IL-6 Augments The Invasiveness Of Human Glioblastoma Multiforme Cells Via Up-regulation Of MMP-2 And Fascin-1

Human glioma is the most common primary tumor of central nervous system. Malignant gliomas account for approximately 70% of all the new cases of malignant primary brain tumors that are diagnosed in adults each year. Although relatively uncommon, malignant gliomas are associated with disproportionately high morbidity and mortality. Despite the great advances made in the treatments including surgical resection, radiotherapy and chemotherapy, the multimodality therapy has achieved only modest gains for malignant gliomas in the last fifty years. Glioblastoma multiforme (GBM) account for approximately 60 to 70% of malignant gliomas, the median survival is only 12 to 15 months for patients with that. The prognosis for recurrent malignant gliomas is even worse, with median survivals of 3 to 9 months.It is generally believed that invasion and angiogenesis both play critical roles in the formation of recurrent tumors. GBM is hard to remove completely because of the cells' infiltration into adjacent normal brain parenchyma. And the residual glioma cells will proliferate and form new tumor masses via induction of angiogenesis in the microenvironment. Antiangiogenic therapies have gained encouraging preliminary survival results, but GBM eventually acquires resistance to these drugs. The major cause appears that cutting off the blood supply with antiangiogenic therapy may inadvertently promote invasive perivascular growth of malignant gliomas. Therefore, it is necessary to further characterize the mechanisms involved in invasion by GBM in order to discover new therapeutic targets.IL-6 (interleukin-6) is a kind of important multifunctional cytokine, contributes to the regulation of hemopoiesis, immunity and inflammatory response, also contributes to regulate physiological function of central nervous and cardiovascular system, and associates with cell survival, proliferation and apoptosis. High levels of circulating IL-6 have been observed in almost every type of tumor investigated (including multiple myeloma, lymphoma, prostate cancer and brain glioma), and are associated with poor prognosis. Recently, some studies have shown that IL-6 directly influences the invasiveness of colorectal, head and neck cancers. In GBM, the autocrine regulation of IL-6 has been recognized since 1990, and its aberrant expression and secretion have been found to be associated with GBM formation, angiogenesis, grade and prognosis. However, the role of IL-6 in the malignant progression of GBM remains unclear.The multifunction of IL-6 is mediated by its receptors. When the complex of IL-6 and gp80 (IL-6R, a chain) binds to gp130 (βchain), gp130-associated janus kinases (JAKs) are activated. JAKs induce STAT3 (Signal Transducers and Activators of Transcription 3) phosphorylation and dimerization to promote its translocation to the nucleus where it regulates STAT3 transcriptional activation. The transcription factor STAT3 which controls the transcription activation of its downstream genes, including cyclinD1, survivin, and VEGF etc., is involved in the survival and differentiation, and pathogenesis of malignancies through in Jak2/STAT3 signaling pathway. Like many other cancers, aberrant STAT3 activation was found in primary malignant gliomas too. The constitutive activation of STAT3 contributes to the apoptosis inhibition and cell proliferation by up-regulation of various oncogenes, which lead to the transformation and progression of glioma. So STAT3 has been regarded as one of transcription factor-like oncogenes. IL-6 can also induce the secretion of MMPs (matrix metalloproteinases) such as MMP-2 and MMP-9 in normal and tumor cells. In melanoma, phosphorylated STAT3 regulates the secretion of MMP-2 by directly binding to its promoter. Thus, we hypothesized that the JAK/STAT3 pathway was involved in the invasiveness of GBM promoted by IL-6.Fascin-1 is a 55 kDa actin bundling protein which increases cell motility in multiple human malignancies. In GBM, fascin-1 expression is correlated with histologic grade, prognosis, motility and invasiveness. In vitro invasiveness of U87 MG and U251 glioma cells was inhibited by fascin-1 siRNA treatment. Whether fascin-1 is involved in the pathway of invasion promoted by IL-6, and whether fascin-1 expression is regulated through the JAK/STAT3 pathway, were both investigated in this study.In the current study, the influence of IL-6 on the invasiveness of human glioma cells and the possible STAT3 pathway involved in the regulation were investigated. It might be helpful to define some possible novel anti-invasion targets. The study was divided into three parts.Part I The Invasiveness Augmented of Human GBM Cell Lines by IL-6ObjectiveTo study the influence of various concentrations of IL-6 on the invasiveness of human glioma cellsMethods1. CCK-8 assay was used to analyze the proliferation promoting effects of various concentrations of IL-6 in U87 MG and U251 cells.2. Wound Healing assay was used to analyze the acceleration of the wound healing in U87 MG, U251 and U343 cells stimulated by various concentrations of IL-6.3. Transwell invasion assay was used to investigate the number difference of U87 MG, U251 or U343 cells invaded across the Matrigel matrix and filter stimulated by various concentrations of IL-6 during the same period.Results1. At an IL-6 dose range of 0 to 100 ng/ml, there were no significant differences between the proliferation rates of cells treated for 12 h or for 24 h in human GBM cell lines U87 MG or U251 (P>0.05)2. Wound healing by U87 MG and U251 glioma cells was accelerated by IL-6 treatment for 9 h and 16 h respectively in a dose-dependent manner (P<0.05) However, the wound healing by U343 cells was not accelerated by IL-6 (P> 0.05)3. Transwell invasion by U87 MG and U251 glioma cells was enhanced by IL-6 treatment for 16 h in a dose-dependent manner (P<0.05). However, the invasion by U343 cells was not enhanced by IL-6 (P> 0.05)Conclusion1. IL-6 accelerates the wound healing by U87 MG and U251 glioma cells in a dose-dependent manner, but does not accelerate that by U343 cells.2. IL-6 enhances the transwell invasion by U87 MG and U251 glioma cells in a dose-dependent manner, but does not enhance that by U343 cells..Part II Activation of STAT3 in Human GBM Cell Lines Required in Up-regulation of MMP-2 by IL-6ObjectiveTo study the activation of JAK/STAT3 signaling pathway by IL-6, the influence of IL-6 on the secretion of MMP-2 in GBM cells, and to investigate the change of the secretion of MMP-2 and invasiveness in GBM cells when blocking the STAT3 pathway with JSI-124.Methods1. After treating U87 MG cells with various concentrations of IL-6 for 9 h, total RNA was extracted and RT-PCR was performed to investigate change of the mRNA levels of MMP-2.2. After treating U87 MG cells with various concentrations of IL-6 for 16 h, the supernatants were collected and Western blotting was used to investigate the change of the production of MMP-2.3.After treating U87 MG cells with various concentrations of IL-6 for 16 h, the supernatants were collected and zymography assay was performed to investigate the change of the activity of MMP-2.4. Western blotting was used to analyze the pSTAT3 (Tyr705, Ser727) levels in U87 MG cells after treatment with various concentrations of IL-6 for 16h.5. Western blotting was used to analyze the pSTAT3 (Tyr705, Ser727) levels in U87 MG cells after treatment with 100 ng/ml IL-6 and various concentrations of JSI-124 for 16h.6. After treating U87 MG cells with 100 ng/ml IL-6 and various concentrations of JSI-124 for 16 h, the supernatants were collected and zymography assay was performed to investigate the change of the activity of MMP-2.7. Transwell invasion assay was used to investigate the number difference of U87 MG cells invaded across the Matrigel matrix and filter after treatment with 100 ng/ml IL-6 and various concentrations of JSI-124 for 16h..8. At 24 h after STST3 siRNA transfection, the whole U87 MG cell extracts were analyzed by Western blotting to examine total STAT3 expression.9. IL-6 were added into the media (final concentration 100 ng/ml) at 24 h after STAT3 siRNA transfection, and more 16 h later Matrigel invasion analysis was performed to investigate the change of the number of invaded U87 MG cells.Results1. The mRNA levels of MMP-2 in U87 MG cells were up-regulated by IL-6 after 9 h treatment in a dose-dependent manner (P<0.05)2. The production of MMP-2 in the supernatant of U87 MG cells were up-regulated by IL-6 after 16 h treatment in a dose-dependent manner (P<0.05)3. The activity of MMP-2 in the supernatant of U87 MG cells were up-regulated by IL-6 after 16 h treatment in a dose-dependent manner (P<0.05)4. The up-regulation of pSTAT3 (Tyr705, Ser727) levels in U87 MG cells by IL-6 after 16 h treatment remained in a dose-dependent manner (P<0.05)5. The up-regulation of pSTAT3 (Tyr705, Ser727) levels in U87 MG cells by 100 ng/ml IL-6 were suppressed by JSI-124 after 16 h concomitant treatment in a dose-dependent manner (P<0.05)6. The production and activity of MMP-2 in the supernatant of U87 MG cells were up-regulated by 100 ng/ml IL-6 was suppressed by JSI-124 after 16 h concomitant treatment in a dose-dependent manner (P<0.05)7. The number of U87 MG cells stimulated by 100 ng/ml IL-6 invaded across the Matrigel matrix and filter was reduced by JSI-124 in a dose-dependent manner after 16 h concomitant treatment (P<0.05)8. The STAT3 siRNA depleted the total STAT3 significantly in U87 MG cells at 24 h after transfection (P<0.05)9. At 24 h after STAT3 siRNA transfection, IL-6 (final concentration 100 ng/ml) treatment for more 16 h, the number of invaded U87 MG cells was reduced (P< 0.05)Conclusion1. IL-6 induces constitutively activation of STAT3 signaling pathway (both Tyr-705-phospho-STAT3 and Ser-727-phospho-STAT3 levels) and up-regulates the expression and secretion levels of MMP-2 in U87 MG cells. They are both in a dose-dependent manner.2. JSI-124 effectively suppresses the levels of phosphorylated-STAT3 (Tyr705, Ser727) in GBM cells up-regulated by IL-6 and the levels of active MMP-2 induced by IL-6 in the cell supernatants. And JSI-124 inhibited the invasion of U87 MG cells promoted by IL-6. They are all in a dose-dependent manner.3. The depletion of the total STAT3 expression in U87 MG cells with STAT3 siRNA can abate the invasiveness promoted by IL-6 too.4. The up-regulation of MMP-2 induced by IL-6 that requires STAT3 activity is involved in the promotion the invasiveness of human GBM cells by IL-6.Part III Up-regulation of Fascin-1 by IL-6 in Human GBM CellsObjectiveTo study the influence of IL-6 on fascin-1 expression levels in U87 MG cells, and to further investigate the change of fascin-1 expression levels in U87 MG cells stimulated by IL-6 when blocking STAT3 signaling pathway by JSI-124.Methods1. Western blotting was used to analyze the fascin-1 levels in U87 MG cells after treatment with various concentrations of IL-6 for 15 min.2. Western blotting was used to analyze the fascin-1 levels in U87 MG cells after treatment with various concentrations of IL-6 for 16 h.3. At 24 h after STAT3 siRNA transfection, IL-6 (final concentration 100 ng/ml) treatment for more 15 min, Western blotting was used to analyze the fascin-1 levels in U87 MG cells.4. Western blotting was used to analyze the fascin-1 levels in U87 MG cells after treatment with 100 ng/ml IL-6 and various concentrations of JSI-124 for 16h.Results1. The fascin-1 levels in U87 MG cells were up-regulated rapidly by IL-6 after 15 min treatment in a dose-dependent manner (P<0.05)2. The expression in U87 MG cells reached the same levels after treatment with different concentrations of IL-6 for 16 h (P>0.05)3. The fascin-1 levels activated by IL-6 (final concentration 100 ng/ml) treatment for 15 min in U87 MG cells transfected with STAT3 siRNA were not suppressed (P>0.05)4. The fascin-1 levels in U87 MG cells activated by IL-6 (100 ng/ml) was not suppressed by JSI-124 (0-100 nM) after 16 h concomitant treatment (P>0.05)Conclusion1. IL-6 up-regulates the fascin-1 levels in GBM cells rapidly in a dose-dependent manner. When the expression of fascin-1 reaches a high level, it does not response to exogenous IL-6 any more.2. The up-regulation of fascin-1 activated by IL-6 is not mediated by the STAT3 pathway.3. Fascin-1 is involved in the invasion promoted by IL-6. In summary, IL-6 not only induces the up-regulation of MMP-2 expression that requires STAT3 activity but also activate the up-regulation of fascin-1 not mediated by the STAT3 pathway to promote the invasiveness of human GBM cells Therefore, anti-IL-6 strategies targeting its downstream pathways and proteins may be worth exploring for the treatment of recurrent GBM.