Apoptosis-mediated anti-proliferative activity of Calligonum comosum against human breast cancer cells, and molecular docking of its major polyphenolics to Caspase-3

Abstract

Due to poor diagnosis breast cancer in women has emerged as the most common cause of death disease in developing countries. Medicinal plants have been used for thousands of years and can be useful in healthcare, especially in developing countries. Ethanol extracts of leaves of fire bush or arta (Calligonum comosum; EECC), exhibited significant anticancer potencies against two breast cancer cell lines, MCF-7 and MDA 231. These in vitro effects of EECC indicated potential anticancer activities that were determined to be specific since minimal toxicity was recorded against MCF-12, a non-cancerous breast cell line used as a reference. EECC also induced cell cycle arrest in MCF-7 and MDA 231 as revealed by the increased proportions of sub-G1 cells. Fluorescence-activated cell sorter analysis (FACS), utilizing double staining by annexin V-FITC/propidium iodide, revealed that the observed cytotoxic effects were mediated via apoptosis and necrosis. FACS measurement of thegreater in fluorescence intensity, linked with oxidation of DCFH to DCF, revealed that apoptosis was attributable to production of free radicals. EECC-mediated apoptosis was further validated by observation of up-regulation in the "executioner" enzyme, caspase 3. The current findings reveal that EECC exhibits significant, selective cytotoxicity to breast cancer cells, that proceeds via the generation of ROS, which culminates in apoptosis. The anti-proliferative effects of EECC weres further verified by use of a structure-based, virtual screening between its major bioactive polyphenolic constituents and the apoptosis executioner marker enzyme, caspase-3. Based on their glide score values against the active site of caspase 3, some phyto-constituents present in EECC, such as DL-alpha-tocopherol and campesterol, exhibited distinctive, drug-like potential with no predicted toxicity to non-target cells. Taken together, the usefulness of natural phenolic and flavonoid compounds contained in Calligonum comosum were suggested to be potent anticancer agents.

Keywords: Calligonum comosum; anticancer; apoptosis; cytotoxicity; fire bush; gas chromatography mass spectrometry; molecular docking; natural products.

FIGURE 1

Ethanolic extract of Colligonum comosum (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, 24 h; E, for 48 h; F, for 72 h). Cancer cells were treated with 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 Materials and Methods part. *p < 0.05, **p < 0.01, ***p < 0.0001 vs. control. Data represent mean ± SD of eight technical well-replicates.

FIGURE 2

Flow cytometry analysis of MCF-7 cells following the treatment with various concentrations of ethanolic extracts of C. comosum. MCF-7 cells were treated with EECC 50, 150 or 300 μg/ml for 48 h. Apoptosis and necrosis were detected by use of FACS Annexin V and PI staining. *p < 0.05 vs. control. Data represent mean ± SD of triplicates.

FIGURE 3

Ethanol extract of Colligonum comosum (EECC) induces apoptosis and necrosis in MDA 231 cells. MDA 231 cells were treated with indicated concentrations of EECC for 48 h. Cells were then labeled with Annexin V and PI and analyzed by flow cytometry as described in the Methods section. **p < 0.01 vs. control. Data represent mean ± SD of triplicates.

FIGURE 4

Ethanol extract of Colligonum comosum (EECC) causes cell cycle arrest in MCF-7 and MDA 231 cells. Following treatment for 48 h with 300 μg/ml of EECC, cells were analyzed for DNA content as detailed in the Methods section.

FIGURE 5

Ethanol extract of Colligonum comosum (EECC) causes oxidative stress in MCF-7 cells. MCF-7 cells were treated for 48 h with indicated concentrations of EECC. Cells were analyzed for DCF fluorescence (ROS generation) after having been stained with DCFDA. *p < 0.05, ***p < 0.0001 vs. control. Data represent mean ± SD of triplicates.

FIGURE 6

Caspase-3 enzyme activity in MCF- cells following exposure to different concentrations of ethanol extracts of Calligonum comosum for 48 h *p < 0.05 vs. control. Data represent mean ± SD of eight technical well-replicates.

FIGURE 7

Gas chromatography mass spectrometry analysis of ethanol extract of Colligonum comosum. Image depicts a representative chromatograph of constituents identified by GC/MS in in EECC. Peaks represent absolute abundances, whereas numbers on the x-axis represent retention times in min.

FIGURE 8

Molecular docking of four major polyphenolics to caspase-3. (A) The 2D (left) and 3D (right) diagrams of the 1-Hexacosanol interactions with the active site of caspase-3 (PDB: 3GJQ). (B) The 2D (left) and 3D (right) diagrams of the 9,12-Octadecadienoic acid (Z,Z)- interactions with the active site of caspase-3 (PDB: 3GJQ). (C) The 2D (left) and 3D (right) diagrams of the Campesterol interactions with the active site of caspase-3 (PDB: 3GJQ). (D) The 2D (left) and 3D (right) diagrams of the DL-alpha-Tocopherol interactions with the active site of caspase-3 (PDB: 3GJQ).