| Backgroud: In the solid tumor tissues, in addition to a large number of tumor cells, it also includes fibroblasts, endothelial cells, extracellular matrix and immune cells. Infiltration of immune cells into tumor tissue include: macrophages, dendritic cells(DC) and lymphocytes, these immune cells form an inflammatory microenvironment in the tumor tissue. The macrophages can helps the growth and proliferation of tumor cells, and inhibit tumor immune response, leading to tumor cells immune escape.Macrophages are clustered in most tumor tissues, which is directly involved in the function of endothelial cells by secreting cytokines, growth factors, chemokines and matrix degrading enzymes, and promotes the migration of endothelial cells through the extracellular matrix reconstruction. At present, tumors of various treatment methods(including radiotherapy, chemotherapy and anti-angiogenesis drugs) rendered tolerant state, which may be because: these methods can increase the infiltration of macrophages in tumor tissues, thereby breaking the balance between of the pro-angiogenic and anti-angiogenic signal pathway in the tumor microenvironment.Tumor associated macrophages(TAMs) are derived from the mononuclear cells of the bloos system, it differentiate into TAMs in the tumor tissue under the action of chemokines. Colony stimulating factor secreted by tumor cells can prolong the survival time of TAMs. When TAMs is moderately activated(M1 type macrophages) in the tumor environment, it can kill tumor cells and destroy vascular endothelial cells, thereby inhibiting tumor development. However, if this stimulation is not inhibited in a short time, TAMs will be polarized into M2 type which under the action of many cytokines secreted by tumor cells, this is also the reason of TAMs most of the M2 type in tumor tissues. In contrast to M1-like macrophage, the role of M2-like includes the secretion of growth factor, angiogenesis factor and protease, which can stimulate tumor cell proliferation, degradation of extracellular matrix, promote angiogenesis and tumor cell invasion and migration, and escape the surveillance of anti-tumor immunity. Thus, in recent years the TAMs is induced to re-polarization in tumor tissues that TAMs is shift from M2-like to M1-like is a important target for tumor therapy.Endostatin is one of the most effective angiogenesis inhibitors, which can inhibit tumor angiogenesis and inhibit tumor growth and metastasis by inhibiting the proliferation and migration of endothelial cells. In recent years, studies have found that in addition to the inhibition of angiogenesis, Endostatin also have some immunological effect. Endostatin treated mice tumor model can induce a large number of leukocyte infiltration, and increased the infiltration of cytotoxic lymphocytes, inhibit tumor immune escape cells-Treg cells activation, suggesting that Endostatin can enhance anti-tumor immune response in tumor microenvironment.TAMs is prefers the anoxic environment, and is often clustered in the avascular area and the necrotic area. Vascular endothelial growth factor(VEGF) can induce monocytes to the tumor site and differentiate of M2-like TAMs. TAMs can autocrine of VEGF, platelet-derived factor(PDGF) and fibroblast growth factor(FGF), and other vessels growth factor, thus forming a vicious cycle. In addition, placental growth factor(PIGF) can also activate the generation of abnormal angiogenesis in tumor, and inhibit the anti-tumor immune response mediated by T cells, and promote TAM polarized to M2-like that can promote tumor growth and angiogenesis. Therefore, we hypothesized that the inhibition of Endostatin on tumor in addition to affecting tumor angiogenesis, but also by inhibiting the expression of PIGF and VEGF affect the polarization of TAMs.Objective: The expression of Endostatin was up-regulated by p Endostatin over-expression plasmid. To observe the effect of Endostatin on the polarization of macrophages in vivo and in vitro, and the effect of Endostatin induced macrophage activation to tumor cells, and the possible mechanism was preliminarily discussed.Method: Murine macrophage cell RAW264.7 and bone marrow-derived macrophages(BMDMs) were cultured in vitro, and the cells were divided into Control group, p Scramble group and p Endostatin group that were transiently transfected with empty plasmid and p Endostatin plasmid that over-expressing Endostatin, respectively. MTT and HE staining method for the detection of proliferation activity and morphology of macrophages; the expression of M1 and M2 related markers were detected by flow cytometry; the expressin of protein and m RNA in macrophage polarization related markers were detected by ELISA and q RT-PCR; Luciferase reporter gene assay activity of NF-κB in macrophages; Western blot method to detect changes in the expression of NF-κB-related gene and its downstream gene.4T1 cells and B16 cells were co-cultured with normal RAW264.7 cells(Control group) and RAW264.7 cells transfected with p Endostatin(M1 group) in 48 h, respectively. 4T1 cells and B16 cells were re-collected for the following experiments. The effect of M1-like macrophages on the proliferation activity of tumor cells was observed by MTT and colony formation assay; the effect of M1-like macrophages on the migration and invasion of tumor cells was observed by scratch and Transwell invasion assay; Flow cytometry to observe the effect of M1-like macrophages on apoptosis and cell cycle of tumor cells; Western blot detect the changes in the expression levels of related proteins.Construction of mouse breast cancer xenograft model, the mice was divided into Control group, PQ group, SL/p Scramble group and SL/p Endostatin group. p Scramble plasmid and p Endostatin plasmid were delivered by Attenuated Salmonella Typhi Ty21 a and mice were treated by intraperitoneal. The tumor growth index and tumor index were observed and calculated; the effects of Endostatin on tumor blood vessels were observed by immunohistochemical staining and scanning electron microscopy; the proportional change of M1-like and M2-like in TAMs was observed by flow cytometry; ELISA and q RT-PCR method to observe the expression of macrophage polarization related markers in tumor.Result: The expression level of Endostatin in RAW264.7 cells and BMDMs was significantly increased after transfection of p Endostatin expression plasmid. Upregulation of Endostatin expression can induce RAW264.7 and BMDMs polarization to M1 type macrophages, enhanced transcription activity of NF-κB in the cells, and the expression of p-p65 and p-STAT1 were up-regulated, but the expression of PIGF, VEGF-A and p-STAT3 were down-regulated.Endostatin induced M1-type macrophages can inhibit the proliferation and migration of 4T1 cells and B16 cells, and the expression levels of MMP-9, MMP-2 and PCNA were decreased; at the same time, it can promote the apoptosis of B16 cells and up-regulate the expression of Bax, c-Caspase8 and c-Caspase3, down-regulate the expression of Bcl-2, but no effect on apoptosis of 4T1 cells; Endostatin induced M1-type macrophages can induce G2 phase arrest in 4T1 cells and up-regulate the expression of p21, CDK1 and Cyclin B1, but no effect on cell cycle of B16 cells.Endostatin can inhibit the growth of mouse breast cancer xenograft, reduce the proportion of M2-like TAMs and increase the proportion of M1-like TAMs in tumor tissues; inhibit the expression of VEGF-A and PIGF and reduce angiogenesis and make it morphology normalization in tumor tissues. These results indicate that Endostatin can change the polarization type of TAMs in vivo.Conclusion: Increased expression of Endostatin can enhance the transcriptional activity of NF-κB and affected the expression of the downstream genes, induced RAW264.7 cells and BMDMs to produce M1 type polarization, which can inhibit the proliferation of 4T1 and B16 cells, so that make it happen cell cycle arrest and apoptosis, respectively. Endostatin over-expression can inhibite the expression of VEGF-A and PIGF, so that make TAMs to M1 type of polarization, thereby enhancing the anti-tumor immune response. |
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