In vitro antioxidant and antiproliferative effects of ellagic acid and its colonic metabolite, urolithins, on human bladder cancer T24 cells
Graphical abstract
Introduction
Phenolic compounds are the secondary metabolite of fruits and vegetables and it is confirmed that their beneficial effects for human health related to antioxidant activity, anti-inflammatory, potential tumorous prevention (Larrosa et al., 2010, Nandi et al., 2007, Rice-Evans et al., 1997). The consumption of vegetables and fruits with high content polyphenols is considered to be the prevention of various forms of tumor.
Intestinal flora plays an essential role in not only deconjugation of glycosyl or glucuronosyl moiety from the phenolic backbone, but also in structural modification, such as decarboxylation, demethylation and dehydroxylation (Gusman et al., 2001). As the hydrolysis product of ellagitannins (ET), Ellagic acid (EA) was further metabolized to urolithins, a family of metabolites of the 6H-dibenzo-[b, d] pyran-6-one structure with different phenolic hydroxylation patterns, in gastrointestinal tract (Mertens-Talcott et al., 2006a, Mertens-Talcott et al., 2006b).
Recently, anti-tumor activity of EA and urolithins has been explored in human or other mammal cells. A recent research has found EA could inhibit the proliferation of Caco-2, MCF-7, Hs 578T and DU 145 cells. There antiproliferative activities were related to the decrease of ATP (Losso et al., 2004). Ellagic acid was confirmed to be effective in decreasing the lipid peroxidation and increasing the GSH. This antioxidant effect is essential for Anticarcinogenic potential of ellagic acid (EA) against N-nitrosodiethylamine-induced lung tumorigenesis in mice (Khanduja et al., 1999). Activation of the cdk inhibitory protein p21 by EA suggests an important role for EA in cell cycle regulation of cancer cells (Narayanan et al., 1999). EA also shows its anti-tumor activity by eliminating the multiple drug resistance (MDR), inducing apoptosis and the expression of caspase-3 (Hayeshi et al., 2007, Larrosa et al., 2006; Mertens-Talcott et al., 2006a, Mertens-Talcott et al., 2006b). Researchers found that urolithins released in the colon could potentially curtail the risk of colon cancer development, by inhibiting cell proliferation and inducing apoptosis (Kumar et al., 2010). Selma showed that urolithin A could inhibit the canonical wnt signal pathway and interfer with β-catenin/TCF-dependent transcription in human colon cancer cell lines HT29 (Selma et al., 2009). In other study, it has been showed that urolithins exhibits inhibiting activity on 22Rv1 prostate cancer cells by interfered with the expression of CYP1B1 protein (Kasimsetty et al., 2009).
Nowadays, finding low toxicity and efficient anticancer compounds from natural drugs is one of the hotspot of medical research. Bladder cancer is the most common malignant tumor of the urinary system. The biological behavior of bladder tumors is so complicated and changeable that it makes the bladder cancer vulnerable to relapse, invasion and metastasis. In addition to surgery, systemic chemotherapy has become the standard treatment of metastatic bladder cancer. Chemotherapy drugs commonly used in the treatment of bladder cancer include doxorubicin, hydroxycamptothecin, mitomycin and gemcitabine. However, due to the drug resistance, the tumor cells showed insensitive to these drugs. Mitogen-activated protein kinase (MAPK) is the transmitter of cell signal conduction from the cell surface to the nucleus. Research has shown that p38, one of four MAPK subfamilies, is mainly involved in malignant invasion and metastasis of bladder neoplasms (Roux and Blenis, 2004). The gene encoding MEK kinase 1 (MEKK1) has important biological functions, such as cell survival and apoptosis, also involved in the movement and migration of bladder cancer. Activated MEKK1 can also activate its downstream pathway, such as p38, JNK, and ERK signal pathway (Yujiri et al., 1998). The p53 gene is one of the most systematic and widely studied tumor suppressor genes. The wild-type p53 involves in cell cycle regulation, DNA repair, apoptosis and angiogenesis. Decreased expression or mutation of p53 gene makes the loss of function, leading to formation of bladder tumor (Roychowdhury et al., 2012).
The aim of present study was to investigate antiproliferative properties of urolithin A, urolithin B, 8-OMe-urolithin A and ellagic acid in T24 human bladder cancer cells. We also assayed the antioxidant ability of these compounds against H2O2 induced intracellular oxidative stress, their effects on caspase-3 activation and mRNA and/or protein of p38-MAPK (mRNA, phosphorylated protein), MEKK1 (mRNA, protein), c-Jun (mRNA, phosphorylated and non-phosphorylated protein), p53 (mRNA), cleaved caspase-3 (protein) and peroxisome proliferator-activated receptors Gamma (PPAR-γ, protein).
Section snippets
Reagents and compounds
Commercial standards of ellagic acid (EA) and chemicals used for syntheses of the urolithin derivatives (2-bromobenzoic, 2-bromo-5-methoxybenzoic and acetic acids, resorcinol, etc.) were obtained from Sigma–Aldrich (St. Louis, MO). Urolithins (Urolithin A, UroA; Urolithin B, UroB; 8-OMe-Urolithin A, 8-OMe-UroA) were synthesized according to methods previously reported (Bialonska et al., 2009) and identified by nuclear magnetic resonance (NMR). The NMR data was showed as follow: UroA 1H NMR: (400
Urolithins and EA could inhibit the proliferation of T24 cells
Antiproliferative activities of urolithin compounds on the growth of T24 cells in vitro were summarized in Fig. 1. When the T24 cells were administrated with different urolithin compounds and EA concentrations in vitro from 1 to 120 μM, all compounds showed consistent concentration-dependent but nonlinear growth inhibition in CCK assay. The IC50 values for T24 cell inhibition were 43.9, 35.2, 46.3 and 33.7 μM for UroA, UroB, 8-MeO-UroA and EA, respectively. In the crystal violet staining test,
Discussion
In recent days, polyphenols in diet, such as pomegranate and raspberry (Jean-Gilles et al., 2011; Mertens-Talcott et al., 2006a, Mertens-Talcott et al., 2006b) has been demonstrated to have potential biological activity. In fact, most of polyphenols have been metabolized to other absorptive compound in gastrointestinal tract before intake by human or other mammals (Selma et al., 2009).
Due to the constituent complexity and chemical instability of hydrolyzable tannins, we focus on urolithins and
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by grants from the Young Scientists Project of Health Department of Hubei Province of China (No. QJX2010-12) for Dr. F Zhu; the National Natural Sciences Foundation of China (No. 30570182); the Science and Technology Department Supported Program of Jiangxi Province of China (No. 2010BSA13500); the Science and Technology Project of Education Department of Jiangxi Province of China (No. GJJ11570); Hubei Provincial Natural Science Foundation for Innovative Research Team
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These authors contributed equally to this work.