Antioxidant, Anti-Proliferative Activity and Chemical Fingerprinting of Centaurea calcitrapa against Breast Cancer Cells and Molecular Docking of Caspase-3

Abstract

Centaurea calcitrapa has been intensively utilized in ethnomedicinal practices as a natural therapeutic recipe to cure various ailments. The current study aimed to chemically characterize ethanolic extract of C. calcitrapa (EECC) aerial parts (leaves and shoots) by use of gas chromatography-mass spectrometry analyses (GC-MS) and investigate its antioxidant and in vitro anticancer activities, elucidating the underlying molecular mechanism by use of flow cytometry-based fluorescence-activated cell sorting (FACS) and conducting in silico assessment of binding inhibitory activities of EECC major compounds docked to caspase-3. CG-MS profiling of EECC identified a total of 26 major flavonoids and polyphenolic compounds. DPPH and ABTS assays revealed that EECC exhibits potent antioxidant activity comparable to standard reducing agents. Results of the proliferation assay revealed that EECC exhibit potent, dose-dependent cytotoxic activities against triple-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cell models, with IC50 values of 1.3 × 102 and 8.7 × 101 µg/mL, respectively. The observed cytotoxic effect was specific to studied cancer cells since EECC exhibited minimal (~<10%) cytotoxicity against MCF-12, a normal breast cell line. FACS analysis employing annexin V-FITC/propidium iodide double labeling demonstrated that the observed anti-proliferative activity against MCF-7 and MDA-MB-231 was mediated via apoptotic as well as necrotic signaling transduction processes. The increase in fluorescence intensity associated with DCFH oxidation to DCF, as reported by FACS, indicated that apoptosis is caused by generation of ROS. The use of caspase-3-specific fluorogenic substrate revealed a dose-dependent elevation in caspase-3 substrate-cleavage activity, which further supports EECC-mediated apoptosis in MCF-7 cells. The major EECC compounds were examined for their inhibitory activity against caspase-3 receptor (1HD2) using molecular docking. Three compounds exhibited the highest glide score energy of −5.156, −4.691 and −4.551 kcal/mol, respectively. Phenol, 2,6-dimethoxy established strong binding in caspase-3 receptor of hydrogenic type, with residue ARG 207 and of PI-PI stacking type with residue HIS 121. By contract, hexadecenoic acid showed 3 H-bond with the following residues: ASN 615, ASN 616a and THR 646. Taken together, the current findings reveal that EECC exhibits significant and specific cytotoxicity against breast cancer cells mediated by the generation of ROS and culminating into necrosis and apoptosis. Further investigations of the phytoconstituents-rich C. calcitrapa are therefore warranted against breast as well as other human cancer cell models.

Keywords: CG-MS profiling; antioxidant potential; apoptosis; breast cancer; caspase-3; purple star thistle.

Figure 1

Dose- and time-dependent anti-proliferation activity of the ethanolic extract of C. calcitrapa (EECC). EECC inhibits proliferation of two breast cancer cells, MCF-7 ((A), for 24 h; (B), for 48 h; (C), for 72 h) and MDA 231 ((D), for 24 h; (E), for 48 h; (F), for 72 h). Cancer cells were treated with the indicated concentrations of EECC with DMSO control for 24, 48 and 72 h followed by determination of proliferation by MTT assay, as detailed in the Methods part. Data represent mean ± SD of 8 technical well-replicates.

Figure 2

Evaluation of the apoptosis- and necrosis-inducing potential of ethanol extract of C. calcitrapa (EECC) in MCF-7. (A): FACS analysis of by Annexin V-FITC/PI double staining of MCF-7 cell after treatment with EECC at different concentrations for 48 h. EECC induced apoptotic and necrotic cell death in MCF-7cells. The Y-axis represents the PI-labeled population, whereas the X-axis represents the FITC-labeled annexin V positive cells. The four quadrants are representative of the following: Q1 quadrant (PI+/Annexin V−)—necrotic cells, Q2 quadrant (PI+/Annexin V+)—early apoptotic cells, Q3 quadrant (PI−/Annexin V+)—late apoptotic cells, Q4 quadrant (PI−/Annexin V−)—viable cells. (B,C): Cell population analysis (%) according to early apoptotic, late apoptotic and necrotic MCF-7 cells treated after exposure to EECC (37.5, 75 and 150 µg/mL), relative to non-treated control cells at different concentrations. ** p < 0.01, *** p < 0.001, **** = p < 0.0001 vs. control. Data represent mean ± SD of triplicates.

Figure 3

Evaluation of the apoptosis- and necrosis-inducing potential of ethanol extract of C. calcitrapa (EECC) in MDA-MB-231. (A): FACS analysis of by Annexin V-FITC/PI double staining of MDA-MB-231 cells after treatment with EECC at different concentrations for 48 h. EECC induced apoptotic and necrotic cell death in MDA-MB-231 cells. The Y-axis represents the PI-labeled population, whereas the X-axis represents the FITC-labeled annexin V positive cells. The four quadrants are representative of the following: Q1 quadrant (PI+/Annexin V−)—necrotic cells, Q2 quadrant (PI+/Annexin V+)—early apoptotic cells, Q3 quadrant (PI−/Annexin V+)—late apoptotic cells, Q4 quadrant (PI−/Annexin V−)—viable cells. (B,C): Cell population analysis (%) according to early apoptotic, late apoptotic and necrotic MDA-MB-231 cells treated after exposure to EECC (25, 50 and 100 µg/mL), relative to non-treated control cells at different concentrations. * p < 0.05, **** = p < 0.0001 vs. control. Data represent mean ± SD of triplicates.

Figure 4

(A): Ethanol extract of C. calcitrapa (EECC) causes oxidative stress in MCF-7 cells. (A): Histogram represents relative DCF fluorescence intensity (FL1-H) vs. cell counts plot showing intracellular reactive oxygen species (ROS) generation in MCF-7 cells after 48 h exposure to EECC at the indicated concentration (150 µg/mL), relative to the control. (B): Quantitation of mean DCF fluorescence (ROS) in MCF-7 cell lines exposed to different concentrations of EECC (37.5, 75 and 150 µg/mL). * p < 0.05, **** = p < 0.0001 vs. control. Data represent mean ± SD of triplicates.

Figure 5

Caspase-3 enzyme activity of MCF-7 cells following exposure to different concentrations of ethanol extract of C. calcitrapa extract for 48 h. (*) denoted p < 0.05 relative to control. Data represent mean ± SD of 8 technical well-replicates.

Figure 6

The 2D (A) and 3D (B) diagrams of the 1H-Cycloprop[e]azulen-7-ol, decahydro-1,1,7-trimethyl-4-methylene-, [1ar-(1a.alpha.,4a.alpha.,7.beta., 7a.beta.,7b.alpha.)]- interactions with the active site of caspase-3 (PDB: 3GJQ). The ligand-caspase glide score energy values were −5.156 kcal/mol.