| Gliomas are the most common type among malignant tumors in the central nervous system, accounting for35.26-60.96percent (average44.69%) of all primary tumors in the central nervous system. Gliomas grow rapidly and invade diffusely, which result in an unfeasible complete surgical resection of gliomas. Moreover, gliomas are not sensitive to radiotherapy and chemotherapy. These featrures of gliomas lead to tumor relapse after conventional therapy and even threaten patients’life and their survival quality seriously. Despite surgical techniques and adjuvant radiation and chemocherapies techniques have been developed, the prognosis of patients with gliomas remains poor, with a median survival time of14.6months. Traditional view of oncology believes that cancer cells have the capacity to proliferate infinitely. However, in recent years, oncologists noticed that only a subpopulation of tumor cells in a kind of tumor is endowed with the ability of self-renewal, multilineage differentiation, extensive proliferation and tumor initiation; meanwhile, the majority of tumor cells could not proliferate, or it only proliferates for a very short time. Such tumor cells were named "tumor stem cells". Up to now, tumor stem cells have been separated and identified from many kinds of tumors including malignant digestive system tumors, malignant hematopoietic system tumors, malignant genitourinary system tumors, malignant brain tumors, malignant breast tumor, cutaneous tumors, etc. Tumor stem cells in glioma were named "glioma stem cells" by oncologists of brain tumor research field.Glioma has heterogeneous subpopulations, every rank of tumor cells in a glioma are different in their ability for differentiation, proliferation and sensitivity to anti-tumor drugs. Most of tumor cells in a glioma could be eradicated by traditional therapies; however, glioma stem cells express higher levels of genes related to drug-resistance and anti-apoptosis, which make them resistant to traditional radiation and chemotherapy. These tumor stem cells survive and glioma would recur sooner or later. So, glioma stem cells were taken for tumor initiation, tumor relapse, tumor metastasis and treatment resistance of giloma. Therefore brain tumor stem cells represented a critical target for tumor therapy; in view of the situation that cancer may be cured by eradicating cancer stem cells, it is very necessary to study the molecular mechanism of giloma stem cells proliferation and to develop novel therapies to eliminate cancer and cancer stem cells.Hypoxia is a common feature of glioma. Necrosis by serious hypoxia usually be found in glioma tissues. However, glioma stem cells adapt this hypoxic niche. Meanwhile, hypoxia blocks glioma stem cells differentiation and also shows a stimulative effect on glioma stem cell proliferation. Therefore, study of proliferation of glioma stem cells should be conducted under hypoxia condition. Study has also found that hypoxic conditions help release of extracellular ATP and AMP from tumor cells to exert anti-tumoral effects, like inhibiting growth of tumor cells and inducing apoptosis of tumor cells by stimulating purinoceptor P2X7. By contrast, ATP and AMP can be hydrolyzed by ectonucleotidases to produce extracellular adenosine, which is rich in hypoxic tumor tissues, promotes angiogenesis and protects against radiotherapy and ischemia/reperfusion injury. Therefore ectonucleotidases may play an important regulatory role in converting an unfavorable cellular microenvironment where tumor cells survive. The only two ectonucleotidases that have been discovered so far are CD73and prostatic acid phosphatase (PAP). CD73has been reported to convert AMP into extracellular adenosine and eventually enhance the proliferation of glioblastoma cell lines, rat C6and human U138MG PAP, the other ectonucleotidase, generates extracellular adenosine too, but little has been revealed about its role in proliferation of glioblastoma cell lines.Therefore, the present study was to explore whether PAP may promote the proliferation of GSCs by converting AMP into extracellular adenosine under a hypoxic condition.The study includes three chapters:Chapter Ⅰ Establishment of glioma stem cell lines from human U87and GL1glioma cellsObjective:To establish glioma stem cell lines, we adopted and modified a functional stem cells isolation method to isolate and characterize tumor stem cells from human U87and GL1glioma cell lines.Methods:We adopted an improved functional isolation approach to separate and purified glioma stem cells from U87and GL1glioma cells. Roughly, U87and GL1glioma cells were cultured in serum-free stem cell proliferation medium (SCM, containing DMEM/F12+40ng/mL bFGF+40ng/mL EGF+4%B27) under hypoxic condition to proliferate as tumorspheres. Formed tumorspheres were dissociated and plated at a density of1000cells/well into6-well plates. After10 days under hypoxia, the second generation spheroids were passaged at the same density again. Finally, after tumor stem cell property identification, the fifth generation of tumorspheres were dissociated and plated onto6-well plates coated with Matrigel where they grew as a monolayer culture in SCM medium under hypoxiaThe morphology and self-renew was observed by optic microscope, the immunophenotype and the ability for differentiation were tested by immunocytochemistry.Results:Tumorspheres formed from U87and GL1glioma cells in a low cell density by the improved functional isolation approach. In random10tumorspheres of the fifth generation, CD133possitive cells accounted for95%, and Nestin possitive cells accounted for89%. Cells in tumorspheres of the fifth generation could adhesively grow on Matrigel coated plates and show good cell viability. Cells in tumorspheres of the fifth generation could differetiate into cells expressing β Ⅲ-Tublin, GFAP or O1proteins.Conclusion:Cells in tumorspheres of the fifth generation derived from U87or GL1glioma cells expressed glioma stem cell marker and possessed the ability of stem cells for proliferation, multilineage differentiation.Chapter II Expression of PAP on glioma stem cells and its regulation by hypoxiaObjective:To compare the expression levels of PAP or CD73in hypoxic and physioxic common glioma cells or glioma stem cells, PAP and CD73protein and mRNA levels were detected in these cells. This study was also aimed to explore whether PAP was regulated by hypoxia. Methods:Western blot and QRT-PCR were employed to detect the protein and mRNA levels of PAP and CD73in hypoxic and physioxic U87or U87GSC. The protein and mRNA levels of PAP were further compared between hypoxic and physioxic non-tumor brain cells (NT1) and human glioma cells (U87and GL1). Four hypoxia time point were set to compare the protein and mRNA levels of HIFla, HIF2a and PAP in U87and U87GSC cells. the expression of HIFla and HIF2a were silenced respectively to explore which one response to the upregulation of PAP.Results:CD73was weakly expressed in U87and U87GSC cells. PAP was weakly expressed in non-tumor brain tissue cells (NT1), neural stem cells (NTINSC) and human glioma cells (U87and GLl) too, but it highly expressed in human glioma stem cells (U87GSC and GL1GSC). Furthermore, the expression of PAP increased when human glioma stem cells were transfered to hypoxic condition.We found that in U87GSC cells, HIFla was slightly increased but HIF2a and PAP were significantly upregulated in a time-dependent manner. Also, immunohistochemistry assay confirmed such expression patterns of HIF2a and PAP in U87GSC-derived tumorspheres. However, the levels of both PAP and HIF2a in U87cells were extremely low, and no significant increase of these proteins was detected with the increase of hypoxia time. Knockdown of HIF2a, rather than of HIFla, led to formation of smaller tumorspheres from hypoxic U87GSC and GL1GSC cells and failed increase of PAP.Conclusion:CD73hardly expressed in both human glioma cells and glioma stem cells, while PAP highly expressed in glioma stem cells with a hypoxia induced upregulation featrue. In hypoxia, HIF2a was overexpressed to promote glioma stem cells proliferation and upregulate PAP. Our findings strongly suggest that HIF2a is an important regulator in hypoxic GSCs and takes PAP as a downstream gene. Chapter Ⅲ The function of PAP and its molecular mechanismObjective:This study was aimed to investigate the functions of PAP and its molecular mechanism.Methods:To investigate the functions of PAP and its molecular mechanism, we detected the concentration of adenosine after PAP was silenced in hypoxic glioma stem cells. Glioma stem cells were divided into four groups to compare effect of silencing PAP or additing adenosine in hypoxic glioma stem cells. After explore the function of PAP, the expression levels of adenosine receptors in hypoxic glioma stem cells were further detected. RNA silence technique and specific chemical blocker of adenosine receptors were employed in this study to judge the functional adenosine receptors and its downstream pathways.Results:Silence of PAP significantly decreased the concentration of adenosine in liquid supernatant of glioma stem cells. While, adding AMP increased the concentration of adenosine in liquid supernatant of glioma stem cells. Tumorsphere forming assays showed that U87GSC and GL1GSC formed smaller and fewer tumorspheres (both p<0.01). In contrast, addition of adenosine had the opposite effect (in GSC-Ado and GSC-siP-A groups)(both p<0.01). Moreover, our limiting dilution assay showed that U87GSC-siPAP needed more cells to form a new tumorsphere than the control, but U87GSC-siP-A needed fewer cells. In addition, our cell-cycle progression assay showed that the knockdown of PAP in hypoxic GSCs contributed to a significant decrease in SPF, an index of proliferation, while the addition of adenosine led to a significant increase of SPF (p<0.01). Animal assays found that tumors derived from U87GSC-siPAP were significantly smaller in volume and grew markedly slower than those in the control group (both p<0.01). Tumors derived from U87GSC with addition of adenosine grew significantly faster than those without addition of adenosine (U87GSC-Ado vs. U87GSC-Ctrl, p<0.01; U87GSC-siP-A vs. U87GSC-siPAP, p<0.01).Our Western blot analysis showed no significant difference regarding the phosphorylation level of JNK between the four hypoxic U87GSC groups. Differently, the phosphorylation levels of Akt and Erk-1/2were increased in U87GSC-Ado but decreased in U87GSC-siPAP, compared with the control group (Fig.6E). Furthermore, to determine the relationship between the activation of these pathways and the stimulation of adenosine receptors, we re-detected these pathways after blockage or knockdown of A2B or A3receptors. Results showed that blockage and knockdown of A2B, not of A3, significantly downregulated the phosphorylation of Akt and Erk-1/2.Conclusion:These results support that PAP enhances the proliferation of GSCS in vitro and promotes tumorigenesis in vivo by producing adenosine. Our study also suggest that PAP may activate Akt and Erk-1/2pathways by stimulating A2B receptor. |
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