Plasma protein affinity, antioxidant, and anti-lung cancer properties of O-methylated flavonol rhamnazin

2.1 Materials

Rhamnazin; 99 % (HPLC), HSA, Dulbecco's modified Eagle's medium (DMEM)/Ham's F12, fetal bovine serum (FBS), and tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (Saint Louis, MO, USA). Primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FITC Annexin V Apoptosis Detection Kit I was obtained from BD Biosciences (USA).

2.2 Sample preparation

The stock solution (100 μM) of HSA was freshly prepared in phosphate buffer (100 mM, pH 7.4). UV–visible spectroscopy was used to determine the protein concentration using ε_1cm^% of 5.30 at 280 nm (Alam, Chaturvedi et al. 2015). Rhamnazin solution (4 mM) was prepared freshly in phosphate buffer (100 mM, pH 7.4) or cell culture medium with the addition of 1 % DMSO.

2.3 Intrinsic fluorescence quenching study

The intrinsic fluorescence quenching study was done using a fluorescence spectrophotometer (RF-5301 Shimadzu, Japan). The analysis was done at three different temperatures of 298/310/315 K with a quartz cell of 0.1 cm path length. Excitation and emission slits were set at 5 and 10 nm, respectively, where the samples were excited at 280 nm and emission signals were read in the range of 300–420 nm. The titration of rhamnazin (5–40 μM) to HSA (5 μM) solution was performed at three different temperatures. The maximum fluorescence intensity was used to calculate the quenching, binding and thermodynamic parameters. All spectra were corrected against off-signals and inner filter effect (Ali and Al-Lohedan 2018).

2.4 Circular dichroism study

The far-UV CD study (200–260 nm) was performed using a JASCO-J 813 spectropolarimeter at room temperature. The quartz cell with a path length of 0.1 cm was used to detect the ellipticity changes in the HSA solution (5 µM) upon interaction with increasing concentrations of rhamnazin (5, 10, 15, 20, 30, 40). The CD data read from 200 to 260 nm were analyzed using CDNN.

2.5 Molecular docking study

Vina Autodock was used to perform the docking study. The 3D structures of HSA (PDB ID: 1AO6) and rhamnazin (PubChem CID: 5320945) were obtained from Protein Data Bank and PubChem, respectively. Water molecules as well as ions were removed, and hydrogen atoms and partial Kollman charges were added/assigned to rigid HSA. The grid size was set at 51 Å, 108 Å, 90 Å along X, Y, and Z axes, respectively. Spacing, mode number, energy range and exhaustiveness were fixed at 1, 10, 5 and 8, respectively.

2.6 Cell culture

Human proximal renal tubular epithelial cells (HK2, purchased from Procell Life Science and Technology Co., Wuhan, China) and lung adenocarcinoma A549 cells (purchased from ATCC) were cultured in DMEM/Ham's F12 and DMEM, respectively, supplemented with 10 % FBS, penicillin (1000 U/ml) and streptomycin (1000 U/ml) in 5 % CO₂ at 37 °C.

2.7 Treatments

A fixed concentration of H₂O₂ (0.5 mM) for 24 h was used to trigger apoptosis in HK2 cells based on the previous studies (Small, Morais et al. 2014, Shahzad, Small et al. 2016). Also, concentration-response assays (range 1–50 µM) were done to determine the maximum rhamnazin concentration that did not stimulate cytotoxicity in HK2 cells. Based on this pilot assay, a maximum safe concentration of 10 µM rhamnazin for HK2 cells at an incubation time of 24 h was determined. HK2 cells were then incubated for 24 h with H₂O₂ (0.5 mM) alone or co-incubated with H₂O₂ (0.5 mM) and rhamnazin (10 µM). For lung adenocarcinoma A549 cells, the cells were incubated with different concentrations of rhamnazin (1–50 µM) for 24 h.

2.8 MTT assay

MTT assay was done to assess the cell viability based on the previous study (Shahzad, Small et al. 2016). Briefly, the culture medium was replaced with 100 μL of fresh medium supplemented with MTT (0.5 mg/ml), incubated for 60 min, and followed by removal of the medium and replacement with 100 μL of DMSO. The absorbance was then determined at 570 nm using a microplate reader (RT-2100C Microplate Reader, China).

2.9 Determination of oxidative stress

To analyze the formation of intracellular ROS, the HK2 cells incubated with different samples were added by 2,7-dichlorodihydrofluorescein diacetate acetyl ester (H₂DCFDA) (10  mM) for 60  min at 37 °C. Then, the cells were washed with PBS and the fluorescence intensity of samples was detected using a fluorescence microplate reader (Bio-Tek Instruments, Winooski, USA) by excitation and emission wavelengths of 485 and 528  nm, respectively. CAT activity (Abcam, ab83464), SOD activity (Abcam, ab65354), and GSH content (Abcam, ab239709) in HK2 cells were examined following the product manual.

2.10 Quantitative real-time PCR

The relative expression of Bax, Bcl-2, and caspase-3 mRNA in HK2 cells and relative expression of Bax, Bcl-2, caspase-3, and p53 mRNA in A549 cell was assessed by quantitative real-time PCR following standard methods (Kong, Zhang et al. 2017, Yuan, Yang et al. 2021) Briefly, RNA from cells were extracted using Trizol kit (Invitrogen, CA, USA) and was reverse-transcribed using a Revert Aid First Strand cDNA Synthesis kit (Bio-Rad, CA, USA) following the product manual. The relative expression of mRNA was assessed through real-time PCR using SYBR green master mix. The primer sequences were used as reported in the previous studies (Kong, Zhang et al. 2017, Yuan, Yang et al. 2021). The data was analyzed through the 2^−ΔΔCt method and GAPDH was used as the target gene to normalize data.

2.11 Western blot analysis

Western blot analysis was done based on the previous study (Shahzad, Small et al. 2016). Briefly, the HK2 cells were washed, scraped with RIPA cell lysis buffer supplemented with phosphatase and protease inhibitors, sonicated, and centrifuged (3,000g, 20 min, 4 °C). Protein concentration was calculated based on the Bradford assay. Then protein samples were run on an SDS-polyacrylamide gel (12 %), transferred onto PVDF membrane, soaked in a blocking buffer for 60 min for 1 h, and incubated with primary antibodies (1:1000) 24 h at 4 °C. Afterwards, the membrane was washed and added by anti-rabbit HRP-conjugated secondary antibody (1:2000). Finally, protein bands were detected using enhanced chemiluminescence.

2.12 Apoptosis induction

The apoptotic A549 cells after exposure to rhamnazin (IC₅₀ concentration) for 24 h was assessed using FITC Annexin V Apoptosis Detection Kit I according to the manufacturer’s protocol and instructions reported previously (Akram, Al-Saffar et al. 2022). Briefly, after exposure, the A549 cells were collected, resuspended in the binding buffer, stained, and incubated in the dark place for 15 min. at room temperature. Cells were screened using flow cytometry (FACS ARIA III, BD Biosciences).

2.13 Determination of the level of expression of p53, Bcl-2, and Bax

Rhamnazin (IC₅₀ concentration)-treated and −untreated A549 cells were lysed. The level of expression of caspase-3, p53, Bax, and Bcl-2 was analyzed through colorimetric assays using human p53 ELISA kit (ab171571), human cleaved caspase-3 (Asp175) ELISA Kit (ab220655), human Bcl-2 ELISA kit (ab119506) and human Bax ELISA kit (ab199080), according to the protocols provided by the manufacturer. The expression of proteins was determined by detecting the absorbance at 450 nm using a microplate reader (RT-2100C Microplate Reader, China).

2.14 Statistical analysis

Data were presented as mean ± standard deviation of triplicate experiments for cellular assays (n = 3). Statistical analyses were analyzed with one-way ANOVA, followed by Tukey’s test. Statistical significance was set at P < 0.05.

3.1 Intrinsic fluorescence spectroscopy

Fluorescence spectroscopy is widely used to characterize ligand-macromolecule (protein/DNA) interaction mechanisms to provide details on the quenching mechanisms, binding parameters, as well as thermodynamic changes (Van de Weert and Stella 2011, dos Santos Rodrigues, Delgado et al. 2023). Principally, alterations in the intrinsic emission intensity of proteins are determined at the λmax. The addition of rhamnazin to HSA results in the fluorescence quenching of HSA (excitation at 280 nm) at three different temperatures of 298/310/315 K with different quenching rates (Fig. 1).

Close

Fig. 1

Fluorescence quenching study of HSA (5 µM) upon interaction with different concentrations of rhamnazin ranging from 5 to 40 µM at three different temperatures of 298/310/315 K.

Export to PPT

The observed changes in fluorescence intensity patterns at different temperatures could be attributed to the reorganization of intra and intermolecular forces that contribute to protein stability and folding.

3.1.1

3.1.1 Quenching mechanism

Titration of rhamnazin to the HSA solution can lead to the potential interaction of this ligand with HSA and may form a complex (Kenoth and Kamlekar 2022). HSA fluorescence intensity was found to be quenched upon the addition of rhamnazin, concentration-dependently (Fig. 1). Quenching of HSA fluorescence emission with the addition of rhamnazin was assessed by the well-known Stern–Volmer Eq. (1) (Leilabadi-Asl, Divsalar et al. 2024):

(1)

$$F_o/F=1+K_{\mathit{sv}}[C]$$ where F_o and F are defined as emission intensities of HSA before and after interaction with rhamnazin, respectively. K_SV and [C] are the Stern–Volmer constant and the concentration of rhamnazin, respectively.

The Eq. (1) in Fig. 2a2a was plotted to determine the K_SV at three different temperatures of 298/310/315 K.

Close

Fig. 2

(a) Stern-Volmer plot, (b) modified Stern-Volmer plot, (c) Van’t Hoff plot for the interaction of HSA (5 µM) with different concentrations of rhamnazin ranging from 5 to 40 µM at three different temperatures of 298/310/315 K.

Export to PPT

Fluorescence quenching can be categorized into three different classes: static, dynamic, or combined mechanisms, where they show different behaviors against temperature (Fossum, Johnson et al. 2023). The quenching constants under a dynamic quenching mechanism increase upon elevating temperatures, leading to large K_SV values. Nevertheless, higher temperatures can lead to a reduction in the stability of the formed complex, which can result in a decline in the K_SV values for static quenching. The formation of the static or dynamic complex is also can be verified using the calculation of quenching rate constant (k_q) values (Eq. (2)) (Roy, Tapan Kumar Das et al. 2016):

(2)

$$\mathit{kq}=Ksv/{\tau }_o$$ where τ₀ is known as a constant revealing the average lifetime of the fluorophore in the excited state (10^–8 s) (Roy, Tapan Kumar Das et al. 2016).

Fig. 2a displays the Stern–Volmer plot at three different temperatures of 298/310/315 K for the interaction of rhamnazin and HSA, and the resultant calculated K_SV and k_q values based on Eq. (1) and Eq. (2) are summarized in Table 1. Decreasing the K_SV values after increasing the system temperature revealed that the interaction of rhamnazin and HSA results in a static quenching mechanism. Furthermore, the order of magnitude of the k_q for rhamnazin-complex was found to be 10¹³, which was greatly higher than the bimolecular dynamic quenching rate constant (2 × 10¹⁰ L mol⁻¹ s⁻¹) (Patar, Jalan et al. 2024), indicating a static quenching of the binding of rhamnazin to HSA.

Table 1 Quenching, binding and thermodynamic parameters of HSA upon interaction with different concentrations of rhamnazin at 298/310/315 K.

T (K)	Ksv (105 L/mol)	kq (1013 L/mol s−1)	logKb (L/mol)	n	ΔH° (kJ/mol)	ΔS° (J/mol K−1)	ΔG° (kJ/mol)
298	2.12	2.12	7.64	1.53	147.922	641.258	−43.172
310	1.63	1.63	8.42	1.73	147.922	641.258	−50.867
315	0.65	0.65	9.12	1.97	147.922	641.258	−54.073

3.2 Circular dichroism study

Circular dichroism (CD) has been utilized as a sensitive spectroscopic technique to analyze the secondary structural change of protein in the presence of ligands (Tongkanarak, Loupiac et al. 2024). In order to explore the secondary structural alterations of HSA triggered by rhamnazin, a far-UV CD (200–260 nm) study was performed. In fact, upon interaction of rhamnazin, intermolecular forces involved in the stability of HSA structure may undergo some partial or substantial rearrangement, leading to conformational changes in the protein which is detected by monitoring the chirality changes (Tongkanarak, Loupiac et al. 2024). Fig. 3 depicts the far-UV CD signals of HSA without and with increasing concentrations of rhamnazin. It is evident that ellipticity changes of native HSA show two minima centered at 208 and 222 nm, representing the characteristic minima of typical α-helix structure (Tongkanarak, Loupiac et al. 2024).

Close

Fig. 3

Far-UV CD (200–260 nm) for the interaction of HSA (5 µM) with different concentrations of rhamnazin ranging from 5 to 40 µM at room temperature.

Export to PPT

Upon the addition of rhamnazin, a notable increase in the ellipticity changes at minima was detected without any significant shift, indicating an increase in the helical content and stability of HSA. In fact, it was deduced that the α-helix content of HSA was increased from 51.35 % to 59.77 % upon increasing the concentration of rhamnazin from 0 to 40 µM, respectively, determined by CDNN software.

3.3 Molecular docking study

A theoretical simulation tool, molecular docking study, can be used to evaluate the nature of the ligand-receptor interaction (Fan, Wang et al. 2024). A molecular docking study was run to support experimental data about binding affinity and forces as well as amino acid residues at the binding pocket (Fig. 4a). Outcomes are detected from this assay, indicating that the rhamnazin shows a strong interaction and binding affinity (ΔG°= − 33.89 kJ/mol) with HSA at the domain B (Fig. 4b). The rhamnazin conformer was revealed to interact with the active site residues R186, R145, R114, L112, E425, P110, L463, D108, V426, Y148, P147, H146, K190, S193, and F148 at domain B (Fig. 4c). Moreover, it was displayed that rhamnazin provided hydrophobic interaction with HSA through residue R145 (CB), whereas H146 is involved in the cation-pi interaction, D108, R145 (NH1), and R197 are involved in the formation of hydrogen bonding. Data obtained from the molecular modeling study is in good agreement with the experimental outcomes, indicating the formation of rhamnazin-HSA binding mostly through hydrophobic forces.

Close

Fig. 4

(a) Rhamnazin and HSA structures, (b) binding site of HSA upon interaction with rhamnazin, (c) amino acid residues in the binding pocket.

Export to PPT

3.4 Rhamnazin protects HK2 cells against H₂O₂ –induced cytotoxicity

Fig. 5a displays the data obtained from the MTT assay, expressed as a percentage of cell viability in HK2 cells. For rhamnazin, it was shown that the maximum safe concentration at 10 µM was achieved, while further increasing the rhamnazin concentration up to 50 µM induced a significant reduction in cell viability (Fig. 5a). Therefore, rhamnazin with a concentration of 10 µM was used for further studies. It was also shown that the HK2 cell viability was significantly (***P < 0.001) decreased after incubation of cells with 0.5 mM H₂O₂ for 24 h competed to control cell culture, as it was in good agreement with the previous study (Shahzad, Small et al. 2016). However, co-incubation of HK2 cells with rhamnazin with a concentration of 10 µM for 24 h markedly (^$$P < 0.01) restored the cell viability compared with the H₂O₂-treated sample (Fig. 5b). Based on these data, it can be deduced that the rhamnazin (10 µM) can protect cells against cytotoxicity induced by H₂O₂ (0.5 mM).

Close

Fig. 5

Cell viability was assessed by the MTT assay. (a) Incubation of HK2 cells with increasing concentrations of rhamnazin for 24 h. (b) Incubation of cells with H₂O₂ (0.5 mM), or co-incubation of HK2 cells with H₂O₂ (0.5 mM) and rhamnazin (10 µM) for 24 h. All data are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, relative to untreated negative control cells; ^$$P < 0.01 relative to H₂O₂-treated cells.

Export to PPT

3.5 Rhamnazin protects HK2 cells against H₂O₂–induced oxidative stress

The potential of rhamnazin to inhibit the generation of ROS was assessed using the DCFH-DA assay. As illustrated in Fig. 6a the significant intracellular ROS generation in the cells exposed to H₂O₂ (0.5 mM) was potentially inhibited upon co-incubation of cells with H₂O₂ (0.5 mM) and rhamnazin (10 µM) compared to the H₂O₂-treated group. Furthermore, the activities of the antioxidant enzymes, including CAT and SOD in HK2 cells were analyzed after 24 h of incubation with H₂O₂ (0.5 mM) and co-incubation with H₂O₂ (0.5 mM) and rhamnazin (10 µM). As expected, H₂O₂ decreased SOD (Fig. 6b) and CAT (Fig. 6c) activities in HK2 cells and co-incubation of cells with H₂O₂ (0.5 mM) and rhamnazin (10 µM) restored significantly the enzyme activities. Also, the analysis of GSH content (Fig. 6d) verified the antioxidant capacity of rhamnazin.

Close

Fig. 6

Antioxidant properties of rhamnazin after incubation of HK2 cells with H₂O₂ (0.5 mM) or co-incubation with H₂O₂ (0.5 mM) and rhamnazin (10 µM) for 24 h. (a) ROS activity, (b) SOD activity, (c) CAT activity, (d) GSH content. All data are expressed as means ± SD. **P < 0.01, ***P < 0.001, relative to untreated negative control cells; ^$P < 0.05, ^$$P < 0.01 relative to H₂O₂-treated cells.

Export to PPT

ROS production can be triggered by some mechanisms, including disruption of the mitochondrial membrane and activation of NADPH oxidase (Magnani and Mattevi 2019). ROS overproduction can result in some serious side effects against biomacromolecules, eventually triggering apoptosis (Juan, Pérez de la Lastra et al. 2021). As a result, any agent that reduces ROS production can be used as an antioxidant to counteract the cytotoxic effects of oxidative stress. In this study, we showed that rhamnazin can reduce ROS production by restoring the activity of antioxidant enzymes and biomolecules maintaining a redox state. It has been also shown that quercetin and rhamnazin can mitigate lipopolysaccharide-induced oxidative stress and inflammation in porcine intestinal cells (Karancsi, Kovács et al. 2022). Moreover, rhamnazin was shown to reduce traumatic brain injury through the inhibition of apoptosis and oxidative stress (Yang, Zhang et al. 2022).

3.6 Rhamnazin protects HK2 cells against H₂O₂–induced apoptosis

It was shown that incubation of HK2 cells with H₂O₂ (0.5 mM) induced apoptosis by upregulating caspase-3 and Bax mRNA and protein expression and downregulating Bcl-2 mRNA and protein expression (Fig. 7). However, rhamnazin (10 µM) co-incubation decreased Bax mRNA expression (Fig. 7a) and increased Bcl-2 mRNA expression (Fig. 7b). Also, it was shown that rhamnazin (10 µM) co-incubation mitigated the overexpression of caspase-3 mRNA induced by H₂O₂ (0.5 mM) in HK2 cells (Fig. 7c). Western blot analysis also verified that rhamnazin (10 µM) co-incubation resulted in the downregulation of apoptotic proteins (Bax and cleaved caspase-3) and upregulation of anti-apoptotic protein (Bcl-2) (Fig. 7d).

Close

Fig. 7

Antiapoptotic properties of rhamnazin after incubation of HK2 cells with H₂O₂ (0.5 mM) or co-incubation with H₂O₂ (0.5 mM) and rhamnazin (10 µM) for 24 h. (a) Bax mRNA expression assay, (b) Bcl-2 mRNA expression assay, (c) caspase-3 mRNA expression assay, (d) western blot analysis. All data are expressed as means ± SD. **P < 0.01, ***P < 0.001, relative to untreated negative control cells; ^$P < 0.05, ^$$P < 0.01 relative to H₂O₂-treated cells.

Export to PPT

In fact, this data revealed that rhamnazin can inhibit apoptosis through mitigation of ROS-associated Bax/Bcl-2 and caspase-3 signaling pathway stimulated by H₂O₂ in HK2 cells. Therefore, it may be discussed that rhamnazin can mitigate the induction of apoptosis in HK2 cells by regulating the mitochondrial pathway, decreasing Bax expression, and increasing Bcl-2 expression at both mRNA and protein levels as already shown by some other therapeutic compounds (Chang, Shen et al. 2014, Cao, Xu et al. 2021, Chen, Xie et al. 2023, Zhang, Tian et al. 2024). In accordance with our results, some other reports have shown that treatment with rhamnazin can control the induction of oxidative stress and apoptosis (Wu, Wang et al. 2022, Yang, Zhang et al. 2022, Ijaz, Mustafa et al. 2023).

3.7 Rhamnazin-induced cytotoxic effect in lung adenocarcinoma A549 cells

To assess the anti-lung cancer activity of rhamnazin, we assessed the MTT assay after interaction of lung adenocarcinoma A549 cells with different concentrations of rhamnazin for 24 h. The data suggested that rhamnazin induces cytotoxicity in lung adenocarcinoma A549 cells. Indeed, A549 cells were treated with different concentrations (1, 5, 10, 20, and 50 μM) of rhamnazin, and the cell viability decreased from 100 % to 98.35 %, 71.74 %, 62.27 %, 52.18 %, and 34.32 %, respectively. Then the calculated IC₅₀ concentration was determined to be 10.03 μM (Fig. 8a).

Close

Fig. 8

(a) Cytotoxic effects of rhamnazin on lung adenocarcinoma A549 cells after incubation for 24 h, (b) flow cytometry analysis of lung adenocarcinoma A549 cells after interaction with IC₅₀ concentration of rhamnazin (10.00 μM) for 24 h. All data are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, relative to untreated negative control cells.

Export to PPT

To detect whether the rhamnazin-induced cytotoxicity was mediated by apoptosis, the A549 cells were incubated with IC₅₀ concentrations of rhamnazin for 24 h, then the cells were subjected to flow cytometry.

As shown in Fig. 8b, the control cells (cells treated without rhamnazin) had 91.17 % lived cells with 6.21 % apoptotic cells. A549 cells treated with IC₅₀ concentration had a cell viability of 53.18 % upon treatment. The decrease in cell viability was accompanied by an elevation in the percentage of apoptotic cells to 27.15 % (Fig. 8b). Activation of the programmed cell death signaling pathway has been reported as an important mechanism for reducing tumor formation and associated progression; thus, several anticancer agents have been recognized to potentially trigger apoptosis as a primer way of effect for their cytotoxic behavior (Carneiro and El-Deiry 2020, Biswas, Roy et al. 2024). Accordingly, in the present study, we found that the pathway of rhamnazin-induced anticancer effects is mediated by the induction of apoptosis.

3.8 Rhamnazin-induced signaling pathway in lung adenocarcinoma A549 cells

Following DNA damage, the tumor suppressor gene p53 could regulate the expression of several downstream proteins such as Bax, Bcl-2, and caspases (Amaral, Xavier et al. 2010, Wei, Wang et al. 2023). A high ratio of Bax/Bcl-2 can lead to the permeabilization of the outer mitochondrial membrane, causing the release of cytochrome c and the corresponding activation of caspase-9 and -3, to trigger the process of apoptosis (Kaloni, Diepstraten et al. 2023, Saddam, Paul et al. 2024, Singh, Anand et al. 2024). Hence, it is presumed that rhamnazin triggers DNA damage in A549 cells, resulting in the overexpression of p53 which then lead to regulation of the Bax, Bcl-2, and caspases.

A significant increase in the p53 mRNA expression was detected in the A549 cells treated with rhamnazin after 24 h of incubation (Fig. 9a). Bax (Fig. 9b) and Bcl-2 (Fig. 9c) mRNA expression in the treatment with rhamnazin after 24 h of incubation increased and reduced, respectively, in comparison with the control. Also, there was a significant change in the upregulation of apoptotic caspase-3 mRNA in the treatment with rhamnazin after 24 h of incubation (Fig. 9 d).

Close

Fig. 9

Effect of IC₅₀ concentration of rhamnazin (10.00 μM) in lung adenocarcinoma A549 cells for 24 h on the (a) p53 mRNA expression, (b) Bax mRNA expression, (c) Bcl-2 mRNA expression, (d) caspase-3 (cas-3) mRNA expression, (e) p53 protein expression, (f) Bax protein expression, (g) Bcl-2 protein expression, (h) caspase-3 (cas-3) protein expression. All data are expressed as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, relative to untreated negative control cells.

Export to PPT

The same data was also detected in the p53, Bax, Bcl-2, and cleaved caspase-3 protein levels in the A549 cells treated with rhamnazin after 24 h of incubation (Fig. 9 e-h).

Therefore, the data demonstrated that the anticancer effect of rhamnazin on lung adenocarcinoma A549 cells is linked with its capability to trigger apoptosis in the cells. The apoptosis induction by rhamnazin in lung adenocarcinoma A549 cells might be based on a p53-dependent manner, via upregulation of apoptotic Bax and caspase-3 and down-regulation of anti-apoptotic Bcl-2. However, the cytotoxicity of rhamnazin should be evaluated against several normal cell lines in vitro and in vivo to recognize the selectivity of this compound on cancerous cells. Therefore, we might suggest that rhamnazin could be a potential O-methylated flavonol for the treatment of lung cancer in the future.