Cell culture and treatments by Avastan:
Human breast cancer cell line (MCF-7) was provided from American Type Culture Collection (ATCC, Rockville, MD, USA) and were cultured in DMEM medium (Gibson, Paisley, U.K.) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 100 U/100 μg/ml penicillin-streptomycin (Gibco, Paisley, U.K.). The cells were trypsinized and sub cultured twice a week. In logarithmic phase of growth, 2.0 ×104 the cells were seeded in every wells of 6 well plates (250 ×103 cells/well) and/or 2.0 ×104 cells/well in 96 flat bottom micro wells (Nunc, Denmark). The cells were incubated over night for complete adherence. Then the cells were treated by 12.5-500 µg/ml of Avastin (Bevacizumab) (Roche, Hafteman-La, Basel, Switzerland). The cells were incubated in DMED medium, supplemented by 10% of FBS for 96 hr at 37°C, 5% of CO2. After 96 hr, the cells pellet and supernatants were separately collected and the total cell number, viability, MTT cell proliferation assays, VEGF expression and secretion were done as below.
siRNAs transfection
The MCF-7 cells were cultured for 24 hours and transfected by 3double-stranded RNA and oligo targeting VEGF which were purchased from Santa Cruz (Santa Cruz Biotechnology Inc, Bergheimer, Heidelberg, Germany). Oligoes which used as scramble and VEGF siRNA have sequences as:
Scramble siRNA sense: 5/ -GCG GAG AGG CUU AGG UGU A[TT]-3/.
Antisense: 5/-UAC ACC UAA GCC UCU CCG C[TT]-3/.
hVEGF-siRNA, sense strand RNA, 5/-GGA GUA CCC UGA UGA GAU C[TT]-3/.
hVEGF antisense strand RNA, 5/-GAU CUC AUC AGG GUA CUC C[TT]-3/.
The cells (with 60% of confluency) were incubated in 800 µL of antibiotic and serum free DMEM in six-well plate per well. 10 µL of 3 siRNAs stock (10µmol) and 10 µL of an X-termGENE (Roche) transfection reagent were diluted to 100 µL antibiotic and serum free DMEM and then incubated separately at room temperature for 5 min. The two mixtures were combined and incubated at room temperature for 30 min. The siRNAs, X-term mixtures were added drop wise to cells in well. The cells were incubated for 6 hours. Then 1000 µL of 20% FBS DMEM were added to cells, after 72 hours’ cell detached and analyzed for cell proliferation, viability, qRT-PCR and ELISA assay as described below. To analyze the percent of transfection of cells, the experiment was performed by control siRNA conjugate with FITC (Invitrogen) and the percent of FITC positive cells were analyzed by flow cytometry according to the manufactures recommendations.
For combination study the cells were treated by 25 µg/ml of Avastin and VEGF siRNA.
Cell proliferation and growth
Cell number counting and viability (Trypan blue dye exclusion) were enumerated using a Neobar hemocytometer.
MTT assay: After 96 hr of incubation, 10µl of freshly prepared (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, U.S.A.) solution (5 mg/ml in PBS) was added to each microwell and were incubated for 4hr. Then, 50µl of MTT lysis solution (20% Sodium Dodcyl Sulphate W/V and 50% Dimethy Formamide V/V) was added to each well and incubated overnight. Absorbance was read at 580 nm using a Titretek multiscan ELISA reader (Labsystems Multiskan, Roden, Netherlands).
Semi-quantitative detection of VEGF mRNAs by RT-PCR
Avastin and/or VEGF siRNA treated cells were collected and total RNA was extracted by using the Tripur isolation reagent (Roche, Mannheim, Germany), according to the manufacture’s protocol. RNA yield and purity were quantitated by measuring optical density (OD260/280) using a Nanodrop (Beckman Coulter Inc. CA, USA). First-strand cDNA was synthesized from 1 μg of total RNA using Maurine Maloney Leukemia virus (M-MLV, Fermentas) and reverse transcriptase (Fermentas Gmbh, Leon-Rot, Germany) with oligo-dT primer (Fermentas Gmbh, Leon-Rot, Germany), according to the manufacturer’s instructions. The VEGF and GAPDH cDNA were amplified by the following primers:
hVEGF forward: 5/-CCA TGA ACT TTC TGC TGT CTT-3/, and reverse: 5/-ATC GCA TCA GGG GCA CAC AG-3/; GAPDH forward: 5/-AAT CCC ATC ACC ATC TTC CA-3/ and reverse: 50/GTC ATC ATA TTT GGC AGG TT-3/.
The expected sizes of the RT-PCR product were 121 bp for GAPDH and 250 bp for VEGF. The thermal cycling conditions for amplification of those fragments were as follows: 94 °C for 10 min., followed by 35 cycles at 94 °C for 50s; 54 °C for 30s; 72 °C for 60s. This was followed by re-extension at 72 °C for 10 min. The PCR products were separated on a 2% agarose gel (using 0.5× TBE buffer) and visualized by ethidium bromide staining. For quantitation each band of gel was scanned by image analysis program (ImageJ. Exe). The expression of the target genes was quantified and then normalized by an endogenous reference housekeeping gene (GAPDH) relative to the calibrator (untreated cells). Relative intensity of each gene mRNA and fold of change referred to untreated control cells.
Real-Time PCR quantitative detection of the VEGF mRNAs:
Quantitative real-time PCR was carried out on cDNAs prepared for conventional RT-PCR. We used the ABI PRISMTM 7700 Sequence Detector System (PE Applied Biosystems) and the fluorescent dye SYBR® Green and the amount of PCR products was determined based on the fluorescence produced during the extension step of each cycle in a closed tube. To normalize the amount of total RNA present in each reaction, we used GAPDH as housekeeping gene an internal control. The threshold cycle (Ct) value for each sample was proportional to the log of the initial amount of input cDNA. Relative expression levels of VEGF in each treatment group were derived from normalizing the Ct value of genes against that of an endogenous reference and a calibrator, where by GAPDH was used as a normalizer against untreated cells as a calibrator. Quantitative data were analyzed and relative quantification of VEGF mRNAs were derived by the 2-ΔΔCT methods.
Quantitative ELIZA for VEGF
The amount of secreted VEGF in response to Avastin and/or VEGF siRNA by cells were measured in supernatants by using commercial Calbiochem human VEGF ELISA kits. (Calbiochem-Merck-Bioscience, Darmstadt, Germany) according to the procedure manual of the kits. The method is a ‘‘sandwich’’ enzyme immunoassay employing monoclonal and polyclonal antibodies. Quantitation is achieved by construction of a standard curve using known concentrations of VEGF calibrators of the kit.
Scratching assay
Cell migration and quantification was done using the wound healing assay as described by Bobadilla and co workers [22]. Briefly, equal numbers of cells (5×105) were plated and kept overnight in 6-well plates. After that the monolayer of cells was carefully wounded by manual scratching with a micro-pipette tip head. Exactly, the cells were treated with different concentration of Rosuvastatin and/or Avastin. Treated cells were cultured in complete growth medium and incubated for 96 hr. The wounded regions were photographed at different times interval (0-96hr) using a phase contrast microscope (Nikon). The photographs of the cells were analyzed using a Cell science software program. The distance between the two sides of scratch layers of images taken from migration of all concentrations at specified times was determined by the Cell science software.
Statistical analysis
Each experiment was minimally performed three times for all data, each carried out in duplicated sequences. Data were analyzed using a One-Way Analysis of variance (ANOVA) Values were given as the mean ± Standard Deviation (SD) and analytical variables were compared by using the students’ t-Test. By convention, a α-level of p<0.05 was considered to be statistically significant