Apoptotic Effect Induced By Combined Treatment Of Engineered Antibody Plus Paclitaxel On BT474 Cells And Its Emerging Mechanism

HER2/neu receptor is the second member of EGFR family.It has been reported that overexpression of HER2/neu receptor plays a key role in pathological such as tumorigenesis and progression,especially breast cancer,ovarian cancer,prostate cancer, nasopharyngeal carcinoma and other neoplasmas etc.HER2/neu gene encodes p185KDa protein composed of an extracellular ligand binding domain,a short transmembrane domain,and an intracellular domain,which has tyrosine kinase activity.After autophosphorylation of HER2/neu induced by binding of EGF-like ligand to HER2/neu, it can activate downstream effectors including PI3K/AKt pathway and Ras/MAPK pathway etc,which is also thought to play a critical role in malignant transformation, proliferation and migration.It is found that HER2/neu is amplified and overexpressed in～30%of human breast carcinomas.Overexpression of HER2/neu has also been shown to correlated with tumor grade,size and lymph node metastasis.HER2 overexpression provides the important implication for malignant transformation,and thereby leads to aberrant physiological actions of cell migration and invasion.Studies of individuals with HER2 overexpressing tumors have shown that they have a significantly chemoresistance after therapy,having a shorter time to relapse and a lower overall survival rate or disease free survival rate than those patients whose tumors do not overexpress HER2.Paclitaxel is a potent and highly effective antineoplastic agent in the treatment of advanced,drug-refractory,metastatic breast cancers,but HER2 overexpression in breast cancer ceils confers increased resistance to chemotherapeutic agent.Therefore,a novel approach to adjuvant therapy in cancer treatment is increasingly needed.Because of the tight correlation of HER2 gene amplification with several disease parameters including survival rate,time to relapse and other prognostic indexs.Immunotherapeutic project using antibody against HER2 have been proposed.At present,HER2 have been considered as an attractive target of cancer immunotherapy not only because it is overexpressed in many cancers and cannot be detected in normal adult issue,but also because it is an accessible target in the tumor cell surface.In previous study they have constructed anti-p185^c-erbB2/neumonoclonal antibody A21.They cloned its V_H and V_L genes and constructed the single-chain Fv(scFv)by joining them through a peptide linker.Based on the structure of scFv A21,then it was humanized by inserting the complementary determing regions of scFvA21 into the framework of a consensus human IgG₁.The resulting anfi-p185^c-erbB2/neuengineered antibody has a growth inhibitory effect against breast cancer cells overexpressing HER2.In my study,I investigate whether combined anti-p185^c-erbB2/neuengineered antibody plus paclitaxel treatment,which renders HER2-overexpressing breast cancer cells more susceptible to paclitaxel-induced apoptosis,and understanding of the mechanism of HER2-mediated paclitaxel,resistance.I has a dream that this anti-p185^c-erbB2/neu engineered antibody has promise to be as the tumor targeting drug in the future,with a view to application in the diagnosis and treatment of human breast cancer.The proliferative inhibitory effect on BT474 cells is assessed by MTS assay.The TUNEL-positive cells are identified using the in situ cell apoptosis detection kit.Cells stained with AnnexinV-FITC and PI are used to qualify the apoptotic cell number by FACS.These data indicates that combined engineered antibody plus paclitaxel treatment leads to synergistic effect on proliferative inhibition and better apoptotic induction compared with engineered antibody treatment or paclitaxel treatment alone in BT474 cells.The HER2 is immunoprecipitated using anti-HER2 antibody,and then is blotted with anti-Tyr antibody.The AKt,p44/p42MAPK,NF-κ3 are blotted with phospho-Akt(Ser473),phospho-p44/p42MAPK(Thr202/Tyr204),phospho-NF-κ3 (p65subunit).These data indicates that AKt,p44/p42MAPK,NF-κB are activated in paclitaxel treatment compared with combined engineered antibody plus paclitaxel treatment or engineered antibody treatment alone in BT474 cells.NF-κB-DNA binding activity is demonstrated by EMSA.These data indicates that NF-κ3-DNA binding activity is activated in paclitaxel treatment compared with combined engineered antibody plus paclitaxel treatment or engineered antibody treatment alone in BT474 cells.DNA content and cell cycle distribution are determined by FACS.The relationship between percentage of S phase and proliferation index is determined by SAS(9.0).CyclinD1, MMP-9,VEGF expression levels are determined by Westernblot.These data indicates that the relationship between percentage of S phase and proliferation index is classical positive relatedness(r=0.963).Percentage of S phase,CyclinD1,MMP-9 and VEGF expression levels are increased in paclitaxel treatment compared with combined engineered antibody plus paclitaxel treatment or engineered antibody treatment alone in BT474 cells.