| Section I Mechanism of CCL22 downregulation by fucoidan and the effect on macrophages functionBackgroundThe immune system is one of the important systems. Through the unique identification of "self and non-self component, immune play the role in immune defense (against pathogenic microbial infection), immune surveillance (monitoring and removal of malignant tumor cells, etc.) and maintain internal balance. A large number of studies have confirmed that the immune system dysfunction related to the occurrence and progress of body infections, autoimmune diseases, tumors and many other diseases. In cellular level, immune cells (lymphocytes, monocytes and macrophages, etc.) and immune molecules (cytokines, chemokines, etc.) compose the differentiation and functional status, which maintain the basis of normal immune response. Macrophages are one of the key groups in immune cells, which connect the inmate immune response and adaptive immune response, and involved in the regulation of inflammation, angiogenesis, tissue repair and tumor invasion and metastasis. The normal or abnormal function of macrophages is usually related to their differentiated phenotypes, and play a key role in the progression of many immune-related diseases such as autoimmune diseases, tumors and atherosclerosis. Therefore, the mechanism of macrophage function’ regulation has great theoretical and practical value in the treatment of macrophages through targeting immune intervention.Cancer has become the number one killer which threat to human health, so the research hotspot in the field of life sciences is finding effective therapeutic targets and therapeutic drug. In the complex tumor microenvironment, the interaction of tumor cells, stroma, blood and lymph vessels and infiltrating immune cells provide favorable conditions for tumor survival, proliferation, migration and metastasis. Affected by microenvironment, the killing effect of tumor cell by infiltrating immune cells are significantly inhibited, which even promote tumor growth. Macrophages are the most common cell population in tumor infiltrating immune cells, which accounts for 30%-50%, known as tumor associated macrophages (TAM). A number of studies have shown that the degree of invasion closely related to tumor prognosis. Accordingly, disclosure the internal regulatory mechanisms of TAM and finding the right formulation of its regulatory function are the new target for cancer therapy, which will improve the current cancer treatment.Macrophages represent plasticity through changing the polarization state to accommodate different physiological or pathological conditions. Currently, according to the immune function, macrophages can be divided into "classically-activated macrophages (M1)" and "alternatively-activated macrophages (M2)" subtypes. The research for many types of tumors found that TAM has the M2 phenotype induced by the tumor cells and T lymphocytes produced cytokines, which released type II cytokines, promoted the formation of blood and lymphatic vessels, enhanced tumor cell proliferation, migration and exerted immunosuppressive properties. M2 macrophages can be converted into M1, such activation is sufficient on its own to cause tumor rejection. Chemokines belong to a superfamily of small proteins with the role of cell chemoattractant in immune and inflammatory reactions, which has been a new dimension of transcriptional profiling to the characterization of different forms of macrophage activation. Among them, being the typical chemokine of M2 phenotype, the high level of CCL22 can cause poor prognosis of various types of cancer patients, which attracted widespread attention. As CCL22 receptor, CCR4 was expressed on the membrane of many types’tumor cells and T lymphocytes. On the one hand, M2 macrophages promote tumor cell migration via CCL22/CCR4 axis. On the other hand, since T lymphocytes are the core of the immune response, the study on the chemotaxis of CCL22/CCR4 mainly focuses on CCR4+T lymphocytes (including: Th2 and Treg cells, which belong to CD4+T lymphocytes). In tumor microenvironment, a large number of Treg cells infiltration and Th1/Th2 drift can protect tumor cells from immune surveillance and attack, which can promote the development of tumor.Chemotherapy has been widely used in the clinical treatment of cancer since 1960s. The toxic and side effects of chemotherapy drugs have been a key problem in the research and development of new chemotherapeutic drugs and the effect of tumor therapy. Therefore, natural medicine as a tumor therapy drug or adjuvant chemotherapy drugs has great application prospects. Being a kind of water soluble dietary fiber, fucoidan is extracted from brown algae surface. A number of studies have shown that fucoidan has anti-tumor, anti-inflammatory, hypolipidemic, antioxidant and other biological effects. The antitumor effect of fucoidan is mainly manifested as inhibition of tumor cell growth and promoting lymphocyte proliferation and anti-tumor cytokine secretion. There is no study on the effect of fucoidan on M2-like TAMs functions. Whether there exists possible pathways and mechanisms of macrophages as a tumor suppressor by fucoidan regulation, is urgent problem to be solved. Therefore, the research contains four parts to explore the effect of fucoidan on macrophages, especially M2 subtype, in order to provide new ideas and targets for antitumor research about TAMs.Part I. Effect of Fucoidan on macrophage polarizationObjective:To assess the effect of fucoidan on the polarization of macrophages and specific cytokine expression.Methods:THP-1 cells were induced to MO (unpolarized), Ml and M2 subtypes. Under fucoidan treatment, we observed the morphological changes in three types of macrophages and detected the M1 and M2 subtype cytokines by quantitative real-time polymerase chain reaction (qRT-PCR).Results:1. In THP-1 derived macrophages, fucoidan has no significant effect on macrophage morphology.2. As M1 markers, the expression of TNF-a was significantly down-regulated by fucoidan during Ml macrophages polarization process.3. For M2 type cytokines, the fucoidan significantly down-regulated expression of TGF-β in M2 macrophages polarization process.Summary:During polarization process, fucoidan affected part of M1 and M2-type cytokines, but there was no significant effect to the M1 or M2 polarization under fucoidan treatment.Part II. Fucoidan regulates the CC chemokines profiles and particularly down-regulate the CCL22 expression and secretion level in M2 macrophagesObjective:To study the regulation of fucoidan on CC-chemokines in macrophage polarization process. Focus on the chemokines CCL22 and explore the factors inducing CCL22 high expression and the regulation of fucoidan.Methods:qRT-PCR detect the transcription of CC-chemokines in macrophage polarization process under fucoidan treatment. Using qRT-PCR and ELISA techniques, analyzed the difference effect of M2-inducible factor IL-4, IL-13 and fucoidan in the regulation of CCL22 expression in THP-1 derived M2 polarized model. Using CD 14 MACS(?) technology, monocyte isolated from human peripheral blood mononuclear cells and then differentiated into macrophages for further verification.Results:1. CCL2 and CCL22, which were respectively described as Ml and M2 markers, were significantly higher in Ml or M2-like macrophages.2. Fucoidan down-regulated the mRNA expression of CCL2, CCL4, CCL5 and especially CCL22 in M0, M1 and/or M2-like macrophages.3. The expression of CCL22 was up-regulated by IL-4 and IL-13, significantly. Among them, IL-4 played the major role in the up-regulation of CCL22 expression and secretion. And IL-13 may play a synergic effect.4. Fucoidan notably reduced the expression and extracellular concentrations of CCL22 in THP-1/monotype-derived M2 polarized macrophages.Summary:Fucoidan significantly inhibited IL-4& IL-13 induced high expression and secretion level of CCL22 in M2 macrophages.Part Ⅲ. The influence of fucoidan on the M2 macrophages’ characteristic of recruitmentObjective:On the basis of chemokine regulation by fucoidan, explore the influence of tumor cells migration and T lymphocytes cells recruitment mediated by macrophages under fucoidan treatment.Methods:1. Using transwell assay, detect the role of fucoidan on M2 macrophages media MHCC-97H cells migration and using CCL22 neutralizing antibodies and recombinant proteins to validate the association with CCL22 level.2. Using transwell chambers, flow cytometry analysis the T lymphocytes cells recruitment mediated by macrophages under fucoidan treatment.3. Magnetic beads isolated CD4+T lymphocytes, analysis the regulatory T lymphocytes (Treg) recruitment mediated by macrophages under fucoidan treatment.Results:1. CCL22 neutralizing antibody or fucoidan pretreatment significantly inhibited the number of migrated MHCC-97H cells.2. Fucoidan pretreatment significantly inhibited the proportion and number of M2 condition media recruited CD4+T lymphocytes.3. Medium from the fucoidan pretreated THP-1-derived M2 macrophages recruited significantly less CD4+CD25+FoxP3+(Treg) cells, which related to the supernatant CCL22 level.Summary:Fucoidan inhibited the effect of M2 supernatant on tumor cell migration and CD4+T lymphocytes, especially Tregs, recruitment via the down-regulation of CCL22.Part IV. CCL22 down-regulation in THP-1-derived M2 macrophages by fucoidan treatment via NF-κB pathwayObjective:Explore the cellular regulatory mechanisms in fucoidan media CCL22 downregulation.Methods:1. Western blot analyzed the change of AKT, p38-MAPK and p65-NF-κB phosphorylation levels under fucoidan treatment.2. Using qRT-PCR and ELISA to detect the CCL22 expression under pathway inhibitor treatment.3. Analyzed the p65-NF-κB nuclear translocation to verify the impact of fucoidan on NF-κB activity.Results:1. the phosphorylation of p38-MAPK were up-regulated by fucoidan stimulation in macrophages. At the same time, the phosphorylation of AKT and p65-NF-κB were inhibited.2. Wortmannin (PI3K-AKT pathway inhibitor) can significantly increase the expression and secretion level of CCL22, p38-MAPK pathway inhibitor SB203580 and NF-κB pathway inhibitor BAY 11-7082 can significantly down-regulate the expression and secretion level of CCL22.3. Wortmannin which inhibited the PI3K-AKT pathway, promoted the phosphorylation of p65-NF-κB and p38-MAPK; SB203580 which inhibited the p38-MAPK pathway, inhibited the phosphorylation of p65-NF-κB and AKT at the same time. Similar results of NF-κB activation were observed by immunofluorescence which detected the p65-NF-κB nuclear translocation.Summary:Fucoidan can activate p38-MAPK signal pathway, inhibit PI3K-AKT signal pathway and the activity of NF-κB. Among them, the down-regulation of CCL22 mainly through inhibition of NF-κB transcription activity, and p38-MAPK, PI3K-AKT pathway also has correlation to some extent.Conclusion:Taken together, we reveal an interesting result that fucoidan can inhibit tumor cell migration and lymphocytes recruitment by suppressing CCL22 in M2 macrophages via NF-κB-dependent transcription, which may be a novel and promising mechanism for tumor immunotherapy.Section Ⅱ The influence factor analysis of preoperative neutrophil-lymphocyte ratio as a tumor prognostic indexBackgroundThe basic, clinical and epidemiological studies have confirmed that inflammation is an important risk factor for the occurrence and development of tumor. Innate immune response and activation of inflammation promote the occurrence and development of tumor. At present, the relevant parameters based on peripheral blood testing are often used to assess the inflammatory state of the system, such as:C reactive protein (CRP), albumin, neutrophil count and platelet count. These parameters have important significance in the evaluation of the prognosis of many advanced tumors. In recent years, peripheral blood neutrophil and lymphocyte proportion (neutrophil-lymphocyte ratio, NLR) has been proved to be an independent prognostic indicator for several types of tumor. Studies have indicated that the prognostic accuracy of NLR is better than the traditional tumor staging evaluation.Renal tumor is one of the most common malignant tumors. Renal cell carcinoma (RCC) originated in the proximal tubule, is the most common type of renal tumors, accounting for 90%-95% of renal tumors. The latest NCCN data show that the rate of RCC has increased by 1.7% per year for the past 10 years. Analysis of the SEER database indicates that the 5-year survival rate of kidney cancer at localized disease stage is up to 91.8%, but only 12.3% at advanced disease stage. At present, the treatment of RCC is mainly through the partial or radical nephrectomy. But the survey shows that about 30% of patients had recurrence after nephrectomy. Therefore, as the treatment of tumor come into the "precise medical" model, it is of great significance to evaluate the prognosis of patients, in order to establish a targeted treatment strategy. At present, the accepted RCC prognosis evaluation index come from tumor related factors including:TNM stage, Fuhrman nuclear grade, local infiltration and regional lymph node metastasis or distant metastasis. In order to improve the accuracy of prognostic evaluation, a variety of prognostic scoring systems have been set up to evaluate the prognosis of RCC patients. But at present, like other host-related factors, NLR has not been included in any RCC prognostic scoring system. As a systemic inflammatory index, NLR has been proved to be related to various clinical parameters. There was a positive correlation between NLR and hypertension. One of the clinical indications of RCC is hypertension, and hypertension is also one of the causes of RCC. Therefore, this study aims to evaluate the effect of hypertension on the prognosis of patients with RCC.Objective:Establish the prognostic evaluation method for RCC patients based on NLR. Correlation analysis between NLR and RCC clinical parameters. Using the NLR prognostic evaluation criteria which have been established to compare the recurrent-free survival of RCC patients with or without hypertension, respectively. The evaluation of preoperative NLR’s clinical application values a RCC prognostic indicator.Materials and methods:1. Patient characteristics:Establish selection criteria, the early treatment of 401 cases of renal cell carcinoma patient information included in the study.2. The extraction and processing of sample information:summarized the information contained patients of basic information, pathology information, peripheral information and follow-up reports.3. Calculation of NLR cut-off value:The NLR and follow-up information added to cut-off value analysis platform. Using log-rank test, ROC curves and Cox regression model as the basis for analysis.4. Statistical analysis: Kaplan-Meier survival curves were compared by using log-rank test. Correlation analysis between clinical parameters and NLR was used Pearson chi square test, exact test for qualitative data, and t test for quantitative data. The factors with significant association in univanate analysis were included in multivariate analysis using Cox hazards regression model.Results:1. Study samples included 276 males and 125 females, with an average age of 54 years. There were 175 subjects with history of hypertension, accounting for 43.64%. The average follow-up was 45.5 months.106 cases were recurrence, accounting for 26.43%, and the average follow-up time was 26.4 months.2. The cut-off value of NLR is 3.319. Recurrence-free survival was significantly worse among patients with a preoperative NLR, Larger tumor size, higher T stage, non-clear cell histology and NLR>3.139 were independent factors associated with poor recurrence-free survival.3. Tumor histology, tumor size, TNM stage, Fuhrman nuclear grade and platelet count were all associated with NLR. But NLR was not associated with the history of hypertension.4. In normotensive subgroup, low NLR patients had a better recurrence-free survival. Clinical T stage, tumor histology, NLR and platelet count were independent prognosis factor.5. In hypertensive subgroup, there were no significant difference of recurrence-free survival between high-and low-NLR subjects. NLR should not be used as the indicator for prognostic.Conclusion:After dividing the patients with RCC into normotensive and hypertensive subgroup, the accuracy of NLR on prognosis was affected by hypertension. Being the tumor-related factors, clinical T stage and tumor histology can still indicate the prognosis in this case. So NLR being used to indicate prognosis has limitation. Clinician should take into account including NLR and other pathophysiology factors for predicting tumor outcomes. |
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