The Effect And Molecular Mechanism Of Tumor-associated Macrophages On Drug Resistance Of Breast Cancer

PART IINDUCTION AND PHENOTYPIC IDENTIFICATION OFTUMOR-ASSOCIATED MACROPHAGESObjective: To induce normal macrophages into tumor-associatedmacrophages (TAM) in vitro, and analyze their biological phenotypiccharacteristics.Methods: In general, TAM are regarded as M2-phenotypemacrophages, with normal macrophages as M1-phenotype macrophages. Inthis study, we induced TAM or M1macrophages by macrophagecolony-stimulating factor (MCSF) or LPS/IFN-γ respectively, following thatTHP-1cells were differented into macrophages by PMA. Furthermore, TAMin human breast cancer tissues were also collected using immunomagneticbeads. The expression of surface molecule CD163in different types ofmacrophages was detected by flow cytometry. Arginase-1expression andactivity in different types of macrophages were measured by western blot or arginase activity assay respectively. In addition, the mRNA and proteinexpression of IL-10, IL-12and IL-23in different types of macrophages wasanalyzed by real-time PCR or ELISA respectively.Results: Compared with that in PMA-induced M0macrophages orLPS/IFN-γ-induced M1macrophages, the expression of CD163, arginase-1and IL-10, and arginase activity were increased in MCSF-inducedmacrophages with decreased IL-12and IL-23, which was in concidence withthe phenotypic characteristics of TAM collected from human breast cancertissues.Conclusion: We successfully induced normal macrophages into TAMwhich was M2macrophages characterized by higher CD163, arginase-1andIL-10, and lower expression of IL-12and IL-23. PART IITHE EFFECT AND PRELIMINARY MECHANISM OFTUMOR-ASSOCIATED MACROPHAGES ON DRUGRESISTANCE OF HUMAN BREAST CANCER CELLSObjective: To investigate the effect and preliminary molecularmechanism of tumor-associated macrophages (TAM) on the multidrugresistance of breast cancer.Methods: We first investigate the effect of TAM on the sensitivity ofbreast cancer MDA-MB-231and MCF-7cells to various drugs such asadriamycin (ADM), cisplatin and taxol by CCK-8kit, with relativeresistance index (RI) calculated. Cell apoptosis induced by ADM wasdetected by flow cytometry, and caspase-3activity was measured by thecaspase-3activity kit. The concentration of ADM and rhodamine efflux inbreast cancer cells were analyzed by spectrophotometry or flow cytometryrespectively. Moreover, the expression of drug resistance-related proteinssuch as P-gp, MRP, LRP and BCRP, and apoptosis-related proteins such asBcl-2, Bcl-xl, Bax, Bad, and Bid in breast cancer cells were measured byrealtime PCR and/or western blot.Results: Compared with that in wild-type breast cancer cells, TAMinduced a decreased sensitivity of breast cancer cells to drugs such as ADM,paclitaxel or cisplatin, with reduced cell apoptosis and caspase-3activity after treatement with ADM. Furthermore, decreased ADM concentrationand increased rhodamine efflux were confirmed in cancer cells coculturedwith TAM. More importantly, the higher expression of P-gp was verified inbreast cancer cells cocultured with TAM. Inhibiting the function of P-gp byP-gp inhibitor C-4partially reversed the drug resistance induced by TAM.Further experiments showed increased Bcl-2and decreased Bax in breastcancer cells cocultured with TAM.Conclusion: TAM induced drug resistance of breast cancer cells byoverexpressing P-gp and Bcl-2, and down-regulating Bax. PART ⅢTHE EFFECT AND MOLECULAR MECHANISMS OFCOX-2/PGE2/AKT SIGNALING PATHWAY ON TUMORASSOCIATED MACROPHAGES INDUCED DRUGRESISTANCE OF HUMAN BREAST CANCER CELLSObjective: To further explore the effect and potential mechanisms ofrelated signaling pathways in tumor associated macrophages(TAM)-induced drug resistance of breast cancer.Methods: The activation of PI3K/Akt, NF-κB and GSK-3β/β-cateninsignaling pathways in breast cancer cells was measured by western-blot andimmunofluorescence analysis. After inhibition of the PI3K/Akt pathway bywortmannin or knockout of β-catenin by adenovirus expressing siRNAβ-catenin in breast cancer cells cocultured with TAM, cell apoptosisinduced by ADM and rhodamine efflux were detected by flow cytometry,with the expression of P-gp, Bcl-2and Bax measured by western blot.TGF-β, TNF-α, IL-1β and PGE2in cell supernatants were then detected byELISA or EIA assays. After PGE2signaling pathway inhibitors AH6809andAH23848B were used to block PGE2signal transduction, cell apoptosisinduced by ADM and rhodamine efflux were detected by flow cytometry,with the activation of Akt and GSK-3β/β-catenin pathways, and theexpression of P-gp, Bcl-2and Bax measured by western blot. Furthermore, the expression of COX-1and COX-2in breast cancer cells or differenttypes of macrophages was measured by western blot. After coculturingbreast cancer cells with TAM transfected with or without adnovirusexpressing siRNA COX-2, cell apoptosis induced by ADM and rhodamineefflux in breast cancer cells were detected by flow cytometry, while theactivation of Akt and GSK-3β/β-catenin pathways, and the expression ofP-gp, Bcl-2and Bax in cancer cells were measured by western blot.Results: Compared with that in wild-type breast cancer cells, therewas a significant activation of PI3K/Akt and GSK-3β/β-catenin signalingpathways in breast cancer cells cocultured with TAM. Inhibiting PI3K/Aktpathway by wortmannin or knockout of β-catenin by adenovirus expressingsiRNA β-catenin in breast cancer cells reversed the drug resistance inducedby TAM. Intrestingly, inhibiting PI3K/Akt pathway regulated theexpression of p-GSK-3β, active β-catenin, P-gp, Bcl-2, and Bax, whileknockout of β-catenin modulated the expression of P-gp, Bcl-2, and Bax,without affecting p-Akt and p-GSK-3β. Furthermore, compared with that insupernatants cultured alone, a significant increase of PGE2was found incocultured supernatants, without significant changes of TGF-β,TNF-α andIL-1β. Blocking PGE2signaling pathway by AH6809and AH23848B inbreast cancer cells cocultured with TAM significantly improved thesensitivity of cancer cells to ADM, and modulated the activation of Akt andβ-catenin pathways, and downstream molecules. Subsequent western blot analysis showed that there was no significant change in the expression ofCOX-1and COX-2in breast cancer cells cocultured with TAM, comparedwith that in breast cancer cells cultured alone. However, a marked increaseof COX-2was observed in TAM, compared with that in M0or M1macrophages. Knockout of COX-2in TAM reversed TAM-mediated drugresistance of breast cancer cells by regulating Akt and β-catenin pathways.Conclusion: PGE2secreted by TAM over-expressing COX-2, inducedthe activation of PI3K/Akt pathway which regulated GSK-3β/β-cateninsignaling pathway and downstream molecules including P-gp, Bcl-2andBax in breast cancer cells, resulting in drug resistance of breast cancercells.