Introduction

Lung cancer is mainly associated with metastatic tumor growth, which has the ability to differentiate into adjacent cells. Lung cancer can be divided into small-cell lung cancer and non-small cell lung cancer (NSCLC) by histological methods. At the time of diagnosis, approximately 75% of NSCLC patients present with locally advanced or metastatic disease. NSCLC accounts for 80% of all lung cancers, and these patients have a poor prognosis [1]. Lung cancer is the most prevalent cancer in China, with 870,982 new reports and 766,898 deaths, according to GLOBOCAN 2022 [2]. Many approaches have been adopted in an attempt to eliminate cancer. However, the incidence of lung cancer continues to rise year after year with limited effectiveness. This encourages researchers to develop a novel chemotherapeutic method to effectively address lung cancer incidences.

Biochanin-A is a class of phytochemicals and is an O-methylated isoflavone. It can be found in red clover [3] and in several other legumes, such as soy, alfalfa sprouts, peanuts, and chickpea (Cicer arietinum). It can be classified as a phytoestrogen and has putative benefits in dietary cancer prophylaxis. It is of potential clinical interest for cardiovascular diseases due to its vasodilatory effect [4]. In addition, biochanin-A has anti-inflammatory, antioxidant, neuroprotective, antimicrobial, and hepatoprotective activities [5,6,7,8,9]. Biochanin-A has also been reported to inhibit cellular growth, angiogenesis, and metastasis and trigger apoptosis in various cancer model systems, such as bladder, breast, pancreatic, prostate, osteosarcoma and liver cancers [10, 11]. It inhibits the NF-kB (nuclear factor kappa β) pathway and prevents cellular proliferation in head and neck cancer [12]. Despite all the activities, certain drawbacks, such as drug resistance, insufficient cytotoxicity, and poor bioavailability, limit the efficacy of flavonoids, which can be improved by metal chelation of flavonoids.

Metal complexes play an essential role in cancer chemotherapy because of their physico-chemical characteristics, variable oxidation states, lipophilicity, high aqueous solubility and positively charged nature [13, 14]. Considering the above findings, a ruthenium-based organometallic complex was synthesized, and the chemotherapeutic effect of the ruthenium biochanin-A complex was evaluated against lung carcinoma through in vivo and in vitro approaches.

There are various etiological variables that contribute to the onset and progression of lung carcinoma, including multiple genetic and epigenetic anomalies that essentially activate oncogenes and suppress tumor suppressor genes. The genetic mutations associated with chronic inflammation have been identified as important stimulatory events that are responsible for the onset of malignancies [15]. Studies have reported that inflammation could promote lung cancer through the generation of reactive oxygen species (ROS) originating from inflammatory cells, which form DNA adducts and cause alterations in genetic sequences [16]. The inflammatory mediator tumor necrosis factor-alpha (TNF-α) is directly related to cancer initiation and progression through the regulation of inflammatory responses in lung carcinoma [17]. The PI3K (phosphoinositide 3-kinase) signalingcascade is a critical intracellular pathway that results in cancer development through genomic mutations and upregulation of downstream proteins that regulate the PI3K cascade [18]. In lung cancer, the PI3K pathway is frequently activated, resulting in oncogenesis by inducing the growth and proliferation of cancer cells [19]. Furthermore, the activation of the TGF-β (transforming growth factor beta) receptor is also responsible for the activation of the PI3K pathway, which promotes the tumor-inducing function of TGF-β. In addition, TGF-β-mediated activation of the PI3K pathway promotes a pro-cell survival response with the stimulation of cancer progression [20]. Peroxisome proliferator-activated receptor-γ (PPARγ), a member of the PPAR family of ligand-activated nuclear receptors, is activated by synthetic agents that inhibit cancer progression in the colon, breast, prostate, and lung [21]. Nevertheless, the role of PPARγ in lung cancer progression is still under investigation. Recent studies have shown that the activation of PPARγ has an inhibitory role on lung cancer cells through the induction of apoptosis and inhibition of cellular growth [22, 23].

In this research, we synthesized a novel ruthenium biochanin-A complex, and the chemotherapeutic activity of the complex against lung carcinoma was evaluated in vitro and in vivo by investigating the TGF-β/PPARγ/PI3K pathway and the regulation of inflammatory mediators with the p53/caspase-3-mediated intrinsic apoptotic cascade. The chemotherapeutic effect of the ruthenium biochanin-A complex was further correlated by in vivo and in vitro approaches to demonstrate its anticancer activity.

Materials and methods

Chemicals and reagents

Biochanin-A (product id: D2016), ruthenium trichloride (product id: 208,523), benzo-α-pyrene (product id: B1760), 3,3′- diaminobenzidine (DAB) (product id: D8001) and proteinase K (product id: 1.07393) were procured from Sigma Aldrich Chemical Co. (St. Louis, MO). HRP conjugated goat anti-rabbit IgG (cat. no. GTX213110-01), anti-rabbit p53 (cat. no. GTX50438), PI3K (cat. no. GTX100462), TNF-α (cat. no. GTX110520),TGF-β (cat. no. GTX21279), PPARγ (cat. no. GTX02531), Bcl2 (cat. no. GTX100064), caspase-3 (cat. no. GTX110543), cleaved caspase-3 (cat No. GTX86952), and PCNA (proliferating cell nuclear antigen) (cat. no. GTX100539) were obtained from GeneTex International Corporation (Global). The remaining reagents were purchased from the local store in the purest quality.

Prediction of drug-likeness

The properties of drug likeness were evaluated by the molinspiration software at https://www.molinspiration.com/cgi-bin/properties. When H-bond donors are > 5, H-bond acceptors are > 10, and logP is > 5, the molecule shows poor absorption and permeation properties. The ideal range of molecular weight should be from 130 to 725 Da [24].

Identification of protein targets

The sequences of target proteins, such as PI3K, PPARγ, and caspase-3, were obtained from running protein BLAST (tool of NCBI) submitted in NCBI, and the CastP calculation server was used to identify the active site amino acids through the evaluation of surface area and surface volume of the 3D protein structure [25].

Molecular docking

Molecular docking was performed on the homology modeled proteins by using AutoDock 4.2.6 and AutoDock Vina 4.2. The AutoDock energy calculation was used to calculate the docking energy of all ligand molecules and proteins [26].

Synthesis of ruthenium biochanin-A complex

Approximately 0.2 g of biochanin-A and 0.1 g of ruthenium trichloride were dissolved in ethanol in separate beakers. The ruthenium solution was then added to the biochanin-A solution dropwise with continuous stirring for 24 h. The pH of the solution was maintained at 7.4 by the addition of 1 M NaOH solution. After 24 h, a black colored solution was obtained, which was then filtered, and the filtrate was dried over calcium chloride in vacuum desiccators to obtain solid product. The product was then lyophilized and stored at 4 °C. The percentage yield of the product was found to be 94% (Fig. 1A, B).

Fig. 1
figure 1

Structures of the A biochanin-A and B ruthenium biochanin-A complexes. Molecular docking study of the ruthenium biochanin-A complex with C PI3K, D PPARγ and E caspase-3

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UV‒vis spectroscopy

UV‒Vis spectroscopy (UV-1800 Shimadzu double beam spectrophotometer) was performed for both biochanin-A and the complex in methanol to investigate the mode of chelation.

Fourier transform infrared spectroscopy

The formation of metal oxide bonds was investigated by performing FTIR spectroscopy (ALPHA-T, Buker, Rheinstetten, Germany) of both biochanin-A and the complex.

ESI (electrospray ionization) mass spectrometry

Mass spectrometry of the complex was carried out by using electron ionization tandem mass spectrometry (1200 L/MS/MS, varian, mass spectrometer).

Scanning electron microscopy

The surface morphological characteristics of the complex were determined by scanning electron microscopy at an accelerating voltage of 20 kV. The images were captured at 500×, 1000×, 1500×, and 2000× magnification using SEM (JEOL, UK, JSM-6360).

DNA binding assay

The DNA intercalative property of the complex was determined by investigating the intercalation of the complex with ctDNA (calf thymus DNA) using UV‒visible spectroscopy. The ctDNA stock solution, composed of ctDNA in 5 mM Tris-HCl buffer, was used to check the purity of ctDNA. The absorbance ratio of 1.8–1.9:1 at 260 and 280 nm indicated that the ctDNA stock solution was protein free. Then, the UV spectrum was recorded after each successive addition of DNA to the 5 µM stock solution of the complex, and the intrinsic binding constant was evaluated by the following equation:

$${\text{DNA/}}\varepsilon _{a} - \varepsilon _{f} \,{\text{ = }}\,{\text{DNA/}}\varepsilon _{b} - \,\varepsilon _{f} {\text{ + 1/Kb}}\,{\text{(}}\varepsilon _{b} - \,\varepsilon _{f} {\text{)}}$$

DNA is the concentration of ctDNA, εa is the observed extinction coefficient Aobs/Ru, εf represents the extinction coefficient of the free compound, εb represents the extinction coefficient of the bound compound. εf is determined from the calibration curve of the complex.

Cell culture

The A549 (ATCC number: CCL-185) lung cancer cell line was obtained from ATCC (American Type Culture Collection), United States, and incubated in DMEM, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were cultured at 37 °C in 5% CO2 and 95% relative humidity.

Cell viability assay

The cell viability assay was performed by MTT (3-(4,5 dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. A549 cells were seeded in 96-well plates and incubated at 37 °C in 5% CO2 for 24 h. The cells were then exposed to different concentrations (5–40 µM) of ruthenium biochanin-A complex and incubated for 24 h. After incubation, 0.5 mg/ml MTT solution in DMEM was added to the cells and incubated for 3 h. Following MTT addition, DMSO was added to dissolve the formazan crystals, and the absorbance was measured at 560 nm. The percentage of cell viability was determined as follows:

$$\begin{aligned}& \% \,{\text{cell}}\,{\text{viability}} = \,[{\text{Mean}}\,{\text{absorbance}}\,{\text{of}}\,{\text{treated}}\,{\text{cells}}\\&\quad / {\text{Mean}}\,{\text{absorbance}}\,{\text{of}}\,{\text{untreated}}\,{\text{cells}}]\, \times \,{\text{100}} \end{aligned}$$

The half-maximal inhibitory concentration (IC50) value was calculated from the Equation y = mx + c, where y = 50, and the values of m and c were determined from the viability graph.

Flow cytometry

Cellular apoptosis was investigated by flow cytometry with an Annexin V-FITC apoptosis detection kit. A549 cells were seeded in 6-well plates and treated with different concentrations (IC25, IC50, and IC75) of the complex for 24 h. After incubation, the cells were labeled with Annexin V-FITC and propidium iodide (PI). The cells were then analyzed by a FACSCelesta flow cytometer (BD Biosciences, San Jose, CA, United States), and cell cycle events were also evaluated. The results obtained were analyzed by Modfit tools, and the percentage of apoptotic cells was determined.

Determination of caspase-3 expression

The expression of caspase-3 in A549 cells was investigated by flow cytometry. A549 cells were seeded in 6-well plates and treated with different concentrations (IC25, IC50, and IC75) of the complex for 24 h. The cells were then treated with anti-rabbit caspase-3 polyclonal antibody for 30 min at 37 °C in the dark. The percentage of cells labeled with caspase-3 antibody was analyzed by flow cytometry.

Western blotting

Western blot analysis was performed to evaluate the expression of target proteins in the A549 cell line that are associated with lung cancer development and progression. The A549 cells were treated with IC25, IC50, and IC75 concentrations of the complex and incubated for 24 h. The total cell lysate from each group was obtained, further resolved by SDS‒PAGE (8–12%) electrophoresis and transferred to PVDF (polyvinylidene fluoride) membranes. The membrane was then blocked with 5% nonfat dried milk in PBST (phosphate buffered saline with Tween 20) and incubated with anti-rabbit PI3K, PPARγ, TGF-β, TNF-α, and cleaved caspase-3 antibodies overnight in a humidified chamber. After incubation, the membrane was treated with HRP-conjugated goat anti-rabbit secondary antibodies, and the expression was detected by a chemiluminescent (ECL Western Blotting) kit with β-actin as the loading control.

Animals

The in vivo experiments were carried out as per the Institutional Animal Ethics Committee and the Animal Regulatory Body of the Government (1458/PO/E/S/11/CPCSEA dated 12.05.2011).

The animals were obtained from a registered animal breeder (1828/PO/Bt/S/15/CPCSEA dated 14.09.2015). Swiss albino mice of both sexes (18–20 g), which were 6–8 weeks old, were utilized for the toxicity study, and the carcinogenic study was performed on 6-week-old male Balb/c mice (20–25 g). The animals were placed in polypropylene cages at 20–25 °C with 20% relative humidity in a 12 h light/12 h dark cycle. The animals were given adequate food and drinking water for a 7-day acclimatization period.

Acute oral toxicity study

The acute oral toxicity study of the ruthenium biochanin-A complex was performed as per the OECD guideline TG 420 (adopted December 2001). In this study, five experimental groups (6 animals per group, three of each sex) were selected. Group I is the vehicle control group, which receives 0.5% CMC (carboxy methyl cellulose) in distilled water. Group II, III, IV, and V animals were treated with 50, 100, 200, and 400 mg/kg of complex respectively by dissolving it in distilled water and administered through oral gavage. Following drug treatment, the animals were supplied with adequate food and water and observed for mortality or any abnormal clinical appearance for 3 consecutive days.

Subacute toxicity study

The subacute oral toxicity study was performed according to the OECD guideline TG 407 (adopted December 2001). In this study, five experimental groups (6 animals per group, three of each sex) were selected. Group I was the vehicle control group, which received 0.5% CMC in distilled water. The animals in groups II, III, IV, and V were given 50, 100, 200, and 400 mg/kg of complex, respectively, for 28 days. The animals were monitored continuously for clinical appearance, body weights, and mortality during the experiment. After 28 days, the vital organs (liver, kidney, stomach and testis) were excised from the euthanized animals for haematological, serum biochemical, and histopathological evaluations.

Body weight and water consumption

The body weights of each animal were recorded at weekly intervals. The water consumption of each animal (ml/animal/day) was also measured on a weekly basis.

Haematology and serum biochemistry

The blood samples were collected from the Retro-Orbital Plexus of rats and placed into EDTA-containing tubes (Merck Pvt. Ltd Mumbai, India) which were then analyzed immediately. The hematological parameters such as RBC count, hemoglobin (Hb) concentration, MCH, MCV, MCHC, Platelet (PLT) count, WBC count, and WBC differential counts (lymphocyte, monocyte, basophil, and eosinophil) were determined by MedonicCA-20 cell analyzer systems (Boule Medical, Stockholm, Sweden). The serum was separated by centrifugation of the blood samples at 3000 rpm for 10 min and then analyzed by Microlab 3000 auto-analyzer to determine serum biochemical parameters including ALT, AST, ALP, GLU, creatinine, and blood urea nitrogen (BUN).

Organ weight analysis

The animals were euthanized and the macroscopic abnormalities were carefully observed upon the completion of the study period. The weights of key organs such as the stomach, kidney, pancreas, liver, testis, and ovary were assessed in both absolute and relative terms (organ to body weight ratio).

Histopathology

The organs were fixed in 10% formalin overnight and then dehydrated with graded alcohol. Tissues with a thickness of 5 microns were placed on glass slides. The slides were rehydrated with alcohol and stained using hematoxylin and eosin (H&E). The histopathological analysis was carried out under a light microscope.

Carcinogenic study

In the carcinogenic study, seven experimental groups (6 animals per group, 3 of each sex) were selected, and the animals were given (except Group I) benzo-α-pyrene (100 mg/kg body weight, i.p.) in corn oil to induce lung cancer. After 2 weeks, chemotherapeutic treatment was started and continued for 25 weeks. The groups were determined as follows:

Group I (vehicle control) received 0.5% CMC in distilled water, Group II (carcinogen control) received benzo-α-pyrene, Group III was treated with carcinogen + 50 mg/kg complex, Group IV was treated with carcinogen + 100 mg/kg complex, Group V was treated with carcinogen + 200 mg/kg complex, Group VI was treated with carcinogen + 100 mg/kg biochanin-A, and Group VII was treated with carcinogen + 100 mg/kg ruthenium trichloride.

The animals were sacrificed after the experiment, and lung tissues were excised for histopathology and immunohistochemistry.

Histopathology

The lungs were kept in formalin overnight and set in paraffin wax after dehydration of the tissue samples by graded alcohol. Tissues with a thickness of 5 μm were placed on slides, rehydrated and stained using H&E. The lung tissues were observed for histopathology under a light microscope [27].

Immunohistochemistry

The excised lung tissues mounted on poly-l-lysine-coated glass slides were deparaffinized and treated with 3% H2O2. The slides were treated with 20 µg/ml proteinase K for 15 min for antigen retrieval. The slides were then treated with blocking solution for 1 h and incubated with anti-mouse p53, anti-rabbit Bcl2, and caspase-3 antibodies at a dilution of 1:500 overnight at 4 °C. The slides were then treated with HRP (horseradish peroxidase)-conjugated secondary antibody for 2 h. Nuclear staining was performed withDAB (3,3′-diaminobenzidine), and hematoxylin was used for background staining. The slides were then observed under a light microscope [27].

Cell proliferation assay

The excised lung tissues mounted on poly-l-lysine-coated glass slides were deparaffinized and treated with 3% H2O2. The slides were then treated with blocking solution for 1 hand incubated with anti-rabbit PCNAat a dilution of 1:500 antibody overnight at 4 °C. The slides were then treated with HRP-conjugated goat anti-rabbit secondary antibody for 2 h. Nuclear staining was performed with DAB, and hematoxylin was used for background staining. PCNA expression was examined under a light microscope [27].

Tunel assay

The poly-l-lysine-coated slides of colon tissues were used for the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The slides were treated with proteinase-K followed by H2O2 to block the activity of endogenous peroxidase. The slides were then treated with terminal deoxynucleotidyl transferase (TdT) buffer at 37 °C for 90 min. The tissues were then treated with saline-citrate solution (Sigma‒Aldrich) for 10 min to stop the reaction and incubated with antidigoxigenin peroxidase. Tissue was treated with DAB and stained with hematoxylin. The slides were placed under a light microscope, and images were captured, followed by quantification of the TUNEL-positive cells.

The labeling index (LI) designated the percentage of PCNA-positive cells. The apoptotic index (AI) represents the percentage of TUNEL-positive cells to the total number of cells.

Statistical analysis

The data are depicted as the mean ± SEM. Statistical analysis was carried out by ANOVA associated with Tukey’s multiple comparison test using GraphPad Prism (Version 5). The differences were designated as statistically significant at p < 0.05.

Results

Ruthenium biochanin-A efficiently binds with target proteins

The results of the molecular docking study are depicted in Fig. 1C–E; Table 1. The ruthenium biochanin-A complex showed considerable binding affinity with the target proteins. Among all the target proteins, PI3K and PPARγ demonstrated the most significant docking scores of −7.14 and − 7.18, respectively.

Table 1 Molecular docking of ruthenium biochanin-A complex
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Characterization of complex

The UV‒visible spectroscopic data of biochanin-A and the complex are shown in Fig. 2A. For biochanin-A, two absorption peaks were identified at 272 nm and 257 nm, which represented band I (cinnamoyl band) and band II (benzoyl band). After formation of the complex, band I and band II shifted to 273 nm (band III) and 262 nm (band IV), respectively. The IR spectra of biochanin-A and the complex are shown in Fig. 2B; Table 2. The characteristic peak for ν (Ru-O) was found at 527.38 cm−1 in the IR spectrum of the complex, which was absent in the IR spectrum of biochanin-A. Moreover, the ν (O–H) peak of biochanin-A at 3747.44 cm−1 was shifted to 3386.69 cm−1. The peaks for ν (C–H), ν (C = O), ν (C = C), and ν (C–O–H) at 2884.27 cm−1, 1652.48 cm−1, 1565.19 cm−1, and 1247.97 cm−1 were shifted to 2833.42 cm−1, 1617.63 cm−1, 1513.63 cm−1, and 1247.42 cm−1, respectively, in the case of the complex. The mass spectra of the complex are depicted in Fig. 2C. The molecular ion peak at m/z 773.04 suggested the formation of a complex. The other peaks represented molecular fragments of the complex, and the fragmentation pattern is presented in Fig. 2D. The scanning electron microscopy images of the complex at different magnifications (500×, 1000×, 1500×, and 2000×) are illustrated in Fig. 2E–H. The SEM images of the complex demonstrated the surface morphology and crystalline nature of the complex.

Fig. 2
figure 2

Characterization of the complex. A UV‒visible spectroscopy of the biochanin-A and ruthenium biochanin-A complexes. B FTIR spectroscopy of the biochanin-A and ruthenium biochanin-A complexes. C Mass spectrometry of the ruthenium biochanin-A complex. D Mass fragmentation analysis of the ruthenium biochanin-A complex. Scanning electron microscopic images of the ruthenium biochanin-A complex at E 500X, F 1000X, G 1500X, and H 2000X

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Table 2 FTIR Spectra of biochanin-A and ruthenium biochanin-A complex (band position in cm−1)
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Intercalative binding of ruthenium biochanin-A

The results of the ctDNA binding assay are shown in Fig. 3. The results showed that the successive addition of ctDNA decreased the absorbance of the ruthenium biochanin-A complex. The continuous decrease in the absorbance suggested the binding of the complex to ctDNA, which leads to alteration of the DNA double helix structure. The hypochromic effect suggested the intercalative mode of binding of the complex within the base pairs of DNA.

Fig. 3
figure 3

ctDNA binding activity of the ruthenium biochanin-A complex

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Ruthenium biochanin-A decreased viability of A549 cells

The cell viability assay of the complex demonstrated a significant inhibition of cell viability that was dose dependent (Fig. 4A). The viabilities of A549 cells were 89.54%, 72.86%, 51.29%, 38.43%, and 12.59% at concentrations of 5 µM, 10 µM, 20 µM, 40 µM, and 60 µM, respectively. The IC50 of the complex was then calculated to be 20 µM. The subsequent doses for the chemotherapeutic study were further determined to be 10 µM (IC25), 20 µM (IC50), and 30 µM (IC75).

Fig. 4
figure 4

Effect of the complex on the A549 cell line. A Effects of the ruthenium biochanin-A complex on the viability of A549 cells at 24 h, where *represents p < 0.05 compared to the untreated group. Similarly, # represents p < 0.05 compared to the 5 µM concentration, $ represents p < 0.05 compared to the 10 µM concentration, α represents p < 0.05 compared to the 20 µM concentration, and β represents p < 0.05 compared to the 40 µM concentration. B Detection of apoptosis in A549 cells by flow cytometry after treatment with ruthenium biochanin-A complex. C Percentage of apoptotic cells versus concentration in A549 cells, where *represents p < 0.05 compared to the untreated group. Similarly, # represents p < 0.05 compared to IC25, $ is p < 0.05 compared to IC50. D Percentage of apoptotic cells in the early and late apoptosis stages in A549 cells. * represents p < 0.05 compared to the untreated group. Similarly, # represents p < 0.05 compared to IC25, $ is p < 0.05 compared to IC50. E Quantitative distribution of A549 cells in different phases of the cell cycle. F Analysis of the cell cycle phase distribution of A549 cells after treatment with the ruthenium biochanin-A complex. G Determination of caspase-3 expression in A549 cells after treatment with the ruthenium biochanin-A complex. H Western blot analysis of the target protein. Data represent the means ± SEM from three different experiments in triplicate. The results were compared using ANOVA, followed by Tukey’s multiple comparison posthoc analysis

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Ruthenium biochanin-acaused cell cycle arrest with the induction of apoptosis

Treatment with the ruthenium biochanin-A complex increased the percentage of apoptotic cells in the A549 cell line in a dose-dependent manner (Fig. 4B, C). The % apoptotic cells were 15.64%, 38.24%, and 52.81% at the IC25, IC50, and IC75 of the complex, respectively. The cell population was also increased in a dose-dependent manner in the early and late apoptotic stages due to complex therapy (Fig. 4D).

The flow cytometric data for cell cycle phase distribution are depicted in Fig. 4E and F. The cell population was significantly decreased at the G0/G1 phase, whereas a significant increase in S phase and G2/M phase was noted in the complex-treated group. The cell population at the G0/G1 phase was found to be 65.28%, 61.45%, 50.29%, and 41.51% in the untreated, IC25, IC50, and IC75 groups, respectively. However, at the S phase and G2/M phase, the cell population was found to be 4.21%, 7.02%, 13.87%, 16.26% and 21.47%, 23.85%, 29.24%, 36.47% in the untreated, IC25, IC50, and IC75 groups, respectively.

Ruthenium biochanin-A increases caspase-3 expression

Treatment with the ruthenium biochanin-A complex showed a dose-dependent increase in caspase-3 expression in the A549 cell line (Fig. 4G). The untreated group showed caspase-3-labeled cells in the M1 quadrant. However, a significant increase in the population of caspase-3-labeled cells in the M2 quadrant was observed in the complex-treated group.

Ruthenium biochanin-A alters the expression of PI3K, PPARγ, TGF-β, TNF-α, and cleaved caspase-3

Western blot analysis showed the expression of PI3K, PPARγ, TGF-β, TNF-α, and cleaved caspase-3 in A549 cells (Fig. 4H). The untreated group showed increased expression of PI3K, PPARγ, TGF-β, and TNF-α in A549 cells. On the other hand, a significant decrease in the expression of cleaved caspase-3 was detected in the untreated group. However, treatment with the ruthenium biochanin-A complex downregulated the expression of PI3K, PPARγ, TGF-β, and TNF-α and upregulated cleaved caspase-3 expression in a dose-dependent manner.

Toxicity study

The LD50 of the ruthenium biochanin-A complex was determined from the acute oral toxicity study, which was 500 mg/kg body weight. The doses for the subacute toxicity study were selected to be 50, 100, 200, and 400 mg/kg of complex as per the LD50 value. In the 400 mg/kg group, mortality was observed with a significant decrease in food and water intake. Moreover, a significant loss of body weight was also noted in the 400 mg/kg complex-treated group.

Body weight and water consumption

Figure 5A–C displays the animals’ average body weight and water consumption rate. As comparison to the control and other complex treatment groups, the mean body weight and water consumption rate of the animals in the 400 mg/kg group were significantly lower.

Fig. 5
figure 5

A Graphical representation of mean water consumption rate of mice. Bar diagram depicting mean body weight of B male mice and C female mice. Bar diagram depicting absolute organ weight of D male mice and e female mice. Bar diagram depicting relative organ weight of F male mice and G female mice.*represented p < 0.05 as compared to normal control. Similarly #represented p < 0.05 as compared to 50 mg/kg group, $ p < 0.05 as compared to 100 mg/kg group, α p < 0.05 as compared to 200 mg/kg group. Data represent means ± SEM (n = 6). The results were compared using ANOVA, followed by a Tukey’s multiple comparison post-hoc analysis

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Haematology and serum biochemistry

When compared to the control group, the group receiving 400 mg/kg of the complex considerably worsed the haematological parameters, particularly the WBC count (Tables 3 and 4). When compared to other complex treatment groups, the serum biochemical examination showed that the 400 mg/kg group had significantly higher levels of ALT, AST, and ALP (p < 0.05) (Tables 5 and 6). Also, the 400 mg/kg group considerably outperformed the control group in terms of glucose and blood urea nitrogen levels (p < 0.05).

Table 3 Haematological findings in male Swiss Albino mice treated with ruthenium biochanin-A complex for 28 days repeated-dose oral sub-acute toxicity study
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Table 4 Haematological findings in female Swiss Albino mice treated with ruthenium biochanin-A complex for 28 days repeated-dose oral sub-acute toxicity study
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Table 5 Serum biochemistry findings in male Swiss Albino mice treated with ruthenium biochanin-A complex for 28 days repeated-dose oral sub-acute toxicity study
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Table 6 Serum biochemistry findings in female Swiss Albino mice treated with ruthenium biochanin-A complex for 28 days repeated-dose oral sub-acute toxicity study
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Gross observation and organ weight

Following the 28-day repeated dosage toxicity research, the weight of the vital organs, such as the stomach, kidney, pancreas, liver, testis, and ovary, was determined. The absolute and relative weight of the organs in the 50, 100, and 200 mg/kg groups did not show any such notable alterations. Nonetheless, a significant rise in the organs’ absolute and relative weight was found in the 400 mg/kg treated group (Fig. 5D–G).

Histopathology of vital organs

Histopathological analysis of vital organs, such as the kidney, liver, stomach, and testis, was performed after the subacute toxicity study. In the vehicle control group, the histopathology of the liver showed a central vein, portal vein, bile duct, hepatic artery, sinusoids, Kupffer cells, and lymph vessels (Fig. 6(i)A). The 400 mg/kg treated group exhibited sinusoidal dilution, periportal mononuclear infiltrates, and hepatocyte degeneration along with focal inflammation (Fig. 6(i)E). However, the 50, 100, and 200 mg/kg treatment groups did not exhibit any histological alterations in liver tissues (Fig. 6(i)B–D). In the vehicle control group, the morphological architecture of the kidney demonstrated Bowman’s capsule, proximal convoluted tubules, distal convoluted tubules, macula densa, juxtaglomerular cells, and podocytes (Fig. 6(ii)A). The 400 mg/kg group exhibited a thick capsular membrane, desquamation of the epithelial cells, and infiltration of inflammatory cells (Fig. 6(ii)E). The 50, 100, and 200 mg/kg treatment groups were not associated with any alterations in kidney tissue (Fig. 6(ii)B–D). In the vehicle control group, the histology of the stomach tissues showed muscularis mucosa, submucosa, lamina, and muscularis propria, along with circular muscle (Fig. 6(iii)A). The 50, 100, and 200 mg/kg treated groups (Fig. 6(iii)B–D) denoted no histopathological alterations in the stomach tissue. On the other hand, the 400 mg/kg group showed vascular congestion, thickening of the submucosal area, vacuolization, and glandular widening (Fig. 6(iii)E). In the vehicle control group and complex-treated group (50, 100, and 200 mg/kg), the morphological architecture of the testis showed seminiferous tubules, Sertoli cells, and spermatogonia, along with spermatozoa, Leydig cells, and round and elongated spermatids (Fig. 6(iv)A–D), while the 400 mg/kg group exhibited degeneration of seminiferous tubules, edema in interstitial tissues, and vacuolization (Fig. 6(iv)E). A semi-quantitative histological scoring has been demonstrated in Table 7 for all the vital organs.

Fig. 6
figure 6

Histopathology of vital organs. i Liver, ii kidney, iii stomach, and iv testis of Swiss albino mice for the A control group, B 50 mg/kg complex, C 100 mg/kg complex, D 200 mg/kg complex, and E 400 mg/kg complex. Liver tissue of the control group showing (pv) portal vein, (bd) bile duct, (ha) hepatic artery, (lv) lymph vessel, (cv) central vein, and (kc) Kupffer cells and the 400 mg/kg complex showing (fi) focal inflammation, (sd) sinusoidal dilation, and (hd) hepatocyte degeneration. Kidney tissue of the control group showing (g) the glomerulus, (bc) Bowman’s capsule, (dct) distal convoluted tubule, (pct) proximal convoluted tubule, (bs) Bowman’s capsular space, (p) podocytes, (up) urinary pole, and (md) macula densa and the 400 mg/kg complex showing (h) hemorrhages, (vc) vascular congestion, (tcm) thickening of the capsular membrane, (iic) infiltration of inflammatory cells, and (dec) desquamated epithelial cells. Stomach tissue of the control group showing (gp) gastric pits, (gc) gastric cavity, (bv) blood vessel, (s) submucosa, (mp) muscularis propria, (lp) lamina propria, (cm) circular muscle, (lm) longitudinal muscle and the 400 mg/kg complex showing (vc) vascular congestion, (v) vacuolization, (gw) glandular widening, and (d) desquamation of the gastric mucosa. Testis of the control group showing (sg) spermatogonia, (lu) lumen, (st) spermatid, (sc) sertolli cells, and (sz) spermatozoa and the 400 mg/kg complex-treated groupshowing (el) the empty lumen, (oe) edema, (v) vacuolization, and (mng) multinucleated giant cells. (H&E) 10× magnification [inset 40×]

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Table 7 Semi-quantitative histology scoring of vital organs
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Ruthenium biochanin-A treatment alters the histopathological lesion in lung carcinoma

The histopathology of the lung is shown in Fig. 7; Table 8. The vehicle control group showed bronchiole (b), pulmonary alveoli (p.a.), alveolar sac (as), blood vessels (bv), and pulmonary capillary (pc) of the lung tissue (Fig. 7A). Severe histopathological alterations were noted in the carcinogen control, followed by thickened alveolar walls (t), congested alveolar cavities (cac), lymphocyte infiltration (li), fibrosis (f) and granulomas (g) (Fig. 7B). The 50 mg/kg group showed fibrosis (f), lymphocyte infiltration (li), thickened alveolar walls (t) and congested alveolar cavities (cac) (Fig. 7C). The 100 mg/kg group showed slight lymphocyte infiltration (li) and widening in the alveolar wall (t) (Fig. 7D). However, the 200 mg/kg group showed normal lung morphological architecture (Fig. 7E). The 100 mg/kg biochanin-A-treated group demonstrated congested alveolar cavities (cac) and lymphocyte infiltration (li) (Fig. 7F). The 100 mg/kg ruthenium trichloride-treated group exhibited fibrosis (f), granuloma (g) and thickened alveolar walls (t) (Fig. 7G).

Fig. 7
figure 7

Histological appearance of lung tissue. A The vehicle control showing the bronchiole (b), pulmonary alveoli (p.a.), alveolar sac (as), blood vessels (bv), and the pulmonary capillary (pc). B Carcinogen control shows thickened alveolar walls (t), the congested alveolar cavity (cac), lymphocyte infiltration (li) in alveolar space, severe fibrosis (f) and formation of the granuloma (g). C Lung tissue of the benzo-α-pyrene induced group treated with the 50 mg/kg complex showing severe fibrosis (f), lymphocyte infiltration (li) in the alveolar space, thickened alveolar walls (t) and the congested alveolar cavity (cac). D Lung tissue of the benzo-α-pyrene induced group treated with 100 mg/kg complex showing slight lymphocyte infiltration (li) and minor thickening of the alveolar walls (t). E Lung tissue of the benzo-α-pyrene-induced group treated with 200 mg/kg complex having almost normal architecture. F Lung tissue of the benzo-α-pyrene-induced group treated with 100 mg/kg biochanin-A showing lymphocyte infiltration (li) and the congested alveolar cavity (cac). G Lung tissue of the benzo-α-pyrene-induced group treated with 100 mg/kg ruthenium trichloride showing fibrosis (f) and granuloma (g)

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Table 8 Semi-quantitative histology scoring of lung
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Ruthenium biochanin-A increased the expression of apoptotic proteins with significant downregulation of antiapoptotic protein expressionin vivo

The expression profile of p53, caspase-3, and Bcl2 in the lung tissue was determined by immunohistochemistry (Fig. 8). The vehicle control group showed increased p53 expression in the bronchiolar and alveolar walls. In addition, cytoplasmic localization of p53 in the bronchiolar cavity was identified (Fig. 8(i)A). However, the carcinogen control showed reduced p53 expression (Fig. 8(i)B). In the complex-treated group, the 50, 100 and 200 mg/kg doses showed significantly (p < 0.05) higher p53 levels in the alveolar walls and bronchiolar epithelial membrane (Fig. 8(i)C–E) than the carcinogen control (Table 9). The 100 mg/kg biochanin-A- and ruthenium trichloride-treated groups (Fig. 8(i)F, G) also showed significantly low p53 expression. The vehicle control group showed expression of caspase-3 in the bronchiolar and alveolar walls (Fig. 8(ii)A). In the carcinogen control, caspase-3 expression was significantly reduced (Fig. 8(ii)B). The complex-treated groups (50, 100, 200 mg/kg) demonstrated significantly (p < 0.05) higher caspase-3 expression (Fig. 8(ii)C–E) than the carcinogen control (Table 9). Low expression of caspase-3 in the lung tissue was also noted in the 100 mg/kg biochanin-A- and ruthenium trichloride-treated groups (Fig. 8(ii)F, G). Moderate Bcl2 expression was noted in the vehicle control (Fig. 8(iii)A) at the alveolar wall and capillary lumen. The carcinogen control showed high Bcl2 expression (Fig. 8(iii)B) in hyperplastic lung tissue. Conversely, the complex-treated group showed a significant (p < 0.05) reduction in Bcl2 expression (Fig. 8(iii)C–E) (Table 9). The 100 mg/kg biochanin-A- and ruthenium trichloride-treated groups (Fig. 8(iii)F, G) demonstrated significantly high expression of Bcl2 in lung tissue.

Fig. 8
figure 8

The effect of the complex on protein expression in lung tissue. Immunohistochemical analysis of i p53, ii caspase-3, iii Bcl2 iv and PCNA expression in the lung tissues of different groups of mice: A vehicle control, B carcinogen control, C 50 mg/kg complex-treated group, D 100 mg/kg complex-treated group, E 200 mg/kg complex-treated group, F 100 mg/kg biochanin-A-treated group, and G 200 mg/kg ruthenium trichloride-treated group. All images at 10X. Arrows represent the expression of specific antibodies

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Table 9 Effect of ruthenium biochanin-A complex on the expression of p53, caspase-3, and Bcl2 in lung tissues
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Ruthenium biochanin-A decreased the proliferation of cancer cells in vivo

The cells with PCNA labeling showed discrete nuclear localization and brown staining, which were used for cellular quantification. The vehicle control showed a low number of PCNA-labeled cells (Fig. 8(iv)A). The percentage of PCNA-labeled cells was measured by the labeling index (LI). The carcinogen control group (Fig. 8(iv)B) showed the maximum LI (Table 10). However, a significant (p < 0.05) reduction in LI was noted for the complex-treated group (Fig. 8(iv)C–E) (Table 10). Moreover, significantly high cellular proliferation was also noted in the 100 mg/kg biochanin-A- and ruthenium trichloride-treated groups (Fig. 8(iv)F, G).

Table 10 Cell proliferation and apoptosis in lung tissue
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Ruthenium biochanin-A promotes apoptosis of the cancer cells in vivo

An apoptosis assay was conducted to evaluate the incidence of apoptosis in lung cancer. Apoptotic cells were identified by brown staining due to chromogen treatment (Fig. 9). The carcinogen control group (Fig. 9B) showed the lowest number of positive cells, an average of 2–3 TUNEL-positive cells, in a field of approximately 500 cells in comparison to the vehicle control group (Fig. 9A). Significantly (p < 0.05) higher numbers of TUNEL-positive cells were noted in the complex-treated group (Fig. 9C–E) (50, 100, 200 mg/kg) than in the carcinogen control. An average of 9–10 apoptotic cells in a field of 500 cells were observed in the complex-treated group. However, the 100 mg/kg biochanin-A- and ruthenium trichloride-treated groups also demonstrated a low number of apoptotic cells in lung tissue (Fig. 9F, G). The percentage of TUNEL-positive cells was measured by the apoptotic index (AI) (Table 10). The R value is denoted as the ratio of cell proliferation to apoptosis. In the carcinogen control group, the increased R value indicated the enhanced proliferative activity of the cancer cells, but treatment with the ruthenium biochanin-A complex significantly decreased the R value due to the induction of apoptosis.

Fig. 9
figure 9

Immunohistochemistry of TUNEL-positive apoptotic cells in the lung tissues of different groups of mice at 40× magnification. A Vehicle control, B carcinogen control, C 50 mg/kg complex-treated group, D 100 mg/kg complex-treated group, E 200 mg/kg complex-treated group, F 100 mg/kg biochanin-A treated group, and G 200 mg/kg ruthenium trichloride-treated group. Arrows represent the expression of TUNEL-positive apoptotic cells

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Discussion

In recent times, standard chemotherapeutic drugs have become ineffective due to their serious complications and the development of chemoresistance, which prompted the design of novel chemotherapeutic medications to prevent the prevalence of the disease. In this circumstance, natural remedies might be the most plausible alternative due to their excellent anticancer efficacy and few side effects [28]. In addition, these natural compounds, specifically bioflavonoids, possessed excellent metal chelating properties from organometallic complexes with various metal ions. These organometallic complexes exhibited significant antioxidant efficacy, which in turn provides strong protection against oxidative damage and ROS-mediated stress [29].

In the current study, the chemotherapeutic activity of a novel ruthenium biochanin-A complex was investigated against lung carcinoma through both in vitro and in vivo approaches. Moreover, pharmacophore analysis was also carried out to investigate the binding affinity of the novel complex against the target proteins. The molecular docking study signified that the ruthenium biochanin-A complex had a strong binding affinity for PI3K, PPARγ, and caspase-3, which enabled effective binding of the target proteins. Additionally, it promotes the respective downstream signaling cascade by acting as an agonist or antagonist to inhibit lung cancer incidence.

In connection with this, the ruthenium-based organometallic complex has been synthesized, which was further characterized by various spectroscopic techniques to confirm the formation of the ruthenium biochanin-A complex. The UV spectroscopy of the complex showed that the maximum shift occurred for band II or the benzoyl band (biochanin-A), which in turn confirmed the formation of a metal chelate at the 4-oxo and 5-hydroxy positions of biochanin-A in a molar ratio of 1:2. IR spectroscopy of the complex showed the appearance of the peak for the ruthenium-oxide bond at 613 cm−1, which was absent in the IR spectrum of biochanin-A, suggesting the formation of a metal complex. Moreover, a major shift in the ν (O–H) band from 3677 to 3390 cm−1 demonstrated chelation of biochanin-A with ruthenium. The mass spectrometry analysis depicted the presence of the molecular ion peak of the complex at m/z 773.04, which further indicated the formation of a metal complex. Last, the images from scanning electron microscopy at various magnifications (500×, 1000×, 1500×, and 2000×) showed the surface morphology and the crystalline structure of the complex. The ctDNA binding assay demonstrated a hypochromic effect upon consecutive DNA addition to the complex, which is due to the interaction of the complex with DNA that leads to the alteration of the DNA double helix structure. The continuous decrease in the absorbance suggested that the ruthenium biochanin-A complex was able to intercalate within the DNA base pairs.

In addition, the chemotherapeutic activity of the ruthenium biochanin-A complex was investigated against the A549 lung cancer cell line. The cell viability assay demonstrated that the complex significantly inhibited the viability of A549 cells in a dose-dependent manner. Furthermore, the IC50 value was also calculated from the cell viability assay, which was found to be 20 µM. Subsequently, the chemotherapeutic doses of the complex were determined, which were 10 µM (IC25), 20 µM (IC50), and 30 µM (IC75). Flow cytometric analysis demonstrated the induction of apoptosis in the A549 cell line upon treatment with the ruthenium biochanin-A complex. A significant number of cells at the apoptotic stage were identified in the complex-treated group, where treatment with the 30 µM complex showed maximum apoptosis (52.81%) in A549 cells. The loss of cell cycle regulation followed by uncontrolled cellular growth is one of the important aspects of cancer cells [30]. Thus, the regulation of the cell cycle through the induction of cell cycle arrest is a major chemotherapeutic strategy that allows the accumulation of DNA damage and promotes apoptosis in cancer cells. The effect of ruthenium biochanin-A on cell cycle phase distribution was also investigated in A549 cells through flow cytometry. The results from the cell cycle analysis indicated that treatment with the complex caused cellular arrest in the S phase and G2/M phase of the cell cycle and ultimately stimulated apoptotic events in lung cancer cells.

In lung cancer, the PI3K pathway is the most commonly deregulated pathway due to genomic mutation, which causes increased activation of the PI3K signaling cascade [31]. Increased genomic expression of PIK3CA, the gene encoding the p110a catalytic subunit of PI3K, was also observed in NSCLC tumors and preinvasive lesions [32]. Several studies have reported that chronic inflammation plays a significant role in cellular homeostasis, metabolism, and genomic regulation, which leads to the onset of tumorigenesis [33]. There are various inflammatory mediators associated with the regulation of metastasis and invasion of cancer cells [34], such as tumor necrosis factor α (TNF-α) and tumor growth factor-β (TGF-β). The overexpression of TNF-α promotes DNA damage and induces tumor growth, angiogenesis, and invasion [35]. In addition, TGF-β is responsible for cancer development through the suppression of the anticancer immune response [36]. The role of PPARγ in different cancers is still unclear. Recent studies have demonstrated that PPARγ is associated with tumor progression [37]. Moreover, the activation of PPARγ also caused the upregulation of vascular endothelial growth factor (VEGF) transcription, which subsequently activated PI3K/Akt signaling and induced endothelial cell proliferation and angiogenesis [38]. Western blot analysis demonstrated increased expression of PI3K, TNF-α, TGF-β, and PPARγ, whereas decreased expression of cleaved caspase-3 in the untreated group was observed in A549 cells. Nevertheless, treatment with ruthenium biochanin-A significantly reduced the expression of PI3K, TNF-α, TGF-β, and PPARγ proteins and increased cleaved caspase-3 expression in a dose-dependent manner.

In the in vivo study, the toxicological profile of the ruthenium biochanin-A complex was assessed to determine the LD50 and subsequent chemotherapeutic doses. The LD50 of the complex was determined by an acute oral toxicity study, which was 500 mg/kg body weight. A subacute toxicity study was performed to investigate the side effects at higher doses. As per the subacute toxicity study the 400 mg/kg dose showed significant lose of body weight and decrease rate of water intake as compared to vehicle control group. Moreover, the absolute and relative organ weight also found to be decreased in the 400 mg/kg group with an exception of liver. The increase in liver weight can be linked to severe histopathological alteration of liver tissue at the higher dose i.e. 400 mg/kg. Conversely, the 50, 100, and 200 mg/kg doses did not show any histopathological anomalies in the vital organs, which suggested that the 50, 100, and 200 mg/kg doses of the complex were nontoxic and safe dose which makes us to conclude that the 50, 100, and 200 mg/kg doses were no observed adverse effect level (NOAEL). Thus, the 50, 100, and 200 mg/kg doses of the complex could be used for chemotherapeutic study.

The in vivo chemotherapeutic study was carried out on Balb/c mice after induction of lung cancer in the animals by the induction of benzo-α-pyrene (100 mg/kg i.p.). The histopathology of the lung showed severe pulmonary fibrosis and granuloma formation in the carcinogen control group. In addition, congestion in the alveolar cavity and severe lymphocytic infiltration in the alveolar space were identified in the carcinogen control. However, ruthenium biochanin-A complex treatment significantly decreased pulmonary fibrosis and restored the normal cellular architecture of lung tissue. These findings suggested the anticancer efficacy of the complex against benzo-α-pyrene-induced lung carcinoma.

The expression profile of p53, caspase-3, and Bcl2 was determined in the lung tissue by immunohistochemical analysis. Several studies have demonstrated that mutation of the tumor suppressor protein p53 is responsible for malignant lesion development through inhibition of the tumor suppressive pathway [39]. Increased expression of p53 is directly associated with caspase-3-mediated intrinsic apoptosis in cancer cells [40]. The suppression of apoptosis can directly facilitate neoplastic transformation by promoting tumor growth, metastasis, survival, and therapeutic resistance. In these processes, increased expression of the pro-survival protein Bcl2 has been identified in several cancers, which prevents cancer cells from p53-mediated apoptosis [41, 42]. The immunohistochemical analysis of lung tissue exhibited decreased expression of p53 and caspase-3 in the carcinogen control group with increased expression of Bcl2. Treatment with the ruthenium biochanin-A complex showed significant upregulation of p53 and caspase-3 expression with downregulation of Bcl2 expression.

Studies have reported that the increased expression of PCNA is associated with abnormal epithelial cell proliferation [43]. The cell proliferation assay exhibited an upregulation of PCNA expression with an increased labeling index in the carcinogen control group. In contrast, the complex-treated group significantly downregulated the expression of PCNA and reduced the labeling index in the lung tissue. Previous studies also suggested the induction of apoptotic events in cells with low PCNA expression [44]. In accordance with this report, this study also demonstrated that treatment with the ruthenium biochanin-A complex showed increased levels of apoptotic cells with low levels of PCNA, while the carcinogen control group depicted a significant decrease in TUNEL-positive cells (apoptotic cells). Consistent with this in vivo report, the in vitro study also depicted the induction of cellular death in A549 cancer cells through caspase-3-mediated apoptosis.

Taken together, it is concluded that the ruthenium biochanin-A complex showed potential chemotherapeutic activity against both the A549 lung cancer cell line and the benzo-α-pyrene-induced lung cancer mouse model through the induction of p53/caspase-3-mediated intrinsic apoptotic events and cell cycle arrest in the S phase and G2/M phase. Furthermore, the complex also inhibited the TGF-β/PPARγ/PI3K/TNF-α pathway and prevented cancer cell growth, survival, and differentiation (Fig. 10). On the other hand, this work has a number of limitations, including cellular uptake assay, the exploration of several more protein expressions, testing on other cell lines, and lack of computation and justification of sample size (animals). However, one important utilization of this study is cost-effective and targeted delivery of anticancer drugs with decreased side effects and easy administration, which is associated with a novel chemotherapeutic mechanism to alter the cellular growth and survival of cancer cells. Considering all the aspects, this complex may serve as a potent chemotherapeutic agent against lung cancer in clinical settings.

Fig. 10
figure 10

Schematic diagram representing the possible mechanism of action of the ruthenium biochanin-A complex in lung cancer

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