
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00234-1
10.1016/j.tranon.2024.102107
102107
Original Research
Unraveling the chemotherapeutic potential of taxifolin ruthenium-p-cymene complex in breast carcinoma: Insights into AhR signaling pathway in vitro and in vivo
Das Abhijit
Bhattacharya Barshana
Gayen Sakuntala
Roy Souvik souvik.roy@nshm.com
⁎
Department of Pharmacy, NSHM Knowledge Campus- Kolkata, 124 BL. Saha Road, Kolkata, West Bengal 700053, India
⁎ Corresponding author. souvik.roy@nshm.com
23 8 2024
11 2024
23 8 2024
49 1021073 9 2023
21 1 2024
18 8 2024
© 2024 Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• Chemotherapeutic evaluation of complex by in vitro and in vivo study.

• Inhibition of AhR signalling pathway.

• Inhibition of EMT mediated metastasis by the complex.

• Induction of apoptosis and reduction of cell proliferation by the complex.

Background

Mammary carcinoma is the most frequently diagnosed form of carcinoma in women worldwide. The organometallic compounds showed a prospective anticancer activity. This research explored the anticancer efficacy of taxifolin ruthenium-p-cymene counter to breast cancer.

Methods

The anticancer efficacy of the novel organometallic compound was investigated via various in vitro and in vivo techniques using breast cancer cell lines and breast cancer model of rat.

Results

Target proteins were identified via pharmacophore analysis, which revealed a high binding affinity towards AhR, EGFR, and β-catenin. The compound induced apoptotic events and prevented cancer cell colony formation. Furthermore, decreased expression of AhR, EGFR, and N-cadherin inhibited cancer cell growth, migration, and proliferation. The compound provoked the cell cycle arrest at sub G0/G1 phase, S phase and G2/M phase and inaugurated the caspase-3 dependent apoptotic events. The in-vivo experimentation displayed the fruitful restoration of breast tissue since the complex treatment in DMBA persuaded breast carcinoma in rat. Moreover, the upstream of p53 and caspase-3 expression along with substantially downstream of vimentin, β-catenin, m-TOR and Akt expression.

Conclusions

In conclusion, the compound repressed the cancerous cellular viability, migration, and EMT via modulating the AhR/EGFR/ PI3K transduction pathway and the expression of EMT biomarkers such as N-cadherin, E-cadherin, thus eventually revoked the EMT facilitated metastasis of malignant cells.

Graphical abstract

Image, graphical abstract

Keywords

Ruthenium-p-cymene
Taxifolin
Epithelial-to-mesenchymal transition
Breast carcinoma
AhR pathway
==== Body
pmcIntroduction

Mammary carcinoma is a particularly common type of malignancy in women and the leading cause of cancer-related deaths worldwide. In 2020, 2,261,419 new instances of mammary carcinoma and 684,996 fatalities have been reported worldwide [1]. As a consequence, novel treatment strategies for the management of mammary carcinoma are clearly required.

Breast carcinoma is brought about by unrestrained proliferation of breast cells, which predominantly arise from the inner lining of the lobules, as a result of abnormalities in the genes which govern mammary cell proliferation [2]. As per earlier findings, numerous proteins have been identified that regulate breast carcinoma development, including aryl-hydrocarbon receptor (AhR), a ligand mediated transcription factor. The expression of AhR was found to be elevated in mammary carcinoma cells in association with the healthy mammary cell [3] and it is primarily involved in xenobiotic metabolism by activating multiple AhR sensitive genes [4,5], thus forming DNA adducts which transforms a normal cell into cancerous one [6]. In addition, AhR also regulates the cellular processes like cellular proliferation, migration, and adhesion by triggering Wnt/β‐catenin or MAPK through non‐genomic pathway [7].

In the process of cancer progression and migration epithelial-to-mesenchymal transition (EMT) portrays a pivotal part. It is a biological process that happens throughout healthy embryonic development as well as during the regeneration of tissues, the fibrosis of organs, and the healing of wounds. Epithelial cells have the ability to change into mesenchymal phenotypes through an extremely fluid procedure. However, it is also involved in the cancerous cell proliferation with stem cell characteristics which is important for the resistance in cancer therapies [8]. In this instance, Wnt/β-catenin transduction pathway can play pleiotropic characteristic from tumor commencement to metastasis and recurrence, through provoking the epithelial to mesenchymal transition [9] and it has also been identified that β-catenin certainly modulates the AhR mediated signal by stimulation of AhR expression and augmentation of AhR-dependent gene induction [10]. Additionally, abnormal buildup of β-catenin in cancers is frequently linked to mutational deactivation of the p53 tumor suppressor that prevents cancer cells from dying naturally through apoptosis [11]. Disruption of apoptotic regulation extends the cancerous cellular survival which facilitates the accumulation of mutations, thus may participate in angiogenesis, cellular propagation, enhance invasiveness and interrupt differentiation during the cancer propagation [12]. Several investigations were suggested that AhR has negative impact on apoptosis. Increased AhR expression has anti-apoptotic effect which has been investigated on various cancer cells [13].

It has been also identified that Wnt pathway promotes cell homeostasis, cell proliferation, and cell survival by attenuating apoptosis [14]. Additionally, there is a linkage among the Wnt/β-catenin and EGFR signaling in malignancy, indicating that Wnt ligands may stimulate EGFR expression whereas EGFR may trigger β-catenin through the tyrosine kinase receptor-PI3K/Akt signaling pathway. It was also demonstrated that EGFR forms a complex with β-catenin, which promotes cancer cell invasion and metastasis [15].

Recently an active flavonoid, taxifolin (3,5,7,3′, 4′-pentahydroxyflavanone or dihydroquercetin) has gained the attention due to its diverse pharmacological activities such as antioxidant, anti-inflammatory, hepatoprotective, anti-diabetic, cardioprotective, neuroprotective, anti-alzheimer's and antiangiogenic efficacy, also possessing antitumor properties [16]. In today's world, natural compounds are already functionalized with metals to create organometallic complexes with considerable chemotherapeutic efficiency to increase therapeutic outcomes. In our previous study the complexation of ruthenium-p-cymene with taxifolin significantly increased the antioxidant activity of taxifolin. In addition, a strong anticancer efficacy was noted against lung cancer when treated with the complex which significantly reduces the cancer progression via the interruption with EMT and cancer stemness associated signaling pathway [17].

In this current research work, the anticancer activity of taxifolin ruthenium-p-cymene complex on breast cancer has been explored. We have hypothesized that the inhibition of AhR signaling may elevate the intrinsic apoptosis through upregulation of p53 expression along with the downregulation of anti-apoptotic markers including EGFR and PI3K via inhibiting Wnt/β-catenin pathway which prevent the cancer progression and metastasis.

Materials and methods

Chemicals, reagents and antibodies

AhR (Cat. No GTX637885), E-cadherin (Cat. No GTX100443), N-cadherin (Cat. No GTX127345), vimentin (Cat. No GTX100619), β-catenin (Cat. No GTX101435), p53 (Cat. No GTX70214), Akt (Cat. No GTX121937), mTOR (Cat. No GTX101557), caspase 3 (Cat. No GTX110543), EGFR (Cat. No GTX121919) have been purchased by GeneTex, Inc (Irvine, CA, USA). DMBA (Product No 185582), MTT (Product No 475989), Annexin V (Product No A9460) and propidium iodide (PI) (Product No 537059) (purchased by Sigma-Aldrich Chemical Co). The additional supplementary chemicals have been found through the commercial retail providers which is high purity analytical grade.

Pharmacophore study

The pharmacophore analysis methods have been displayed in Supplementary File 1.

In-vitro studies

Cell culture

The mammary carcinoma cell lines MCF-7 and MDA-MB-231 have been found by the National Centre for Cell Science (NCCS), University of Pune. The cells have been seeded into DMEM high glucose media along with 10 % foetal Bovine Serum (FBS) and 1 % antibiotic-antimycotic solution. Then incubated in 18–20 % O2, 5 % CO2, 95 % relative humidity at 37 °C in CO2 incubator and have been sub-cultured on every two days.

Cellular viability assessment

Cellular viability analysis has been executed via MTT (3-(4, 5 dimethylthiozol-2-yl)−2, 5- diphenyltetrazolium bromide) analysis. Several concentrations of compound been applied to the cells (25, 50, 100, 200, 400 µg/ml) and cellular viability has beenestimated at 48 h. The cellular viability has been considered via the metabolism of MTT to form formazan crystals by mitochondrial succinate dehydrogenase.% of the cellular viability been quantified as:

% Cellular viability= [Mean absorbance of treated cells/Mean absorbance of untreated cells] × 100

The IC50 (Half-maximal inhibitory concentration) value has been measured thru the linear regression equation i.e., y= mx+ C, where, y = 50, m and C values has been achieved by the cellular viability graph.

Olegonucleosomal fragmentation assessment

The cells were stained with fluorescent nuclear dye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) which allowed researchers to see how the compound affected the cancer cells. The stained cells been visualized through ZEISS LSM 880 Fluorescence live cell imaging system (Confocal Microscopy) and filter cube together with Excitation and Emission wavelength 358 nm and 461 nm respectively. The pictures have been examined through ZEN Blue Software and recorded.

Clonogenic analysis

The complex activity on cancerous cellular propagation as well as colony formation has been assessed by using a clonogenic test followed by incubation at 48 h. The colonies of the malignant cells have been marked by using 0.5 % crystal violet solution and discriminate the colonies by visualization. Then the following equation has been used to quantify the clonogenicity:

Clonogenicity = (Cloning number/500x) ×100

This experimentation has been executed in triplicate ways.

Transwell migration assessment

The malignant cellular activity on the MCF-7 and MDA-MB-231 cells been evaluated through the transwell migration analysis. The cancerous cells have been cultured (5 × 104 cells/well) in superior compartment of the transwell and supplemented the 0.1 % foetal bovine serum (FBS) in DMEM growth media. The inferior compartment has been occupied by DMEM medium, supplemented thru 10 % foetal bovine serum (FBS). The cancerous cells in both compartments of transwell have been introduced with the various concentrations of the compound. The cells in the inferior compartment been fixed with 4 % paraformaldehyde as well as stained thru 0.25 % crystal violet after 48 h of incubation. Then the transwells have been analyzed with four random fields and the numbers of invaded cells been quantified via bright field microscope at 40X magnification.

Flow cytometry

On the MCF-7 and MDA-MB-231 cell lines, the apoptotic assay as well as cell cycle distribution assessment was carried out since the compound treatment by flow cytometry. The DNA has been labeled by Annexin-V conjugated-FITC and propidium iodide (PI). This study has been completed through BD FACSCalibur analyser and data been examined via BD Cell Quest Pro Software (Version: 6.0).

Determination of caspase-3 proliferation

The caspase-3 proliferation on MCF-7 and MDA-MB-231 cell lines been analyzed via flow cytometry. IC25, IC50, and IC75 concentrations of compound have been given to the cells for 48 h after they had been seeded in a 6-well plate. The anti-rabbit caspase-3 polyclonal antibody was then used to treat the cells in a dark condition at 37 °C. Using flow cytometry, the percentage of the cells marked with the caspase-3 antibody been quantified.

Western blot

The AhR, N-cadherin, E-cadherin and EGFR expression on the MCF-7 and MDA-MB-231 cell lines have been determined by western blotting analysis. The cancerous cell lines been incubated in various concentrations (IC25, IC50, and IC75) of compound for 48 h. The cancerous cell lysate obtained been fixed in SDS-PAGE (8–12 %) electrophoresis which was transferred to PVDF membrane. The PVDF membrane been incubated overnight thru anti-rabbit AhR, N-cadherin, E-cadherin and EGFR antibodies (1:500 dilution). After that, this membrane has been incubated by goat anti-rabbit secondary antibody that were HRP-conjugated, and chemiluminescent (ECL) kit been used to analyze the expression, where β-actin serving as the loading control.

In-vivo assessment

Animals

The in-vivo experimentations have been conducted according to the Institutional Animal Ethical Committee and the Animal Regulatory Body of the Government (1458/PO/E/S/11/CPCSEA dated 12.05.2011). The entire techniques accomplished during the investigation were complied as per the ethical guidelines which have been mentioned in institutional ethical protocol.

The experimental animals been accomplished by the registered animal breeder (Regd.no. −1458/PO/E/S/11/CPCSEA Dated- 12.05.2011). For toxicological assessment, 18–20 gm, 6 to 8 weeks old Swiss albino mice of both sexes have been taken. The carcinogenicity assessment been executed on eight-week-old female Wistar rats (70–120 gm). The animals been kept in polypropylene cages at 20 to 25 °C temperature and 20–25 % relative humidity in 12 h light/dark sequence. The experimental animals have been provided the sufficient food and water for one week of acclimatization period.

Experimentation procedure

The experimental animals have been dispersed arbitrarily after the acclimatization period into 7 groups where in each group contained 6 experimental animals. 7–8 weeks old of Wistar rats (exception with group I) has been given 7,12-dimethylbenz[a]anthracene (DMBA) (0.5 mg/100 g body weight) in corn oil via tail vein single i.v injection and the mammary carcinoma were developed. Subsequently, the chemical carcinogen inducible mammary carcinoma of Wistar rats been introduced with treatment by taxifolin ruthenium-p-cymene via oral gavages that was sustained for twenty-four weeks. All experimental animal sets been divided as:

Group I- vehicle control animal group, Group II- introduced the chemical carcinogen DMBA which was designated as carcinogen control, Group III- DMBA induced carcinoma + administered with 50 mg kg-1 complex, Group IV- DMBA persuaded carcinoma + administered with 100 mg kg-1 compound, Group V- DMBA persuaded carcinoma + administered with 200 mg kg-1 compound, Group VI- DMBA persuaded carcinoma + administered with 50 mg kg-1 body weight taxifolin, Group VII- DMBA persuaded carcinoma + administered with 50 mg kg-1 body weight ruthenium-p-cymene.

At the end of the chemotherapeutic analysis, the mammary tissues have been isolated from the euthanized (using Pentobarbital sodium) experimental animals and preserved within 10 % formalin solution for fixation. Mammary tissues have been evaluated for demonstrating cancer architecture. Breast tissues have been assessed through histological study, immunohistochemical analysis and cellular proliferation assay method.

Histopathology of breast

The thoracic and abdominal inguinal mammary tissues have been isolated from the animals after euthanized them by using diethyl ether and then the tissue fixed into 10 % neutral buffered formalin (NBF) solution for 24 h. The mammary tissues were dehydrated and set into paraffin wax. The tissues were incised into 5 µm thickness which were mounted onto glass slides, after that the rehydration been done by the graded alcohol. Hematoxylin and eosin (H&E) have been used for staining the slides, subsequently the slides were examined by a light microscope for histological analysis.

Antioxidant assessment of breast tissues

The in-vivo antioxidant assessment of breast tissues was demonstrated in supplementary file 1.

Immunohistochemical assessment

The isolated mammary tissue was embedded into the paraffin. After that, the tissues were incised into 5 µm width and fixed onto the poly-l-lysine covered glass slides. The mammary tissues have been deparaffinized before being immersed in H2O2 solution. Then the breast tissue sections have been treated by protein blocking solution for 1 hour after that the sections were incubated in primary antibodies (anti-mouse vimentin, p53, m-TOR, Akt, caspase-3, β-catenin) with appropriate dilution (1:500) at 4 °C temperature for 24 h. Then the slides been washed with phosphate buffer solution (PBS) and introduce HRP conjugated secondary antibody on the slides for half an hour. DAB was utilized for the color development of the section and counterstaining has been completed by using hematoxylin and eosin (H&E) staining. Then, the tissue section has been visualized by light microscope (OLYMPUSCX 21i TR) and pictures been taken for analysis of immune-responsive cells through ImageJ software (version 1.8.0).

Cellular proliferation assessment

The mammary tissue section of 5 µm width has been fixed onto the poly‐L‐lysine covered glass slides. Then, the slides been deparaffinized, rehydrated and introduced into the H2°2 solution. Next, the slides were introduced in anti-rabbit Ki-67 antibody (1:500) at 4 °C overnight. Then, these glass slides have been introduced in HRP conjugated secondary antibody for half an hour at room temperature. DAB was used for color development on the tissue sections and counterstaining by H&E that have been visualized under light microscope.

Apoptosis assessment through TUNEL assay

The occurrence of apoptosis was investigated through TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) assessment. The proteinase-K solution been supplemented into the tissue sections which was introduced in H2O2 in order to obstruct the endogenous peroxidase action. Then, the tissue sections have been treated by terminal deoxynucleotidyl transferase (TdT) associated with TdT and dUTP comprised of TdT, reaction happened for an hour at 37 °C. After that, the saline citrate has been supplemented to abort. Further, the glass slides have been incubated with anti-digoxigenin peroxidase enzyme (1:1000) at 37 °C for 30 mins and subsequently the tissues were stained by DAB and hematoxylin. Finally, the tissue sections been visualized by the light microscope and TUNEL-responsive cells been quantified.

The percentage of cells that were Ki-67 positive was shown by the labeling index (LI). The% of the TUNEL-responsive cells per total number of cells been expressed by the apoptotic index (AI).

Statistical assessment

The experimental data have been articulated by the mean ± standard deviation (SD). The normality of distribution has not performed due to small sample size. The entire statistical analysis has been completed via ANOVA since Tukey's post hoc multiple comparison assessment with Graph Pad Prism (Version 5). The variations have been obtained as a statistically significant with p value <0.05 (p < 0.05).

Results

Induction of cytotoxicity by the complex therapy

Taxifolin ruthenium-p-cymene complex inhibits the cell viability of MCF-7 and MDA-MB-231 mammary carcinoma cells in dose dependently (Fig. 1a, b). The cellular viability of MCF-7 been appeared to be 78.07 %, 69.32 %, 59.45 %, 47.22 %, 13.43 % and for MDA-MB-231 cells obtained as 79.48 %, 61.92 %, 46.24 %, 25.69 %, 3.32 % at the concentrations of 25 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml, and 400 µg/ml correspondingly. The results found that the IC50 concentration of compound been estimated as 176.33 µg/ml and 78.28 µg/ml for MCF-7 and MDA-MB-231 cells correspondingly. For the chemotherapeutic investigation, the working concentrations of compound been evaluated as IC25, IC50 and IC75. The IC25, IC50 and IC75 concentrations of the complex on MCF-7 cells been found to be 88.17 µg/ml, 176.33 µg/ml, 264.5 µg/ml and for MDA-MB-231 cells the concentrations been obtained as 39.14 µg/ml, 78.28 µg/ml, 117.42 µg/ml respectively.Fig. 1 Effect of taxifolin ruthenium-p-cymene complex on the cell viability of (a) MCF-7 cells and (b) MDA-MB-231 cells at 48 h of incubation. * represented p < 0.05 as compared to untreated cells. Similarly, # represented p < 0.05 as compared to 25 µg/ml concentration, $ represented p < 0.05 as compared to 50 µg/ml concentration, α represented p < 0.05 as compared to 100 µg/ml concentration and β represented p < 0.05 as compared to 200 µg/ml concentration. DAPI staining of (c) MCF-7 cells and (d) MDA-MB-231 cells after 48 h of taxifolin ruthenium-p-cymene complex treatment denoting viable cells (white arrows), nuclear fragmentations (red arrows) and membrane blebbing (red arrowheads). (e) Clonogenic assay of MCF-7 and MDA-MB-231 cells after 48 h of treatment with taxifolin ruthenium-p-cymene complex. Quantification of (f) colony number, (g) colony size, and (h) plating efficiency for MCF-7 cells. Quantification of (i) colony number, (j) colony size, and (k) plating efficiency for MDA-MB-231 cells. * represented p < 0.05 as compared to untreated cells. Similarly, # represented p < 0.05 as compared to IC25, $ represented p < 0.05 as compared to IC50. Quantification of colony number and size was performed using Image J public domain software. Data represented as means ± SD from different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey's multiple comparison post-hoc analysis.

Fig 1

Nuclear fragmentation caused via taxifolin ruthenium-p-cymene complex treatment

The complex encouraged nuclear fragmentation and membrane blebbing in MCF-7 and MDA-MB-231 cells (Fig. 1c, d). The supreme nuclear condensation has been obtained at the concentration of 264.5 µg/ml and 117.42 µg/ml in MCF-7 and MDA-MB-231 cells correspondingly.

Taxifolin ruthenium-p-cymene complex abrogated cellular propagation

The compound treatment in both MCF-7 and MDA-MB-231 cells caused significantly the inhibition of colony formation (Fig. 1e). The complex treated cells demonstrated a substantially decreases the number of colony (Fig. 1f, i), colony size (Fig. 1g, j) and plating efficacy (PE) (Fig. 1h, k) in both cell lines respectively.

Epithelial-mesenchymal-transition inhibited caused via taxifolin ruthenium-p-cymene compound treatment

The invasiveness characteristics of mammary carcinoma cells (MCF-7 and MDA-MB-231) have been inhibited by the taxifolin ruthenium-p-cymene compound treatment (Fig. 2a-c) in dose dependently. Additionally, the western blot assessment (Fig. 2d) suggested that the obstruction of epithelial-mesenchymal transition via the downstream and upstream effect of N-cadherin and E-cadherin mutation individually in both MCF-7 and MDA-MB-231 cells.Fig. 2 (a) Effect of taxifolin ruthenium-p-cymene complex on the invasion of MCF-7 and MDA-MB-231 cells at 48 h of incubation. Estimation of invasion rate of (b) MCF-7 (c) MDA-MB-231 cells. (d) Western blot analysis of E-cadherin and N-cadherin on MCF-7 and MDA-MB-231cells. (e) Quadrangular plots representing the Annexin V/PI expression in MCF-7 cells upon culturing in the presence and absence of taxifolin ruthenium-p-cymene complex with IC25, IC50 and IC75 concentrations. Percentage of (f) apoptosis, (g) early apoptosis, (h) late apoptosis, (i) live cells, and (j) dead cells in MCF-7 cells at different culture conditions. (k) Distribution of cell cycle phases in MCF-7 cells upon culturing in the presence and absence of taxifolin ruthenium-p-cymene complex with IC25, IC50 and IC75 concentrations (l) Quantitative analysis of cell cycle phase distribution in MCF-7 cells. * represented p < 0.05 as compared to untreated cells. Similarly, # represented p < 0.05 as compared to IC25, $ represented p < 0.05 as compared to IC50. Data represented as means ± SD from different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey's multiple comparison post-hoc analysis.

Fig 2

The introduction of apoptotic events and cell cycle arrest caused through taxifolin ruthenium-p-cymene compound treatment

The initiation of apoptotic events in MCF-7 and MDA-MB-231 cells followed by the compound treatment for 48 h were shown in Fig. 2e and Fig. 3a correspondingly. The% of apoptotic cells for MCF-7 cells been determined as 9.07 %, 70.6 %, 93.89 % and for MDA-MB-231 cells it was 33.58 %, 52.11 %, 80.92 % at IC25, IC50 and IC75 concentrations of compound correspondingly (Fig. 2f, 3b). The% of early and late apoptotic pathway been substantially escalated in dose dependent manner for MCF-7 and MDA-MB-231 followed by the complex treatment for 48 h (Fig. 2g, h and Fig. 3c, d). A substantially lower% of viable cells been observed in the cells treated with complex (Fig. 2i, 3e). The% of the dead cells has also been exhibited in compound introduced cells. The IC50 concentration of compound evaluated the higher% of dead cells in both MCF-7 and MDA-MB-231 cell lines (Fig. 2j, 3f).Fig. 3 (a) Quadrangular plots representing the Annexin V/PI expression in MDA-MB-231 cells upon culturing in the presence and absence of taxifolin ruthenium-p-cymene complex with IC25, IC50 and IC75 concentrations. Percentage of (b) apoptosis, (c) early apoptosis, (d) late apoptosis, (e) live cells, and (f) dead cells in MDA-MB-231 cells at different culture conditions. (g) Distribution of cell cycle phases in MDA-MB-231 cells upon culturing in the presence and absence of taxifolin ruthenium-p-cymene complex with IC25, IC50 and IC75 concentrations. (h) Quantitative analysis of cell cycle phase distribution in MDA-MB-231 cells. * represented p < 0.05 as compared to untreated cells. Similarly, # represented p < 0.05 as compared to IC25, $ represented p < 0.05 as compared to IC50. Data represented as means ± SD from different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey's multiple comparison post-hoc analysis.

Fig 3

The dispersal of cell cycle regulation in untreated cells and compound introduced cells in MCF-7 and MDA-MB-231 cells is represented in Fig. 2k, l and Fig. 3g, h. The cell cycle examination on MCF-7 cells based on apoptotic phase (sub G0/G1 phase) showed as 1.53 %, 0.97 %, 2.75 % and 9.63 % of cell become arrested in IC25, IC50 and IC75 concentration of untreated cells correspondingly. Additionally, the growth phase (G0/G1 phase) demonstrated that 73.72 %, 57.62 %, 45.35 % and 40.27 % of cells, in synthetic phase (S phase) depicted as 1.72 %, 8.45 %, 9.15 % and 12.81 % of cells, in G2/M phase, 20.02 %, 28.91 %, 35.76 % and 33.27 % of cell become arrested in IC25, IC50 and IC75 concentration of untreated cells correspondingly (Fig. 2k, l). In MDA-MB-231 cells, the study of cell cycle based on the sub G0/G1 interphase displayed as 1.44 %, 1.45 %, 1 % and 44.55 % of cell become arrested in IC25, IC50 and IC75 concentration of untreated cells correspondingly. Likewise, the G0/G1 phase was showed that 75.26 %, 73.67 %, 54.65 % and 33.04 % of cell, in S phase displayed the 1.72 %, 2.05 %, 10.17 % and 2.6 % of cell, in G2/M phase demonstrated that 19.21 %, 18.88 %, 28.31 % and 17.6 % of cell become arrested in IC25, IC50 and IC75 concentration of untreated cells correspondingly (Fig. 3g, h).

Taxifolin ruthenium–p-cymene compound amplified the caspase-3 expression

The caspase-3 expression on MCF-7 and MDA-MB-231 cells after introduction of various concentration of compound for 48 h is displayed in Fig. 4a, b individually. The highest quantity of caspase-3 marked cells in M1 quadrant been represented in untreated cells. While, the frequency of caspase-3+ cells in M2 quadrant were suggestively amplified after the cells introduced in compound.Fig. 4 Effect of taxifolin ruthenium-p-cymene complex on the expression of caspase-3 in (a) MCF-7 cells and (b) MDA-MB-231 cells. (c) Western blot analysis of AhR and EGFR on MCF-7 and MDA-MB-231 cells. * represented p < 0.05 as compared to untreated cells. Similarly, # represented p < 0.05 as compared to IC25, $ represented p < 0.05 as compared to IC50. Data represented as means ± SD from different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey's multiple comparison post-hoc analysis.

Fig 4

Consequence of taxifolin ruthenium-p-cymene compound on AhR and EGFR expression

The study of western blot represented the complex treated group substantially abrogated the AhR and EGFR expression in MCF-7 and MDA-MB-231 cells as comparison to untreated cells (Fig. 4c).

Histopathology of breast tissue

At the end of the chemotherapeutic investigation was demonstrated the histopathological modifications of normal architecture of breast tissue (Fig. 5). The vehicle control group (Fig. 5a) showed the typical morphology of breast tissue such asalveoli (a), lobular terminal duct (td), alveolar septa (as), connective tissue (c), serous gland (sg). The DMBA induced carcinogen control group (Fig. 5b) exhibited the ductal carcinoma (dc), periductal stromal fibrosis (psf), microinvasive cancer cells (ic). Besides that, the complex administered group (Fig. 5c-e) got back the typical cellular morphology of the breast tissue. However, the minor stromal fibrosis (sf) was appeared in 50 mg kg-1 compound administered group (Fig. 5d). Additionally, the 200 mg kg-1 complex treated group (Fig. 5e) has not been shown any modifications in DMBA persuaded breast carcinoma and fruitfully reinstated the typical architecture of breast tissue. In the other hand, 50 mg kg-1 taxifolin administered group (Fig. 5f) displayed microinvasive cancer cells and stromal fibrosis. 50 mg kg-1 body weight doses of ruthenium-p-cymene administered group (Fig. 5g) exhibited the microinvasive cancer cells and stromal fibrosis.Fig. 5 (i) Histopathological appearance of breast at 10X [inset 40X]. (a) Normal control group showed alveoli (a), alveolar septa (as), lobular terminal duct (td), connective tissue (c), serous gland (sg). (b) Carcinogen control group depicted ductal carcinoma (dc), periductal stromal fibrosis (psf), microinvasive cancer cells (ic). (c 50 mg kg-1 taxifolin ruthenium-p-cymene complex treated group showed stromal fibrosis (sf). (d) 100 mg kg-1 taxifolin ruthenium-p-cymene complex treated group showed normal cellular morphology. (e) 200 mg kg-1 taxifolin ruthenium-p-cymene complex treated group restored normal morphological architecture of carcinogen induced breast tissue. (f) 50 mg kg-1 taxifolin treated group depicted microinvasive cancer cells (ic) and stromal fibrosis (sf). (g) 50 mg kg-1 ruthenium-p-cymene treated group depicted microinvasive cancer cells (ic) and stromal fibrosis (sf).

Fig 5

Taxifolin ruthenium-p-cymene activity on vimentin, p53, m-TOR, Akt, caspase-3, β-catenin expression

Fig. 6 and Table 1 demonstrated the expression of vimentin, p53, m-TOR, Akt, caspase-3, β-catenin on the breast carcinoma tissue of rat. A suggestively lower level of p53 as well as caspase-3 expression have been depicted in DMBA induced carcinogen control group (Fig. 6(i)b, (ii)b) as compared with the vehicle control (Fig. 6(i)a, (ii)a) (p < 0.05). While, in the compound administered group represented a higher proliferation of p53 and caspase-3 in dose dependently (Fig. 6(i) c-e, (ii) c-e). In 200 mg kg-1 complex treated group (Fig. 6(i)e, (ii)e) exhibited substantial increment of p53 and caspase-3 mutation on terminal ducts as well as alveolar area. A modest mutation of p53 and caspase-3 been denoted in 50 mg kg-1 taxifolin (Fig. 6(i)f, (ii)f) and 50 mg kg-1 ruthenium-p-cymene administered group (Fig. 6(i)g, (ii)g). Whereas, the DMBA persuaded carcinogen control group showed a substantial increases the vimentin (Fig. 6(iii)b), Akt (Fig. 6(iv)b), m-TOR (Fig. 6(v)b) and β-catenin (Fig. 6(vi)b) expression on terminal end buds and alveolar duct area as comparison with the vehicle control (Fig. 6(iii)a-(vi)a) (p < 0.05). The compound (50 mg kg-1, 100 mg kg-1, and 200 mg kg-1) therapy substantially cause the (p < 0.05) downregulation of vimentin (Fig. 6(iii) c-e), Akt (Fig. 6(iv) c-e), m-TOR (Fig. 6(v) c-e) and β-catenin (Fig. 6(vi) c-e) expression. At 50 mg kg-1 of taxifolin (Fig. 6(iii)f-(vi)f) and 50 mg kg-1 of ruthenium-p-cymene administered group (Fig. 6(iii)g-(vi)g) demonstrated a significant expression of vimentin, Akt, m-TOR and β-catenin in breast tissues.Fig. 6 The immunohistochemical analysis of [i] p53 [ii] caspase-3, [iii] vimentin, [iv] Akt, [v] mTOR, and [vi] β-catenin expression in the breast tissues at 10X magnification of (a) normal control, (b) carcinogen control, (b) 50 mg kg-1 complex, (d) 100 mg kg-1 complex, (e) 200 mg kg-1 complex, (f) 50 mg kg-1 taxifolin, and (g) 50 mg kg-1 ruthenium-p-cymene treated group, represented by black arrows.

Fig 6

Table 1 The effect of taxifolin, ruthenium-p-cymene and taxifolin ruthenium-p-cymene complex on the expression of vimentin, p53, caspase-3, Akt, mTOR and β-catenin.

Table 1Groups	p53§	Caspase-3§	vimentin§	Akt§	mTOR§	β-catenin§	
Normal control	8.25 ± 0.1	5 ± 0.5	7.68 ± 0.1	8.83 ± 0.1	7.35 ± 0.2	4.68 ± 0.1	
Carcinogen control	3.82 ± 0.6*	2.17 ± 0.1*	21.59 ± 0.1*	22.22 ± 0.1*	25.97± 0.1*	22.98 ± 0.1*	
50mg kg-1 complex	4.07 ± 0.1*	9.85 ± 0.1*,#	15.32 ± 0.2*,#	14.98 ± 0.1*,#	14 ± 0.1*,#	18.85 ± 0.1*,#	
100mg kg-1 complex	6.1 ± 0.1*,#,$	9.95 ± 0.1*,#	11.02 ± 0.1*,#,$	12.87 ± 0.1*,#,$	12.2 ± 0.1*,#,$	12 ± 0.1*,#,$	
200mg kg-1 complex	7.87 ± 0.1#,$,α	13.75 ± 0.1*,#,$,α	9.05 ± 0.1*,#,$,α	8.23 ± 0.2*,#,$,α	8.03 ± 0.1*,#,$,α	8.87 ± 0.1*,#,$,α	
50mg kg-1 taxifolin	3.92 ± 0.1*,α,β	4.17 ± 0.1*,#,$,α,β	16 ± 0.1*,#,$,α,β	17 ± 0.1*,#,$,α,β	20.1 ± 0.1*,#,$,α,β	20.01 ± 0.1*,#,$,α,β	
50mg kg-1 ruthenium-p-cymene	3.18 ± 0.1*,α,β	2.98 ± 0.1*,#,$,α,β,γ	18.65 ± 0.2*,#,$,α,β,γ	19.13 ± 0.1*,#,$,α,β,γ	23.15 ± 0.1*,#,$,α,β,γ	21.05 ± 0.1*,#,$,α,β,γ	
§ Each score represents the results of 6 slides per rat and 6 rats per group, mean ± SD (n = 6). Each field was selected randomly for evaluation of percentage of immune-positive cells.

⁎ Significant difference as compared to normal control (p < 0.05).

# significant difference as compared to carcinogen control group (p < 0.05).

$ significant difference as compared to 50 mg kg-1 complex treated group (p < 0.05).

α significant difference as compared to 100 mg kg-1 complex treated group (p < 0.05).

β significant difference as compared to 200 mg kg-1 complex treated group (p < 0.05).

γ significant difference as compared to 50 mg kg-1 taxifolin treated group (p < 0.05).

The downregulation of Ki-67 levels via taxifolin ruthenium-p-cymene compound treatment

For the cellular quantification, the cancerous cells along with Ki-67 marking displayed a distinct nuclear localization and a brown stain. The labeling index (LI) been used to calculate the percentage of Ki-67 labeled cells (Table 2). A significantly higher expression of Ki-67 labelled cells was showed in carcinogen control group (Fig. 7(i)b) as compared with vehicle control (p < 0.05) (Fig. 7(i)a). While, in the compound administered group showed a substantially lower appearance of Ki-67 labelled cells (Fig. 7(i) c-e). Additionally, a significant increase the cellular propagation been appeared in both 50 mg kg-1 of taxifolin (Fig. 7(i)f) and 50 mg kg-1 of ruthenium-p-cymene (Fig. 7(i)g) administered group (p < 0.05).Table 2 Cell proliferation and apoptosis in breast tissue.

Table 2Groups	Ki-67-LI§	AI (%)§	R= Ki-67-LI/AI	
Normal control	15.5 ± 0.1	0.11 ± 0.01	148 ± 11.23	
Carcinogen control	36.88 ± 0.1*	0.03 ± 0.01*	1689 ± 445.59*	
50mg kg-1 complex	23.88 ± 0.1*,#	0.06 ± 0.005*	439 ± 41.75#	
100mg kg-1 complex	15 ± 0.4#,$	0.09 ± 0.01#,$	170 ± 10.95#	
200mg kg-1 complex	6.96 ± 0.1*,#,$,α	0.12 ± 0.004#,$,α	56 ± 1.36#	
50mg kg-1 taxifolin	29.06 ± 0.1*,#,$,α,β	0.04 ± 0.01*,α,β	1142 ± 375.34*,α,β	
50mg kg-1 ruthenium-p-cymene	33 ± 0.1*,#,$,α,β,γ	0.03 ± 0.01*,α,β	1481 ± 405.58*,α,β	
LI= Labelling index, Ki-67-LI= percentage of Ki-67 labelled cells/total number of cells counted, AI= Apoptotic index. R= Ki-67-LI/AI. AI was calculated as the percentage of TUNEL positive cells/total number of cells counted. Values represent mean ± SD (n = 6).

§ Total number of six slides was evaluated per rat and 6 rats per group. Each field consisted of 700 cells.

⁎ Significant difference as compared to normal control (p < 0.05).

# significant difference as compared to carcinogen control group (p < 0.05).

$ significant difference as compared to 50 mg kg-1 complex treated group (p < 0.05).

α significant difference as compared to 100 mg kg-1 complex treated group (p < 0.05).

β significant difference as compared to 200 mg kg-1 complex treated group (p < 0.05).

γ significant difference as compared to 50 mg kg-1 taxifolin treated group (p < 0.05).

Fig. 7 The immunohistochemical analysis of [i] Ki-67 expression and [ii] TUNEL-positive apoptotic cells in the breast tissue at 40X magnification of (a) normal control, (b) carcinogen control, (c) 50 mg kg-1 complex, (d) 100 mg kg-1 complex, (e) 200 mg kg-1 complex, (f) 50 mg kg-1 taxifolin, and (g) 50 mg kg-1 ruthenium-p-cymene treated group, represented by black arrows. Approximately 700 cells were counted per field, 10 fields were examined per slide.

Fig 7

The initiation of apoptosis caused by the taxifolin ruthenium-p-cymene compound treatment

The purpose of apoptotic analysis was to assess the prevalence of apoptotic events in breast cancer. The chromogen-induced brown staining of the apoptotic cells allowed for their identification (Fig. 7(ii)). Along with, the vehicle control (Fig. 7(ii)a) represented the moderate mutation of TUNEL-responsive cells. In contrast, the DMBA induced carcinogen control (Fig. 7(ii)b) displayed a considerably (p < 0.05) lower numeral of apoptotic cells than normal control group. Fig. 7(ii) c-e shows that the compound-treated group had substantially (p < 0.05) augmented the proportion of apoptotic cells. The 50 mg kg-1 of taxifolin (Fig. 7(ii)f) and 50 mg kg-1 of ruthenium-p-cymene (Fig. 7(ii)g) administered group exhibited a lesser number of apoptotic cells. Within the field of 700 cells, an average of 4 to 5 apoptotic cells was denoted in carcinogen control group. On contrary, 11 to 12 TUNEL-responsive cells have been measured at 200 mg kg-1 of compound administered group (Table 2).

The R value has been defined as the cellular propagation to apoptotic ratio. The complex treatment dramatically decreased R value because of the initiation of apoptotic events, consequently fostering the prevention of cellular propagation, whereas the raised R value in the carcinogen control group suggested that the heightened proliferative activity of the cancerous cells.

Discussion

In last decades, breast carcinoma was recognized as the utmost predominant carcinoma which in turn mandates the establishment of a newer therapeutic approach against carcinoma associated death. In paradigm, the heterogenic characteristics of malignant cells restrain the inhibitory action of conventional chemotherapeutic drugs [18]. Hence, in the forthcoming year the researcher has designing the versatile targeted antineoplastic agents which is the important characteristics of carcinoma chemotherapeutic study. In the recent research has been aimed on synthesizing a newer flavonoid based organometallic compound to evaluate its chemotherapeutic effects against mammary carcinoma.

The anticancer activity of taxifolin ruthenium-p-cymene compound has been examined in both MCF-7 and MDA-MB-231 mammary cancer cells from the perspective of EMT and AhR expression. The cellular viability assessment exhibited the compound suppressed the viability of MCF-7 and MDA-MB-231 cells in dose dependently. The IC50 value determined through MTT analysis as found to be 176.33 µg/ml and 78.28 µg/ml for MCF-7 and MDA-MB-231 cells correspondingly. Since the chemotherapeutic doses of compound (IC25, IC50 and IC75) have been quantified. The initiation of cell death has been observed through DAPI examination that represented the superior nuclear condensation at IC75 concentration of compound on both MCF-7 and MDA-MB-231 cells individually. Moreover, the colony formation capability of the malignant cell has been substantially abrogated since the cells treated with taxifolin ruthenium-p-cymene compound.

As per recent studies, AhR may possess crucial role in carcinogenesis, angiogenesis, cell cycle regulation and proliferation control. Furthermore, AhR is also important for the regulation of immune cells which react to endogenous and exogenous substances [19]. AhR suppresses apoptosis, which acts as a prime role in emergence of mammary carcinoma. This raises the possibility that pairing AhR antagonists with chemotherapy could efficiently synergistically kill breast carcinoma cells [20]. In the recent investigation, the taxifolin ruthenium-p-cymene compound treatment attenuating the downregulation of AhR expression in both MCF-7 and MDA-MB-231 cells in dose dependent manner and thus, it abrogates the AhR mediated initiation and progression of the cancer.

It was observed that, the epithelial-to-mesenchymal transition may act as a prime role that take place prior to the commencement of tumor metastasis [21]. Present study suggested that, the tumor cell undergo metastasis via the alteration of PI3K/Akt/mTOR, and Wnt/β-catenin transduction scheme [18]. Herein, unusual stimulation of AhR either through endogenic or exogenic activation can interrupt with the different biological and pathological progressions which promote the invasiveness of tumor cells [22]. The EMT is also controlled through the several growth factor stimulations like EGFR. In this research, the EMT inhibition in tumor cells has been considered by the transwell migration analysis. The result of this study portrayed that the management by complex in dose dependently abrogated the invasive property of both MCF-7 and MDA-MB-231 cells. Additionally, the deregulation of N-cadherin as well as upstream of E-cadherin expression has been denoted, thus further inhibited the EMT expression in cancer cell. The downstream effect of EGFR mutation is significantly correlated with the decreasing EMT facilitated metastasis via the obstruction of EMT mediating transduction pathway in cancer.

In this regard, the flow cytometric assessment reported that the initiation of apoptotic events in MCF-7 and MDA-MB-231 cells upon the management with the novel compound. In the complex-treated group, many cells were found to be in the apoptotic stage, wherein the management by IC75 represented a highest apoptotic event was 264.5 µg/ml in MCF-7 and 117.42 µg/ml in MDA-MB-231 cell lines. Additionally, a substantial percentage of early and late apoptotic events as well as dead cells have been exhibited in both cell lines of compound treated group. While, a substantially lower the% of live cells have been denoted in compound treated cells in a dose dependent manner.

The malfunction of cell cycle control, which results in unchecked cellular proliferation, may play as most significant hallmarks of malignant cells [23]. Hence, inducing cell cycle corruption is a fundamental chemotherapeutic technique that settles for build-up of DNA double strand breaks and endorses death in tumour cells. The initiation of apoptosis in tumor microenvironment has also been evaluated through the examination of caspase-3 proliferation. The flow cytometry assay showed that increasing caspase-3 marked cells followed by the cancer cells treated with compound that confirmed an indication of the initiation of caspase-3 facilitated apoptotic events in tumor cells. Flow cytometry was carried out to evaluate the cytotoxicity activity of complex on cell cycle phase distribution in MCF-7 and MDA-MB-231 cells. The cell cycle study revealed that the complex therapy caused cellular arrest in sub G0/G1 phase, S phase, and G2/M phase of the cell cycle, eventually stimulating apoptotic events in breast carcinoma cell.

In in-vivo assessment have been performed in DMBA persuaded breast carcinoma model of Wistar rats. The histological analysis of breast tissues showed that the ductal carcinoma with periductal stromal fibrosis and microinvasive cancer cells in the carcinogen control group. Nevertheless, the taxifolin ruthenium-p-cymene compound treatment signified the reconstitution of the natural cellular architecture of the breast tissue. These data revealed that the combination has increases the anticancer activity against DMBA-induced breast malignancy.

Immunohistochemical analysis was used to assess the activation profiles of vimentin, caspase-3, p53, m-TOR, Akt, and β-catenin in breast tissue. Vimentin, an intermediate filament cytoskeleton, was recognized as an effective EMT indicator of malignant cells, thus further associated with the carcinoma cell invasion and metastasis. Expression of vimentin can also accompany with tumor formation as well as lymph node metastasis [24]. Numerous researches have been shown the mutations in tumor suppressive gene p53 are significantly associated with the progression of cancerous abrasions by inhibiting the apoptotic events [25]. A significant increment of p53 mutation is accountable for caspase-3 facilitated intrinsic apoptotic pathway in malignant cells [26]. The inhibition of apoptotic pathway may promote carcinogenic conversion through boosting cancer survival, growth, metastasis, and treatment resistance. The present research suggested that, the activation of PI3K/Akt/m-TOR transduction pathway is directly associated with the progression of breast cancer [27] followed by the modification of several up-stream regulator like EGFR as a resulting of the constitutive stimulation of the intracellular transduction pathway. As such, the phosphorylation of Akt was shown to be modified in various breast carcinoma occurrences [28]. Moreover, the upstream effect of PI3K/Akt/mTOR expression can also accompany by the advancement of drug resistance counter to EGFR targeted treatment [29]. The expansion of EGFR mutation in breast cancer can also accountable for the aberrant β-catenin expression [30]. Additionally, the current research confirmed the stimulation of Wnt/β-catenin signaling scheme may responsible for the initiation of metastasis in breast carcinoma [31]. Therefore, the abrogation of β-catenin expression cans significantly accomplishes with revoking the breast carcinoma progression, metastasis, and drug resistance. The immunohistochemistry study of breast tissue revealed that the DMBA persuaded carcinogen control group had lower expression of p53 and caspase-3 while having higher expression of vimentin, m-TOR, Akt, and β-catenin. The taxifolin ruthenium-p-cymene compound treatment resulted in substantial increases the p53 and caspase-3 level while suppressing vimentin, m-TOR, Akt and β-catenin expression in breast, implying that breast cancer can be prevented by inducing p53/caspase-3 facilitated apoptotic events, while in other hand, the suppressing vimentin/m-TOR/ Akt/β-catenin transduction scheme signified the cellular survival strategy.

The Ki-67 was acknowledged as a potent prognostic biomarker of the mammary carcinoma that associated with the regulation of cellular propagation [32]. Recent study suggested that an amplified the Ki-67 mutation which considered as lower apoptotic index (AI) in cancer progression [33]. In carcinogen control group, the cellular proliferation analysis revealed a rise in Ki-67 level with an elevated labelling index (LI). The DMBA induced carcinogen control group, the TUNEL analysis revealed a much lower frequency of TUNEL responsive cells along with a lower apoptotic index. However, the compound administered group demonstrated that drastically decreases the Ki-67 expression which causes the reduction of labelling index along with an elevation of apoptotic cells in breast tissue. Hence, the management with the compound caused the reduction of cancerous cellular proliferation and consequently provoked apoptotic events. Consistent with the in-vivo findings, and the in-vitro investigation demonstrated that the cell death by p53/caspase-3 facilitated apoptotic pathway and restrains the cellular growth through the down-regulation of the AhR/PI3K/EGFR transduction scheme in MCF-7 and MDA-MB-231 cell lines. Despite the desired outcome of the study there are certain limitations that should be taken into consideration for better scientific approach such as absence of sample size analysis. Moreover, other different breast cancer cell lines and biomarkers should be evaluated for obtain a wide spectrum of action and to understand the mechanistic pathway more precisely.

Conclusions

Taking all consideration, it has been concluded that, the taxifolin ruthenium-p-cymene compound demonstrated a potent antineoplastic activity on both MCF-7 and MDA-MB-231 cells and DMBA persuaded mammary cancer model on rat by inducing p53/caspase-3 regulated apoptosis and cell cycle arrest at sub G0/G1 phase, S phase and G2/M phase. In addition, the treatment with taxifolin ruthenium-p-cymene compound obstructed cancerous cellular viability and EMT expression in both in-vitro and in-vivo model via modulating upregulation of AhR/PI3K/EGFR expression that ultimately causes the inhibition of EMT facilitated metastasis in carcinoma cells. Considering the in-vivo as well as in-vitro investigation, the probable versatile targeted molecular mechanism scheme of the compound counter to breast carcinoma is depicted in Fig. 8. These results strongly suggested that the prospective effectiveness of taxifolin ruthenium-p-cymene compound combating to breast carcinoma which rigorously supported the potential activity of this innovative chemotherapeutic drug in future clinical research.Fig. 8 The possible mechanism of action of taxifolin ruthenium-p-cymene complex against breast carcinoma.

Fig 8

Availability of data and materials

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Ethics approval and consent to participate

The in-vivo experimentations have been conducted according to the Institutional Animal Ethical Committee and the Animal Regulatory Body of the Government (1458/PO/E/S/11/CPCSEA dated 12.05.2011). The entire techniques accomplished during the investigation were complied as per the ethical guidelines which have been mentioned in institutional ethical protocol.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

Abhijit Das: Conceptualization, Methodology, Writing – original draft. Barshana Bhattacharya: Data curation, Investigation, Visualization. Sakuntala Gayen: Writing – review & editing. Souvik Roy: Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no competing interests.

Appendix Supplementary materials

Image, application 1

Acknowledgments

The NSHM Knowledge Campus in Kolkata provided the authors with ongoing assistance and encouragement during the project, for which they are thankful.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102107.
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