Inhibition of stemness and EMT by taxifolin ruthenium-p-cymene complex via downregulating the SOX2 and OCT4 expression on lung cancer

2.2.1 Protein structure prediction

The overall research is based on the understanding of proteins that are involved in lung cancer. The protein sequences such as PI3K (P42336), β-catenin (CAA61107), EGFR (P00533) were retrieved from UniProt knowledge database. These protein sequences were used to predict 3D protein structure based on the structural similarity to construct homology models by predicting similar templates using BLASTp.

2.2.2 Ligand preparation and drug likeliness analysis

The compound taxifolin ruthenium-p-cymene complex was designed using ChemSketch 2020.1.1. The chemical structures were saved in. mol format and further used for drug-likeliness to understand the druglike properties of the compounds. Pharmacokinetic analysis of the selected compound was predicted using AdmetSAR tool that show high throughput virtual screening based on the traditional medical chemistry properties.

2.2.3 Molecular docking and virtual screening

Based on pharmacophore and pharmacokinetic properties, the selected chemical is further used for molecular docking against target lung cancer proteins. Using AutoDock4.2 and AutoDockVina software to predict the protein–ligand interactions based on binding energy and hydrogen bond formation.

2.4.1 UV–Visible spectroscopy

The UV–visible spectrum of pure taxifolin and taxifolin ruthenium-p-cymene complex was recorded using UV-1900i Shimadzu double beam spectrophotometer.

2.4.2 Job’s method

The stoichiometric ratio of taxifolin to ruthenium-p-cymene in the complex was analyzed by continuous variation technique also known as Job’s method. Taxifolin of 0.1 mM (1, 2…9 mL) was taken into nine 10 mL volumetric flasks and an aliquot (9, 8…1 mL) of 0.1 mM [Ru(p-cymene) Cl₂] was added respectively at which a constant mole fraction of solution was attained. All the measurements were made at λ_max which was found to be at 241 nm.

2.4.3 Fourier-transform infrared spectroscopy (FTIR)

FTIR spectrophotometer (IR Affinity-1S, Shimadzu) was used to record the IR spectrum of taxifolin and taxifolin ruthenium-p-cymene complex over the range of 500–4000 cm⁻¹ wavenumber to distinguish the metal oxide bond as a validation of complexation.

2.4.4 Mass spectroscopy

The molecular weight and the fragmentation pattern of the complex were evaluated by mass spectroscopy using electrospray ionization mass spectroscopy (ESI-MS) (Xevo G2 Xs Q-Tof ESI Mass).

2.4.5 NMR spectroscopy

The ¹H NMR spectrum of the complex was recorded using Bruker-Avance-600 MHz spectrometer in methanol-D4. Tetramethylsilane (TMS) was used as an internal reference.

2.4.6 Scanning electron microscopy

The morphological features of the complex were evaluated using scanning electron microscopy (JEOL MAKE, (UK) Model-JSM-IT800 Schottky Field Emission Scanning Electron Microscope) at an accelerating voltage of 10 kV. The surface characteristics and morphological appearance of the complex was also examined at different magnifications (50X, 4000X, 6000X and 10000X).

2.4.7 X-ray diffractometry

For taxifolin ruthenium-p-cymene complex, no single crystal was formed. To detect the degree of crystallinity of this metal complex, X-ray powder diffraction was carried out. The X-Ray diffraction pattern of the complex was assessed by ULTIMA IV XRD System (Rigaku) operating at 45 kV and 40 mA using CuKα radiation (1.540562 Å) with the Bragg-Brentano geometry with step size 0.02 (2θ), step time 3 sec from 5 to 95. The average size of the crystallites has also been estimated using the Scherer’s equation; $$\mathrm{D}=\mathrm{K}\mathrm{\lambda }/\mathrm{\beta }\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{\theta }$$

Where, D represents average crystallite size, K is Scherrer constant, λ represents X-ray wavelength, β is line broadening FWHM (full width half maxima) in radians, θ is Bragg’s angle in degree. FWHM has been calculated based on Single Line profile Analysis using X'pert Organizer (PANalytical) software.

2.5.1 DPPH assay

The DPPH assay was performed as per the method of Rahman and co researchers (Rahman et al., 2015). The absorbance of test solutions was measured at 517 nm for both taxifolin and taxifolin ruthenium-p-cymene complex, in which 0.1 mM DPPH solution was considered as control. The % of radical scavenging activity (% RSA) was calculated as $$\%RSA=\{(A_0-A_1)/A_0\}\times 100\%$$

Where A₀ is the absorbance of control, A₁ is the absorbance of test solution.

2.5.2 ABTS assay

The ABTS assay was performed as per the protocol mentioned by Pennycooke and coworkers (Pennycooke et al., 2005). In this method taxifolin and the complex was dissolved in ABTS solution separately for 15 min at room temperature and the absorbance was then taken at 734 nm. The % radical scavenging activity (% RSA) was then measured by the equation mentioned below $$\%RSA=(1-A_f/A_0)\times 100\%$$

Where A₀ = Absorbance of free radical cation, A_f = Absorbance observed 15 min after incorporation of the complex.

2.5.3 FRAP assay

The antioxidant activity of the complex was also analysed using FRAP method according to the protocol described by Benzie and Strain (Benzie and Strain, 1996) which was developed by Griffin and Bhagooli (Griffin and Bhagooli, 2004). The antioxidant potential was measured by the ability of the complex to reduce Fe³⁺ ion. In this assay, the FRAP reagent was prepared by mixing TPTZ solution with FeCl₃ in freshly prepared acetate buffer. The bluish colour of the Fe³⁺ ion in TPTZ (2,4,6-Tripyridyl-S-triazine) solution changes its colour to dark as it reacts with the antioxidant substance and reduced to Fe²⁺ ion. The experiment was conduct by using different concentration of the taxifolin ruthenium-p-cymene complex as well as by busing free taxifolin. The absorbance of the solution was measure at 593 nm.

2.7.1 Cell culture

The lung cancer cell lines A549 and NCI-H460 were procured from National Centre for Cell Science (NCCS), University of Pune. The cancer cells were seeded in DMEM high glucose media, supplemented with 10 % FBS (Foetal Bovine Serum) along with the 1% antibiotic–antimycotic solution and incubated in 5% CO₂, 18–20% O_2, 95% relative humidity at 37 °C in the CO₂ incubator and sub-cultured for every 2 days.

2.7.2 MTT assay

The cell viability assay of the complex was conducted using MTT (3-(4, 5 dimethylthiozol-2-yl)-2, 5- diphenyltetrazolium bromide) reagent. The cells were treated with different concentrations of the complex (43.6, 87.3, 174.5, 349, 698.1 µM) and cell viability was observed at 48 h. The cell viability was investigated through mitochondrial succinate dehydrogenase which metabolizes MTT to produce formazan crystals. % Cell viability was measured as: $$\%\text{Cell}\text{viability}=\left[\text{Mean}\text{absorbance}\text{of}\text{treated}\text{cells}/\text{Mean}\text{absorbance}\text{of}\text{Untreated}\text{cells}\right]\times 100$$

The IC₅₀ (Half-maximal inhibitory concentration) value was determined by using linear regression equation i.e., y = mx + C, where, y = 50, m and C values were derived from the viability graph.

2.7.3 Assessment of olegonucleosomal fragmentation

The effect of the complex on the cancer cells was observed by staining the cells with fluorescent nuclear dye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). The cells were observed under ZEISS LSM 880 Fluorescence live cell imaging system (Confocal Microscopy) with filter cube with Excitation 358 nm and Emission 461 nm. Images were analysed by ZEN Blue Software and recorded.

2.7.4 Colony formation assay

The effect of the complex on the cancer cell proliferation and colony formation was evaluated by clonogenic assay after 48 h of incubation. The colonies of the cancer cells were stained with 0.5% crystal violet solution and visually quantified to distinguish the colonies which comprises > 50 cells/colony. The clonogenicity was then evaluated by using the formula below: $$\text{Clonogenicity}=\left(\text{Cloning}\text{number}/500\text{x}\right)\times 100$$

The experiment was conducted in triplicate manner.

2.7.5 Transwell migration assay

The cell invasion activity of the A549 and NCI-H460 cell lines were assessed by transwell migration assay. The cells were seeded (5 × 10⁴ cells/well) in the upper chamber of the transwell and supplemented with 0.1% FBS containing DMEM medium. The lower chamber was filled with DMEM, supplemented with 10% FBS. The cells of both chambers were treated with different concentrations of the complex. After 48 h of incubation the cells in the lower chamber was fixed by 4% paraformaldehyde and stained with 0.25% crystal violet. The wells were then scanned at four random fields and the numbers of invaded cells were counted using bright field microscope at 40X magnification.

2.7.6 Sphere formation assay

A549 and NCI-H460 cells were seeded in the ultra-low attachment multiwall plate at the concentration of 1 × 10⁴ cells/ml in 3dGRO spheroid medium. The cells were treated with various concentrations (IC₂₅, IC₅₀, and IC₇₅) of complex and incubated for 10 days at 37 °C in 5% CO_2. Then the pictures of the spheres were taken and the sphere numbers were counted in four random fields under the microscope.

2.7.7 Flow cytometry

The apoptosis assay and cell cycle distribution analysis were performed on the A549 and NCI-H460 cells after the treatment with the complex through flow cytometric analysis. The cells were suspended and the nuclear DNA was tagged with Annexin-V conjugated-FITC and propidium iodide (PI). Acquisition was done by using BD FACSCalibur and data were analysed by BD Cell Quest Pro Software (Version: 6.0).

2.7.8 Detection of caspase-3 expression

The expression of caspase-3 in A549 and NCI-H460 cells were investigated by flow cytometry. The cells were seeded in 6 well plate and treated with different concentrations (IC₂₅, IC₅₀, and IC₇₅) of the complex for 48 h. The cells were then treated with anti-rabbit caspase-3 polyclonal antibody at 37 °C in dark environment. The percentage of cells labeled with caspase-3 antibody was investigated by flow cytometric analysis.

2.7.9 Western blot

The expression of PI3K, EGFR, SOX2, OCT4, N-cadherin, E-cadherin on the A549 and NCI-H460 cells was detected by using the western blot analysis. The cells were treated with different concentrations (IC₂₅, IC₅₀, and IC₇₅) of the complex and incubated for 48 h. The total cell lysate from each group was obtained that was further resolved in SDS-PAGE (8–12%) electrophoresis and transferred to PVDF membrane. The membrane was then blocked with 5% non-fat dried milk in PBST and incubated with anti-rabbit PI3K, EGFR, SOX2, OCT4, N-cadherin and E-cadherin antibodies (1:500 dilution) overnight in humidified chamber. After incubation the membrane was treated with HRP conjugated goat anti-rabbit secondary antibodies and the expression was detected by chemiluminescent (ECL Western Blotting) kit with β-actin as loading control.

2.8.1 Animal

For the toxicity study Swiss albino mice (15–20 g), 6–8 weeks old of both sexes (females should be nulliparous and nonpregnant) and for chemotherapeutic study 6 weeks old male Balb/c mice (20–30 g) were purchased from registered animal breeder (Regd.no. −1458/PO/E/S/11/CPCSEA Dated- 12.05.2011). The animals were placed in polypropylene cages in 12 h light/dark cycle at 22 °C (±3°C) with relative humidity of about 50–58%. Animals were given free access to food and demineralized water. The experimental protocol was approved by the Institutional Animal Ethics Committee and by the Animal Regulatory Body of the Government (1458/PO/E/S/11/CPCSEA dated 12.05.2011) and experiments have been conducted in accordance to ARRIVE guidelines. The animals were acclimatized to the laboratory condition for one week prior to the initiation of the experiment.

2.8.2 Toxicity study

The toxicity study has been demonstrated in Supplementary file 1.

2.8.3 Experimental protocol

After the acclimatization period, the animals were randomly distributed into seven groups (I, II, III, IV, V, VI, VII) and each group consist of six animals. When the mice were 7–8 weeks old, all the animals (except group I) were given a single i.p. injection of 100 mg/kg Benzo(α) pyrene in 0.2 mL of corn oil to induce the lung cancer. Treatment with taxifolin ruthenium-p-cymene complex was started after 2 weeks of benzo(α)pyrene (B(α)P) induction and continued for 20 weeks. The experimental design is followed as:

Group I- Normal untreated vehicle control animals, Group II- B(α)P treated animals assigned as carcinogen control, Group III- B(α)P treated animals receives 50 mg/kg complex, Group IV- B(α)P treated animals receives 100 mg/kg complex, Group V- B(α)P treated animals receives 200 mg/kg complex, Group VI- B(α)P treated animals receives 50 mg/kg taxifolin, Group VII- B(α)P treated animals receives 50 mg/kg ruthenium-p-cymene.

The tumor volume was assessed via caliper two times per week and tumor sizes were calculated by using the following equation (Dethlefsen et al., 1968): $$\mathrm{T}\mathrm{u}\mathrm{m}\mathrm{o}\mathrm{r}\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}(\mathrm{V})=\mathrm{\pi }/6\times (\mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h})\times (\mathrm{w}\mathrm{i}\mathrm{d}\mathrm{t}\mathrm{h})2$$

The tumor volume and body weight were assessed two times per week over the course of the treatment. At the end of the experiment the animals were sacrificed using pentobarbital sodium. The lungs were excised, fixed in formalin, transferred to 70% alcohol, and examined for further experimentations.

2.8.4 Histopathology of lungs

An initial incision was made at the ventral midline which extends from the chin to the pubis. The sub cutis of the ventral and lateral thorax and abdomen was completely exposed. Subsequently, the rib cage was removed and the entire thoracic cavity was exposed. Then all dorsal attachments were cut and the tongue, esophagus, trachea, heart, and lungs were removed from the thoracic cavity. Heart and the other organs were separated from trachea and lungs and the whole tissue was fixed into 10% neutral buffered formalin (NBF) solution for 24 h. The excised tissues were dehydrated and embedded in paraffin wax. The tissues of 5 µm thickness were kept on slides and rehydrated by graded alcohol. The slides were stained using haematoxylin and eosin (H&E), which were observed under the light microscope for histological evaluation.

2.8.5 Antioxidant assay of lung

The in vivo antioxidant assay has been depicted in Supplementary file 1.

2.8.6 Immunohistochemistry

The excised lung tissues, mounted on poly-L-lysine coated glass slides were deparaffinized and treated with 3% H₂O_2. The blocking solution was added to the slides and then incubated with appropriately diluted (1:500) primary antibodies (anti-rabbit p53, Akt, mTOR, caspase-3, β-catenin) overnight at 4 °C in a humidified chamber. After incubation, the HRP conjugated goat anti-rabbit secondary antibodies were added to the slides for 2 h. The nuclear staining was done by DAB and hematoxylin was utilized for background staining. The slides were then observed under light microscope and the immune-positive cells were quantified by ImageJ software (version 1.8.0).

2.8.7 Cell proliferation assay

The excised lung tissues, mounted on poly-L-lysine coated glass slides were deparaffinized and treated with 3% H₂O_2. The blocking solution was added to the slides and then incubated with appropriately diluted (1:500) anti-rabbit Ki-67 antibody overnight at 4 °C. The slides were then treated with HRP conjugated goat anti-rabbit secondary antibody for 2 h. The nuclear staining was done by DAB and hematoxylin was used for background staining. The Ki-67 expression was examined under light microscope and the immune-positive cells were quantified by ImageJ software (version 1.8.0).

2.8.8 Apoptosis assay by TUNEL method

The occurrence of apoptotic events in the lung was assessed by TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) assay. The proteinase-K solution was added to the tissue sections and further treated with H₂O₂ to abrogate the peroxidase activity. poly-L-lysine coated slides of colon tissues were used for TUNEL assay. The slides were treated with proteinase-K followed by H₂O₂ to block activity of endogenous peroxidase. The tissue sections were supplemented with terminal deoxynucleotidyl transferase (TdT) accompanied by TdT and dUTP containing TdT reaction at 37 °C for an hour. Later on, saline citrate was added to abort. The slides were further incubated antidigoxigenin peroxidase (1:1000) at 37 °C for half an hour and finally stained with DAB and hematoxylin. The slides were then observed under microscope followed by quantification of the TUNEL-positive cells.

The labeling index (LI) designated the percentage of Ki-67-positive cells. The apoptotic index (AI) represented the percentage of TUNEL-positive cells to the total number of cells.

3.1.1 Bioactive properties

The bioactive properties of the complex was predicted based on the probable activity against different target receptors (GPCR ligand, Ion channel modulator, Kinase inhibitor, Nuclear receptor ligand, Protease inhibitor and Enzyme inhibitor) (Table 1) and the results showed the increasing positive values which represents the best active potential against target receptors. The complex showed highest positive value for nuclear receptor ligand (0.29) suggesting great binding affinity to the DNA.

3.1.2 Pharmacokinetic profile

The pharmacokinetic profile of the complex has been documented in the Tables 2, 3. According to the ADMET profile, the complex is a P-glycoprotein substrate and showed a great absorption from GI tract and through blood–brain-barrier. The toxicity profile of the complex reported that the complex is not carcinogenic but having high fish toxicity and honey bee toxicity probability. It is also non AMES toxic and having grade III acute oral toxicity i.e. slightly toxic and slightly irritating.

3.1.3 Molecular docking

Docking interaction of proteins with ligand molecules exhibited that lung cancer proteins such as PI3K, β-catenin and EGFR were strongly interacted with Taxifolin ruthenium-p-cymene complex (Table 4). The PI3K and EGFR protein structure were forming 4 hydrogen bonds with the interaction energy of −7.2 and −6.9 kcal/mol respectively within active site of amino acids such as Ser753, Glu849, Arg852 and Glu738, Met769, Gly772 respectively. β-catenin was forming 3 hydrogen bonds with the energy of −7.2 kcal/mol within active site of amino acids Arg474, Arg515 and Tyr654 (Fig. 1B, C, D).

3.2.1 UV–Visible spectroscopy

The absorption spectra of taxifolin represent two characteristics absorption band at 287 nm and 224 nm for band I and band II respectively. The absorption spectra of the taxifolin ruthenium-p-cymene complex showed significant shifts in their absorption band position (Fig. 2A). The band I shift from 287 nm to 295 nm and band II shifts from 224 nm to 227 nm. The occurrence of the maximum shift occurs for band I position (Fig. 2B).

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Fig. 2

(A) UV–visible spectra of taxifolin and taxifolin ruthenium-p-cymene complex (B) taxifolin and its different bands (I and II) related to UV–vis spectroscopy (C) Plot of absorbance vs. mole fraction of taxifolin at 241 nm (D) FTIR spectra of taxifolin and taxifolin ruthenium-p-cymene complex.

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3.2.2 Job’s method

The interaction between taxifolin and ruthenium-p-cymene dimer molecule has been evaluated by spectrophotometric analysis through Job’s method. The solution containing taxifolin and ruthenium-p-cymene dimer at a ratio of 1:1.5 or approximately 1:2 at a pH of 6.8 shows the maximum absorbance (0.854) (Fig. 2C).

3.2.3 FTIR study

The FTIR spectra of taxifolin and taxifolin ruthenium-p-cymene complex have been depicted in (Fig. 2D) and evaluated Table 5. A distinguishable peak at the region of 628.6 cm⁻¹ has been appeared in the IR spectra of the complex that is absent in the IR spectrum of taxifolin. This peak corresponds to the (Ru-O) bond due to the complex formation. The appearance of V (O—H) band at 3440.03 cm⁻¹ for taxifolin is shifted to 3451.19 cm⁻¹ for the complex. The V (C⚌O) stretching band is shifted from 1621.41 cm⁻¹ to 1634.64 cm⁻¹.

3.2.4 Mass spectroscopy

The mass spectrum of taxifolin ruthenium-p-cymene complex is depicted in (Fig. 3A). The highest molecular abundance was observed for the molecular ion peak with m/z value 573.0148. The other peaks that appeared in the mass spectrum were due to the fragmentation. The mechanism of the fragmentation has shown in the (Fig. 3B).

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Fig. 3

(A) Mass spectroscopy of taxifolin ruthenium-p-cymene complex (B) The fragmentation mechanism of the complex (C) ¹H NMR spectra of taxifolin ruthenium-p-cymene complex.

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3.2.5 NMR spectroscopy

6.96 (d, 1H, J = 1.8 Hz); 6.85 (dd, 1H, J = 7.8, 1.8 Hz); 6.8 (d, 1H, J = 7.8 Hz); 5.92 (d, 1H, J = 1.8 Hz); 5.89 (d, 1H, J = 2.4 Hz); 5.87 (d, 2H, J = 6 Hz); 5.64 (d, 2H, J = 6 Hz); 5.62 (d, 2H, J = 4.8 Hz); 5.49 (s, 1H); 5.42 (d, 1H, J = 5.4 Hz); 4.92 (d, 1H); 4.51 (d, 1H, J = 11.4 Hz); 2.77 (m, 1H); 2.19 (s, 3H); 1.21 (d, 6H). (Fig. 3C) depicted the ¹H NMR spectrum of the taxifolin ruthenium-p-cymene complex.

3.2.6 Scanning electron microscopy

The surface morphology of the complex is evaluated by scanning electron microscopy with various magnifications (50X, 4000X, 6000X and 10000X) which has been demonstrated in the (Fig. 4 A-D). The orthorhombic crystal structure of the complex can be clearly visualized from the SEM images with sharp edges and symmetrical structures.

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Fig. 4

Scanning electron microscopy (SEM) of the complex at (A) 50X (B) 4000X (C) 6000X (D) 10000X. (E) X-ray diffractogram of taxifolin ruthenium-p-cymene complex.

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3.2.7 X-ray diffractometry

X-ray Diffractogram of taxifolin ruthenium-p-cymene complex has been depicted in Fig. 4E. The X-ray Diffractogram of the complex exhibited three major high intensity peaks at three diffraction angles which include 31.74°, 45.51° and 56.54° with its relative intensity 100%, 74.35%, and 19.81% respectively. The maximum intensity was found at 31.74° and minimum intensity was recorded at 71.53°. The interplanar spacing (d) as well as their relative intensities in relation to acquired peaks at different diffraction angles has been depicted in Table 6. The crystallite sizes for the major three peaks have been recorded in Table 6. The average crystallite size of taxifolin ruthenium-p-cymene complex was found to be 49.81 nm.

3.3.1 DPPH activity

The absorption spectra of different DPPH solution containing different concentration of free taxifolin and the complex has been depicted in (Fig. 5A). The % radical scavenging activity (% RSA) of free taxifolin increases very slightly with increasing concentration and the maximum % RSA was detected to be 99.23%. But for the complex it showed a drastic increase in the % RSA with increasing concentration which reaches up to 98.85%. The complex shows less DPPH radical scavenging activity than the free taxifolin at same concentration.

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Fig. 5

Measurement of antioxidant potential of the complex by (A) DPPH method (B) ABTS method (C) FRAP method.

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3.3.2 ABTS activity

The ABTS assay of taxifolin and the complex has been depicted in the (Fig. 5B). It was found that the absorbance of ABTS solution decreases with increase in the concentration of the complex. Along with that the complex showed more radical scavenging activity than that of free taxifolin.

3.3.3 FRAP activity

The absorbance of free taxifolin and the complex of different concentrations have been estimated at 593 nm wavelength in presence of Fe³⁺-TPTZ. The complex exhibited enhanced antioxidant capacity than free taxifolin which has been depicted in the (Fig. 5C).

3.4.1 Induction of cytotoxicity through taxifolin ruthenium-p-cymene complex treatment

Taxifolin ruthenium-p-cymene complex inhibits the cell viability of A549 and NCI-H460 lung cancer cell lines in a dose dependent manner (Fig. 6A, B). The cell viability of A549 was found to be 91.28%, 72.35%, 49.82%, 32.98%, 13.52% and for NCI-H460 the cell viability was 76.25%, 51.64%, 34.75%, 20.88%, 12.10% at the concentrations of 43.6, 87.3, 174.5, 349, 698.1 µM respectively. From the above experiment the IC₅₀ value of the complex was calculated as 187.19 µM and 108.62 µM for A549 and NCI-H460 cell lines respectively. For chemotherapeutic study, the working concentrations of the complex were determined as IC₂₅, IC₅₀ and IC₇₅. The IC₂₅, IC₅₀ and IC₇₅ values for A549 was 93.6 µM, 187.19 µM, 280.78 µM and for NCI-H460 cell lines the values were 54.31 µM, 108.62 µM, 162.93 µM respectively.

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Fig. 6

Effect of taxifolin ruthenium-p-cymene complex on the cell viability of (A) A549 cells and (B) NCI-H460 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) A549 cells and (D) NCI-H460 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 A549 and NCI-H460 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 A549 cells. Quantification of (I) colony number, (J) colony size, and (K) plating efficiency for NCI-H460 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 ± SEM from different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey’s multiple comparison post-hoc analysis.

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3.4.2 Taxifolin ruthenium-p-cymene complex caused nuclear fragmentation

Taxifolin ruthenium-p-cymene complex induced nuclear fragmentation and membrane blebbing in A549 and NCI-H460 cell line (Fig. 6C, D). The maximum nuclear condensation was observed at the concentration of 280.78 µM and 162.93 µM in A549 and NCI-H460 cell line respectively.

3.4.3 Taxifolin ruthenium-p-cymene complex inhibited cellular proliferation

The treatment with the complex significantly inhibited the colony formation in both A549 and NCI-H460 cell lines (Fig. 6E). The cells treated with the complex exhibited the inhibition of colony number (Fig. 6F, I), colony size (Fig. 6G, J) and plating efficiency (PE) (Fig. 6H, K) as compared to the untreated cells in both the cell lines respectively. In comparison to the untreated cells the complex treated cells displayed lowest PE at the concentrations of 280.78 µM and 162.93 µM in A549 and NCI-H460 cell lines respectively.

3.4.4 Taxifolin ruthenium-p-cymene complex inhibited EMT

Taxifolin ruthenium-p-cymene complex treatment inhibited the invasive cells in both A549 and NCI-H460 cell lines (Fig. 7A, B) in a dose dependent manner. The western blot analysis (Fig. 7C) further confirmed the inhibition of EMT through the downregulation and upregulation of N-cadherin and E-cadherin expression respectively in both the cell lines.

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Fig. 7

(A) Effect of taxifolin ruthenium-p-cymene complex on the invasion of A549 and NCI-H460 cells at 48 h of incubation (B) Estimation of invasive cells per field (C) Western blot analysis of E-cadherin and N-cadherin on A549 and NCI-H460 cells (D) Effect of taxifolin ruthenium-p-cymene complex on the sphere formation of A549 and NCI-H460 cells at 10 days of incubation (E) Estimation of sphere diameter and (F) Number of spheres per 200 cells (G) Western blot analysis of SOX2 and OCT4 on A549 and NCI-H460 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 ± SEM from different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey’s multiple comparison post-hoc analysis.

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3.4.5 Taxifolin ruthenium-p-cymene complex inhibited stemness

Taxifolin ruthenium-p-cymene complex treated cells exhibited dose dependent inhibition of sphere formation (Fig. 7D) in respect to both the diameters of spheres (Fig. 7E) and number of spheres per 200 cells (Fig. 7F) in both the cell lines. In addition, the expressions of stem cell markers such as SOX2 and OCT4 were also investigated in both the cell lines through western blotting (Fig. 7G). The western blot analysis depicted marked downregulation in the expression of SOX2 and OCT4 followed by the complex treatment in both the cell lines.

3.4.6 Taxifolin ruthenium-p-cymene complex induce apoptosis and cell cycle arrest

The induction of apoptosis in A549 and NCI-H460 cell line due to the treatment with the complex for 48 h has been demonstrated in Fig. 8A and Fig. 9A respectively. The total percentage of apoptotic cells in A549 cells was found to be 90.87%, 92.42%, 97.07% and for NCI-H460 cells it was 33.28%, 42.87%, 82.62% at the concentration of IC₂₅, IC₅₀ and IC₇₅ of the complex respectively (Fig. 8B, 9B). The percentage of early apoptosis and late apoptosis was increased dose dependently in both the cell lines followed by the treatment with complex for 48 h (Fig. 8C, D and Fig. 9C, D). A significant decrease in the percentage of live cells was noted in the complex treated cells (Fig. 8E, 9E). The percentage of dead cells was also evaluated in the complex treated cells. The IC₅₀ of the complex exhibited maximum percentage of dead cells in both the cell lines (Fig. 8F, Fig. 9F).

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Fig. 8

(A) Quadrangular plots representing the Annexin V/PI expression in A549 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 A549 cells at different culture conditions. (G) Distribution of cell cycle phases in A549 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 A549 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 ± SEM from different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey’s multiple comparison post-hoc analysis.

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Fig. 9

(A) Quadrangular plots representing the Annexin V/PI expression in NCI-H460 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 NCI-H460 cells at different culture conditions. (G) Distribution of cell cycle phases in NCI-H460 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 NCI-H460 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 ± SEM from different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey’s multiple comparison post-hoc analysis.

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The distribution of cell cycle phases on untreated cells and complex treated cells in both the cell lines have been depicted in Fig. 8G, H and Fig. 9G, H. In A549 cell line, the cell cycle analysis subjected to sub G0/G1 phase (apoptotic phase) demonstrated that 1.46%, 6.09%, 13.97% and 9.61% of cell got arrested in untreated, IC_25, IC₅₀ and IC₇₅ group respectively. Similarly, in G0/G1 phase (growth Phase), 66.37%, 60.76%, 51.5% and 40.71% of cells, in S phase (synthetic phase), 3.79%, 6.99%, 4.68% and 13.6% of cells, in G2/M phase, 22.76%, 24.78%, 27.85% and 34.07% of cells got arrested in untreated, IC_25, IC₅₀ and IC₇₅ group respectively (Fig. 8G, H). In NCI-H460 cell line, the cell cycle analysis subjected to sub G0/G1 phase (apoptotic phase) demonstrated that 1.93%, 4.84%, 12.01% and 21.27% of cells got arrested in untreated, IC_25, IC₅₀ and IC₇₅ group respectively. Similarly, in G0/G1 phase (growth Phase), 70.69%, 57.55%, 47.56% and 30.41% of cells, in S phase (synthetic phase), 1.94%, 10.08%, 12% and 2.83% of cells, in G2/M phase, 20.72%, 24.44%, 27.36% and 42.29% of cells got arrested in untreated, IC_25, IC₅₀ and IC₇₅ group respectively (Fig. 9G, H).

3.4.7 Taxifolin ruthenium–p-cymene complex increased caspase-3 expression

The expression of caspase-3 in A549 and NCI-H460 cell line followed by the treatment with various concentration of taxifolin ruthenium-p-cymene complex for 48 h has been demonstrated in Fig. 10A, B respectively. The untreated group exhibited highest number of caspase-3 labeled cells in M1 quadrant. Whereas, the number of caspase-3 labeled cells in M2 quadrant was significantly increased followed by the drug treatment in both the cell lines.

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Fig. 10

Effect of taxifolin ruthenium-p-cymene complex on the expression of caspase-3 in (A) A549 cells and (B) NCI-H460 cells. (C) Western blot analysis of PI3K and EGFR on A549 and NCI-H460 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 ± SEM from different experiments in triplicate. The results were compared using ANOVA, followed by a Tukey’s multiple comparison post-hoc analysis.

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3.4.8 Effect of taxifolin ruthenium-p-cymene complex on the expression of PI3K, EGFR

The western blot analysis demonstrated that the treatment with the complex significantly inhibited the expression of PI3K and EGFR in the both A549 and NCI-H460 cell line as compared to the untreated cells (Fig. 10C).

3.5.1 Taxifolin ruthenium-p-cymene rehabilitates carcinogenicity and reduces tumor burden

The histopathological alterations of lung at the end of chemotherapeutic study have been depicted in Fig. 11. The normal control group (Fig. 11A) exhibited the normal morphological architecture of lungs which includes bronchiole (b), pulmonary alveoli (pa), alveolar sac (as), blood vessels (bv), pulmonary capillary (pc), columnar epithelium (ce), smooth muscle (sm). The carcinogen control group (Fig. 11B) showed thickened alveolar walls (t), congested alveolar cavity (cac), nodular lymphocytic collection (nlc), lymphocyte infiltration (li) in alveolar space, severe fibrosis (f) and formation of granuloma (g). On the other hand, taxifolin ruthenium-p-cymene treated group (Fig. 11C-E) restored the normal cellular architecture of the lung tissue. Though slight lymphocyte infiltration (li) and minor thickening of alveolar walls (t) were observed in 100 mg/kg group (Fig. 11D). The 200 mg/kg group (Fig. 11E) did not show any alterations in the B(α)P induced lung and successfully restored the morphological features of healthy lung. Whereas, 50 mg/kg taxifolin treated group (Fig. 11F) showed lymphocyte infiltration (li) in alveolar space, thickened alveolar walls (t) and congested alveolar cavity (cac). 50 mg/kg ruthenium-p-cymene treated group (Fig. 11G) demonstrated thickened alveolar walls (t), congested alveolar cavity (cac), nodular lymphocytic collection (nlc) and lymphocyte infiltration (li) in alveolar space. In addition, 200 mg/kg taxifolin ruthenium-p-cymene treatment significantly (p < 0.05) reduces the tumor volume and tumor weight as compared to the carcinogen control group (Fig. 11H, I).

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Fig. 11

Histopathological appearance of lung at 10X [inset 40X]. (A) Normal control group showed bronchiole (b), pulmonary alveoli (pa), alveolar sac (as), blood vessels (bv), pulmonary capillary (pc), columnar epithelium (ce), smooth muscle (sm). (B) Carcinogen control group depicted thickened alveolar walls (t), congested alveolar cavity (cac), nodular lymphocytic collection (nlc), lymphocyte infiltration (li) in alveolar space, severe fibrosis (f) and formation of granuloma (g). (C) 50 mg/kg taxifolin ruthenium-p-cymene complex treated group showed thickened alveolar walls (t), nodular lymphocytic collection (nlc) and lymphocyte infiltration (li) in alveolar space. (D) 100 mg/kg taxifolin ruthenium-p-cymene complex treated group showed thickened alveolar walls (t) and lymphocyte infiltration (li) in alveolar space. (E) 200 mg/kg taxifolin ruthenium-p-cymene complex treated group restored normal morphological architecture of carcinogen induced lung tissue. (F) 50 mg/kg taxifolin treated group depicted thickened alveolar walls (t), congested alveolar cavity (cac) and lymphocyte infiltration (li) in alveolar space. (G) 50 mg/kg ruthenium-p-cymene treated group depicted thickened alveolar walls (t), congested alveolar cavity (cac), nodular lymphocytic collection (nlc) and lymphocyte infiltration (li) in alveolar space. Effect of taxifolin ruthenium-p-cymene complex on the (H) tumor volume (mm³) and (I) tumor weight (g).

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3.5.2 Effect of taxifolin ruthenium-p-cymene complex on the expression of p53, caspase-3, Akt, mTOR and β-catenin

Fig. 12 and Table 7 depicted the expression profiling of p53, caspase-3, Akt, mTOR and β-catenin on the cancerous lung tissue of mice. A significantly low expression of p53 and caspase-3 was noted in the carcinogen control group (Fig. 12(i)B, (ii)B) in comparison to the normal control (Fig. 12(i)A, (ii)A) (p < 0.05). Whereas, a dose dependent increase in the expression of p53 and caspase-3 was exhibited in the complex treated group (Fig. 12(i) C-E, (ii) C-E). The 200 mg/kg group (Fig. 12(i)E, (ii)E) showed significant expression of p53 and caspase-3 on the alveolar wall and bronchiolar epithelial region. A moderate expression of p53 and caspase-3 was noted in 50 mg/kg taxifolin (Fig. 12(i)F, (ii)F) and 50 mg/kg ruthenium-p-cymene treated group (Fig. 12(i)G, (ii)G). Carcinogen control group exhibited significant upregulation of Akt (Fig. 12(iii)B), mTOR (Fig. 12(iv)B) and β-catenin (Fig. 12(v)B) expression on the alveolar wall, bronchiolar epithelial region with bronchiolar and vascular cytoplasmic localization in comparison to normal control (Fig. 12(iii)A-(v)A) (p < 0.05). The complex (50, 100, 200 mg/kg) treatment significantly (p < 0.05) downregulates the Akt (Fig. 12(iii)C-E), mTOR (Fig. 12(iv)C-E) and β-catenin (Fig. 12(v)C-E) expression. A significant expression of Akt, mTOR and β-catenin was also observed for 50 mg/kg taxifolin (Fig. 12(iii)F-(v)F) and 50 mg/kg ruthenium-p-cymene treated group (Fig. 12(iii)G-(v)G).

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Fig. 12.

The immunohistochemical analysis of [i] p53 [ii] caspase-3, [iii] Akt, [iv] mTOR, and [v] β-catenin expression in the lung tissues at 10X magnification of (A) normal control, (B) carcinogen control, (C) 50 mg/kg complex, (D) 100 mg/kg complex, (E) 200 mg/kg complex, (F) 50 mg/kg taxifolin, and (G) 50 mg/kg ruthenium-p-cymene treated group, represented by black arrows.

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3.5.3 Taxifolin ruthenium-p-cymene complex downregulates Ki-67 level

The cells with Ki-67 labeling showcased a discrete nuclear localization and brown stain which were used for the cellular quantification. The %Ki-67 labeled cells was measured by the labeling index (LI) (Table 8). The carcinogen control group (Fig. 13(i)B) showed highest expression of Ki-67 as compared to the normal control group (p < 0.05) (Fig. 13(i)A). The expression of Ki-67 was significantly downregulated in complex treated group (Fig. 13(i)C-E). Moreover, a significantly high cellular proliferation was also noted in the 50 mg/kg taxifolin (Fig. 13(i)F) and 50 mg/kg ruthenium-p-cymene (Fig. 13(i)G) treated group (p < 0.05).

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Fig. 13

The immunohistochemical analysis of [i] Ki-67 expression and [ii] TUNEL-positive apoptotic cells in the lung tissue at 40X magnification of (A) normal control, (B) carcinogen control, (C) 50 mg/kg complex, (D) 100 mg/kg complex, (E) 200 mg/kg complex, (F) 50 mg/kg taxifolin, and (G) 50 mg/kg ruthenium-p-cymene treated group, represented by black arrows. Approximately 700 cells were counted per field, 10 fields were examined per slide.

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3.5.4 Taxifolin ruthenium-p-cymene complex induces apoptosis

The apoptosis assay was conducted to evaluate the incidences of apoptosis in the lung cancer. The apoptotic cells were identified by their brown staining due to chromogen treatment (Fig. 13(ii)). The normal control group (Fig. 13(ii)A) denoted a moderate expression of TUNEL-positive cells. Whereas, significantly (p < 0.05) low number of apoptotic cells were noted in the carcinogen control (Fig. 13(ii)B) in comparison to normal control. A significantly (p < 0.05) high number of apoptotic cells were noted in the complex treated group (Fig. 13(ii)C-E) as compared to carcinogen control. The 50 mg/kg taxifolin (Fig. 13(ii)F) and 50 mg/kg ruthenium-p-cymene (Fig. 13(ii)G) treated group showed very few apoptotic cells. An average of 4–5 apoptotic cells in the field of 700 cells were identified in carcinogen control. On the other hand, 11–12 TUNEL-positive cells were quantified in 200 mg/kg complex treated group (Table 8).

The R value is expressed as the ratio of cell proliferation to apoptosis. In the carcinogen control group, the increased R value designated the enhanced proliferative activity of the cancer cells but treatment the complex significantly decreased R value due to induction of apoptosis and inhibition of cell proliferation.