C-myc Monocular Antibody (McAb) on Gastric Cancer Cells

C-myc Monocular Antibody (McAb) on Gastric Cancer Cells I ntroduction Gastric cancer (GC) is estimated to be one of the mos t common and frequent malignant tumor of the digestive system. The incidence and mortality of GC have ranked the second among all tumor diseases worldwide [ 1-5 ]. However, it ranks in first place in China[ 6 ]. Complete surgical resection is still the standard for all patients with resectable GC. It remains highly problematic for the regional and less common systemic recurrences[ 7 ]. Recent improvement in surgical technique, adjuvant chemotherapy and radiotherapy has increased the survival rate of patients with early-stage, but the patients who have advanced GC are difficult to cure. With more and more research of molecular biological mechanisms known by us, molecular targeted therapies including cell growth, cell cycle, apoptosis and invasion have become a popular tumor comprehensive therapy[ 8 ]. Some of single-targeted spots are mainly Human epidermal growth factor receptor (HER-1, HER-2), Vascular endothe lial growth factor (VEGF), Human epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), Cyclin-dependent kinase inhibitor (CDKI), Human proto-oncogene (c-MET)[ 9 , 10 ]. However, it needs a huge space to develop the targeted anticancer drugs. An elegant way to accumulate therapeutic agents at the tumor site is their specific antibodies[ 11 ]. Oncogenes are well documented to be involved in mediating apoptosis and cell cycle resulting in cancers[ 12 , 13 ], its activation can play an important role in the progress of cancer. C-myc is an important member of the c-myc family and a master regulator of genes involved in diverse cellular processes in GC[ 14 ]. The c-myc is a nuclear transcription factor which centrally regulates cell proliferation, differentiation, cell cycle and apoptosis, once c-myc is activated in vivo or in vitro , it is easy to make the cells far from the normal growth and promote cell malignant transformation to cancer finally[ 15-17 ]. It was reported that the expression of c-myc is an important consideration in the biological characteristic of GC [ 18-20 ]. The previous studies also have proved that c-myc has tight relation with Brest cancer, lung cancer, colon cancer, hematopoietic cancer [ 21-24 ]. Currently few data exist on the occurrence of the c-myc McAb targeting against GC. In this study, we assessed the effects of c-myc McAb on the Balb/e2nu/2nu nude mice model of GC and the human gastric cancer SGC-7901 cells, and tried to investigate the function of c-myc McAb for targeting against GC. Materials and M ethods P reparation of c-myc monocular antibody (McAb) All experiments involving animals were approved by the Institutional Animal Care and Use Committee of Renji Hospital Affiliated to Shanghai Jiao Tong University of Medicine. Mice were used in this study from Animal Science Laboratory of Shanghai Jiao Tong University, and all effects were made to minimize distress.T he c-myc proteins prepar ed in E.coli BL21 were used as immunogens. [U1] Before McAb preparation, the c-myc proteins were mixed with equal volume of complete Freund's adjuvant (CFA). Female Balb/c mice aging from 6-8 weeks [U2] were immunized intraperitoneally with 50 ÃŽ ¼g c-myc proteins (1v:1v) in CFA. The immunization was repeated with the same amount of immunogens [U3] in incomplete Freund's adjuvant (IFA) at 14d. A final immunization was performed with 100ÃŽ ¼g mixture of c-myc proteinsand IFA at 28 d. Then, t he blood was drawn from the caudal vein and serum titers were measured by ELISA at 35 d. A booster injection was given intraperitoneally at the antibody titers of 640,000 [U4] tested by ELISA at 35 d. Five days after boost, spleen cells were isolated and mixed [U5] with SP2/0 myeloma cells. When the Hybrid cells grew to 50%, the positive clones were collected by ELISA. The hybridomas processed by Silica gel H was inoculated intraperitoneally into unsexed Balb/c mice. Then, the m ice were scarified and the ascetic fluid was collected. The McAb was purified and the concentration was determined by bicinchoninic acid assay (BCA) Protein Assay Reagent Kit.