Introduction: DNA is a genetic compound responsible for vital processes in creatures. Therefore, change of DNA by any factor can modify the biological processes (1). Studies have shown that some small molecules can modify, inhibit or activate the DNA function and act as medicine by changing the feature and power of binding of regulating proteins (2). It is crucial to recognize the binding method and function of small molecules with DNA to better comprehend the DNA-drug bindings, design new medicine, and develop chemical probes (3). During the evaluation of binding between DNA and drugs, the most attention is paid to the binding mode. Drugs bind to DNA both through covalent and non-covalent bonds (4).
The 3-hydroxy-2-oxindole compounds have a variety of biological and medicinal activities, such as anticancer, anti-HIV and antioxidants (5). The objective of the research was the investigation of the interaction between the synthesized compound of 5-(2-hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-1,3thiazolidine-2,4-dione and DNA. In addition, the cytotoxicity was determined by MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay.
Methods: 1. UV absorbance Measurement
The absorbance experiments were conducted in a fixed drug concentration (15.2μM); but the DNA concentration was increasing from 0 to 226μM. UV spectra were recorded from 220 to 300 nm in Tris–HCl buffer by the Shimadzu UV -2550 spectrophotometer.
2. Molecular Docking
The docking studies were performed with ArgusLab software package. The DNA duplex receptor structure was obtained from Protein Data Bank (PDB code: 1DSC). The structure of compounds has been drawn with ChemOffice and a preliminary study of stabilization was performed by this software. The docking runs were performed on the Argus Dock docking engine.
3. MTT Assay
The MTT assay was used to evaluate the cytotoxicity effects of 3-hydroxy-2-oxindole on the cellular genome of human prostate cancer. This test was carried out in 48 hours, where cells were cultured in a 96-well plate. In this assay, untreated cells (control) and cells treated with the compound at concentrations of 25, 50, 100 and 200 µM were applied. At the end, the absorption of samples was read at 570 nm using ELISA device after addind DMSO.
Results: 1. UV Spectroscopy Studies
The absorption spectrum of this compound was derived in various DNA concentrations. The spectrum related to oxindole A experienced hyperchromesis by adding DNA to the compound. This increase was indicative of electrostatic interaction to ctDNA (6).
According to the half-reciprocal equation, the binding constant was calculated by having the free molecular absorption coefficient and absorption amounts in increasing concentrations of DNA.
The diagram of [DNA]/(εb-εf) against [DNA] was drawn. The binding constant of oxindole A to ctDNA was equal to 26666 M-1. With regard to the binding constant of Tris(1,10-phenanthroline)ruthenium(II), which is a compound that binds to the groove (7), and based on other compounds that bind to the DNA groove, including 3-(Pyridine-2-yl)-5,6-diphenyl-as-triazine (pdtb) (8.62×103 M-1) (8),[(Py-3')TPP-Ru(phen)2Cl]Cl (1.35×105 M-1), [Ni(Py-3')TPP-Ru(phen)2Cl][PF6] (1.29×105 M-1), [Cu (Py-3')TPP-Ru(phen)2Cl](PF6) (1.22×105 M-1) (9), it seems that the compound in the current research bound to ctDNA through DNA grooves.
2. Molecular Docking Studies
Molecular docking is an interesting tool for determination of the binding site of biopolymers and ligands on double-strand DNA (10). As observed, the evaluation of docking results showed the binding of oxindol compound to the major groove of DNA.
3. MTT
To investigate the viability of cells in 48 hours after treatment, MTT assay was performed with three times repeat (11, 12). The statistical analysis was assessed by SPSS software. According to MTT results, oxindole compound (200 µM) had a significant anti-proliferative effect on the DU145 prostate cancer cells after 48 hours of treatment (P<0.05).
Conclusion: In this research, the interaction of calf thymus DNA (ctDNA) with oxindole compound was investigated using UV absorption, molecular docking and MTT assay. UV absorption demonstrated that hyperchromism occurred by adding various DNA concentrations to a fixed concentration of oxindole compound. The binding constants for this compound with DNA was similar to the binding constants of DNA groove-binding compounds, which was 26×103 M-1. Docking also predicted binding to the major groove of DNA for this compound. According to the results of the MTT assay, the highest level of anti-reproduction effect of this compound on DU145 cells was observed at 200 µM concentration and after 48 hours of treatment.