The Role Of Tumor Associated Macrophages In Gastric Cancer By Secreting CCL5 And Its Mechanism

Gastric cancer is ranked in the fourth place among the world’s most common malignant tumors, although the incidence is decreased, but the number of gastric cancer in China still accounts for 42% of the world, ranking the first of the East Asia common cancers. At present, gastric cancer patients can choose chemotherapy and surgical treatment, but the 5 year survival rate is still around 50%, one of the main reason leading to death is the invasion and metastasis of cancer. Therefore, it is very useful to study the specific mechanisms of gastric cancer development, development and metastasis, and to make early prediction and intervention.The relationship between gastric cancer and tumor associated macrophages is increasingly close, but there is less research on the mechanism of tumor associated macrophages promoting the proliferation, migration and invasion of gastric cancer. Wu H reported that tumor associated macrophages promoted angiogenesis and lymph node metastasis of gastric cancer through the expression of VEGF and VEGF-C.Some evidence suggests that chemokines and their receptors played a role in the regulation of tumor local inflammation and inhibition of immune system response to anti tumor response. Chemokine ligand 5(CCL5/RANTES)belongs to the CC chemokine family, CCR1, CCR3 and CCR5 are its receptor.CCL5 is mainly expressed in T cells, macrophages, platelets, synovial fibroblasts, renal tubular epithelial cells and some types of tumor cells. CCL5 plays an important role in tumor and inflammatory diseases. Recent studies have found that the expression of CCL5 was directly related to the disease progression in breast cancer patients, In addition, the expression of CCL5 was associated with disease progression in cervical cancer, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer and melanoma in tissue and serum. But now there is less study on the relationship between the expression of CCL5and gastric cancer.Based on the above theory, we conducted a study as follows: First, we contrasted the difference expression of CCL5 in gastric cancer tissues vs adjacent tissues and gastric cancer patients vs healthy volunteers serum, and analyzed the relationship between CCL5 and CD68 which was the marker of tumor associated macrophages, at the same time, the relationship between the pathological factors and the biological behavior of gastric cancer vs the expression of CCL5 in tissue and serum was analyzed. The results showed that CCL5 was highly expressed in tumor stroma of gastric cancer, and its expression level was closely related to the stage, prognosis and distant metastasis of gastric cancer. Therefore, we did deep research into the mechanism about how CCL5 secreted by tumor associated macrophages played a role on gastric cancer cells in vitro. So we can provide new ideas and targets for clinical treatment of gastric cancer, This has a very important value for the design of the anti cancer program.As there is an upward trend in the incidence and mortality, tumor becomes a serious threat to human health. At present, the efficacy of chemotherapy is not ideal. The main reason is the side effects of traditional antitumor drugs in the clinical treatment. Therefore,looking for new high efficiency and low toxicity of antitumor drugs is a major subject in the field of biomedicine. Natural product chemistry has been the important way in development of new pharmaceutical industry. Natural products have the advantages of novel structure, high curative effect and side effects, so the natural products chemistry and its research results have been widely used in pharmaceutical.The research contents and results are as follows:Part I The expression of CCL5 in tumor associated macrophages(TAM)of ganstric cancer and the study of their correlationship Objective: In order to study the expression of CCL5 in tumor associated macrophages(TAM)of ganstric cancer and explore the relationship between them.Methods: 48 cases patients with completed clinical and pathological dataof Gastric cancer paraffin block specimens were selected.cancer tissue and paracancerous tissues were used as control, using SP immunohistochemical method to detect CD68 and CCL5 in gastric cancer tissues and adjacent tissues, and using the Spearmann correlation statistics statistical methodsfor the correlation.Results:1 CD68 was positive expressed in macrophages of gastric stroma. Clear yellow or brown particles in cytoplasm were considered as positive. In paracancerous tissues only 1 case showed weak positive expression, analysied by paired T test according to the pathological grading, with significant difference(P<0.01).The expression level of CD68 in gastric cancer and the depth of invasion, lymph node metastasis, TNM staging and tumor differentiation were correlated(P<0.05).2 CCL5 was positive expressed in macrophages of gastric stroma. Clear yellow or brown particles in cytoplasm were considered as positive. In paracancerous tissues showed negative expression, analysied by paired T test according to the pathological grading, with significant difference(P<0.01).The expression level of CCL5 in gastric cancer and the depth of invasion, lymph node metastasis, TNM staging and tumor differentiation were correlated(P<0.05).3 By Spearmann correlation analysis, the expression of CD68 and CCL5 in gastric cancer tissues were significantly correlated, and was positively correlated(r=0.759,P<0.01).Conclusion:CD68 and CCL5 in invasion and metastasis of gastric cancer occurrence, may play an important role in promoting, and the expression of CCL5 and CD68 showed a positive correlation, prove that CCL5 secretion may be one of the important reasons that TAM promote the invasion and metastasis of gastric cancer.Part II The expression of CCL5 in serum of patients with gastric cancer and its clinical significance Objective: In order to detect the expression of CCL5 in serum of patientswith gastric carcinoma and to explore its clinical significance.Methods: Serum specimens were extracted from 50 cases of newly diagnosed patients with gastric cancer(from none underwent operation and chemotherapy) and 16 healthy volunteers, ELISA was used to detect the expressionof CCL5 in gastric cancer patients and healthy volunteers in the serum,the differences between the expression, relationship between pathologicalfactors and biological behavior of gastric cancer and expression of CCL5 were studied.Results:1 Compared with control group, serum CCL5 expression level in gastric cancer patients was significantly higher than that in control group(P<0.001),and the expression level of CCL5 was significantly higher in patients with tumor invasion, tumor thrombus formation, tumor thrombus formation, and distant metastasis, tumor thrombus formation, and distant metastasis,with statistical difference(P<0.01).Conclusion: The high expression of CCL5 in serum of gastric cancer patients increased significantly, the expression of CCL5 and gastric cancer invasion depth, lymph node metastasis, TNM staging and tumor differentiationwere correlated, no correlation with gender and age of patients.Part III The effection of tumor associated macrophages on the biological behavior of gastric cancer cells by high expression of CCL5Objective: In order to investigate the effect of CCL5 which coming from Tumor associated-macrophages(TAM) on the biological behavior of gastric cancer cells, and to investigate the mechanism of TAM in the invasion and metastasis of gastric cancer.Methods: In vitro,the human monocytic cell line THP-1 Cell was induced by IL-4,then formed M2 type macrophages, cocultured with gastric cancer cell line AGS,then formd gastric cancer microenvironment, to verify the TAM can promote the proliferation, invasion and metastasis of gastric cancer cells by secretion of CCL5.RT-PCR detected the expression of cytokines and their receptors in gastric cancer cells and macrophages, Westernblot was used to detect STAT3 signaling activity changes and downstream target protein expression changes.Results:1 MTS cell proliferation assay showed that the proliferation rate of gastric cancer cells co-cultured with supernatant of macrophages induced by IL-4 was(397.57 + 28.82)%, after 5 days of co-culture, and it was higher than that of co-cultured group with THP-1 supernatant(385.95±32.56)% and group of cells not co-cultured( 320.49±7.62) %,(P<0.05), respectively, with statistical significance. The number of gastric cancer cells co-cultured with supernatant of macrophages induced by IL-4 were significantly higher than that of co-cultured group with THP-1 supernatant and group of cells not co-cultured(P<0.05).2 Compared with group without co-cultured with the supernatant, gastric cancer cells co-cultured with supernatant of macrophages induced by IL-4 and THP-1 supernatant throuth the chamber increased significantly, and their ability of migration was enhanced(P<0.01), with statistical significance.3 Compared with group without co-cultured with the supernatant, the number of colonies formed of gastric cancer cells co-cultured with supernatant of macrophages induced by IL-4 and THP-1 supernatant increased significantly(P<0.01), respectively,38.00±7.21/HP and 58.33±3.79/HP. The number of colonies in gastric cancer cells co-cultured with supernatant of macrophages induced by IL-4 was the most(P<0.01), with statistical significance.4 It was shown that the m RNA expression of CCL2,CCL4,CCL17 and CCL22 were unaffected.Treatment with IL-4 after culturing with AGS cells on THP-1 cells,significantly increased CCL5 m RNA expression(P<0.01),and modestly increased MMP2 and MMP9 m RNA expression(P<0.01).Treatment with IL-4 after culturing with supernant of THP-1 resulted in a significantly increased of CCR5 m RNA expression(P<0.01).5 Compared with group without co-cultured with the supernatant, the relative expression of CCL5 receptor CCR5 m RNA in gastric cancer cells wassignificantly increased in group co-cultured with supernatant of macrophages induced by IL-4 and THP-1 supernatant(P<0.01). Group co-cultured with supernatant of macrophages induced by IL-4 was the most(P<0.01), with statistical significance.6 CCL5(0μM/ml、10μM/ml、50μM/ml) were added to AGS, and the number of cells that migrated increased with the increase of CCL5concentration(P<0.01), and the number of cells migrated at CCL5(50μM/ml),which was 28.33 + 5.13/ HP higher than that of other groups(P<0.01), with statistical significance(P<0.01).7 The expression of Stat3 and p-Y705(p-S727 and p-Stat3) was significantly increased after co-cultured with TAM derived from THP-1 cells,higher than that of the gastric cancer cell line and the THP-1 co-culture group(P<0.01), P-Stat3(p-S727 and p-Y705) trends were consistent with Stat3(P>0.05).Conclusion: After co-cultured with the supernatant of THP-1,The proliferation and clone forming ability of gastric cancer cells were enhanced,and the expression of CCL5 m RNA in THP-1 cells was the highest, while the expression of CCR5 m RNA was the highest in AGS cells.The number of gastric cancer cells increased with the increase of CCL5 concentration. Stat3 signal and its target protein expression increased after co culture of AGS.