Evening Primrose Oil Enhances Tamoxifen’s Anticancer Activity against Breast Cancer Cells by Inducing Apoptosis, Inhibiting Angiogenesis, and Arresting the Cell Cycle

2. Materials and Methods

2.1. Chemicals

The 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 Line

Human 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 Approval

This study was approved by the ethical committee of the Faculty of Pharmacy, Damanhour University (Ref. No. 719PB11).

2.4. Cell Viability Assay

The 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.

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The 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) [16].

2.5. Treatment of MCF-7 and MDA-MB-231 Cells for Biomolecular Investigations

Cells 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:
All treatments were applied to MCF-7 and MDA-MB-231 cells at 70–80% confluence, and the cells were incubated in a CO2 incubator for 72 h; then, the cells were harvested and portioned into aliquots. Finally, the aliquots were kept at −80 °C for further investigations.

2.6. Preparation of Cell Lysates

Cell 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 Assay

Protein 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 Technique

VEGF 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 Assay

A 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-2

Total 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 Detection

Cell 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 Cytometry

Following 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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2.13. Characterization of EPO Using Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

The chemical composition of EPO in the supplied softgel was analyzed using a Trace GC-TSQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG–5MS (30 m × 0.25 mm × 0.25 µm film thickness).An amount of 0.35 g EPO was added to 6 mL 0.5 mol/L NaOH-methanol in a 250 mL flask, and it was shaken well. Then, 7 mL 12.5% BF3-methanol was added and it was shaken again. The mixture was refluxed at 70 °C for 2–3 min, then 5 mL hexane was added through the reflux system and heating continued for an additional 2 min. During reflux, the mixture was shaken occasionally until the methylation process was completed. After cooling, 30 mL saturated NaCl solution was added and extracted vigorously. The aqueous phase was transferred into a 250 mL separatory funnel and extracted with two 50 mL petroleum ethers. The combined organic layer was washed with Aqua Bidest until free of base, and the solvent was evaporated using a rotary evaporator. The concentrate was dissolved with 1 mL hexane prior to injection [17].The column oven temperature was initially held at 100 °C for 1 min, then increased by 10 °C/min to 160 °C, then increased again to 220 °C with 2.5 °C/min, then increased once more to 240 °C with 10 °C/min. The injector temperature was kept at 270 °C. Helium was used as a carrier gas at a constant flow rate of 1 ml/min. The solvent delay was 4 min and diluted samples of 1 µL were injected automatically using an Autosampler AS3000 coupled with GC in split mode. EI mass spectra were collected at 70 eV ionization voltages over an m/z range of 50–650 in full-scan mode. The ion source and transfer line were set at 250 °C and 280 °C, respectively. The components were identified by comparing their mass spectra with those in the WILEY 09 and NIST 14 mass spectral database.

2.14. Statistical Analysis

Values were analyzed using Graph Pad Prism 8 (Graph Pad Software, Inc., San Diego, CA, USA) using one-way ANOVA, followed post hoc by Tukey’s multiple comparisons tests. The results were expressed as the mean ± standard deviation of the mean (SD). Significant differences among means were estimated at p < 0.05.


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About the Author: Tung Chi