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. 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.
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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. 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. 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. 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-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
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 μg 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 100μg 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.
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). The assay was carried out in 96-well polystyrene plates according to the standard procedures . Briefly, c-myc protein (10μg) 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 DH5α and Cells were lysed inice-cold radioimmunoprecipitation buffer (RIPA) for 30 min and centrifuged to collect the supermanant. Cell lysates were blent with 3× 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. 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 μg/mL) at 22 oC for 1 h.
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 μg/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 2×107 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 4μm. 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.
C-myc McAb (1 μg/mL, 2 μg/mL, 4 μg/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 μg/mL, 2 μg/mL, 4 μg/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 μL 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 10μg/mL fibronectin and 10 μg/mL poly-L-lysine overnight, the wells were washed with PBS-T and blocked with 5 % BSA in PBS-T. 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 μL/well) and incubated at 37oC for 12 h. When cells were grown to approximately 90 % confluence, the c-myc McAb(1 μg/mL, 2 μg/mL, 4 μg/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. 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 5×105 cell/mL in serum-free bascal culture medium (EBM) containing 0.1 % BSA. The suspension (100 μL), which were seeded on the upper chamber, were mixed with c-myc McAb (1 μg/mL, 2 μg/mL, 4 μg/mL, respectively). The lower chamber was filled with 500μL 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 × 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 μg/mL, 2 μg/mL, 4 μg/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 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<0.05 was considered to be statistical significance.
Production of c-myc Monoclonal Antibody (McAb)
One stable hybridoma cell lines excreting McAb was obtained by ELISA. Ascetic fluid was obtained from mice which were injected with positive hybridoma cells and c- myc McAb reached a purity of 97 % and a concentration of 2.3 mg/mL after purification (Figure 1). Isotype of c-myc McAb were determined by testing purified McAb, type of immunoglobulin was shown on the Figure 2A (Figure 2). C-myc McAb were IgG1 and asceties were mainly IgG1, IgG2a and IgM, compared with the condition medium of SP2/0 cell. Western blot results were shown that a single protein band of 62 kD was observed in the lane of c-myc protein and E.coil DH5α bacterial lysate (Figure 3), it was suggested that c-myc protein could be identified by c-myc McAb specifically.
C-myc McAb affect growth of tumor
The anti-tumor rates of middle-dose (40.26 ± 4.25%) and high-dose groups (44.81 ± 5.74%) were significantly larger than the low-dose (7.79 ± 0.92%) and control group (P < 0.05) (Table 2). Furthermore, the potential for inhibiting tumor growth was dependent on the dose in vivo.
The positive signals of IHC were mostly located in the nucleus (Figure 4). The number of positive signals was decreased obviously in tissues treated with the C-myc McAb of middle-dose and high-dose, compared with control group and saline group. C-myc McAb inhibited the expression of c-myc in a dose-dependent manner in vivo.
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SGC-7901cells were collected to examine the expression level of c-myc in 24 h, 48 h, 72 h (Figure 5). Compared with the expression of β-actin, the amount of protein expression was evidently decreased in the middle-dose and high-dose groups. However, the expression of c-myc had no changes nearly at the each point in time. The expression of c-myc were highly depentent on the dose, but not time in vitro.
The performance of SGC-7901cells were influenced by c-myc McAb
Compared with the HFE-145 cells, the proliferation of SGC-7901cells was decreased by the increase in concentration of c-myc McAb (Figure 6). After cultured with c-myc McAb, the adhesion rates of SGC-7901cells in the middle-dose and high-dose groups were reached as 56.32±8.24 % and 48.22±7.15%. C-myc McAb were significantly reduced the adhesion rates, compared with the blank control group (P < 0.05) (Table 1). The cell migration were examed under the microscope, the migration rates of SGC-7901cells in the middle-dose and high-dose groups was obviously lower than the blank control group (P < 0.05) (Table 2).
Cell cycles and apoptosis of SGC-7901cells were analyzed by Elite ESP. In the middle-dose and high-dose groups, SGC-7901cells were increased in the G0/G1 phase and reduced in the S phase of the cell cycle after treatment with c-myc McAb (P < 0.05), but there was no significantly difference in HFE-145 cells . It was suggested that c-myc McAb can inhibit proliferation of SGC-7901cells by stagnation in G0/G1 phase (Table 2). Compared with the control group of HFE-145 cells, the apoptosis rates of SGC-7901cells were highly increased after cultured along with c-myc McAb in the middle-dose and high-dose groups (P < 0.05), the apoptosis of SGC-7901cells was suggested by a promotion of c-myc McAb (Table 3).
The GC is one of the most malignant tumor in the world, especially in South America and South-East Asia, where it shows the highest frequency and morbility[5, 31]. Several preclinical studies, including both in vitro and in vivo studies are committed to explore a novel efficient treatment method for GC. Our findings show that c-myc McAb which was pepared with c-myc proteins can remarkably inhibit proliferation, migration, adhesion and promote apoptosis in vitro, and affect the growth of transplanted tumors of GC in vivo as well.
With the intensive study of oncogenes and cancer chemotherapy drug toxicity, more scholars have paid attention to the targeted therapy of oncogenes, their corresponding McAb are mainly acted as targeted drugs such as Trastuzumab, Cetuximab, Panitumumab and Figitumumab and so on. Overexpression and amplification of c-myc were observed in gastric cancer cells for the first time in 1985 and also have close relation with biological behavior of cancers, such as cancer of biliary duct, lung cancer, liver cancer, nasopharynx carcinoma and so on [33-35].
As known, the healing process of transplanted tumors is a chronic and complex course, involving cell migration, adhesion, invasion, as well as immunityconditions. Of them, cell migration is also a prominent component of tissue repair, regeneration and immune surveillance, in which leukocytes from the circulation migrated into the surrounding tissue to destroy invading microorganisms and infected cells to clear debris[37, 38]. The tissue repair, regeneration and tissue remodeling require active cell motility, which is an adhesion-dependent process involving cell migration, angiogenesis, and regrowth of severed nerve ends . Cell migration and cell adhesion have been taken as gold indicators to determine the success of cancer treatments in a mass of studies. Hence, the influence on cell migration and adhesion will directly affect on the healing in GC patients. The degradation in cell migration, proliferation and adhesion caused by the suppression of expression of c-myc is obviously detrimental for healing for GC. The suppression of expression of c-myc also affected the cell cycle, accelerated cell apoptosis, and then led to a rapid alteration of SGC-7901 cells. Therefore, the expression of c-myc can give rise to the degradation in cell migration, proliferation and adhesion, but acceleration in cell apoptosis, as well as changes in cell cycle. All the variations in cell function can further make for the persistent course of GC.
C-myc is overexpressed in most cases of primary GC, metastatic lesions also have high levels of expression, which suggest that overexpression of c-myc may play a role in GC and tumor metastasis. Previous study has reported that abnormal c-myc expression is an important factor in biological behavior of GC and can regulate apoptosis[18, 41]. It was also previously confirmed that overexpression of c-myc in the cells of various malignant tumors could result in migration and resistance against cell apoptosis and cell growth. As shown in the results, the expression of c-myc protein was significantly down-regulated after incubated with c-myc McAb. It has reported that down-regulation of expression of c-myc will result in active apoptosis but weakened cell migration, adhesion and proliferation. Moreover, activation of expression of c-myc has been shown to overcome cell cycle arrest in G0/G1 phases in cancer cells [43-45]. All of above are consistent with the results in our experiment.
.In conclusion, a dose of c-myc McAb could weaken cell proliferation, adhesion and migration, promote apoptosis, interfere cell cycle of SGC-7901cells, and affect the normal growth of tumor. The effect of c-myc McAb in vivo and in vitro was mainly generated by suppression of expression of c-myc protein. Our findings have an important implication that c-myc McAb may be a novel molucar target for GC treatment. However, this study only can provide an experimental basis for clinical trials, further study is imperative to quantify the dose of c-myc McAb in clinical trials.
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