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

C-myc Monocular Antibody (McAb) on Gastric Cancer Cells Introduction Gastric cancer (GC) is estimated to be one of the most 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 endothelial 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 da ta exist on the occurrence of the c-myc McAb targeting against GC. In this study, we assessed the effects of c-mycMcAb on the Balb/e2nu/2nunude mice model of GC and the human gastric cancer SGC-7901 cells, and tried to investigate the function of c-myc McAbfor targeting against GC. Materials and Methods Preparation 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.Thec-myc proteins prepared in E.coliBL21 were used as immunogens.[U1] Before McAb preparation, the c-myc proteins were mixed withequal 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, the 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 mice 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. Characteristic Identification of c-myc McAb The subtype of purified McAb was determined by antibody chips according to the manufacturer's instructions (Raybitech Company, USA) and antibody titers were measured by ELISA kit (Cistron Biotechnology, Pine Brook, NJ)[25]. The assay was carried out in 96-well polystyrene plates according to the standard procedures [26]. Briefly, c-myc protein (10ÃŽ ¼g) were loaded onto plate in 0.1 M carbonate buffer (PH 9.6) and reacted with McAb at 37oC for 2 h. After washed, the mixture was monitored with horseradish peroxidase (HRP)-conjugated rabbit-anti-mouse IgG (diluted 1:100; Sigma) at 37oC for 1 h. Nonspecific antibody binding sites wereblocked with 2% FCS in blocking reagent for 15min.The OD450vaule was read with a 96-well plate reader. The antigenic specificity of McAb was determined by Western blot. Purified c-myc protein was transferred into E.coil DH5ÃŽ ± and Cells were lysed inice-cold radioimmunoprecipitation buffer (RIPA) for 30 min and centrifuged to collect the supermanant[27]. Cell lysates were blent with 3Ãâ€" loading buffer (6 % SDS, 15 % 2-mercaptoethanol, 30 % glycerol, and 0.3 mg/mL bromphenol blue in 188 Tris-HCl, pH 6.8), heated at 90oC for 10 min, and then separated by 16 % SDS-PAGE. Separated proteins in the gels were electrophoreticaly transferred onto nitrocellulose membrane, boiled in phosphate buffered saline for 4 min, and blocked with 5 % nonfat dry milk for 20 min[28]. After several rinses, the membranes were incubated with c-myc McAb overnight at 4 oC. McAb were detected by HRP-conjugated goat-anti-mouse IgG (50 ÃŽ ¼g/mL) at 22 oC for 1 h. The establishment and treatment of nude mice model of GC Four-to-five-weeks old Balb/c nu/nu mice (body weight was 18 ±1.5g) were purchased for the establishment of nude mice model of GC . The human gastric cancer cell line (SGC-7901) was grown in 10% DMEM (Gibco) supplemented with FCS (100 mL/L), penicillin sodium (100 U/mL) and streptomycin sulfate (100 ÃŽ ¼g/mL), and cultured at a 5% CO2 incubator at 37 oC. Exponentially growing SGC-7901 cells were trypsinized resuspended to make a cell suspension of 2Ãâ€"107 cellls/mL. The nude mouse was injected subcutaneously with the suspension (0.2 mL) into the right and left root. Tumor masses were obvious at 10 d. Mice were randomized into 4 groups including low-dose group, middle-dose group, high-dose group and saline group (10 mice/group). Mice of each treatment group were inoculated intraperitoneally with c-myc McAb weekly (10 mg/kg, 20 mg/kg, 30 mg/kg, respectively), and mice of saline groups were injected with normal saline (0.2 mL) instead. Four weeks after injection, the mice were sacrificed and tumors were examined to calculate the tumor inhibition rates (). Immunohistochemistry (IHC) The procedures of SP immunohistochemical kit (SP kit, Maxim Biotech) were as follows: The tissue of tumors was fixed in 10 % phosphate-buffered Formalin, embedded in paraffin, and sectioned at a thickness of 4ÃŽ ¼m. Tissue sections were deparaffnized, hydrated and washed in PBS. Antigen retrieval was performed by combining the tissue with 10mM citrate buffer (pH 6.0) in a microwave for 10 min. Nonspecific protein bindings of tissues was blocked with 5 % normal sheep serum (NSS) for 10 min. after rinsing in PBS, sections were incubated with c-myc McAb at 4 oC overnight at a dilution of 1:100. Secondary antibody (Carpinteria, goat anti-mouse biotenylated, 1:50 in PBS) was applied at room temperature for 30min after washed, and then HRP-conjugated streptavidin were added. The slides were visualized by diaminobenzidine (DAB) (Dako, Carpinteria, CA, USA) for 5 min and counterstained with hematoxylin for 2 min, terminated, dehydrated, transparentized, sealed and photographed step by step. Negative controls were prepared by replacing primary antibody with PBS. Western blot analysis C-myc McAb (1 ÃŽ ¼g/mL, 2 ÃŽ ¼g/mL, 4 ÃŽ ¼g/mL, respectively) were added into SGC-7901cells andcultured for 24 h, 48 h, 72 h, for blank controls, the SGC-7901cells were omitted and HFE-145 cells were used instead. Cells were collected and lysed in ice-cold RIPA,and then following sections were mainlysimilar with the procedures of the Characteristic Identification of c-myc McAb. MTT (3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide) assay Cells were seeded into 96-well plates (10, 000 cells/well) and cultured at 37 oC in a 5% CO2 incubator after HGC-7901cells and normal gastric cell line HFE-145 were trypsinized. The culture medium was washed with PBS 3 times, and thenc-myc McAb (1 ÃŽ ¼g/mL, 2 ÃŽ ¼g/mL, 4 ÃŽ ¼g/mL,) were added respectively, 20 mL (5 mg/mL) At the indicated time points (1 d, 2 d, 3 d, 4 d, 5 d), each well were added with 20 mL MTT cultured at 37 oC for 4 h. Then 150 ÃŽ ¼L DMSO was added again to stop the reaction after the supernate were dropped, The plate was read on multiwall plate reader (Thermo Fisher, Basingstoke, United Kingdom) at 570nm. . A dose-response curve was plotted for the HGC-7901cells and HFE-145 cells. Cell adhesion assay Before cell adhesion and migration assays, SGC-7901 cells and HFE-145 cells were serum starved in bascal culture medium overnight. In brief,6-well tissue culture plates were coated with 10ÃŽ ¼g/mL fibronectin and 10 ÃŽ ¼g/mL poly-L-lysine overnight, the wells were washed with PBS-T and blocked with 5 % BSA in PBS-T[29]. SGC-7901cells and HFE-145 cells were released with trypsin to prepare of single cell suspensions. The cells were applied to 6-well tissue culture plates (50 ÃŽ ¼L/well) and incubated at 37oC for 12 h. When cells were grown to approximately 90 % confluence, the c-myc McAb(1 ÃŽ ¼g/mL, 2 ÃŽ ¼g/mL, 4 ÃŽ ¼g/mL,) were added respectively. Cells were allowed to attach for 2 h, and the culture medium were discarded. Before released with trypsin, cells were washed twice with PBS and 1mM Ethylene Diamine Tetraacetic Acid (EDTA). The formula of was used to calculate the adhesion rates. Cell migration assay Cell migration assays were performed in transwell bicameral chambers as described[30]. Matrigel (Becton Dickinson Company, Bedford) at a dilution of 1:100 were coated with culture medium without serum at 37oC for 30 min in the apical chamber. Cells were released with trypsin, washed ,and resuspended at a final concentration of 5Ãâ€"105 cell/mL in serum-free bascal culture medium (EBM) containing 0.1 % BSA. The suspension (100 ÃŽ ¼L), which were seeded on the upper chamber, were mixed with c-myc McAb (1 ÃŽ ¼g/mL, 2 ÃŽ ¼g/mL, 4 ÃŽ ¼g/mL, respectively). The lower chamber was filled with 500ÃŽ ¼L mouse embryonic fibroblasts (NIH3T3) which were cultured with RPMI (10mM Hepes, 0.5% BSA, pH 7.4). Migration chambers were incubated at 37oC in 5% CO2 incubator for 72h. After removing stationary cells from the upper side of the membrane with a cotton-tipped swab, migrated cells were fixed and stained with 1% crystal violet. Cells were counted in three fields at a magnification of Ãâ€" 400. Cell apoptosis and cell cycle assays HGC-7901cells and HFE-145 cells were seeded into 6-well plates containing Dulbecco's Modified Eagle's Medium (DMEM) with 10% FCS and cultured at 37 oC in a 5% CO2 incubator overnight. Supernatants were discarded, before c-myc McAb (1 ÃŽ ¼g/mL, 2 ÃŽ ¼g/mL, 4 ÃŽ ¼g/mL,) were added respectively and attached for 72 h. cells were fixed with pre-cooled ethanol (75%) and stained with Propidium Iodide (PI, Sigma) in the dark at room temperature for 15 min , after centrifuged at 1,000 rpm and rinses in PBS several times. The cell cycle and apoptosis rates were analyzed by FACS (Elite ESP, Beckman Coulter, Brea, CA). Statistical analyses Statistical analysis was performed using Software version SPSS11.0. All data was expressed as mean  ± SD (standard deviation). Comparisons were made by Student's t-test and comparisons of parameters were made using one-way analysis of variance among 4 groups. A value of P