Major Resveratrol Metabolite And Its Associated Metabolic Enzymes In Human Glioblastomas In Vitro And In Vivo

Objective:1) To study resveratrol sensitivities of human glioblastoma cell lines U251 and LN18;2)To compare the resveratrol metabolites between U251 and LN18 cells and to correlate the metabolic pattern and the metabolic enzyme expression with chemosensitivities;3)To analyse the metabolic enzymes which responded to resveratrol metabolites by multiple molecular biology approaches and to clarify the expression of related metabolic enzyme in the U251 and LN18 cells; 4) To explore whether there was some degree of correlation between the sensitivity of resveratrol in human glioblastomas and expression of metabolic enzymes resveratrol, so as to determine the effect of resveratrol in the different cancers and offer individual treatment.Methods:Human glioblastomas cell lines U251 and LN18 were cultured in DMEM (Dulbecco's Modified Eagle's Medium) culture medium containing 10% fetal bovine serum (FBS). The cells were treated with resveratrol (100μM) for 48 hours, and then the culture media and cells were collected from the cells without and with 100μM resveratrol treatment respectively. The chemosensitivity of U251 and LN18 cells were determined by HE morphological staining. After purification by solid phase extraction (SPE), the resveratrol metabolites in U251 and LN18 cells were separated and identified by HPLC. The SULTs expression in U251 and LN18 cells with/without resveratrol treatment was evaluated by ICC, RT-PCR and Western-blotting. The expression of SULT1A1,1C2 and 4A1 were detected in human glioblastomas tissues by tissue microarray-based immunohistochemical staining (IHC), and were compared with the SULTs in tumor-surrounding noncancerous cerebella.Results:1) HE results showed that resveratrol led human glioblastomas U251 cells to growth arrest and apoptosis, while no obvious morphological change was observed on LN18 cells; 2) Resveratrol metabolite(s) in U251 and LN18 cells were separated and identified by HPLC and resveratrol monosulfate is the major metabolites in LN18 cells, but no metabolite was found in the samples of U251 cells; 3) RT-PCR, Western-blotting and ICC analyses showed that the expression levels of SULT1A1,1C2 and 4A1 were up-regulated in resveratrol-treated U251 and LN18 cells,and more distinct up-regulation of 1C2 and 4A1 was found in resveratrol-insensitive LN18 cells; 4) Tissue microarray based immunohistochemical staining showed that the expression of SULT1A1,1C2 and 4A1 were found in most of glioblastomas with the rate of 70.27%,81.08% and 86.49% respectively,and were found in all of tumor-surrounding noncancerous brain tissues.Conclusions:1) Human glioblastomas U251 cells were sensitive to resveratrol, while LN18 cells were resistant to it; 2) The resveratrol metabolic may not occur in resveratrol-sensitive U251 cells,but it was happened in resveratrol-insensitive LN18 cells;3) More distinct up-regulation of SULTs was found in resveratrol-insensitive LN18 cells; 4) the overall levels of the brain-associated SULTs in human glioblastomas tissues were lower than that in human noncancerous tumour-surrounding cerebella.therefore,we deduce that the metabolic efficiency would be the main parameters for evaluating the chemosensitives of Human glioblastomas cells to resveratrol. The reason(s) of undetectable resveratrol metabolite in SULT-positive U251 cells remains to be addressed.