Regulation Of Curcumin On Polarization Of Tumor-associated Macrophages And Its Mechanism In Oral Squamous Cell Carcinoma

Objective: Tumor-associated macrophages(TAMs),the majority of the immune cells in the oral squamous cell carcinoma(OSCC)tumor microenvironment,display two major phenotypes,M1 and M2.TAMs are predominantly the M2 phenotype that fosters the pace of cancer initiation and progression.Whereas M1 phenotype macrophages have anticancer characteristics.TAMs are expected to be a cell target for the treatment of OSCC.Promoting the repolarization of macrophages towards antitumor phenotypes is one of the main strategies in the pursuit of developing macrophage-targeted therapies in cancer.Curcumin(CUR)is an active ingredient extracted from the Curcuma longa,which can be used to treat a variety of chronic diseases,including cancer.Curcumin can suppress cancer epithelial-mesenchymal transition,metastasis,as well as proliferation.Whether and how curcumin affects macrophages in the tumor microenvironment remains unclear.The purpose of this study is to investigate the role of curcumin in regulating the phenotypes and functions of TAMs,as well as the potential mechanism.A thorough understanding of the antitumor effects of curcumin may improve the prognosis of patients with OSCC.Methods:1.After exposure to the conditional medium of the CAL27 cells(CAL27-CM)for 6-24 h,qRT-PCR was used to detect the mRNA level of cytokines in macrophages.RAW264.7cells stimulated by CAL27-CM were treated with curcumin at different concentrations.The mRNA level of cytokines for M1 and M2 macrophages was measured by qRT-PCR.2.RAW264.7 cells were co-cultured with CAL27 cells in the 6-well Transwell plates and polarized into TAMs.TAMs were further treated with 20 μM curcumin.qRT-PCR was used to detect the mRNA levels of the markers for M1 and M2 macrophages.Flow cytometry was used to detect the expression of CD86 and CD206 in macrophages.3.20 ng/m L IL-4 was employed to induce RAW264.7 cells differentiation into M2 subset macrophages.M2 macrophages were treated with 20 μM curcumin.qRT-PCR was used to detect the mRNA level of the markers for M1 and M2 macrophages.ELISA assay was employed to analyze the secretion level of IL-6 and TNF-α.Western blot was used to assay the relative protein level of arginase-1(Arg-1)in macrophages.The percentages of CD86 and CD206 were measured through flow cytometry.4.M2 macrophages stimulated with or without 20 μM curcumin were further co-cultured with CAL27 cells in a 24-well Transwell plate.The migration and invasion of CAL27 cells induced by macrophages were detected by Transwell assays.CAL27 cells were cultured in a supernatant of macrophages treated by curcumin.The wound-healing assay was employed to detect the migration ability of CAL27 cells.5.The mRNA and protein levels of monoamine oxidase A(MAO-A)in M2 macrophages treated with or without curcumin were measured by qRT-PCR and Western blot.Intracellular reactive oxygen species(ROS)was measured through flow cytometry.After20 μM curcumin or 100 μM and 200 μM moclobemide treatment,the expression of signal transducer and activator of transcription 6(STAT6)and p-STAT6 in macrophages was detected by Western blot.Results:1.CAL27-CM upregulated the mRNA level of the markers for M1(IL-12,iNOS,TNF-α)and M2 macrophages(TGF-β,Arg-1,IL-10)(P < 0.05).20 μM curcumin can increase the levels of TNF-α and IL-6,and decrease the expression of IL-10(P= 0.013,P < 0.001,P <0.001 respectively).2.Curcumin increased the mRNA levels of IL-6,TNF-α and iNOS(P= 0.025,P= 0.016,P < 0.001 respectively),and decreased the mRNA levels of IL-10,CD206 and Arg-1 in TAMs(P < 0.001,P= 0.028,P < 0.028 respectively).Curcumin promoted the expression of the membrane protein CD86 and reduced the expression of CD206 in TAMs(P= 0.043,P= 0.024 respectively).3.Curcumin increased IL-6,IL-12,TNF-α,and iNOS(P < 0.001,P < 0.001,P < 0.001,P= 0.003 respectively),and decreased IL-10,Arg-1,TGF-α,and PD-L1 in M2macrophages(P= 0.014,P < 0.001,P= 0.004,P < 0.001 respectively).Curcumin promoted the secretion of cytokines(IL-6 and TNF-α)in M2 macrophages(P < 0.001),and decreased the protein level of Arg-1 in M2 macrophages(P= 0.028).The percentage of CD86 in M2 macrophages was upregulated after curcumin treatment,while CD206 was downregulated(P < 0.001).4.Transwell assay demonstrated that curcumin could suppress the migration and invasion behaviors of CAL27 cells induced by M2 macrophages(P < 0.001).The healing rate of the CAL27 cells induced by the CM from the curcumin-pretreated M2 macrophages was decreased(P < 0.001).CM of Curcumin-stimulated M2 macrophages increased the protein expression of E-cadherin(P= 0.023),and decreased the gene level of Vimentin and Ncadherin in CAL27 cells(P= 0.003,P= 0.017 respectively).5.Curcumin suppressed the mRNA and protein expression of MAO-A(P= 0.001,P=0.035 respectively)and decreased the level of ROS in M2 macrophages.Curcumin,100μM moclobemide,and 200 μM moclobemide treatment resulted in the inactivation of STAT6 signaling(P= 0.002,P < 0.001,P < 0.001 respectively).Conclusion: Curcumin could promote the TAMs to express the makers for M1 macrophages and facilitate the polarization of macrophages from the M2 towards the M1 phenotype.Curcumin suppressed the migration and invasion behaviors of CAL27 cells induced by M2 macrophages.Curcumin suppressed the mRNA and protein expression of MAO-A and decreased the level of ROS in M2 macrophages to further inactivate STAT6 signaling,which resulted in the repolarization of TAMs from a protumor phenotype towards an antitumor phenotype.These findings identified curcumin as a key modulator of TAMs.A thorough understanding of the antitumor effects of curcumin may improve the prognosis of patients with OSCC.