Anti-tumor Mechanism Of Magnesium Isoglycyrrhizinate In Regulating The Inflammatory Microenvironment Of Gastric Cancer

The occurrence,development,invasion and metastasis of tumor are closely related to inflammation.Almost every aspect of tumor progression is affected by immune cells and inflammatory factors.Tumor-associated inflammation,which has been included in the seventh feature of tumor,can promote tumorigenesis and progress by promoting angiogenesis and tumor metastasis,inhibiting antitumor immune responses,and altering the sensitivity of tumor cells to chemotherapeutic agents,and this effect is mainly dependent on tumor microenvironment(TME).So,regulating tumor inflammatory microenvironment may take something new to the cancer treatment strategy.Gastric cancer,as the fifth most common malignancy in the world,was always diagnosed in late stage and difficult to treat.Considering its association with Helicobacter pylori(Hp)infection and chronic gastritis,we are trying to find a way to control gastric cancer by regulating inflammatory microenvironment.Magnesium isoglycyrrhizinate(MgIG),which found to have anti-cancer effect recently,may be that one we look for.Magnesium isoglycyrrhizinate is the fourth generation of glycyrrhizic acid,which has effects of anti-inflammatory,anti-virus,anti-oxidation,anti-allergy,regulating immune and anti-cancer.In this study,we firstly verify the anti-cancer effect of magnesium isoglycyrrhizinate on gastric cancer by animal experiments.Then we examined the expression of inflammatory cytokines and the density of immune cells in mouse tumor tissues.After analysis and discussion,we selected macrophages to go on the study.We investigated the effect of magnesium isoglycyrrhizinate on the macrophage polarization in tumor microenvironment in vitro so as to explore whether magnesium isoglycyrrhizinate interfere gastric cancer by regulating the inflammatory microenvironment of gastric cancer.PART 1 Effects of Magnesium Isoglycyrrhizinate on the Expression of Inflammatory Cytokines and Immune Cells in Gastric Cancer MicroenvironmentBackground:It is known that magnesium isoglycyrrhizinate treatment can reduce tumor weight,tumor volume and prolong lifetime of mice in vivo.Besides,the regulation effects of magnesium isoglycyrrhizinate on inflammatory factors and immune cells as well as the effects of inflammatory microenvironment on the progress of gastric cancer are definite.Objective: We aim to detect immune cells and inflammatory factors in gastric cancer tissues of mice,and to find out whether magnesium isoglycyrrhizinate can inhibit the development of gastric cancer by regulating the inflammatory microenvironment of gastric cancer.Methods: First,we selected a PCR Array which can detect the expression levels of 96 factors associated with inflammation and autoimmunity,mostly are Chemokines and receptors,interleukins and receptor as well as key molecules in NF-κB pathway,to test the effect of magnesium isoglycyrrhizinate on the expression of these important cytokines in the gastric cancer xenografts of the mice.Then we verified the chip results with qPCR.Finally,we use immunohistochemistry to stain the key inflammatory cells in the tumor microenvironment.Result: The expression of CCL3,CCL5,CCL11,CCL13,CCL19,CCL21,CXCL1,CXCL2,CXCL8,CCR1,CCR3,CCR4,CCR7,CXCR1 and CXCR2 in gastric carcinoma tissues were down-regulated by magnesium isoglycyrrhizinate,so as the expression of IL1A、IL1B、IL1R1、IL1RAP、IL1RN、IL5、IL6、IL6R、IL10、IL10RB、IL15、IL17A、IL18、IL22、IL23A、IL23R and TLRs、MyD88、Tollip、NF-κB.While magnesium isoglycyrrhizinate increased the expression of IL-2 and IL-12.And The density and activity of Treg cells in gastric cancer tissues were decreased while the density of CD8 + T cells and macrophages in gastric cancer tissues was increased(p <0.05).Conclusion: Magnesium isoglycyrrhizinate may inhibit the development of gastric cancer by regulating the expression of chemokines,interleukins and their receptors,and key molecules in NF-κB pathway as well as the density(and activity)of Treg cells and CD8+ T cells in gastric cancer tissues.PART 2 Effects of Magnesium Isoglycyrrhizinate on the Macrophages Polarization in Gastric Cancer MicroenvironmentBackground:In the first part,we found that the CD68+ macrophage density was increased after magnesium isoglycyrrhizinate treatment.However,except in pancreatic cancer,it was generally accepted that the higher the density of macrophages is,the worse the prognosis is.It seems a contradiction.However,macrophages are a heterogeneous cell population with a high degree of plasticity that can differentiate into M1(classical activation)and M2(selectively activated)macrophages in different environments.And the role of different types is different,too.Macrophages in the tumor microenvironment will be divided into M2 type.Objective: We aim to elucidate whether magnesium isoglycyrrhizinate inhibit gastric cancer by inducing macrophages in tumor microenvironment differentiate into M1 type.Methods: The conditioned medium was used to simulate the microenvironment of gastric cancer.The mouse gastric cancer cells MFC were treated with different concentrations of magnesium isoglycyrrhizinate solution.The supernatant was collected to cultured mouse macrophages RAW264.7.The expression of iNOS,Arg1 and IL12 in RAW264.7were detected by western blotting.The levels of IL-6,IL-10,IL-12,TGF-β1,TNF-α and IL-12 in supernatant were detected by ELISA.Result: MFC cells could induce mouse macrophages RAW264.7 differentiate into M2(high expressing Arg1,low expressing iNOS and IL12),while macrophages RAW264.7 were differentiated into M1(high expressing iNOS and IL12,low expressing Arg1)when adding magnesium isoglycyrrhizinate to the culture medium of MFC cells.The levels of IL-6,IL-10,TGF-β,TNF-α and VEGF-A in the RAW264.7 supernatant cultured by MFC CM was significantly higher than that by DMEM,and IL12 was significantly lower.After adding different concentrations of magnesium isoglycyrrhizinate to MFC culture medium,the level of IL-6,IL-10,TGF-β,TNF-α and VEGF-A were significantly decreased while the level of IL12 increased in the supernatant of RAW264.7.Conclusion: Magnesium isoglycyrrhizinate can regulate the secretion of tumor cells indirectly,and directly regulate the tumor-associated macrophages in microenvironment to M1 type macrophage repolarization,which is likely to be the role of magnesium isoglycyrrhizinate intervention in the development of gastric cancer.