| The theory of"cancer stem cells (CSCs)"was proposed more than 150 years ago. However, the studies on CSCs once developed rather slowly due to the lack of effective isolated methods. Fortunately, in the past decades, due to the rapid development in the research field of stem cells, CSCs have been the focal spot of the research field. Recently, CSCs have been successfully isolated and identified from acute myeloid leukemia (AML) (1) and various solid malignancies of the breast, brain, and prostate (2-4), etc. CSCs have been considered as the origin of tumorgenesis, tumor metastasis and recurrence.Recently, studies on CSCs indicated that CD133+ tumor cells produced more vascular endothelial growth factor (VEGF) than CD133- subpopulations, and they also induced the migration and tube formation of endothial cells. The secretion of VEGF could be further up-regulated by hypoxia (5). Ours previous study showed that glioma stem cells (GSCs) expressed higher CXCR4 and its activation promoted the secretion of VEGF and interleukin-8 (IL-8), hinting that GSCs possessed stronger ability to promote angiogenesis (6). In addition, GSCs exhibited more powerful invasive capacity. Yuan et al. found that there were more GSCs (both CD133+ and nestin+ cells) in the marginal zone and the junctional zone of the GSCs xenogafts than that in the center zone (7). Lee et al. also observed that there were a number of nestin+/Sox2+ cells in the normal brain tissue of tumor border, indicating that GSCs were more invasive than differentiated cells (8). All these findings demonstrated that GSCs played important roles in the angiogenesis and invasion of glioma.Our previous study found GSCs expressed more functional CXCR4. Because CXCR4 plays a key role in the proliferation, survival, apoptosis, migration, invasion and angiogenesis, we suppose that CXCR4 is involved in the process of the angiogenesis and invasion of GSCs. CXCR4 is a number of G-protein-coupled cell suface receptors that have 7 transmembrane domain. CXCR4 can be activited by its ligand, CXCL12, thus induces the activation of the singnaling of PI3K-Akt, Ras-Raf-MEK-ERK, PLCγ-DAG-PKC, PLCγ-IP3-Ca2+. Previous studies indicated that the singnaling of PI3K/Akt and Ras-Raf-MEK-ERK were participated in angiogenesis, especially in the secretion of VEGF, cells migration and cells invasion. The activation of CXCL12/CXCR4 axis was involved in the biological function of normal and maligant cells through different singnaling, which was cell type dependent. In breast cells, the activation of CXCL12/CXCR4 axis promoted VEGF secretion which was involved in angiogenesis through the PI3K/Akt singnaling (9). However, the activation of CXCL12/CXCR4 axis promoted diverse angiogensis factors respectively through the singnaling of PI3K/Akt and Ras-Raf-MEK-ERK in the different cell line of prostate cancer (10). At the same time, some cells promoted the migration and invasion through the activation of one of the singnaling of PI3K/Akt and Ras-Raf-MEK-ERK (11-14), but other cells involved in this process through both of them (15). Accordingly, it is significant to study which kind of singnaling is involved in GSCs-induced invasion and angiogenesis after CXCR4 activation.In the present study, GSCs sphere from U87 cell line was isolated with the conditional medium as previously described (16). We detected the expression of neural stem cells markers with immuofluorescence. We then tested the activation of CXCL12/CXCR4 axis in GSCs with Western-Blot, measured the secretion of VEGF in different conditions with ELISA and assessed the ability of the migration and invasion of GSCs in inverse condition with Transwell cell body. The main reaults and conclusions are as follows:1. CXCL12/CXCR4 axis activated the PI3K/Akt and MAPK/ERK singaling pathway in GSCs. We found that the immunofluorescene results showed that the spheres of glioma cells which were harvested from U87 cell line with the conditional medium expressed the neural stem cell markers CD133 and nestin. CXCL12 increased Akt and ERK1/2 phosphorylation in GSCs that were detectable after 5 min's treatment and the effect of CXCL12 reached a maximum level at 30 min. CXCR4 specific antagonist AMD3100 reduced the level of Akt and ERK1/2 phosphorylation in dose-dependent manner. Neither CXCL12 nor AMD3100 affected the expression of Akt and ERK1/2. The results indicated that the binding of CXCR4 with CXCL12 activated the PI3K/Akt and MAPK/ERK pathway.2. The PI3K/Akt and MAPK/ERK signaling pathway did not affect with each other in GSCs when CXCR4 was activated. LY294002, a PI3K specific inhibitor, blocked the activation of PI3K/Akt singnaling pathway, but it didn't affect the level of p-ERK1/2. In addition, PD98059, a MEK specific inhibitor, inhibited the activation of MAPK/ERK but did not change the level of p-Akt. These results showed that the downstream singnaling of PI3K/Akt and MAPK/ERK activated by CXCR4 were independent.3. The PI3K/Akt singnaling pathway induced the migration and invasion of GSCs. We detected the migration and invasion of the GSCs using the Transwell cells body coated with or without Matrigel. The results showed that CXCL12 induced GSCs migration in a dose-depent manner and the ability of migration was the strongest at 100 ng/ml. The group pretreated with AMD3100, LY294002 strikingly inhibited the migration and invasion of GSCs induced by CXCL12. However, pretreatment with PD98059 have no significant inhibiting influence on GSCs migration and invasion.4. The PI3K/Akt singnaling pathway promoted VEGF secretion in GSCs. ELISA showed that VEGF secretion was significantly inhibited in the groups pretreated with AMD3100, LY294002. In constrast, PD98059 pretreatment did not inhibit CXCR4-mediated VEGF production, indicating that the activation of downstream PI3K/Akt but not MAPK/ERK singnaling pathway was involved in CXCR4-induced VEGF secretion in GSCs.In summary, CXCL12/CXCR4 axis activited the downstream PI3K/Akt and MAPK/ERK singnaling pathway in GSCs. The two pathways did not dependent each other. The activation of CXCL12/CXCR4 axis promoted VEGF secretion and cell migration and invasion through the downstream PI3K/Akt singnaling. |
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