Fig 1.
Flowchart of research methodology.
Table 1.
Specific primers of 18S rRNA, caspase-9, caspase-3 and survivin genes.
Table 2.
Physicochemical data of seventh active compounds.
Table 3.
Energy values obtained in molecular docking calculations with survivin.
Fig 2.
Molecular docking of andrographolide and rocaglamide with survivin.
(A) (left) Molecular docking of andrographolide (purple) at the Thr34 phosphorylation site of survivin (brown). At phosphorylation site Thr34, andrographolide (H-donor) interacts with Thr34 and Glu36 residues. (right) Ligand interaction between andrographolide and survivin. (B) (left) Molecular docking of rocaglamide (blue) with survivin (brown) at phosphorylation site Thr34. It interacts (pi-H/hydrogen acceptor) with Ile19 residue. (right) Ligand interaction between rocaglamide and survivin. Rocaglamide has no interaction with Thr34 but reacts with Ile19 residue.
Table 4.
Interaction of andrographolide and rocaglamide with survivin on Thr34 residue.
Table 5.
Molecular docking between andrographolide and survivin.
Fig 3.
Molecular docking of andrographolide with caspase-9 and caspase-3.
(A) (left) Molecular docking of andrographolide (purple) with caspase-9 (blue). (right) Ligand interaction between andrographolide and caspase-9. Andrographolide interacts with Asp228 as an H-donor and with Lys278 and Lys409 as an H-acceptor. (B) (left) Molecular docking of andrographolide (purple) with caspase-3 (yellow). It acts as an H-donor to Phe275. (right) Ligand interaction between andrographolide and caspase-3.
Table 6.
Binding energy of molecular interactions of andrographolide with survivin, caspase-9, and caspase-3.
Fig 4.
Cytotoxic activity of andrographolide.
(A) CC50 of andrographolide in BCSCs; (B) CC50 of andrographolide in MSCs. Andrographolide was diluted with DMSO 0.01% to obtain final concentrations of 0.075, 0.15, 0.3, and 0.6 mM, respectively. C: control cells treated with DMSO 0.01%. Percentage of cell viability is shown as the mean ± SD from three independent experiments (** p<0.01; *** p<0.001 compared to control). Following 24-h andrographolide treatment, BCSC morphology was observed under an inverted microscope (OPTIKA Srl, Ponteranica, Italy) with 100x magnification. (C) BCSC morphology untreated with andrographolide; (D) BCSC morphology treated with 0.6 mM andrographolide.
Fig 5.
Effect of andrographolide on the expression levels of survivin, caspase-9, and caspase-3 in BCSCs.
(A) mRNA expression levels of survivin, caspase-9, and caspase-3 in BCSCs treated with various concentrations of andrographolide; (B) Protein levels of total survivin and Thr34-phosphorylated survivin in BCSCs treated with various concentrations of andrographolide analyzed using ELISA; (C) Immunoassay results of survivin, Thr34-phosphorylated survivin, active caspase-9, and active caspase-3; C: control cells treated with DMSO 0.01% (vehicle). Data (A) and (B) were shown as the mean ± SD from three independent experiments. One-way ANOVA followed by Tuckey’s multiple comparison tests were used to determine mean differences between groups. Statistical significance is shown in the figure as follows: **p<0.01 and ***p<0.001 compared to control.
Fig 6.
Apoptosis analysis of andrographolide-treated BCSCs using flow cytometry.
(A) Cells were double stained with Annexin V-FITC and PI after treatment with andrographolide in various concentrations for 24 hours; (B) Ratio of early and late apoptosis compared to control; C: control cells treated with DMSO 0.01%.
Fig 7.
Proposed mechanism of andrographolide on the intrinsic apoptosis pathway.
Andrographolide interacts with survivin, phosphorylated survivin, caspase-9, and caspase-3. Andrographolide treatment could inhibit the phosphorylation of survivin and the binding of survivin and p-survivin to caspase-9 and caspase-3, as shown by the dashed red line. As a consequence, intrinsic apoptosis could be induced through activation of caspase-9 and caspase-3, as shown by the continuous red line in the pathway.