A herbal formulation inhibits growth and survival of lung cancer cells through DNA damage and apoptosis - in vitro and in vivo studies.

1
Introduction
Cancer continues to be one of the leading causes of death worldwide despite improvements in diagnostic techniques for its treatment. GLOBOCAN 2022 data showed 2,480,301 new cases and 1,817,172 fatalities due to lung cancer globally. To treat and manage cancer and lengthen the lives of cancer patients, surgery, radiation, chemotherapy, and immunotherapy are employed alone or in combination [1,2]. Moreover, chemotherapeutic drugs damage healthy cells and possess high toxicity, including nausea, diarrhea, vomiting, headache, constipation, mucositis, alopecia, etc [3]. To control the side effects of chemotherapies, alternative and complementary medicines could be an option for finding more effective and safe medicines derived from medicinal plants and also for anticancer drug development [4,5]. Plants and herbs are key in the deployment of novel anticancer drugs [6,7]. Alkaloids, polyphenols, flavonoids, terpenoids, and polysaccharides are used against a variety of cancers [[8], [9], [10]].
The Unani system of medicine is an ancient traditional medicine that originated in Greece [11]. Medicinal plants are used in Unani medicine, which describes cancer and its management [12]. Here, we discussed the plants and their extracts, which are the components of the SL formulation employed in the current investigation. The SL formulation, a fine powder, consists of 13 ingredients described here, although Lazurite gemstone has not been well studied. Plant extracts of Cuscuta epithymum and Operculina turpethum have shown anticancer properties by disrupting cell growth and migration, and promoting apoptosis in human cancer cells [13,14]. Melissa axillaris is not well explored for its anticancer properties while Lavandula stoechas is an evergreen shrub known for its anti-inflammatory and cytotoxic effects against human cancers [15]. The aqueous and organic extracts of Cassia angustifolia contain quercimeritrin, scutellarein, and rutin, which are known to have antioxidant and anticancer activities [8]. The ethanolic leaf extract of another medicinal plant Terminalia chebula contains a high concentration of phenolics/flavonoids and shows effective cytotoxic activities [16]. Polypodium vulgare is a fern with a high amount of shikimic acid, caffeoylquinic acid derivatives, epicatechin, and catechin, and has anticancer activities against human cancers [17]. Rheum emodi is a leafy herb that shows diuretic, antioxidant, antibacterial, antifungal, and anticancer activities and protects DNA from damage by UV [6]. Rosa damascene, an ornamental plant, possesses antiproliferative, anticlonogenic, and antimigratory properties against human cancer [18]. In the Unani system of medicine, Agaricus albus is traditionally referred to as Ghariqoon, exhibits anticancer potential through cytotoxic effects and free radical scavenging activity [19]. Our previous study revealed that Habb-e-Ustukhuddus, a Unani herbal formulation, induced apoptosis and inhibited cell migration potential in lung and breast cancer cell lines with no adverse effects on mice [20].
In the present study, we investigated Safoof Lajward (SL), a Unani Pharmacopoeial drug, for its in vivo toxicity, hepatomodulatory enzymes, antioxidant parameters, and in vitro and in vivo efficacy and associated mechanisms against lung tumorigenesis. Safoof is called the powder medicine. The SL extract was also chemically analysed by HPLC and GC-MS.

2
Materials and methods
2.1
Reagents and antibodies
The pharmacopoeial composition of Safoof Lajward is given in Table 1 and was obtained from Hamdard Laboratories, India, which maintains authentication of the components present in the formulation. The reagents used for HPLC and GC-MS analyses, and cell culture were of analytical and molecular biology grades and were from Sigma Aldrich, USA. Trypan blue dye from HiMedia, Annexin V- FITC/PI kit from Invitrogen, JC-1 dye from Cayman Chemical, and Matrigel from Corning. Primary antibodies for Bcl-2, #2872; Bax, #2772; Chk1, #2360; Chk2, #3440; Rad51, #82263; PCNA, #2586; Ki-67, #9449; Cleaved caspase3, # 9664; Cleaved PARP, #5625; and Beta-actin, #A3853, and secondary antibodies, anti-mouse, #7076 and anti-rabbit, #7074P2, were acquired from Cell Signaling Technology (CST), USA.

2.2
SL extract
SL formulation was added to methanol with a concentration of 0.1 mg/ml following overnight shaking at 37 °C. The solution was sonicated for 30 min at 37 °C and spun for 15 min at 5000 rpm and the supernatant was taken. After that, the solution was dried at 40 °C in the oven and the extract was dissolved in autoclaved deionized water for the studies.

2.3
GC-MS analysis
GC-MS (Gas chromatography and mass spectrum) analysis of a methanolic extract of SL was conducted at the AIRF by GC-MS QP-2010 ultra-model at Jawaharlal Nehru University, India. As the carrier gas, helium was used at a steady flow rate of 1 ml/min (split ratio = 10:0). The oven was set to start at 50 °C and finish at 300 °C with a hold duration of 17 min. The temperatures of the injection, ion source, and interface were 260 °C, 220 °C, and 270 °C, respectively. The solvent cut time was 4.50 min and the total GC running time was 45 min.

2.4
HPLC analysis
Utilizing a C18 reverse-phase chromatography column, the methanolic extract of SL was subjected to HPLC (High-Performance Liquid Chromatography) analysis. % aqueous acetic acid with methanol served as the phase of gradient mobility. Separation of the compounds in SL was carried out with a 20 μl injection volume and a 1 ml/min flow rate at a wavelength of 278 nm.

2.5
In vivo toxicity study
Male C57BL/6 of 6-week old mice were used for the toxicity study. Approval for this study was given by the IAEC, JNU, New Delhi, India. The mice were housed and maintained at CLAR, JNU, under controlled room temperature and humidity throughout the experiment. Six mice (n = 6/group) were taken in each group for the study and given SL (50 and 100 mg/kg) orally for fifteen days daily after being randomly divided into groups. The SL formulation was suspended in saline and fed by oral gavage. Doxorubicin (Dox) was used as positive control and given to the mice with 5 mg/kg through intraperitoneal injection on the first, sixth, and eleventh day of treatment. On every other day, the body weight of mice was noted along with diet and water consumption as described earlier [21]. A priori power calculation using G∗Power 3.1 (power = 0.80, α = 0.05, effect size d = 0.9) indicated that a minimum of n = 6 mice per group were required which was extrapolated from the study [22,23]. Group comparisons were performed using one way ANOVA. To assess the robustness of our findings, we reported both p-values and effect size estimates.

2.6
Hepatotoxicity studies
Following mouse euthanasia in the above experiment, the entire liver was promptly perfused using saline and washed with 150 mM Tris-KCl buffer (PH 7.4) on ice. Further, the liver was homogenized in 150 mM Tris-KCl buffer (10 %), and immediately performed sulfhydryl group (-SH) assay and the remaining homogenate was used for fractions isolation (cytosolic and microsomal) as mentioned in Ref. [21]. Serum from mice was used for SGPT or serum glutamic pyruvic transaminase and SGOT or serum glutamate oxaloacetate transaminase tests as mentioned in Ref. [24]. TBARS or thiobarbituric acid reactive compounds were used to determine the amount of microsomal peroxidative damage as mentioned in Ref. [25]. The level of GSH or reduced glutathione was determined as the total amount of non-protein sulfhydryl groups as mentioned in Ref. [21]. SOD or superoxide dismutase activity was measured at the wavelength of 420 nm in the cytosolic fraction [26]. The catalase-specific activity was determined in the cytosolic fraction at the wavelength of 240 nm as mentioned in Ref. [27]. Both NADH cytochrome b5 reductase and NADPH cytochrome P450 reductase assays were performed with microsomal fraction as described in Ref. [28]. GST or glutathione S-transferase-specific activity was determined in cytosolic fraction as described earlier [29]. Bradford's reagent was used to measure the total protein level at a wavelength of 595 nm, using BSA as a reference.

2.7
Human cancer cell culture
Lung cancer A549 and H1299 cells were procured from NCCS Pune, India. The culture media used for these cells were DMEM (Dulbecco modified Eagle's medium) with 10 % fetal bovine serum (FBS) (Gibco Life Technology, USA) and antibiotic solution (0.1 %) containing penicillin, streptomycin, and amphotericin (HiMedia). The cell culture was maintained in an incubator with 37 °C and 5 % CO2 up to 15 passages for the experiments.

2.8
Trypan blue staining assay
Trypan blue staining assay was employed to assess the effect of SL on the survival of the A549 and H1299 cells, as previously mentioned [20]. In brief, six-well plates were used to seed cells and treated with SL (50–400 μg/ml) for 48 h, and after trypsinization, all of the cells were collected and suspended in 1X phosphate buffer saline. Trypan blue stain was used to stain the cells and then the number of living and dead cells was determined using a hemocytometer under a phase contrast microscope.

2.9
Clonogenic assay
To assess the clonogenicity of lung cancer cells, A549 and H1299 cells were exposed to SL at concentrations ranging from 50 to 100 μg/ml. The culture medium was replaced every three days. On the eighth day, the cells were fixed using methanol, stained with crystal violet, and washed with 1x phosphate buffer saline before being photographed and counted [20].

2.10
Cell cycle phase distribution analysis
Cell cycle phase distribution was determined on A549 cells that were treated with SL (100–400 μg/ml) for 48 h. Total cells were collected and analysed in the flow cytometer (FACS Aria III, BD Biosciences, USA) as mentioned in Ref. [30].

2.11
Annexin V-FITC/PI staining
After A549 cells were subjected to 100–400 μg/ml of SL for 48 h, the total cells were processed following the Annexin V-FITC apoptosis detection kit manufacturer's protocol (BD Pharmingen, USA) and analysed in the flow cytometer [20].

2.12
Mitochondrial membrane potential assay
After A549 cells were treated with 200–400 μg/ml of SL for 48 h, total cells were stained with 1.25 μl of JC-1 (1 mg/ml stock) for 15 min at 37 °C in dark condition. Cells were washed with 1x phosphate buffer saline following the staining to remove extra JC-1 stain and then analysed the cells in a flow cytometer to examine the JC-1 monomer/dimer ratio [20].

2.13
Acridine orange/ethidium bromide (AO/EB) assay
After A549 cells were treated with 200–400 μg/ml of SL for 48 h total cells were mixed in 1x phosphate buffer saline. The cell suspension of 25 μl was gently mixed with 1 μl of AO/EB cocktail and photographs were taken at 200x magnification under the fluorescence microscope.

2.14
DNA damage analysis by DNA fragmentation assay
Cells undergoing apoptosis can be analysed by DNA damage detection on agarose gel electrophoresis. A549 cells were treated with 200–400 μg/ml of SL for 48 h, and then total cells were collected and processed for the extraction and determination of cellular DNA damage as described earlier [31]. DNA electrophoresis was performed on 1 % agarose gel and the DNA was visualized and pictured by using the UV gel doc system (Applied Biosystems).

2.15
Western blotting
A549 cells were subjected to 200–400 μg/ml of SL for 48 h. A non-denaturing lysis buffer was used to prepare the total cell lysate and the Bradford method was used to estimate the amount of protein. Proteins were transferred to the PVDF membrane following the SDS-PAGE run of cell lysates and they were subsequently blocked using a 5 % blocking buffer as mentioned in Ref. [32]. A specific primary antibody was applied to the membrane and it was then treated for 1–2 h at room temperature with secondary antibodies. Additionally, the membrane was prepared for ECL detection, and ImageJ software was used to quantify the band intensity on the film.

2.16
Lung cancer xenograft mouse model
The in vivo effectiveness of SL was investigated in male athymic nude mice of six weeks old. As previously described, 5x106/200 μL of the A549 cell suspension was subcutaneously injected into each mouse's right flank after the cells were suspended in serum-free DMEM media and matrigel (1:1) [33]. Upon the tumor volume reaching approximately 200 mm3, the mice were divided randomly into two groups, each with five mice (n = 5/group). Then, throughout the 20-day treatment, oral doses of saline (control group) and SL formulation (100 mg/kg) were given and the tumor was measured with a Vernier caliper every 2nd day. Also, the weight of mice, water, and diet consumed by mice were measured on every 2nd day. For the tumor volume measurement following formula was used: tumor volume (mm3) = length x width2 x 0.5. A priori power calculation using G∗Power 3.1 (power = 0.80, α = 0.05, effect size d = 2.1) revealed a minimum of 5 mice per group was required according to the sample size informed by the study [[22], [34]]. Group comparisons were performed using the Student's t-test (two-tailed). We reported both p-values and effect size estimates to assess the robustness of our findings.

2.17
H&E and IHC analysis of tumor xenograft
Tumor tissues from nude mice were used for immunohistochemical analysis as mentioned previously [[35], [36]]. Briefly, tumor samples were fixed with neutral buffered formalin buffer (10 %) at 4 °C for 24 h and cut 5 μm thick paraffin-embedded tumor sections followed by hematoxylin and eosin (H&E) staining and images were photographed at 200x magnification. To identify the apoptotic markers cleaved PARP and caspase 3, as well as the cell proliferation marker Ki-67, through immunohistochemistry (IHC), tumor sections were processed for antigen retrieval and quenching of the endogenous peroxidase activity by 0.3 % H2O2. Next, sections were incubated with 5 % horse serum for 1 h at 20–25 °C and subsequently probed with primary antibodies against Ki-67, cleaved caspase 3, and cleaved PARP overnight at 4 °C. Next, sections were treated with biotinylated secondary antibody (Sigma, MO) for 2 h and after that, diaminobenzidine (DAB) (Sigma, MO) was added and incubated for 30 min with streptavidin peroxidase to show the peroxidase activity, counterstained with hematoxylin, and then mounted. Finally, images were photographed at 400x magnification and quantified.

2.18
Statistical analysis
Graph Pad Prism 8 software was used to perform statistical analysis and compare two groups using Student's t-test, whereas multiple groups comparison was done by one-way ANOVA test followed by Tukey as post hoc test. The difference was considered statistically significant at P < 0.05.

3
Results
3.1
SL analyses by HPLC and GC-MS
The well-known GC-MS technique which combines mass spectrometry and gas-liquid chromatography is used to profile the secondary metabolites found in plants. The GC-MS analysis of SL revealed the presence of various compounds as shown in Fig. 1A and B. The standard library of the National Institute of Standards and Technology was used to determine the chemicals in SL. A total of sixty chemicals were found, out of which the most abundant ten compounds are shown in Fig. 1B. On the other hand, HPLC is one of the convenient and comprehensive analytical methods to identify, quantify, and purify the phytoconstituents present in medicinal plants [37]. We also did HPLC profiling of SL to identify the presence of active components as shown in Fig. 1C and D. We found that SL (100 mg/ml) had epicatechin (1257 μg/ml) followed by epicatechin gallate (399 μg/ml) and vanillin (302 μg/ml) in abundant concentration (Fig. 1D).

3.2
Effects of oral SL on mice
3.2.1
Systemic and hepatic toxicity studies
SL (50 and 100 mg/kg) was orally given to mice daily for fifteen days. Mice's body weight, feed consumption, and water intake did not show any significantly change during the treatment (Fig. 2A–C). Further, the effects of SL on the liver were measured by analysing cytosolic and microsomal fractions.
Intraperitoneal injection of doxorubicin, a chemotherapeutic drug and positive control for toxicity, caused liver damage significantly by increasing the levels of SGOT and SGPT (P < 0.001), while SL did not show any significant impact on these enzymes (Fig. 2D and E). Analysis of peroxidative damage was carried out by lipid peroxidation assay using liver homogenate microsomal fraction. Malondialdehyde (MDA) formation, an indicator of lipid peroxidation, was increased with doxorubicin treatment (P < 0.001), however, SL treatment to the mice kept the MDA level near to control with no significant change (Fig. 2F).

3.2.2
Antioxidant enzyme studies
GSH, SOD, and catalase are the antioxidant enzymes that play a crucial role in the body's defense system. GSH is well known for preserving redox equilibrium and protecting cells from oxidative damage. At the same time, the SL treatment in the mice did not alter the GSH level while doxorubicin caused a 21 % (P < 0.05) reduction in GSH level (Figs. 2G) and 30 % (P < 0.01) reduction in SOD activity (Fig. 2H). Whereas the specific activity of SOD and catalase levels were not altered with SL treatment (Fig. 2H and I). Consequently, SL had no negative effects on the mice's levels of antioxidant enzymes.

3.2.3
Drug-metabolizing enzymes
The microsomal fraction of mouse liver homogenate was used to assess the drug-metabolizing enzyme (phase I) cytochrome P450 reductase (Cyt P450R) and cytochrome b5 reductase (Cyt b5R) and SL treatment (100 mg/kg) to the mice significantly increased the Cyt P450R specific activity by 1.9-fold (P < 0.01) in comparison with the control (Fig. 2J). SL treatment to the mice induced Cyt b5R specific activity by 1.7-fold (P < 0.01) (Fig. 2K). In the cytosolic fraction of liver homogenate, the modulatory impact of SL (100 mg/kg) on the drug-metabolizing enzyme (phase II) glutathione S-transferase (GST) was evaluated by 1.8 fold (P < 0.05) (Fig. 2L) and doxorubicin treatment revealed an increase in GST specific activity from the basal level (Fig. 2L).

3.3
SL inhibited cell survival and clonogenicity of lung cancer cells
Lung cancer cells were used in trypan blue staining experiment to assess the anticancer potential of SL and it was observed that SL (50–400 μg/ml) decreased the total live cells significantly (P < 0.05–0.001) (Fig. 3A and C) and caused cell death by 3–37 % (P < 0.05–0.001) in both A549 and H1299 cells (Fig. 3B and D). SL (50–100 μg/ml) also strongly inhibited the colony formation of A549 and H1299 cells (20–100 %, P < 0.001) (Fig. 3E–H). These findings revealed that SL extract significantly decreases human lung cancer cells' ability to proliferate, survive, and become colonies. However, SL powder without extraction did not show any considerable effect on the viability of A549 cells (data not shown). Therefore, the methanolic extract of SL was used for further studies with A549 cells.

3.4
SL promoted sub-G1 cancer cell accumulation
To examine SL growth inhibitory mechanisms in lung cancer A549 cells, the distribution of cell cycle phases was analysed in the flow cytometer. We found sub-G1 cell accumulation by 19–24 % (P < 0.01–0.001) after 48 h of treatment with 200–400 μg/ml of SL in A549 cells (Fig. 4A and B).

3.5
SL caused apoptosis and mitochondrial membrane depolarization in cancer cells
The treatment with SL increased the number of dead cells, allowing us to determine if the death was caused by apoptosis. Annexin V-FITC/PI analysis showed a dose-dependent increase in the apoptotic cells by 2–47 fold (P < 0.01) at 100–400 μg/ml of SL in A549 cells (Fig. 5A and B). One of the signs of apoptosis induction is also the loss or alteration of mitochondrial membrane potential, which was determined by the JC-1 stain that makes dimer in healthy cells. As opposed to this, in apoptotic cells, it exits the mitochondria as a result of diminished mitochondrial potential and breaks into the monomers [38]. After 48 h of treatment with SL (200–400 μg/ml), A549 cells evidenced the dissociation of dimer to monomer form of JC-1 dye and an increase in the monomer/dimer ratio of up to 2–7 fold (P < 0.01–0.001) (Fig. 5C and D). We also looked into the expression of apoptosis-associated proteins, which are produced by mitochondria and promote apoptosis. Among these molecules are pro-apoptotic Bax and anti-apoptotic Bcl-2. We observed that A549 cells treated with 200–400 μg/ml of SL for 48 h showed an increased expression of Bax protein by 1.52–1.57 fold and a decreased expression of Bcl-2 protein by 0.60 to 0.06 fold change (Fig. 5E). These results indicated the involvement of mitochondrial-mediated apoptosis as one of the processes underlying SL-induced A549 cell death.

3.6
SL induced DNA damage in cancer cells
Acridine orange (AO) is a vital dye that is very spectrally similar to fluorescein and intercalates between the nucleotides. It can penetrate live membranes and can stain both live and dead cells. DNA is stained red by the intercalating dye ethidium bromide (EB), which enters dead cells that have lost their ability to selectively permeabilize their membrane. Live cells appear as fluorescent green upon being excited by blue light, and apoptotic cells with compromised membrane integrity allow the incorporation of EB and therefore look reddish or yellow-orange with green fluorescence after binding to fragmented chromatin. Here, A549 cells treated with SL (200 and 400 μg/ml) showed a significant increase in apoptotic cells (53–62 %, P < 0.001) after 48 h compared to the control group (Fig. 6A and B). The fluorescent cells are photographed at 200x magnification (Fig. 6A) and quantified (Fig. 6B).

3.7
SL caused DNA fragmentation and decreased DNA damage-repair proteins in cancer cells
DNA fragmentation at the inter-nucleosomal linker regions by caspase-activated DNases is one of the characteristic hallmarks of apoptosis. Therefore, to further confirm the apoptotic effect of SL, a DNA fragmentation assay was performed. The electrophoresis of DNA from A549 cells was treated with 200–400 μg/ml of SL for 48 h and showed induced DNA fragmentation and maximum effect was observed at the higher dose of SL as seen from the ladder pattern (Fig. 6C). Further, western blotting was used to analyze the expression levels of proteins related to DNA damage and repair. We found that A549 cells treated with 200–400 μg/ml of SL for 48 h showed no expression of Chk1, however, Chk2 expression was reduced by 0.15 to 0 fold and Rad51 expression was decreased by 0.09 to 0.01 fold, suggesting that SL inhibits the DNA repair pathway in lung cancer cells (Fig. 6D).

3.8
SL inhibited human lung tumor growth in nude mice
To further explore the anticancer effect of SL in vivo, we used 6-week old male athymic nude mice, and subcutaneous injection of A549 cells was given on the right flank of the nude mice. When tumor size reached ∼200 mm3, mice were orally gavaged with saline or 100 mg/kg of SL formulation daily for 20 days. We observed that the tumor growth was suppressed significantly by 45 % (P < 0.01) (Fig. 7A) and tumor weight by 53 % (P < 0.05) (Fig. 7C) in the SL treated group as compared to the control group. The tumor images at the end of the experiment are shown in Fig. 7B. Additionally, throughout the experiment, the SL treatment in nude mice did not significantly alter their diet, water intake, or body weight (Fig. 7D–F).

3.9
SL inhibited cell proliferation, induced apoptosis, and decreased DNA repair proteins in tumor
H&E staining of tumor xenograft tissues showed reduced tumor size and lesser cellular density in SL treated group in comparison with the control group (Fig. 8A). Further, we performed IHC analysis of the tumor samples for the cell proliferation marker, Ki-67, where SL (100 mg/kg) treated group of tumors showed 19 % (P < 0.001) Ki-67 positive cells than the control group showing 53 % (Fig. 8A and B). Further, an increased number of positive cells were observed for cleaved caspase 3 (40 %, P < 0.001) and cleaved PARP (45 %, P < 0.001) in SL treated group of tumors than the control group showing 7 % and 8 %, respectively (Fig. 8A–C-D). Overall, with no negative health effects in nude mice, SL was shown to have a potent anticancer impact against lung cancers generated by A549 cells. We further analysed xenograft tumor tissues by Western blot and observed that the decreased protein expression for PCNA (78 %, P < 0.05), Chk1 (61 %, P < 0.05), Chk2 (68 %, P < 0.05) and Rad51 (65 %, P < 0.001) whereas the ratio of Bax/Bcl-2 was enhanced by 26 fold (P < 0.05) in SL-treated group as compared to the control group (Fig. 8E and F). These observations supported the translation of in vitro cell culture results into the in vivo tumor model of lung cancer.

4
Discussion
The notion of humors, which holds that the body consists of four humors: sawda (black bile), safra (yellow bile), balgham (phlegm), and dam (blood), is the basis of Unani medicine, while science acknowledges inflammation as a critical factor in the growth of tumors [39,40]. The Safoof Lajward (SL) used for the study is extensively mentioned for its anti-inflammatory (Mohallil-e-Warm) nature and managing black bile (Mukhrij-e-Sawda) in Unani medicine. However, no study reports that SL possesses anticancer properties. Thus, using human lung cancer cells, we investigated the anticancer effectiveness of SL and related mechanisms for the first time in vitro and in vivo.
We performed a chemical profiling of SL to evaluate its phytochemical constituents and contents through GC-MS as well as HPLC. SL profiling using GC-MS revealed the various chemicals present, including fatty acid hexadecanoic acid, which is known for its anti-bacterial and anti-fungal, anti-inflammatory, antioxidant, antiandrogenic, anticancer, and anti-tumor properties [41]. Lazurite present in the powdered formulation may not be available in the methanoloic extract. Another substance detected was 9,12-Octadecadienoyl chloride, which has been linked to blocking AT1 receptors, which are associated with the microvascular anomaly in diabetic retinopathy [42]. The other prevalent compound in SL was methyl jasmonate, which is a lipid derivative and has anti-inflammatory and anticancer effects. The octadecanoic acid present in SL also exhibits cancer-preventive effects [43]. The methanolic extract of SL included several anticancer substances, as revealed by the HPLC chromatogram. It has been shown that epicatechin, a phenol that is most abundant in SL, has antiproliferative, antiangiogenic, antioxidant, and apoptotic properties toward cancerous cells [44,45]. For its potential to treat cancer, resveratrol, another polyphenol found in SL, is currently undergoing clinical trials [46]. The additional bioactive substances found in SL, including salicin, vanillin, acetylsalicylic acid, and epicatechin gallate [47] are reported to show anticancer properties. Thus, the profiling of SL helped us to understand the broad chemical composition and thereby exert the anticipated anticancer properties.
Next, we studied the modulatory effects of orally administered SL on hepatic enzymes and associated toxicity using a mouse model. To evaluate the chemopreventive potential of SL further, the enzymes involved in the metabolism of xenobiotics and pharmaceuticals were investigated. Doxorubicin, a cancer chemotherapeutic drug also known for its toxicity, was used as a positive control in male C57BL/6 mice [48]. SL, given to mice at 50 and 100 mg/kg every day for 15 days, had no adverse effects observed on animals throughout the experiment. Since SL therapy did not affect SGOT, SGPT, or MDA production, there was no sign of liver injury. The oxidative damage of the liver is shown by the decrease in glutathione, SOD, and catalase activity that results from doxorubicin treatment.
Phase I and phase II enzymes that make up the cytochrome P450 enzyme system are essential for drug metabolism [49]. In the present study, giving SL to mice increased the specific activity of Cyt P450R and Cyt b5R (phase I enzymes). Toxic substances are transformed into hydrophilic metabolites by phase I metabolic enzymes. Phase II enzymes then use these metabolites to transform them into water-soluble compounds, making it easier for the body to remove them [21,28]. The phase II enzyme GST's specific activity was elevated by SL, suggesting that it has a protective impact against a range of cytotoxic and cancer-causing agents. The regulation of the cytochrome P450 system by SL provided evidence for its chemopreventive nature towards carcinogenic and toxic xenobiotic compounds.
We carried out several studies in vitro and in vivo tumor xenografts, the findings of which strongly supported the anticancer potential of SL. A strong decrease in cell viability and cell growth by SL in lung cancer cells was indicative of such efficacy. Cell growth and survival are controlled by maintaining the cell cycle [50]. An increase in A549 cells at the sub-G1 phase by SL suggested the slow-down of cell cycle progression with possible increases in apoptotic cell death. This was further validated by the SL treatment of cancer cells, showing inhibition of survival through the induction of apoptosis by Annexin V-FITC assay. Apoptosis is an orchestrated process to kill cancer cells. Hence, we further investigated the mechanism behind apoptosis induction by SL.
The mitochondrial membrane depolarization is an early indicator of apoptosis mediated by mitochondria and JC-1 staining can be used to confirm this [51]. The membrane integrity was disrupted in cancer cells as shown by the considerable depolarization of the mitochondrial membrane potential after SL treatment. During apoptosis, the Bcl-2 protein family controls the permeability of the mitochondrial membrane [52,53]. As anticipated, SL reduced the expression of the Bcl-2 protein and enhanced the Bax protein in lung cancer cells. It has been observed that the loss of mitochondrial membrane potential caused by the alterations in Bax and Bcl-2 expressions results in the release of cytochrome c to the cytoplasm of the cell, which further induces caspases and subsequent breakdown to trigger apoptosis [54]. Hence, the apoptotic effect of SL was mediated through the mitochondrial pathway activation in lung cancer cells.
Further, morphological as well as quantitative evidence of apoptosis in the cancer cells can be analysed through acridine orange/ethidium bromide (AO/EB) staining [55]. Fluorescence imaging of cancer cells treated with SL revealed the induction of apoptosis characterized by the shrinkage of cells, blistering, and membrane blebbing. SL showed a sharp decrease in total cell number and increased presence of more cells in the early and late apoptotic stages. During apoptosis, the formation of oligo-nucleosomes as a result of DNA fragmentation appeared as a series of DNA bands called DNA ladders on agarose gel [31]. SL showed in a dose-dependent manner a laddering pattern of DNA, suggesting the characteristic of apoptosis in lung cancer cells. Furthermore, the DNA damage pathway-related proteins, including the expression of Chk1 and Chk2 were found to be decreased, indicating that the DNA damage checkpoint has been compromised by the SL in lung cancer cells. Interestingly, the expression of DNA repair protein Rad51 which plays a key role in homologous recombination repair of damaged DNA was also decreased in cancer cells after the SL treatment. These results confirmed that apoptosis induction in lung cancer cells by SL was accompanied by increased DNA damage as well as a decrease in the DNA repair pathway.
To validate the translational significance of cell culture findings in an in vivo model, a lung tumor xenograft study was conducted in athymic nude mice. First of all, the oral treatment of SL (100 mg/kg) for 20 days to athymic mice did not have any detrimental effects on water and food intake or body weight gain. These findings further substantiated the C57BL/6 mice study showing its non-toxic nature at the systemic level. The kinetics of lung tumor growth was significantly decreased by SL treatment to tumor-bearing athymic nude mice, showing a strong decrease in tumor volume and tumor weight, and thus providing evidence for its in vivo antitumor efficacy. The decrease in tumor growth by SL was further supported at the molecular level by histological analysis of tumor tissues that showed reduced expression of cell proliferation marker Ki-67 and increased expression of cleaved caspase 3 and cleaved PARP. Further, immunoblotting was done in tumor tissues to assess the expression of molecular markers for cell survival, DNA damage and repair, and proliferation. SL treatment showed reduced expression of proteins, including PCNA, Chk1, Chk2, and Rad51, while elevating the ratio of Bax/Bcl-2, and thus validated our in vitro findings in an in vivo tumor condition.
In summary, the current work for the first time revealed that SL possesses chemopreventive as well as strong anticancer potential for lung cancer cells both in vitro and in vivo with underlying molecular pathways, without causing any hepatotoxicity in mice. SL triggered apoptotic cell death through mitochondria and caspase pathways and caused DNA damage by down-regulating the checkpoint kinases and inhibiting the DNA repair pathway in tumor cells. Overall, these findings provided evidence for the mechanism-based anticancer potential of SL and have translational relevance for human lung cancer control.

Authors’ contribution
MA, RP and NS: Visualization, Investigation, Methodology, Formal analysis and software; MA, RP: Writing- original draft; YS, RPS: Conceptualization, Formal analysis, Reviewing, Editing and Funding acquisition; RPS: Supervision and Project Administration. All authors read and approved the final manuscript.

Ethics approval
All animal experiments were conducted in accordance with and after the approval of the Institutional Animal Ethics Committee (IAEC), JNU, New Delhi, India. The mice were housed and maintained at CLAR, JNU, under controlled room temperature and humidity throughout the experiment.

Data statement
All the data are included in the manuscript; however, the raw data will be available upon reasonable request.

Declaration of generative AI in scientific writing
None.

Funding sources
This project was financially supported by 10.13039/100030887CCRUM, Ministry of AYUSH, Government of India. The work supported in part by UPE-2, DST-PURSE, DRS-10.13039/501100001501UGC, DBT-Builder, DST-FIST, 10.13039/100017896JNU, India, and Centre for Excellence on Ayurveda and Systems Medicine, 10.13039/100020374Ministry of AYUSH are gratefully acknowledged.

Conflict of interest
The authors declare that none of the work reported in this study could have been influenced by any known competing financial interests or personal relationships.