Phytochemicals from Ajwa dates pulp extract induce apoptosis in human triple-negative breast cancer by inhibiting AKT/mTOR pathway and modulating Bcl-2 family proteins

Abstract

Ajwa dates (Phoenix dactylifera L.) have been described in traditional and alternative medicine to provide several health benefits, but their mechanism of apoptosis induction against human triple-negative breast cancer MDA-MB-231 cells remains to be investigated. In this study, we analyzed the phytoconstituents in ethanolic Ajwa Dates Pulp Extract (ADPE) by liquid chromatography-mass spectrometry (LC-MS) and investigated anticancer effects against MDA-MB-231 cells. LC-MS analysis revealed that ADPE contained phytocomponents belonging to classes such as carbohydrates, phenolics, flavonoids and terpenoids. MTT assay demonstrated statistically significant dose- and time-dependent inhibition of MDA-MB-231 cells with IC₅₀ values of 17.45 and 16.67 mg/mL at 24 and 48 h, respectively. Hoechst 33342 dye and DNA fragmentation data showed apoptotic cell death while AO/PI and Annexin V-FITC data revealed cells in late apoptosis at higher doses of ADPE. More importantly, ADPE prompted reactive oxygen species (ROS) induced alterations in mitochondrial membrane potential (MMP) in ADPE treated MDA-MB-231 cells. Cell cycle analysis demonstrated that ADPE induced cell arrest in S and G2/M checkpoints. ADPE upregulated the p53, Bax and cleaved caspase-3, thereby leading to the downregulation of Bcl-2 and AKT/mTOR pathway. ADPE did not show any significant toxicity on normal human peripheral blood mononuclear cells which suggests its safe application to biological systems under study. Thus, ADPE has the potential to be used as an adjunct to the mainline of treatment against breast cancer.

Figure 1

Total Ion Chromatogram (TIC) of 95% ethanolic ADPE obtained using LC–MS. A 5 mL sample was loaded on Silica C18 Reversed Phase column (150 × 2.1, 2.6 μm), with 0.45 mL min⁻¹ flow rate using mobile phase solvents (a): acetonitrile/water (5:95, v/v), (b): acetonitrile, (c): methanol and (d): 5 mM ammonium acetate (95:5 H₂O:Acetonitrile, pH 6.5). Mass spectrometry was performed by ESI and MS data was recorded in the mass range m/z 200–2000 from 0–30 min under ionization mode of ES⁺ and ES⁻. Y-axis is relative abundance and X-axis is retention time.

Figure 2

Microscopic observation and cytotoxic activity of different concentrations (10–25 mg/mL) of ADPE against MDA-MB-231 and PBMCs. (a) and (b) Photomicrograph of MDA-MB-231 cells treated with 10 to 25 mg/mL concentrations of ADPE at 24 and 48 h, respectively. Photomicrographs were taken with an inverted phase contrast microscope (Scale bar = 100 μm). (c) Percent cell viability of ADPE at various concentrations against MDA-MB-231 cells after 24 and 48 h incubation. (d) Dose response curve (Log concentration vs % cell viability) representing IC₅₀ values of ADPE at 24 and 48 h incubation. (e) Photomicrograph of PBMCs treated at various concentrations of ADPE at 24 h (Scale bar = 100 μm). (f) Percent cell viability of ADPE at various concentrations against PBMCs after 24 h incubation. Values are expressed as mean ± SEM of three independent experiments. *p < 0.05 as compared to control.

Figure 3

Chromatin condensation and apoptosis-inducing activity of ADPE in MDA-MB-231 treated cells (a) Chromatic condensation of MDA-MB-231 treated cells at 15, 18 and 20 mg/mL of ADPE for 48 h by Hoechst 33342 staining (Scale bar = 100 μm). (b) Fluorescent micrographs of AO/PI-double-stained MDA-MB-231 cells at 15, 18 and 20 mg/mL of ADPE at  48 h. (i) Untreated MDA-MB-231 cells depict normal  structure (ii) Early apoptosis features such as chromatin condensation and membrane blebbing were observed at 15 mg/mL of ADPE (iii) Late apoptosis cells were observed at 18 mg/mL (iv) Late apoptosis and secondary necrosis were observed at 20 mg/mL of extract (Scale bar = 100 μm), VC: Viable cells; CC: Chromatin condensation; LA: Late apoptosis and SN: Secondary necrosis . (c) DNA fragmentation assay in MDA-MB-231 cells as an index of apoptosis. Lane 1: showing control MDA-MB-231 cells; Lane 2, 3, and 4: cells treated with 15, 18, and 20 mg/mL of ADPE, respectively. (d) Flow cytometry analysis of MDA-MB-231 cells after 48 h of treatment using annexin V/FITC & PI double stain. Representative figures showing the population of viable (annexin V⁻ PI⁻), early apoptotic (annexin V⁺ PI⁻), late apoptotic (annexin V⁺ PI⁺) and necrotic (annexin V⁻ PI⁺) cells.

Figure 4

Intracellular ROS generation and mitochondrial membrane potential of human MDA-MB-231 cells (a) Photomicrographs showing intracellular ROS generation induced by three effective concentrations (15, 18 and 20 mg/mL) of ADPE after 12 h incubation. Photomicrographs were taken with a fluorescence microscope (Scale bar = 100 μm). (b) The fluorescence in the cells is represented as the percentage of ROS production analyzed using flow cytometry. (c) Photographs indicate a decrease in MMP, an early event in apoptosis with increased concentrations of ADPE. Photomicrographs were taken with a fluorescence microscope (Scale bar = 100 μm). (d) Fluorescence in the cells is represented as the percentage of MMP reduction in MDA-MB-231 cells analyzed by flow cytometry.

Figure 5

DNA content analysis by flow cytometry. Pictorial graph showing the mean proportion of cells in different phases of cell cycle (a) Untreated control (b) 15 mg/mL (c) 18 mg/mL and (d) 20 mg/mL of ADPE at 48 h.

Figure 6

Western blot analysis of apoptotic proteins and signaling molecules in MDA-MB-231 cells. (a) Immunoblot analysis showing expression levels of p53, Bax, Bcl2, cleaved Caspase-3, mTOR, p-mTOR, AKT and p-AKT. The MDA-MB-231 cells were treated at two effective concentrations of ADPE (15 and 20 mg/mL) for 48 h. Equal amounts of total proteins (30 μg/lane) were subjected to 10–12% SDS-PAGE. Expression of p53, Bax, Bcl2, cleaved Caspase-3, mTOR, p-mTOR, AKT, p-AKT and β-actin were detected using specific antibodies. Lane 1: 0 mg/mL (Untreated); Lane 2: 15 mg/mL; Lane 3: 20 mg/mL of ADPE. (b) Graph representing relative intensity of various proteins vs concentration. The data represents mean ± SEM of three independent experiments.

Figure 7

Schematic diagram summarizing the proposed mechanism of ADPE action on MDA-MB-231 cells. Bioactive components from ADPE arrest G2/M and S phase checkpoints and induce apoptosis by upregulation of p53, Bax and cleaved caspase-3, thereby leading to downregulation of Bcl-2 family proteins and AKT/mTOR pathway.