The Role Of Macrophages In Resistance To 5-FU In Colorectal Cancer Cell CT-26

Colorectal cancer(CRC)is one of the most common malignant tumors in clinic,which cause serious damage to human health.At present,as a single or combination drug therapy,5-FU is still the first choice for clinical practice to treat colorectal cancer.Recently,it is reported that more than a half of patients developed resistence to 5-FU,and the majority is due to the reduced susceptibility to 5-FU after sustained using.It is well established that chemotherapy drugs can play its anti-tumor effect via inducing tumor cell apoptosis.Hence,inhibiting the apoptosis of tumor cell induced by chemotherapy drugs will lead to the chemoresistance.Consistently,our recent study in vitro had found that 5-FU played its anti-tumor effect by activating P-JNK-dependent caspase-3 signaling in colorectal cancer cells CT-26.At present,it is becoming increasingly clear that the tumor microenvironment plays a key role in mediating drug resistance and apoptosis resistance.Macrophage,which is an important component of tumor microenvironment,plays a crucial role in promoting tumor growth,metastasis and invasion through complex autocrine and paracrine.But the relationship between macrophage and chemoresistance is little known.In a colorectal cancer model,5-FU treated macrophage conditioned medium(CM(5-FU))could induce 5-FU chemoresistance via inhibiting P-JNK/caspase-3 signaling both in vitro and in vivo.Further through the metabonomics analysis,we found that 5-FU could induce the rise of polyamine synthesis in macrophages which further induced resistance to 5-FU in CT26 cells.Therefore,we identified that polyamine metabolism in macrophage as a dominator to regulate sensibility to 5-FU through inhibiting P-JNK-dependent caspase-3 signaling in colorectal cancer.Our findings revealed a novel mechanism linking apoptosis and polyamine metabolism in macrophage to the resistance to 5-FU in colorectal cancer cell CT-26.These results might have important guiding significance on the therapeutic strategies of this malignancy.Purpose:Revealing the role of macrophage in secondary chemoresistance to 5-FU of colorectal cancer cell line CT26 and exploring the mechanism of it.Method1.5-FU play its anti-tumor effect via activating P-JNK/caspase-3 signaling in CT26 cells.In order to further verify the role of apoptosis in the anti-tumor effect of 5-FU,we treated CT26 cells with or without 5-FU.Then PI/Annexin-V assays were used to detect cell apoptosis and Western Blot were used to detect the expression level of cleaved caspase-3,P-JNK.To further verify the role of apoptosis,we used P-JNK specific inhibitors to inhibit P-JNK/caspase-3 signaling in mice transplanted tumor model,then observe curative effect of 5-FU.2.5-FU-stimulated macrophages induced resistance to5-FU in CT26 cells.For researching the role of macrophages in 5-FU resistance,CT26 cells were cultured in normal medium(NM),macrophage conditioned medium(CM)or 5-FU pretreated macrophage conditioned medium CM(5-FU),followed by 5-FU treatment.PI/Annexin-V assays were used to detect cell apoptosis and CCK-8 assays were used to detect cell proliferation.In vivo,BABL/C mice were seeded with CT26 cells and treated with NM?CM?CM(5-FU)and 5-FU.Size of tumor was detected.3.5-FU-stimulated macrophages induce 5-FU resistance via inhibiting P-JNK/caspase-3 signaling in colorectal cancer cells.In order to further verify the detailed mechanisms,CT26 cells were cultured in normal medium(NM),macrophage conditioned medium(CM)or 5-FU pretreated macrophage conditioned medium CM(5-FU),followed by 5-FU treatment.PI/Annexin-V assays were used to detect cell apoptosis and Western Blot were used to detect the expression level of cleaved caspase-3,P-JNK.4.5-FU-stimulated macrophages attenuated P-JNK-dependent caspase-3 activation in CRCs via polyamine.In order to reveal the specific metabolite of macrophages induced 5-FU resistance in CT26 cells.Macrophages were treated with or without 5-FU,then the cultural supernatant was analysed by metabolomic profiling.Further we use the specific inhibitor of this metabolite and 5-FU treated macrophages and collected the culture supernatant,marked as CM(5-FU + inhibitor).Then CT26 cells were cultured in NM,CM(5-FU)and CM(5-FU + inhibitor),followed by 5-FU treatment.Western Blot were used to detect the expression level of cleaved caspase-3,P-JNK.In vivo,BABL/C mice were seeded with CT26 cells and treated with NM?CM(5-FU)?CM(5-FU + inhibitor)and 5-FU.Size of tumor was detected.Conclusion:1.Compared with control group,CT26 cells treated with 5-FU had a increased cell apoptosis.Meanwhile the expression level of cleaved Caspase-3 and P-JNK is higher in the group treated with 5-FU compared to that in control group.P-JNK specific inhibitors inhibit suppressed anti-tumor effects of 5-FU in a colorectal cancer model.Thus,we concluded that 5-FU play its anti-tumor role via activating P-JNK/ caspase-3 signalling in CT26 cells.2.In CCK8 assays,CT26 cells treated with CM(5-FU)had a decreased sensitivity to 5-FU compared with NM and CM group.In the subcutaneous tumor model,the tumor size of the groups treated with CM(5-FU)and 5-FU were much larger than the group treated with NM?CM and 5-FU.Taken together,these results demonstrated that 5-FU-stimulated macrophages could induced 5-FU resistance in colorectal cancer cells.3.In PI/Annexin-V assays,CM(5-FU)could dramaticlly reduce the number of apoptotic cells induced by 5-FU.Consistently,the expression level of cleaved Caspase-3 and P-JNK in CT26 cells treated with CM(5-FU)and 5-FU were much lower than that in CT26 cells treated with NM ? CM and 5-FU.Altogether,these data indicated that 5-FU-stimulated macrophages induced 5-FU resistance via attenuating P-JNK-dependent caspase-3 activation in CT26 cells.4.Metabolic chip analysis found that polyamine synthesis in the macrophages which were treated with 5-FU was remarkedly elevated,which was associated with 5-FU resistance in CT26 cells.The expression level of cleaved Caspase-3 and P-JNK in CT26 cells treated with CM(5-FU+DFMO)and 5-FU were higher than that in CT26 cells treated with CM(5-FU)and 5-FU.Consistently,DFMO could markedly increase the sensibility to 5-FU in a colorectal cancer model.All of these indicated that 5-FU can stimulate the rise of polyamine synthesis in macrophages which further induced 5-FU resistance via inhibiting P-JNK-dependent caspase-3 signaling in CT26 cells.