Anti‐proliferative effect of rhein, an anthraquinone isolated from Cassia species, on Caco‐2 human adenocarcinoma cells

Gabriella Aviello,Angelo A. Izzo,Ian Rowland,Francesco Capasso,Mark McCann,Francesca Borrelli,Christopher I. Gill,Raffaele Capasso,Angela Maria Acquaviva

doi:10.1111/j.1582-4934.2009.00815.x

Abstract

In recent years, the use of anthraquinone laxatives, in particular senna, has been associated with damage to the intestinal epithelial layer and an increased risk of developing colorectal cancer. In this study, we evaluated the cytotoxicity of rhein, the active metabolite of senna, on human colon adenocarcinoma cells (Caco-2) and its effect on cell proliferation. Cytotoxicity studies were performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and trans-epithelial electrical resistance (TEER) assays whereas 3H-thymidine incorporation and Western blot analysis were used to evaluate the effect of rhein on cell proliferation. Moreover, for genoprotection studies Comet assay and oxidative biomarkers measurement (malondialdehyde and reactive oxygen species) were used. Rhein (0.1–10 μg/ml) had no significant cytotoxic effect on proliferating and differentiated Caco-2 cells. Rhein (0.1 and 1 μg/ml) significantly reduced cell proliferation as well as mitogen-activated protein (MAP) kinase activation; by contrast, at high concentration (10 μg/ml) rhein significantly increased cell proliferation and extracellular-signal-related kinase (ERK) phosphorylation. Moreover, rhein (0.1–10 μg/ml): (i) did not adversely affect the integrity of tight junctions and hence epithelial barrier function; (ii) did not induce DNA damage, rather it was able to reduce H2O2-induced DNA damage and (iii) significantly inhibited the increase in malondialdehyde and reactive oxygen species (ROS) levels induced by H2O2/Fe2+. Rhein was devoid of cytotoxic and genotoxic effects in colon adenocarcinoma cells. Moreover, at concentrations present in the colon after a human therapeutic dosage of senna, rhein inhibited cell proliferation via a mechanism that seems to involve directly the MAP kinase pathway. Finally, rhein prevents the DNA damage probably via an anti-oxidant mechanism.

Highlights

Constipation, a complaint conceptually regarded as disordered movement of stool through the large intestine, afflicts many people in the Western countries [1, 2]
Monoclonal primary antibodies for phosphorylated extracellular-signalrelated kinase ERK1/2 and ERK2 were obtained from Santa Cruz Biotechnology Inc. (CA, USA) while peroxidase-conjugated (HRP) antimouse IgG antibody was obtained from Amersham Biosciences Inc. (GE Healthcare, Milan, Italy)
Deoxycholic acid (DCA, 250 ␮M), a secondary bile acid used as positive control, significantly (P Ͻ 0.001) reduced the cell viability in proliferating Caco-2 cells

Summary

Introduction

Constipation, a complaint conceptually regarded as disordered movement of stool through the large intestine, afflicts many people in the Western countries [1, 2]. Three clinical studies have evaluated the potential side effects of long-term use of anthraquinone laxatives on colonic nerve tissue [7,8,9]. Van Gorkom and colleagues reported three clinical studies showing increased cell proliferation in colonic tissue of patients treated with a single high dose of sennosides [10,11,12]. There is no conclusive clinical evidence that senna is dangerous, there are still doubts and unresolved questions on the safety of this drug and its components On this basis, physicians often underuse anthraquinones laxatives and are more willing to use far more expensive drugs that have a much shorter track record concerning potential long-term consequences to the constipated patient

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Conclusion