Molecular Mechanisms Of Cyclovirobuxine D(CVB-D) Regulating Tumor Associated Macrophages Against Colorectal Cancer Metastasis

Objective Natural monomeric compounds derived from traditional Chinese medicine have various biological activities and are widely used in clinical practice,and many of them have been found to have anti-tumor efficacy,so finding high-quality compounds with anti-tumor activity and figuring out their molecular mechanisms is an effective way to develop potential anti-tumor drugs.Colorectal cancer(CRC)has a high incidence and mortality rate,and is characterized by easy recurrence and strong metastasis,and the clinical treatment of CRC still lacks effective drugs.Cyclovirobuxine D are derived from the rhizomes and leaves of Buxus sinica var.parvifolia M.Cheng.Studies have shown that the alkaloid components of Buxus sinica var.parvifolia M.Cheng show good biological activities in anti-tumor,anti-bacterial and anti-arrhythmia,but there are few reports on its anti-tumor effects in colorectal cancer treatment and how it exerts its anti-tumor effects.This study is intended to investigate the role of alkaloids from Buxus sinica var.parvifolia M.Cheng in regulating colorectal cancer metastasis and to elucidate its mechanism.Methods Cyclovirobuxine D inhibits macrophage M2 phenotype polarization: 1.The effect of Cyclovirobuxine D on the proliferation of colorectal cancer cell lines CT26 and SW480 was examined by MTT assay at different doses;the effect of Cyclovirobuxine D on the proliferation of macrophage lines RAW264.7 and THP-1 was examined by MTT assay at different doses.2.The exogenous cytokines IL-4 and IL-13 were added to induce the M2 phenotype polarization of macrophages RAW264.7 and THP-1,and the effect of Cyclovirobuxine D on the morphology of macrophages treated with cytokines IL-4 and IL-13 was observed under microscope,and the effect of Cyclovirobuxine D on the proliferation of macrophages was investigated by quantitative real-time fluorescence reverse transcription-PCR(q-RT-PCR).The effect of macrophage M2 phenotypic indicators,interleukin-10(IL-10),transforming growth factor-β(TGF-β),and IL-13,on the morphology of macrophages after treatment with IL-4 and IL-13,was examined by quantitative real-time fluorescence reverse transcription-PCR(q-RT-PCR)and Western blot(WB).(TGF-β),and mannose receptor(CD206)to observe the effect of Cyclovirobuxine D on the regulation of macrophage phenotypic repolarization.The effect of Cyclovirobuxine D on the morphology of macrophages after treatment with cytokines IL-4 and IL-13 was also observed under microscope.Cyclovirobuxine D reverses tumor-associated macrophage phenotype to inhibit cell metastasis: 1.The effect of Cyclovirobuxine D on the morphology of macrophages after co-culture treatment with CT26,RAW264.7 co-culture system and SW480,THP-1 co-culture system was observed under the microscope.The m RNA expression levels of macrophage M2 phenotypic indicators IL-10,TGF-β and CD206 were detected by quantitative real-time fluorescence reverse transcription-PCR to observe the effect of Cyclovirobuxine D on macrophage phenotype.2.The expression levels of low affinity receptor(Fce R II,also known as CD23)protein of macrophage M2 phenotypic markers IL-10,TGF-β,CD206,Ig E were detected by Western blot in CT26,RAW264.7 co-culture system and SW480,THP-1 co-culture system treated with Cyclovirobuxine D to observe the effect of Cyclovirobuxine D on macrophage phenotype.The effect of Cyclovirobuxine D on macrophage phenotype.3.The effects of Cyclovirobuxine D on the migration of co-cultured colorectal cancer cells and tumor-associated macrophages were investigated by Transwell assay and scratch assay,given Cyclovirobuxine D treated CT26,RAW264.7 co-culture system and SW480,THP-1 co-culture system.The mechanism of Cyclovirobuxine D reversing macrophage polarization to inhibit colorectal cancer: 1.Prediction of the relationship between immune checkpoint Siglec-10 and macrophage M1 and M2 phenotypes in Cibersort database using TIMER database.2.Predict the potential binding ability of Cyclovirobuxine D to Siglec-10 using molecular docking assay.3.To examine the binding ability of Cyclovirobuxine D to Siglec-10 using CETSA assay.4.To examine the expression level of Cyclovirobuxine D for Siglec-10 in tumor-associated macrophages after co-culture using flow cytometry(FCM).5.To detect the m RNA and protein expression levels of Siglec-10 in tumor-associated macrophages after co-culture with Cyclovirobuxine D treatment by quantitative real-time fluorescence reverse transcription-PCR(q-RT-PCR)and Western blot.6.The phagocytic ability of Cyclovirobuxine D-treated tumor-associated macrophages for colorectal cancer cells was detected by immunofluorescence assay(Immunofluorescence,IF)and flow cytometry.7.Siglec-10 was overexpressed in macrophages and subsequently co-cultured with tumor cells.Transwell assay and M4 laser holographic cell imaging analysis system were applied to detect the migration ability of cells after Cyclovirobuxine D treatment,and Western blot was applied to examine the expression level of M2 phenotypic marker protein in macrophages.8.The candidate genes with high coefficient of interaction with Siglec-10 were predicted using Genemania database,and the ability of Cyclovirobuxine D to bind Siglec-10 to the candidate gene protein was examined by Co-Immunoprecipitation(Co-IP).9.A mouse model of CRC in situ was constructed using AOM/DSS,and the effects of Cyclovirobuxine D on colorectal cancer induced by chronic enteritis were detected by colon length,mouse body weight,Hematoxylin-eosin staining(H&E staining),and the effects of Cyclovirobuxine D on macrophages and colorectal cancer in the tumor microenvironment were detected by Western blot.The expression levels of macrophage and colorectal cancer cell metastasis-related proteins and Siglec-10 and SHP-1 in the tumor microenvironment were detected by Western blot.The effects of the compounds on macrophage phenotypes in the tumor microenvironment and spleen were detected by flow analysis,and the effects of Cyclovirobuxine D on macrophage markers and T-cell markers in colorectal and spleen tissues were detected by immunohistochemistry.Results Cyclovirobuxine D inhibits macrophage M2 phenotype polarization: 1.The results of MTT assay showed that the maximum non-toxic concentration of Cyclovirobuxine D on colorectal cancer cell lines CT26 and SW480 was 10 μM;at a concentration of 10 μM Cyclovirobuxine D was also safe and non-toxic on macrophage cell lines RAW264.7 and THP-1.2.The m RNA expression levels of IL-10,TGF-β and CD206 were significantly down-regulated after the administration of Cyclovirobuxine D to the M2 phenotype of RAW264.7 and THP-1 macrophages induced by IL-4 and IL-13.3.Administration of safranine interfered with IL-4 and IL-13-induced M2 phenotype of RAW264.7 and THP-1 macrophages,and the results of WB showed that the protein expression levels of cytokine-induced macrophage M2 phenotypic indicators IL-10,TGF-β,and CD206 were significantly down-regulated after safranine administration.Cyclovirobuxine D reverses tumor-associated macrophage phenotype to inhibit cell metastasis: 1.The results of quantitative real-time fluorescence reverse transcription-PCR on RAW264.7 macrophages co-cultured with CT26 colorectal cancer cells and THP-1macrophages co-cultured with SW480 colorectal cancer cells showed that the M2 phenotypic markers IL-10,TGF-β,and CD206 were significantly reduced after co-culture of macrophages with Cyclovirobuxine D.β and CD206 were elevated,and the administration of safranin suppressed the m RNA levels of the M2 phenotypic markers in macrophages.The results of microscopic observation also showed that Cyclovirobuxine D could reverse the effect of cytokines IL-4 and IL-13 on macrophage morphology.2.The results of Western blot of RAW264.7 macrophages co-cultured with CT26 colorectal cancer cells and THP-1 macrophages co-cultured with SW480 colorectal cancer cells showed that the expression levels of macrophage M2 phenotypic markers IL-10,TGF-β,CD206,CD23 proteins were significantly increased after co-culture,while the expression levels of macrophage M2 phenotypic markers were significantly increased after administration of The levels of macrophage M2 phenotypic marker proteins were significantly decreased after administration of Cyclovirobuxine D.3.The results of Transwell assay and scratch assay of RAW264.7 macrophages co-cultured with CT26 colorectal cancer cells and THP-1 macrophages co-cultured with SW480 colorectal cancer cells showed that Cyclovirobuxine D reversed the upregulation of migration ability of co-cultured colorectal cancer cells and tumor-associated macrophages.Cyclovirobuxine D reverses the migratory capacity of colorectal cancer cells and their co-cultured tumor-associated macrophages.The mechanism of Cyclovirobuxine D reversing macrophage polarization to inhibit colorectal cancer: 1.TIMER2.0 database prediction,which showed a correlation between the immune checkpoint Siglec-10 and macrophage M1 and M2 phenotypes in the Cibersort database.2.Molecular docking assay showed the potential binding ability between Cyclovirobuxine D and Siglec-10.3.CETSA assay found that Cyclovirobuxine D has good binding ability with Siglec-10.4.Flow cytometry results showed that Cyclovirobuxine D inhibited the upregulation of Siglec-10 expression in tumor-associated macrophages after co-culture.5.The ability of Cyclovirobuxine D to inhibit the elevated expression of Siglec-10 in tumor-associated macrophages after co-culture was confirmed by quantitative real-time fluorescence reverse transcription-PCR and Western blot using Cyclovirobuxine D treatment of tumor-associated macrophages.6.Immunofluorescence assay and flow cytometry showed that the phagocytic ability of macrophages for colorectal cancer cells increased after the administration of Cyclovirobuxine D compared to the blank group.7.Siglec-10 overexpressing macrophages were co-cultured with tumor cells,and the co-culture system was treated with Cyclovirobuxine D,and the Transwell assay and M4 laser holographic cell imaging analysis system showed that the migration ability of Siglec-10 overexpressing cells was significantly up-regulated for macrophages and could reach above the migration level under co-culture conditions,and for colorectal cancer cells,the migration rate was up-regulated under co-culture conditions,while the migration ability of macrophages co-cultured with Siglec-10 overexpressed cells also increased,while the migration ability was inhibited by the administration of Cyclovirobuxine D.Western blot revealed that the expression of M2 phenotypic marker protein in macrophages was significantly upregulated after co-culture and further upregulated after overexpression of Siglec-10,and the expression of M2 phenotypic marker protein was inhibited after administration of Cyclovirobuxine D.8.The Genemania database was used to predict the genes with interrelationship with Siglec-10,among which PTPN6 and PTPN11 were ranked high,and the effect of Cyclovirobuxine D on the binding ability of Siglec-10 to the proteins SHP-1 and SHP-2expressed by PTPN6 and PTPN11 was examined by immunoprecipitation assay,and it was found that Cyclovirobuxine D did not inhibit their binding ability.9.By constructing an AOM/DSS-induced CRC mouse model we found that Cyclovirobuxine D was able to reverse the elevated expression of macrophage and colorectal cancer cell metastasis-associated proteins and Siglec-10 and SHP-1 in the tumor microenvironment after modeling.By the results of colon length,mouse body weight,and H&E staining,we found that Cyclovirobuxine D could alleviate colorectal cancer induced by chronic enteritis,mainly manifested by prolonging colon length,slowing down weight loss in mice,and reducing inflammatory and cancerous tissues in colon.10.By constructing an AOM/DSS-induced CRC mouse model,the results of flow analysis showed that Cyclovirobuxine D could increase the expression level of M1 phenotypic markers of macrophages and inhibit the expression of M2 phenotypic markers in the tumor microenvironment and spleen.Immunohistochemical assays revealed that Cyclovirobuxine D inhibited macrophage M2 phenotypic markers and elevated T cell markers in colorectum and spleen.Conclusion In this study,we found that Cyclovirobuxine D,an alkaloid component of Populus chrysanthemi,which is derived from the genus Populus of the family Populus,acts on the immune checkpoint Siglec-10 to regulate the effect of tumor microenvironment on macrophage phenotype and inhibit the metastasis and recruitment of macrophages in colorectal cancer,thus inhibiting the occurrence and development of colorectal cancer,with the aim of improving the theoretical basis for the use of Cyclovirobuxine D,a natural and effective small molecule,as a potential therapeutic agent for colorectal cancer.This study aims to improve the theoretical basis for the use of natural and effective small molecule Cyclovirobuxine D as a potential therapeutic agent for colorectal cancer.