| Glioma is one of the most common intracranial primary malignant tumors, accounting for2%of all adult malignant tumors, with high incidence rate, high recurrence rate and poor prognosis. Despite the current clinical joint treatments including operation, postoperative radiotherapy and chemotherapy, and even immunotherapy and vascular targeting therapy, have been made great progress in the treatment of malignant glioma, the malignant tumor can not be controlled to stop developing. We need to research it on and seek new targets for treatment, so as to prolong life of patient until gliomas could be completely cured. Therefore, a new target for diagnosis and treatment of gliomas can provide a theoretical basis for the clinic with very important significance.Malignant tumors cannot survive and grow without sufficient blood supply. For a long time, new blood vessels in tumor were believed to exclusively occur through the mechanism of angiogenesis and vasculogenesis. Until vasculogenic mimicry (VM) proposed, it is gradually realized that there is another form of blood supply without endothelial cells involved, but containing red blood cells and plasma, which could be an alternative therapeutic target for cancer. There have been an increasing number of studies have shown that vasculogenic mimicry exists in tumor tissues from different tumors. While Maniotis and his colleagues first defined vasculogenic mimicry, they explored the relationship between vasculogenic mimicry and tumor prognosis, and there was a strong statistical separation in survival between patients whose tumors lacked loops and networks and those whose tumors contained these structures by the Kaplan-Meier survival analysis. Then, growing researches confirm that the existence of vasculogenic mimicry marks a poor prognosis for the patients. Therefore, a depth study on the vasculogenic mimicry and the targeted therapy has important practical significance.Traditional vascular targeting therapies for the treatment of malignant tumor are aimed to inhibit endothelial cells, which will prevent tumor blood supply and suppress the tumor growth. In recent years, vascular targeting therapies have been gradually accepted. The anti-angiogenesis drug, Avastin (bevacizumab) has become one of the most popular vascular targeting therapy drugs for some tumors, including glioma. However, studies show that the treatment is not effective in the treatment of tumors, and will accelerate to metastasis and the formation of vasculogenic mimicry with hypoxia. Compared with the normal endothelium, vasculogenic mimicry, as an alternative tumor blood supply, can supply nutrition and metabolism for tumors. It forms a resistance to chemotherapy and promotes tumor metastasis. In order to prevent the ischemia and hypoxia after endothelial vascular targeting therapy, which is prone to form vasculogenic mimicry and to accelerate the metastasis, some scholars suggest that therapy with small and multiple doses of anti-angiogenic drugs (such as bevacizumab) would get good effect. Vasculogenic mimicry has deepened the understanding of tumor microcirculation and transfer mechanism, therefore, some scholars considered that it was because of endothelial vascular targeting therapy, which induced a tumor escape mechanism without endothelial cells participation in the formation of vasculogenic mimicry, the simple anti-endothelial vascular targeting therapies may be meaningless, and the anti-vasculogenic mimicry should be considered. Therefore, inhibition of vasculogenic mimicry formation may be one of novel directions for the tumor vascular targeting therapies.Many laboratories have been discussed the mechanism of signal transduction of mimicry formation. So far, a number of molecules involved in the formation of vasculogenic mimicry have been confirmed in different tumors, including HIF-1α, VE-cadherin, EphA2, MMP-14, MMP-2and Laminin. Along with the related molecules involved in vasculogenic mimicry above found, a classical model of vasculogenic mimicry signal pathway formed gradually. In this model, hypoxia is considered to be a switch of vasculogenic mimicry formation, which can directly regulate the gene expression of EphA2(through HIF-1α) or regulating the expression of VE-cadherin indirectly (through the activation of intermediary protein), then activated the rest of the signaling cascade. In the final stage of vasculogenic mimicry signaling pathway, expression and activation of MMP-14can activate MMP-2. MMP-2binding with the activated MMP-14promote the decomposition of laminin, resulting that the extracellular matrix contract and form the so-called lumens, which are the vasculogenic mimicry. Simultaneously it also increases the ability of migration and invasion of tumor cells. Although the classical pathway interprets the formation process of vasculogenic mimicry well, the specific mechanism is still unclear.Hypoxia, either persistent or transient, is a hallmark of most tumors and has been shown to regulate pathways in the maintenance of the stem cell-like phenotype, cellular differentiation, invasion, metastasis, apoptotic resistance, genomic instability, angiogenesis, and VM. Molecularly, protein stabilization and nuclear localization of hypoxiainducible factor-la (HIF-1α)/HIF-2a transcription factors and binding to hypoxia response elements (HRE) in promoter and enhancers of effector genes occurs in response to low oxygen, oncogenes, or inactivated tumor suppressor genes. Hypoxia has been shown to induce VM in hepatocellular carcinoma, Ewing sarcoma, and melanoma. Moreover, hypoxia can induce a dedifferentiated phenotype in breast carcinoma. Pertinent to VM, hypoxia has been shown to either directly modulate VEGF-A, VEGFR, EphA2, Twist, Nodal, and COX-2gene expression (via HIF-1/HRE binding) or indirectly modulate VE-cadherin and TF expression (via activation of an intermediary protein). Hypoxia can also modulate the expression of Notch-responsive genes; specifically, hypoxia stabilizes the NICD protein, which interacts with HIF-1α and activates genes with Notch-responsive promoters, including Nodal. This noncanonical cross-talk between HIF-la and Notch-signaling pathways is thought to promote an undifferentiated cell state, further illuminating the possible etiology of tumor cell plasticity underlying VM. Based on the numerous studies showing hypoxia-induced VM and/or VM-associated genes, it is conceivable that therapeutic use of antiangiogenic agents may promote tumor plasticity and metastatic progression.In addition to the above mechanism, in recent years, a variety of regulatory factors of vasculogenic mimicry formation are found. For example, the expression of tissue factor pathway inhibitor-2(TFPI-2) gene is significantly higher in invasive melanoma cells than the non invasive ones, while the low invasive melanoma cell cultured in a three-dimensional matrix containing the recombinant TFPI-2can be converted into highly invasive cells and exhibit phenotypes like endothelial cells, which form some vessel-like channel. In addition, cyclooxygenase-2(COX-2) is involved in the regulation of vasculogenic mimicry formation in breast cancer. These studies confirmed that the molecular mechanisms of tumor vasculogenic mimicry formation are very complex, which is a process with multi-factor involved and multi-path regulated.In addition to discuss on the mechanism of vasculogenic mimicry formation, at the same time, researchers also explored some targeting therapies for the vasculogenic mimicry. Inhibition of moleculars involved in vasculogenic mimicry formation can reduce the blood supply of tumor, which provides more theoretical basis for the treatment of vasculogenic mimicry with a good clinical significance.However, the molecular mechanism of tumor vasculogenic mimicry formation is very complex, multi path is a multi factor involved in the regulation of, development. The mechanism of formation of vasculogenic mimicry is not completely understood; for the treatment of vasculogenic mimicry has certain effect, still need to be further studied.TOR is found as a target gene of rapamycin in yeast in1991. Then people also discovered this gene in mammalian cells and called it the mammalian target of rapamycin (mTOR). mTOR is a molecular weight of289kDa serine-threonine kinase, which is a central molecule regulating the initiation of protein translation and plays an important role in many physiological processes such as cell growth, proliferation, differentiation and apoptosis, and could become a target for glioma therapy.Studies have shown that excessive activation of mTOR signaling plays a very important role in the development of gliomas. mTOR signaling pathways is an important signal pathway within cells found recently, which is highly conserved in evolution, and regulates cell growth mainly by controlling protein synthesis. In the mTOR signaling pathway, various cellar signals including the mitogen, growth factor, hypoxia or cellular energy level can affect the mTOR signaling pathway. Studies have confirmed that, mTOR is closely related to tumor angiogenesis. However, the relationship between mTOR and vasculogenic mimicry is unclear. In addition, mTOR is close related to multiple moleculars in the formation of vasculogenic mimicry, especially the hypoxia-inducible factor (HIF). Hypoxia can promote HIF-la to express VEGF-A, VEGFR-1, EphA2, etc. Most importantly, some scholars using mTOR inhibitor rapamycin to inhibite HIF1α discovered that vasculogenic mimicry was also inhibited. In summary, mTOR are closely related to the formation of vasculogenic mimicry, and there is a crosstalk between signal pathways in a variety of molecules involved in vasculogenic mimicry formation. If we can further clarify that the role of mTOR in in the formation of vasculogenic mimicry in glioma, and identify the correlation between mTOR and vasculogenic mimicry formation, it will be beneficial for identifying new mechanisms of vasculogenic mimicry formation and new therapeutic targets and methods, which has a very important significance in improving the prognosis of patients with glioma.Chapter one:Clinical significance of vasculogenic mimicry and the correlation analysis between mTOR expression and vasculogenic mimicry in gliomaObjective To verify the presence of vasculogenic mimicry in glioma, and investigate the relationship between mTOR and vasculogenic mimicry in glioma.Methods127cases of glioma were collected from Department of Pathology, Zhujiang Hospital, Southern Medical University between2009and2012. All specimens are preserved in paraffin embedded by standard procedures after surgery. And all pathological specimens are need to be re-sliced and stained by immunohistochemistry, then diagnosis by two pathologists according to the2007World Health Organization classification of tumors of the central nervous system without knowledge of the patient’s clinical data. Cases inclusion criteria:1) Patients underwent surgery without radiation and chemotherapy before.2) Postoperative pathological diagnosis is glioma.3) Complete information of patient can be collected. Clinical data of patients including gender, age, KPS score, tumor size and tumor grading were sorted out. All slices would be stained by CD34-PAS, and according to the results, patients were divided into two groups, which are vasculogenic mimicry positive group and vasculogenic mimicry negative group. For experimental data processing, χ2test was carried out to compare the clinical and pathological features of patients in two groups, including gender, age, KPS score, tumor size, histological grade and level of expression of mTOR using SPSS13.0statistical software. In the statistical analysis process, P<0.05was considered as statistically significant difference.Results In the total127cases of glioma, vasculogenic mimicry structures were found in34cases, accounting for26.8%. Compared to low-grade gliomas, vasculogenic mimicry structures are more iditified in high-grade gliomas (gradeⅢ and grade IV were33.3%and39.4%, while grade I and II were0.0%and15.6%)(χ2=9.051, P=0.029). In the glioma slices with more vasculogenic mimicry structures, mTOR expression was significantly higher than that of vasculogenic mimicry negative (-/+10.0%++21.7%, and+++was44.1%)(χ2=7.748, P=0.021). These results suggest that vasculogenic mimicry in gliomas is related to the level of mTOR expression (P<0.05).Conclusion This study verified the presence of vasculogenic mimicry in gliomas and suggested that vasculogenic mimicry is not only positively correlated with tumor grade, but also with the level of mTOR expression in gliomas, and there is no correlation with patient’s general clinicopathological features such as gender, age, KPS score, tumor size, etc.Chapter two:The effect of hypoxia and mTOR-specific inhibitor rapamycin on the formation of vasculogenic mimicry of malignant gliomaObjective To establish dimensional culture model of U87malignant glioma (U87-MG), and observe the effect of hypoxia and the specific mTOR inhibitor rapamycin on the formation of vasculogenic mimicry in vitro.Methods the ability of vasculogenic mimicry formation by U87-MG cells was studied under normoxia and hypoxia condition in vitro using three-dimensional culture and tube formation assay. Vasculogenic mimicry formation by U87-MG cells was observed with different concentrations of mTOR specific inhibitor rapamycin treatment in vitro. The expression of HIF-1α was studied after mTOR inhibition under normoxic and hypoxic conditions by technique of Western Blotting.Results U87-MG cells are capable of forming a kind of tubular structures analogous to the endothelial cells on Matrigel. However, this structure is incomplete under normoxic condition, some lumens is not closed, whereas a more complete lumen structure formed under hypoxic condition. The mTOR-specific inhibitor rapamycin were able to decrease the structures of vasculogenic mimicry formed by U87-MG in vitro whether under normoxic or hypoxic condition. Furthermore, with rapamycin concentrations increasing, the ability of vasculogenic mimicry formed U87-MG cells was gradually weakened. Western Blotting showed that rapamycin can inhibit both mTOR and HIF-la expression under normoxic condition and hypoxic condition.Conclusion Through three-dimensional culture in vitro, U87-MG cells can form tubular structure-like similar to the HUVEC on Matrigel, which confirmed that malignant glioma cells have the ability to form vasculogenic mimicry in vitro. Hypoxia promotes U87-MG to form vasculogenic mimicry. mTOR was involved in vasculogenic mimicry signaling pathways via HIF-1α.Chapter three:Study on the molecular mechanisms of mTOR involved in vasculogenic mimicry of malignant gliomaObjective To study the effect of the vasculogenic mimicry related molecules expression in U87-MG by mTOR gene interference technology.Methods Four mTOR siRNAs were designed and synthesized, and one of which knockdown maximally mTOR expression was chosen to screen the downstream signaling of VM. The differential expressions of vasculogenic mimicry-related molecules were detected by Western Blotting technique and Cell migration assay.Results We successfully designed and synthesized four mTOR siRNA, and chose one which knockdown maximally mTOR expression to screen the downstream signaling of VM. When mTOR expression significantly was decreased by the siRNA, as expected, HIF-la expression was also significantly downregulated, either under normoxic or hypoxic condition. By western blotting, both MMP-14and MMP-2expressions were extremely lower in U87-MG cells transfected with mTOR siRNA than the control cells, even under hypoxia. MMP-2was associated with cell migration. Migration abilities of U87-MG cells increased after hypoxia for24h. However, this increase did not recur after siRNA interference.Conclusion HIF-la and vasculogenic mimicry-related genes (such as MMP-14and MMP-2) expression were inhibited after mTOR gene interference, either under normoxic or hypoxic conditions. Through mediating HIF-la, mTOR is one of important molecules in the vasculogenic mimicry signaling pathway of glioma. |
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