Study On Anti-tumor Effect Of Human Blood Group Antigen A Glycosyltransferase Expression Recombinant Plasmid Transfected Human Breast Cancer Cell MDA-MB-231 In Vitro

Background:Of all types of breast cancer,triple-negative breast cancer(TNBC)is the one with the three types of receptors estrogen(ER),progesterone(PR),and the HER-2/neu gene(HER2)negative in testing.This type accounts for 15 to 24% of all breast cancers with a poor response to routine therapies.Patients of TNBC can't benefit from endocrinology or targeted therapy due to lack of corresponding targets.TNBC is characterised by high early recurrence and metastasis rate and poor prognosis.So it has become a challenge or a problem in clinical treatments.Antibody-targeted immunotherapy has sprung up in recent years,and it showed that trastuzumab,a drug of this therapy,can markedly improve the prognosis of breast cancer patients with over expression of HER-2,which provided us a new insight in TNBC treatment.Such as PRAP inhibitor,EGFR inhibitor,anti-VEGF antibody,m TOR inhibitor and TRAIL inducer,many targets in TNBC treatment has come up.But only a small part of TNBC patients could benefit from it,and the efficacy and safety need long-term assessement.Thus,efforts have been consistently made to find more effective targets.Studies have shown that breast cancer cells highly express the precursors of blood group antigen H,which could be converted into blood group antigen A or B by glycosylation of BGA glycosyltransferase.The change in the expression of blood group antigens(BGA)is associated with malignanc,which can affect the proliferation,differentiation and metastasis abilities.Meanwhile,BGA are a group of allo-antigens with strong immunogenicity and antigenicity,which could be identified by blood group antibodies and activate complement-dependent cytotoxicity(CDC)and antibody-dependent cell-mediated cytoxicity(ADCC)such as the hemolytic reaction.Moreover,the micro environment in breast cancer patients are under a state of immunosuppression.Antigen presenting cells(APC)aften failed to present the tumor antigens to immune system.BGA are natural antigens,which could be identified directly by blood group antibodies of the same type.It is unclear that whether we could target BGA as a therapeutic target for TNBC treatment.Objectives:We will start from the establishment of blood group antigen A highly expressed MDA-MB-231 cell line and study on the anti-tumor effect of B type plasma on the transfectant.Meanwhile,we will observe the influence of blood group antigen A on MDAMB-231 cells and investigate the mechanisms.This will provide the fundamental basis for the use of blood antigen as a potential target by blood antibodies in treatment of TNBC.Methods:1.We transfected blood group antigen A glycosyltransferase into MDA-MB-231 cell line by lenti-virus tansfection.2.The blood group antigens expression and the cell cycle change of MDA-MB-231 cells before and after transfection,and the apoptosis of transfectant after dealing with B type plasma were detected by flow cytometry.3.Proliferation rate of MDA-MB-231 cells before and after transfection together with transfectant after dealing with B type plasma was measured by WST-1 method,and the synergistic effect of different concentration of 5-FU and NK cells were also evaluated by WST-1 method.4.The invasive and metastasis ability and colony forming ability were respectively evaluated by scratch injury experiment and colony forming unit assay.5.The change of ATP and HMGB1 level before and after transfection were measured using ELISA assays to evaluate the ICD.6.Western blot was used to measure the expression of cell cycle related proteins,cell cycle regulatory proteins and integrin signaling pathway related proteins to investigate the probable mechanism of changes of biological behavior after transfection.Results:1.We successfully transfected blood group antigen A glycosyltransferase into MDA-MB-231 cell line by lenti-virus and the high expression of antigen A on the cells were proved by flow cytometry assay.2.The proliferative capacity,invasive and metastasis ability and colony forming ability of transfectant were all reduced.3.There was a S phase arrest in the transfectant,and 5-FU had an synergistic anti-tumor effect on the transfectant.4.The level of ATP and HMGB1 increased obviously after transfection.5.The apoptosis increased,proliferative capacity,invasive and metastasis ability increasd of transfectant after dealing with ype B plasma.6.The downstream signal pathway of integrin such as ERK1/2 and p-PI3Kwere inhibited,and p-p38 MAPK increased.Conclusions:1.Type B plasma had an anti-tumor effect,which killed the transfected MDA-MB-231 cells via CDC and ADCC effect.2.Blood group antigen A glycosyltransferase led to reduction of proliferative capacity,invasive and metastasis ability and colony forming ability via inhibition of integrin-FAK signal pathway.3.Blood group antigen A glycosyltransferase led to S phase synchronization and improved the chemotherapy sensitivity of 5-FU.4.Blood group antigen A glycosyltransferase enhanced the immunogenicity of MDA-MB-231 cells by induction of ICD.