Metabolic Patterns, Bioactive Form And Major Molecular Target Of Resveratrol In Human Primary Brain Tumors: Relevance With Anticancer Effacacy

Background and objectives:Medulloblastoma (MB) is the commonest primary malignant brain tumor in children, while glioblastoma (GB) is the most common primary brain tumor in adults. The patients with MB and GB have very poor prognosis because of the high recurrent rates due to the difficulty to remove the highly invasive tumor masses radically. A large amount of clinical data showed that MB had the highest mortality rate among childhood malignant tumors although its morbidity rate was lower than leukemia's. It is known that MB is less sensitive to conventional radiotherapy or chemotherapy, and children patient couldn't tolerate stosstherapy with high-dose chemotherapy. Even though some patients survived after multi-treatment, different kinds of sequela such as mental retardation, secondary tumors in his adolescence, ploycystic renal disease, pulmonary fibrosis, developmental malformation could be seen frequently among them. Similarly, patients suffered from glioblastoma multiforme, which is the most malignant tumor in astrocytomas, could only live 12 to 15 months and die of tumor recurrence even though they received adjunctive therapy such as post-operative chemotherapy and radiotherapy. So far, there is still no reliable adjuvant therapeutic means for these two primary brain tumors at home and abroad. Therefore, exploration of more effective and less toxic therapeutic approach for MB and GB is urgently required and is of distinct academic value and clinical significance.Retinoic acid (RA), as a differentiation inducer, has been commonly used clinically to promote differentiation of certain types of leukemia. However, the data from medulloblastoma Med-3 cells suggest that RA by itself is insufficient to induce apoptosis unless it is used in combination with conventional chemotherapeutic drugs such as cisplatin. We recently found that the sensitivity of MB to RA is largely dependent upon the expression of intra-cellular ratinoic acid binding protein-Ⅱ(CRABP-Ⅱ) and CRABP-Ⅱwas down-regulated or silenced in 60% of MB cases due to DNA Methylation, leading to the defection of RA signal transmission from cytoplasm to the nucleus. Similarly, RA by itself neither inhibits growth nor induces apoptosis of glioblastoma cells; it induces GB cell apoptosis only when combined with immunotherapy agents such as INF-y, which undoubtedly increases the side effects of the treatment and additional injury to the patients. Therefore, it is urgent to explore more effective and safer anti-MB and anti-GB drugs and to analyze their metabolic activity forms and their anti-tumor molecule mechanisms for primary malignant brain tumor treatment, and it would be of clinical significance.Resveratrol (Res) is a plant polyphenol secondary metabolite existing in grapes, strawberries, peanuts and many other natural foods, which possesses a wide range of biological activities such as cardiovascular protection, antioxidative activity, anti-inflammatory property, and cancer preventive and therapeutic effects. More importantly, this compound has little cytotoxic effect and is able to penetrate blood-brain barrier, suggesting its potential therapeutic values in the management of brain tumors especially intracranial malignancies. We observed systematically five medulloblastoma cell lines in different differentiation degrees, and the results showed that resveratrol could induce differentiation and apoptosis of MB cells in time-and dose-dependent fashions. We also found that resveratrol could up-regulate CYP1A1 but down-regulate CYP1B1 expression, and change the expression of some cancer-associated genes by altering the activities of Wnt, Notch and Stat3 signal pathways. The above evidence suggests that resveratrol may be an ideal drug for MBs in clinical therapy. Compared to MBs, GBs have different responses to resveratrol. Some cell lines such as U251 are sensitive to resveratrol, while others are not sensitive even tolerated to resveratrol. So that, it would be worthwhile to elucidate the internal molecular mechanism leading to different responses to resveratrol, which would help to identify the drug value of resveratrol for GB treatment, and provide a cue for personalized cancer therapy. Although more and more attention has been paid to resveratrol's chemotherapeutic value, it is still unclear whether resveratrol exerts its anticancer effects directly or through its metabolites. 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 free of metabolism in vivo and to fully transform resveratrol to an identical conjugate in vitro. And this is a neck for resveratrol's rational adminis-tration. As an alternative approach, it would be worthwhile to elucidate the potential therapeutic implications of resveratrol metabolites, So far, the research about identification of resveratol's effective metabolic form in central nervous system's tumour has seldom reported, while studying in this field would be of potential values in exploring tumor-selective and personalized application of resveratrol in cancer prevention and treatment.In view of this, our work was carried out using a pair of ideal and reliable resveratrol-sensitive MB and insensitive GB tumour cells model.1) Identification the metabolites in MB and GB cells by HPLC (high performance liquid chromatography), LC/MS/MS (liquid chromatography tandem with mass spectrum) and HRMS (high resolution mass spectrum);2) Analyse on the metabolic enzyme which responed to resveratrol metabolites by multiple molecular biology skills, to clarify the expression of raleted metabolic enzyme in MB UW229-3 and GB LN-18 cells.3) Comparing the resveratrol's metabolites and the metabolic enzymes between the MB UW228-3 and GB LN-18 cells, outline the metabolites and the metabolic enzymes which related to resveratrol's anticancer activity;4) Evaluate the STAT3 signaling and its related gene expression with/without resveratrol's treatment in UW228-3 and LN-18 cells. The findings would be of translational values in exploring individual cancer prevention and treatment of resveratrol.Materials and methods:Medulloblastoma paraffin embedded speci-mens and their surrounding noncancerous counterparts were provided by the Pathology Department in First Affiliated Hospital of Dalian Medical University and Shengjing Affiliated Hospital of China Medical University. Human MB cell line, UW228-3 was established and kindly provided by the Department of Neurological Surgery, University of Washington at Seattle, USA. GB cell line, LN-18 was provided by Department of Neurological Surgery, University of Lausanne Medical University, Switzerland. Wistar rat was provided by Experimental Animal Centre of Dalian Medical University. Human MB cell line UW228-3 was cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum, then the culture media and cells were collected respectively after with/without 100μM resveratrol treatment; After purification by solid phase extraction (SPE), the resveratrol's metabolites in MB and GB cells were separated and identified by HPLC, LC/MS/MS and HRMS; Based on the resveratrol's metabolites, we presumed the related metabolic enzymes sulfotransferases (SULT). The expression of SULT1A1,1C2 and 4A1 were detected in MB and GB tissues by paraffin embedded tissue array and immunohisto-chemistry (IHC), and were compared with the SULTs in normal rat brain tissues for the impossibility to obtain the normal human brain tissues; The chemosensitivity of UW228-3 and LN-18 cells were detected by flow cytometry and trypan blue counts; The SULTs expression in MB and GB cells with/without resveratrol treatment were evaluated by immuno-cytochemistry (ICC), RT-PCR and Western-blotting (WB), then the STAT3 signaling and its upstream/downstream gene expressions were evaluated by RT-PCR, WB, ICC and immunofluorescence (IF). The experimental data were expressed as mean±S.D. and were analyzed with SPSS 11.5 statistic software. Group differences of PCR and WB grey density and viable cell numbers were analyzed by a one-way ANOVA. Mann-Whitney tests were used to analyze the statuses of SULT1A1,1C2 and 4A1 expression in different histological groups. It was considered statistically significant if the p-value is less than 0.05.Results:1. Resveratrol-sensitive features and metabolic form in MB UW228-3 cells1) Resveratrol promoted neuronal-like differentiation and apoptosis of MB UW228-3 cellsHE and ICC results showed that UW228-3 cells were elliptical without synaptophysin expression under normal culture condition; after being treated by 100μM resveratrol for 48 hours, the cells exhibited elongated fibrous phenotype with synaptophysin expression and showed distinct signs of apoptosis. Flow cytometry analyses 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 with 100μM resveratrol for 48h. The percentages of apoptosis in UW228-3 cells were 3.67% before and 31.56% after 48-hour resveratrol treatment. Accordingly, the 0.25% trypan blue evaluation revealed that after 100μM resveratrol treatment for 0, 12,24,36, and 48h, the percentages of unviable cells were 0.01%,11.2%, 30.89%,35.36%, and 42.86% respectively in the UW228-3.2) Resveratrol monosulfate is the major metabolites in MB UW228-3 cellsResveratrol's metabolites in UW228-3 cells were separated and identified by HPLC and LC/MS/MS using a combination of full and selected ion scanning techniques. The peaks of M1, M2 and M3 from TIC (total ion chromatogram) represented trans-resveratrol, cis-resveratrol and resveratrol monosulfate, respectively. The negative ion mass spectrum of Ml showed a stable molecular ion at m/z 226.9 which generated m/z 184.9 and m/z 142.9 fragment ion; The spectrum of M2 was identical to M1, and the fragment ions showed m/z at 184.8 and 142.9 generated from m/z 226.7; The [M-H]-ion of M3 showed the deprotonated molecule ions of m/z 307.1 and 227.0, and the m/z 227.0 was fragmented to m/z 184.9 and 143.0, respectively. The identities of the compounds were further confirmed by HRMS that gave the [M-H]- molecular ion exact massas 227.0698 (C14H11O3, caculated m/z 227.0708),227.0697 (C14H11O3, caculated m/z 227.0708) and 307.0261 (C14H11SO6, caculated m/z 307.0276), which corresponded to trans-resveratrol, cis-resveratrol and resveratrol monosulfate, respectively3) Down-regulated SULTs in medulloblastoma tissuesImmunohistochemical staining and Western blotting showed the three brain-associated SULTs were constitutively expressed in tumour and normal tissues. The expression difference of SULT 1A1 and 4A1 between MB and noncancerous tissues/rat cerebellum was considered statistically signaifi-cant (P<0.01); the distinct difference of SULT 1C2 only existed between MB tissues and rat cerebellum (P<0.01), but the difference between MB and noncancerous tissues was not significant (P>0.05).4) Resveratrol-enhanced SULTs'expression in MB UW228-3 cellsRT-PCR, Western-blotting and ICC showed in UW228-3 cells, SULT 1C2 was expressed in low and SULT4A1 in extremely low levels, whereas SULT1A1 was expressed in relatively normal level. After resveratrol treatment, the level of SULT1A1 remained almost unchanged, while both SULT4A1 and 1C2 were enhanced, although their levels were still lower than that in the normal cerebella.5) cis-Resveratrol and resveratrol monosulfate were less bioactive in UW228-3 cellsHE and trypan blue counts showed neither cis-resveratrol enriched s.olution nor resveratrol monosulfate mixture exhibited antimedulloblastoma activity in terms of growth inhibition, neuron-like differentiation and apoptosis induction.2. Metabolic form in Resveratrol-insensitive glioblastoma LN-18 cells1) Unlike UW228-3 cells, LN-18 cells showed neither growth arrest nor apoptotic death after resveratrol treatmentHE and TUNEL results showed that LN-18 cells were oval in shape under normal culture condition; 100μM resveratrol treatment for 48 hours didn't cause morphologic change and apoptosis. Flow cytometry analyses demonstrated that the G1 and S fractions were 37.13% and 52.50% in normally cultured LN-18 cells, and changed to 72.85% and 26.72% after 100μM resveratrol treatment for 48h. The 0.25% trypan blue evaluation revealed that the percentages of unviable LN-18 cells were 0.11%,1.20%, 5.63%,8.72% and 10.55% after 100μM resveratrol treatment for 0,12,24, 36, and 48h; accordingly, the percentages of unviable cells in normal culture were 0.09%,0.11%,2.68%,4.66%and 5.69%, respectively.2) Resveratrol monosulfate is the major metabolites in LN-18 cellsResveratrol metabolites in LN-18 cells were identified by HPLC, LC/MS/MS and HRMS. The results showed that the major metabolite is resveratrol monosulfate, and its metabolic pattern was similar to UW228-3 cells.3) Resveratrol up-regulated brain-associated SULTs'expression in GB LN-18 cellsRT-PCR, Western-blotting and ICC showed in LN-18 cells, SULT 1C2 was expressed in low and SULT4A1 in extremely low levels, whereas SULT1A1 was expressed in relatively normal level. After resveratrol treatment, the level of SULT1A1 remained almost unchanged, while both SULT4A1 and 1C2 were enhanced and their overall level was similar than that in the normal cerebella.3. Effects and significance of STAT3 signaling pathway in MB and GB cells1) Resveratrol inhibited STAT3 transcription and activation, and altered the expression of STAT3 upstream/downstream genes, which was accompanied with neuron-oriented differentiationThe effects of resveratrol on STAT3 signaling were elucidated by RT-PCR, Western-blotting and ICC. STAT3 was expressed in normally cultured UW228-3 cells, and was down-regulated after resveratrol treatment; STAT3 was distributed in cytoplasm and nuclei of UW228-3 cells under the normal culture condition, which became apparently reduced in the nuclei after resveratrol treatment; Resveratrol altered STAT3 downstream gene expressions, c-Myc, survivin, Cox-2, and cyclin D1 were expressed in normally cultured UW228-3 cells and down-regulated after resveratrol treatment. However, Bcl-2 transcription and production was enhanced by resveratrol incubation, and resveratrol also promoted STAT3 upstream gene LIF expression and secretion; IF and ICC showed that PIAS3 was constitutively expressed and distributed in the cytoplasm, and apparent nuclear translocation of PIAS3 was observed in resveratrol-treated UW228-3 cells.2) In difference with UW228-3 cells, resveratrol neither inhibited STAT3 transcription and activation nor altered STAT3 upstream/downstream gene expressions in LN-18 cellsThe RT-PCR, Western-blotting and ICC results showed that STAT3 was found in both cytoplasmic space and the nuclei of normally cultured LN-18 cells together with c-Myc, survivin, Cox-2 and cyclin D1 expression; this situation remained unchanged after 48h resveratrol treatment. LIF expression was upregulated by resveratrol, while PIAS3 was constitutively expressed and defined in the cytoplasm irrespective to resveratrol treatment.3) AG490, a selective inhibitor of STAT3 phosphorylation, was used to treat UW228-3 and LN-18 cells. ICC staining demonstrated that STAT3 phosphorylation was inhibited in 60μM AG490-treated cells in terms of the reduction of p-STAT3 nuclear labeling. Meanwhile, the proliferations of UW228-3 and LN-18 cells were apparently suppressed by AG490 and most of the treated cells died of apoptosis after a 48-hour AG490 treatment. RT-PCR and ICC staining revealed that the expression levels of c-Myc, Cox-2, cyclin D1 and survivin were decreased in AG490-treated cells.Conclusions:1. Resveratrol could promote MB UW228-3 cells neuronal-like differentiation, and induce cells apoptotic death; However, LN-18 cells showed neither neuronal-like differentiation nor apoptotic death after resveratrol treatment.2. The resveratrol metabolic pattern in resveratrol-sensitive UW228-3 cells was similar to resveratrol-insensitive LN-18 cells, and its major metabolite was resveratrol monosulfate.3. The resveratrol monosulfate could be detected in UW228-3 cells as early as 10 min of drug treatment, and its intracellular amount increased to the platform level at 12-h time point, which suggesting that the metabolic machinery pre-existed in the tumor cells and its efficiency was enhanced in response to resveratrol treatment.4. SULT 1A1,1C2 and 4A1 were preferably expressed in human and rodent brains, and the overall bevels of three brain-associated SULTs in MB tissues were lower than that in human noncancerous tumour-surrounding cerebella, rat normal cerebella as well as LN-18 cells.5. Resveratrol could up-regulate the three brain-associated SULT 1A1, 1C2 and 4A1 expression in UW228-3 and LN-18 cells, but their overall level in UW228-3 cells was still lower than that in the rat normal cerebella.6. Resveratrol was chemically unstable and formed an isomer cis-resveratrol in natural light, the cis-form reduced the anti-meullo-blastoma efficacy in terms of less differentiation and apoptosis tendencies in UW228-3 cells. 7. Resveratrol monosulfate, a major metabolite in resveratrol-sensitive UW228-3 and resveratrol-insensitive LN-18 cells, exhibited attenuated cell crisis, which might refleect an active extracelluar excretion due to the increased the solubility of resveratrol.8. Resveratrol inhibited STAT3 transcription and activation, and altered STAT3 upstream/downstream gene expressions, thus leading UW228-3 cells to neuron-oriented differentiation. But the situation in UW228-3 cells was different because resveratrol neither inhibited STAT3 transcription and activation nor altered STAT3 upstream/downstream gene expressions.9. AG490 inhibited STAT3 phosphorylation and induced UW228-3 and LN-18 cells to apoptotic death, accompanied with decreased expression levels of c-Myc, Cox-2, cyclin D1 and survivin in both cell lines.10. Taken together, the metabolic efficiency and the respond manner of STAT3 to resveratrol would be the two parameters for evaluating the chemosensitivities of primanry brain cancer cells to resveratrol and, therefore, would be of translational values in selective application and personalized therapy of resveratrol.