| BackgroundGliomas are the most frequent primary brain tumors and account for about 70% of all primary brain tumors, of these, glioblastoma is the most malignant histological type with the features of high incidence, recurrence and death rate and low curative rate. Recently, despite the advances in microsurgery, radiotherapy, chemotherapy and immunotherapy, the overall prognosis of glioblastoma patients is still disappointing, with the two-year survival rate of 27%. Infiltration of the tumor cells along the white tracts is the fundamental reason for recurrence of the tumor. Therefore, illuminating the mechanism of invasion and migration will benefit the anti-invasion treatment of glioblastoma and the prognosis of the patients with glioblastoma.Tumor invasion and migration is a complex process, including degradation of the extracellular matrix, attachment and detachment of the cells to(from) the extracellular matrix, the formation of invasion associated structures, deformity of the cytoskeleton and so on. Among these steps, degradation of the extracellular matrix was seen as the speed limiting step of tumor invasion. This process depends on several types of proteolysis enzymes, particularly matrix metalloproteinase (MMPs). MMPs were considered as the main proteolysis enzymes in tumor invasion. MMP2 and MMP9 are the most important members of MMPs family. They could degrade typeⅣcollagen, which constitutes of the main component of extracellular matrix. Matrix metalloproteinase inhibitor (MMPI) could dramatically inhibit the invasion and migration of tumor cells in a 3D in vitro culture system. In clinical practice, MMPI could to some extent inhibit the invasion and migration of several tumor types. However, the overall prognosis of the patients did not increase. These results drive us to reconsider the anti-invasion strategy of malignant tumor. Nowadays, the role of cytoskeleton deformity in tumor cell invasion and migration has drawn great attention. The cytoskeleton of the cell is constituted of microfilaments, microtubes and intermittent filaments. Cell migration could be divided into two types, named mesenchymal or amoeboid movement depending on the differences in migration strategy, the manner of ECM breaking and the interaction between tumor cells and the ECM. Glioblastoma exerts mesenchymal movement to migrate. The microfilaments formed structures involved in mesenchymal movement are filopodia, lamellipodia and stress fibers. Filopodia are tiny finger-like protrusions of the cells which constituted of parallel arranged microfilaments. These structures could sniff the extracellular signals. Lamellipodia are constituted of branched arranged microfilaments, which could supply sufficient supportive force for the formation of cell protrusions. Stress fibers are contractile structures constituted of anti-parallel arranged microfilaments and myosin II. With the help of adhesion structures, stress fibers could supply the strength for cell contraction. In mesenchymal movement, firstly filopodia sniff the extracellular signals, then lamellipodia supply the force for membrane protrusion, and then, with the help of adhesion structures, stress fibers contract to pull the body of the cell to move forward, finally, the caudal end of the cell detaches from the ECM. Repeatedly, the cell fulfills the crawling on the planar substrate. All factors that affect the proteolysis of the ECM and deformity of the cytoskeleton could affect the invasion and migration of the cells.Tumor microenvironment could dramatically affect tumor cell biological properties, leading to genetic instability, resistance to radiotherapy and chemotherapy, as well as changes in tumor cell proliferation, apoptosis and invasion. Cytokines existing in the tumor microenvironment and the low oxygen pressure are important factors that initiate changes in tumor cell biological properties.Interleukin-6(IL-6) is a multifunctional cytokine, which is involved in haematogenesis, immune response and inflammation. Recently, IL-6 was found to be highly expressed in several types of tumor cells including prostate, breast and lung cancers, as well as glioblastoma. IL-6 could regulate the biological properties of the cells in proliferation, apoptosis and migration. In patients with glioblastoma, serum IL-6 level could somewhat indicate the prognosis of the patients, which could be partially explained by the proliferation promotive effect of IL-6. In addition, the tumor incidence rate in IL-6 knockout mice is much lower than their parent control. All these findings indicate the important role of IL-6 in glioma tumorigenesis. Although the effect of IL-6 on human glioblastoma proliferation has been investigated many years ago, its effects on glioblastoma invasion and migration has not yet been elucidated.Hypoxia is a significant feature of tumor mass with the diameter lager than 2mm. Although glioblastoma is one of the most vascularized tumors, the high metabolic rate of the tumor renders the cells in a hypoxia conditions. Necrosis often occurs in the centre of glioblastoma due to extreme hypoxia. Hypoxia could promote the invasion and migration in many tumor cell types, but the mechanism is not very well understood. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine which is upregulated by hypoxia. It takes part in immune response and inflammation of the organism. Recently, MIF was found to promote invasion and migration in a variety of tumors. Whether MIF is involved in hypoxia induced tumor invasion and its mechanism is not clear.This paper includes two parts. We will discuss the effect and mechanism of IL-6 and hypoxia on glioblastoma invasion and migration. The findings may benefit the anti-invasion strategy for glioblastoma.Objective1. To detect the expression of IL-6 in glioma specimens and cell lines2. To detect the effect of IL-6 on invasion and migration in glioblastoma cell lines T98G and U2513. To study the molecular mechanism underlying IL-6 induced glioblastoma invasion and migration4. To study the intracelluar signal transduction for IL-6 induced glioblastoma invasion and migration5. To study the effect of IL-6 on glioma angiogenesis Methods1. The expression of IL-6 in glioma specimens and cell lines Collect the specimens of 3 cases of glioma,1 case of paratumor tissue,1 case of normal brain tissue and 1 case of hypertensive hematoma invaded brain tissue. RT-PCR was performed to detect the gene expression of IL-6 in the spcimens and 3 glioblastoma cell lines including T98G, U87 and U251.2. The effect of IL-6 on invasion and migration in T98G and U251 cell lines①T98G and U251 cells were serum starved for 6h, then the cells were suspendend in serum free DMEM. The density of the cells were diluted to 1×105/ml with various concentration of IL-6.200μl such suspension was added into the uper chamber of the Transwell system. Serum containing DMEM(T98G 20%, U251 10%) was added into the lower chamber for chemotaxis. After incubation for 12h, the uper chambers were picked out and the cells were fixed and stained. The basement membrane was viewed and photographed under a microscope. Five random view fields were chosen to count the invading cells and statistical analysis was done.②T98G and U251 cells were seeded into the six-well plates at the density of 105/well. When the cells grow to 70% confluent, serum starvation was performed for 6h. Three parallel wounding lines were made using the tip of a 10μl pipette. Photoes were taken at the same sites at 0,12 and 24h. The width of the wounding lines were measured and statistical analysis was done.③T98G and U251 cells were serum starved for 6h, then 3×103 cells were seeded into the wells of a 96-well plate and treated with various concentration of IL-6. MTT was done at 12 and 24h after cell seeding to detect the effect of IL-6 on glioblastoma proliferation.3. The molecular mechanism of IL-6 induced glioblastoma invasion and migratioin①T98G and U251 cells were serum starved for 6h, and treated with various concentration of IL-6.24h later, the effect of IL-6 on MMP2 and MMP9 gene expression was measured by RT-PCR.②T98G and U251 cells were serum starved for 6h, and treated with various concentration of IL-6.24h later, the effect of IL-6 on MMP2 and MMP9 protein expression was measured by Western blot.③T98G and U251 cells were serum starved for 6h, and treated with serum free DMEM containing various concentration of IL-6 for 24h. The supernatant was collected and the effect of IL-6 on MMP2 and MMP9 activities was measured by gelatine zymography.④T98G and U251 cells were serum starved for 6h, and treated with various concentration of IL-6.24h later, the effect of IL-6 on Fascin-1 protein expression was measured by Western blot.⑤Coverslips were placed into the wells of a 24-well plate, then 2×104 T98G and U251 cells were seeded into each well and cultured overnight. Fresh complete DMEM culture medium with various concentration of IL-6 were replaced into the wells.24h later, The distribution of Fascin-1 was measured by immunofluorescence staining.⑥Coverslips were placed into the wells of a 24-well plate, then 2×104 T98G and U251 cells were seeded into each well and cultured overnight. Then the cells were cultured in 1% serum containing DMEM for 18h, followed by serum free DMEM for another 6h. Finally, the cells were stimulated with various concentration of IL-6 for 10min. The cytoskeleton of the cells was viewed under fluorescence microscope after rhodamine-phalloidine staining.4. The intracelluar signal transduction mechanism for IL-6 induced glioblastoma invasion and migration①T98G and U251 cells were serum starved for 6h, and treated with various concentration of IL-6 for 20min. The effect of IL-6 on JAK/STAT3, MAPK/ERK and PI3K/AKT signal pathways was detected by Western blot.②T98G and U251 cells were cultured in 1% serum DMEM for 18h, and then in serum free DMEM for 6h. Finally, the cells were treatd with various concentration of IL-6 for 10min. The activities of RhoGTPases members RhoA, Rac1 and Cdc42 was measured by G-LISA.5. The effect of IL-6 on glioblastoma angiogenesis ①U251 cells growing well were changed for serum free RPMI 1640 for 6h, the supernatant was collected.②ECV-304 vascular endothelial cells were suspended in serum free RPMI 1640, U251 supernatant, RPMI 1640+IL-6 and U251 supernatant+IL-6 Ab. The density of the cells was diluted to 1×105/ml.200μl of each suspension was added into the uper chamber of the Transwell system, serum containing RPMI 1640 was added into the lower chamber for chemotaxis.12h later, the uper chambers were picked out and cells fixed. The basement membranes were viewed under a microscope and photoes were taken. The invading cells in five random view fields were counted and statistical analysis was done.③ECV-304 cells were collected and IL-6 and IL-6R subunits gp80 and gp130 gene expression were analyzed by RT-PCR.Results1. The expression profile of IL-6 in glioma specimens and cell lines In 3 cases of glioma and 1 case of paratumor specimens, IL-6 expression was detected. IL-6 gene expression was not detected in normal brain tissue. In the specimen of hypertensive hematoma invaded tissue, IL-6 gene expression was also detected. All of the 3 glioma cell lines tested were positive for IL-6 gene expression.2. The effect of IL-6 on glioblastoma invasion and migration in T98G and U251 cell linesTranswell invasion and migration results revealed that IL-6 could promote T98G and U251 invasion and migration in a dose dependent manner, it is statistical significant at high IL-6 concentration. Results from the wound healing assay revealed that IL-6 could promote the random migration of U251 cell line in a dose dependent manner, but inhibit the random migration of T98G cell line. Results form MTT assay demonstrated that under the conditions employed for invasion and migration assay in this experiment, IL-6 could slightly promote U251 but inhibit T98G proliferation, but it was not statistical significant.3. The molecular mechanism of IL-6 promoting glioblastoma invasion and migration Results from RT-PCR and Western blot revealed that IL-6 could promote the expression of MMP9 in T98G and U251 cell lines but not affect MMP2 expression. Zymography showed high MMP2 activity in T98G and U251 supernatant, but it was not affected by IL-6 stimulation. IL-6 could promote MMP9 activity in U251 supernatant, but MMP9 activity in T98G supernatant was not so obvious.Western blot and immunofluorencence results revealed IL-6 could not only promote the expression of Fascin-1 but also change its distribution style.Cytoskeleton immunofluorencence showed IL-6 could promote lamellipodia formation in T98G and U251 glioblastoma cell lines.4. The intracelluar signal pathways that mediate IL-6 induced glioblastoma invasion and migrationPhosphorylation of JAK/STAT3 at Tyr 705 was not influenced by IL-6 stimulation, while phosphorylation of Ser 727 showed a dose dependent relation with IL-6 stimulation in T98G and U251 cell lines. The basic phosphorylation of AKT was very high and was not affected by IL-6 stimulation in these two cell lines. IL-6 promoted the phosphorylation of p42/44MAPK in U251 cell line but inhibited its phosphorylation in T98G cell line. G-LISA showed that IL-6 could induce Rac1-GTPase activity but not RhoA and Cdc42.5. The effects of IL-6 on glioma angiogenesisTranswell assay revealed that the supernatant from glioblastoma could promote endovascular cell migration in ECV-304 cell line, and the promotive effect could be reversed by IL-6 polyclone antibody. RT-PCR showed IL-6 and IL-6R were expressed by ECV-304 cell line.Conclusion1. IL-6 was expressed in glioma specimens and cell lines2. IL-6 could promote glioblastoma invasion and migration in T98G and U251 cell lines3. IL-6 could promote MMP9 expression in T98G and U251 cell lines4. IL-6 stimulatioin could increase Fascin-1 expression and change its distribution style in T98G and U251 cell lines 5. IL-6 could promote lamellipodia formation in T98G and U251 cell lines6. IL-6 could promote glioma angiogenesis in a paracrine mannerObjective1. To study the effect of hypoxia on glioblastoma invasion and migration2. To study the molecular mechanism of hypoxia induced glioblastoma invasion and migration3. To study the correlation between MIF and hypoxia induced glioblastoma invasion and migration4. To study the expression of key molecules of MIF signal pathway in glioblastomaMethods1. Effect of hypoxia on glioblastoma invasion and migration T98G and U87 cells were resuspended in serum free DMEM and diluted to the density of 1×105/ml.200uμl of such suspension was added into the uper chamber of Transwell system, then serum containing DMEM(T98G 20%, U87 10%) was added into the lower chamber for chemotaxis. The systems were cultured in normoxia incubator, and specific chambers were moved into hypoxia inculbtor (37℃,5% CO2,1% O2) at 4 and 8h. The experiment ended 12h after cell seeding. All chambers were picked out, and cells were fixed and stained. The invading cells on the basement membrane were viewed and photographed under a microscpoe. The invading cells of five random view fields were counted and statistical analysis was done.2. The melecular mechanism of hypoxia induced glioblastoma invasion and migration①T98G and U87 cells were cultured in serum containing DMEM, when the cells grew to 70% confluent, serum free DMEM was changed. Specific groups were moved into hypoxia incubator at 4 or 8h. At 12h, the supernatant was collected and zymography was performed to detect the activities of MMP2/9 in the supernatant. ②Coverslips were placed into the wells of 24-well plates, then T98G and U87 cells were seeded into the wells at the density of 2×104/well. After incubation under normoxia or hypoxia for 24h, the coverslips were viewed and photographed under light microscope. Then the cytoskeleton was stained with rhodamine-phalloidine and viewed under fluorescence microscope. Fascin-1 distribution style was detected by immunofluorescence and viewed under fluorescence microscope.③T98G and U87 cells were seeded into the wells of six-well plates. After incubation for overnight, the cells in hypoxia group were moved into hypoxia incubator for 24h. G-LISA was performed to detect the activities of RhoA, Racl and Cdc42.3. The correlation between MIF and hypoxia induced glioblastoma invasion and migration①T98G and U87 cells were seeded into the wells of six-well plates. After incubation under normoxia for overnight, the cells in hypoxia group were moved into hypoxia incubator for 24h. Cells were collected and MIF gene and protein expression were detected by RT-PCR and Western blot.②T98G and U87 cells were resuspended in serum free DMEM and diluted to the density of 1×105/ml with various concentration of MIF.200μl of such suspension was added into the uper chamber of Transwell system, then serum containing DMEM(T98G 20%, U87 10%) were added into the lower chamber for chemotaxis. 12h later, all chambers were picked out, fixed and stained. The invading cells on the basement membrane were viewed and photographed under a microscpoe. The invading cells of five random view fields were counted and statistical analysis was done.③Coverslips were placed into the wells of 24-well plates, then T98G and U87 cells were seeded into the wells at the density of 2×104/well. After incubation for overnight, the medium was chaged for fresh medium containing various concentration of MIF.24h later, the coverslips were viewed and photographed under a light microscope. Then the cytoskeleton was stained with rhodamine-phalloidine and viewed under fluorescence microscope. ④3 siRNA sequences targeting MIF were Synthesized and transfected into T98G and U87 cell lines with lipofectamineTM 2000.24h later, RT-PCR and Western blot were performed to test the silencing effect of MIF siRNA. The most effective sequence of MIF siRNA was chosen.⑤MIF siRNA was transfected into T98G and U87 cell lines.24h later, Transwell migration assay was done under nomorxia and hypoxia to test the effect of MIF siRNA on glioblastoma migration.⑥MIF siRNA was transfected into T98G and U87 cell lines.24h later, the cells were collected and seeded into 24-well plates after coverslips were placed. Four experiment groups including control, control siRNA, MIF siRNA and MIF siRNA+MIF were set. After incubation under hypoxia for 24h, the cells were stained with rhodamine-phalloidine to view the cytoskeleton changes after MIF silencing.4. The expression of key molecules in MIF signal pathway in glioblastomaT98G and U87 cells were cultured under normoxia and hypoxia respectively. RT-PCR was performed to test the expression of the key molecules in MIF signal pathway, such as CD74, CD44, Tiam-1, Vav2, p115RhoGEF, RhoA, Racl and Cdc42.Results1. The effect of hypoxia on glioblastoma invasion and migrationThe invasion and migration ability of glioblastoma cell lines T98G and U87 increased under hypoxia as measured by Transwell system. The longer the cultured time under hypoxia, the more of the cells invaded the basment membrane. The difference was statistical significant.2. The molecular mechanism of hypoxia induced glioblastoma invasion and migration①The activities of MMP2 and MMP9 in the supernatant of T98G and U87 was not influenced by hypoxia as detected by zymography.②The cells cultured under hypoxia were much thinner and longer than those under normoxia. The polarity of the cells was more obvious. Cytoskeleton immunofluorescence showed more stress fibers formed under hypoxia. ③As detected by G-LISA, Racl activity was found to be upregulated under hypoxia while RhoA activity decreased. The changes of Cdc42 activity was not so obvious.3. The correlation of MIF and hypoxia induced glioblastoma invasion and migration①MIF gene and protein expression increased under hypoxia conditions as measured by RT-PCR and Western blot.②Transwell migration assay showed that MIF could promote the migration of glioblastoma in T98G and U87 cell lines.③Cells became thinner and longer after MIF stimulation. The polarity of the cells was more obvious. Cytoskeleton immunofluorescence showed more stress fibers formed after MIF stimulation.④RT-PCR and Western blot showed that MIF siRNA sequence 2 was effective in silencing MIF expression.⑤MIF siRNA transfection could dramatically inhibit T98G and U87 migration under normoxia and hypoxia conditions as measured by Transwell migration assay.⑥Cytoskeleton immunofluorescence showed MIF siRNA could reverse hypoxia induced stress fibers formation in glioblastoma.4. The expression of key molecules in MIF signal pathway in glioblastomaKey molecules in MIF signal pathway such as CD74, CD44, Tiam-1, Vav2, p115RhoGEF, RhoA, Racl and Cdc42 were all expressed in T98G and U87 glioblastoma cell lines.Conclusion1. The invasion and migration ability of glioblastoma cell lines T98G and U87 increased under hypoxia2. The activities of MMP2 and MMP9 in T98G and U87 supernatant was not affected under hypoxia3. The cytoskeleton of T98G and U87 cells rearranged under hypoxia with the cells became thinner and longer and with more stress fibers formed4. MIF was responsible for hypoxia induced glioblastoma invasion and migration5. Key molecules in MIF signal pathway were expressed in glioblastoma...
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