Cell Lines
T-47D, MDA-MB-231, and MCF-10A (a normal breast epithelial cell line) were obtained from the American Type Culture Collection (ATCC) [18]. Cells were maintained in Roswell Park Memorial Institute RPMI 1640 medium (Invitrogen-Gibco), supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen-Gibco), and incubated in a humidified atmosphere containing 5% CO2 in the air.
Reagents and Antibodies
The supramolecular coordination polymer (SCP) [CuCN./ Me3SnCN./pyz] (pyz./pyrazine], was obtained as described by Etaiw et al. [19] and graciously provided by Dr. Ahmed Badr Eldin (Tanta University). It was dissolved in 100% DMSO (Sigma-Aldrich) as stock solutions and stored at 20°C. The concentration targeted of DMSO in the culture medium under 0.2% (v/v). As same concentration was the control. Anti-P53, anti-Bax, anti-Bak, anti-Bcl-2, anti-caspase-3, anti-caspase-8, and anti-caspase-9 antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).
The Molecular Modeling studies
Essential Pharmacophoric Feature
Choosing the target by comparing our tested compound with the Crystal ligand and determining the essential feature that can bind with the target site. The main criteria should be present i) planner aromatic system (electron-dense region) ii) tail containing hydrogen bond donor or hydrogen bond acceptor act as groove binding group iii) basic amine protonated in physiological PH and converted to cationic center that can interact with phosphate group in DNA backbone.
Docking Process
Using Biovia discovery studio 2019 Software, various steps should have been done after selecting the protein of the target site to provide insights into the molecular binding modes of the tested drug inside the pockets of (Human topoisomerase II alpha bound to DNA). The binding sites were created inside crystal protein (PDB codes: 5GWK) (https://www.rcsb.org) using the co-crystallized ligand. Water molecules were initially removed from the compound. Then, employing protein report and utility, as well as clean protein options, crystallographic abnormalities, and unfilled valence atoms were repaired. MMFF94 force fields were used to reduce protein energy. By using a fixed atom constraint, the protein's stiff binding site structure was produced. The essential amino acids in proteins are determined and prepared for the docking process. 2D structures of tested compounds were created in Chem-Bio Draw Ultra17.0 and saved in MDL-SD file format. The saved file was opened in Biovia discovery studio 2019 Software, 3D structures were protonated, and energy was reduced using the.05 RMSD kcal/mol. An MMFF94 force field. Then, following the prepared ligand methodology, the reduced Structures were prepared for docking. The CDOCKER protocol was used to carry out the Molecular Docking procedure. During the refinement, the receptor was kept stiff while the ligands were permitted to be flexible. Each molecule was given ten alternative interaction postures with the protein. Then, using Discovery Studio 2019 Client software, docking scores (-CDOCKER interaction energy) of the best-fitted poses with the active site at Human topoisomerase II alpha coupled to DNA were recorded, and a 3D image was created. All of these procedures are used to predict the suggested binding mode, affinity, preferred orientation of each docking pose, and binding Free energy (G) of the drugs studied (Human topoisomerase II alpha bound to DNA).
Validation of Molecular Docking
The hypothesized docking approach was first tested for reliability and stability by redocking co-crystallized ligands into the active site of the appropriate receptor and calculating root mean square deviation (RMSD). When the crystal ligand was docked to Human topoisomerase II alpha coupled to DNA (5GWK), the RMSD 1.46 exceeded 2.00 Å, demonstrating that the algorithm was verified.
Cell Culture
In 75cm2 culture flasks, cells were plated and cultured in RPMI-1640 medium (Invitrogen-Gibco) supplemented with 10% fetal bovine serum (Hyclone), 2 mmol/L l-glutamine (Invitrogen), and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO2. For the MCF-10A cell line, a normal breast epithelial cell line was cultured in MEBM media from Lonza along with 100 ng/mL cholera toxin.
Cytotoxicity Assay (MTT assay)
MTT assay was performed for both cell lines (T-47D, MDA-MB-231, and MCF-10A) as described before [20]. Cells were seeded into 96-well culture plates at a density of 5×104 cells/well and allowed to attach. Cells were treated with different concentrations (2, 4, 6, 8, and 10 mM) of SCP compound (tested in triplicate), and incubated at 37°C for 48 h in 5% CO2. To each well, 20μL of MTT was added and further incubated in the same conditions for 2 hours. Media in each well was removed, and 100μL DMSO was added to solubilize formazan. Wells containing untreated cells served as positive controls. The absorbance was measured at 570 nm with a reference wavelength of 630 nm. Cell viability was stated as a percentage of the value for the control. The concentration of the compound with 50% cell growth inhibition was expressed as the half-maximal inhibitory concentration (IC50) value.
LDH Assay
For investigating the growth inhibitory effect of SCP compound, the leakage of lactate dehydrogenase (LDH) into the culture medium was estimated. After treatment with SCP at IC50 concentrations were incubated for 48 hours. Lyophilized LHD positive control was used according to manufacturer instructions. Untreated cells were used as a negative control. Media was then collected, centrifuged and the LDH reaction was performed for supernatants. Formazan salt generation and red color intensity were considered LDH activity indicators in both treated and untreated cells. Lastly, using a Tecan Infinite® 200 pro microplate reader, the absorbance was measured at 490 nm. The level of LDH released in treated cells is a percentage of the positive control.
Apoptosis Assay by ELIZA
Apoptosis in cells after SCP treatment was determined by Cell Death Detection ELISA PLUS Kit (Roche-Applied Science, Indianapolis, USA). After seeding cells (T-47D and MDA-MB-231) at a density of 2 × 104 cells/ well in a 96-well plate for 24h, both types of cells were treated with SCP compound at a dose of IC50 (4µM for MDA-MB-231 and 6µM for T-47D) for further 48 h. Untreated cells are used as control. This assay measures fragmented DNA and histone released from apoptotic cells using mouse monoclonal antibodies directed against DNA and histones in the cytoplasm. Therefore, cell lysate was prepared using 200-µL lysis buffer for 30 min at RT. 20 μl of cell lysate supernatant was incubated with anti-histone biotin and anti-DNA peroxidase for 2 h at RT. 100 μl of 2,2′azino-di (3-ethylbenzthiazolin-sulphuric acid) was added to each well and incubated for 15 min at RT. The absorbance was measured using an ELISA reader (Spectra Max Plus) at 405 nm. Each assay was done in triplicate and the standard deviation was calculated.
ROS Assay
The fluorescent probe 2′, 7′-dichlorofluorescein diacetate DCFH-DA was used to detect ROS generation. Cells were treated with IC50 concentrations of SCP compound for 48 hours. According to protocol, 5 mM DCFH-DA stock solution was prepared by dissolving the dye in 2.5 mL DMSO. This solution was diluted with PBS to prepare a 5μM working solution. Cells treated with 2mM H2O2 were used as a positive control. Untreated cells were considered negative control. After treatment, 5μL of DCFH-DA working solution was added to each well and incubated at 37°C for 30 minutes. Following that, formaldehyde 16% was used to fix cells for 10–15 minutes and then the plate was washed with PBS three times. Fluorescence intensity was determined using a fluorescence microplate reader with an excitation wavelength of 485 and an emission wavelength of 530nm. For quantitative evaluation of intracellular ROS production efficacy, the fluorescence was measured by the software modules supplied with the InCell 2000.
Real-Time PCR
Cells have been allowed to grow for a day, and then treated with SCP compound for 48 h at IC50. Untreated cells served as a control. RNA was obtained from cells via Qiagen RNeasy. cDNAs were synthesized from RNA, via GeNeiTM, Bangalore kit. For RT-PCR reaction, cDNAs were used as a template for amplification to quantify the steady-state mRNA levels of the tested genes. GAPDH gene was amplified as an internal control. The expression level of the apoptotic markers under the effect of SCP was also calculated in terms of relative fold change. A list of genes and primers were summarized in Table 1 [23-26].
Protein Extraction and Immunoblotting Analysis
Western blot analysis for investigating the effect of SCP on the regulation of apoptotic proteins was performed as reported previously [27]. After culture with SCP for 24 h, T-47D cells (SCCP: 6 µM, 3 µM) and MDA-MB-231 cells (SCP: 4µM and 2µM) were harvested, lysed, centrifuged and supernatants were aliquoted, heated and loaded on SDS-PAGE. Proteins in gels were then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA).
Membranes were blocked with 5% fat-free dry milk for 1 h, overnight incubated with a corresponding primary antibody for P53, Bax, Bak, Bcl-2, caspase-3, caspase-8, caspase-9, and β-actin (Santa Cruz, CA) at 4°C and later incubated for 2 h with HRP-conjugated secondary antibody. Protein bands were visualized using an enhanced chemiluminescent reagent kit (Amersham, ECL advance, Western blotting detection kit, UK), as per the manufacturer’s protocol. Quantification of the Western blots was performed using ImageJ software. Secondary antibodies were a peroxidase-conjugated affinity-purified anti-rabbit or mouse IgG (Rockland).
Statistical Analysis
Statistical data analysis was done using the statistical package for the social sciences (SPSS, Chicago) version 16. Results were expressed as mean ± standard deviation of three individual experiments. Statistical analysis was performed by a one-way ANOVA and t analysis followed by post hoc testing. *P<0.05 was considered statistically significant.