Correlation Of Notch And Wnt Signaling Pathways With Proliferation And Treatment Of Medullobastoma

Background and objective:Medulloblastoma (MB) is originated from granule cell-precursor cells in the external germinal layer of the developing cerebellum with lesser-known molecular mechanism(s). As the most common malignant brain tumour, MB accounts for more than 25% of cancer-related death among child patients. Surgery, radiotherapy, systemic chemotherapy or their combination are conventional clinical managements for MBs. Although 70% of MB patients can survive from dieth by the routine treatments, great majority of them suffers from both direct damages and long-term drug side effects . Therefore, more reliable and less toxic therapeutic approach is urgently needed in clinical management of medulloblastomas.Since MBs were derived from neuroectodermal stem cells on their way toward fate specification and maturation, they sustain the unmatured and proliferation status. So MB maintained the potential for further differentiation upon the treatments of differentiation promoters. All-trans retinoic acid (RA) could inhibit growth and promote differentiation of human MB cells without inducing apoptotic cell death, its effect on MB could be reversed. Our previous studies demonstrated that resveratrol, a nontoxic cancer chemopreventive polyphenols compound, which was contained in grapes and many natural foods, exhibited ideal anti-medulloblastoma effects by promoting either differentiation or apoptosis. It was found more recently that resveratrol could suppress c-Myc expression and arrest MB cells at S-phase, indicating that inhibition of c-Myc expression might be a critical molecular event of resveratrol-treated MB cells. However, unlike resveratrol treatment, blockage of c-Myc expression via antisense oligonucleotide transfection directly committed MB cells to apoptosis without inducing differentiation-like phenotypes, suggesting that additional molecular element(s) may involve in the resveratrol-induced differentiation of MB cells.Through recently discoveries in cell molecular biology, it has become increasingly evidenced that normal development and tumorigenesis share some similar properties. Both processes involve in the proliferation, differentiation, motility and death of cells under the guidance of multiple signaling pathways. Genes that involved in normal development processes might, therefore, contribute to tumourigenesis if their expression patterns are changed. Both Notch and Wnt signaling transduction protein families play important roles in the differentiation of primitive neural stem cells during central nervous system developments. They exert effects on target cells through the activation of signaling pathway. The liberation and the translocation to nucleus of notch intracellular domain (NICD) is the key points in Notch signaling pathway. The translocation to nucleus of NICD could initiation the target gene transcription, such as Hes1.Notch signaling plays important roles in regulating cell proliferation, differentiation and survival under normal and pathological conditions, and, alternatively, the anti-cancer effects of resveratrol are largely reflected through the above cellular events. However, the influence of resveratrol in Notch signaling has not been reported either in MBs or in other cancer types. This study thus aimed to check the status of Notch1 and Notch2 expression/activation in medulloblastoma cells, the response of Notch signaling to resveratrol treatment, and the fate of MB cells when Notch signaling was blocked.Many signaling pathways involved in the tumor formation. Similar with the Notch signaling pathway, Wnt signaling was involved in neural stem self-renewal and neural differentiation. In addition to its roles in neurogenesis, Wnt signaling has been shown to contribute to the carcinogenesis of medulloblastoma, as well as in other tumors such as colon carcinomas, breast carcinomas and prostate. its functions rely on the successive molecular events, up-regulation of Wnt signals, and nuclear translocation ofβ-catenin, binding ofβ-catenin/TCF/LEF complex to the special DNA sequence of target genes, relief of the transcription repression and initiation of target gene transcription.Most of Wnt mutations reported inβ-catenin, less common mutations have been described in APC and the axin gene in medulloblastoma. All these mutation could cause aberrant Wnt signaling by blocking the degradation ofβ-catenin. Although most evidence showed that Wnt signaling is concerned with tumorigenesis of medulloblastoma, recently some investigator revealed that Wnt pathway activation were multifarious and tissue specifics. Some researchers have shown that the activation of Wnt signaling predict a favorable prognosis in medulloblastoma.Accumulating evidence showed that overexpressed c-Myc oncoprotein was closely related with MB formation and progression. However, the mechanism leading to elevated c-Myc in MB cells remians unknown. Our recent data showed that resveratrol treatment suppressed c-Myc expression. Since c-Myc is one of the target genes of Wnt signaling, and some of the Wnt family members were expressed in human medulloblastomas, we assumed that resveratrol-induced c-Myc downregulation might be due to the suppression of Wnt signaling. To clarify this issue is the another aim of this study.Materials and methods:Medulloblastoma paraffin embedded speciments and their surrounding noncancerous counterparts were provided by pathology department in First Affiliated Hospital of Dalian Medical University and Shengjing Affiliated Hospital of China Medical University. Human MB cell lines, UW228-2, UW228-3 and Med-3 were established and kindly provided by the Department of Neurological Surgery, University of Washington at Seattle, USA and the Department of Neurological Surgery in Kobe University in Japan. By the methods of paraffin embedded tissue array, immunohisto- chemistry was used to detect the status of Notch and Wnt signaling pathway and their associated genes in MB tissue samples; Total cell numbers and cell viability under different culture conditions were determined by FCM, MTT and trypan blue; by the methods of immunocytochemistry, immunofluorescence, RT-PCR and Western blotting, the status of Notch and Wnt signaling pathway and their associated genes in human MB cell lines(UW228-2, UW228-3 and Med-3)with or without resveratrol were detected to analysis the correlation between Notch and Wnt signaling pathways and proliferation and therapy in medullobastoma. SPSS 12.0 software was adopted to analyze the results.Results:1. The status of Notch and Wnt signaling pathway in Medulloblastoma1) The expressions of Wnt2 ,β-catenin , TCF4 and Hes1 in MB are significantly lower than that in MB surrounding noncancerous counterparts (p<0.05).2) The expressions of Notch1 and Notch2 in MB are lower than that in MB surrounding noncancerous counterparts (p>0.05).3) The expressions of Wnt target genes,cyclin D1, c-Myc,VEGF and survivin in MB are significantly higher than that in MB surrounding noncancerous counterparts (p<0.05).4) The difference of the expressions of Wnt5a between MB and MB surrounding noncancerous counterparts is insignificantly (p>0.05).2. The effects of resveratrol on MB cell lines.1) Resveratrol induced differentiation, growth arrest and apoptosisFlow cytometry evaluation revealed that in accompany with increased severity of apoptosis, resveratrol-treated UW228-2, UW228-3 and Med-3 cells exhibited G1 arrest in time-related fashion.2) Appearance of synaptophisin in resveratrol-treated cellsImmunocytochemical staining and RT-PCR for synaptophisin (SYP) indicated that SYP expression was up-regulated in Med-3 cells and became detectable in UW228-2 and -3 cells after resveratrol treatments.3) Appearance of the serine peptidase inhibitor B6 (SPIB6) in cells without resveratrolPCR using specific primers for SPIB6 generated a 570bp band in UW228-2 and -3 cells, which was diminished in resveratrol-treated ones.3. The status of Notch signaling pathway in resveratrol-treated cells.1) Resveratrol elevated expression and nuclear translocation of Notch1 and Notch2Immunocytochemistry, immunofluorescence, RT-PCR and Western blotting results demonstrated that Notch1 and Notch2 were up-regulated in the UW228-2 and -3 cells accompanied with nuclear translocation. Notch1 and Notch2 remained unchanged in Med-3 cells after resveratrol treatment.2) Nuclear translocation of Hes1 in resveratrol-treated cellsBoth ICC and IF stainings for Hes1 showed that enhanced Hes1 immuno-labeling was observed in cytoplasm and nuclei of resveratrol- treated UW228-2 and -3 cells, especially those exhibiting distinct differentiation. The level and intracellular distribution pattern of Hes1 in Med-3 cells remained unchanged upon resveratrol treatment.4. The correlation between the activation of Notch and the anti- medulloblastoma effect of resveratrol.1) Notch signaling pathway could be blocked byγ-secretase inhibitor.Notch activation was efficiently blocked by 8μM L-685, 458 and 10μM DAPT at the time points of 36h and 48h in terms of reduced cytoplasmic distribution and almost diminished nuclear translocation of Hes1 proteins.2) No effect ofγ-secretase inhibitors on medulloblastoma cells growth.MTT and Typran Blue results showed that both inhibitors neither influenced the normal growth nor apoptosis of the three MB cell lines.3) No effect ofγ-secretase inhibitors on anti-medulloblastoma of resveratrol.Both inhibitors neither influenced the normal growth nor interfered with resveratrol-induced growth arrest, neuronal differentiation and apoptosis of the three MB cell lines.5. The effects of resveratrol on Wnt signaling pathway in MB cell lines.1) Wnt signaling pathway is activated in medulloblastoma cell lines especially after treated by resveratrol.RT-PCR results demonstrated that Wnt2 and TCF4/LEF1 were upregulated in resveratrol-teated UW228-2 and -3 cells; ICC and IF indicated that enhancedβ-catenin staining and TCF4 nuclear translocation in resveratrol-treated cells.2) Appearance of Ngn1 in resveratrol-treated cellsRT-PCR results showed that Ngn1 was up-regulated in the UW228-2 and -3 cells, which is more significantly in UW228-3 cell. Conclusion1. Notch and Wnt signaling pathways were inactivation in 77.8% and 84.2% MB, which suggest that the defected of these two pathways may link with the development of medulloblastomas.2. In accompany with increased severity of apoptosis, resveratrol-treated UW228-2, UW228-3 and Med-3 cells exhibited G1 arrest in time-related fashion. Resveratrol induces or further promotes neuronal differentiation of these three cell lines .3. Notch1, Notch2 and Hes1 were up-regulated in the UW228-2 and -3 cells accompanied with nuclear translocation suggest that resveratol could promote the activation of Notch in MB cells.4. Notch signaling pathway could be blocked byγ-secretase inhibitor. Both inhibitors neither influenced the normal growth nor interfered with resveratrol-induced growth arrest, neuronal differentiation and apoptosis of the three MB cell lines.5. Enhancedβ-catenin staining and TCF4 nuclear translocation in resveratrol-treated cells suggest that resveratrol could promote the activation of Wnt in MB cells.6. The downregulaton of c-Myc may be the cooperation results of many signaling pathway network, which suggest that the effects of Wnt in MB may be cell context-dependent.7. Although the extra finding of SPIB6 is unrelated with the topic of current study, it provides a cue of potential relation of inverse SYP and SPIB6 expressions with neuronal differentiation and apoptosis of MB cells.