A newfangled coordinated ruthenium phloretin complex reprogramming breast cancer microenvironment interceded by modulation of PI3K/Akt/mTOR/VEGF pathway and modifying the antioxidant status correlated with intensied apoptotic events.

Background: Our recent investigation directed to synthesize and characterize a novel ruthenium– phloretin complex accompanied by the study of antioxidant in addition to DNA binding capabilities, and to determine the chemotherapeutic activity against breast carcinoma in vitro and in vivo approach. Methods: Ruthenium–phloretin complex was synthesized and characterized using various spectroscopic methods. The complex was further investigated to determine its ecacy in both MCF-7 and MDA-MB-231 human cancer cell lines and nally in an in vivo model of DMBA induced mammary carcinogenesis in rats Results: Our studies conrm that the chelation of the metal and ligand was materialize by the 3-OH and 9-OH functional groups of the ligand and the complex is found crystalline and was capable of intercalating with CT-DNA. The complex was capable of reducing cellular propagation and initiate apoptotic events in MCF-7 and MDA-MB-231 breast carcinoma cell lines. Additionally, ruthenium-phloretin complex could modulate p53 intervene apoptosis in the breast carcinoma, initiated by the intrinsic apoptotic trail facilitated by the Bcl2 and Bax and at the same time down regulating the PI3K/Akt/mTOR pathway coupled with MMP9 regulated tumor invasive pathways.


Background
Breast cancer is the most frequent of all carcinomas occurring in women, living in both developed and developing countries and estimated that worldwide over 6,26,679 women died in 2018 due to breast cancer (1). Breast cancer incidence in the Asian continent is still lower than that of Europe or America, but the participation of Asia to the global burden of breast cancer is increasing rapidly due to expressed economic growth and urbanization (2). Owing to their side effects and minimal effectiveness on tumors, current therapies have limitations leading to clinical resistance and therapy-related morbidity. Therefore, new medications are desperately needed to address emerging breast cancer cases including fewer side effects, effective tumor regression and decreased mortality.
The broad range of symmetries and coordinating numbers along with available redox states, thermodynamic/kinetic qualities as well as the variety of coordinating ligands, enables us to explore the unique characteristics of metal ions in the development of modern anti-cancer therapies (3;4). Cisplatin, is amongst the most widely used metal-based therapies for the treatment of malignant breast cancers, but these molecules typically have a variety of severe side effects owing to their absence of selectivity against normal and cancerous tissues (5). In addition to platinum-based chemotherapies, many endeavors have been dedicated to the development of ruthenium-based compounds, as these molecules recorded a smaller number of side effects owing to their alternative modes of action (6;7). Ruthenium complexes have been found to exhibit high cytotoxicity against platinum-resistant cancer cell lines in many cases, making them ideal targets for additional research (8;9). It should be noted that many complexes containing ruthenium such as RAPTA-C (10), NAMI-A, and KP1019 (11) have currently reached clinical trials for the management of various cancers.
Foods rich in bers and a variety of colorful fruits and vegetables are considered part of a healthy diet in general, and prevent the development or progression of breast cancer and possess anti-carcinogenic, antimetastatic and immuno-modulatory activities, making them prospective contenders in cancer prevention and treatment (12). The role of avonoids as antioxidants is already well-established and avonoids interact directly with proteins, making them perfect small molecules in the regulation of enzymes, transcription factors and receptors (13). Amongst several polyphenols, phloretin [2′,4′,6′-Trihydroxy-3-(4hydroxyphenyl)-propiophenone] is present in apple and in other plants, including Pieris japonica (14).
Phloretin was capable of preventing the neoplastic progression of BALB/3T3 cells (15). Phloretin also has an inhibitory role on dextran sulfate sodium-induced ulcerative colitis in mice by altering nuclear factor-κB, toll-like receptor 4 and PPARγ pathways (16). In addition, phloretin possesses diverse pharmaco-therapeutic effects on human malignancies, including the suppression of oral squamous cell carcinoma by modulating the glucose uptake (17), apoptosis in human gastric cancer by arresting the cell cycle of G2/M phase, suppression of cell invasion by diminution of JNK activity (18), attenuation of triple-negative breast cancer cell proliferation and migration (19) and promote apoptosis in human esophageal cancer (20). A very recent literature suggested that phloretin loaded chitosan nanoparticles augments the mitochondrial mediated intrinsic apoptosis in human oral cancer cells (21). Substantial evidence states that development of cancer manifested unregulated proliferation along with impaired apoptosis (22; 23) where Bcl2 and the tumor suppressor like p53 genes are thoroughly researched (24;25;26). Bax and Bcl-2 are the main pro-apoptotic regulators that can further enhance the PI3K / AKT related pathway and other cell survival and death associated trail (27). By regulating the matrix metalloproteinases (MMPs), natural agents can also in uence invasion and metastasis of cancer cells. Among them, MMP-9 is of special concern as it has been reported that patients with enhanced MMP-9 expression will end up with weaker prognosis (28). Furthermore, the breast cancer progression involves mutations of p53, VEGF (modulated by angiogenic pathway), mTOR related signaling cascade, pro-apoptotic protein like Bax, and enhanced activation of Bcl2 like anti-apoptotic protein and proliferating cell nuclear antigen (PCNA) (26;29;30;31).
In addition, the current work utilizes molecular docking, a commonly employed bioinformatics tool, (32;33) especially applied herein to determine how the ruthenium-phloretin complex binds and interacts with the target proteins Bcl-2. Autodoc is one such software consisting of a set of automated docking tools and designed to determine how molecules bind to a speci c targeted protein with a pre-determined structure (34).
To the best of our understanding, chemotherapeutic behavior in the model for breast cancer and the fundamental mechanistic approach of the ruthenium-phloretin complex has not yet been thoroughly studied. The present research aims at synthesizing, characterizing the complex of ruthenium-phloretin together with the investigation of antioxidant status, along with DNA binding properties and further examining the chemotherapeutic action against mammary cancer in both in vitro and in vivo experiments.

Pharmacophore Analysis
Target protein selection The apoptotic signaling protein BCL2 (UID: P10415) sequence was recuperate from UniProt database (UID) and protein sequence used to predict sequence similarity and sequence templates by PSI-BLAST.
Ligand structure design and pharmacophore analysis Chemical structures were designed the using ACD/ChemSketch software to add all chemical compositions and the nal output was saved in MOL2 format.
Molecular Docking AutoDock 4.6 software was used to predict the protein-ligand interaction using various parameters such as preparation of protein properties, addition of Gaussian charges, addition of hydrogen atoms with polar region of amino acids, preparation of ligand molecule with interaction of bonds with rotatable angles etc.
The lowest energy conformations were regarded as the binding conformations between the ligands and the proteins.

Synthesis and Characterizations of ruthenium-phloretin complex
Detailed synthesis and characterization of the complex is described in the supplementary section.
Evaluation of antioxidant status of ruthenium phloretin complex by DPPH, FRAP, ABTS methods

DPPH assay
The complex ability to scavenge DPPH radicals was analysed using the method described by Dolatabadi (35). (A c ) denotes blank and (As) denotes absorbance at every 5 minutes.

FRAP assay
The test was performed using Benzie and Strain's protocol (36).
ABTS assay ABTS radical scavenging activity of ruthenium phloretin complex was analysed using the procedure as described by Pennycooke and colleagues (37).
Binding mode of DNA with ruthenium-phloretin complex Intercalation of CT-DNA with the compound was determined by using UV-Visible spectrophotometer (UV-1800 Shimadzu), based on method reported by Dehghan (38). The binding constant was computed as: DNA represents the number of base pairing of DNA, ε a represents the extinction coe cient (A obs /Ru) factor, ε f is extinction coe cient of free drug and ε b is extinction coe cient of bound drug 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 MCF-7and MDA-MB-231 breast cancer cell lines were purchased from NCCS, Pune and maintained under appropriate conditions.

Cell viability assay
The MCF-7 and MDA-MB-231 cells were plated for 24 hours in a 5% CO 2 humidi ed incubator and treated with various concentrations of ruthenium phloretin complex for 24 hours in 96 well plate. Upon treatment the medium was withdrawn and MTT solution was applied to each well and incubated for 3 hours. The optical density of solubilized crystals was estimated at 560 nm. % viability = 100-% of cytotoxicity DAPI staining apoptotic cells The cells were examined for nuclear blebbing and condensation of chromatin by dyeing them with DAPI using the process established by Li (39).

Clonogenic assay
Trypsinization of the cells were performed to produce a single cell suspension and were seeded in six well plate at a density of 500 cells /well. After 24 hours, culture was replaced with fresh media containing three different concentrations of complex with 2% FBS and incubated for two weeks then stained with 0.5% crystal violet in 25% (v/v) methanol. The colonies were visually counted to contain > 50 cells / colony. Clonogenicity = (Cloning number/500x) x100.
Cell cycle analysis and measurement of apoptotic assay by ow cytometry MCF-7 and MDA-MB-231 cells were suspended and their nuclear DNA was marked with propidium iodide (PI). The distribution of nuclear DNA in the cell cycle process was conducted on FACS ( uorescenceactivated shorter cell). A total of 10,000 events were acquired and ow-cytometric data processing was conducted using Mod t tools using the tool originated by Li (39).
Detection of Caspase-3 protein expression by ow cytometry MCF-7 and MDA-MB-231cells (5×10 5 cells/well) were cultured on six well plate and subsequently treated to three concentrations of ruthenium phloretin complex for 24 hours. Cell were incubated further by rabbit anti active caspase 3 polyclonal antibody (351-68655X, BD Pharmingen) for thirty minutes and analyzed by ow cytometry (BD Accuri C6 Plus ow cytometer).
Detection of Akt, mTOR, p13K, VEGF, pro Caspase-3 and Active Caspase-3 protein expressions by western blot The cultured cells were treated with three separate doses of ruthenium-phloretin complex for 24 hours and cultured for 6 hrs. Cell lysates were processed and comparable protein quantities were analyzed by SDS-PAGE electrophoresis, accompanied by shifting to a PVDF (polyvinylidene di uoride) membrane.
The membranes were incubated with the primary antibodies followed by the secondary antibody labeled with horseradish peroxidase for 1hr (40).

In-vivo experimentation
Animal husbandry and maintenance Sprague Dawley rat (120-125 gm) and 28 days old virgin female Sprague-Dawley rats (80-100 grams) were acquired from IICB, Kolkata, India. The whole animal testing procedure was carried out in compliance with the endorsement of the Institutional Animal Ethics Committee & the Government's Regulatory Body (Regd No. 1458/PO/a/11/CPCSEA).

Toxicological investigation
Details of the toxicological investigation of the complex is described in the supplementary section.

In-vivo experiments Experimental Protocol
During acclimatization, the animals were grouped into seven groups and once the rats were 50 days old, DMBA was given as a single tail vein shot to animals of group II to VII at a dosage of 0.5 mg per 100 g body weight in an oil emulsion. Group I normal untreated controls. Group II carcinogen (DMBA) treated animals. Group III-V carcinogen induced animals accompanied by 50 mg/kg, 100 mg/kg and 200 mg/kg treatment with the Ru-phloretin complex. Group VI carcinogen induced animals with 100 mg/kg ruthenium treatment. Group VII carcinogen induced animals with 100 mg/kg phloretin treatment. After 16 weeks, the animals were sacri ced followed by midline incision from the pubis to the sub-maxillary Histopathology of mammary tissue Breast tissue was xed in 10% neutral buffered formalin, coated in para n, cut in 5μm thickness, and mounted on slides. For histopathological investigations the tissue segments were treated with hematoxylin and eosin (H&E).

Antioxidant status of mammary tissues
Collected mammary tissues were compressed and homogenized (10% w / v) in 0.1 M phosphate buffer (pH 7.0). The homogenized mixture was centrifuged for 10 minutes to evaluate the antioxidant activity. The procedure of Jagatheesh was employed to obtain the activity of the supernatant (41). Catalase activity was carried out in the process described by Sinha and his colleagues (42). The activity of superoxide dismutase was conducted by the method of Awasthi (43). GPx activity was assessed using the Rotruck method (44).

Immunohistochemical analysis of mammary tissue
Para n embedded mammary tissue segments were depara nized accompanied by submersion in H 2 O 2 .
The segments were covered with goat serum for 1 hour, followed by exposure to anti-mouse p53, Bcl2-, Bax-and MMP-9 antibodies (1:50 ratio) and kept overnight following labelling with the HRP-conjugated secondary antibody streptavidin biotin for 30 min. DAB were used to stain the segments and counterstained with hematoxylin.
Cell proliferating assay Tissue samples processed as above were covered with goat serum for 1 hour, followed by exposure to anti-mouse Ki-67 antibody at 4°C overnight. Tissues were stained with DAB and counter stained with hematoxylin (26).
Apoptotic assay by TUNEL method Tissue samples processed as above were treated with proteinase K (20 μg/ml in PBS) for 15 minutes then treated with the terminal deoxynucleotidyl transferase (TdT) buffer accompanied by TdT and dUTP at 37°C for 90 min. After washing the tissue were stained with DAB and counter stained with hematoxylin (26).

Assessment of labelling and apoptotic index
The labelling index (LI) was assessed as the percentage of Ki-67-positive nuclei /total number of cells counted. The apoptotic index (AI) was determined by calculating the percentage of TUNEL-positive cells / total number of cells.

Statistical Analysis
The results were de ned as mean ± standard error mean (SEM). Statistical evaluation was conducted by t-test and one-way variance analysis (ANOVA) discrepancy was found to be statistically relevant when P<0.05 was used.

Results
Pharmacophore Analysis The docking results shows that Bcl-2 protein is strongly binds with ruthenium-phloretin within active site amino acids of both polar and electrostatic interactions within target amino acids of Tyr9, Asn182, Arg183, Thr187 and Gln190, with strong hydrogen bonding of -9.52 kcal/mol of energy. (Table 1 and Supplementary Figure 1B  In vitro antioxidant activity of ruthenium phloretin complex Ruthenium phloretin complex scavenges DPPH, FRAP and ABTS radicals It has been ascertained that the absorbance of ABTS at 734 nm declined dramatically by uctuating the concentrations complex in the solution (Figure 1 A). Compared to free phloretin the complex was capable of scavenging free radical quite successfully in the presence of ABTS, which might be directly linked to the hydroxyl functional group and their ability to donate hydrogen atoms. Figure 1(B) demonstrated the power of phloretin and ruthenium-phloretin complex in scavenging of radicals, where the chart implied that phloretin scrounged free radical to almost 43 per cent, while the complex was able to scrounge to about 79 percent of free radicals.
Reduction in Fe+3-TPTZ absorption spectrum was ascertained at 593 nm with the interaction of phloretin and ruthenium-phloretin complex. Figure 1(C) reinforces that the ruthenium phloretin has an antioxidant capacity greater than just phloretin.     Figure 4B). Complex treatment groups of 50 and 100 mg / kg showed slight hyperplasia of serous and mucous glands (Figure 4 C&D), whereas the highest dose group displayed perfect histological pro le with no signs of hyperplasia or cell proliferation in breast tissue, and standard architecture of the cells that line the ducts. (Figure 4 E). Phloretin-treated group showed standard histopathologic pattern of rat breast tissue ( Figure 4G) while ruthenium-treated DMBA group represented atrophy of glands with encompassing fatty tissue (ag) and necrosis of serous glands (asg) ( Figure 4F).

Antioxidant activity
The homogenized mammary tissue of the DMBA-treated rats was found with a decline in SOD, CAT and diminished glutathione levels. The animals treated with 200 mg / kg of the complex showed a prominent increase in the levels of SOD, CAT and glutathione in the homogenized mammary tissues as opposed to carcinogen control and other groups (Figure 4 H).

Immunohistochemical analysis
The presence of cellular biomarkers were assessed by the immunohistochemical staining techniques ( Figure 5) ( Table 2). It was observed that DMBA administration increased the levels of Bcl-2 [ Figure 5

Suppression of Ki-67 by Ruthenium-phloretin complex
The e cacy of the ruthenium-phloretin compound in the proliferation of mammary tissues is designate in Figure 6 (i) ( Table 3)  Ruthenium-phloretin complex promotes apoptosis TUNNEL activity was assayed to nd out the e cacy of ruthenium-phloretin therapy on cellular death in breast cancer [ Figure 6 Table 3. Animals taking 200 mg / kg of the complex, represented a considerable rise in cell death, as compared against the carcinogen control group. The interpretation R is that it shows the proportion of cell death to tumor growth.

Discussion
Recent clinical trials in phase II have shown that the anticancer effects of phytochemicals or whole plant foods (45;46;47) are manifested by low toxicity and can deliver a pragmatic therapeutic solution for cancer victims or high-risk individuals (48). In addition, avonoids interact with transition metals creating complexes which shows greater oxidation potential with anticarcinogenic activity and has attracted increasing attention in recent years (49). In the past decades, ruthenium therapeutics have been used successfully in clinical research exhibited their profound anticancer activities (50). The notion of ruthenium-based cancer treatments is powered by the possibilities that several ruthenium compounds preferably aggregate in tumor tissue as well as have extended coordination sites contrasted to platinum (II), and a majority of ruthenium complexes exhibit redox action in the biological system (51). Our current study encompasses the synthesis of a novel ruthenium-phloretin complex and evaluating its anticarcinogenic property against breast cancer in vivo and in vitro. Studies reveal that approximately 75% of primary breast cancer expresses elevated levels of Bcl-2 and considered to be involved a crucial role in the regulation of apoptotic events and cellular proliferation in breast cancer cells (52), thus it may be a promising candidate for anticancer therapy. Several small molecular inhibitors of Bcl-2, including ABT-737 and ABT-199, have been investigated extensively and offer potential as a prospective drug target (53;54). Prompted by this evidence and systemically address this issue, we took advantage of molecular docking studies to investigate the mode of interaction and to explore the binding mechanism of the novel complex against a target protein Bcl-2. Our results denoted that the free binding energy for the complex was small thus favoring the binding orientation of compounds in the Bcl-2 binding pocket surrounding the active site, which resulted in inhibition of enzyme activity. The complex was observed as an inhibitory candidate of Bcl-2, which may be considered as a potential ligand for treatment of breast cancer.
Moreover, our study included synthesis and characterization of the complex. Result con rmed that the chelation materializes by the 3-OH and 9-OH functional groups of the ligand and the complex is found crystalline. The study of antioxidant activity revealed that the property of free radical scavenging of phloretin is considerably intensi ed on subsequent complexation with metal. Therefore, ruthenium facilitates to modify the oxidative capacity of phloretin following complexation by increasing the shifting of electrons from phloretin and hence escalating its redox potential. The reaction of the complex through CT-DNA ensued in a decline in spectrum of absorption of uncombined DNA, evidence of the phloretin complex intercalated with CT-DNA.
MTT analysis found that ruthenium phloretin complex could minimize cell proliferation and activate apoptosis. An explanation of the complex's mechanism involved in apoptosis initiation was achieved by ow cytometry experiments that introduced Annexin-V and PI staining. Research ndings found that a signi cant percentage of early apoptotic events on both MCF-7 and MDA-MB-231 cancer cells are labelled with annexin V PI following ruthenium phloretin therapy, which leads to cell death by restricting the G0 / G1 stage (Figure 3 I & J).
A cell signaling cascade concerned with regulation of growth, proliferation, survival, motility, metabolism and immune response is the PI3K/Akt/mTOR pathway (55;56). The mammalian target of rapamycin (mTOR) is actively involved in tumor progression by triggering the PI3K/ Akt signaling trails (57). Variations to this pathway are observed in virtually all human tumors, along with breast cancer (58).
Misregulation of this pathway is associated with unregulated proliferation, genomic instability and metabolic reprogramming in tumor cells (59;60). Additionally, activation of the PI3K / Akt / mTOR pathway is among the leading causes of resistance to current cancer chemotherapy (61) and the contribution of this pathway as a prospective therapeutic strategy and the prognostic and diagnostic value in breast cancer patients (62;63). Prompted this evidence our studies in the manuscript denoted that ruthenium-phloretin complex effectively, downregulates PI3K, Akt and mTOR in both MCF-7 and MDA-MB-231 cells.
Besides these, the growth factor of the epidermis and its receptors (EGFR) belongs to a transmembrane tyrosine kinase receptor which modulates cell proliferation and epithelial cell survival through PI3K / Akt / mTOR and (MAPK) pathways (64) and constantly over expressed in breast cancer (65), has been extensively explored in cancer chemotherapeutics (66;67;68). Therefore, targeting the EGFR and VEGF signaling pathways is a recognized strategy for treating cancer (69). Our western blot results provide conclusive evidence that the complex acts on both EGFR and VEGF by down regulation their expressions in both MCF-7 and MDA-MB-231 cells.
Apoptotic stimulation has been considered to be a standard and best strategy in cancer therapeutics (70). In our current study, alteration of nuclear morphology was investigated by DAPI staining method using uorescence microscopy and treatment with ruthenium phloretin complex clearly depicted the induction of apoptosis in these cells.
Newfound interest has lately been focused on the role of p53 in controlling cell growth (71) which triggered and regulates the target genes associated with cell cycle arrest, DNA repair, senescence and apoptosis (72). Reactivation of p53 in tumors has been reported to provoke tumor regression triggered by senescence induction (73). At molecular standpoint, DNA damage modulate P53 related signals which further leads to pro-apoptotic signals (74;75). Pro-apoptotic proteins including Bax damage the mitochondrial membranes and promote the secretion of cytochrome c and other pro-apoptotic factors, while anti-apoptotic proteins such as Bcl-2 and BclxL interfere with this action (76). Our western blot and immunohistochemical results indicated that there was an up-regulation of the activity p53, caspase-3 and Bax, while down regulating the activity of Bcl-2 proteins, thus con rming our hypothesis that the novel complex acts via the p53 induced intrinsic apoptotic pathway regulated by Bax and Bcl-2.
Research revealed that a substantial increase in MMP-9 expression in cancerous tissues (77), occupies a crucial role in tumor invasion, metastasis and epithelial-to-mesenchymal transformation in breast cancer (78). Our studies denoted that ruthenium phloretin complex can effectively downregulate the MMP-9 expression and thus halt the breast cancer progression.
Numerous studies tend to show redox equilibrium and redox signaling are directly associated with breast cancer and drug resistance (79). It can therefore be predicted that the up-regulation of SOD, GSH and CAT will lead to an increase in H 2 O 2 levels in the mitochondria (80). Already many researchers have discovered that mitochondrial H 2 O 2 is an appropriate and e cient stimulator of the apoptotic process (81). Ruthenium-phloretin complex therapy markedly increased the signals of SOD , CAT, and GSH in breast cancer, presumably by inducing ROS to induce cell death.
Unregulated proliferation is a hallmark feature of carcinoma and studies revealed that quanti cation of proliferation by immunohistochemical evaluation of Ki-67 antigen, often considered a prognostic factor for breast cancer and has been investigated in several studies (82; 83; 84). Our study reveals that, the carcinogen control animals exhibited an upsurge in the number of cells labeled with Ki-67 and subsequently, a decrease of cells labeled with Ki-67 and consequent increment of AI was observed post the treatment with ruthenium-phloretin complex.

Conclusion
In a nutshell, the ruthenium phloretin complex is remit for the p53 mediated apoptosis in the breast cancer, activated by the apoptotic events expedited by the Bcl2, Bax signaling and down-regulating the PI3/Akt/mTOR pathway coupled with MMP9 regulated breast cancer signaling cascade. Furthermore, the complex halted the angiogenic process by down-regulating the EGFR and VEGF signaling. Besides, the complex successfully established the enormous capabilities on the free radical scavenging activities of breast carcinoma cells and the attenuation of Ki-67 signaling which further intensify the fact that apoptosis was achieved by reducing the cellular proliferation of cancerous cells. The experimental ndings provide conclusive evidence that ruthenium phloretin chemotherapy could halt, abrogate or delay the progression of breast carcinoma by interacting with different biomarkers corresponded with altering apoptotic events along with the interrupting angiogenesis process, therefore performing the function of potential contender in future cancer chemotherapeutics.

Declarations
Author Contributions CH and GY conceived the study and participated in its design and co-ordination. AKM performed cell culture and cellular assays. SR and TC carried out animal experiments. All authors read and approve the nal manuscript.

Funding
This work was funded by the National Natural Science Foundation of China (81560461) and was supported by medical high-level talent training plan and thousands of young and middle-aged backbone teachers cultivation plan of Guangxi province.

Availability of data and materials
All relevant data are within this published paper.

Ethics approval and consent to participate
The whole animal testing procedure was carried out in compliance with the endorsement of the Institutional Animal Ethics Committee & the Government's Regulatory Body (Regd No. 1458/PO/a/11/CPCSEA).

Consent for publication
The publication was read and approved by all the authors participating in the study. The views, opinions, and ndings contained in this report are those of the authors and do not re ect o cial policy of any institute or funding agency.       The molecular mechanistic pathway of Ruthenium-phloretin complex in mammary cancer.

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