Unraveling the therapeutic potential of Rutin against osteosarcoma cells: Targeting TNF-α and VEGF signaling pathways

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Gnanamathy, S. Nancy Sheela, R. Jeevitha, P. Elumalai, M. Sridevi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4191813/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Rutin is a flavonoid that is widely distributed in plants and has been identified as having medicinal qualities as well as potential benefits for treating a range of ailments. In this work, we examined rutin's anticancer effects, specifically with regard to osteosarcoma, a type of bone cancer. Methods and results We evaluated the cytotoxic activity of rutin using MTT and LDH tests on the MG-63 osteosarcoma cell line, and the results showed a notable cytotoxic effect. Following rutin treatment, morphological alterations, such as membrane blebbing and cell shrinkage, were noted, which are typical of anticancer medications. Additionally, an in vitro assessment employing the wound healing assay revealed rutin's anti-migratory action on MG-63 cells. The results of the RT-PCR gene expression research pointed to possible pathways of rutin-induced apoptosis, including downregulation of the anti-apoptotic gene BCL-2 and elevation of pro-apoptotic genes including p53, Bax, and caspase-3. Additionally, the migration-causing genes VEGF and EGF were downregulated by rutin. Moreover, the relationship between rutin and proteins linked to osteosarcoma, like VEGF and TNF-α, was evaluated using in silico models. Conclusion The findings demonstrated effective binding at various binding sites, pointing to rutin's possible therapeutic use in the treatment of osteosarcoma. Although this work uses the MG-63 cell line to provide light on the anticancer activity of rutin against osteosarcoma, more preclinical research is necessary to establish the best dosages and assess safety profiles for the possible development of medications for the treatment of osteosarcoma. Osteosarcoma Rutin TNF- α EGF VEGF Apoptosis Insilico analysis Molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. INTRODUCTION Osteosarcoma, primary malignant paediatric bone cancer that has a high probability of metastatic spread and medication resistance. The most common malignant bone-related cancer in teenagers, with a complicated heterogeneity and an improperly generated juvenile osteoid matrix (Liu et al., 2022). The diagnostic incidence of osteosarcoma in adolescents and children is 67%, according to data from the American Cancer Society (ACS) and the Surveillance, Epidemiology and End Results Programme (SEER) 3,970 new cases and 2,140 fatalities from bone and joint cancer were predicted for 2023 (Siegel et al., 2023). Chemotherapy, once the second line of treatment after surgical amputation, has been a common method for treating cancer since the 1970s. The National Cancer Institute (NCI) has mentioned surgery, chemotherapy, radiotherapy, the usage of samarium, and targeted cancer therapies as common methods for treating cancer. However, these techniques have their disadvantages and side effects. Successful chemotherapeutic drugs include doxorubicin and cisplatin, which damage cancer cells' DNA and induce death by forming DNA adducts. Researchers are now exploring alternative treatments, as chemotherapy can cause side effects, increased recurrence rates, and medication resistance (Harrison et al., 2017).Since natural compounds have long been employed as therapeutic agents in traditional Indian and Chinese medicine, many academics worldwide are concentrating their research on substances and extracts from natural sources (Pan et al., 2021). Over half of anticancer drugs contain natural products with phytochemical components that offer antibacterial, anticancer, and neuroprotective effects (Choudhari et al., 2019). When focused specifically on osteosarcoma, studies documenting the anti-cancer capabilities of plant-derived metabolites reveal a range of processes and pathways involved in creating these effects (Kazantseva et al., 2022). These phytochemicals act on several apoptotic pathways and augment the therapeutic advantages of conventional medications, they are chosen in combination therapy (Rodriguez et al., 2021). Phytochemicals from plants can impact various molecular targets, including cancer cells, causing cell cycle arrest and death through various signal transduction channels (Almatroodi et al., 2021). Flavonoids, which are found in plant nutritional supplements, suppress cancer by preventing the growth of new cells, causing the cell cycle to be detected, causing apoptosis, preventing the growth of new blood vessels, and having anti-inflammatory and antioxidant actions (Ahmadi et al., 2019). Rutin is a naturally occurring secondary metabolite and flavonol glycoside is also referred to as rutoside, quercetin-3-O-rutinoside, sophorin, and vitamin P. Chemically, rutin is a yellowish powder and known as 2-(3,4-dihydroxyphenyl).-5,7-dihydroxy-3-[L-rhamnopyranosyl-( 16 )-D-glucopyranosyloxy].-4H-chromen-4-one with a molar mass of 610.521 g/mol. Rutin melts at 125°C and has a pKa value of 6.17 whereas it does not dissolve in water as quickly as pyridine. Rutin is an essential chemopreventive agent, as various studies have recently demonstrated (Semwal et al., 2021 & Shahbaz et al., 2023). In terms of pharmacological effects, it has been shown to have antioxidant, cytoprotective, vasoprotective, anticarcinogenic, neuroprotective, and cardioprotective effects (Pandey et al., 2022). Various studies on rutin have shown its potential against several cancers including breast cancer towards two different cell lines (Iriti et al., 2017) and in combination therapy berberine and rutin incorporation onto chitosan polymer nanoparticles were found more effective (Padmavathy et al., 2023). Researchers also examined colorectal cancer by comparing rutin micelles with reference proapoptotic cisplatin and raw rutin, as well as advanced outstanding dual cytotoxicity-antiinflammation bioefficacies in significantly unique submicro nano affinities (Ibrahim et al., 2023). Metastasis is an inefficient process that requires a coordinated choreography of events to prevent failure. It involves detachment of malignant cells, EMT, invasion, migration, intravasation, lymph fluid and bloodstream travel, extravasation, and growth reestablishment at a distant site, preventing the elimination of emigrating cancer cells (Gandhi et al., 2023). TNF-α, a key cytokine, can cause severe health issues like tissue damage, septic shock, and inflammation, potentially linked to apoptosis in vulnerable cells. Gene polymorphisms in TNF receptor or protein encoding genes may impact OS patients, potentially limiting TNF-α's ability to replicate the disease (Zhou et al., 2022). Studies also revealed that Rutin also showed a reduction of TNF-α production in an in vitro L929 bioassay (Guruvayoorappan et al., 2007). Host macrophage production of TNF-α keeps osteosarcoma cells in an undifferentiated state, which is necessary for tumor growth and this study is strong evidence to prove that cytokines and TNF- α were correlated with osteosarcoma progression (Mori et al., 2013). The trans-membrane receptor tyrosine kinase known as epidermal growth factor receptor (EGFR) is essential for controlling growth factor signalling. In epithelial malignancies, EGFR activation is strongly linked to angiogenesis, metastasis, and cancer development (Zhang, 2023). High EGFR expression is linked to poor prognosis in OS patients as well as increased proliferative activity and metastatic potential. In clinical practice, EGFR overexpression is presently employed as a target for several cancer treatments and is typically present in a wide variety of human cancer types. It has been discovered that EGFR overexpression significantly correlates with a poor prognosis in patients with specific malignancies, including those of the head and neck, bladder, cervical, and ovarian regions (Zubair & Bandyopadhyay, 2023). A member of the angiogenic factor family, VEGF stimulates the growth and migration of endothelial cells and controls capillary permeability by attaching to its receptors, which is crucial for tumour angiogenesis (Yang et al., 2023). There is strong evidence indicating that tumour metastasis and prognosis are directly related to the degree of VEGF expression (Jianzhang et al., 2023). In osteosarcoma, VEGF signalling suppression induces apoptosis and halts cell development (Velayutham et al., 2023). Singh et al, (2023) have investigated the anti-cancer effects in both invitro and invivo studies by encapsulated into nanoparticles and found that the MgO-NH-PBA-Rutin nanohybrid significantly reduced the growth and migration of MDA-MB-231 cells by inducing apoptosis and intracellular production of reactive oxygen species in breast cancer. Although studies involving rutin in various cancers were reported to the best of our knowledge, the anticancer activity of rutin in human osteosarcoma has not yet purused. Consequently, the purpose of this study was to evaluate the underlying apoptosis mechanism, cell migration and invasion by signaling pathways that are most directly associated with OS in order to ascertain the possible mechanism by which rutin functions as an anticancer agent. In addition, we also examine the interaction between drug and the proteins related to osteosarcoma were studied using molecular docking and confirmed by molecular docking simulations. 2. MATERIALS AND METHODS 2.1 Maintenance of MG – 63 Cell line Human osteosarcoma cell lines (MG-63) were obtained from the National Centre for Cell Science (NCCS), Pune. In T25 culture flasks containing RPMI and Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were kept at 37 0 C in a humid environment with 5% CO 2 . The cells were trypsinized and passaged after they had reached confluence. 2.2 Cell viability (MTT) assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the viability of an osteosarcoma cell line treated with rutin. The cells were plated in 96-well plates, then starved, incubated in serum-free medium, washed twice, and exposed to rutin at varying concentrations for 24 hours. After treatment, the media was discarded and 100µl of MTT-containing DMEM was added to each well. The cells were placed in a CO2 incubator and kept at 37°C for 4 hours. After decanting the MTT-containing media, 1x PBS was used to wash the cells. The formazan crystals were dissolved in 100µl of DMSO and measured using a Micro ELISA plate reader. The quantity of viable cells was calculated as a proportion of control cells grown in serum-free media. The cells were cultured in serum-free media and the numbers of viable cells were expressed in percentage. In the untreated control medium, 100% of the cells were viable. Using the following formula, the cell viability is determined: % cell viability = [A570 nm of treated cells/A570 nm of control cells] ×100. 2.3 Lactate dehydrogenase (LDH) assay LDH is a stable cytosolic enzyme that is present in the cytosol when the cell membrane is intact. When the cell membrane is damaged or loses its integrity, it is released, and the LDH assay allows for its quantitative measurement. The conditioned medium and the identical MTT assay treatment methods were employed for the LDH leakage assay. A water bath was maintained at 37°C with 0.1 ml of condition media added to 1 ml of buffered substrate. After a 15-minute incubation period at 37°C, 0.2 ml of NAD + solution was added and mixed. Next, 1 ml of DNPH solution was added and incubated for another 15 minutes. Lastly, 10 ml of 0.4N sodium hydroxide were added, and after a 1–5 mints of incubation, the absorbance at 440 nm was determined. Standard graph preparation involves the use of sodium pyruvate as a standard. LDH activity = OD of unknown/OD of known ×standard concentration = µg of Lactate liberated/ml of conditioned media. 2.4 Cell Morphological characteristics The optimal doses (IC-50- 150µM/ml) was selected based on MTT assay for the osteosarcoma cell line, 100 and 150µM/ml concentrations used for further studies. The morphology of the cells is examined with a phase contrast microscope. In 6 well plates, 2×10 5 cells were seeded and treated with rutin for 24 hours. The cells were removed from the media after incubation period and given one wash in phosphate buffer saline (PBS, pH 7.4). The plates were examined using a phase contrast microscope. 2.5 Determination of mode of cell death by acridine orange (AO)/ethidium bromide (EtBr) dual staining The AO/EtBr dual staining, as previously mentioned, was used to ascertain the effects of rutin on osteosarcoma cell death. After 24 hours of rutin treatment, the cells were harvested, and washed with ice-cold PBS. In 5 µl of acridine orange (1 mg/mL) and 5 µl of EtBr (1 mg/mL), the pellets were redissolved. The apoptotic transformations of the labelled cells were observed using a fluorescence microscope. 2.6 Assaying Cell cycle analysis The MG-63 cells treated with rutin for 24 hours were cultured in (1 × 10 6 cells/per plate) 100-mm culture plates with 0.25% trypsin, centrifuged at 3000xg for 5min, washed with PBS, fixed in 70% ice-cold ethanol overnight − 20°C, and incubated with 50 µg/ml of propidium iodide and 1mg/ml of ribonuclease in PBS for 30 minutes. This flow cytometer procedure was adopted and modified (Elumalai et al., 2014). Cell cycle analyses were performed on a BD FACSCanto TM II (Becton and Dickinson Biosciences, Mountain View, CA, USA), and the data were analysed using BD FACSCanto clinical software. 2.7 Real Time PCR The gene expression of apoptosis signaling molecules was analysed using real-time PCR. The total RNA was isolated by the standardized protocol using Trizol Reagent (Sigma). 2µg of RNA is used for cDNA synthesis using reverse transcription using a PrimeScript, 1st strand cDNA synthesis kit (TakaRa, Japan). The targeted genes were amplified using specific primers. The PCR reaction was performed with GoTaq® qPCR Master Mix (Promega), which contains SYBR green dye and all the PCR components. Real-time PCR was performed in a CFX96 PCR system (Biorad). The results were analyzed by comparative C T method and 2 −∆∆C T method was used for fold change calculation described by (Schmittgen and Livak, 2008). 2.8 Scratch wound healing assay Onto six-well culture plates, MG-63 cells (2×10 5 cells/well) were planted. An inverted microscope was used to take pictures of the scratched cell monolayer after it had been cleaned with PBS and created a wound using a 200µl tip. Following a 24-hour rutin treatment period, the injured area was captured on camera using the same microscope. The control cells were given vehicle DMSO (0.01%) in culture media without rutin. Additionally, each treatment group underwent three replicate runs of the studies. 2.9 Protein Preparation The crystal structures of Vascular endothelial growth factor (VEGF) (PDB ID: 1VPP) (Wiesmann et al., 1998) and Tumour Necrosis Factor-alpha (TNF- α) (PDB ID: 2E7A) (Shibata et al., 2008) were acquired from the RCSB PDB. The resolution of the VEGF structure is 1.90Å, while the TNF- α structure has a resolution of 1.80Å determined using the X-ray diffraction method. After being retrieved, the structures went through preprocessing steps which involved removing complexes bound to the protein receptor molecules and determining the protonation statuses of ionizable residues. This was done to ensure that proper electrostatic interactions would occur during docking. To optimize the system, water molecules and unnecessary ligands were removed from the protein structures. Subsequently, force field parameters were utilized to replicate the protein's behavior in docking simulations. The protein structures were then subjected to optimization and refinement processes to prepare them for molecular docking investigations. 2.10 Ligand preparation The molecular structures of the rutin compound known for their therapeutic potential, were sourced from PubChem (Kim et al., 2019) ( http://pubchem.ncbi.nlm.nih.gov ). Chimera (Pettersen et al., 2004) converted the structure from the SDF to the PDB format, enabling a detailed analysis of their three-dimensional configurations. This essential step applies accurate force fields to rutin's 3D structure, preparing it for target protein simulations. 2.11 Molecular docking Autodock (Morris et al., 2009) analyses different binding orientations and interactions and is utilized to align the ligand with the target protein. Subsequently, a scoring method is employed to evaluate the most appropriate ligand-protein complex identified during this analysis. This method enables precise projections of the ligand's compatibility with the active site of the protein, offering a crucial understanding of the ligand as a therapeutic agent. 2.11 Statistical analysis One-way ANOVA and Student's t-test SPSS were used to evaluate the obtained data. The statistical analysis on all experimental data, with P < 0.05 being deemed statistically significant. 3. RESULTS 3.1 Rutin inhibits cell proliferation and induces cytotoxicity in osteosarcoma cells. The MTT assay was employed to determine the anti-proliferative effects of rutin on the MG-63 cells after different dosages of rutin were administered for 24 hours to test for the inhibition of the growth of human osteosarcoma cells. The treatment of rutin caused a notable decrease in cell proliferation in a concentration-dependent manner as depicted in Fig1A. 3.2 Lactate dehydrogenase (LDH) assay LDH release assays were used to assess the cytotoxicity of rutin in MG63 cells. Additionally, MG63 cells were exposed to rutin at various doses (25, 50, 100, 150, 200, and 250 μM). After a 24-hour incubation period, the amount of cell death was assessed using an LDH release assay. This assay offers a reliable estimate of the cell death triggered on by rutin because the amount of LDH released into the culture media by the dying cells is proportionate to the degree of cell death. When compared to the control, Fig. 1B clearly illustrates the dose-dependent, significant increase in rutin-induced LDH release (P<0.05). 3.3 Rutin alters the cell morphology of osteosarcoma cells After cell proliferation, the MG-63 cells treated with rutin of two concentrations (100 &150μM/ml) for 24 hrs, were undergone morphology studies to determine the alteration of cell morphology. It was observed that after treatment with rutin the cells were showed reduction in number and recognized with blebbed membrane and shrinkage of cells in Fig 2(A). 3.4 Rutin induces apoptotic cells in osteosarcoma cells The induction of apoptosis by rutin on osteosarcoma cell line MG-63 was assessed with AO/EtBr staining and the results were showed in Fig. 2 (B). AO/EtBr staining was used to distinguish the live and dead cells. The green cells are live cells where the acridine orange stain penetrates into both living and dead cells incontrast, ethidium bromide can only infiltrate dead cells. MG-63 cells treated with rutin showed increased number of dead cells in our AO/EtBr staining. 3.4 Rutin suppresses cell growth and causes cell cycle arrest in osteosarcoma cells. We examined the impact of rutin on the cell cycle proliferation in order to validate the correlation between growth inhibition and cell cycle arrest decipited in Fig 3. 3.6 Rutin modulates the mRNA expression of apoptotic genes in osteosarcoma cells Rutin's impact on apoptotic genes (p53, Bcl-2, Bax, and Caspase-3) in MG-63 cells was assessed. Rutin (100 and 150 μM/ml) enhanced the mRNA expression of apoptotic genes in the MG-63 cell line much more than the untreated control cells, as seen in Fig. 4. In this MG-63 osteosarcoma cell line, rutin upregulated the pro-apoptotic genes, p53, Bax, and caspase-3 while downregulating the anti-apoptotic gene, Bcl-2. 3.7 Scratch wound healing assay The migratory capacity of MG-63 cells was initially identified by scratch assay, which allowed for the confirmation of rutin's impact on migratory behaviour. Following a 24-hour stimulation of MG63 cells with varying rutin concentrations (100 and 150 µM/ml), the number of cells moving to the centre of the scratch decreased as the concentration of rutin increased, as seen in Figure 5. According to these results, it proved that rutin could inhibit OS cell motility in vitro. Rutin effects on VEGF and EGF migration gene were assessed by mRNA expression for rutin concentrations 100 and 150 μM/ml along with control group (Fig 6). Following EGF and VEGF suppression, this suggests that rutin have downregulated the genes responsible for migration. 3.8 Molecular docking The molecular docking study examines rutin's interaction with TNF-α and VEGF proteins, which play a crucial role in inflammation and angiogenesis. Rutin exhibits binding energies of -3.01 kJ/mol with TNF-α and -4.24 kJ/mol with VEGF, suggesting a stronger affinity for VEGF. It forms hydrogen bonds with TNF-α at LEU26 (3.01236 Å) and GLN27 (2.90033 Å) and with VEGF at GLN89 (2.82538 Å), indicating significant interactions (Figure 7 & Table 1). Furthermore, rutin engages in an electrostatic Pi-Anion interaction with VEGF's ASP11 (3.66573 Å), and hydrophobic interactions with LEU16 (3.98319 Å) and HIS86 (4.94879 Å). Additional interactions include a Pi-Donor Hydrogen Bond with VEGF's GLN89 (4.12276 Å) (Figure 8 & Table 1). These findings suggest rutin's potential to inhibit TNF-α and VEGF, highlighting its therapeutic prospects in inflammation and angiogenesis-related diseases. 4. DISCUSSION Osteosarcoma a prevalent primary malignant bone tumor and its clinical features include a high morbidity rate and a high risk of recurrence. Adolescents and children's health are significantly impacted by osteosarcoma. The overall prognosis for people with osteosarcoma remains dismal, despite significant advancements in the clinical diagnosis and surgical treatment of the disease. Therefore, the need to identify efficient molecular therapy targets is important. The active components of natural medicines are also heavily utilised in the treatment of osteosarcoma (Daher et al., 2023). Rutin exhibits enormous activities including antioxidant, antiaging, neuroprotective, cardioprotective and anticancer. According to previous studies, rutin serves as a potent anticancer agent in breast, liver, lung, cervical, and prostate as well as tends to induce apoptosis and causes cell cycle arrest (Liga et al., 2023). Here, we evaluated rutin, a flavonoid which suppresses the growth of osteosarcoma by inactivating the signaling pathway by inhibiting proliferation and migration of cells with enhancing the apoptosis of cell in OS cell line, MG- 63 which was further validated by molecular docking. At first, the OS cell line MG- 63 was treated with rutin at different concentrations (25–250 µM/ml) for 24 hrs to assess the inhibition of rutin on growth of the osteosarcoma cells and the rutin cytotoxic effects were evaluated using MTT assay. Fang et al. (2023) have used MTT assay to detect osteosarcoma cell growth and also found that there is a reduction in the proliferation, migration and invasion of cells by targeting miR-340-5p gene expression via promoting FGF23. The release of lactate dehydrogenase enzyme into the medium, a sign of cell damage in treated cells, may be measured with the sensitive and accurate LDH test. In the presence of NADH, these enzymes catalyse the conversion of pyruvate to lactate (Farhana & Lappin, 2024). The LDH cytotoxicity assay was used on osteosarcoma cells in order to more thoroughly characterise the impact of rutin. That is well recognised that the release of LDH enzyme into the medium from the cytoplasm of injured cells, together with a rise in absorbance, is evidence of membrane damage-induced cell death. LDH release increased dose-dependently when rutin was applied to osteosarcoma cells. We observed a notable release (**p˂0.05) of LDH in the culture medium compared to the control after MG-63 cancer cells were treated for 24 hours with 100 and 150µM/ml rutin. Our results are similar to other authors that found a significant increase of LDH in osteosarcoma cell lines (MG-63). Chandrasekaran et al. (2023) have evaluated that NH extract against MG-63 cells and have concluded the cell damage was increased in response to dose concentrations. Higher concentrations resulted in more cell damage, which was a result of LDH leaking into the medium at a higher level. This demonstrates the phenomenon of NH extract-induced apoptosis causing cell death. Khan et al. (2021) have estimated the cytotoxicity of rutin by the release of LDH by disrupting its cell membrane in cervical cancer caski cells. They have found that rutin treatment of 24h have significantly increased in cell death so it’s strongly confirms the anticancer potential of rutin. After being treated with rutin significantly fewer cell proliferation were observed in a concentration dependent manner. From those different concentrations the IC 50 value of 150µM/ml used to assess the inhibitory impact and in addition, the morphology was also studied using phase contrast microscope to verify its anticancer potential. Here, the cancer cells were significantly reduced after treatment with rutin for 24 hrs and the cells exhibited in the indications of cytotoxicity by shrinking and blebbing of the ctoplasmic membrane. Cao et al. (2022) have investigated the role of flavonoid alpinetin in osteosarcoma cell lines 143B and U2OS under different concentrations and proved that it inactivated the signaling pathways PI3K/AKT and ERK and inhibited the cell proliferation and metastasis in osteosarcoma. Alyami et al. (2023) have evaluated that Rutin suppressed the development and proliferation of hepatic and pancreatic cancer cells, with HepG-2 being the most impacted. It also inhibited the GST enzyme, lowering chemotherapeutic drugs anticancer effectiveness, and the CYP3A4 enzyme, showing the possibility for combination-based therapies. Thus proved that flavonoids are the alternate treatment for cancer. We evaluated the apoptosis induction in MG-63 cell line on treatment with rutin (100 & 150µM/ml) for 24hrs along with control group to analysed their morphological changes which were observed under florescence microscope after stained with AO/EtBr dual staining. The results outcome indicated that rutin induce apoptosis in MG-63 cells shows green fluorescence for viable cells and yellowish orange and orange in hue for early and late apoptotic cells, and also showed morphological changes including cell shrinkage and blebbing of membrane. Alzahrani et al. (2023) have identified apoptotic induction in the MDA-MB-435s cells treated with albumin–CGA NPs and paclitaxel for 24 hours by AO/EtBr analysis. They have also showed a significant increase in AO/EtBr stained cells, similar to paclitaxel-treated cells and was observed no staining in untreated cells. Liu et al. (2023) have observed morphological changes in the apoptosis cells due to AO/EB dual staining. The OS control cells shows green live cells which were untreated whereas, the increased cell apoptosis showed in CL (30 µM/ml) with condensed chromatin and membrane blebbing and showed orange membrane integrity loss in late apoptotic cells. Apoptosis is the regulated and orderly death of cells that requires the activation, expression, and regulation of a number of distinct genes. Extrinsic and intrinsic apoptosis pathways are the two main signalling routes that trigger apoptosis. Binding death ligands and death receptors, such as TNF-R1, Fas, DR3, TRAIL-R1, DR6, EDAR, and nerve growth factor, starts extrinsic apoptosis. Intracellular stressors such oxidative stress, growth factor deprivation, chemotherapeutic drugs, or radiation might trigger intrinsic apoptotic pathways. The 20 members of the Bcl-2 family of proteins, which are divided into three subfamilies, control the intrinsic apoptotic pathway (Li et al., 2016). The family of proteins known as B-cell lymphoma protein-2, or BCL-2, controls this extremely conserved mechanism. Based on their unique functions in the process of apoptosis, these proteins can be categorised into three major classes: pro-apoptotic, anti-apoptotic, and effector proteins. Effector proteins are activated by a variety of intracellular and extracellular signals, such as DNA damage, the absence of growth factors, hypoxia, chemo- and radiotherapy, by up- or down-regulating pro-apoptotic and anti-apoptotic proteins, respectively. This activation leads to the formation of multimeric pores in the outer membrane of the mitochondria (Alipour et al., 2023). Since flow cytometry study revealed cell cycle arrest in MG-63 cells treated with rutin. The efficacy of rutin to induce cell cycle arrest was explored to identify potential mechanisms of action. It was observed that there in control G0-G1: 90.88%, S: 4.23%, and G2/M: 5.00% whereas the cells which were treated with rutin showed G0-G1: 73.56%, S: 11.78% and G2/M: 14.66%. This result indicates that the G2/M phase shows an increased number of cells whereas cells were decreased in the S phase. Kelly et al. (2023) have investigated the potential mechanisms of action of RL71 on cell cycle arrest. The results revealed that RL71 caused an arrest in the G2/M phase by increasing the number of cells in that phase and decreasing those in the G1 phase. The effect of rutin on pro-apoptotic and anti-apoptotic genes p53, Bcl-2, Bax and Caspase-3 expressions were studied in MG- 63. The anti-apoptotic gene Bcl-2 decreased; however, the pro-apoptotic genes p53, Bax and Caspase-3 were significantly increased during apoptosis. There is a significant increase observed in both concentrations (100µM/ml & 150µM/ml) when compared to control group in the pro-apoptotic genes: p53 with 3 and 4 fold; Bax with 2 and 4 fold and Caspase- 3 with 1.4 and 2.5 fold. Whereas, there is a decrease in the anti-apoptotic gene Bcl-2 with 0.7 and 0.5 fold when compared with control group. Hajimehdipoor et al. (2023) have investigated the rutin expression of MKI67, VEGFA, VIM, CDH2, and FN1 in MDA-MB-231 and MCF-7 cell lines compared to untreated control cells. They proved that rutin activated the EMT process by suppressing CDH1 expression and increasing VIM, FN1, and CDH2 expression and promoted VEGFA expression in MCF-7 cells without significant effect on THBS1. A scratch well test was then performed to investigate the effects of rutin (100 and 150µM/ml) on the invasion and migration of osteosarcoma cell line, MG- 63. Following a 24-hour incubation period, rutin significantly reduced the migration of cells in a dose-dependent manner, with no effect on the control group. Rutin may have an inhibitory effect on the migratory potential of MG-63 cells, as evidenced by the dose-dependent suppression of cell migration. Rutin significantly inhibits cell migration, suggesting that these substances may obstruct cellular mechanisms involved in migration, including cytoskeleton rearrangements and cell signalling pathways. It implies that rutin influences not just the survivability of cells but also their functional behaviour, including their capacity to migrate and take part in the processes involved in wound healing. Qi et al., 2023 have proved that piperine (PIP), alkaloid which has the capacity to inhibit the effects of doxorubicin (DOX) against OS cell migration in U2OS and 143B cells. They have concluded that when compared to the control group, PIP and DOX dramatically decreased U2OS and 143B cell migration. Cell migration decrease was greatly enhanced when PIP and DOX were combined. The wound area was greater in all pharmacological groups than in the control group. The PIP + DOX group showed noticeably larger percentages of surviving wound area after 24 hours, indicating a decrease in cell migration and development. Huo et al. (2022) have evaluated the wound healing activity of rutin (5 µg/mL and 10 µg/mL) for pancreatic cell line (PANC-1). In that they explored that migration of PANC-1 cell as well as in SW 1990 cells and MIA PaCa-2 cells was significantly inhibited after treatment of rutin for 48hrs. The effect of rutin on migration genes EGF and VEGF expressions were studied in MG- 63.. There is a significant decrease observed in both concentrations (100µM/ml & 150µM/ml) when compared to control group in the migration genes: EGF with 0.7 and 0.3 fold and VEGF with 0.8 and 0.5 fold when compared with control group. This proved that rutin have the capability to down regulate the EGF and VEGF mRNA expression. Furthermore, using the AutoDock tools, an in silico molecular docking research was carried out to look at the binding interactions of rutin with TNF- α and VEGF proteins. Remarkably, every active component of rutin demonstrated a strong affinity for interacting with the proteins TNF- α and VEGF. The molecular interactions are represented by interacting residues in binding sites with certain physico-chemical characteristics that point to particular target protein functions. According to this finding, the complex structure was stabilised by the interactions between participating amino acids and ligand molecules through the use of H-bond, alkyl, pi-alkyl, and van der Waals forces. 5. CONCLUSION In conclusion, it has been proved that Rutin, a flavonoid that is commonly found in plants that is thought to have numerous medicinal benefits and is used to treat variety of illness. This is an effective anticancer medication that works by preventing the formation of tumor-promoting proteins and cancer cells. The osteosarcoma cell line (MG-63) was used in the current investigation to assess the cytotoxicity activity of rutin. The MTT and LDH assays demonstrated a significant cytotoxic effect. The cell line underwent morphological alterations, including membrane blebbing and shrinkage of the cells, upon close inspection following rutin treatment. A characteristic of anticancer drugs, the in vitro evaluation of the wound healing assay revealed an anti-migratory effect. The MG-63 cell line was used in the study's mRNA gene expression analysis by RT-PCR. The results showed that the anti-apoptotic gene BCL-2 was downregulated and the pro-apoptotic genes p53, Bax, and caspase-3 were upregulated. Additionally, the study evaluated the interaction between rutin and the proteins linked to OS using in silico models, and it demonstrated efficient interaction at different TNF-α and VEGF binding sites. Since the osteosarcoma MG-63 cell line was the sole focus of the current analysis, further preclinical research is required in light of this study in order to ascertain the safe and effective dosages for potential medication development for the treatment of osteosarcoma. Declarations Data availability No associated data in the manuscript Acknowledgements We thank to Gold Lab and the Department of Pharmacology,Saveetha University for offering the study's research lab facilities. Funding None Author information Authors and Affiliations Department of Biotechnology, Vinayaka Mission’s Kirupananda Variyar Engineering College, Vinayaka Mission’s Research Foundation (Deemed to be University), Salem - 636 308, Tamil Nadu, India G. Gnanamathy, S. Nancy sheela & M. Sridevi Cancer Genomics Laboratory, Centre for Global Health Research, Saveetha Medical College, Saveetha Institute of Medical and Technical Science, Chennai, Tamil Nadu, India Elumalai Perumal & R. Jeevitha Contributions GG, RJ conducted the experiments and involved in the manuscript preparation. GG, SN were involved in experimental design and in preparation of manuscript. MS and EP reviewed and finalized the manuscript. All authors have read and approved the manuscript. Corresponding author Correspondence to M. Sridevi Ethics declarations Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical approval The present study is not involved animal/human studies. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. 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Molecules 27(7):2293. 10.3390/molecules27072293 PMID: 35408691; PMCID: PMC9000526 Table Table 1: Interaction of rutin with TNF- α and VEGF protein Compound Name Protein name Binding energy (kJ/mol) Amino acid Distance Interaction type Interaction bond Rutin TNF-α -3.01 LEU26 3.01236 Hydrogen Bond Conventional Hydrogen Bond GLN27 2.90033 Hydrogen Bond Conventional Hydrogen Bond VEGF -4.24 GLN89 2.82538 Hydrogen Bond Conventional Hydrogen Bond ASP11 3.66573 Electrostatic Pi-Anion GLN89 4.12276 Hydrogen Bond Pi-Donor Hydrogen Bond LEU16 3.98319 Hydrophobic Pi-Sigma HIS86 4.94879 Hydrophobic Pi-Alkyl Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4191813","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288509378,"identity":"8f1722cb-e1de-4fce-a1f0-34cf7bec3700","order_by":0,"name":"G. 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Sridevi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYDCCA0CcwMbAwAbiMDYwMPCDGAkFpGiRbACJGBDQAlEP1WIAFsGjhe/28WcfHpTZJPaJnTH7zLvDLs/4/OrEDw8MGOT5xQ5g1SJ5Lsd4RsK5tMQ26Rzj2bxnkovNbrzdLAF0mOHM2QlYtRic4WFmSGw7bMwG1MLM28acuO3G2Q0gLQkGt3FpYX8M1PIfpqU+cfOMs5t/4NfCYAzUckAOquVw4gb+3m14bZE8w2PMkHAuGaglrZhxbtvxxBk3eLdZJBhI4PQLH9BhjD/K7HjkZydvZnjbVp3Y3392880fFTby/NLYtWABEmCVEsQqBwH+A6SoHgWjYBSMghEAAFbXXSW6qsMEAAAAAElFTkSuQmCC","orcid":"","institution":"Vinayaka Mission’s Kirupananda Variyar Engineering College, Vinayaka Mission’s Research Foundation (Deemed to be University)","correspondingAuthor":true,"prefix":"","firstName":"M.","middleName":"","lastName":"Sridevi","suffix":""}],"badges":[],"createdAt":"2024-03-30 10:29:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4191813/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4191813/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54265893,"identity":"ea8d99e7-142d-42d1-92c6-1f464e20dfc6","added_by":"auto","created_at":"2024-04-08 04:59:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e(A) The cytotoxic effects of Rutin on cell growth and proliferation of osteosarcoma cells. MTT assay was employed to evaluate the cell viablity for the cells treated with Rutin (25 - 250μM/ml) for 24. Data are shown as means ± Standard Deviation (n = 3).* compared with the control blank group, p \u0026lt; 0.05.(B) Effect of Rutin on the LDH assay in MG-63 cells. The cytotoxic effect was determined by LDH assay in MG-63 cells. Each bar represents the mean ± SEM of three independent observations. * Represents statistical significance between control vs Rutin treatment. Significance at P\u0026lt;0.05 level using t-test.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4191813/v1/9c33411c01495c30f2492f67.png"},{"id":54265894,"identity":"f6fea9d4-af85-4111-9081-91f98fa5ad27","added_by":"auto","created_at":"2024-04-08 04:59:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":361064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e(A). Rutin cell morphology impact on human osteosarcoma cells (MG-63). Following a 24-hour treatment with Rutin (100 \u0026amp; 150μM/ml), the cells were examined using an inverted phase contrast microscope. After being exposed to Rutin, the number of cells shrank along with their cytoplasmic membrane blebbing. (B): Both the control group and human osteosarcoma cells were subjected to rutin treatment (100 \u0026amp; 150μM/ml) for a whole day. The cells were treated with AO/EtBr dual staining following the treatment. A phase-contrast inverted fluorescence microscope was used to capture the images.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4191813/v1/3c47c8781f08ebc240db75a8.png"},{"id":54265896,"identity":"bd33fe98-f1ca-4e99-8da8-846cc6277f61","added_by":"auto","created_at":"2024-04-08 04:59:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEffect of Rutin (150 µM/ml) on cell cycle analysis in osteosarcoma cells. The cell cycle analysis was evaluated by flow cytometric analysis after propidium iodide (PI) staining. A. Representative plots showing PI staining of osteosarcoma cells treated with the rutin. B.\u003c/em\u003e \u003cem\u003eThree independent experiments were carried out. The data represent the mean and standard deviation of three independent experiments.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4191813/v1/1f0cf9c93e323bddd59c1a03.png"},{"id":54265902,"identity":"cd1c39f7-a7ff-4f2a-8ae1-a4a02f816115","added_by":"auto","created_at":"2024-04-08 04:59:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEffect of Rutin (100 and 150μM/ml) on pro-apoptotic genes (p53, Bcl-2, Bax and Caspase-3) expression in the osteosarcoma cell line. Once the target gene expression has been normalised to GAPDH mRNA expression, the results are expressed as fold change from control. Each bar represents the mean + SEM of three independent observations. '*' denotes statistical significance at the p\u0026lt;0.05 level between the control and drug treatment groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4191813/v1/020d9eb638e7ef5f656c32f6.png"},{"id":54265897,"identity":"7eff28c5-728d-4b84-9096-5eb9cc61cb52","added_by":"auto","created_at":"2024-04-08 04:59:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":250131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro scratch wound healing assay. Human osteosarcoma cells were injured and cell migration assay with and without treatment of Rutin (100 and 150μM/ml) was performed at 24h. Images were obtained using an inverted Phase contrast microscope.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4191813/v1/7499e354af38105da50c7713.png"},{"id":54265899,"identity":"c3d8f8bc-80cb-4401-85a3-87502e1adfb4","added_by":"auto","created_at":"2024-04-08 04:59:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":25887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEffect of Rutin (100 and 150μM/ml) on migration genes (EGF \u0026amp; VEGF) expression in the osteosarcoma cell line. The results are expressed as fold change from control after normalising the target gene expression to GAPDH mRNA expression. The mean + SEM of three independent observations is represented by each bar. '*' denotes statistical significance between the control and drug treatment groups at the p\u0026lt;0.05 level.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4191813/v1/b4b7c04f5558e1aeda43d6b8.png"},{"id":54265895,"identity":"c77a6416-da81-47a6-acbb-1b98e98afee5","added_by":"auto","created_at":"2024-04-08 04:59:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":163622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVisualization of docking analysis of rutin binding with TNF- α (A) Interaction of rutin with TNF- α (B) 3D representation of rutin with TNF- α (C) 2D representation describing bindings of rutin with active site of TNF- α\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4191813/v1/7bb1a0941062f4f8cc4d5ea8.png"},{"id":54265891,"identity":"0475a403-7f5d-4f4e-9dfd-ee28d101c768","added_by":"auto","created_at":"2024-04-08 04:59:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":124156,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVisualization of docking analysis of rutin binding with VEGF (A) Interaction of rutin with VEGF (B) 3D representation of rutin with VEGF (C) 2D representation describing bindings of rutin with active site of VEGF\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4191813/v1/973e4d0019f6fbf6c564dee0.png"},{"id":57504659,"identity":"28d1786c-496d-4f9c-9540-b36dc01b34a5","added_by":"auto","created_at":"2024-05-31 14:50:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1965214,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4191813/v1/77a5102b-3adb-4a33-91e8-f765696cb0a7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unraveling the therapeutic potential of Rutin against osteosarcoma cells: Targeting TNF-α and VEGF signaling pathways","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eOsteosarcoma, primary malignant paediatric bone cancer that has a high probability of metastatic spread and medication resistance. The most common malignant bone-related cancer in teenagers, with a complicated heterogeneity and an improperly generated juvenile osteoid matrix (Liu et al., 2022). The diagnostic incidence of osteosarcoma in adolescents and children is 67%, according to data from the American Cancer Society (ACS) and the Surveillance, Epidemiology and End Results Programme (SEER) 3,970 new cases and 2,140 fatalities from bone and joint cancer were predicted for 2023 (Siegel et al., 2023).\u003c/p\u003e \u003cp\u003eChemotherapy, once the second line of treatment after surgical amputation, has been a common method for treating cancer since the 1970s. The National Cancer Institute (NCI) has mentioned surgery, chemotherapy, radiotherapy, the usage of samarium, and targeted cancer therapies as common methods for treating cancer. However, these techniques have their disadvantages and side effects. Successful chemotherapeutic drugs include doxorubicin and cisplatin, which damage cancer cells' DNA and induce death by forming DNA adducts. Researchers are now exploring alternative treatments, as chemotherapy can cause side effects, increased recurrence rates, and medication resistance (Harrison et al., 2017).Since natural compounds have long been employed as therapeutic agents in traditional Indian and Chinese medicine, many academics worldwide are concentrating their research on substances and extracts from natural sources (Pan et al., 2021).\u003c/p\u003e \u003cp\u003eOver half of anticancer drugs contain natural products with phytochemical components that offer antibacterial, anticancer, and neuroprotective effects (Choudhari et al., 2019). When focused specifically on osteosarcoma, studies documenting the anti-cancer capabilities of plant-derived metabolites reveal a range of processes and pathways involved in creating these effects (Kazantseva et al., 2022). These phytochemicals act on several apoptotic pathways and augment the therapeutic advantages of conventional medications, they are chosen in combination therapy (Rodriguez et al., 2021). Phytochemicals from plants can impact various molecular targets, including cancer cells, causing cell cycle arrest and death through various signal transduction channels (Almatroodi et al., 2021). Flavonoids, which are found in plant nutritional supplements, suppress cancer by preventing the growth of new cells, causing the cell cycle to be detected, causing apoptosis, preventing the growth of new blood vessels, and having anti-inflammatory and antioxidant actions (Ahmadi et al., 2019).\u003c/p\u003e \u003cp\u003eRutin is a naturally occurring secondary metabolite and flavonol glycoside is also referred to as rutoside, quercetin-3-O-rutinoside, sophorin, and vitamin P. Chemically, rutin is a yellowish powder and known as 2-(3,4-dihydroxyphenyl).-5,7-dihydroxy-3-[L-rhamnopyranosyl-(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)-D-glucopyranosyloxy].-4H-chromen-4-one with a molar mass of 610.521 g/mol. Rutin melts at 125\u0026deg;C and has a pKa value of 6.17 whereas it does not dissolve in water as quickly as pyridine. Rutin is an essential chemopreventive agent, as various studies have recently demonstrated (Semwal et al., 2021 \u0026amp; Shahbaz et al., 2023). In terms of pharmacological effects, it has been shown to have antioxidant, cytoprotective, vasoprotective, anticarcinogenic, neuroprotective, and cardioprotective effects (Pandey et al., 2022).\u003c/p\u003e \u003cp\u003eVarious studies on rutin have shown its potential against several cancers including breast cancer towards two different cell lines (Iriti et al., 2017) and in combination therapy berberine and rutin incorporation onto chitosan polymer nanoparticles were found more effective (Padmavathy et al., 2023). Researchers also examined colorectal cancer by comparing rutin micelles with reference proapoptotic cisplatin and raw rutin, as well as advanced outstanding dual cytotoxicity-antiinflammation bioefficacies in significantly unique submicro nano affinities (Ibrahim et al., 2023).\u003c/p\u003e \u003cp\u003eMetastasis is an inefficient process that requires a coordinated choreography of events to prevent failure. It involves detachment of malignant cells, EMT, invasion, migration, intravasation, lymph fluid and bloodstream travel, extravasation, and growth reestablishment at a distant site, preventing the elimination of emigrating cancer cells (Gandhi et al., 2023). TNF-α, a key cytokine, can cause severe health issues like tissue damage, septic shock, and inflammation, potentially linked to apoptosis in vulnerable cells. Gene polymorphisms in TNF receptor or protein encoding genes may impact OS patients, potentially limiting TNF-α's ability to replicate the disease (Zhou et al., 2022). Studies also revealed that Rutin also showed a reduction of TNF-α production in an in vitro L929 bioassay (Guruvayoorappan et al., 2007). Host macrophage production of TNF-α keeps osteosarcoma cells in an undifferentiated state, which is necessary for tumor growth and this study is strong evidence to prove that cytokines and TNF- α were correlated with osteosarcoma progression (Mori et al., 2013).\u003c/p\u003e \u003cp\u003eThe trans-membrane receptor tyrosine kinase known as epidermal growth factor receptor (EGFR) is essential for controlling growth factor signalling. In epithelial malignancies, EGFR activation is strongly linked to angiogenesis, metastasis, and cancer development (Zhang, 2023). High EGFR expression is linked to poor prognosis in OS patients as well as increased proliferative activity and metastatic potential. In clinical practice, EGFR overexpression is presently employed as a target for several cancer treatments and is typically present in a wide variety of human cancer types. It has been discovered that EGFR overexpression significantly correlates with a poor prognosis in patients with specific malignancies, including those of the head and neck, bladder, cervical, and ovarian regions (Zubair \u0026amp; Bandyopadhyay, 2023).\u003c/p\u003e \u003cp\u003eA member of the angiogenic factor family, VEGF stimulates the growth and migration of endothelial cells and controls capillary permeability by attaching to its receptors, which is crucial for tumour angiogenesis (Yang et al., 2023). There is strong evidence indicating that tumour metastasis and prognosis are directly related to the degree of VEGF expression (Jianzhang et al., 2023). In osteosarcoma, VEGF signalling suppression induces apoptosis and halts cell development (Velayutham et al., 2023). Singh et al, (2023) have investigated the anti-cancer effects in both invitro and invivo studies by encapsulated into nanoparticles and found that the MgO-NH-PBA-Rutin nanohybrid significantly reduced the growth and migration of MDA-MB-231 cells by inducing apoptosis and intracellular production of reactive oxygen species in breast cancer.\u003c/p\u003e \u003cp\u003eAlthough studies involving rutin in various cancers were reported to the best of our knowledge, the anticancer activity of rutin in human osteosarcoma has not yet purused. Consequently, the purpose of this study was to evaluate the underlying apoptosis mechanism, cell migration and invasion by signaling pathways that are most directly associated with OS in order to ascertain the possible mechanism by which rutin functions as an anticancer agent. In addition, we also examine the interaction between drug and the proteins related to osteosarcoma were studied using molecular docking and confirmed by molecular docking simulations.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Maintenance of MG \u0026ndash; 63 Cell line\u003c/h2\u003e \u003cp\u003eHuman osteosarcoma cell lines (MG-63) were obtained from the National Centre for Cell Science (NCCS), Pune. In T25 culture flasks containing RPMI and Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were kept at 37\u003csup\u003e0\u003c/sup\u003eC in a humid environment with 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were trypsinized and passaged after they had reached confluence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cb\u003eCell viability (MTT) assay\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the viability of an osteosarcoma cell line treated with rutin. The cells were plated in 96-well plates, then starved, incubated in serum-free medium, washed twice, and exposed to rutin at varying concentrations for 24 hours. After treatment, the media was discarded and 100\u0026micro;l of MTT-containing DMEM was added to each well. The cells were placed in a CO2 incubator and kept at 37\u0026deg;C for 4 hours. After decanting the MTT-containing media, 1x PBS was used to wash the cells. The formazan crystals were dissolved in 100\u0026micro;l of DMSO and measured using a Micro ELISA plate reader. The quantity of viable cells was calculated as a proportion of control cells grown in serum-free media. The cells were cultured in serum-free media and the numbers of viable cells were expressed in percentage. In the untreated control medium, 100% of the cells were viable. Using the following formula, the cell viability is determined: % cell viability = [A570 nm of treated cells/A570 nm of control cells] \u0026times;100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Lactate dehydrogenase (LDH) assay\u003c/h2\u003e \u003cp\u003eLDH is a stable cytosolic enzyme that is present in the cytosol when the cell membrane is intact. When the cell membrane is damaged or loses its integrity, it is released, and the LDH assay allows for its quantitative measurement. The conditioned medium and the identical MTT assay treatment methods were employed for the LDH leakage assay. A water bath was maintained at 37\u0026deg;C with 0.1 ml of condition media added to 1 ml of buffered substrate. After a 15-minute incubation period at 37\u0026deg;C, 0.2 ml of NAD\u0026thinsp;+\u0026thinsp;solution was added and mixed. Next, 1 ml of DNPH solution was added and incubated for another 15 minutes. Lastly, 10 ml of 0.4N sodium hydroxide were added, and after a 1\u0026ndash;5 mints of incubation, the absorbance at 440 nm was determined. Standard graph preparation involves the use of sodium pyruvate as a standard.\u003c/p\u003e \u003cp\u003eLDH activity\u0026thinsp;=\u0026thinsp;OD of unknown/OD of known \u0026times;standard concentration\u0026thinsp;=\u0026thinsp;\u0026micro;g of Lactate liberated/ml of conditioned media.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell Morphological characteristics\u003c/h2\u003e \u003cp\u003eThe optimal doses (IC-50- 150\u0026micro;M/ml) was selected based on MTT assay for the osteosarcoma cell line, 100 and 150\u0026micro;M/ml concentrations used for further studies. The morphology of the cells is examined with a phase contrast microscope. In 6 well plates, 2\u0026times;10\u003csup\u003e5\u003c/sup\u003ecells were seeded and treated with rutin for 24 hours. The cells were removed from the media after incubation period and given one wash in phosphate buffer saline (PBS, pH 7.4). The plates were examined using a phase contrast microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Determination of mode of cell death by acridine orange (AO)/ethidium bromide (EtBr) dual staining\u003c/h2\u003e \u003cp\u003eThe AO/EtBr dual staining, as previously mentioned, was used to ascertain the effects of rutin on osteosarcoma cell death. After 24 hours of rutin treatment, the cells were harvested, and washed with ice-cold PBS. In 5 \u0026micro;l of acridine orange (1 mg/mL) and 5 \u0026micro;l of EtBr (1 mg/mL), the pellets were redissolved. The apoptotic transformations of the labelled cells were observed using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Assaying Cell cycle analysis\u003c/h2\u003e \u003cp\u003eThe MG-63 cells treated with rutin for 24 hours were cultured in (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/per plate) 100-mm culture plates with 0.25% trypsin, centrifuged at 3000xg for 5min, washed with PBS, fixed in 70% ice-cold ethanol overnight \u0026minus;\u0026thinsp;20\u0026deg;C, and incubated with 50 \u0026micro;g/ml of propidium iodide and 1mg/ml of ribonuclease in PBS for 30 minutes. This flow cytometer procedure was adopted and modified (Elumalai et al., 2014). Cell cycle analyses were performed on a BD FACSCanto\u003csup\u003eTM\u003c/sup\u003eII (Becton and Dickinson Biosciences, Mountain View, CA, USA), and the data were analysed using BD FACSCanto clinical software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Real Time PCR\u003c/h2\u003e \u003cp\u003eThe gene expression of apoptosis signaling molecules was analysed using real-time PCR. The total RNA was isolated by the standardized protocol using Trizol Reagent (Sigma). 2\u0026micro;g of RNA is used for cDNA synthesis using reverse transcription using a PrimeScript, 1st strand cDNA synthesis kit (TakaRa, Japan). The targeted genes were amplified using specific primers. The PCR reaction was performed with GoTaq\u0026reg; qPCR Master Mix (Promega), which contains SYBR green dye and all the PCR components. Real-time PCR was performed in a CFX96 PCR system (Biorad). The results were analyzed by comparative C\u003csub\u003eT\u003c/sub\u003e method and 2\u003csup\u003e\u0026minus;∆∆C\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e method was used for fold change calculation described by (Schmittgen and Livak, 2008).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Scratch wound healing assay\u003c/h2\u003e \u003cp\u003eOnto six-well culture plates, MG-63 cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were planted. An inverted microscope was used to take pictures of the scratched cell monolayer after it had been cleaned with PBS and created a wound using a 200\u0026micro;l tip. Following a 24-hour rutin treatment period, the injured area was captured on camera using the same microscope. The control cells were given vehicle DMSO (0.01%) in culture media without rutin. Additionally, each treatment group underwent three replicate runs of the studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Protein Preparation\u003c/h2\u003e \u003cp\u003eThe crystal structures of Vascular endothelial growth factor (VEGF) (PDB ID: 1VPP) (Wiesmann et al., 1998) and Tumour Necrosis Factor-alpha (TNF- α) (PDB ID: 2E7A) (Shibata et al., 2008) were acquired from the RCSB PDB. The resolution of the VEGF structure is 1.90\u0026Aring;, while the TNF- α structure has a resolution of 1.80\u0026Aring; determined using the X-ray diffraction method. After being retrieved, the structures went through preprocessing steps which involved removing complexes bound to the protein receptor molecules and determining the protonation statuses of ionizable residues. This was done to ensure that proper electrostatic interactions would occur during docking. To optimize the system, water molecules and unnecessary ligands were removed from the protein structures. Subsequently, force field parameters were utilized to replicate the protein's behavior in docking simulations. The protein structures were then subjected to optimization and refinement processes to prepare them for molecular docking investigations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Ligand preparation\u003c/h2\u003e \u003cp\u003eThe molecular structures of the rutin compound known for their therapeutic potential, were sourced from PubChem (Kim et al., 2019) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"http://pubchem.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Chimera (Pettersen et al., 2004) converted the structure from the SDF to the PDB format, enabling a detailed analysis of their three-dimensional configurations. This essential step applies accurate force fields to rutin's 3D structure, preparing it for target protein simulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Molecular docking\u003c/h2\u003e \u003cp\u003eAutodock (Morris et al., 2009) analyses different binding orientations and interactions and is utilized to align the ligand with the target protein. Subsequently, a scoring method is employed to evaluate the most appropriate ligand-protein complex identified during this analysis. This method enables precise projections of the ligand's compatibility with the active site of the protein, offering a crucial understanding of the ligand as a therapeutic agent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eOne-way ANOVA and Student's t-test SPSS were used to evaluate the obtained data. The statistical analysis on all experimental data, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 being deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e\u003cstrong\u003e3.1 Rutin inhibits cell proliferation and induces cytotoxicity in osteosarcoma cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MTT assay was employed to determine the anti-proliferative effects of rutin on the MG-63 cells after different dosages of rutin were administered for 24 hours to test for the inhibition of the growth of human osteosarcoma cells.\u0026nbsp; The treatment of rutin caused a notable decrease in cell proliferation in a concentration-dependent manner as depicted in Fig1A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Lactate dehydrogenase (LDH) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLDH release assays were used to assess the cytotoxicity of rutin in MG63 cells. Additionally, MG63 cells were exposed to rutin at various doses (25, 50, 100, 150, 200, and 250 μM). After a 24-hour incubation period, the amount of cell death was assessed using an LDH release assay. This assay offers a reliable estimate of the cell death triggered on by rutin because the amount of LDH released into the culture media by the dying cells is proportionate to the degree of cell death. When compared to the control, Fig. 1B clearly illustrates the dose-dependent, significant increase in rutin-induced LDH release (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Rutin alters the cell morphology of osteosarcoma cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter cell proliferation, the MG-63 cells treated with rutin of two concentrations (100\u0026nbsp;\u0026amp;150μM/ml)\u0026nbsp;for 24 hrs, were undergone morphology studies to determine the alteration of cell morphology. It was observed that after treatment with rutin the cells were showed reduction in number and recognized with blebbed membrane and shrinkage of cells in Fig 2(A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Rutin induces apoptotic cells in osteosarcoma cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe induction of apoptosis by rutin on osteosarcoma cell line MG-63 was assessed with AO/EtBr staining and the results were showed in Fig. 2 (B). AO/EtBr staining was used to distinguish the live and dead cells. The green cells are live cells where the acridine orange stain penetrates into both living and dead cells incontrast, ethidium bromide can only infiltrate dead cells. MG-63 cells treated with rutin showed increased number of dead cells in our AO/EtBr staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Rutin suppresses cell growth and causes cell cycle arrest in osteosarcoma cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe examined the impact of rutin on the cell cycle proliferation in order to validate the correlation between growth inhibition and cell cycle arrest decipited in Fig 3. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Rutin modulates the mRNA expression of apoptotic genes in osteosarcoma cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRutin's impact on apoptotic genes (p53, Bcl-2, Bax, and Caspase-3) in MG-63 cells was assessed. Rutin (100 and 150\u0026nbsp;μM/ml) enhanced the mRNA expression of apoptotic genes in the MG-63 cell line much more than the untreated control cells, as seen in Fig. 4. In this MG-63 osteosarcoma cell line, rutin upregulated the pro-apoptotic genes, p53, Bax, and caspase-3 while downregulating the anti-apoptotic gene, Bcl-2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7\u0026nbsp;\u0026nbsp;Scratch wound healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe migratory capacity of MG-63 cells was initially identified by scratch assay, which allowed for the confirmation of rutin's impact on migratory behaviour. Following a 24-hour stimulation of MG63 cells with varying rutin concentrations (100 and 150 µM/ml), the number of cells moving to the centre of the scratch decreased as the concentration of rutin increased, as seen in Figure 5. According to these results, it proved that rutin could inhibit OS cell motility in vitro. Rutin effects on VEGF and EGF migration gene were assessed by mRNA expression for rutin concentrations 100 and 150 μM/ml along with control group (Fig 6). Following EGF and VEGF suppression, this suggests that rutin have downregulated the genes responsible for migration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.8\u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eMolecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe molecular docking study examines rutin's interaction with TNF-α and VEGF proteins, which play a crucial role in inflammation and angiogenesis. Rutin exhibits binding energies of -3.01 kJ/mol with TNF-α and -4.24 kJ/mol with VEGF, suggesting a stronger affinity for VEGF. It forms hydrogen bonds with TNF-α at LEU26 (3.01236 Å) and GLN27 (2.90033 Å) and with VEGF at GLN89 (2.82538 Å), indicating significant interactions (Figure 7 \u0026amp; Table 1). Furthermore, rutin engages in an electrostatic Pi-Anion interaction with VEGF's ASP11 (3.66573 Å), and hydrophobic interactions with LEU16 (3.98319 Å) and HIS86 (4.94879 Å). Additional interactions include a Pi-Donor Hydrogen Bond with VEGF's GLN89 (4.12276 Å) (Figure 8 \u0026amp; Table 1). These findings suggest rutin's potential to inhibit TNF-α and VEGF, highlighting its therapeutic prospects in inflammation and angiogenesis-related diseases.\u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eOsteosarcoma a prevalent primary malignant bone tumor and its clinical features include a high morbidity rate and a high risk of recurrence. Adolescents and children's health are significantly impacted by osteosarcoma. The overall prognosis for people with osteosarcoma remains dismal, despite significant advancements in the clinical diagnosis and surgical treatment of the disease. Therefore, the need to identify efficient molecular therapy targets is important. The active components of natural medicines are also heavily utilised in the treatment of osteosarcoma (Daher et al., 2023). Rutin exhibits enormous activities including antioxidant, antiaging, neuroprotective, cardioprotective and anticancer. According to previous studies, rutin serves as a potent anticancer agent in breast, liver, lung, cervical, and prostate as well as tends to induce apoptosis and causes cell cycle arrest (Liga et al., 2023). Here, we evaluated rutin, a flavonoid which suppresses the growth of osteosarcoma by inactivating the signaling pathway by inhibiting proliferation and migration of cells with enhancing the apoptosis of cell in OS cell line, MG- 63 which was further validated by molecular docking.\u003c/p\u003e \u003cp\u003eAt first, the OS cell line MG- 63 was treated with rutin at different concentrations (25\u0026ndash;250 \u0026micro;M/ml) for 24 hrs to assess the inhibition of rutin on growth of the osteosarcoma cells and the rutin cytotoxic effects were evaluated using MTT assay. Fang et al. (2023) have used MTT assay to detect osteosarcoma cell growth and also found that there is a reduction in the proliferation, migration and invasion of cells by targeting miR-340-5p gene expression via promoting FGF23.\u003c/p\u003e \u003cp\u003eThe release of lactate dehydrogenase enzyme into the medium, a sign of cell damage in treated cells, may be measured with the sensitive and accurate LDH test. In the presence of NADH, these enzymes catalyse the conversion of pyruvate to lactate (Farhana \u0026amp; Lappin, 2024). The LDH cytotoxicity assay was used on osteosarcoma cells in order to more thoroughly characterise the impact of rutin. That is well recognised that the release of LDH enzyme into the medium from the cytoplasm of injured cells, together with a rise in absorbance, is evidence of membrane damage-induced cell death. LDH release increased dose-dependently when rutin was applied to osteosarcoma cells. We observed a notable release (**p˂0.05) of LDH in the culture medium compared to the control after MG-63 cancer cells were treated for 24 hours with 100 and 150\u0026micro;M/ml rutin. Our results are similar to other authors that found a significant increase of LDH in osteosarcoma cell lines (MG-63). Chandrasekaran et al. (2023) have evaluated that NH extract against MG-63 cells and have concluded the cell damage was increased in response to dose concentrations. Higher concentrations resulted in more cell damage, which was a result of LDH leaking into the medium at a higher level. This demonstrates the phenomenon of NH extract-induced apoptosis causing cell death. Khan et al. (2021) have estimated the cytotoxicity of rutin by the release of LDH by disrupting its cell membrane in cervical cancer caski cells. They have found that rutin treatment of 24h have significantly increased in cell death so it\u0026rsquo;s strongly confirms the anticancer potential of rutin.\u003c/p\u003e \u003cp\u003eAfter being treated with rutin significantly fewer cell proliferation were observed in a concentration dependent manner. From those different concentrations the IC\u003csub\u003e50\u003c/sub\u003e value of 150\u0026micro;M/ml used to assess the inhibitory impact and in addition, the morphology was also studied using phase contrast microscope to verify its anticancer potential. Here, the cancer cells were significantly reduced after treatment with rutin for 24 hrs and the cells exhibited in the indications of cytotoxicity by shrinking and blebbing of the ctoplasmic membrane. Cao et al. (2022) have investigated the role of flavonoid alpinetin in osteosarcoma cell lines 143B and U2OS under different concentrations and proved that it inactivated the signaling pathways PI3K/AKT and ERK and inhibited the cell proliferation and metastasis in osteosarcoma. Alyami et al. (2023) have evaluated that Rutin suppressed the development and proliferation of hepatic and pancreatic cancer cells, with HepG-2 being the most impacted. It also inhibited the GST enzyme, lowering chemotherapeutic drugs anticancer effectiveness, and the CYP3A4 enzyme, showing the possibility for combination-based therapies. Thus proved that flavonoids are the alternate treatment for cancer.\u003c/p\u003e \u003cp\u003eWe evaluated the apoptosis induction in MG-63 cell line on treatment with rutin (100 \u0026amp; 150\u0026micro;M/ml) for 24hrs along with control group to analysed their morphological changes which were observed under florescence microscope after stained with AO/EtBr dual staining. The results outcome indicated that rutin induce apoptosis in MG-63 cells shows green fluorescence for viable cells and yellowish orange and orange in hue for early and late apoptotic cells, and also showed morphological changes including cell shrinkage and blebbing of membrane. Alzahrani et al. (2023) have identified apoptotic induction in the MDA-MB-435s cells treated with albumin\u0026ndash;CGA NPs and paclitaxel for 24 hours by AO/EtBr analysis. They have also showed a significant increase in AO/EtBr stained cells, similar to paclitaxel-treated cells and was observed no staining in untreated cells. Liu et al. (2023) have observed morphological changes in the apoptosis cells due to AO/EB dual staining. The OS control cells shows green live cells which were untreated whereas, the increased cell apoptosis showed in CL (30 \u0026micro;M/ml) with condensed chromatin and membrane blebbing and showed orange membrane integrity loss in late apoptotic cells.\u003c/p\u003e \u003cp\u003eApoptosis is the regulated and orderly death of cells that requires the activation, expression, and regulation of a number of distinct genes. Extrinsic and intrinsic apoptosis pathways are the two main signalling routes that trigger apoptosis. Binding death ligands and death receptors, such as TNF-R1, Fas, DR3, TRAIL-R1, DR6, EDAR, and nerve growth factor, starts extrinsic apoptosis. Intracellular stressors such oxidative stress, growth factor deprivation, chemotherapeutic drugs, or radiation might trigger intrinsic apoptotic pathways. The 20 members of the Bcl-2 family of proteins, which are divided into three subfamilies, control the intrinsic apoptotic pathway (Li et al., 2016). The family of proteins known as B-cell lymphoma protein-2, or BCL-2, controls this extremely conserved mechanism. Based on their unique functions in the process of apoptosis, these proteins can be categorised into three major classes: pro-apoptotic, anti-apoptotic, and effector proteins. Effector proteins are activated by a variety of intracellular and extracellular signals, such as DNA damage, the absence of growth factors, hypoxia, chemo- and radiotherapy, by up- or down-regulating pro-apoptotic and anti-apoptotic proteins, respectively. This activation leads to the formation of multimeric pores in the outer membrane of the mitochondria (Alipour et al., 2023).\u003c/p\u003e \u003cp\u003eSince flow cytometry study revealed cell cycle arrest in MG-63 cells treated with rutin. The efficacy of rutin to induce cell cycle arrest was explored to identify potential mechanisms of action. It was observed that there in control G0-G1: 90.88%, S: 4.23%, and G2/M: 5.00% whereas the cells which were treated with rutin showed G0-G1: 73.56%, S: 11.78% and G2/M: 14.66%. This result indicates that the G2/M phase shows an increased number of cells whereas cells were decreased in the S phase. Kelly et al. (2023) have investigated the potential mechanisms of action of RL71 on cell cycle arrest. The results revealed that RL71 caused an arrest in the G2/M phase by increasing the number of cells in that phase and decreasing those in the G1 phase.\u003c/p\u003e \u003cp\u003eThe effect of rutin on pro-apoptotic and anti-apoptotic genes p53, Bcl-2, Bax and Caspase-3 expressions were studied in MG- 63. The anti-apoptotic gene Bcl-2 decreased; however, the pro-apoptotic genes p53, Bax and Caspase-3 were significantly increased during apoptosis. There is a significant increase observed in both concentrations (100\u0026micro;M/ml \u0026amp; 150\u0026micro;M/ml) when compared to control group in the pro-apoptotic genes: p53 with 3 and 4 fold; Bax with 2 and 4 fold and Caspase- 3 with 1.4 and 2.5 fold. Whereas, there is a decrease in the anti-apoptotic gene Bcl-2 with 0.7 and 0.5 fold when compared with control group. Hajimehdipoor et al. (2023) have investigated the rutin expression of MKI67, VEGFA, VIM, CDH2, and FN1 in MDA-MB-231 and MCF-7 cell lines compared to untreated control cells. They proved that rutin activated the EMT process by suppressing CDH1 expression and increasing VIM, FN1, and CDH2 expression and promoted VEGFA expression in MCF-7 cells without significant effect on THBS1.\u003c/p\u003e \u003cp\u003eA scratch well test was then performed to investigate the effects of rutin (100 and 150\u0026micro;M/ml) on the invasion and migration of osteosarcoma cell line, MG- 63. Following a 24-hour incubation period, rutin significantly reduced the migration of cells in a dose-dependent manner, with no effect on the control group. Rutin may have an inhibitory effect on the migratory potential of MG-63 cells, as evidenced by the dose-dependent suppression of cell migration. Rutin significantly inhibits cell migration, suggesting that these substances may obstruct cellular mechanisms involved in migration, including cytoskeleton rearrangements and cell signalling pathways. It implies that rutin influences not just the survivability of cells but also their functional behaviour, including their capacity to migrate and take part in the processes involved in wound healing. Qi et al., 2023 have proved that piperine (PIP), alkaloid which has the capacity to inhibit the effects of doxorubicin (DOX) against OS cell migration in U2OS and 143B cells. They have concluded that when compared to the control group, PIP and DOX dramatically decreased U2OS and 143B cell migration. Cell migration decrease was greatly enhanced when PIP and DOX were combined. The wound area was greater in all pharmacological groups than in the control group. The PIP\u0026thinsp;+\u0026thinsp;DOX group showed noticeably larger percentages of surviving wound area after 24 hours, indicating a decrease in cell migration and development. Huo et al. (2022) have evaluated the wound healing activity of rutin (5 \u0026micro;g/mL and 10 \u0026micro;g/mL) for pancreatic cell line (PANC-1). In that they explored that migration of PANC-1 cell as well as in SW 1990 cells and MIA PaCa-2 cells was significantly inhibited after treatment of rutin for 48hrs.\u003c/p\u003e \u003cp\u003eThe effect of rutin on migration genes EGF and VEGF expressions were studied in MG- 63.. There is a significant decrease observed in both concentrations (100\u0026micro;M/ml \u0026amp; 150\u0026micro;M/ml) when compared to control group in the migration genes: EGF with 0.7 and 0.3 fold and VEGF with 0.8 and 0.5 fold when compared with control group. This proved that rutin have the capability to down regulate the EGF and VEGF mRNA expression. Furthermore, using the AutoDock tools, an in silico molecular docking research was carried out to look at the binding interactions of rutin with TNF- α and VEGF proteins. Remarkably, every active component of rutin demonstrated a strong affinity for interacting with the proteins TNF- α and VEGF. The molecular interactions are represented by interacting residues in binding sites with certain physico-chemical characteristics that point to particular target protein functions. According to this finding, the complex structure was stabilised by the interactions between participating amino acids and ligand molecules through the use of H-bond, alkyl, pi-alkyl, and van der Waals forces.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eIn conclusion, it has been proved that Rutin, a flavonoid that is commonly found in plants that is thought to have numerous medicinal benefits and is used to treat variety of illness. This is an effective anticancer medication that works by preventing the formation of tumor-promoting proteins and cancer cells. The osteosarcoma cell line (MG-63) was used in the current investigation to assess the cytotoxicity activity of rutin. The MTT and LDH assays demonstrated a significant cytotoxic effect. The cell line underwent morphological alterations, including membrane blebbing and shrinkage of the cells, upon close inspection following rutin treatment. A characteristic of anticancer drugs, the in vitro evaluation of the wound healing assay revealed an anti-migratory effect. The MG-63 cell line was used in the study's mRNA gene expression analysis by RT-PCR. The results showed that the anti-apoptotic gene BCL-2 was downregulated and the pro-apoptotic genes p53, Bax, and caspase-3 were upregulated. Additionally, the study evaluated the interaction between rutin and the proteins linked to OS using in silico models, and it demonstrated efficient interaction at different TNF-α and VEGF binding sites. Since the osteosarcoma MG-63 cell line was the sole focus of the current analysis, further preclinical research is required in light of this study in order to ascertain the safe and effective dosages for potential medication development for the treatment of osteosarcoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo associated data in the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank to Gold Lab and the Department of Pharmacology,Saveetha University for offering the study's research lab facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Biotechnology, Vinayaka Mission’s Kirupananda Variyar Engineering College, Vinayaka Mission’s Research Foundation (Deemed to be University), Salem - 636 308, Tamil Nadu, India\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG. Gnanamathy, S. Nancy sheela \u0026amp; M. Sridevi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCancer Genomics Laboratory, Centre for Global Health Research, Saveetha Medical College, Saveetha Institute of Medical and Technical Science, Chennai, Tamil Nadu, India\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElumalai Perumal \u0026amp; R. Jeevitha\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGG, RJ conducted the experiments and involved in the manuscript preparation. GG, SN were involved in experimental design and in preparation of manuscript. MS and EP reviewed and finalized the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;M. Sridevi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study is not involved animal/human studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher's Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRights and permissions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eLiu W, Zhao Y, Wang G, Feng S, Ge X, Ye W, Wang Z, Zhu Y, Cai W, Bai J, Zhou X (2022) TRIM22 inhibits osteosarcoma progression through destabilizing NRF2 and thus activation of ROS/AMPK/mTOR/autophagy signaling. 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Molecules 27(7):2293. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/molecules27072293\u003c/span\u003e\u003c/span\u003ePMID: 35408691; PMCID: PMC9000526\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cem\u003eTable 1: Interaction of rutin with TNF- \u0026alpha; and VEGF protein\u003c/em\u003e\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"745\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.21476510067114%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompound Name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.724832214765101%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.738255033557047%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBinding energy (kJ/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.87248322147651%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmino acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.798657718120806%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDistance\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.986577181208055%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInteraction type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.664429530201343%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInteraction bond\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.21476510067114%\" rowspan=\"7\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eRutin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.724832214765101%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.738255033557047%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e-3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.87248322147651%\" valign=\"top\"\u003e\n \u003cp\u003eLEU26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.798657718120806%\" valign=\"bottom\"\u003e\n \u003cp\u003e3.01236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.986577181208055%\" valign=\"bottom\"\u003e\n \u003cp\u003eHydrogen Bond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.664429530201343%\" valign=\"bottom\"\u003e\n \u003cp\u003eConventional Hydrogen Bond\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.913555992141454%\" valign=\"top\"\u003e\n \u003cp\u003eGLN27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.341846758349705%\" valign=\"bottom\"\u003e\n \u003cp\u003e2.90033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.326129666011788%\" valign=\"bottom\"\u003e\n \u003cp\u003eHydrogen Bond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.418467583497055%\" valign=\"bottom\"\u003e\n \u003cp\u003eConventional Hydrogen Bond\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.938837920489297%\" rowspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eVEGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.232415902140673%\" rowspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e-4.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.385321100917432%\" valign=\"top\"\u003e\n \u003cp\u003eGLN89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.162079510703364%\" valign=\"top\"\u003e\n \u003cp\u003e2.82538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.489296636085626%\" valign=\"top\"\u003e\n \u003cp\u003eHydrogen Bond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.79204892966361%\" valign=\"top\"\u003e\n \u003cp\u003eConventional Hydrogen Bond\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.913555992141454%\" valign=\"top\"\u003e\n \u003cp\u003eASP11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.341846758349705%\" valign=\"bottom\"\u003e\n \u003cp\u003e3.66573\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.326129666011788%\" valign=\"bottom\"\u003e\n \u003cp\u003eElectrostatic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.418467583497055%\" valign=\"bottom\"\u003e\n \u003cp\u003ePi-Anion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.913555992141454%\" valign=\"top\"\u003e\n \u003cp\u003eGLN89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.341846758349705%\" valign=\"bottom\"\u003e\n \u003cp\u003e4.12276\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.326129666011788%\" valign=\"bottom\"\u003e\n \u003cp\u003eHydrogen Bond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.418467583497055%\" valign=\"bottom\"\u003e\n \u003cp\u003ePi-Donor Hydrogen Bond\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.913555992141454%\" valign=\"top\"\u003e\n \u003cp\u003eLEU16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.341846758349705%\" valign=\"bottom\"\u003e\n \u003cp\u003e3.98319\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.326129666011788%\" valign=\"bottom\"\u003e\n \u003cp\u003eHydrophobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.418467583497055%\" valign=\"bottom\"\u003e\n \u003cp\u003ePi-Sigma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.913555992141454%\" valign=\"top\"\u003e\n \u003cp\u003eHIS86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.341846758349705%\" valign=\"bottom\"\u003e\n \u003cp\u003e4.94879\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.326129666011788%\" valign=\"bottom\"\u003e\n \u003cp\u003eHydrophobic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.418467583497055%\" valign=\"bottom\"\u003e\n \u003cp\u003ePi-Alkyl\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteosarcoma, Rutin, TNF- α, EGF, VEGF, Apoptosis, Insilico analysis, Molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-4191813/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4191813/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRutin is a flavonoid that is widely distributed in plants and has been identified as having medicinal qualities as well as potential benefits for treating a range of ailments. In this work, we examined rutin's anticancer effects, specifically with regard to osteosarcoma, a type of bone cancer.\u003c/p\u003e\u003ch2\u003eMethods and results\u003c/h2\u003e \u003cp\u003eWe evaluated the cytotoxic activity of rutin using MTT and LDH tests on the MG-63 osteosarcoma cell line, and the results showed a notable cytotoxic effect. Following rutin treatment, morphological alterations, such as membrane blebbing and cell shrinkage, were noted, which are typical of anticancer medications. Additionally, an in vitro assessment employing the wound healing assay revealed rutin's anti-migratory action on MG-63 cells. The results of the RT-PCR gene expression research pointed to possible pathways of rutin-induced apoptosis, including downregulation of the anti-apoptotic gene BCL-2 and elevation of pro-apoptotic genes including p53, Bax, and caspase-3. Additionally, the migration-causing genes VEGF and EGF were downregulated by rutin. Moreover, the relationship between rutin and proteins linked to osteosarcoma, like VEGF and TNF-α, was evaluated using in silico models.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe findings demonstrated effective binding at various binding sites, pointing to rutin's possible therapeutic use in the treatment of osteosarcoma. Although this work uses the MG-63 cell line to provide light on the anticancer activity of rutin against osteosarcoma, more preclinical research is necessary to establish the best dosages and assess safety profiles for the possible development of medications for the treatment of osteosarcoma.\u003c/p\u003e","manuscriptTitle":"Unraveling the therapeutic potential of Rutin against osteosarcoma cells: Targeting TNF-α and VEGF signaling pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-08 04:59:12","doi":"10.21203/rs.3.rs-4191813/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"183a1a54-6d98-4146-8b8b-2c6f1d174aa9","owner":[],"postedDate":"April 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-31T14:42:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-08 04:59:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4191813","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4191813","identity":"rs-4191813","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}