Study On The Role Of Amillariella Mellea Polysaccharides In Modulating Antitumor Response By Promoting The Transformation Of M2-type Macrophages To M1-type

Macrophages have plasticity and can differentiate into M1-type or M2-type macrophages under different conditions.M1-type macrophages can release more pro-inflammatory factors such as TNF-α,IL-6 and IL-1β,and can prevent tumor proliferation;M2-type macrophages can release more anti-inflammatory factor such as IL-4,IL-8 and IL-10,which can promote tumor proliferation and angiogenesis.Colon cancer is one of the diseases that plague human health,and its development is closely related to the reaction in the tumor microenvironment.Most of the tumor-associated macrophages(TAMs)present in tumor microenvironment are M2-type.Therefore,the finding of effective drugs that can reverse M2-type macrophages to M1-type has become a hot research topic.Armillaria mellea is a common edible fungi.The polysaccharides,extracted from Armillaria mellea,exhibit various biological activities.Our previous researches have demonstrated that Armillaria mellea polysaccharide AAMP-A70 activated macrophages and showed no cytotoxicity on normal cells.However,the effect on macrophage polarization and tumor suppression by polysaccharides from Armillaria mellea has not been reported.In this study,we detected the effect of AAMP-A70 on the polarization of TAMs(M2-type macrophages)and the subsequent cell viability suppression effect on colon cancer cells.Furthermore,the mechanism which was accounted for the effect was also investigated.First,we established the induction methods of M2 and M1 macrophage using the murine mononuclear macrophage cell line RAW264.7 and the human monocyte cell line THP-1.The results showed that AAMP-A70 decreased the expression of M2-type specific markers such as Arg-1,CD206 and VEGF decreased significantly,and increased the expression of M1-type specific markers such as TNF-α,IL-6 and IL-1βin M2-type macrophages.These results indicating that AAMP-A70 can inhibit the polarization of M2-type macrophages and promote their transformation to M1-type macrophages.Secondly,in order to investigate the mechanism of AAMP-A70 promoting the transformation of M2-type macrophages to M1-type macrophages,we examined the expression of Akt,NF-κB and MAPK signaling pathway related proteins,respectively.The result showed that AAMP-A70 could promote the phosphorylation of NF-κB,Akt,ERK and JNK in M2-type macrophages.When the cells were pretreated with NF-κB,Akt,ERK and JNK inhibitors(BAY11-7082,LY294002,U0126 and SP600125),the inhibitory effect of AAMP-A70 on the CD206 expression in M2-type macrophages was reversed.These results suggested that AAMP-A70 inhibited the polarization of M2-type macrophages via NF-κB,Akt,ERK and JNK signaling pathways.In addition,we found that the inhibition of phosphorylation of Akt,ERK and JNK was the upstream of NF-κB activation induced by AAMP-A70.In addition,since our previous studies found that Toll-like receptor 2(TLR2)is the recognition receptor of AAMP-A70 on the macrophage activation.In the present,we treated M2-type macrophages with TLR2 blocking antibody and inhibitor(C29),respectively.The results showed that after TLR2 was blocked or inhibited,the inhibitory effect of AAMP-A70 on the CD206 expression in M2-type macrophages was inhibited,indicating that TLR2 is the recognition receptor of AAMP-A70 on the M2-type macrophages.Finally,we co-cultured the colon cancer cells DLD-1 and HCT116 with the supernatant of each group.The viability of DLD-1 and HCT116 were detected using MTT assay and crystal violet staining assay.The results showed that AAMP-A70 itself had no cytotoxicity to colon cancer cells.The culture supernatant of M1-type macrophages could significantly inhibit the proliferation of colon cancer and promote the apoptosis of colon cancer.However,the culture supernatant of M2-type macrophages had no effect on colon cancer cell viability.Notably,culture supernatant of M2-type macrophages treated with AMMP-A70 could inhibite the viability of colon cancer cells and promote the apoptosis.These results suggested that AAMP-A70 exerted an anti-tumor effect by promoting the transformation of M2-type macrophages to M1-type macrophages.We also found that the cell viability suppression effect was decreased after inhibiting the phosphorylation of NF-κB,Akt,ERK and JNK,or TLR2 was blocked or inhibit.The result further suggested that AAMP-A70 through TLR2 to activating Akt/NF-κB,ERK/NF-κB and JNK/NF-κB pathway to promote the transformation of M2-type macrophages to M1-type,thereby inhibit the proliferation of colon cancer cells and promote their apoptosis to play the role of anti-tumor.In conclusion,we found that AAMP-A70,a non-cytotoxic polysaccharide extracted from Armillaria mellea,inhibited the polarization of M2-type macrophages and promoted their transformation to M1-type macrophages through TLR2 to activating Akt/NF-κB,ERK/NF-κB and JNK/NF-κB pathway,thereby inhibited the viability of colon cancer cells.