The Molecular Mechanisms Of A Novel Agonistic Anti-human DR5 Monoclonal Antibody Inducing Apoptosis In Esophageal Squamous Carcinoma Cell Lines

Esophageal carcinoma (EC) is one of the sixth malignant tumors worldwide. EC is featured by geographic variation in incidence and poorest prognosis. Epidemilogical studies have indicated that incidence in highest region of EC could be 500-fold higher than lowest area of EC. The five-year survival rate for EC is only 10% for advanced EC patients. Patients with Esophageal squamous cell carcinoma(ESCC) are common in China.The present treatment modalities of EC comprise combination therapy, e.g. surgery, radiotherapy, chemotherapy and biological therapy. However, the efficiency of modalities above is still ominous and we need to explore effective ways to treat EC. Neoplastic growth results from loss of balance of cell growth and apoptosis. A large body of evidence has shown that aberrant mechanisms of apoptosis contribute to pathogenesis of EC and thus many anti-malignant treatments implicate apoptotic pathway. Accordingly, the identification of agents which can trigger apoptosis of EC is necessary, and this will promise to be one of effective treatments for EC.TRAIL (TNF-related apoptosis inducing ligand) was identified by Wiley in 1995 ,which is one of members of TNF super-family and to be able to induce apoptosis in tumor cells with no harmful effects on normal cells. TRAIL can bind to death receptors on cell membrane following by apoptotic pathway cascades activation. The receptors binding to TRAIL fall into five types, in which TRAIL receptor 1/DR4 (death receptor 4) and TRAIL receptor 2/DR5 (death receptor 5) posses the intracellular death domain contributing to apoptotic activation. However, other three types of TRAIL receptors, i.e. TRAIL receptor 3 (DcR1), TRAIL receptor 4 (DcR2) and OPG (Osteoprotegerin) were revealed to be decoy receptors due to lacking intracellular death domain. Where TRAIL is different from TNF and FasL in TRAIL inducing apoptosis in tumor cells and cells infected by virus ,as well as without harm to normal cells. In this regard, TRAIL can be developed as an ideal anti-tumor substance. Conversely, other investigations revealed that some recombinant human TRAIL (rhTRAIL) can induce apoptosis in normal liver cells, which gives rise to its safety in clinical application. In addition, multi-types of TRAIL receptor could reduce activity of TRAIL and hinder its clinical application. Further studies have found that monoclonal antibody against TRAIL receptor 1 and 2 (DR4 and DR5) has the abilities of TRAIL and can induce apoptosis in tumor cells. In normal physiological conditions, DR5 is the main functional receptor due to its binding affinity of TRAIL stronger than DR4. Currently, monoclonal antibody targeting DR5 receptor with apoptotic activity has great promise to become choice of effective anti-tumor treatment and has drawn much attention in this field.Objectives: To procure DR5 monoclonal antibody with apoptotic activity in tumor cells as well as to determine its effects and mechanisms on esophageal tumor cells, immortal esophageal epithelial cells and Scid tumor mouse model inoculated with esophageal tumor cells.Methods: Mouse monoclonal anti-DR5 antibody, designated as mDRA-6, was prepared by BALB/c mouse immunized by sDR5,when mouse spleen cell was fused with myeloma NS-1 cells at presence of 50% PEG (polyethylene glycol). specific binding of mDRA-6 by Western blot analysis, and mDRA-6 binding cell surface DR5 by flow cytometry. mDRA-6 was purified by protein G affinity chromatography. Flow cytometry was used to describe the DR5 distribution on esophageal tumor cells and immortal cells. Suppression of cell growth was determined by MTT assay, Hoechst 33258 staining was used to determine changes of cell morphology under fluorescence microscope, and JC-1 single staining and Annexin V-FITC/PI double staining were used to quantify apoptotic outcome after mDRA-6 exposure. Caspase 8, caspase 3, caspase 9 as well as Bid, bax, bcl-2 and cytochrome c were analyzed by Western blotting analysis after mDRA-6 treatment. Scid tumor model inoculated with esophageal cancer cells EC109 was injected with mDRA-6 in abdominal cavity to value mDRA-6's effects on tumor growth.Results:1. Mouse monoclonal DR5 antibody (mDRA-6) with specific ability binding to DR5 is generated by cell fusion of myeloma NS-1 cells with BALB/c mouse spleen cells follwing sDR5 immunization.2. DR5 cell membrane expression is distinct on cells with different biological behavior, which are 91.31%, 96.56%, 45.81%, 5.64% on a poorly differentiated ESCC cell line EC9706,a well differentiated ESCC cell line EC9706, esophageal immortal cells NE6 and NEC, respectively. 3. MTT analysis showed that esophageal cancer cell growth is suppressed after mDRA-6 treating at 20μg/mL for 24 hrs, i.e. EC9706 (56.53%), EC109 (68.82%), while immortal esophageal epithelial cell after mDRA-6 treating showed little growth inhibition, i.e. NE6 (26.82%) and NEC (4.56%).4. Esophageal cancer cells EC9706 and EC109 displayed typical apoptotic morphology after mDRA-6 treatment at 20 ug/ml for 6 hrs, 12 hrs and 24 hrs when observed under fluorescence microscope. The apoptotic frequency was 20.58%, 39.90%, 50.09% for EC9706 and 27.47%, 33.28%, 57.46% for EC109 at 6 hrs, 12 hrs and 24 hrs, respectively. Mitochondria membrane potential was 15.71%, 24.40%, 44.93%for EC9706 and 36.60%, 40.48%, 52.31% for EC109 at the above time points, respectively.5. Apoptotic pathway induced by mDRA-6 in esophageal cancer cells is initiated by DR5 and mDRA-6 binding followed by exogenic and later endogenic cell apoptotic cascades activation as revealed by caspase 8, caspase 3, caspase 9 and Bid, bax, bcl-2, cytochrom c by western blotting, respectively.6. Tumor growth in Scid tumor model is suppressed markedly by mDRA-6 injection in abdominal cavity compared with control group.Conclusions:1. We procured a anti-DR5 monoclonal antibody with apoptotic activity derived from BALB/c mouse which is designated as mDRA-6.2. DR5 expression on esophageal cancer cells (EC9706 and EC109) is strikingly higher than immortal esophageal epithelial cells (NE6 and NEC).3. mDRA-6 induced apoptosis in esophageal cancer cells EC9706 and EC109 through binding to DR5 followed by apoptotic cascades activation, i.e. caspase 8, caspase 3, caspase 9 and Bid, bax, bcl-2, cytochrom c.4. In vivo Powerful anti-tumor effects of mDRA-6 on Scid tumor model paved grounds for mDRA-6 humanization and its clinical application.