Identification of metabolic pattern and bioactive form of resveratrol in human medulloblastoma cells
Graphical abstract
trans-Resveratrol but not its cis- and sulfate counterparts exhibits anti-medulloblastoma efficacy (A) and medulloblastoma UW228-3 cells show less metabolic capacity due to the lower brain-associated sulfotransferase/SULT1A1, 1C2 and 4A1 levels than that expressed in the rat normal brain (B) (*) p < 0.001.
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.
Section snippets
Cell culture and treatments
Human medulloblastoma cell line UW228-3 [25] were cultured in Dulbecco's modified Eagles medium (DMEM; Invitrogen Co., Grand Island, NY, USA) containing 10% fetal bovine serum (Invitrogen Co., Grand Island, NY, USA) under 37 °C and 5% CO2 condition. The cells (5 × 104/mL) were plated to 100 mm dishes (Nunc A/S, Roskilde, Denmark) and incubated for 24 h before further experiments. For morphologic evaluation and ICC staining, the coverslips were put into the dishes before initial cell seeding and
Resveratrol-sensitive features of UW228-3 cells
As shown in Fig. 1A, UW228-3 cells were elliptical without synaptophysin expression under normal culture condition; after being treated by 100 μM resveratrol for 48 h, the cells exhibited elongated fibrous phenotype with synaptophysin expression and showed distinct signs of apoptosis. Flow cytometry analyses (Fig. 1B) demonstrated that the G1 and S fractions were 48.50% and 45.97% in normally cultured UW228-3 cells, and changed to 97.62% and 1.26% in the cells treated by 100 μM resveratrol for 48
Discussion
An ideal cancer therapeutic agent should have minimal cytotoxicity to normal tissues, meanwhile, exerts crucial effects on cancer cells. Resveratrol is such candidate, because of its nontoxic property and anticancer activities in a variety of human and rodent cancers [40]. It has been recognized that resveratrol undergoes intracellular enzymatic biotransformation that generates one or more metabolites [36], [41]. However, the pharmaceutical potentials of those metabolic products have not yet
Acknowledgments
We gratefully acknowledge Hui Wang, Jing-Ping Cao and Ying Gao for their assistance with the HPLC and LC/MS detection. We thank Drs. Xin-Feng Zhao and Peng Gao for their assistance with the HRMS analysis and Professor Xiang-Hong Yang for providing human medulloblastoma specimens. This work is supported by the grants from National Natural Science Foundation of China (Nos. 30527002, 30670946 and 30971038) and by the special grants of Liaoning Department of Education for the key laboratory (
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