Cancer Letters

Cancer Letters

Volume 291, Issue 1, 1 May 2010, Pages 46-58
Cancer Letters

Aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway

https://doi.org/10.1016/j.canlet.2009.09.016Get rights and content

Abstract

Aloe-emodin (AE), a natural, biologically active compound from the rhizome of Rheum palmatum, has been shown to induce apoptosis in several cancer cell lines in vitro. However, its molecular mechanism of action in the apoptosis induction of human nasopharyngeal carcinoma (NPC) cells has not been explored. This study shows that AE induced G2/M phase arrest by increasing levels of cyclin B1 bound to Cdc2, and also caused an increase in apoptosis of NPC cells, which was characterized by morphological changes, nuclear condensation, DNA fragmentation, caspase-3 activation, cleavage of poly (ADP-ribose) polymerase (PARP) and increased sub-G1 population. Treatment of NPC cells with AE also resulted in a decrease in Bcl-XL and an increase in Bax expression. Ectopic expression of Bcl-XL but not Bcl-2 or small interfering RNA (siRNA)-mediated attenuation of Bax suppressed AE-induced apoptotic cell death. AE-induced loss of mitochondrial membrane potential (MMP) and increase in cellular Ca++ content, reactive oxygen species (ROS) and apoptotic cell death were suppressed by the treatment of cyclosporin A (CsA) or caspase-8 inhibitor Z-IETD-FMK. Co-treatment with caspase-9 inhibitor Z-LEHD-FMK could inhibit AE-induced cell death and the activation of caspase-3 and -9. In addition, suppression of caspase-8 with the specific inhibitor Z-IETD-FMK inhibited AE-induced the activation of Bax, the cleavage of Bid, the translocation of tBid to the mitochondria and the release of cytochrome c, apoptosis-inducing factor (AIF) and Endo G from the mitochondria and subsequent apoptosis. Taken together, these results indicate that the caspase-8-mediated activation of the mitochondrial death pathway plays a critical role in AE-induced apoptosis of NPC cells.

Introduction

Nasopharyngeal carcinoma (NPC) is a poorly differentiated carcinoma that arise from the epithelium of the nasopharynx [1]. It occurs with high frequency in Southeast Asian populations, especially among Chinese people. The incidence of NPC among people in Taiwan is about 8.6 for men and 4.35 for women per 100,000 in the year 2000 [2]. The average age was 10 years younger in NPC patients compared to those who with other head and neck cancers. Clinically, this cancer exhibits a high incidence of lymph node spread as well as distant metastasis that contribute to its poor prognosis [3].

Apoptosis is a physiological mechanism for eliminating malignant cells or cancer cells without eliciting damage to normal cells or surrounding tissues. Thus, induction of apoptosis in target cells is a key mechanism by which anti-cancer therapy works [4]. It is now recognized that mitochondria play a crucial role in regulating cell death, which seems to be the main target for apoptosis induction in response to a variety of stress stimuli such as growth factor withdrawal, γ-irradiation and chemotherapeutic drugs. The disruption of the mitochondrial membrane integrity results in the release of cytochrome c, second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO), apoptosis-inducing factor (AIF) and endonuclease G (Endo G) from mitochondria to the cytosol [5]. Cytosolic cytochrome c can trigger the processing of pro-caspase-9 initiating an apoptosome formation composed of Apaf-1, dATP, caspase-9 and cytochrome c[6]. Apoptosome formation leads to the activation of executioner caspase-3, -6 and -7 [7]. Nuclear translocation of AIF and Endo G occurs and can induce DNA fragmentation and apoptotic cell death in a caspase-independent manner [8]. However, it was shown that the release of Endo G and AIF from the mitochondria in response to pro-apoptotic stimuli occurs in a caspase-dependent manner [9]. Members of the Bcl-2 family such as Bcl-2/Bcl-XL and Bax/Bak can both negatively and positively regulate mitochondrial events of apoptotic cell death through suppression of cytochrome c release [10]. Several studies have shown that chemotherapeutic agent-induced apoptosis involves the cleavage of cytosolic Bid to truncated Bid (tBid) by caspase-8 [11], [12], [13]. tBid could induce pro-apoptotic protein Bax oligomerization and the interaction of mitochondrial permeability transition pore leading to the loss of mitochondrial membrane potential (MMP) and to cytochrome c release [14].

Aloe-emodin (AE) (1,8-dihydroxy-3-(hydroxymethyl) anthraquinone), isolated from the rhizomes of Rheum palmatum, has been shown to inhibit the growth of cancer cells in vitro[15], [16], [17]. Animal studies show that AE pre-treatment significantly reduced acute hepatocellular injury of rat induced by carbon tetrachloride [18]. In addition, it has also been shown to induce apoptosis in several cancer cell lines such as human lung squamous carcinoma CH 27 cell line, lung nonsmall carcinoma H460 cell line and hepatoma HepG2 cell line [15], [19], [20]. These results suggest that AE should have a great potential serving as a cancer chemopreventative agent. Although the mechanisms underlying AE-induced cancer apoptosis have been studied, however, to our knowledge, the involvement of caspase-8 mediated activation of the mitochondrial death pathway in AE-induced apoptosis of NPC cells has not been identified. In this study, we investigated the molecular mechanism by which AE induces apoptotic effects in NPC cell lines and how these effects were modulated by caspase-8 and mitochondria.

Section snippets

Chemicals and reagents

AE, cyclosporin A (CsA), dantrolene dimethyl sulfoxide (DMSO), NP-40, paraformaldehyde, propidium iodide (PI), Tris–HCl, 4′-6-diamidino-2-phenylindole (DAPI) and Triton X-100 were obtained from Sigma–Aldrich (St. Louis, MO, USA). AE was dissolved in and diluted with DMSO, and stored at −20 °C as 100 mM stocks. Potassium phosphate was purchased from Merck (Darmstadt, Germany). DMEM, MEM, FBS, trypsin–EDTA and glutamine were obtained from Gibco BRL (Grand Island, NY, USA). Caspase-3 activity assay

AE induces apoptosis of NPC cells via caspase-3 activation

The first study was conducted to determine whether AE exhibited cytotoxicity on human head and neck cancer cells as well as normal cells. The quantitation of the effect of AE on cell viability was determined by PI staining and flow cytometric analysis. As shown in Fig. 1A, panel I, AE significantly reduced cell viability in a dose- and time-dependent fashion in NPC cells, but did not affect the viability of other human head and neck cancer cells including RPMI 2650, Detroit 562, SCC-4, CE

Discussion

Radiation therapy and chemotherapy are widely used for the treatment of cancer. However, the problems for both types of treatments are not selective in their action against rapidly growing cells, so that can also damage normal cells in patients. Many natural compounds found in dietary plants, such as extracts of herbs and fruit extracts, have been shown to inhibit carcinogenesis in vitro and in vivo[31]. The results seem to suggest that natural products have potential benefits in treating

Conflict of interest

We (the authors) disclose that there is no financial and personal relationships with other people or organizations that could inappropriately influence (bias) our work ‘‘Aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway’’.

Acknowledgements

This work was supported by the Grant from Central Taiwan University of Science and Technology (CTU98-LS-001). We would like to thank Dr. Wu-Tse Liu for critical reading of the manuscript.

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