Preparation Of Anti-HPA Monoclonal Antibody And Effects Of Anti-HPA Monoclonal Antibody Combined With Paclitaxel On Proliferation And Invasion Of Breast Cancer Cells

Objective: Heparanase (heparanase, HPA) as the only endogenous D-glucuronidaseenzymes, is capable of degrading heparan sulfate proteoglycan (HSPG). The HSPG is amajor component of glycosaminoglycans, and plays an important role in cancerproliferation and metastasis. HPA degrades heparan sulfate side chains, and destroys theintegrity of tissue, promotes more active molecules (growth factors, cytokines, etc.)release, so has an important role in the tumor invasion, tumor angogenesis and metastasis.The objective of the experiment was to obtain some anti-HPA monoclonal antibodieswith high specificity. To investigate the inhibitory effects of anti-HPA monoclonalantibody alone or combined with paclitaxel in human breast cancer cells on cellproliferation and metastasis in vitro. Lay a foundation for development of anti-metastaticdrugs.To provid material for the studies of mechanism of HPA in tumor occurrence anddevelopment, and effects of HPA in cancer diagnosis, treatment and prognosis. Lay thetheoretical foundation for the clinical treatment of breast cancer by select paclitaxel incombination with anti-HPA monoclonal antibodies.Methods: BABL/c mice were immunized with prokaryotic expression HPA50kDaprotein, and anti-HPA monoclonal antibody was obtained by hubridoma technology.Anti-HPA monoclonal antibody was purificated with PEG6000precipitation andmolecular sievr chromatography after identified the mouse antibody Ig subclasses bycapturing ELISA and test strip. Specificity of monoclonal antibody was identified byindirect ELISA and Western blot. The effects of paclitaxel, anti-HPA monoclonalantibody and combination of paclitaxel and anti-HPA monoclonal antibody onproliferation of breast cancer cells were examined by MTT assay and cell cycle wereanalyzed by flow cytometry. The cells scratch test and Transwell assay were used to evaluate the invasion of breast cancer cells.[Result]:(1) The HPA50kDa prokaryotic protein, that was dialyzed and concentrated,immunized five BALB/c mice. After three times, No.3and No.4mice had higher titerswhich were detected by indirect ELISA. Their titers achieved1:64000above, so the No.4was chosen. The4th mouse was choosed to obtain spleen cells which was used withmyeloma cells in cell fusion by PEG. The fusion rate was90%, and the positive rate was82%. Using limited dilution method to subclone, hybridoma cell lines secreting thedesired antibody were obtained, named2A4-2、2B9-2、2C3-2、2C8-2、2F6-2、2G11-2、3B8-2、4B11-3、4D4-2、4E5-2、4G11-3、5C7-2、5F2-2、6D3-2. Ig subclasses of antibodywere IgM by capture ELISA and test strip. The cells (2B9-2、2C3-2、2C8-2、3B8-2、4B11-2、4D4-2、5C7-2) were choosen to pruduce ascites.The specificity of monoclonal antibody was identified by indirect ELISA andWestern Blot, and there were three kinds of monoclonal antibody (2C3-2、3B8-2、4D4-2)had high specificity. In the progress of purification, ascites were precipitated by PEG6000preliminarily, and the purity of antibody was about80%. Then, samples were furtherpurified by the molecular sieve column. The protein consentration was0.116mg/ml.(2) The purified anti-HPA monoclonal antibody (3B8-2) was used to interfere breastcancer cells (MCF-7cell、MDA-MB-231cell), and found that all the cell morphologyhad significant changed. When anti-HPA monoclonal antibody was used alone, thegrowth of breast cancer cells（MCF-7cell、MCF-7/F5cell、MDA-MB-231cell、SK-BR-3cell）were inhibited. MCF-7/F5had the maximum inhibition rate of cell growth. Whenthe concentration of anti-HPA monoclonal antibody was5μg/ml, the inhibition rate was22.37%. Paclitaxel which was used alone had the concentration-dependent inhibition inbreast cancer cells. The combination of paclitaxel and anti-HPA monoclonal antibody hadsignificantly higher inhibition rates of cell proliferation in breast cancer cells. In cellcycle, the combination of paclitaxel and anti-HPA monoclonal antibody for MCF-7cell、MCF-7/F5cell、MDA-MB-231cell inducted15%,33.7%,53.6%apoptosis. In the cellmigration and invasiveness of breast cancer MCF-7/F5, the penetration rate was75%inthe anti-HPA monoclonal antibody group. In combination of paclitaxel and anti-HPAmonoclonal antibody group, the penetration rate was reduced to20%from30% (penetration rate of paclitaxel group). Combination of paclitaxel and anti-HPAmonoclonal antibody group compared with anti-HPA monoclonal antibody groupwere highly significant difference (P<0.01), while compared with paclitaxel group weresignificant difference (P<0.05).Conclusions:(1)Hybridoma cells were obtained secreting highly specific anti-HPAmonoclonal antibody, the purity of the sample was more than90%of anti-HPAmonoclonal antibody.(2) The anti-HPA monoclonal antibody in combination withpaclitaxel on inhibition of cell proliferation, cell invasion were more significant than thetwo drugs used alone. The ability to inhibit the proliferation and invasion of breast cancercells of paclitaxel was enhanced by anti-HPA monoclonal antibody.