Study About DARC Expression Regulatory Mechanism In Breast Cancer Cell Line MDA-MB-231 And Correlation On Duffy Blood Group And Clinical Pathological Characteristics In Breast Diseases Patients

Purpose:DARC is the abbreviation of Duffy antigen/Receptor of Chemokine, which is a negative regulator to breast cancer.Duffy Blood Group(DBG) is determined by Duffy antigen.Duffy antigen and DARC is the identical protein which share the same structure and function.The only difference between Duffy antigen and DARC is the former is located in the membrane of red bolld cell and the latter is located in the membrane of breast cancer cells and other cells such as endothelial cells.Our lab previous study suggested that DARC correlated to breast cancer malignancy.Besides,DARC is up-regulated in several inflammatory diseases. In this study we test the hypothesis that whether pro-inflammatory cytokines can up-regulate DARC in breast cancer cell lines based on such background.Another hypothesis we test is that clinical patients' DBG phenotypes affect on breast cancer occurrence and correlate to malignancy based on the epidemiological facts that different races has definitely different DBG phenotypes distribution and to them, there is a various breast cancer mobidity and mortality.Methods:Real-Time PCR and Western-Blot analysis was used to test four pro-inflammatory cytokines TNF-α, IL-1β,IFN-γand IL-8 effects on MDA-MB-231 cell lines DARC mRNA and protein expression.After we founded that TNF-αincrease DARC expression at both mRNA and protein level,we tested TNF-αantibody and NF-κB inhibitor AEA(Anadmide) effects on DARC by Real-Time PCR and Western-Blot analysis.Concentration of endogenous TNF-αwas determined by Enzyme Linked Immunosorbent Assay (ELISA).Finally,ELISA measured the concentration of CXCL8(CXC chemokine ligand 8,interleukin-8,IL-8),CCL2(CC chemokine ligand 2,two major DARC ligands,in conditioned media were done to determine DARC chemotactic clearance, and Transwell migration assays towards CCL2 as well as in vitro invasion assay were done to determine whether the TNF-α-induced increase in DARC expression was functional.In the study about correlation between DBG and pathological diagnosie,we investigated DBG phenotypes of 253 cases consecutive hospitalized female patients suffering breast disease in Shanghai Cancer Hospital and analysized its relationship with clinical pathological diagnosis.DBG phenotypes were examined by indirect antiglobulin-test with anti-Fya and anti-Fyb reagents and were classified into Fya+Fyb-,Fya-Fyb+,Fya+Fyb+,Fya-Fyb- according agglutination.Results: TNF-α(0.5～1ng/ml) and IL-1β(4～8ng/ml) obviously increased DARC expression by 4 to 8 folds and 7 folds respectly.TNF-αantibody and NF-κB inhibitor AEA(25μM) attenuated TNF-αup-regulation.IFN-γand IL-8 effects on DARC expression were weak.No correlation between concentration of endogenous TNF-αand DARC expression.DARC expression level in mRNA and protein was paralleled.TNF-αinduced DARC expression enhanced clearance of chemokines,moreover,mobility of MDA-MD-231 cell line to CCL2 was increased and invasion capacity of MDA-MD-231 cell line was decreased.DARC increased by TNF-αwas functional. In the study about correlation between DBG and pathological diagnosie,we found neither DBG phenotypes distribution difference existed in breast disease patients nor DBG phenotypes distribution difference existed in breast cancer patients compared to general Chinese Han population.To breast cancer occurring possibility,Fya-Fyb-and Fya-Fyb+ demonstrated more susceptibility to breast cancer than Fya+Fyb- and Fya+Fyb+,but no statistical significant difference.Fya- group(57.14%) had more malignant incidence than that of Fya+ group(39.02%),but no statistical significance (P=0.28).Neither significant difference had been observed in ER,PR,Her-2 status and P53,PCNA,PS2,nm23,P450 status between every DBG phenotypes,nor statistical difference was found to tumor grades in various DBG phenotypes.More patients were involved in auxiliary lymph nodes metastasis in Fya- than that of Fya+ group and reached a statistical significance(100%and 39.13%respectively,P＜0.05, Fisher exact test).Conclusion:Both TNF-αand IL-1βare two important regulators to breast cancer cell lines MDA-MB-231 DARC expression and the function may via NF-κB pathway.Endogenous TNF-αwas not an important redulator to DARC expression.Fya antigen weights more than Fyb antigen in breast cancer lymphnodes metastasis.Fya negative maybe a clinically useful factor to identify the potential axillary lymph nodes metastasis in breast cancer patients.