Capsaicin induced cell cycle arrest and apoptosis in human esophagus epidermoid carcinoma CE 81T/VGH cells through the elevation of intracellular reactive oxygen species and Ca2+ productions and caspase-3 activation

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Abstract

Capsaicin (N-vanillyl-8-methyl-1-nonenamide) is found in pungent fruits, especially in red pepper. Many studies have focused on the anticarcinogenic, antimutagenic or chemopreventive activities of capsaicin. However, the effects of capsaicin on human esophagus epidermoid carcinoma cells have never been investigated. In this study, we investigated the effects of capsaicin on esophagus epidermoid carcinoma cells in vitro and further examined the molecular mechanisms of capsaicin-induced apoptosis in esophagus epidermoid carcinoma cells. Capsaicin decreased the percentage of viable cells of CE 81T/VGH cells, via induction of G0–G1 phase cell cycle arrest and apoptosis. Capsaicin induced G0–G1 phase arrest underwent the promotion of p53 and p21, which is an inhibitor of Cdk2 and cyclin E complex before leading to the inhibitions of both compounds. Capsaicin induced apoptosis in time-dependent manners. Capsaicin-induced apoptosis was in association with the elevation of intracellular reactive oxygen species and Ca2+ productions and BAPTA, an intracellular Ca2+ chelator, which significantly inhibited capsaicin-induced apoptosis. Collectively, these results suggest that the capsaicin-induced apoptosis in the CE 81T/VGH cells may result from the activation of caspase-3 and intracellular Ca2+ release pathway, and it is further suggested that capsaicin has potential as a novel therapeutic agent for the treatment of esophagus epidermoid carcinoma cells.

Introduction

Oesophagus cancer is the ninth most common malignancy in the world and the number of incidences are rising [1], [2]. Two major types of oesophageal cancer are squamous carcinoma and adenocarcinoma. In USA, the most prevalent oesophageal cancer is oesophageal adenocarcinoma and this incidence is increasing [3]. The age of individuals with oesophageal cancer is around 30–70 years old. The highest morality rates are found in China with about 26.5% in males and 19.7% in females [4]. In Taiwan, the crude incidence of oral esophageal cancer is 14.04 per 100,000 persons. Oesophageal cancer is a multifactorial disease; the cause of oesophageal cancer include smoking, alcohol, mycotoxins, human papilloma virus and Barret's oesophagus [5], [6], [7].

Normal cells enter cell cycle, which is divided into G0/G1, S and G2/M phase based on the DNA content. Cell cycle represents a series of tightly integrated events that allow the cell to grow and proliferate even in cell death, and cell cycle serves to protect the cell from genotoxic stress. An essential part of the cell cycle machinery is the cyclins and cyclin-dependent kinases (CDKs) and even some of the inhibitors of cyclins and CDKs are involved in cell cycle. After activated, the CDKs provide a means for the cell to move from one phase of the cell cycle to the next (G1 to S or G2 to M). If compounds affect the cyclin and/or CDKs, it may lead to cell cycle arrest. If a cell continues to cycle with its damaged DNA intact, the apoptotic machinery is triggered and the cell will undergo apoptosis.

It is well known that numerous naturally occurring substances are considered to have potential value as antioxidants and cancer preventive agents or even as a cancer therapy drug such as taxol. The glycopeptides isolated from Chinese medicinal mushrooms exert anti-tumor effects, and combined with other anticancer therapies, it has been reported to be effective [8]. Importantly, most such bioactive substances have been reported to exert their anti-cancer activity by blocking cell cycle progression and triggering tumor cell apoptosis. Therefore, cell cycle arrest and the induction of apoptosis in tumor cells becomes the major indicator of anti-cancer effect [9], [10].

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is the active principle ingredient of the hot chilli pepper capsicum, which contains about 0.1–1.0% of capsaicin [11]. Although capsaicin extracts have been extensively investigated for their effects on genotoxicity and mutagenicity in vitro as well as in vivo, the study results are conflicting as some results showed capsaicin exists in tumor-initiating or -promoting potential [12], [13], [14], whereas in others, it showed that capsaicin has chemo-protective effects [15], [16], [17], [18]. Recently, it was reported that capsaicin effectively inhibited tumor growth and induced apoptosis in vivo using NOD/SCID mice with no toxic effects and it was suggested that capsaicin has potential as a novel therapeutic agent for the treatment of leukemia [19]. However, there is no available information to address the effect of capsaicin on human esophagus carcinoma cells.

In the present study, we show that capsaicin suppressed the growth of human esophagus carcinoma CE 81T/VGH cells, through the induction of G0–G1 cell cycle arrest undergoing the inhibition of cyclin E and the induction apoptosis through the activation of caspase-3.

Section snippets

Chemical and reagents

Capsaicin, propidium iodide (PI), Triton X-100, Tris–HCl, trypan blue, ribonuclease-A and were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Potassium phosphates, dimethyl sulfoxide (DMSO), and TE buffer were purchased from Merck Co. (Darmstadt, Germany). Ham's Dulbecco's modified Eagle's medium, glutamine, fetal bovine serum (FBS), penicillin–streptomycin, and trypsin–EDTA were obtained from Gibco BRL (Grand Island, NY, USA).

Human esophagus epidermoid carcinoma CE 81T/VGH cell line

Human esophagus epidermoid carcinoma CE 81T/VGH cell line

Capsaicin decreased the percentage of viable CE 81T/VGH cells

After CE 81T/VGH cells were treated with or without various concentrations of capsaicin for various time periods, viable cells were examined by flow cytometric methods. The results demonstrated that capsaicin decreased the percentage of viable cells and is of a dose-pendent (Fig. 1A) and time-dependent (Fig. 1B) manner.

Capsaicin induced cell cycle arrest and apoptosis in CE 81T/VGH cells

CE 81T/VGH cells were treated with or without various concentrations of capsaicin for various time periods before cell cycle and apoptosis (sub-G1 group) were examined and

Discussion

Although the anticancer function of capsaicin is still controversial, our results clearly demonstrated that capsaicin induces G0/G1 phase arrest and apoptosis in examined human esophagus carcinoma CE 81T/VGH cells. This is in agreement with other reports that demonstrated that capsaicin induces G0/G1 phase arrest and apoptosis in examined human leukemia cells. Our data also showed capsaicin-induced apoptosis through the release of levels of ΔΨm and caspase-3 activation.

Cell cycle checkpoints

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