2. Materials and Methods
2.1. ChemicalsThe EPO purchased was Puritan’s Pride Cold-Pressed Evening Primrose Oil 1000 mg Softgels, from Puritan’s Pride, Inc. (Oakdale, NY, USA). The content of each softgel containing 1000 mg of EPO, according to the manufacturer, was 730 mg linoleic acid and 90 mg γ-linolenic acid.TAM (CAS # 10540–29–1) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) was procured from Serva (Heidelberg, Germany). Dulbecco’s modified Eagle’s medium (DMEM), trypsin, phosphate-buffered saline (PBS), and a penicillin/streptomycin mixture were procured from Lonza® (Basel, Switzerland).All other chemicals and materials were commercially available and of standard quality.
2.2. Experimental Cell LineHuman breast adenocarcinoma MCF-7 and MDA-MB-231 were provided by the American Type Culture Collection (ATTC, Manassas, VA, USA). Cells were grown in DMEM with 1% penicillin/streptomycin and 10% FBS, and incubated at 37 °C with humidified air and 5% CO2.
2.3. Ethical ApprovalThis study was approved by the ethical committee of the Faculty of Pharmacy, Damanhour University (Ref. No. 719PB11).
2.4. Cell Viability AssayThe effects of EPO and TAM on cell viability were evaluated using an MTT assay. MCF-7 and MDA-MB-231 were separately seeded in a 96-well plate [1 × 104 cells/well]. Each well contained 100 μL DMEM medium supplemented with 10% FBS, incubated at 37 °C for 24 h, under 5% CO2 and 95% air, until reaching 70–80% confluence.The old media were discarded, then 0.1 mL of DMEM containing different drug concentrations was added and incubated for another 72 h.
- TAM concentrations (1, 0.5, 0.25, 0.125, and 0.0652 μg/mL) or EPO concentrations (250, 125, 62.5, 31.25, and 15.63 μg/mL) were added to all MCF-7 wells, except the control wells.
- TAM concentrations (8, 4, 2, 1, and 0.5 μg/mL) or EPO concentrations (250, 125, 62.5, 31.25, and 15.63 μg/mL) were added to all MDA-MB-231 wells except the control wells.
Read more Study backs oxybutynin for Hot Flashes in Breast Cancer SurvivorsThe media were then aspirated, and cells were incubated with 200 μL MTT working solution (0.5 mg/mL in DMEM) for four hours in the dark. After removing the supernatant, the resulting formazan crystals were solubilized in 100 μL of DMSO by maintaining agitation for 15 min. Absorbance was recorded at 590 nm using a microplate reader. All experiments were conducted at least three times independently, each performed in triplicate. The viability of the cells was calculated as a percentage relative to that of the control wells. The median inhibitory concentration (IC50) values were calculated using CompuSyn software (CompuSyn, Inc., version 1, Paramus, NJ, USA) .
2.5. Treatment of MCF-7 and MDA-MB-231 Cells for Biomolecular InvestigationsCells were seeded at 500,000 cells per T-25 flask containing complete media, and allowed to adhere overnight at 37 °C (15 flasks for MCF-7 cells and 15 flasks for MDA-MB-231 cells). The next day, cells were segregated into groups (3 flasks in each group), and treated as follows:
2.6. Preparation of Cell LysatesCell lysates were prepared with a RIPA lysis buffer (Boster Biological Technology, Pleasanton, CA, USA, Catalog # AR0105). Briefly, 0.5 mL of chilled RIPA lysis buffer was added to the cell pellet, vortexed, and incubated for 30 min on ice. Then, samples were centrifuged at 14,000× g for 10 min, and the supernatant was transferred to another tube for further analysis.
2.7. Protein Quantitation Using BCA AssayProtein concentration was determined using the SMARTTM BCA protein assay kit (#21071) purchased from iNtRON Biotechnology, Inc. (Gyeonggi, Korea). The absorbance was read at 562 nm.
2.8. Biomarker Analysis Using ELISA TechniqueVEGF and cyclin D1 were evaluated in the cell lysates from different treatment groups using the ELISA technique, using a human VEGF ELISA kit and a human cyclin D1 ELISA kit, supplied by MyBioSource inc. (San Diego, CA, USA). The manufacturer’s protocol was followed for all measurements. Each parameter was assayed in triplicate and was expressed relative to the total protein content in the same sample.
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2.9. Caspase-3 Activity AssayA colorimetric kit obtained from Sigma-Aldrich (St. Louis, MO, USA) was utilized to assess caspase-3 activity following the manufacturer’s procedure.
2.10. qRT-PCR Determination of mRNA Genes Expression of VEGF, Bax, and Bcl-2Total RNA was isolated from MCF-7 and MDA-MB-231 using gene easy-REDTM total RNA extraction Kit (iNtRON Biotechnology, Inc., Gyeonggi, Korea). RNA concentration and purity were determined using nanodrop (Q5000, Quawell, CA, USA) and 1% gel electrophoresis. RNA (5 μg) was reverse-transcripted using the Maxime RT Premix kit (iNtRON Biotechnology, Inc., Gyeonggi, Korea). The cDNA produced was used as a template to determine the relative expression of VEGF, Bax, and Bcl-2 genes using Maxime RT PreMix kit (iNtRON Biotechnology, Inc., Gyeonggi, Korea) and specific primers (Table 1). β-actin was used as an internal control. The thermal cycling conditions, melting curve temperatures and relative expression calculations were performed using 2−ΔΔCt.
2.11. Annexin V—FITC/PI Double Staining for Flow-Cytometric Apoptosis DetectionCell apoptosis was assessed using an annexin V/FITC Apoptosis Detection Kit (Catalog #: K101–25, BioVision, Inc., Milpitas, CA, USA), according to the manufacturer’s instructions. Following trypsinization, MCF-7 and MDA-MB-231 cells from all studied groups were centrifuged to collect 5 × 105 cells. Cell pellets were washed with cold 1X PBS and resuspended in 500 μL annexin-binding buffer. Then, 5 μL propidium iodide and 5 μL annexin V/FITC were added and incubated at room temperature for 5 min in the dark. Apoptotic cells were detected using Attune flow cytometer (Applied Biosystems, Foster City, CA, USA).
2.12. Cell-Cycle Analysis by Flow CytometryFollowing trypsinization, MCF-7 and MDA-MB-231 cells from all studied groups were centrifuged. Cell pellets were washed twice, resuspended in warm PBS, fixed using ice-cold absolute ethanol, and incubated for at least 24 h at −20 °C. Samples were centrifuged and the supernatant was removed. Pellets were homogenized in 5 mL cold PBS and centrifuged; 1 mL PBS-Triton X100 and 100 μL RNase-A (200 μg/mL) were added and incubated at 37 °C for 30 min; then, 100 μL propidium iodide (1 mg/mL) was added and incubated in darkness for 30–60 min at room temperature. Cell-cycle analysis was carried out using an Attune flow cytometer (Applied Biosystems, Foster City, CA, USA).
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