Elsevier

Biochemical Pharmacology

Volume 70, Issue 2, 15 July 2005, Pages 229-241
Biochemical Pharmacology

Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway

https://doi.org/10.1016/j.bcp.2005.04.026Get rights and content

Abstract

Emodin, a natural anthraquinone derivative isolated from Rheum palmatum L., has been reported to exhibit anti-cancer effect on several human cancers such as liver cancers and lung cancers. However, the molecular mechanisms of emodin-mediated tumor regression have not been fully defined. In this study, we show that treatment with 50 μM emodin resulted in a pronounced release of cytochrome c, activation of caspase-2, -3, and -9, and apoptosis in human lung adenocarcinoma A549 cells. These events were accompanied by the inactivation of ERK and AKT, generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential (Δψm), decrease of mitochondrial Bcl-2, and increase of mitochondrial Bax content. Ectopic expression of Bcl-2, or treatment with aurintricarboxylic acid, furosemide or caspase inhibitors markedly blocked emodin-induced apoptosis. Conversely, pharmacologic ERK and AKT inhibition promoted emodin-induced apoptosis. Furthermore, the free radical scavenger ascorbic acid and N-acetylcysteine attenuated emodin-mediated ROS production, ERK and AKT inactivation, mitochondrial dysfunction, Bcl-2/Bax modulation, and apoptosis. Take together, these findings suggest that in A549 cells, emodin-mediated oxidative injury acts as an early and upstream change in the cell death cascade to antagonize cytoprotective ERK and AKT signaling, triggers mitochondrial dysfunction, Bcl-2 and Bax modulation, mitochondrial cytochrome c release, caspase activation, and consequent leading to apoptosis.

Introduction

Lung cancer is the leading cause of male and female cancer death worldwide. Every year, about 7000 people die of lung carcinoma, and the number is increasing rapidly in Taiwan. Lung cancers are classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma). NSCLC is the more common variant (∼80% of lung cancers) and, unlike SCLC, is less sensitive to chemotherapeutic agents. Survival statistics are dismal with an average 5-year survival of 10–15% [1]. Thus, it is crucially important to develop better therapeutic strategies for the management of NSCLC. It is well known both growth inhibition and apoptosis are the important determinants of the response of cancers to chemotherapeutic agent [2], [3], [4]. Therefore, compounds that induce cell-cycle arrest and apoptosis may provide potent anti-cancer effect for cancer treatment.

Natural herbal medicines such as Rheum palmatum L. (Polygonaceae) is traditionally applied in cancer therapy in Chinese medicine. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active constituent isolated from the root of R. palmatum L. [5]. Pharmacological studies have demonstrated that emodin possesses anti-bacterial [6], anti-inflammatory [7], immunosuppressive [8], vasorelaxant [9], anti-ulcerogenic [10], and anti-cancer effects. Previous studies have demonstrated that emodin inhibits cell growth in several type of tumor cells [3], [11], [12], [13], [14], [15], [16]. Relevant to its anti-proliferative activity, emodin is a potent inhibitor of the protein tyrosine kinase [17], was shown to suppress HER-2/neu tyrosine kinase activity in HER-2/neu-overexpressing human breast and lung cancer cells in vitro and inhibition of malignant transformation and metastasis-associated properties of HER-2/neu-overexpression breast cancer cells in vivo. It has also been reported that emodin can enhance the sensitivity of cancer cells to chemotherapeutic agents [13], [18], [19]. However, the molecular mechanism of emodin-mediated chemotherapeutic effect on cancer cells remains unclear. The clarification of the mode of action of emodin may be important in developing its application. In this study, we found that emodin-induced apoptosis was via a ROS- and Bcl-2/Bax-dependent mitochondrial signaling pathway in human lung adenocarcinoma A549 cells.

Section snippets

Reagents

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), furosemide (Furo), and aurintricarboxylic acid (ATA), 4′,6-diamindino-2-phenylindole (DAPI), ascorbic acid (Asc), and N-acetylcysteine (NAC) were purchased from Sigma, St. Louis, MO. Anti-Bcl-2, anti-Bcl-XL–S, anti-Bad, anti-phospho-ERK, anti-AKT, and anti-Bax, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Bak, anti-Bid, anti-caspase-8, and anti-cytochrome c were purchased from PharMingen (San Diego, CA). Anti-phospho-AKT

Emodin caused growth inhibition and apoptotic cell death

The effect of emodin on cell growth and cell death were examined in three human lung cancer cell lines. As shown in Fig. 1A, treatment of lung cancer cells with low concentration of emodin (10 μM) resulted in growth inhibition but not cell death, approximately 40, 60, and 90% growth inhibition was detected after 72 h treatment of A549, H460, and CH27 cells, respectively. However, emodin-mediated cytotoxicity occurred at a concentration greater than 25 μM. Clonogenic assays were used to confirm the

Discussion

The poor prognostic outcome of lung cancer is due to its resistance to current therapies, maintaining as the leading cause of cancer related death. Successful treatment with chemotherapeutic agents is largely dependent on their ability to trigger cell death in tumor cells; therefore, novel inducers of apoptosis provide a new therapeutic approach for anti-cancer design. Several previous studies demonstrate that certain phytochemicals present in medicinal herbs exerts anti-tumorigenic activity by

Acknowledgments

This work is supported by grants from the Taichung Veterans General Hospital TCVGH-927312D and National Science Council NSC92-2311-B-075A-001, Taiwan, Republic of China.

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