Ajwa Date (Phoenix dactylifera L.) Extract Inhibits Human Breast Adenocarcinoma (MCF7) Cells In Vitro by Inducing Apoptosis and Cell Cycle Arrest

Abstract

Introduction: Phoenix dactylifera L (Date palm) is a native plant of the Kingdom of Saudi Arabia (KSA) and other Middle Eastern countries. Ajwa date has been described in the traditional and alternative medicine to provide several health benefits including anticholesteremic, antioxidant, hepatoprotective and anticancer effects, but most remains to be scientifically validated. Herein, we evaluated the anticancer effects of the Methanolic Extract of Ajwa Date (MEAD) on human breast adenocarcinoma (MCF7) cells in vitro.

Methods: MCF7 cells were treated with various concentrations (5, 10, 15, 20 and 25 mg/ml) of MEAD for 24, 48 and 72 h and changes in cell morphology, cell cycle, apoptosis related protein and gene expression were studied.

Results: Phase contrast microscopy showed various morphological changes such as cell shrinkage, vacuolation, blebbing and fragmentation. MTT (2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay demonstrated statistically significant dose-dependent inhibitions of MCF7 cell proliferation from 35% to 95%. Annexin V-FITC and TUNEL assays showed positive staining for apoptosis of MCF7 cells treated with MEAD (15 mg and 25 mg for 48 h). Flow cytometric analyses of MCF7 cells with MEAD (15 mg/ml and 20 mg/ml) for 24 h demonstrated cell cycle arrest at 'S' phase; increased p53, Bax protein expression; caspase 3activation and decreased the mitochondrial membrane potential (MMP). Quantitative real time PCR (qRT-PCR) analysis showed up-regulation of p53, Bax, Fas, and FasL and down-regulation of Bcl-2.

Conclusions: MEAD inhibited MCF7 cells in vitro by the inducing cell cycle arrest and apoptosis. Our results indicate the anticancer effects of Ajwa dates, which therefore may be used as an adjunct therapy with conventional chemotherapeutics to achieve a synergistic effect against breast cancer.

Fig 1. Phase contrast images of MCF7 cells.

Phase contrast images of MCF7 cells showing morphological changes in a dose and time-dependent manner following treatment with MEAD at 5, 10, 15, 20 and 25 mg/ml. Prominent such as included cell shrinkage, membrane blebbing, cell fragmentations and detachment mimicking apoptosis were observed. There were also gross decrease in cell numbers with increase in time and concentration of MEAD. Arrows indicate dead cells which appears round and translucent (Magnification 100X).

Fig 2. Inhibition of growth and proliferation of MCF7 cells.

MTT assay of MCF7 cells treated with MEAD at different concentrations (5, 10, 15, 20 and 25 mg/ml) showed inhibition of cell proliferation compared to untreated controls in a dose dependent manner at 24 h, 48 h and 72 h respectively. (A) The percentage decrease in inhibition were (18.0 ± 40% to 60.80 ± 4.80%) at 24 h, (B) (33.70 ± 3.6% to 95.80 ± 2.0%) at 48 h and (C) (37.36 ± 1.60% to 96.0 ± 2.0%) at 72 h. These decreases in inhibitions were statistically significant. The values are expressed as mean ± SEM from triplicate samples of three independent experiments. * and ** indicate p<0.05 and p<0.001 respectively.

Fig 3. Cell cycle arrest in MCF7 cells.

Representative cell cycle images of MCF7 cells after 24 h of treatment with MEAD at 15, 20 and 25 mg/ml MEAD. The histogram indicates 'S' phase arrest and decrease in cell numbers in 'G1' phase of cell cycle in a dose dependent manner. The sub-G1 phase which is indicative of apoptosis also showed increase in a dose dependent manner.

Fig 4. Apoptosis in MCF7 cells.

Representative dot plot of the Annexin V-FITC and PI assay on MCF7 cells treated with 15 and 25 mg/ml of MEAD for 48 h showed increases in apoptotic cells compared to the control. These increases were statistically significant (p<0.05).

Fig 5. TUNEL Assay DNA fragmentation in MCF7 cells.

MCF7 cells were treated for 48 h with 15 mg/ml and 25 mg/ml concentrations of MEAD respectively and incubated with DNA labeling solution containing dUTP-FITC and TdT polymerase overnight. FITC fluorescence represented the dUTP incorporation and DNA fragmentation. Representative dot plot image shows the fluorescence shift towards the left indicating the increase in TUNEL positive cells.

Fig 6. Apoptotic protein expression in MCF7 cells.

MEAD up-regulate pro-apoptotic proteins (p53, Bax) and down-regulates the anti-apoptotic protein (Bcl-2) in MCF7 cells. MCF7 cells were treated with 15 and 20 mg/ml MEAD and Bcl-2 inhibitor ABT-737 (10 μM) concentrations for 24 h and staining is performed for p53, Bax and Bcl-2. The representative graphs are out of three independent flow cytometric analyses.

Fig 7. MEAD activates the Caspase 3 in MCF7 cells.

MCF7 cells were treated with 15 and 20 mg/ml MEAD and Bcl-2 inhibitor ABT-737 (10 μM) for 24 h and stained with FITC-active caspase 3 antibody. The graphs represents one of the three independent flow cytometric analyses.

Fig 8. MEAD induces mild change in MMP in MCF7 cells.

The scatter plot (representative of three independent experiments) for MCF7 cells showing JC-1 green fluorescence on the x-axis (indicator of loss of MMP) and PE-Cy5 red fluorescence on the y-axis (high MMP). MCF7 cells were treated with 15 and 20 mg/ml MEAD and Bcl-2 inhibitor ABT-737 (10 μM) for 24 h and stained with JC-1 and analyzed by flow cytometry.

Fig 9. Apoptotic gene expression in MCF7 cells.

(A) qRT-PCR analysis of MCF7 cells showing the p53, Bax, Bcl-2, Fas and FasL gene expression profile following treatment with 15 mg/ml and 20 mg/ml of MEAD for 48 h. β-Actin was used as an internal control and the data were calculated using the comparative 2 –ΔΔCt method. (B) The fold change ratio of Bax/Bcl-2 gene expression got increased in a dose dependent manner. The values are expressed as mean ± SEM from triplicate samples of three independent experiments.