Interaction of canadine with thymidylate synthase mediated its promising anticancer activity on MCF-7 human breast cancer cells

In this study, the cytotoxic effects of canadine (xanthopuccine or tetrahydroberberine), a benzylisoquinoline alkaloid with a molecular formula of C
20
H
21
NO
4
, on the proliferation of Michigan Cancer Foundation-7 (MCF-7) human breast cancer cells and MCF-10 human normal mammary epithelial cells were assessed through 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Quantitative real-time PCR (qPCR), Quantitative real-time PCR ROS, Catalase (CAT), Superoxide dismutase (SOD), Glutathione peroxidase (GPx) and glutathione (GSH) content assays. The expression and activity of thymidylate synthase (TS) in canadine-treated MCF-7 cells were also explored using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). Furthermore, the interaction of canadine with TS was investigated through spectroscopy and molecular docking. The results indicated that the IC50 concentrations of canadine were 17.50 μM in MCF-7 cells and >40 μM in MCF-10 cells. Colony-forming rates decreased to 95.23%, 80.95%, 71.42%, 47.61%, 47.69%, and 38.09% following the addition of canadine at the concentrations of 1, 5, 10, 20, 30, and 40 µM, respectively. It was also found that canadine induced cell-cycle arrest through the upregulation of p53 and p21 mRNA and apoptosis
via
the upregulation of the Bcl-2-associated X protein/ B-cell lymphoma 2 (BAX/BCL-2) ratio. Additionally, the data revealed that canadine-induced oxidative stress-mediated apoptosis through the upregulation of reactive oxygen species (ROS), downregulation of enzymatic and non-enzymatic mediators, Matrix Metalloproteinases (MMP) collapse, and cytochrome c release, all of which were modulated by the co-treatment of the cells with n-Acetylcysteine (NAC), a potential antioxidant. Moreover, canadine was found to downregulate Thymidylate synthase (TS) expression and activity, which was further evaluated by the Thymidylate synthase, small interfering RNA (TS siRNA) assay. The theoretical data indicated that there is a potential interaction (-6.80 kcal/mol) between canadine and the CYS195 residue in the active site of the TS, mediated by conventional hydrogen bonding, alkyl, and π-alkyl forces. Fluorescence spectroscopy measurements demonstrated TS’s interaction with canadine, leading to the formation of a static complex governed by hydrophobic forces. Calculations for thermodynamic and binding parameters showed that log

K
a

, Δ
H
°, Δ
S
°, and Δ
G
° values were 5.11 ± 0.21, 66.46 ± 3.61 kJ/mol, 324.43 ± 16.73 J/mol K, and -29.24 ± 1.38, respectively. Spectroscopy measurements indicated substantial secondary and tertiary conformational alterations of the TS upon binding with canadine. These data may provide a new perspective on canadine as a potential anticancer molecule.