Using high-performance liquid chromatography, the peptide synthesis and purification were performed to a 90% purity, followed by analyzing through the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, which were ultimately verified by electrosprayionization mass spectrometry (ESI-MS).
A peptide was constructed with the sequence of CGGSGLPLGHIKC according to that of basic fibroblast growth factor (bFGF2) , manufactured by the Shine Gene Biotechnologies Inc. (Shanghai, China).
Metastatic breast cancer modeling
Weak metastasis in mice can be induced by the human tumor cells, and unanticipated outcomes may ensue in case of metastasis occurrence. In spite of this, murine tumor renders more effective metastasis and features similar to those observable in cancerous patients . Tumor in animals with the normal immune system can be analyzed by conditions provided by this type of tumor. The pivotal contribution the immune system to the emergence and development of cancer  necessitates designing models for application in the perfect immune of mice to assess medicines and peptides. One of the several breast cancer cell lines is the 4T1 cell line, which can effectively induce metastasis to areas engaged in human breast cancer .BALB/c mice are the laboratory- bred strain of albino mice specially used for the study cancer. Original BALB/c, and the 4T1 breast cancer cell lines wereprocured from the Pasteur Institute of Iran, and grown in a glucose-rich DMEM culture medium with 10% fetal bovine serum, and 5% inessential amino acids, as well as penicillin and streptomycin antibiotics, and place in an incubator at 37ºC under 5% carbon dioxide environment. BALB/c was preserved at a 24 ºC, 12 h light/12 h dark photoperiod, and sufficient moisture with adequate food and water for 5–7 weeks. The mice were retained in polycarbonate cages with lace doors.
This research was carried out in accordance with the ethical guidelines of research on the experimental animals of the National Ethics Committee in Biomedical Research of the Islamic Republic of Iran.
Tumor implantation and antitumor activity
Breast cancer was induced by two methods of 4T1 cell injection and cancer tissue transplant. In cancer cell injection procedure, the site of injection in rats was sterilized by cotton and alcohol. In day zero, all mice were hypodermically injected cancer cells (4 T1; 1×106 cells/500 μl or 1×105 cells/50 μl)) at a place targeted next to the lower most right side breast gland. Following 2 weeks, the mice underwent euthanasia and surgery (10). The “Euthanasia" defined as”good death" aiming at providing a quick, pain-free, non-stressful death. A fast unconsciousness is induced by carbon dioxide. After which death occurs. The cage volume should be replaced by CO2 flow rate at 10% to 30% per minute. The animals should be easily observable in the euthanasia chamber. The only suggested sour of co2 are cylinders containing compressed co2 gas because they enable the control of gas inflow into the induction chamber.
Then, the tumor was incised into sections of<0.3 cm3 and hypodermically implanted into the mice’s right flanks after anesthetizing the animals with ketamine (100 mg/kg) and xylazine and xylazine (10 mg/kg). Peptide treatment was applied upon reaching a tumor size of 0.5 to 1 mm3 after a 2 weeks period of tumor implantation. The tumor volume was evaluated daily by a digital Vernier caliper according to the formula: Volume = shortest diameter2× longest diameter× 0.52(10).
Tumor size measurement
The BALB/c mice having 4T1- MCT were then used to evaluate the antitumor activities of the peptide. To this end, rats were assigned at random to four groups of 10 mice each (n total = 40 mice).
bFG suppresses the growing and metastasis of a MCT model
bFG antitumor efficacy was investigated in the murine 4 T1 MCT model, and having built the solid tumors (tumor volume ~400mm3), intraperitoneal (i.p.) administration of the peptide was done one time daily at various doses of 1, 2.5, 10 mg/kg over 2 weeks.
On the 28thday, the tumor mean volume showed a significant increase in the phosphate buffered saline (PBS) treatment group compared to that at different doses of bFG 1 mg/kg, 2.5 mg/kg, and 10 mg/kg groups (P < 0.001). This demonstrates that the bFG-caused tumor reversion had no associations with dose- response between the dosages of 1 and 10 mg/kg. No mortality was recorded in animal groups experienced solid tumors throughout the tests, illustrating that bFG is not toxic at dosages used in this trial. Also, we did not see any decrease in the animals’ weight in bFG-treated groups; in fact, weight gain had been observed.
A highlighting prerequisite for tumor cell transport via the circulations and metastasis is believed to be tumor angiogenesis, and tumor angiogenesis inhibition has been revealed to be suppressed for tumor metastasis. This experiment aimed to examine whether or not bFG was also capable of blocking the metastasis of malignant murine mammary tumor cells (4 T1 cells).
Viability analysis using MTT method
MTT is a method based on the colorimetric for determining survivability and cell toxicity of materials on the basis of recovering yellow soluble crystals of 4, 5 Dimethyl thiazole, 2–5Diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase enzyme of viable cells and formation of formazan purple solution crystals. Using measurement of optical absorption in 570nm usingUltraviolet-Visible Spectrophotometer and using standard curve, a number of viable cells can be determined. The Proliferation of 4T1 cells. The antagonistic activity of the peptide was examined on 4T1 cells by the use of different peptide levels (50–600 ng/ml) and 20 ng/ml of rhbFGF. Seeding4T1 cells was done in 96-well plates with 5 × 103 cells per well. After incubation ofwell-plates under a temperature of 37ºC and 5% CO2, cells were rinsedby PBS and kept serum-starving in serum-free DMEM with 0.02 % FBS within 24 h. Next, cells underwent treatment with peptides of serial dilutions and 20 ng/ml bFGF for 24h. Then, the produced formazan was converted to solution via adding a solvent like Dimethylsulfoxide (DMSO). Finally, solution absorption was read in wavelength of 570 nm to determine number of survived cells.
BFG binding assays
To evaluate the competitive binding of BFG protein, 4 T1 cells were first grown in DMEM in 96-well plates (5000 cells/well) and then subjected to incubation in 5% FBS at 37 °C during nighttime. Following 24 h, the media were altered to the DMEM with no FBS, and all cells, excluding the control group cell, were treated with bFG at 37 °C overnight. At ambient temperature over 5–10 min, cells were fixed using paraformaldehyde 4% and rinsed with PBS.Then, the PE-conjugated anti-bFGFR1 antibody was added, and cells were made permeable using PBS+0.3% Triton X–100, blocked with 10% normal goat serum and BSA 1%/PBST at room temperature for 20 minutes.
Cells were stained with 4′, 6-diamidino–2-phenylindole (DAPI; Invitrogen, Carlsbad, California, USA) (1 μg·mL−1) with FITC-secondary anti-rabbit antibodies, and subjected to treatment using propidiumiodide (PI; Sigma Aldrich, USA, P4170) (1 μg·mL−1) for nuclear staining. They were then stained by fluorescence microscopy in the blue light (DAPI; Invitrogen, Carlsbad, California, USA; 1 μg·mL−1, nuclear staining), and prior to undergoing analysis with the NIH ImageJ plug-in, they were combined (http:// imagej.nih.gov/ij/). Error bars indicate the standard error of the mean (SEM) of estimations made on six samples in each group, and scale bars are 20 μm. A. After 24, 48, and 72 hours, the effects of BFG on the 4 T1cells proliferation were quantified by 3-(4, 5-dimethyl thiazolyl–2)–2, 5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis,Missouri, USA) experiments as meticulously clarified formerly with slight alterations. Using the ELISA reader (Space Fax 2100, A wareness,USA), absorbance was read at 570 nm with background deduction of 630 nm.
Due to immunohistochemical (IHC) analysis, excised tumor tissues were fixated in formalin (4%), imbedded in paraffin, segmented, and exposed to staining by hematoxylin-eosin (H&E). Heat-activated epitope recovery in a buffer at pH 9.0 was doneprior to immunohistochemistry (IHC).
IHC staining was carried out followed by the formalin-fixated paraffin imbedded segments to measure different proteins involved in various procedures such as CD31 and CD34 proteins for assigning MVD and Ki67 to specify the proportion of Ki67-positive cells, and also P53 and Bcl–2 to clarify the fraction of apoptotic cells in relation to the whole cells.
Dead cells were stained by TUNEL (terminal deoxynucleotidyltransferase nick-end labeling), then by developing the enzyme in diaminobenzidine (DAB) (Invitrogen, Carlsbad, California, USA) recognition and counter-staining with hematoxylin. The samples were analyzed using microscopy (Olympus BX–51, Japan), and having analyzed five random tissue samples under scale bars 100 and 20 μm, many positive cells were determined and quantitatively analyzed by ImageJ software.
Apoptosis Examination with Tunel Kit
Apoptosis was identified through the in situ Cell Death Detection Kit, Roche–11684817910. In brief, the sections were deparaffinized and dehydrated, and then rinsed with distilled water. The tissues were stored in a solution of proteinase K (20 mg/mL) at room temperature for 15 min. Endogenous peroxidase activity was also blocked through incubation in 3 mL/L solution of hydrogen peroxide/methanol for 30 min at 37 °C. Sections were exposed to incubation with terminal deoxynucleotidyltransferase at 37 °C for 60 min. Then, digoxygenin-conjugated deoxyuridine triphosphate (dUTP) was added to the 3’-OH ends of the fragmented DNA molecule. Anti-dioxygenin peroxidase antibody was used to detect the labeled nucleotides. The sections were stained with diaminobenzidine and hematoxylin was used for background staining.
In the current research, the impacts of the peptide were investigated on PI3K/AKT and ERK/MAPK signaling pathways.To obtain homogeneous tumor tissue, approximately 150–200 mg of the tumor tissue was powdered in a mortar, lubricated in RIPA buffer (50 mL of Tris hydrochloride, 150 mM sodium chloride, 1% NP–40, sodium deoxycholate, 10% sodiumdodecyl sulfate, 0.01 M phenylmethylsulfonyl fluoride, emulsifiable EDTA) with a ratio of 1:5, and completely homogenized. The solution was then stored at 4 °C for 30 min and then underwent centrifugation at 14,000 rpm at 4 °C for 15 min. The supernatant was collected and kept at –80 °C. The Protein contents of specimens were assessed using the Bradford technique based on the Bovine serum albumin.
To perform western blot, 20 µg of protein was loaded into each well, and the protein was isolated using the SDS PAGE technique with 12.5% gel.The isolated proteins were relocated to PVDF membrane with a pore size of 0.45 µm. Then, the membrane was moved to a solution that contained 50 mM Tris-HCl buffer, 0.1% TWEEN 20 Detergent, 150 Mm NaCl, and 5% skimmed milk and stored for 1.5 hours. Then; the membrane was stored in an initial antibody solution with the concentration of 1 μg/mL overnight, diluted in a buffer containing 1% BSA and 2% skimmed milk. After rinsing the membrane, it was subjected to the secondary HRP to remove the non-attached antibodies and then rinsed again. Protein expression was measured using the ECL method. The membrane was exposed to the radiographic film and the bands appeared by processing the films in the darkroom. Then, the bands density was measured using densitometry.
Data were analyzed statistically and the charts were drawn by the Prism software (Version 6.00). Data are expressed as mean ± SEM. One-way ANOVA and then Tukey’s posthoc test was employed to verify statistically significant differences for manifold comparisons. Two-way repeated measures ANOVA ensued by Tukey’s post-hoc test were applied for treatment effectiveness in influencing tumor development with a statistical probability of P <0.05.