| Background and Objection:Central nervous system was thought to be an immune privilege organ for a long time, lacking of peripheral immune cells, part of immune related cell factors and plasma proteins. However, in1964Scheinberg and his colleagues confirmed that lymphocyte and microglia infiltrated in and surround graft site, prompting that central nervous system is not an absolute immune privilege organ, and microglia plays a key role in the immune surveillance of central nervous system. But with the development of molecular biology in recent years, it was found that microglia is also closely related with the development of glioma.Glioma is one of malignant tumors in the nervous system, of the highest incidence, the most malignant degree and the worst prognosis. With the development of medical technology, although the diagnosis and treatment of glioma had great progress at present, but the effect is not satisfactory, most patients have an average survival time of approximately1y after diagnosis. As early as1925, Wilder first described microglia in detail in the glioma tissues. Glioma was proved to be a kind of immunogenic tumor, but it is strange that no obvious immune responses were found in the process of glioma development. Immunohistochemical experiments of glioma specimens found a lot of microglia cells and macrophage accumulated in glioma tissues, but also with a small amount of lymphocyte. For a high level of microglial cells infiltrating in glioma tissues, the reasons tend to be chemotaxis molecules and growth factors that glioma cells secrete into surrounding environment in tumor growth process.Due to the inhibitions of cell phagocytosis, antigen-presenting function and release of proinflammatory factors in glioma tissue, microglia can’t launch innate and adaptive immune responses to glioma cells. Studies have shown that microglial cells cannot phagocytose glioma cells or cell debris. Glioma associated microglia/macrophages(GIMs) can still express TLR, and the interactions between TLR and its ligands are still ongoing, but GIMs cannot be activated and the phagocytosis of microglia was not enhanced; In addition, as lack of necessary stimulatory molecule CD80(B7), CD86(B7-2) and CD40, GIMs cannot provide enough stimulation to activate T lymphocytes. Glioma associated microglia after receiving stimulus signals does not increase the expression of MHC-Ⅱ, and MHC that microglia expressed is insufficient to activate T cells, but the microglial expressions of MHC-Ⅱ and B7can be obviously enhanced in the case of glioma cell is absent. Glioma cells can downregulate the microglial ability of secreting proinflammatory factor TNF-alpha induced by LPS. Moreover, glioma cells accelerate interleukin10(IL-10) secretion in activated microglia, IL-10can inhibite the microglial expression of MHC molecules induced by IFN-gamma, as a direct result, microglia is difficult to activate T lymphocytes.Recent studies show that microglia is closely related with the immunosuppression mechanism of glioma microenvironment and promoting glioma proliferation, infiltration. The mechanism that matrix degradation induced by metalloproteinases (MMPs) plays a key role in the process of glioma invasion and migration. MMP-2and MMP-9are thought to be most closely related with tumor invasion and migration, and microglial cells and glioma cells are important sources of MMPs. MMPs are released into tumor microenvironment, participate in glioma extracellular matrix degradation, and ultimately pave the way for glioma cell proliferation, invasion and migration processes. Studies have found that glioma can upregulate the expression of MT1-MMP located on microglia cell membranes, and microglial MT1-MMP overexpression, in turn, promotes the activation of pro-MMP-2secreted by glioma cells, the release of active MMP-2into glioma microenvironment ultimately promotes glioma invasion and migration.Atorvastatin is a3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, has been widely applied to lower blood cholesterol levels as a drug for cardiovascular disease, but recently it was found that atorvastatin has antitumor effect. It was observed that statins can inhibite MMP-2, MMP-9and MT1-MMP expressions in endothelial cells and a wide variety of tumors. Therefore, we investigate whether atorvastatin affects the MMPs expressions of glioma and microglia, and its impact on glioma invasion and migration.Methods1. The CCK-8assay was used to test the effect of atorvastatin on U87glioma cell and microglia cell.2. For the preparetion of microglia conditioned medium(MCM), microglia cell was incubated with glioma conditioned medium(GCM) for24h. Cell migration and invasion assays were performed by using transwell chambers to test the invasion and migration capacities of U87glioma cell incubated in MCM with or without atorvastatin.3. Gelatin zymography assay was used to test MMP-2and MMP-9expressions of microglia cell incubated in GCM with or without atorvastatin. And MMP-2and MMP-9expressions of U87glioma cell incubated with atorvastatin were detected.4. Western Blotting was performed to detect MT1-MMP, P-p38MAPK, P-ERK and P-JNK expressions of U87cell incubated with atorvastatin. And the microglial expressions of MT1-MMP, P-p38MAPK, P-ERK and P-JNK were tested after microglia cell was incubated in GCM with or without atorvastatin.5. Quantitative Real-Time polymerase chain reaction(QPCR) was performed to detect MMP-2, MMP-9and MT1-MMP mRNA levels of U87glioma cell treated with atorvastatin. And the microglial mRNA levels of MMP-2, MMP-9and MT1-MMP were test after microglia cell was incubated in GCM with or without atorvastatin.Results1. Atorvastatin significantly decreased cell viability of U87and microglia at a10-4M concentration (P<0.05). In addition, the cell viability of microglia was significantly enhanced at a concentration of10-10M (P<0.001). Treatment of U87and primary microglia cells with atorvastatin at concentrations of10-9to10-5M or with DMSO for24h did not result in evidence of toxicity (P>0.05)2. Atorvastatin (10-5M) slightly reduced the average cell migration number in the transwell control chambers (at40×magnification) to55.7±3.0compared to63.7±3.3control cells (P<0.05). MCM intervened U87glioma cells for24h, the migration cell number was89.4±3.5, a significant increase compared with the control group, the difference was statistically significant (P<0.001). Atorvastatin MCM intervened U87glioma cells for24h, the migration cell number was56.3±3.2, a significant decrease compared with MCM intervention group, the difference was statistically significant(P<0.001)3. MCM intervened U87glioma cells for24h, the invasion cell number was28.2±2.8, a significant increase compared to the invasion cell number of13.2±1.9in the control group, the difference was statistically significant (P<0.001). Atorvastatin MCM intervened U87glioma cells for24h, the invasion cell number was11.3±1.0, significantly reduced compared with MCM intervention group, the difference was statistically significant (P<0.001).10-5mol/L atorvastatin intervened U87cell for24h, the invasion cell number was10.2±3.1, a slight decrease compared with the control group, the difference was statistically significant (P<0.05).4. Gelatin zymography was used to detect the expression of MMP-2and MMP-9in U87and microglia cell supernatant. GCM intervened microglia for24h, MMP-2expression in microglia cell supernatant was significantly increased compared with the control group, the difference was statistically significant (P<0.001).10-5mol/L atorvastatin+GCM intervened microglia cells for24h, MMP-2expression in microglia cell supernatant was significantly decreased compared with GCM intervention group, the difference was statistically significant (P<0.001).10-5mol/L atorvastatin intervened microglia cells for24h, the MMP-2expression of microglia cell supernatant was significantly reduced compared with the control group, the difference was statistically significant (P<0.001).10-5mol/L atorvastatin intervened microglia cells for24h, MMP-9expression was decreased compared with the control group, the difference was statistically significant (P<0.05). In addition, MMP-2expression was decreased after U87cells was incubated with atorvastatin for24h.5. Microglia and U87cells were collected after incubated with10-5mol/L atorvastatin to test MT1-MMP, P-p38MAPK, P-JNK and P-ERK protein expressions. The results show that the MT1-MMP expression of microglia cells incubated with GCM for24h was significantly higher than the control group. After10-5mol/L atorvastatin+GCM incubated microglia for24h, the microglial MT1-MMP expression was significantly decreased compared with GCM intervention group. The MT1-MMP expression of U87cells incubated with 10-5mol/L atorvastatin for24h was slightly decreased compared to the control group. Microglia cells were incubated with GCM for12h and24h, P-p38expression was significantly higher than in the control group.10-5mol/L atorvastatin+GCM incubated microglia for12h and24h, P-p38expression was significantly decreased compared to GCM intervention group,but P-JNK and P-ERK expressions were not affected. U87cells were treated with10-5mol/L atorvastatin for24h, the P-JNK expression of U87cells was decreased compared to the control group, P-ERK and P-p38expressions were not significantly affected.6. Microglia and U87cells were collected after incubated with10-5mol/L atorvastatin to test MMP-2, MMP-9and MT1-MMP gene expressions. The results show that after microglia cells were incubated with GCM,10-5mol/L atorvastatin+GCM or10-5mol/L atorvastatin alone for24h, there were no significant changes in MMP-2gene expression compared to the control group (F>0.05), and no significant differences among groups(P>0.05), the differences were not statistically significant. Microglial cells were incubated with GCM for24h, MT1-MMP gene expression were significantly increased compared with the control group, and the difference was statistically significant (P<0.001). MT1-MMP gene expression were significantly decreased compared to GCM intervention group after microglia were incubated with10-5mol/L atorvastatin+GCM for24h, the difference was statistically significant (P <0.001). U87cells were treated with10-5mol/L atorvastatin for24h, the MMP-2, MMP-9and MT1-MMP gene expressions of U87cells were significantly reduced compared with that in the control group, the differences were statistically significant (P<0.001).ConclusionOverall, the data show that atorvastatin reduces the pro-tumorigenic effect of microglia on glioma migration and invasion, and that this inhibitory effect is achieved by reducing the expression of MT1-MMP in microglia. They also show that the down-regulation of MT1-MMP in microglia may be controlled by p38MAPK pathway. In addition, this research also proves that atorvastatin has certain direct anti-tumor invasion and migration effect, and this effect may be via the inhibitions of MMP-2, MT1MMP and P-JNK in glioma cells. Taken together, our findings provide evidence that atorvastatin targets microglia and may thus be a candidate for concomitant adjuvant therapies for glioma. |
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