue 16, 6 September 2002, Pages 1879-1892
Life Sciences

The antiproliferative activity of aloe-emodin is through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines

https://doi.org/10.1016/S0024-3205(02)01900-8Get rights and content

Abstract

The aim of this study is to investigate the anticancer effect of aloe-emodin in two human liver cancer cell lines, Hep G2 and Hep 3B. We observed that aloe-emodin inhibited cell proliferation and induced apoptosis in both examined cell lines, but with different the antiproliferative mechanisms. In Hep G2 cells, aloe-emodin induced p53 expression and was accompanied by induction of p21 expression that was associated with a cell cycle arrest in G1 phase. In addition, aloe-emodin had a marked increase in Fas/APO1 receptor and Bax expression. In contrast, with p53-deficient Hep 3B cells, the inhibition of cell proliferation of aloe-emodin was mediated through a p21-dependent manner that did not cause cell cycle arrest or increase the level of Fas/APO1 receptor, but rather promoted aloe-emodin induced apoptosis by enhancing expression of Bax. These findings suggest that aloe-emodin may be useful in liver cancer prevention.

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 [3], [4]. Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-9,10-anthracenedione) is an anthraquinone compound that is present in some traditional medicinal plants such as Rhei Rhizoma. Many studies have indicated that aloe-emodin has laxative, antifungal, anitibacterial, antiviral, and hepatoprotective effects [5], [6], [7], [8], [9]. Recent study has indicated that aloe-emodin has a specific in vitro and in vivo antineuroectodermal tumor activity. The anticancer mechanism consists of the induction of apoptosis, and the selectivity against neuroectodermal tumor cells is found on a specific energy-dependent pathway of drug incorporation [10]. However, the underlying mechanism of action of aloe-emodin has remained largely unknown.

Fas/APO1, a type I membrane protein in the tumor necrosis factor (TNF) receptor family of cell surface protein, is the major cell surface molecular protein that mediates external ligand-stimulated apoptosis [11], [12], and is ubiquitously expressed in a variety of tissues, including the liver, thymus, heart, lungs, and ovaries [13], [14]. Many anticancer agents induce apoptosis in hepatoma cells through the Fas-Fas ligand system [15]. In addition, tumor suppressor gene p53 is another important molecule in the process of apoptosis. In fact, a number of studies have raised the possibility that cells lacking p53 activity might be more readily resistant to cancer chemotherapy [16], [17], [18]. Once p53 is activated, the outcome of the cellular response is either cell cycle arrest or apoptosis. In some type of cells, p53 increases the amount of p21/WAF1 protein. p21 is a cell cycle regulator that contributes to the arrest of cell in G1 phase by inhibition of cyclin-cdk complex. In certain cells, the activation of p53 leads to apoptosis. It is mediated by transcriptional transactivation of p53 for many apoptotic genes, including Fas/APO1, Bax, IGF-BP3 and PIG3. Moreover, the level of antiapoptotic factor Bcl-2, is decreased by p53. Therefore, these actions of p53 commit cell to apoptosis. [19].

To further elucidate the antiproliferative activity of aloe-emodin, we examined its effect on cell proliferation, cell cycle kinetics, and apoptosis in two well-characterized human hepatoma cell lines, Hep G2 and Hep 3B. In addition, to establish the anticancer effects of aloe-emodin, we also assayed several molecules, including p53, p21, Bcl-2, Bax, and Fas/APO1, that are strongly associated with the pathway of apoptosis and affect the chemosensitivity of tumor cells to anticancer agents. Our results show that the anticancer activity of aloe-emodin in Hep G2 was through a p53-triggerd apoptotic manner, and in Hep 3B, it was mediated by p21-dependent apoptotic pathway.

Section snippets

Test compound

Aloe-emodin was obtained from Sigma Chemical (USA), dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. Final concentrations of aloe-emodin used for different experiments were prepared by diluting the stock with Dulbecco's modified Eagle's medium (DMEM) (Sigma Chemical). Control cultures received the carrier solvent (0.1% DMSO).

Cell lines and culture

Hep G2 (American Type Culture Collection [ATCC] HB8065) and Hep 3B (ATCC HB 80640) were maintained in DMEM supplemented with 10% FCS (GIBCO BRL), 100 units/ml

Cell proliferation inhibition analysis

Cell proliferation inhibition was determined by XTT assay. As shown in Fig. 1, aloe-emodin treatment induced proliferation inhibition in Hep G2 and Hep 3B. At 48 h, the maximal effect on proliferation inhibition was observed with 20 μg/ml aloe-emodin, which inhibited proliferation in 70.98% of Hep G2 cells, and the IC50 value was 11.77 ± 0.02 μg/ml. However, in Hep 3B, the maximum effect on proliferation inhibition was observed with 20 μg/ml aloe-eomdin, which inhibited proliferation only in

Discussion

Aloe-emodin is one of major compositions of Rhei Rhizoma, used for the treatment of various liver diseases in traditional Chinese medicine. Previous study has reported that aloe-emodin can selectively inhibit human neuroectodermal tumor cell growth and lack of acute or chronic toxicity in animal model [10]. In this paper, we examined the antiproliferative effect in hepatoma cells and did further study of the molecular mechanistic basis of the anticancer properties of aloe-emodin. The results

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