A Novel Ruthenium Fluvastatin Complex Downregulates SNCG Expression to Modulate Breast Carcinoma Cell Proliferation and Apoptosis Via Activating PI3K/Akt/mTOR/VEGF/ MAP Kinase Pathway

Background: Breast cancer is the most common cause of malignancy and cancer related morbidity and death worldwide that requests effective and safe chemotherapy. Evaluation of metallo drug based anticancer agents and statins as chemotherapeutics with fewer side effects is largely unexplored research elds Methods: The synthesis and characterization of Ruthenium uvastatin complex was achieved using multiple spectroscopic techniques and thus further examined to evaluate its chemotherapeutic prospects in both MDA-MB-231 and MCF-7 cancer lines and eventually in vivo models of DMBA induced mammary carcinogenesis in rodents. The viability of cells was tested using MTT assay. The apoptotic assay in cell lines was analyzed by ow cytometry. Western blot technique and immunohistochemical approach were used to investigate complex induced signaling pathways. TUNEL assay had predicted in-vivo apoptosis. Results: Our studies indicate that the metal and ligand chelation was materialized by the ligand's functional groups of carbonyl (= O) oxygen and hydroxyl (-OH) and the complex has been observed to be crystalline and able to chelate with CT-DNA. The complex was able to reduce cell proliferation and activate apoptotic events in breast carcinoma cell lines MCF-7 and MDA-MB-231. In addition, the complex was able modify p53 expressions to interfere with apoptosis in the carcinoma of the breast, stimulated by the intrinsic apoptotic path assisted by Bcl2 and Bax, yet at the same point controlling the PI3K / Akt / mTOR pathway correlated with MMP9 regulated tumor mechanisms. Conclusion: Our research reveals that ruthenium-uvastatin chemotherapy may disrupt, rescind or interrupt breast carcinoma progression by modifying intrinsic apoptosis as well as the antiangiogenic cascade, thereby taking the role of a potential candidate in cancer therapy for the immediate future. performed to establish the inhibition of MCF-7 and MDA-MB-231cell growth by the complex through diverse cell cycle modulatory factors variation. veried the effects of ruthenium– uvastatin complex treatment on various proteins like PI3K, Akt, mTOR, EGFR, VEGF, and cleaved caspase 3 in MCF 7 and MDA-MB-231 cells human breast cancer cells. After 24 hours of exposure to ruthenium – uvastatin complex in both MCF 7 and MDA-MB-231 cells, a dose-dependent down-regulation of PI3 K, Akt, mTOR, EGFR and VEGF was identied Nonetheless, a signicant upregulation of cleaved caspase 3 observed in both MCF 7 and MDA-MB-231 cells after 24 hours of exposure to ruthenium – uvastatin therapy.


Background
Breast cancer in women is the most recurrent cancer in any of the ve continents disgnosed worldwide, with an approximate 2.1 million new cases in 2018 (1). The incident of breast cancer on the Asian continent is still underneath than that of Europe or America, but Asia's participation in the world wide burden of breast cancer is speedily increasing as a result of expressed economic growth and urbanization (2). The situation in China has been alarming partly due to its rapid population growth and socioeconomic development. As a densely populated country, China reports for one fourth of total cancer related death and there has been an increasing incidence in cancer prevalence, mortality rate and emergence among young people (3). China reveals 36.1 percent increase in death rate from  according to numerous breast cancer studies (4). Reports further reveal that, at 2015 in China, the highest rate of breast cancer incidence (127.550 per 100000) was observed in women between 65-69 years of age. In addition, breast cancer incidence rate increased by 69.38% in 65-69 years age group from 75303 to 127550 per 100000 women in 2005 to 2015 respectively, indicating a yearly average growth of 6.9% (5). Breast cancer always diagnosed as an invasive malignant tumor without curable therapy (6). Regretfully, the molecular mechanisms underlying breast cancer are still ambiguous to fact. Hence, a good understanding of the genetic underpinnings of breast cancer has great importance and seeking the alteration of researchers for further in-depth study.
Current treatment strategies have limitations which lead to clinical resistance and morbidity associated with therapy due to their side effects and limited e cacy on tumors. Novel molecules are therefore urgently required to combat emerging cases of breast cancer, and with less adverse effects, reduced tumor recurrence and reduced mortality. An interesting approach called drug repurposing in one hand and drug combination approach in another hand to tackle this problem is progressively being investigated and applied (7,8). Addressing the signi cance, the global market for drug repurposing almost touched nearly 20.7 billion euro in 2015 and is expected to reach 26.6 billion euro by 2020 (9). Due to advantages like decreased toxicity, better e cacy, decreased dosage at an equal or increased level of e cacy and counter drug resistance, drug combination represent an impressive and progressively used approach which has become a standard for treating cancer (8). Particularly in cancer, various clinical trials are conducted with a crescent focus on the combination of cytotoxic drugs (10). The combination of repurposed pharmaceutical agents with other chemotherapeutic agents continue to show impressive outcomes which are bene cial when traditional monotherapy for cancer patients has failed to provide safe and tolerable treatment (11). To systemically address the concern, this project aim to investigate the effect of drugs like statin with potential to be repurposed for breast cancer therapy, in combination of chemotherapeutic metallodrug like ruthenium.
Statin elicit several effects beyond their lipid reducing roles de ned as the pleiotropic effects (12) often caused by blocking the prenylation of a multitude of intracellular signaling proteins and thus affecting the development of cancer among statin user (13,14,15,16,17). Statin has been delineated to be associated with reduced rate of cancer progression and cancer associated mortality (18,19) from gastrointestinal carcinoma (20,21), breast carcinoma (22), hepato-cellular carcinoma (23), prostate cancer (24), lung adenocarcinoma (25) and pancreatic carcinoma (26).
Fluvastatin is one of the members of statin family, broadly used as a lipid lowering agent. In addition, to its cholesterol reducing activities, Fluvastatin exhibited the anticancer activities by inducing apoptosis in glioma, breast and hepatocellular carcinoma cell line (27,28,29). Notably, uvastatin was shown to have a chemo-adjuvant effect on experimental pancreatic cancer (30).
Along with platinum-based cancer drugs, several efforts have been made to design compounds based on ruthenium, since these compounds have reported a lower number of side effects due to their alternate modes of action (31,32). Ruthenium complexes have in many cases exhibited strong cytotoxicity against platinum-resistant tumor cell lines, rendering it as exemplary targets for further investigation (33,34). It may be acknowledged that many rutheniumcontaining compounds including RAPTA-C (35), NAMI-A, and KP1019 (36) have o cially managed to reach clinical trials for diverse therapeutic carcinoma management.
Despite advancement in the treatment the high recurrence rate metastasis is associated with poor prognosis of endocrine related cancer (37). Thus a better understanding of the molecular mechanism underlying breast cancer proliferation and metastasis is necessary to identify the potential target for effective therapy. Synucleins are ubiquitously expressed in neuronal cells and abundantly present in presynaptic terminals, and have also been involved in non-neural diseases, especially hormone-responsive breast cancers (38) and ovarian carcinoma (39). Numerous reports have also suggested that SNCG is not expressed in normal and benign breast tissues, but expressed abnormally in a high percentage of advanced and metastatic cancer (40,41) and stimulates hormone dependent growth of breast cancer cells both in vitro and nude mice (42). Research aimed at elucidating the molecular mechanism that trigger the oncogenic function of this protein reported that SNCG expression in the breast cancer cells have developed a more malignant phenotype with increased motility of the cells (38), intensify the activity of transcriptional factor of ERα (43), enhanced resistance to antimicrotubular drugs (39), expedited the microsomal instability (44). Cumulative ndings suggest SNCG is a novel unfavorable prognostic marker for breast cancer progression and a potential target for breast cancer treatment. On the basis of this data, the current research uses molecular docking, a widely used bioinformatics technique to evaluate how the molecule of ruthenium uvastatin binds and interacts with the SNCG target proteins. These in silico methodologies aids towards classifying drug targets through computer-assisted designing that help to i) envision speci c active sites for evaluating target structures ii) produce potential molecules that identify the target iii) determine their comparative receptor binding a nities and iv) modify molecules to optimize binding abilities (45). Autodoc is one of many softwares that consists of a series of automatic docking techniques developed to evaluate how molecules attach to a particular targeted protein with a preordained arrangement (46). Therefore, our objective is to establish the optimum binding energies of the docked molecules and also to determine the ligand's binding position at the protein binding domain.
A combination of cell proliferation and apoptosis regulates the development of normal breast tissues. Conclusive evidence states that development of cancer is not only a manifestation of unregulated proliferative status along with impaired apoptosis (47,48). In the context of breast cancer, the Bcl2 gene and the tumor suppressor like p53 genes are thoroughly researched (49,50). Bax and Bcl-2 are indeed the key pro-apoptotic modulators which could additionally improve the PI3K / AKT-related cascade and other pathways related to cell survival and death (51). Furthermore, natural agents may also affect cell migration and invasion of tumor cells by modulating the matrix metalloproteinases (MMPs). Between others, MMP-9 is of particular interest, as patients with increased MMP-9 expression have been identi ed to end up with poorer diagnosis (52). In addition, breast cancer development includes genetic variations of p53, modi ed by angiogenic pathway (VEGF), mTOR-related signaling pathway and proapoptotic protein such as Bax corelated with the increased formation of antiapoptotic protein Bcl2 and nuclear proliferating cell antigen (PCNA) (53,54,55). 7,12-Dimethylbenz(α)anthracene may be the most harmful polyaromatic hydrocarbon with the widest environmental application (56). DMBA metabolizes and produces various reactive metabolic intermediates during the carcinogenic cycle, thereby stable DNA adducts formation which are genotoxic and mutagenic that initializing carcinogenic events, close to that in humans (57). The oxidative imbalance in uences different types of proteins and genes that modify multiple signaling cascade such as angiogenic process, apoptotic events, cell formation, repairing of DNA, proliferative events and invasion (58,59) Taking all of it into account, we hypothesized and investigated by well-designed experiments in this current study, that ruthenium-uvastatin could interact with cellular proliferation and induce cell apoptosis by modulating the apoptotic regulators and downstream events such as caspase cascade thus inhibiting the invading and migrating properties of cancer cells. Chemotherapeutic actions in the breast cancer paradigm and the underlying mechanistic approach of the ruthenium -uvastatin complex have not yet been extensively analyzed. The objective of this project is to synthesize, characterize the ruthenium-uvastatin complex along with the study of antioxidant status along with DNA binding features and further investigate the chemotherapeutic activity against mammalian cancer in both in vitro and in vivo studies.

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 speci ed 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 veri cation 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 nal 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 exibility to examine MM3 force elds to examine the HOMO and LUMO to understand new molecular orbital of individual compounds. The scaffolds were identi ed 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.

Molecular Docking
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 identi ed hits really t the generated Pharmacophore models and active sites of targets. All the parameters required for molecular docking and Pharmacophore mapping were xed as used in the regular process.

Synthesis of ruthenium Fluvastatin complex
Approximately 433.45 mg (1 mmol) of uvastatin sodium wasdissolvedin60mlHPLC analytical category ethanol at a room temperature of 27° C, with continuous mixing using a mechanical stirrer. In another conical ask, approximately 103.5 mg (0.5 mmol) of ruthenium chloride dissolved in 40 ml of ethanol and added dropwise to the uvastatin solution with continuous stirring for 24 hours. After complete mixing the resultant solution was re uxed 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-uvastatin complex.

Characterizations of ruthenium-uvastatin complex
The UV-Visible spectrum of ruthenium-uvastatin complex and uvastatin 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 rutheniumuvastatin 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.

FRAP assay
The FRAP assay was carried out by method of Benzie and Strain's idea (61) as developed by Gri n 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 FeCl 3 .6H 2 O 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 Fe 2+ /g of sample.

ABTS assay
The radical scavenging activity of ruthenium uvastatin complex by the ABTS method has been evaluated utilizing the process outlined by Pennycooke and coworkers (63). Following the incorporation of uvastatin 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-A f /A 0 100.
Where, A 0 = Absorbance of free radical cation, A f = Absorbance observed 10 min after incorporation of the complex.
DNA Binding assay of ruthenium-uvastatin 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 represents the number of base pairing of DNA, ε a represents the extinction coe cient (A obs /Ru) factor, ε f is the free drug related extinction coe cient and ε b represents bound drug associated extinction coe cient and complex associated calibration curve is derived from ε f in the aqueous solution. ε a represents the ratio of recorded absorbance to concentration of the complex by Beer's law.

In-vitro experimentation
Cell culture 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% CO 2 humidi ed incubator and exposed to a number of ruthenium uvastatin doses for 24 hours, containing proper growing media in 96 well plate containing 5.0 × 10 3 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.

Assessment of apoptotic cells by DAPI staining
The cell lines were studied for nuclear blebbing and chromatin condensation by staining them with uorescent nuclear dye 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), using the method developed by Li (65).

Clonogenic assay
The inhibitory effect of ruthenium-uvastatin 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 humidi ed 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 uvastatin 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 xation 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 ow 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: The clonogenic assay was performed in triplicate.
Apoptotic assay by ow 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 ( uorescence-activated shorter cell). At least 10,000 incidents were obtained and ow-cytometric data collection was carried out using Mod t software, and a histogram of the DNA content against counts was prepared using the methods described by Li (65).

Caspase-3 protein detection by ow cytometry
MCF-7 and MDA-MB-231cells (5 × 10 5 cells / well) were cultured on twelve well plate and incubated at 37 °C in a humidi ed atmosphere with 5% CO 2 for 24 hour and subsequently treated to three concentrations of ruthenium uvastatin complex for 24 hours. The cells were again washed properly with ice cold PBS and resuspended with BD Cyto x / 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 nally added 300 µl of BD Perm/wash buffer and analyze by ow cytometry (BD Accuri C6 Plus ow 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-uvastatin 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 di uoride) 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).

In-vivo experimentation
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-puri ed 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).
Toxicological studies Acute oral toxicity study (LD 50 ) oAcute toxicity evaluation of the ruthenium-uvastatin 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 LD 50 values of the complex. Thirty numbers of Sprague Dawley rats of both sexes (nulliparous & non-pregnant; 120 ± 5 gm) were identi ed and allocated in ve 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 uvastatin 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 rutheniumuvastatin complex and sacri ced 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 para n wax at low melting point on a 5 micron glass slide. By using xylene the sections were depara nized and rehydrated by graded alcohol and subsequently stained with hematoxyl and eosin (H&E) for microscopic examination.

In-vivo Experiments Experimental Protocol
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 Ruuvastatin complex.
Group IV-Carcinogen (DMBA) induced animals accompanied by 50 mg/kg body weight treatment with the Ruuvastatin complex.
Group V-Carcinogen (DMBA) induced animals accompanied by 75 mg/kg body weight treatment with the Ruuvastatin 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 uvastatin.
Following 16 weeks of treatment, the animals were sacri ced 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, para n-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 de ned by Sinha and his associates (69). The absorbance was recorded at 620 nm; CAT action was reported as µ Mol of H 2 O 2 / 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 para n is sliced into 5 µm thickness to position on the glass slides and depara nized together with immersion in H 2 O 2 . 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 para n is sliced into 5 µm thickness to position on the glass slides performed depara nized accompanied by submersion in H 2 O 2 . 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 xed in formalin, implanted in para n, 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 H 2 O 2 (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.

Statistical Analysis
The ndings 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 veri ed by post-hoc measurement check (Dunnet's t test), difference was found to be statistically signi cant by using P < 0.05.

Pharmacophore Analysis
It is of interest to design the inhibitors for the breast cancer target Synuclein gamma (SNCG) protein, using molecular docking based virtual screening followed by molecular docking. The protein structures of SNCG was retained from PDB and the resultant structures were used for molecular modeling methods to check the resolution of amino acid arranges within the complex protein structures and predicted the active site of the amino acids. The docking results shows that SNCG interacted signi cantly with ruthenium-uvastatin within the active site amino acids of both polar and electrostatic charges within the target amino acids of Thr59, Asn64, Val66, Ser67, Glu68 and Val71 with substantial energy of -23.168 kcal/mol. (Table 1   Instrumental analysis The electronic spectrum or UV spectrum of uvastatin has shown absorption bands in two region 230-240 nm and 300-310 nm due to inter-ligand interaction. The complex only showed charge transfer between ligand to metal and metal to ligand as there was no appreciable change found in the UV-visible spectrum of the complex. So, there is no dd transitions are expected due to the complex formation. The rst range of the wavelength can be assigned to π→π* transitions in the aromaticity of double bond. The other range of the wavelength is most probably due to the n→π* transition of mainly pyrrole, carboxylate and hydroxyl group (Fig. 2A). The FTIR study of the uvastatin and ruthenium-uvastatin complex was done to determine the co-ordination sites and binding properties of uvastatin with ruthenium, as shown in the Fig. 2B & C and the analysis of the data was done in the Table 2 Figure 3A, indicates that the scavenging actions of uvastatin and ruthenium-uvastatin by ABTS methods. Absorption of the effective ABTS solution at 734 nm was observed to substantially decrease in the presence of different concentrations of the complex. The complex 's ABTS radical scavenging activity has been found to be better than the free uvastatin. The presence of hydroxyl groups causes statins to have antioxidant activity. The complex's higher antioxidant activity is due to the hydroxyl groups, and their capacity to contribute hydrogen atoms improved after ruthenium chelation.
In Fig. 3B, illustration of different DPPH absorption pattern of uvastatin and ruthenium-uvastatin complex at 515 nm, with increasing amount of time and concentration. With the increase in concentration and time complex showed higher DPPH absorption than the uvastatin molecule. The complex represented better inhibitory effect compared to the free uvastatin, the radical-sensitive Ru-O bond was introduced to ruthenium-uvastatinn complex which synergistically enhances antioxidant activities of uvastatin. In Fig. 2.2, the plot illustrated the radical scavenging activity of uvastatin and the complex, where it has been found that uvastatin scavenged free radicals to about 47.10% and the complex scavenged about 63.17% at the same given reaction time.
The absorption of uvastatin and ruthenium-uvastatin complex in the presence of Fe + 3-TPTZ was estimated at 593 nm by absorbance variability during 10 min of FRAP reagent interaction with the subjected compound. The absorbance reduction is equivalent to the antioxidant quality. Figure 3C shows that the antioxidant capacity of the complex is improved compared with free uvastatin. These ndings indicate that the complex of uvastatin and ruthenium is capable of making a donation of protons and thus could have the ability to end a chain reaction. Metal chelation further increases the transfer of electrons from uvastatin and thus rises the redox potential of the ruthenium-uvastatin complex.

Ruthenium-uvastatin complex is capable of binding with CT-DNA
The broad absorption range in the presence of CT-DNA (5 microns) as can be seen in (Fig. 3 D). A decline in absorption rate (hypochromism) of the absorption peak is observed following the addition of the concentrations of the complex to CT-DNA. The intensity changes can be recognized within the intra-ligand transition band at 383 nm, after raising the complex concentration in the DNA. These absorption spectra disclose that the complex interacts with DNA by stacking action between the ligand's chromophore via intercalative mode and the DNA base pairs.

In vitro Assessment
Ruthenium uvastatin complex instigates the repression of cell viability MTT assay was performed to investigate the inhibitory effect of ruthenium-uvastatin complex on MCF-7 and MDA-MB-231 cells. The cell viability evaluation revealed that ruthenium -uvastatin complex showed a dose-dependent inhibitory impact on human breast cancer cells MCF-7 and MDA-MB-231 ( Fig. 4 A

Ruthenium-uvastatin complex causes chromatin condensation
Detection of nuclear blebbing and condensation of the chromatin were performed by using DAPI staining. Cells containing condensed chromatin morphologically signify that the cells undergoing apoptosis and uoresce with bright blue color. Complex causes dose-dependent nuclear condensation at both cell lines (Fig. 4

Ruthenium uvastatin complex encourages colony inhibition capability
The relevance of the tumor-colony forming assessment for screening new drugs has often been identi ed as an important tool for research. Ruthenium-uvastatin complex effectively stimulates the capacity of MCF-7 and MDA-MB-231 cells to inhibit colony formation (Fig. 4E). In MCF-7 and MDA-MB-231 cells, ruthenium uvastatin complex was substantially more successful in suppressing the colony number ( Ruthenium-uvastatin complex modulates expression of PI3K, Akt, mTOR, EGFR, VEGF, and cleaved caspase 3. Western blot experimentation was performed to establish the inhibition of MCF-7 and MDA-MB-231cell growth by the complex through diverse cell cycle modulatory factors variation. We veri ed the effects of ruthenium-uvastatin complex treatment on various proteins like PI3K, Akt, mTOR, EGFR, VEGF, and cleaved caspase 3 in MCF 7 and MDA-MB-231 cells human breast cancer cells. After 24 hours of exposure to ruthenium -uvastatin complex in both MCF 7 and MDA-MB-231 cells, a dose-dependent down-regulation of PI3 K, Akt, mTOR, EGFR and VEGF was identi ed (Fig.  5 M). Nonetheless, a signi cant upregulation of cleaved caspase 3 was observed in both MCF 7 and MDA-MB-231 cells after 24 hours of exposure to ruthenium -uvastatin therapy.
Toxicity study Acute and sub-acute toxicity study The LD50 dosage of ruthenium-uvastatin complex was estimated to be 300 mg / kg. 25, 50, 100 and 300 mg / kg were chosen as the sub-acute toxic doses following the LD50 dose evaluation. No treatment-related deaths in animals treated with 25, 50, 100 or 300 mg / kg of the complex were recorded during sub-acute toxicity evaluation (28 days).

Analysis of hematological and serum biochemical parameters
Tables 3, 4, 5 and 6 demonstrate the serum biochemical and hematology analysis of the treated and control animals. WBC, RBC quantities in the ruthenium-uvastatin complex (300 mg / kg) dose groups were substantially improved in comparison to control animals. ALP,ALT and AST were slightly higher than the control group at 300 mg / kg dose(p < 0.05). Glucose and BUN were both changed slightly (p < 0.05) in animals administered with 300 mg / kg drug. the complex's 300 mg / kg dosage induced toxicity in animal models to some extent and was thus not considered a standard for subsequent research.  #Signi cant difference at p < 0.05, when compared with control group.  #Signi cant difference at p < 0.05, when compared with control group.

In vivo carcinogenesis study
Histopathology of mammary tissue The normal control (group I) illustrates normal alveolar septa (as), alveoli (a), acinus (ac), serous gland (sg) and terminal duct lobular units (td),of mammary tissue kept intact as seen in Fig. 7 A. DMBA-treated section of (group II) animals revealed atrophy of the periductal, stromal and fatty tissue glands (psf), atrophy of the underlying fatty tissue (ag), atrophy of the serous glands (asg) surrounding stromal brosis, hyperplasia of the serous and mucous glands (ah) in their mammary tissues (Fig. 7 B). Slight hyperplasia of serous and mucinous glands (Fig. 7 C, D) was seen in the histological analysis of 25 and 50 mg / kg ruthenium -uvastatin complex treated groups (Fig. 7C, D), while in the lowest dose category (75 mg / kg) there has been no evidence of hyperplasia or cell proliferation in mammary tissue and normal morphology of the cells covering the ducts was observed (Fig. 7 E). 50 mg / kg Fluvastatin-treated model revealed typical histological composition of rat mammary tissue (Fig. 7G), while 50 mg / kg ruthenium-treated animals showed gland atrophy with surrounding fatty tissue (ag) and serous gland atrophy (asg) (Fig. 7 F).

Antioxidant evaluation of mammary tissues
The fragmented mammary tissue of the carcinogen control animals has been observed with reduced levels of SOD, CAT and reduced glutathione. The animals treated with 75 mg / kg ruthenim-uvastatin complex reported a marked rise in SOD, CAT and glutathione quantities in the homogenized mammary tissues as compared to carcinogenic control and other groups (Fig. 7 H).

Immunohistochemical evaluation of mammary tissues
To outline the in uence of ruthenium-uvastatin therapy on mammary cancer in rats, immunohistochemical staining approaches have been used to determine the existence of cellular biomarkers such as Bax, Bcl-2, p53 and MMP-9 ( Fig. 8) (Table 7). It was found that DMBA treatment greatly raised levels of Bcl-2 ( Fig. 8 [ii] B) and MMP-9 ( Fig. 8 [iv] B), whilst also downregulating levels of Bax (Fig. 8 [iii] B), p53 (Fig. 8I B) as compared to the control group control group ( Fig. 8I iii, iv] A) (p < 0.05). The expression of Bax (Fig. 8 [iii] C, D, E) and p53 ( Fig. 8 I C, D, E) was greatly improved by ruthenium-uvastatin therapy, but Bcl-2 ( Fig. 8 [ii] C, D, E) and MMP-9 ( Fig. 8 [iv] C, D, E) were signi cantly decreased after ruthenium-uvastatin therapy. The dosage of 75 mg / kg ruthenium-uvastatin complex was effective in raising the concentrations of Bax and p53 while the concentrations of Bcl-2 and MMP-9 (p < 0.01) showed a substantial decrease relative to those of carcinogen treated animals. The presence of the above-mentioned biomarkers allows one to believe that the complex focuses on apoptosis and thus regulates the cell cycle to effectively constrain the progression of disease. Flu 50 mg/kg 5.5 ± 0.9 11.7 ± 1.2 4.9 ± 0.3 16.4 ± 1.6 §Each score represents the results of 6 slides per rat and 6 rats per group, mean ± S.E. (n = 6). Each eld were selected randomly for evaluation of percentage of immune-positive cells. * Signi cant difference between treated and carcinogen control (p < 0.01). ** Signi cance difference between treated and carcinogen control (p < 0.05).

Suppression of Ki-67 by Ruthenium-uvastatin complex in the mammary tissue
The potency of the ruthenium-uvastatin molecule in mammary tissue proliferation has been depicted in Fig. 9 [i]. The LI (labeling index) is measured as a proportion of Ki-67 tagged cells shown in Table 8. A substantial improvement in the Ki-67-LI activity was found in the DMBA treated animals ( Fig. 9 I B) compared with the normal control group (  Fig. 9I A), but a small decrease in the Ki-67-LI value was found in the highest dose of ruthenium-uvastatin complex treated animals (p < 0.01) (Fig. 9I C, D, E) relative to carcinogen control animals. Ruthenium-uvastatin complex promotes apoptosis in mammary tissue TUNNEL assay was performed to evaluate the outcome of apoptosis with ruthenium-uvastatin therapy in mammary carcinogenesis ( Fig. 9[ii]). Apoptosis prompts the nuclear DNA to be fragmented into different segments that create DNA strand breaks that could be identi ed by the brown marks produced by DAB chromogen. Normal control cells undergoing cell death have been displayed in (Fig. 9[ii] A). In the carcinogen control group, the TUNEL labelled cells enduring apoptosis were very limited ( Fig. 9[ii] B), while the TUNEL label cells of ruthenium-uvastatin treated animals dramatically increased (Fig. 9[ii] C, D, E). Typically 3 to 5 apoptotic cells were found in an environment of approximately 700 cells throughout the carcinogen control group, which increased to 10-14 cells per 700 cells in the 75 mg / kg of the complex administered animals. AI speci es the apoptotic index and appears in Table 8. Animals obtaining 75 mg / kg of the drug, when compared with the carcinogen control group, represented a marked increase in apoptosis. R value represents the relationship between cell proliferation and apoptosis. Cell proliferation and TUNNEL evaluation suggest that the new improvements in the tumor's microenvironment could be followed by a parallel rise in cell proliferation and a minimization of cell death. R 's value hits a plateau in the carcinogen control group; however, it gradually decreases with the complex's increasing concentration. Through mentioning both of these hypotheses we can conclude that the complex activates apoptosis and ultimately decreases cell proliferation in a dose dependent manner.

Discussion
Current anticancer drugs obtained from metal complexes, focus entirely on inducing cell apoptosis and offer substantial improvements in pharmacological studies (73). This change was spectacularly motivated by the discovery of platinum-based antitumor drugs but various obstacles such as extreme adverse side effects, drug resistance, mutation aggregation and epimutations cause us to come up with alternative therapies. In their research paper Allardyce and Dyson explained that another platinum group metal, ruthenium, exhibits similar propitious biological properties (74) and is further able to establish strong chemical bonds through variable electronegativity thus rendering it capable to interact with a variety of biomolecules (75).
A lot of emerging chemotherapeutics utilize apoptosis as a mechanism to induce cellular death in tumor cells (76). To prevent apoptosis, a tumor may gain a variety of mutations or alterations. While, escaping from programmed cell death is a crucial feature of tumourigenesis, it does not appear to be a general response to all apoptotic stressors.
Arguably, some of the apoptotic mechanisms and pathways in tumours stay unchanged, making them ideal candidates for therapeutic targeting (77). Among other molecules, statins have been found to have apoptosis-inducing properties. The potency of statins as an anticancer therapy has been explored both in monotherapy and in combined regimens with commonly used chemotherapeutics (78). Several reports have also indicated that statins caused programmed cell death in a subset of tumor-derived cell lines in vitro, indicating that the analogous cancers might be susceptible to statin-speci c apoptosis in vivo (79,80,81). Thus, the present study, focuses on exploring the potential effects of the ruthenium uvastatin complex on in vitro and in vivo breast cancer models.
Synuclein-(SNCG), is a member of the synuclein family, that has been implicated in both neurodegenerative diseases and cancer (82). A collection of functional experiments has shown that SNCG's ectopic expression in human breast cancer cells facilitates proliferation and migration (38). SNGC inhibitors have been investigated extensively based on previous studies and offer signi cant probability as a future drug target (83). Inspired by this data, and extensively exploring this problem, we decided to take bene t of molecular docking analyses to evaluate and explore the novel complex's binding mechanism against SNCG as a target protein.
Our ndings showed that the unrestricted binding energy for the complex was low, thus promoting the binding direction of the compounds in the SNCG binding pocket circling the active site, resulting in enzyme inhibition. The complex has been identi ed as an inhibitory SNCG agent, and could be viewed as a potential ligand for breast cancer therapy. In fact, our research included the synthesis and characterization of the complex. We utilized different spectroscopic evaluations to evaluate uvastatin's antioxidant capacity pre and post complex formation. Results con rm that oxygen group (= O) and hydroxyl group is responsible for the chelation and formation of rutheniumuvastatin complex and that it is crystalline in nature. The analysis of antioxidant activity showed that the mechanism of free radical scavenging of uvastatin on resultant metal complexation is greatly enhanced. Ruthenium thus promotes the modi cation of uvastatins 's oxidative ability after complexation by enhancing the transfer of electrons from uvastatin and thus raising its redox potential. The complex 's reaction towards CT-DNA led to a reduction in the absorption spectra as compared to that of uncombined DNA, thus con rming that the complex bind through intercalation mode with CT-DNA.
The next research section was devoted to evaluating the impact of the ruthenium uvastatin complex on the cancer cell lines MCF-7 and MDA-MB-231. MTT assay showed that complex ruthenium uvastatin can minimize cell proliferation and induce apoptosis. One of most important purposes of anticancer therapies is the modulation of the cell cycle, speci cally the inhibition of phases G1 and G2 plays a key role in the cell cycle cascade (84). To determine the complex's mechanistic approach to the induction of apoptosis, ow cytometric experiments were employed that made use of Annexin-V and PI staining procedures. Furthermore, the results revealed that a higher percentage of early apoptotic events were identi ed on both MCF-7 and MDA-MB-231 cancer cells by ruthenium uvastatin treatment, resulting in the arrest of the G0 / G1 point, amounting to cellular death.
In addition, a cell-oriented reporter analysis was performed to identify the effects of complex treatment on the presence of PI3 K, Akt, mTOR, EGFR and VEGF associated signaling trails. The PI3K / Akt / mTOR pathway is a cell signaling cascade associated with growth modulation, proliferation, reproduction, motility, metabolism and immune response (85,86). The mammalian target of rapamycin (mTOR) strongly engages in various tumor progression processes by activating the signaling pathways PI3K / Akt (87). Alterations are found in nearly all human tumors, especially with breast cancer, of which up to 60% of tumors represent unique con gurations that activate this cascade (88). Dysregulation of this mechanism covers a wide variety of cancer symptoms including unchecked proliferation, genomic disturbance and metabolic recon guring in tumor cells (73,89). In fact, activation of the PI3 K / Akt / mTOR pathway is one of the major causes of current cancer chemotherapy resistance (90). It aims to make the PI3K / Akt / mTOR pathway a critical research target for understanding the development and progression of this disease, the importance of this pathway as a potential therapeutic approach along with the prognostic and diagnostic value of this pathway in patients with breast cancer is undeniable (91,92). Thus, our studies denote that ruthenium-uvastatin complex effectively, downregulates PI3K, Akt and mTOR in both MCF-7 and MDA-MB-231 cells.
Apart from these, the growth factor of the epidermis and its receptors (EGFR) in breast cancer are continuously often over-expressed (93). EGFR is a transmembrane tyrosine kinase receptor that regulates cell proliferation and epithelial cell viability via the PI3K / Akt / mTOR and protein kinase mitogen activated (MAPK) signaling cascade (94). The epidermal growth factor pathway serves as a primary mediator for breast cancer initiation and progression by encouraging the proliferation of cancer cells, their survival and promoting resistance to conventional therapy (95). In cancer chemotherapeutics, epidermal growth factor receptors and their ligands are thoroughly studied due to their mutation and over-expression in a large segment of primary breast carcinomas (96,97,98). Likewise, the growth regulation signaling cascade involving the vascular endothelial growth factor (VEGF), binds to the VEGF receptor 2 (VEGFR2) and activates tumor vasculature (99). Therefore, the inhibition of the signaling pathways EGFR and VEGF is also a promising technique for cancer chemotherapy (100). Our western blot ndings provide de nitive proof that the complex operates both in MCF-7 and MDA-MB-231 cells via the EGFR and VEGF pathways by down regulation of their signals.
Apoptosis is a crucial and essential method of ideally programmed cell death that involves eliminating dysfunctional cells in mammalian development, and retaining tissue homeostasis. Apoptotic activation has been considered an essential and effective cancer treatment approach (101). In our current research, DAPI staining approach used uorescence microscopy in MCF7 and MDA-MB231 cells to explore modi cation of nuclear morphology. Treatment with ruthenium uvastatin complex has shown condensed chromatin and scattered nuclei, which speci cally demonstrate apoptosis induction in these cells.
The global harmonized program for classifying and marking chemicals involves the reporting of a safe dose for a novel anti-cancer molecule (102). Therefore, an acute and subacute toxicological analysis was performed to determine the LD50 value and appropriate doses of the complex. The ndings of our in vivo work further indicate that ruthenium-uvastatin complex functions through the escalation of the proteins Caspase-3 and p53 and also downregulates the expresses PI3 K, Akt, mTOR, EGFR and VEGF. Recent research reportedly centered on the role of p53 in regulating the growth of cells induced by intense oncogenic signals or replicative stress (103). P53 Controls the function of large numbers of target genes related to cell cycle capture, DNA repair, senescence and apoptosis when activated (104). Contrary to other cells, cancer cells exhibit elevated mutation levels in the p53 gene. P53-dependent p21 cip1 upregulation induces misregulation of DNA replication and has been documented in active cancer cells (105).
Upregulation of p53 in tumors has been documented to cause senescence-induced tumor progression (106). In addition, the activation of the pro-survival and anti-apoptotic proteins causes the cancer cells to proliferate and survive. This mechanism facilitates tumor development and the progression of the disease. DNA damage modulates P53 based signals from a molecular point of view, which also contributes to pro-apoptotic stimuli (107,108). Proapoptotic proteins such as Bax disrupt the mitochondrial membranes and promote the release of cytochrome c and other pro-apoptotic stimuli through the use of anti-apoptotic proteins such as Bcl-2 and BclxL (109). Our western blot and immunohistochemical results revealed that expression p53, caspase-3 and Bax were up-regulated, while the role of Bcl-2 proteins was down-regulated, thus endorsing our hypothesis that the novel complex operates via the intrinsic apoptotic pathway Bax and Bcl-2 supported by p53.
Consequently, MMP-9, which belongs to a class of zinc-dependent endopeptidases, was down-regulated with ruthenium-uvastatin treatment. Some of the most frequently observed MMPs is MMP-9, which plays a signi cant role in breast cancer tumor colonization, metastasis and epithelial-to-mesenchymal transition (110). investigators studied the signatures of MMP-9 in healthy and cancer breast tissue with various molecular subtypes (111) which showed a marked increase in the expression of MMP-9 in cancer tissues relative to ordinary ones (112). Additionally, it was discovered that MMP-9 was differentially expressed within different molecular subtypes of breast cancer (111).
New ndings suggest that two common features of tumors are alteration of the redox equilibrium and abolition of redox signals that are closely correlated with malignancy and drug resistance (113). Therefore, it can be predicted that the up-regulation of SOD, GSH and CAT will contribute to a rise in H 2 O 2 levels in the mitochondria, which is a major signaling molecule and a 'reactive oxygen species' (114). Several experiments have shown that mitochondrial H 2 O 2 is a strong and e cient inducer of the apoptotic cycle (115). Treatment with ruthenium-uvastatin complex substantially improved the production of SOD, CAT and GSH in breast cancer, possibly through enabling ROS to induce apoptotic events.
Uncontrolled proliferation is a signature of tumors and can be analyzed using a variety of methods, including the counting of mitotic gures in stained tissue samples, the inclusion of labeled nucleotides in DNA and the cytometric ow measurement of the percentage in the S stage of a cell cycle (116). Dowsett determined that an immunohistochemical test of Ki-67 antigen was one of the most important approaches for quantifying proliferation (117). Ki-67 is predominant in all cancer cells and its role as a proliferation predictor is of great importance. The proliferation biomarker Ki-67 has also been regarded as a diagnostic biomarker for breast cancer in many studies (118,119). Our analysis shows that the carcinogen control animals displayed a rise in the number of cells labelled with Ki-67 by decreasing AI, suggesting cell proliferation in the breast tissue. At the other hand, after treatment with ruthenium-uvastatin complex, a decrease in cells labeled with Ki-67 and consequent rise in AI was observed.
In summary, the ruthenium-uvastatin complex is accountable for p53 interfering apoptosis in breast carcinoma, facilitated by the intrinsic apoptotic path provoked by Bcl2 and Bax and simultaneously regulating the PI3K / Akt / mTOR pathway in conjunction with MMP9 regulated invasive tumor pathways. Moreover, the complex reveals antiangiogenic functions by decreasing the EGFR and VEGF biomarkers as well. At the same time, the complex showed a high activity of its free radical scavenging potential in breast carcinoma cells caused by the release of reactive oxygen species extracted from mitochondrial through p53 regulation. The drop in Ki-67 coupled with stimulation of p53 further increases apoptosis attained by limiting cell proliferation. The observations offer ample proof that low doses of ruthenium-uvastatin chemotherapy that could interrupt, suspend or delay breast carcinoma development by observing that the biomarkers co-related with the inhibition of apoptotic processes in breast carcinoma.

Declarations Acknowledgements
The author's would like to thank Department of Oncology, Nanjing First Hospital Nanjing Medical University for their unending support.
Authors' contributions WL, JS, HX and XW conceptualized and designed the study. WL and JS aided in acquiring and analyzing data, drafted and critically revised the manuscript. WL, JS and HX participated in experiments and the data analysis. XW was involved in study design, analyzing and interpreting the data, and critically revised the manuscript. All authors read and approved the nal manuscript.

Funding
This work was supported by National Natural Science Foundation of China (81702619).

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study protocol was designed and approved by the Animal Ethics Committee of Nanjing Medical University & the Government's Regulatory Body (IACUS-1912129).

Consent for publication
Written informed consent for publication was obtained from all participants.