Chemicals and Reagents
The chemicals used for experimental analysis is of pure analytical category. Extra pure methanol, Fluvastatin ((E,3R,5S)-7-[3-(4-fluorophenyl)-1-propan-2-ylindol-2-yl]-3,5-dihydroxyhept-6-enoic acid), TPTZ (2, 4, 6-Tri(2-Pyridyl)-s-triazine),Ruthenium chloride (RuCl3. xH2O), highly polymerized CT-DNA (calf thymus DNA), TrisHCl, 7, 12-dimethyl benz (α) anthracene (DMBA), foetal bovine serum (FBS), 3,3’- diaminobenzidine (DAB), proteinase K, insulin L-glutamine, sodium pyruvate, streptomycin, penicillin, ABTS (2, 2’-azinobis 3-ethylbenzothiazoline-6-sulphonic acid diammonium salt), streptavidin peroxidase, MTT (3-(4,5- dimethyl thiazole-2-yl)-2,5-diphenyltetrazoliumbromide), DPPH (2, 2 -diphenyl-2-picrylhydrazyl), biotinylated goat anti-rabbit IgG, Annexin V and propidium iodide (PI) were purchased from Sigma Aldrich Chemical Co (St, Louis, Mo, USA). Antibodies specific for p53, VEGF (627501), Caspase-3, Akt, mTOR, PI3K, pro and active caspase-3 were acquired from Biolegend (Sun Diego, CA, USA). Rabbit anti-rat p53, Bax, Bcl-2, Ki-67, MMP-9and goat anti rabbit IgG secondary antibody were bought from ANASPEC Inc. (San Jose, CA, USA). The MCF- 7 and MDA-MB-231 breast cancer cell line were obtained from American Type Culture Collection, 10801 University Boulevard Manassas, VA 20110 USA. Kit diagnosed for apoptosis was purchased from Takara Bio Inc (Japan). Others chemical agents utilized for research purpose were procured from local companies in the finest possible form.
Target protein selection
The reviewed sequence of human gamma synuclein was retrieved from universal protein sequence database UniProt (http://www.uniprot.org/). The selected protein sequence was used to predict sequence similarity and to predict sequence templates by PSI-BLAST. The specified sequence of template was used to construct three-dimensional protein structures utilizing Swiss model. Using homology modeling by Swiss PDB Viewer assist to envisage the orientation of the protein structure and were validated to check the overall quality of protein and stereochemical activity of atoms and amino acids which were predicted by structural analysis and verification server (SAVES). The conformational complexity of protein structures was used to predict active site amino acids that help for ligand binding using CastP calculation server and the best complex protein structures were used for molecular docking.
Ligand structure design and pharmacophore analysis
Using ACD / ChemSketch software, chemical structures were designed to add all chemical compositions and the final output was saved in MOL2 format. The training sets were used to predict pharmacophore using molinspiration server and QSAR properties were predicted using Hyperchem. A training set helps to predict the complex polarity and flexibility to examine MM3 force fields to examine the HOMO and LUMO to understand new molecular orbital of individual compounds. The scaffolds were identified and then accelerated screening; a screened pool is focused for bio-targets to inhibit the diseases. Structural screening, fragment-analysis, and pharmacological analyses were used to screen the ligand based on interaction with target apoptotic proteins.
AutoDock 4.6 software was used to predict protein-ligand exchange utilizing different factors such as preparing protein properties, adding Gaussian charges, adding hydrogen atoms with polar amino acid zone, planning ligand molecule with rotatable angle bond interaction, etc.Grid maps of different grid points, centered on the ligands of the complex structure were used for receptors respectively, to cover binding pockets. A set of the Lamarckian genetic algorithm was used for molecular docking simulations. The population size of 150, the mutation rate of 0.02, and crossover rate of 0.8 were set as the parameters. Simulations were performed using up to 2.5 million energy evaluations with a maximum of 27000 generations. Each simulation was performed 10 times, yielding 10 docked conformations. The lowest energy conformations were regarded as the binding conformations between the ligands and the proteins. Further, the reverse validation processes ensured that the identified hits really fit the generated Pharmacophore models and active sites of targets. All the parameters required for molecular docking and Pharmacophore mapping were fixed as used in the regular process.
Synthesis of ruthenium Fluvastatin complex
Approximately 433.45 mg (1 mmol) of fluvastatin sodium wasdissolvedin60mlHPLC analytical category ethanol at a room temperature of 27° C, with continuous mixing using a mechanical stirrer. In another conical flask, approximately 103.5 mg (0.5 mmol) of ruthenium chloride dissolved in 40 ml of ethanol and added dropwise to the fluvastatin solution with continuous stirring for 24 hours. After complete mixing the resultant solution was refluxed at 80 °C for 3 hours. The reaction mixture was kept in vacuum desiccator over silica gel for seven days. The obtained product was brown in color and was found to be soluble in ethanol & dimethyl sulphoxide (DMSO). (Fig, 1A) represents the possible structure of the ruthenium-fluvastatin complex.
Characterizations of ruthenium-fluvastatin complex
The UV-Visible spectrum of ruthenium-fluvastatin complex and fluvastatin were recorded via UV-1800 Shimadzu double beam spectrophotometer with typical 1.00 cm quartz cell. FT-IR spectroscopy (ALPHA-T, Bruker, and Rheinstetten, Germany) was used to document the infrared spectrum of the complex over the span of 500–4000 cm− 1 wavelength to evaluate the complexation by detecting the metal oxide bond.The molecular structure of the ruthenium-fluvastatin complex was studied by employing tandem mass spectrometry (ESI-MS) techniques with electrospray ionization. Molecular ions (m/z) were scanned over a span of 150–1100. A Bruker- Avance-600 MHz spectrometer was used for the studying of the1H-NMR spectrum of the complex dissolved in DMSO. Tetra methyl silane (TMS) was used as an internal reference.Scanning electron microscopy (JEOL MAKE, (UK) MODEL- JSM6360) was used to examine the morphological appearance of the sample at an accelerating voltage of 17 kV.To examine the surface and morphological features, micrographs were recorded at different magnifications (60X, 200X, 500X, and 1000X). X-ray diffraction (XRD) of the complex was recorded by XPERT-PRO Diffractometer (PANanalytical, Almelo, Netherlands) using X’celerator operating at 40 kV and 30 mA with Bragg-Brentano geometry with step size 0.033(2ɵ), step time 19.43 s.
Evaluation of antioxidant status of ruthenium fluvastatin complex by DPPH, FRAP, ABTS methods
Free radical scavenging ability of the complex was assessed by DPPH radical scavenging assay according to the procedure described by Lim and his co-researcher (60). The hydrogen atom donating ability of the complex was determined by the decolorization of methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH). DPPH produces violet/purple color in methanol solution and fades to shades of yellow color in the presence of antioxidants. Subsequently, 100 µl of the complex as well as ligands at different concentrations (5, 10, 15, 20, 25 µM) were mixed with 0.1 ml of DPPH solution (0.2 mg/ml in ethanol) and the absorbance was measured at 515 nm every 5 minute interval further assessed for 30 minutes (As), whereas a solution containing only DPPH functioned as blank (Ac). The percentage of radical scavenging action (RSA %) was enumerated as
(RSA %) = 100 (AC-AS)/AC
The FRAP assay was carried out by method of Benzie and Strain’s idea (61) as developed by Griffin and Bhagooli (62). The working FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM 2, 4, 6-tripytidyl-s-triazine (TPTZ) solution and 20 mM FeCl3.6H2O at a 10:1:1 ratio prior to use and heated to 37˚C in a water bath. 3 ml of FRAP reagent was added to 100 µl of various concentrations (5–40 µM) of the complex and ligands. Following reaction, light bluish tint color of the FRAP solution shifted to dark blue and the change in absorbance was detected at wavelength 593 nm and expressed as mmol Fe2+/g of sample.
The radical scavenging activity of ruthenium fluvastatin complex by the ABTS method has been evaluated utilizing the process outlined by Pennycooke and coworkers (63). Following the incorporation of fluvastatin and the complex to the ABTS solution (incubated at room temperature for 10–12 minutes) the absorbance was taken at 734 nm. The equation below was used to measure the percentage of radical scavenging action (RSA %):
Radical Scavenging activity at 750 nm (%) = 1- Af /A0 100.
Where, A0 = Absorbance of free radical cation, Af = Absorbance observed 10 min after incorporation of the complex.
DNA Binding assay of ruthenium-fluvastatin complex
CT-DNA intercalation with the complex was calculated using a UV-Visible spectrophotometer (UV-1800 Shimadzu) based on the technique recorded by Dehghan (64). The intrinsic binding constant was calculated as:
DNA/εa-εf = DNA/εb-εf + 1/Kb(εb-εf)
DNA represents the number of base pairing of DNA, εa represents the extinction coefficient (Aobs /Ru) factor, εf is the free drug related extinction coefficient and εb represents bound drug associated extinction coefficient and complex associated calibration curve is derived from εf in the aqueous solution. εa represents the ratio of recorded absorbance to concentration of the complex by Beer’s law.
The breast cancer cell lines MCF-7and MDA-MB-231 were acquired from American Type Culture Collection (ATCC) (Manassas, VA, USA). The tumor cells were normally sustained in DMEM, enriched by 10% FBS (foetal bovine serum) constituting of antibiotics such as penicillin / streptomycin (0.5 mL-1) in an environment of 5% CO2 & 95% air at 37 ° C.
Cell viability assay
The experiment was assessed by using mitochondrial succinate dehydrogenase to metabolize tetrazolium salts MTT (3-(4, 5 dimethylthiozol-2-yl)-2, 5- diphenyl tetrazolium bromide), which is yellow to produce formazan crystals. The MCF-7 and MDA-MB-231 cells were plated in a 5% CO2 humidified incubator and exposed to a number of ruthenium fluvastatin doses for 24 hours, containing proper growing media in 96 well plate containing 5.0 × 103 cells for each well and incubated nightly at 37 ºC. Upon treatment the medium was withdrawn and MTT solution (0.5 mg / ml) was applied to each well and 3 hours incubated at 37 ºC. On a microplate reader, the optical density of solubilised crystals in DMSO was estimated at 560 nm. The cell viability percentage was determined by the equation.
% viability = 100- % of cytotoxicity
Assessment of apoptotic cells by DAPI staining
The cell lines were studied for nuclear blebbing and chromatin condensation by staining them with fluorescent nuclear dye 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), using the method developed by Li (65).
The inhibitory effect of ruthenium-fluvastatin complex on MCF-7 and MDA-MB-231 cells on proliferation was determined by clonogenic assay. The cells were trypsinized to create a single cell suspension and implanted in six well plates with a density of 500 cells / well in 2 ml of medium supplemented with 10% FBS, kept in a humidified compartment having an atmosphere of 95% air, 5% CO2 at 37 ºC. After 24 hours of incubation, culture was replaced with fresh media containing three different concentrations of ruthenium fluvastatin complex along with 2% FBS and cultured for two weeks. After 2 weeks, the cell culture medium was removed and the cells were thoroughly washed with PBS. Cell fixation was done by using 100% methanol kept at -20 °C for 30 minutes. Colonies were stained with 0.5% crystal violet in 25% (v/v) methanol for 1 hour at room temperature. The excess dye was removed by gently rinsing with moderate water flow for 15 minutes. Following washing and drying, the colonies were visually counted to contain > 50 cells / colony. The clonogenicity was measured by the help of the equation below:
Clonogenicity = (Cloning number/500x) x100.
The clonogenic assay was performed in triplicate.
Apoptotic assay by flow cytometry and Cell cycle analysis
MCF-7 and MDA-MB-231 cells were suspended for cell cycle distribution analysis, and their nuclear DNA was tagged with propidium iodide (PI). Nuclear DNA distribution through the cell cycle process was assessed using a FACS (fluorescence-activated shorter cell). At least 10,000 incidents were obtained and flow-cytometric data collection was carried out using Modfit software, and a histogram of the DNA content against counts was prepared using the methods described by Li (65).
Caspase-3 protein detection by flow cytometry
MCF-7 and MDA-MB-231cells (5 × 105 cells / well) were cultured on twelve well plate and incubated at 37 °C in a humidified atmosphere with 5% CO2 for 24 hour and subsequently treated to three concentrations of ruthenium fluvastatin complex for 24 hours. The cells were again washed properly with ice cold PBS and resuspended with BD Cytofix / Cytoperm Solution (51-6896KC, BD Pharmingen) 400 µl. The method was initiated by determining the quantity of BD Perm/wash buffer (51-6897KC, BD Pharmingen) and 20 µl of Rabbit anti active caspase 3 polyclonal antibody (351-68655X, BD Pharmingen) was taken, so that each and every individual test was comprise of 100 ml of BD Perm/wash buffer and 20 µl of anti active caspase 3 antibody. After incubation on ice for 20 minutes, followed by centrifugation and washing with BD Perm/wash buffer. Subsequently, BD Perm/wash buffer was further added followed by incubation with antibody for thirty minutes at room temperature. Each and every individual tube was further rinsed with 1 ml of BD Perm/wash buffer, centrifuged and then finally added 300 µl of BD Perm/wash buffer and analyze by flow cytometry (BD Accuri C6 Plus flow cytometer). Values thus obtained were processed by using FlowJo software.
Western Blot expressions detection of Akt, mTOR, p13K, VEGF, pro Caspase-3 and Active Caspase-3 proteins
Western blot analysis detected the expressions Akt, mTOR, p13 K, VEGF, pro Caspase-3 and Active Caspase-3 in cells MCF-7 and MDA-MB-231. The cells were treated for 24 hours after a medium change with three different doses of ruthenium-fluvastatin complex, and maintained for 6 hours. Cell lysates were extracted and equivalent protein amounts were analyzed using SDS-PAGE electrophoresis, followed by a shift to a PVDF (polyvinylidene difluoride) membrane and afterwards blocked to Tris buffer (25 mM) comprising 0.15 M NaCl, 0.1 per cent around 20 and 2–5 per cent non-fat dry milk. At 4 ºC the membranes were cultured with the primary antibodies Akt, mTOR, p13 K, VEGF, pro Caspase-3 and Active Caspase-3 supplemented by a secondary antibody marked with horseradish peroxidase for 1hr. Chemiluminescent (ECL Western Blotting) kit was then used to recognize protein loading against β actin (66).
Animal husbandry and maintenance
Sprague Dawley rat (120–125 gm) of both sexes and 28-day Sprague-Dawley female rats (80–100 grams) were purchased from Nanjing Medical University, Nanjing, China and quarantined even days before experimentation. Animals were housed in a 12-hour light / dark period in polypropylene containers, 22° C (± 3° C) at room temperature and nearly 50–58% humidity. Each animal was fed a semi-purified basal diet and demineralized water ad libitum. The entire animal research process was performed in conjunction with the permission of the Nanjing Medical University's Animal Ethics Committee and the Government's Regulatory Body (IACUS-1912129).
Acute oral toxicity study (LD50)
oAcute toxicity evaluation of the ruthenium-fluvastatin complex was carried out by incorporating the Recommendations for research on chemicals by the Organization for Economic Co-operation and Development (OECD), TG 420 (adopted in December 2001) to establish the LD50 values of the complex. Thirty numbers of Sprague Dawley rats of both sexes (nulliparous & non-pregnant; 120 ± 5 gm) were identified and allocated in five groups (six animals per group, three of each sex) plus control (dispensed in 0.5% carboxy methyl cellulose prepared as a carrier in drinking water at a dosage of 10 ml / kg body weight) and study groups (2000, 800, 600, 300 and 100 mg / kg ruthenium fluvastatin complex). The rats were allowed food and supplies immediately after drug administration and placed under three days' surveillance (67).
Sub-acute toxicity studies
Sprague Dawley rats, both male and female (120 ± 5 gm) were arbitrarily paired to four experimental groups: complex (25, 50, 100, 200 mg/kg) and vehicle control group. Each unit was composed of 10 rats, 5 per gender. For further hematology, serum biochemistry and histological experiments, the animals were orally administered with ruthenium-fluvastatin complex and sacrificed at 28th day by ether anesthesia.
Histopathological study of rat organs
Primary organs such as liver, kidney, stomach and testis were harvested from each participant after 28 days of analysis and stored in 10% formalin solution. Tissue was drained by graded alcohol and preserved in paraffin wax at low melting point on a 5 micron glass slide. By using xylene the sections were deparaffinized and rehydrated by graded alcohol and subsequently stained with hematoxyl and eosin (H&E) for microscopic examination.
After acclimatization, the animals were grouped into six designated units such as I, II, III, IV, V, VI, VII and each unit consisted of six animals. Once all the animals were 50 days old, DMBA was given in an oil emulsion as a single tail vein injection to group II to IV at a dosage of 0.5 mg per 100 g body weight. The description below denotes the experimental layout of the groups
Group I - Animals constituted the normal untreated controls and received basal diet throughout experiment.
Group II - Comprised of carcinogen (DMBA) treated animals.
Group III- Carcinogen (DMBA) induced animals accompanied by 25 mg/kg body weight treatment with the Ru-fluvastatin complex.
Group IV- Carcinogen (DMBA) induced animals accompanied by 50 mg/kg body weight treatment with the Ru- fluvastatin complex.
Group V- Carcinogen (DMBA) induced animals accompanied by 75 mg/kg body weight treatment with the Ru- fluvastatin complex.
Group VI - Carcinogen (DMBA) induced animals accompanied by 50 mg/kg body weight treatment of ruthenium.
Group VII- Carcinogen (DMBA) induced animals accompanied by 50 mg/kg body weight treatment of fluvastatin.
Following 16 weeks of treatment, the animals were sacrificed in light ether anesthesia from each group preceded by midline incision from the pubis to the sub-maxillary region. Dissection of the skin was undertaken to reveal the six sections of mammary glands
Histopathology of mammary tissue
Ten animals were randomly selected from individual group, excising the thoracic and abdominal inguinal mammary tissue from rats anesthetized with ether. Part of the breast tissue was set in 10% neutral formalin buffered, carefully washed, paraffin-coated, sliced into 5 µm thick segments and mounted on slides. The tissue segments were treated with hematoxylin and eosin for histopathological investigations (H&E).
Antioxidant assay of breast tissues
Mammary tissue was crushed and homogenized (10 percent w / v) in 0.1 M phosphate buffer (pH 7.0) and centrifuged for 10 min, and thus the generated supernatant had been used for the measurement of enzymatic antioxidants (68). The catalase reaction was analyzed using the method defined by Sinha and his associates (69). The absorbance was recorded at 620 nm; CAT action was reported as µ Mol of H2O2 / min / mg protein consumed. Superoxide dismutase activity was assessed using the Awasthi technique (70). This activity was expressed as units/min/mg protein. The GPx evaluation was carried out using the process provided by Rotruck (71). The activity was calculated as µMol of GSH consumed/min/mg protein.
Immunohistochemical analysis of mammary tissue
The tissues coated with formalin and set in paraffin is sliced into 5 µm thickness to position on the glass slides and deparaffinized together with immersion in H2O2. For 1 hour, the segments were coated with goat serum, preceded by exposure to anti-mouse p53, Bcl2, Bax and MMP-9 antibodies (1:50 ratio) and maintained overnight at 4 ºC. The slides were immersed with PBS and subsequently cultivated for about 30 min with the HRP-conjugated secondary antibody streptavidin biotin. DAB was utilized to dye the segments and counter-stained with hematoxylin. The labeling index was measured as the number of positive stained cells with p53, Bcl-2, Bax and MMP-9 to the total cell count.
Cell proliferating assay
The tissues coated with formalin and set in paraffin is sliced into 5 µm thickness to position on the glass slides performed deparaffinized accompanied by submersion in H2O2. The segments were covered with goat serum for 1 hour, followed by exposure anti-mouse Ki-67 antibody at 4 °C overnight. At room temperature the positive test slides were processed for 30 min with streptavidin biotin horseradish peroxidase complex. Tissues were treated with DAB (3,3'- diaminobenzidine) and hematoxylin ( 72).
TUNEL assay of mammary tissues
Tissues fixed in formalin, implanted in paraffin, and coated with poly-L-lysine were screened for 15 minutes with proteinase K (20 µg / ml in PBS) and washed with water distilled twice. The tissues were then soaked with H2O2 (2% in PBS) at room temperature for 5 min, accompanied by treatment with the terminal deoxynucleotidyl transferase (TdT) buffer (30 mM trizma base, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride) accompanied by a TdT reaction solution containing TdT and dUTP at 37 °C for 90 min, 2 percent of the normal saline citrate was then added to the tissues (10 min) at room temperature to interrupt the reaction. After washing with PBS the tissue segments were soaked with anti-digoxigenin peroxidase for 30 minutes at RT. Tissues were stained with DAB and counter stained with hematoxylin. Slides then were cleaned, dehydrated and stored. Apoptotic cells were thus detected by a brown staining of the nuclei (72).
Evaluation of labelling and apoptotic index
The labeling index (LI) was determined by counting the proportion of Ki-67-positive nuclei per total number of cells. The apoptotic index (AI) was calculated by measuring the TUNNEL-positive cell percentage to the total cell number.
The findings were set to mean ± standard mean error (SEM). Statistical assessment was carried out using t-test and one-way variance analysis ( ANOVA) using graph pad prism techniques, further verified by post-hoc measurement check (Dunnet's t test), difference was found to be statistically significant by using P < 0.05.