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

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 (3)H-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 microg/ml) had no significant cytotoxic effect on proliferating and differentiated Caco-2 cells. Rhein (0.1 and 1 microg/ml) significantly reduced cell proliferation as well as mitogen-activated protein (MAP) kinase activation; by contrast, at high concentration (10 microg/ml) rhein significantly increased cell proliferation and extracellular-signal-related kinase (ERK) phosphorylation. Moreover, rhein (0.1-10 microg/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 H(2)O(2)-induced DNA damage and (iii) significantly inhibited the increase in malondialdehyde and reactive oxygen species (ROS) levels induced by H(2)O(2)/Fe(2+). 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.

Fig 1

Trans-epithelial electrical resistance (TEER) of polarized Caco-2 monolayer exposed to rhein (0.1–10 μg/ml) and deoxycholic acid (DCA, 250 μM) for 24 and 48 hrs (A). Effect of rhein (0.1–10 μg/ml) and deoxycholic acid (DCA, 250 μM) on the colonic monolayer permeability after 24 and 48 hrs exposure (B). ***P < 0.001 versus control (Ctrl), n= 3, mean ± S.E.M.

Fig 2

Effect of rhein (0.1–10 μg/ml) on Caco-2 cell proliferation after 24 hrs of incubation. ***P < 0.001 versus control (Ctrl), n= 3, mean ± S.E.M.

Fig 3

pERK₁ (A) and pERK₂ (B) expression (MAP-kinase activation) in Caco-2 cells after 24 hrs of rhein (0.1–10 μg/ml) incubation. Insert: representative Western blot analysis. ***P < 0.001 versus control (Ctrl), n= 3, mean ± S.E.M.

Fig 4

Effect of rhein (0.1–10 μg/ml) on Caco-2 cells exposed (▴) or not (▪) to 75 μM hydrogen peroxide (H₂O₂). Insert: representative comet images of a control cell (a), a severally damaged cell (b) and a partially genoprotected cell (c). ^#P < 0.001 versus untreated cells; *P < 0.05 versus H₂O₂ alone, n= 4, mean ± S.E.M.

Fig 5

Effect of rhein (0.1–10 μg/ml) on Fenton’s reagent (H₂O₂/Fe²⁺ 1 mM)-induced malondialdehyde (MDA) production after 24 hrs exposure in 7 days differentiated Caco-2 cells. ^#P < 0.01 versus control (Ctrl) and ***P < 0.001 versus H₂O₂/Fe²⁺ alone, n= 3, mean ± S.E.M.

Fig 6

Effect of rhein (0.1–10 μg/ml) on Fenton’s reagent (H₂O₂/Fe²⁺ 2 mM)-induced reactive species reagents production after 24 hrs exposure in differentiated Caco-2 cells. ^#P < 0.001 versus control (Ctrl) and *P < 0.05 versus H₂O₂/Fe²⁺ alone, n= 3, mean ± S.E.M.