Identification of metabolic pattern and bioactive form of resveratrol in human medulloblastoma cells

Abstract

Cancer preventive reagent trans-resveratrol is intracellularly biotransformed to different metabolites. However, it is still unclear whether trans-resveratrol exerts its biological effects directly or through its metabolite(s). This issue was addressed here by identifying the metabolic pattern and the bioactive form of resveratrol in a resveratrol-sensitive human medulloblastoma cell line, UW228-3. The cell lysates and condition media of UW228-3 cells with or without 100 μM resveratrol treatment were analyzed by HPLC and LC/MS which revealed (1) that resveratrol was chemically unstable and the spontaneous generation of cis-resveratrol reduced resveratrol's anti-medulloblastoma efficacy and (2) that resveratrol monosulfate was the major metabolite of the cells. To identify the bioactive form of resveratrol, a mixture-containing approximately half fraction of resveratrol monosulfate was prepared by incubating trans-resveratrol with freshly prepared rat brain lysates. Medulloblastoma cells treated by 100 μM of this mixture showed attenuated cell crisis. The overall levels of the three brain-associated sulfotransferases (SULT1A1, 1C2 and 4A1) were low in medulloblastoma cells in vivo and in vitro in comparison with that in human noncancerous and rat normal cerebella; resveratrol could more or less up-regulate the production of these enzymes in UW228-3 cells but their overall level was still lower than that in normal cerebellum tissue. Our study thus demonstrated for the first time that trans-resveratrol is the bioactive form in medulloblastoma cells in which the expression of brain-associated SULTs was down-regulated, resulting in the increased intracellular bioavailability and anti-medulloblastoma efficacy of trans-resveratrol.

Introduction

Resveratrol (3,5,4′-trihydroxy-trans-stilbene, Res) is a plant polyphenol existing in grapes and many other natural foods [1], [2], which possesses a wide range of biological activities such as cardiovascular protection [3], [4], antioxidative activity [5], anti-inflammatory property [6], [7], and cancer preventive and therapeutic effects [8], [9]. It was found that resveratrol affected carcinogenic process by inhibiting cancer-associated gene expression [10], [11], altering multiple signaling pathways and/or modulating epigenetic machineries [12], [13]. More importantly, this compound has little cytotoxic effect and is able to penetrate blood–brain barrier [14], suggesting its potential therapeutic values in the management of brain malignancies especially those occur at childhood.

It has been recognized that resveratrol, as a polyphenol compound, can be biotransformed intracellularly by multiple metabolic enzymes. Although the chemotherapeutic value and molecular effects of resveratrol have been demonstrated in many types of cancers [11], [15], [16], [17], [18], [19], it is still unclear whether trans-resveratrol exerts its anticancer effects directly or through its metabolite(s) [20]. So far, no direct evidence has been available concerning the anticancer activity of the parent and conjugated resveratrol because of the difficulty to maintain resveratrol unmetabolized in vivo and to fully transform resveratrol to an identical conjugate in vitro. As an alternative approach, it would be worthwhile to elucidate the molecular bases and potential therapeutic implications of resveratrol metabolism using a reliable resveratrol-sensitive cancer cells.

Medulloblastoma (MB) is originated from primitive neural precursor cells in the external germinal layer of the developing cerebellum and accounts for more than 25% of cancer-related death among child patients [21]. Great majority of MB patients has poor prognosis and suffers from direct surgical damages, long-term side effects and developmental defects [22]. Therefore, a more effective and less toxic therapeutic approach is urgently required for better treatment of MBs. It has been recognized that MBs maintain the potential for further differentiation when exposed to differentiation promoters [23]. According to our results obtained from four human MB cell lines [11], [15], [16], [24], resveratrol possessed anti-medulloblastoma capacity through promoting both neuronal-like differentiation and apoptosis by arresting cell cycle at G1 phase and down-regulating a panel of cancer-associated gene expression in MB cells presumably through altering the activities of several signaling pathways. The resveratrol-sensitive feature of MB cells thus offers us an ideal model to shed light on the metabolic pattern and the bioactive form of resveratrol in cancer cells.