| Background and purpose:Glioma accounts for the40%～50%of brain tumors, with high invision and high proliferation, the comprehensive treatment method such as the conventional surgery and radiotherapy or chemotherapy mainly targets the tumor cells, therefore the efficacy would be poor. In1999, Maniotis found the phenomenon of vasculargenic mimicry (VM), i.e., the tumor cells could simulate the vascular endothelial cells and form a channel that was similar to the normal vessels in exhibiting the functions, thus the inhibiting this blood supply might become the new target towards the tumor vascular targeting therapy.Vasculogenic mimicry phenomena, provided ideas and methods more for glioma.The formation mechanism of VM is not yet very clear. According to the recent study, VM formation is closely related to the changes of tumor microenvironments. Furtherstudy of the glioma microenvironment change will be helpful to further understanding the mechanism of VM formation, diversity and effective method of treatment.The tumor microenvironment refers to local tumor infiltrating immune cells, stromal cells and the secretion of the active medium with tumor cells together constitute the internal environment, which is composed of various kinds of control of active ingredients, including hypoxia and extracellular matrix remodeling is the tumor microenvironment to two major components. Interstitial cell high-pressure formation, hypoxia and acidosis, a lot of growth factor and proteolytic enzymes produced constitute the biological characters of tumor tissue metabolism environment, this characteristic has important effect on tumor proliferation, invasion, migration, adhesion and angiogenesis. The extracellular matrix (Extracellularmatrix, ECM) in a variety of molecules, including matrix metalloproteinases (Matrix metalloproteinases, MMPs),integrins, cadherins, immunoglobulin class of adhesion molecules, selectins, cell surface proteoglycans family(VE-cadherin) etc.. These molecules are also actively involved in tumor invasion, metastasis, angiogenesis and biological behavior.Hypoxia is one of the micro environment of solid tumors, closely related to the malignant biological behavior of tumor development, invasion and metastasis. The growth and proliferation of tumor cells depends on sufficient oxygen and energy supply.However, inside the tumor tissue, tumor cell uncontrolled growth and proliferation consume large amounts of nutrients and oxygen, so the internal structure can not be establishment of neonatal vascular network timely and effectively.The structure and function of neovascular network temporarily closed or "the blind side". Reduction of tumor blood flow and oxygen supply is far less than the oxygen demand, leading tumor cell in the acute (perfusion deficiency) or chronic (diffuse disorder) hypoxia. In addition, the anemia caused by malignant tumor and its process of antitumor treatment also increased tumor hypoxia.HIF-la is considered to be malignant tumor hypoxia adaptive response center to start the molecule. Research shows that, pipeline HIF-la is involved in several solid tumors VM formation, mainly through the following ways:(1) HIF-1α:EphA2/could be controlled by PI3K/MMPs signal pathway of VM formation. Zhang Shiwu reported a study on melanoma VM formation, found that in the anoxic environment, high expression of HIF-la melanoma cells.lt could up regulate the expression of MMP-2and MMP-9protein expression, degradation of ECM, and promote tumor cell invasion and the formation of VM.(2) Through the regulation of HIF-la can influence B cell lymphoma/leukemia-2gene (B-cell lymphoma-2, Bcl-2) expression, further influence the formation of VM. In2012, Zhao Nan of the Medical University Of Tianjin reported in relation to hypoxia anti apoptotic protein Bcl-2and VM melanoma formation. Experiments show that, the hypoxic microenvironment of Bcl-2and up regulation of VEGF expression, when silence after Bcl-2, VEGF expression was down regulated, VM formation was inhibited, reveals that Bcl-2plays a positive role in melanoma VM formation.(3) The formation of HIF-la can also be affected by the PI3K/AKT/mTOR signaling pathway is involved in the regulation of VM. Studies have found that mTOR specific inhibitor rapamycin can inhibit ovarian cancer cell in the HIF-la, rapamycin through regulation of PI3K/AKT/mTOR and HIF-la of VM formation. Thus, HIF-la formation plays a very important role in the inhibition of VM process, combined with in-depth study of HIF-la and multiple target HIF-la will is an important research direction of our future anti-vasculogenic mimicry formaiton.In recent years, scholars have reported the effects of hypoxia on the formation of VM pipeline of brain glioma. In2011, Zhang Xi discovered the vasculogenic mimicry formation in glioma SHG44cells under hypoxic conditions,VEGF, EphA2, MMP2, MMP9expression was significantly enhanced, suggesting that under hypoxic conditions, HIF-la may be formed to participate in VM by affecting VEGF, EphA2, MMPs molecular expression. In2012, Xi Zai reported hypoxia can promote the formation of VM in glioma U251cells, expression induced by HIF-1α and VEGF-A may be one of its mechanisms. Thus, angiogenesis factor HIF-la, matrix metalloproteinase MMPs family, anti-apoptosis protein and other factors play an important role in glioma VM formation.To treat in a variety of solid tumors of the target, the role of VM related molecules of specific inhibitors of increasing attention. Among them, HIF-la specific inhibitor3-(5’-hydroxymethy1-2’-furyl)-1-phenyl methyl indazole{3-(5-hydroxymethl-2-furyl)-1-benzylindazole, YC-1} is proved to have excellent anti angiogenic activity and tumor therapy activity in several cancers such as pancreatic cancer, bladder cancer, ovarian cancer, and may have a dose dependence sex. While the Bcl-2specific inhibitor is considered to be one of the effective drugs for anti angiogenesis. The Bcl-2gene has been shown to be a proto-oncogene, inhibit the apoptosis of tumor cells. As mentioned earlier, HIF-1a can affect the expression of Bcl-2. Studies have reported, Bcl-2may form by adjusting the VE-cadherin on human melanoma VM under hypoxic conditions, suggesting that Bcl-2also inhibits the target tumor cells VM formation.Angiogenesis is rarely reported about the relationship between YC-1and glioma. In2012the Italy scholar Marizia B. Gariboldi by observing under hypoxic conditions of glioma cell line U87cell growth,angiogenesis and cell biological changes. Experimental results show that the YC-1by inhibiting expression of HIF-la, further inhibit MMPs, thus reducing the U87cells, angiogenesis. Domestic only in some reports YC-1promotes glioma radiation sensitivity. Guo Xinwei reports that the choice of SHG44and U251glioma cell line respectively in normoxia (20%O2), hypoxia (1%O2)12h and24h, hypoxia12h+YC-1(HIF-1α inhibitor) and hypoxia24h+YC-la total of5different culture conditions, found and reduce glioma cells radiosensitivity microenvironment hypoxia through to improve the CD133+stem cell proportion, the mechanism may be that HIF-1α/Notchl signal pathway.There are many studies confirmed that hypoxia promotes tumor formation of VM report. As mentioned earlier, HIF-la as tumor hypoxia adaptive response center to start the molecule, plays a very important role in tumor formation of VM, however, there is no effect of HIF-1alpha specific inhibitor YC-1on VM glioma formation of the relevant reports. Under hypoxic conditions, HIF-la, MMPs, Bcl-2plays an important role in tumor angiogenesis, invasion, metastasis, apoptosis, especially HIF-la, MMPs is an indispensable part of vasculogenic mimicry, while Bcl-2was also confirmed in the play an important role in the formation of melanoma VM. This prompted us, effect of YC-1VM U87cells may be regulated by HIF-la, MMPs, and even Bcl-2molecular expression to complete. Thus, the application of HIF-la specific inhibitor YC-1can control VM glioma cells and its related molecular form, worthy of our in-depth study.This study established the method of chemical simulated hypoxia induced by hypoxia in human glioma U87cell microenvironment, expression of VM in U87cells under hypoxia formation and related molecules. Then, using the appropriate concentration of YC-1inhibits HIF-la, YC-1through targeted therapy and related molecules expression of U87cell VM, and to analyze its possible mechanism. Finally, the combined application of Bcl-2inhibitor, synergistic therapeutic effect of dual drug targets and the possible mechanism. Objective to provide new ideas for improved anti malignant glioma angiogenesis strategies for joint action, lays the foundation of target, thus expanding treatment for glioma treatment, improve survival rate. Capture1Effects of hypoxia on the formation of the glioma U87cells of VM and related moleculesObjective To construct the simulation model of hypoxiaU87glioma cell experiment, observation of vasculogenic mimicry of U87cells, expression of HIF-1α, MMP-2, MMP-14, Bcl-2and other related molecules, mechanism of action and the HIF-1α target for simulation study of vascular U87cells exposed to hypoxia mimicry provides a basis to immunosuppressive therapy.Methods Human glioma U87cells were cultured, three-dimensional culture. A control group and hypoxia group.Hypoxia group was added to each well of two cobalt chloride (CoCI2)100μmol/L. Two groups of24h the end of culture. Observation of tubular structure were observed, and the arrangement of integrity:by inverted microscope total length of random take up and down, left, right, center5vision camera recording and counting the number of tubular structure, average each field of vision. RT-PCR method was used to detect expression of HIF-la mRNA two group, MMP-2mRNA,MMP-14mRNA, Bcl-2mRNA.Results Vascular count:24hours after training control group cell growth sequence disorder, no obvious rules,can only detect the network structure of a few, which are connected with each other in hypoxia group cell, is formed by the fusion of network structure, network like structurewith single ring or multiple rings connected. Under microscope, the control group number per field meshstructure is2946.355±114.795μm, and hypoxia group was4244.161±79.686μm; hypoxia group was significantly increased (P<0.05).Detection of RT-PCR (1):Control group, HIF-1a was2.888±0.187, hypoxia group was6.723±0.419, there was significant difference between groups (t=-14.483,P<0.05).(2) MMP-2in control group was4.077±0.676,hypoxia group was4.636±0.51, there was no significant difference between groups (t=-1.143, P>0.05).(3) MMP-14in control group was2.766±0.074, hypoxia group was4.995±0.313, there was significant difference between groups (t=-12.014, P<0.05).(4) Bcl-2in control group was2.796±0.249, hypoxia group was3.772±0.953, therewas significant difference between groups (t=-6.349, P<0.05).Conclusion Hypoxia promotes the formation of VM U87cells. The change of increasing the expression of HIF-la expression first,further regulate MMP-14expression and the Bcl-2expression, may be associated with the formation of U87glioma cells VM.Chapter2Different concentrations of YC-lon hypoxia U87glioma cells VM expression and related moleculesObjective To investigate the application of different concentration of specific hypoxia inducible factor-1inhibitors (YC-1) effect on the formation of glioma U87cells VM under hypoxic conditions, the effect of different concentration of YC-1experiment expressed molecules related to glioma vascular tumor mimicry.Methods The conventional culture and three-dimensional culture method of human glioma U87cells. Under normoxic or hypoxic conditions by adding0,10,50umol/L YC-1divided into normoxic control group, normoxic group YC-1-10, group YC-1-50, hypoxia and normoxia control group,hypoxia group, YC-1-10hypoxia YC-1-50group six group.Hypoxia group was added to each well of two cobalt chloride (CoCI2)100μmol/L. All cells were cultured24hafter termination of culture. To observe the accession to the YC-1-50group of VM formation and compared with control group. Extraction of U87cells in each group of RNA. Differential expression of RT-PCR HIF-la was detected mRNA, MMP-2a mRNA, MMP-14mRNA, Bcl-2mRNA. Analysis of the active degree of group of U87cell HIF-1α, MMP-2, MMP-14, Bcl-2by Western blot technology.Results Vascular count:24hours after hypoxia training joined the YC-1group can only detect the network structure of a few, than that without control group YC-1decreased; low concentration of YC-1(YC-1-10) group of vascular network structure and the high concentration of YC-1(YC-1-50) vascular reticular structure number group significantly reduce the difference (P<0.05). The normal oxygen24hours after culture, also showed reticular structure is few in the experimental group; low concentration of YC-1(YC-1-10) group of vascular net structure of higher concentration of YC-1(YC-1-50)vascular network structure the number of group decreased significantly (P<0.05).Detection of RT-PCR (1):HIF-1alpha molecules:different concentrations of molecular expression was significantly different (F=354.865, P<0.001). There are significant differences between hypoxia and normoxia group(F=104.001, P<0.001). The normal oxygen group, YC-1was significantly down regulated expression (P<0.05), the dose dependence. Hypoxia group, YC-1was significantly down regulated expression, no dose dependence.(2):MMP-2molecular expression between molecules in different concentration had significant difference(F=74.025, P<0.001); there was no significant difference between hypoxia and normoxia group (F=2.353,P=0.151>0.05). The normal oxygen group, YC-1was significantly down regulated expression (P>0.05), no dose dependence. Hypoxia group, YC-1was significantly down regulated expression (P<0.05), the dose dependence.(3): MMP-14molecular expression between molecules in different concentration had significant difference(F=193.218, P<0.001). There is significant difference between hypoxia and normoxia group (F=35.515,P<0.001). Normoxic group and hypoxic group, different concentrations of YC-1was significantly down regulated expression (P>0.05), there were no dose dependence.(4):Bcl-2molecular expression between molecules in different concentration had significant difference(F=221.429, P<0.001). There is significant difference between hypoxia and normoxia group (F=25.816,P<0.001). Normoxic group and hypoxic group, the expression of YC-1was significantly down regulated molecules (P<0.05), the dose dependence.Detection of Western blot Technology:(1) To join YC-l,normoxia and hypoxia after24h incubation, respectively,compared with normoxia and hypoxia control group,showed that HIF-la, MMP-2, MMP-14, Bcl-2molecular reduction.(2)Under normoxic conditions, no significant difference between the YC-1group of Bcl-2protein in different concentration. Under hypoxic conditions, YC-1group MMP-2, Bcl-2molecular high concentration than the low concentration of YC-1groups.Conclusion Under anoxic conditions the inhibitory effect of YC-1on the formation of U87glioma cell VM related with YC-1concentration positivily.Under anoxic conditions the YC-1coud significantly down regulate the expression of HIF-la, MMP-12, MMP-14, Bcl-2molecules; the inhibitory effect of YC-1on MMP-2, Bcl-2and enhanced with the increasing of YC-1concentration.Chapter3YC-1and Bcl-2inhibitors on hypoxia in human U87glioma cells effect of VM formation and its possible mechanismObjectives To observe the common application of YC-1,Bcl-2inhibitor (ABT-737) on the formation of hypoxic conditions in U87glioma cells VM, expression of HIF-la and Bcl-2specific inhibitor on related molecules, to further explore the mechanism of U87cell VM under hypoxia and multi-targeted treatment effect. Methods Cells were divided into control group:YC-1group, ABT-737group (group YC-1+, ABT-737).24h induced by hypoxia. Observation of tubular structure were observed, and the arrangement of integrity, to calculate the total length of the tubular structure. Application of RT-PCR and Western blot expression were detected HIF-la, MMP-2, MMP-14, Bcl-2molecule.Results:Vascular count:(1)Between the four groups of vasculogenic mimicry of lumen like structure length count(VM total length) had significant difference (F=105.267,P<0.001). Compared with the control group, vasculogenic mimicry of lumen like structure length counting and the experimental group YC-1group, ABT-737group, YC-1+ABT-737cells (VM total length) were significantly decreased (P<0.05), followed by YC-1were2836.638±151.301μm, ABT-737was3815.453±22.917μ m, YC-1+ABT-737group2484.528±164.137μm.(2) To display the results with YC-1+ABT-737dual suppression group,simple YC-1group, join or simple into ABT-737group, VM in total length has significant difference (P<0.05).Detection of RT-PCR (1):Comparison between four groups:HIF-1a expression have significant difference(F=507.654, P<0.001); the expression of MMP-2had significant difference (F=189.797, P<0.001); the expression of MMP-14had significant difference(F=71.245, P<0.001); the expression of Bcl-2had significant difference (F=290.613, P<0.001).(2) In each experimental group compared with the control group, YC-1alone, ABT737or YC-1combined with ABT-737, can significantly down regulated the expression of related molecules (P<0.05).(3) And dual suppression group (YC-1+ABT-737):simple join YC-1group or simply joined the ATT-737group, the expression of HIF-1α molecules were significantly increased (P<0.05); only joined the YC-1group or simply joined the ATT-737group, the expression of MMP-2were increased significantly (P< 0.05); simple to join ATT-737in the group, the expression of MMP-14increased significantly (P<0.05), and simply add expression of MMP-14in YC-1group, no significant difference (P>0.05); only joined the YC-1group or simply joined the ATT-737group, the expression of Bcl-2were increased significantly (P<0.05).Western blot:(1) Compared with the control group, YC-1group, YC-1+ABT-737group, the expression of HIF-1a, MMP-2, MMP-14, Bcl-2molecules are reduced. In ABT-737group, the expression of MMP-2, Bcl-2decreased, but HIF-1a, MMP-14had no obvious change.(2) And(YC-l+ABT-737) compared with group dual suppression, HIF-la, MMP-14molecular graphics simply join group ABT-737expression increased significantly; there was no significant difference between the HIF-1a, MMP-2, MMP-14molecular simple joined the YC-1group, but the expression of Bcl-2was higer.Conclusion Under anoxic conditions that joined the YC-1, pure ABT-737or added YC-1and ABT-737, could significantly inhibit U87cell VM formation; at the same time, the accession to the YC-1and ABT-737were more significantly inhibit VM formation.Bcl-2is an effective target for inhibition of glioma formation of VM; YC-1and Bcl-2inhibitors can down regulate the expression of molecular glioma cells, may be the mechanism of inhibition of glioma formation of VM.Dual suppression in the treatment of multiple target a single inhibitory target therapy can effectively reduce the formation of vasculogenic mimicry, there may be accomplished by the expression was significantly down regulated HIF-la, MMP-2, Bcl-2molecule. |
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