Achillea fragrantissima (Forssk.) Sch.Bip Flower Dichloromethane Extract Exerts Anti-Proliferative and Pro-Apoptotic Properties in Human Triple-Negative Breast Cancer (MDA-MB-231) Cells: In Vitro and In Silico Studies

Abstract

The aggressive triple-negative breast cancer (TNBC) is a challenging disease due to the absence of tailored therapy. The search for new therapies involves intensive research focusing on natural sources. Achillea fragrantissima (A. fragrantissima) is a traditional medicine from the Middle East region. Various solvent extracts from different A. fragrantissima plant parts, including flowers, leaves, and roots, were tested on TNBC MDA-MB-231 cells. Using liquid chromatography, the fingerprinting revealed rich and diverse compositions for A. fragrantissima plant parts using polar to non-polar solvent extracts indicating possible differences in bioactivities. Using the CellTiter-Glo™ viability assay, the half-maximal inhibitory concentration (IC₅₀) values were determined for each extract and ranged from 32.4 to 161.7 µg/mL. The A. fragrantissima flower dichloromethane extract had the lowest mean IC₅₀ value and was chosen for further investigation. Upon treatment with increasing A. fragrantissima flower dichloromethane extract concentrations, the MDA-MB-231 cells displayed, in a dose-dependent manner, enhanced morphological and biochemical hallmarks of apoptosis, including cell shrinkage, phosphatidylserine exposure, caspase activity, and mitochondrial outer membrane permeabilization, assessed using phase-contrast microscopy, fluorescence-activated single-cell sorting analysis, Image-iT™ live caspase, and mitochondrial transition pore opening activity, respectively. Anticancer target prediction and molecular docking studies revealed the inhibitory activity of a few A. fragrantissima flower dichloromethane extract-derived metabolites against carbonic anhydrase IX, an enzyme reported for its anti-apoptotic properties. In conclusion, these findings suggest promising therapeutic values of the A. fragrantissima flower dichloromethane extract against TNBC development.

Keywords: Achillea fragrantissima; carbonic anhydrase; caspase activation; mitochondrial apoptosis pathway; triple-negative breast cancer.

Figure 1

Fingerprinting chromatograms of A. fragrantissima flower-, leaf-, and root-derived solvent extracts using HPLC-UV. The chromatograms are based on a gradient method of methanol and water, and UV set at 275 nm with 5 µL injection volume. The separation of the dimethyl sulfoxide (DMSO)-dissolved compounds was performed using a Kinetex^® C-18 column (250 × 4.6 mm, 5 µm) and revealed diverse polar and non-polar compositions for the different solvent extracts of the herbal medicine.

Figure 2

Inhibition of TNBC MDA-MB-231 cell viability by different solvent extracts of A. fragrantissima plant parts using the CellTiter-Glo™ assay.

Figure 3

Microscopic images of the MDA-MB-231 cells treated with increasing concentrations of A. fragrantissima flower dichloromethane extract displaying apoptosis-related morphological changes in cell shrinkage. The MDA-MB-231 cells display a dose-dependent increase in cell shrinkage, a morphological characterization related to apoptosis. The cell shrinkage that occurred in the MDA-MB-231 cells treated with 200 µg/mL of A. fragrantissima flower dichloromethane extract was similar to the cells treated with the strong apoptosis inducer STS, the positive control.

Figure 4

The increase in caspase-3/7 activation in the A. fragrantissima flower dichloromethane extract-treated TNBC MDA-MB-231 cells using confocal microscopy. The MDA-MB-231 cell treatment with the dichloromethane A. fragrantissima flower dichloromethane extract (25–200 µg/mL) and STS (1 µM) showed an increase in caspase-3/7 activity by more than 1.5-fold in comparison to the DMSO-treated cells, the negative control. The arrow is an example of MDA-MB-231 cells with activated caspase-3/7 (red fluorescence). Hoechst 33342 and fluoromethyl ketone-sulforhodamine (FMK-SR) were used as a nuclear dye and a cell-permeant red fluorescent probe labeling active caspase-3/7, respectively. * p < 0.05 vs. DMSO-treated cells.

Figure 5

Determination of the apoptotic status in the TNBC MDA-MB-231 cells exposed to A. fragrantissima flower dichloromethane extract. Representative scatter plots indicating the percentage of MDA-MB-231 cells in viability (V), in apoptotic status including early apoptosis (E APO) and late apoptosis (L APO), and in necrotic (N) status, based on Annexin V (-FITC) and PI (-PE) double staining measured by FACS. A. fragrantissima flower dichloromethane extract prompted apoptosis in TNBC MDA-MB-231 cells, in a dose-dependent manner, compared to DMSO-treated cells. STS was used as a positive control. The bar graph presents data as mean ± SD of three separate experiments. * p < 0.05 and ** p < 0.01 vs. DMSO-treated cells.

Figure 6

The increase in mitochondrial membrane potential opening activity in A. fragrantissima flower dichloromethane extract-treated TNBC MDA-MB-231 cells using confocal microscopy. The MDA-MB-231 cell treatment with A. fragrantissima flower dichloromethane extract (25–200 µg/mL) showed an increase in the activity of the opening of the mitochondrial transmission membrane pores, as indicated by the arrow. * p < 0.05 and ** p < 0.01 vs. DMSO-treated cells.

Figure 7

The identification of A. fragrantissima flower dichloromethane extract metabolites using an Agilent LC-QTOF in positive and negative modes.

Figure 7

The identification of A. fragrantissima flower dichloromethane extract metabolites using an Agilent LC-QTOF in positive and negative modes.

Figure 7

The identification of A. fragrantissima flower dichloromethane extract metabolites using an Agilent LC-QTOF in positive and negative modes.

Figure 7

The identification of A. fragrantissima flower dichloromethane extract metabolites using an Agilent LC-QTOF in positive and negative modes.

Figure 7

The identification of A. fragrantissima flower dichloromethane extract metabolites using an Agilent LC-QTOF in positive and negative modes.

Figure 8

Docking poses for metabolite 11 (M11) and Colchicine into Tubulin crystal structure.

Figure 8

Docking poses for metabolite 11 (M11) and Colchicine into Tubulin crystal structure.

Figure 9

Docking poses for metabolite 11 (M11) and Y0R into CA IX crystal structure.

Figure 10

Radar diagrammatic representation of A. fragrantissima flower dichloromethane extract-derived identified metabolites (M1–M14) using SwissADME webserver. LIPO: Lipophilicity, Size: Molecular weight, POLAR: solubility, INSOLU: insolubility, INSATU: insaturation, and FLEX: flexibility. The properties involved in the colored zone are preferred for orally active drugs.

Figure 10

Radar diagrammatic representation of A. fragrantissima flower dichloromethane extract-derived identified metabolites (M1–M14) using SwissADME webserver. LIPO: Lipophilicity, Size: Molecular weight, POLAR: solubility, INSOLU: insolubility, INSATU: insaturation, and FLEX: flexibility. The properties involved in the colored zone are preferred for orally active drugs.