Epigallocatechin‐3‐gallate induces mesothelioma cell death via H2O2−dependent T‐type Ca2+ channel opening

Elia Ranzato,Luciano Mutti,Bruno Burlando,Stefano Biffo,Valeria Magnelli,Simona Martinotti,Bruno Murer

doi:10.1111/j.1582-4934.2012.01584.x

Abstract

Malignant mesothelioma (MMe) is a highly aggressive, lethal tumour requiring the development of more effective therapies. The green tea polyphenol epigallocathechin-3-gallate (EGCG) inhibits the growth of many types of cancer cells. We found that EGCG is selectively cytotoxic to MMe cells with respect to normal mesothelial cells. MMe cell viability was inhibited by predominant induction of apoptosis at lower doses and necrosis at higher doses. EGCG elicited H2O2 release in cell cultures, and exogenous catalase (CAT) abrogated EGCG-induced cytotoxicity, apoptosis and necrosis. Confocal imaging of fluo 3-loaded, EGCG-exposed MMe cells showed significant [Ca2+]i rise, prevented by CAT, dithiothreitol or the T-type Ca2+ channel blockers mibefradil and NiCl2. Cell loading with dihydrorhodamine 123 revealed EGCG-induced ROS production, prevented by CAT, mibefradil or the Ca2+ chelator BAPTA-AM. Direct exposure of cells to H2O2 produced similar effects on Ca2+ and ROS, and these effects were prevented by the same inhibitors. Sensitivity of REN cells to EGCG was correlated with higher expression of Cav3.2 T-type Ca2+ channels in these cells, compared to normal mesothelium. Also, Cav3.2 siRNA on MMe cells reduced in vitro EGCG cytotoxicity and abated apoptosis and necrosis. Intriguingly, Cav3.2 expression was observed in malignant pleural mesothelioma biopsies from patients, but not in normal pleura. In conclusion, data showed the expression of T-type Ca2+ channels in MMe tissue and their role in EGCG selective cytotoxicity to MMe cells, suggesting the possible use of these channels as a novel MMe pharmacological target.

Highlights

Malignant mesothelioma (MMe) is a highly aggressive tumour arising from mesothelial cells of the serosal surfaces of body cavities, mainly the pleura
We have previously shown that ascorbate, which is known to produce H2O2 [8], exerts anti-tumour properties on mesothelioma through a stronger oxidative stress in MMe cells compared to normal mesothelial cells [9]
The first step of this research was the evaluation of EGCG cytotoxicity to various MMe cell lines, in comparison to normal mesothelial cells, by using the calcein-AM endpoint

Summary

Introduction

Malignant mesothelioma (MMe) is a highly aggressive tumour arising from mesothelial cells of the serosal surfaces of body cavities, mainly the pleura. The incidence of this tumour is increasing worldwide because of exposure to asbestos, while the prognosis for patients is still poor, with a median survival of about 12 months [1]. In the light of current failures in MMe therapies, we have addressed the possibility of combining anti-cancer active nutrients with chemotherapy drugs, to obtain synergistic treatments along with reduced side effects [7]. We have previously shown that ascorbate, which is known to produce H2O2 [8], exerts anti-tumour properties on mesothelioma through a stronger oxidative stress in MMe cells compared to normal mesothelial cells [9].

Methods

Results

Conclusion