| BackgroundNasopharyngeal Carcinoma (NPC) is a commonmalignant tumor in head and neck, which happens in nasopharyngeal mucosa, accounting for 78.08%of the head and neck malignant tumor and 92.99% of the upper respiratory tract cancer. The incidence rate of nasopharyngeal carcinoma in China is the highest all over the world, there are about 25 to 50 NPC cases in every 100000 people,25 times that of western countries. The incidence of nasopharyngeal carcinoma has evident regional characteristics, especially in places such as Guangdong, Guangxi, Hainan and so on.The pathogenesis of nasopharyngeal carcinoma is concealed in clinical, and the early symptoms of this cancer are not obvious, more than 60% of patients have been developed into the middle-late accompanied by cervical lymph node metastasis by the time NPC is first diagnosed. And EB virus infection can be detected in more than 95% of the undifferentiated type of nasopharyngeal carcinoma tissues.Due to the anatomical location of nasopharyngeal carcinoma is special, surgical method has great restrictions and limitations, so the treatment of nasopharyngeal carcinoma mainly depends on the radiation therapy combined with chemotherapy. Even if it is given a radical cure radiation dose, there are still about 30%of patients developed local tumor recurrence, and about 30% to 60% of the patients had metastatic. Therefore, to clarify the transfer mechanism of nasopharyngeal carcinoma and to use targeted prevention and treatment methods have become urgent problems to be solved now.In recent years, the cancer stem cell (CSC/TSC) has become a research hotspot of cancer. Tumor stem cell theory means that there has a close relationship between CSC and the occurrence, development or metastasis of tumor and drug resistance. CSC is a group of small quantities of cells with basic characteristics as follows:1, the ability of self-renewal, that is important for cancer stem cells to keep the ability of differentiation to precursor cells.2, multiple differentiation potential, it makes the tumor stem cells to form new tumors in the body by generate different differentiation degree of offspring tumor cells. In the same tumor tissue, malignant degree is low in mature differentiated tumor cells, while it is high in poorly differentiated tumor cells. A large number of experiments have show that the CSC/TSC has higher proliferation of tumor cells than ordinary.4, drug resistance, which is the one of the main reason that lead to treatment failure of tumor. Membrane protein of ABC transporter family is expressed in most cancer stem cell membrane. It can transport and excrete substance such as metabolic product, drug, etc, which makes lots of chemotherapy drugs that have the effect of inhibit or kill on cancer stem cells could not play their role or have a significantly decreased function. So it may be an effective way for solving treatment problem by specifically killing nasopharyngeal carcinoma stem cell.There have some reports about research on nasopharyngeal carcinoma stem cells. Zhang and his partners confirmed that there were scattered stem cells in nasopharyngeal membrane base by using Brdu labeled stranded cell, and some scattered label-retaining cells were also found. Nasopharyngeal carcinoma cell line 5-8F labeled by BrdU inoculated into nude mice subcutaneously, scattered label-retaining cells can also be found in transplanted tumor after 8 weeks. Wang isolated side group of cells that similar with stem cells from the nasopharyngeal carcinoma cell lines CNE2 by using Hoechst33342 dye, and they considered CK19 as marker of nasopharyngeal cancer stem cell. In 2013, Sun Yat-Sen University confirmed that surface molecule CD 133 can be used as the specificity molecule of the nasopharyngeal carcinoma stem cells, the same year; Cancer Institute of Southern Medical University also confirmed that ALDHl could also act as specific marker for nasopharyngeal cancer stem cells. Therefore, at present, stem cell surface marker such as CD133, ALDH1 +and fluorescent Hoechst 33342 marked SP cells and so on can be used to identify and isolate cancer stem cells in nasopharyngeal carcinoma.Nitric oxide (NO) is a kind of nitrogen compounds, as a sort of vascular endothelial active factors and signaling molecules, it has extensive physiological and pathological effect. It plays an important role in physiological and pathological physiology of multiple systems such as the breathing, circulation, immune and nerve system of all over the body and it also playing a vital role in relevant clinical diseases. In mammals, NO is catalytic synthesized by nitric oxide synthase (NOS).And nitric oxide synthase is divided into three subtypes including neuronal nitric oxide synthase(nNOS), endothelial nitric oxide synthase (eNOS) and induced nitric oxide synthase (NOS2), studies have found that family members of NOS is overexpressed in a variety of tumors, and they are associated with the poor prognosis of tumor. Cobbs [12] found that eNOS has a high expression in tumor cells and endothelial cells of astrocyte tumors, and it is positively related to the grading of tumor. While NOS2 be detected in tumor endothelial cells rarely. In general, the ratio of the cancer stem cell in well differentiated tumor is often lower than the poorly differentiated tumor. So we hope to provide the foundation to clarify the mechanism of nasopharyngeal cancer stem cells and supply a new idea for its treatment by discussing whether there is correlation between the expression of NOSs and nasopharyngeal cancer stem cells in nasopharyngeal carcinoma, and whether NOSs can regulate the expression of nasopharyngeal cancer stem cells. Method: The expression of NOS in nasopharyngeal carcinoma1. The relationship between the expression of the malignant phenotype of stem cell proportion and NOS in Nasopharyngeal carcinoma cell lines(1) And the expression of NOS in nasopharyngeal carcinoma cell lines (invasion and tumor malignant phenotype correlation)In six nasopharyngeal carcinoma cell lines as the experimental object, growth curve, Boden chamber, clone formation and tumor sphere culture experiments (tumor sphere formation), of nasopharyngeal carcinoma cell invasion and tumor function evaluation, analysis of the difference between the malignant phenotype of NPC cell lines; NOS expression levels was detected by immunofluorescence, real-time RealTime-PCR, Western Blot, flow cytometry in six nasopharyngeal carcinoma cell lines; statistical analysis of correlation between the expression of the malignant phenotype of NPC and six strains of nasopharyngeal carcinoma cell line NOS;(2) Relationship between NOS expression and nasopharyngeal carcinoma cell line CSC frequencyUsing CD 133, ALDEFLOUR ASSAY and SP three have confirmed that the method can be used as nasopharyngeal cancer stem cell markers and separation to detect six nasopharyngeal carcinoma cell lines CSC in nasopharyngeal carcinoma cell line frequency; analysis of correlation between the expression of NOS in nasopharyngeal carcinoma and the frequency of CSC;(3) Differences of NOS expression in CSC and non-CSCUsing NOS1, NOS2, NOS3 monoclonal antibody and fluorescence two resistance, and were labeled CD 133, ALDH stem cell marker, by flow cytometry, detection of CSC and nonCSC (CD133+ and CD133-, ALDH+ and ALDH-cells) expression of NOS1, NOS2 and NOS3, through the statistical analysis of NOS in CD133+and CD133-, expression differences in ALDH+and ALDH-. And flow cytometry results through western technology verification;2. Clinical relevance of NOS expression and nasopharyngeal carcinomaIn western region of Guangdong Provincial People’s Hospital from 2003 January to 2005 May 80 cases of nasopharyngeal carcinoma tissue specimens and 22 cases of epithelial tissue of chronic nasopharyngeal specimens. All patients with nasopharyngeal carcinoma biopsies were without any discharge treatment. Immunohistochemical S-P method, the detection of the expression levels of NOS1, NOS2 and NOS3, the statistical analysis of the relationship between NOS expression and nasopharyngeal carcinoma grading and prognosis of nasopharyngeal carcinoma, so as to clear the relationship between NOS expression with the occurrence and development of nasopharyngeal carcinoma;In vitro experiments, the expression of NOS effects on nasopharyngeal carcinoma CSC funcdon1. Small molecular compounds (L-NAME) inhibiting nasopharyngeal carcinoma CSC function(1) NOS inhibitory control on the proportion of CSC in nasopharyngeal carcinoma:The use of common inhibitors of NOS 1, NOS2 and NOS3 (L-NAME), the inhibition of NOS function, changes of frequency of CSC inhibited nasopharyngeal carcinoma cell line NOS, to investigate the NOS inhibitory effect on nasopharyngeal carcinoma CSC frequency;(2) NOS suppression sphere function on nasopharyngeal carcinoma (tumor sphere formation) Regulation:Using in vitro tumor cells into the sphere experiment, comparison of nasopharyngeal carcinoma cell line L-NAME inhibits NOS and control group, difference sphere ability analysis of nasopharyngeal carcinoma, NOS regulates the expression into a sphere on the ability of;(3) NOS inhibited the cytotoxic effect of glomus cells in nasopharyngeal carcinoma:Nasopharyngeal carcinoma cell line into the sphere after incubation with NOS, pan inhibitors (L-NAME) are killing experiment, compared to L-NAME treatment group and the control group tumor sphere size, regulating effect of NOS on the growth of nasopharyngeal carcinoma tumor function of sphere, to investigate the killing effect of L-NAME on nasopharyngeal carcinoma sphere;(4)Analysis of NOS inhibitory regulation of nasopharyngeal carcinoma stem cell transcription factor:Using the real-time PCR technology and Western Blotting Technology, detection of nasopharyngeal carcinoma in L-NAME group and control group, tumor stem cell factor NANOG, expression of SOX2 and c-MYC, to investigate the regulation of tumor inhibition of NOS expression of stem cell factor;(5)Analysis of NOS1, NOS2 and NOS3 respectively on nasopharyngeal carcinoma CSC:A specific inhibitor of NOS1, NOS2 (NPLA and 1400W) and NOS pan inhibitor L-NAME, NOS inhibition of nasopharyngeal carcinoma cells. In NPLA group,1400W group and L-NAME group and the control group (group PBS) in nasopharyngeal carcinoma CSC frequency, sphere function, and tumor sphere killing function were analyzed and compared, to explore the role of NOS2 NOS1, and NOS 3 respectively on nasopharyngeal carcinoma CSC.2. NOS1 silencing regulating nasopharyngeal carcinoma CSC function(1) The establishment of NOS1 silencing and the control of nasopharyngeal carcinoma cell lines:Using the technology of shRNA and lentiviral vectors, NOS1 silencing on nasopharyngeal carcinoma cell line CNE2, and screening of stably transfected cell lines (sh-NOSl-CNE2) transfected with unrelated sequence, and a control cell line (con-NOS1-CNE2). And the two cell proliferation, cell cycle and transfer function were detected, analysis of biological function of NOS 1 silencing in nasopharyngeal carcinoma cells(2) Analysis of NOS 1 silencing on nasopharyngeal carcinoma cell line CSC and the frequency of sphere function:Using NOS 1 nasopharyngeal carcinoma cell line sh-NOSl-CNE2 silencing and control con-NOSl-CNE2, two strains of cells and the frequency of CSC into the sphere ability, further confirmed the role of NOS in regulation of nasopharyngeal carcinoma CSC function.3. L-name inhibiting nasopharyngeal carcinoma in nude mice(1) L-NAME on the growth of transplanted tumor of nasopharyngeal carcinoma effect:Nude mice model of nasopharyngeal carcinoma cell line CNE2, when the tumor grew to lcm in diameter, intraperitoneal injection of L-NAME (experimental group), the control group were injected with PBS. Comparison of L-NAME group with PBS group, the difference of volume and weight of tumor, to investigate the inhibition of NOS inhibited the growth of transplanted tumor;(2) Effect of L-NAME on nasopharyngeal carcinoma xenografts in the frequency of CSC:Nude mice transplantation tumor samples were taken from the L-NAME group and the PBS group, the proportion of CSC streaming technology, inhibitory effect on nasopharyngeal carcinoma in nude mice CSC frequency level verification NOS;(3) L-NAME on the regulation of the expression of stem cell factor in nasopharyngeal carcinoma xenografts:Nude mice transplantation tumor samples were taken from the L-NAME group and the PBS group, the stem cell factor (NANOG, SOX2 and c-MYC), real-time PCR and Western blotting detection, comparison between L-NAME group and control group differences in dry cytokine expression, inhibit the in vivo experiment in nude mice NOS verification regulation of tumor stem cell factor expression;Mechanism of NOS regulation on nasopharyngeal carcinoma stem cell function1. Expression microarray analysis of genome:Stem cell function related gene regulation of NOS 1 in nasopharyngeal carcinoma cell lines:Choose the relatively high tumorigenicity of three nasopharyngeal carcinoma cell lines; using NOS pan inhibitors (L-NAME) for NOS suppression. The three cell lines in the experimental group (group L-NAME) for whole genome expression profile detection and control group (affimatrix chip). The experimental group and the control group were ANOVA comparative analysis, differential gene screening of NOS regulation. And the use of biological information online software screening the NOS-gene for regulation of stem cell related (NOS-CSC-gene).2. NOS regulation of stem cell related genes involved in pathways:Using DIVAD software, pathway involves the analysis of the NOS-CSC-gene; and analyses the main pathway genes involved in regulation of NOS cells of nasopharyngeal carcinoma stem from.3. Molecular network stem cell gene regulated by NOS:Using string software, the molecular correlation network analysis consisting of NOS-CSC-gene, combining with the main stem cell pathways involved in NOS-CSC-gene, the molecular target of NOS regulation;4. Molecular biological experiment:Verification regulation of NOS pathway in nasopharyngeal carcinoma cells in nasopharyngeal carcinoma cell lines: Nasopharyngeal carcinoma cell line L-NAME suppression (L-NAME group) and the control cells (group PBS), total protein extraction, key molecules in NOS pathway bioinformatics analysis of Western blotting were tested, results in bioinformatics; Nasopharyngeal carcinoma xenografts in nude mice NOS verification regulation of nasopharyngeal carcinoma cells:The inhibition of L-NAME (L-NAME group) and the control group (group PBS) nasopharyngeal carcinoma xenografts in nude mice, Western blotting and immunohistochemical analysis, verify the bioinformatics results, to further clarify the molecular mechanism regulated by NOS in nasopharyngeal carcinoma cells;Results:1. NOS expression in nasopharyngeal carcinoma cell line was positively correlated with the potential of malignant and the frequency of CSC in nasopharyngeal carcinomaReal Time-PCR and Western blot detection showed:six strains of nasopharyngeal carcinoma cell line expressed NOS1 and NOS2, and the expression level of NOS2 is relatively high, but the expression of NOS3 is low, Western blot did not detect significant expression; immune fluorescent display, NOS1 and NOS2 mainly showed as table as part of cytoplasm, nuclear expression. While NOS3 and only trace amounts of cytoplasmic expression;Through the six nasopharyngeal carcinoma cell lines invasion, proliferation and CSC frequency (CD133, ALDH and SP labeled CSC) detection, analysis of nasopharyngeal carcinoma cell lines (malignant phenotype of tumor invasion, function) and CSC frequency, the relationship between the expression of nasopharyngeal carcinoma and NOS, found that six of two cell lines, NOS1 and NOS2 expression volume and cell migration and tumorigenicity of function, and CSC was positively correlated with the frequency, high expression quantity transfer function of 5-8F and high tumorigenic functions of CNE2 NOS1 and NOS2 were significantly higher than that of other four cell lines (p<0.05).2. Expression of NOS1 and NOS2 in nasopharyngeal carcinoma tissuesThe results of immunohistochemistry showed that,80 cases of nasopharyngeal carcinoma,36 cases of NOS1 expression was low, while 44 cases showed high expression, expression sites located in the plasma of cancer cells, the positive expression rate of 55%. In 80 cases of nasopharyngeal carcinomas,21 cases of NOS2 expression was low, while 59 cases showed high expression, the positive expression rate of 73.7%. Expression sites are mainly located in the cell cytoplasm and nuclei.Between the expression of NOS1 and NOS2 protein and clinical pathological factors of nasopharyngeal carcinoma:in nasopharyngeal carcinoma, NOS2 expression and tumor invasion (P= 0.044) and clinical stage (P= 0.016) closely related.χ2 test results showed that, NOS2 and CSC markers OCT4 and Nanog are closely related, suggesting that its expression may be related with nasopharyngeal carcinoma cell dry. While NOS1 expression and patient’s sex, age, histological type, T stage, N stage and M stage, no obvious relationship, and also CSC no statistical significance3. NOS inhibitors and NOS-RNAi decreased CSC function in nasopharyngeal carcinoma cell linesThe NOS pan inhibitors of L-NAME reduce nasopharyngeal carcinoma cell frequency of CSC, its inhibitory effects were dose dependent inhibition of nasopharyngeal carcinoma cells; L-NAME sphere function, and has a killing effect on nasopharyngeal carcinoma cell sphere has growth. Moreover, nasopharyngeal carcinoma cell concentrations of L-NAME killer CSC sphere on conventional culture only growth inhibition but not apoptosis and necrosis phenomenon. L-NAME specific cytotoxic CSC show, NOS has more functions related to CSC cells. Application of NOS1 or NOS2 specific inhibitors (NPLA and 1400W) alone, no effect has been found on nasopharyngeal cancer stem cells inhibition, suggesting that the regulation of NOS1 and NOS2 may be involved in the function of CSC in nasopharyngeal carcinoma.NOS-RNAi also significantly reduced the proliferation and migration of nasopharyngeal carcinoma cell line CNE2, reduced CNE2 and the frequency of CSC clone formation rate, which proves that NOS1 is involved in the regulation of nasopharyngeal cancer stem cell function in gene level. But the NOS2-RNAi experiment has not completed.Western blot experiments showed that NOS inhibitors or NOS1-RNAi were down regulated the expression of CSC gene in nasopharyngeal carcinoma cells such as NANOG, c-myc, NOS in nasopharyngeal carcinoma stem cell function regulation at the molecular level.4. NOS inhibitor (L-NAME) significantly inhibits the growth of nasopharyngeal carcinoma xenograftCNE2 nude mice transplantation tumor model:injection of L-NAME inhibitor significantly inhibited the growth of transplanted tumor of experimental group CNE2, the tumor weight and diameter were significantly less than that of the control group (P<0.05). And the transplanted tumor showed necrosis. Western blot and immunohistochemistry showed:expression levels of NANOG and c-myc in L-NAME xenografts, the treatment group was significantly lower than the control group, suggesting that the antitumor effect of L-NAME by inhibiting the function of stem cells and nasopharyngeal carcinoma.5. NOS promote nasopharyngeal carcinoma CSC function through PI3K/AKT pathwayThrough the comparison of nasopharyngeal carcinoma cell line (SUME1/5-8F/CNE2) of whole genome chip L-NAME treatment group and control group, we screened 1700 differentially expressed genes (ANOVA, P<0.05), and these genes by geneclin software online analysis, screening out the stem cell associated gene 122. These genes were involved in cell growth and differentiation. Expression analysis of DIVID software, these genes are involved in signaling pathways for the growth factor GF/ growth factor receptor GFR/RAS/PI3K/AKT/ stem cell transcription factor. Expression analysis of String software, these genes constitute the molecular network formed by RAS and AKT node as the center, the results support the DIVID software.In order to verify the bioinformatics analysis results, we performed Western blot detection in nasopharyngeal carcinoma cell line L-NAME treated group and the control group in the phosphorylation of AKT protein and PTEN protein, the results showed that L-NAME inhibited the phosphorylation of AKT protein in nasopharyngeal carcinoma cells, the phosphorylation of AKT protein up regulation of PTEN. To prove the mechanism of NOS function through the AKT pathway in regulation of nasopharyngeal carcinoma CSC.Conclusion:1. The expression of NOS in nasopharyngeal carcinoma cell line was positively correlated with the malignant phenotype (invasion and tumor formation) and the CSC frequency. NOS promoting CSC function and the malignant phenotype in NPC;2. NOS promotes phosphorylation of AKT and up-regulates the stem-cell transcription factor in nasopharyngeal carcinoma tissue and cell lines (NANOG and c-myc), suggesting that the NOS induce cancer stem cell function is PI3K/AKT pathway evolved;3. NOS inhibitor (L-NAME) significantly inhibits the growth of nasopharyngeal carcinoma xenograft. Moreover, L-NAME cut the stem cell transplantation factor and the CSC frequency which suggesting that NOS may become the target of nasopharyngeal carcinoma stem cell therapy;4. The expression of NOS2 was significantly negatively correlated the grading and prognosis of nasopharyngeal carcinoma and the expression of stem cell factors, suggesting NOS2 may become a marker of prognosis and treatment in nasopharyngeal carcinoma;... |
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