Silibinin Induces Apoptosis and Suppresses Cell Migration by Targeting the Transforming growth factor-β Signaling Pathway in Osteosarcoma Cells

doi:10.21203/rs.3.rs-4933548/v1

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Silibinin Induces Apoptosis and Suppresses Cell Migration by Targeting the Transforming growth factor-β Signaling Pathway in Osteosarcoma Cells

https://doi.org/10.21203/rs.3.rs-4933548/v1

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Osteosarcoma is the most common primary malignant bone tumor in children and teenagers, followed by lymphomas and brain tumors. Silibinin, a flavonolignan mix from milk thistle, has anticancer, neuroprotective, and anti-diabetic properties. It induces apoptosis in MG-63 cells.; Silibinin treatment of MG-63 cells resulted in a dose-dependent inhibition of cell viability; for the MG-63 cell line, the growth-dependent rate peaked at 40μM/ml and 60μM/ml. Although studies involving Silibinin in various cancers were reported, the anticancer activity of Silibinin in human osteosarcoma has not been reported. Utilising MTT assay, morphological studies, and mode of cell death. Acridine orange (AO)/ethidium bromide (EtBr) dual labeling at the ideal dosage is followed by morphological examinations and a fluorescence microscopy examination of the labeled cells to identify apoptotic alterations and the mode of cell death. Utilising LDH assay, Scratch wound healing assay, and molecular docking. Silibinin promotes apoptosis in MG-63 cell lines and may be a target for treatment in people with osteosarcoma and it may also have a role in the development of osteosarcoma. At 60μM/ml of Silibinin concentration, the prevention of cell division and cell cycle arrest in MG-63 cells was examined. In the MG-63 cells, the impact of Silibinin on the apoptotic genes p53, Bcl-2, Bax, and caspase-3 was assessed. Silibinin promotes apoptosis in MG-63 cell lines and may be a target for treatment in people with osteosarcoma and it may also have a role in the development of osteosarcoma.

Osteosarcoma

silibinin

apoptosis

MG-63

TGF-β

Bcl-2.

Osteosarcoma is the most prevalent cause of primary malignant bone tumors. In children and adolescents, it is also the third most common kind of cancer, behind brain tumors and lymphomas. Mesenchymal in origin, osteosarcoma cells can produce osteoid substances and/or immature bone. Data from the Surveillance, Epidemiology and End Results Programme (SEER) and the American Cancer Society (ACS) show that 67% of children and adolescents with osteosarcoma have received a diagnosis. Estimates predict that in 2023, there will be 3,970 new cases and 2,140 fatal cases of bone and joint cancer [1]. Surgical resection, chemotherapy, and radiation treatment are now used to treat high-grade osteosarcoma. Unfortunately, these treatments have caused a number of individuals to have both short- and long-term side effects, including myelotoxicity, cardiotoxicity, and renal toxicity [2].

Plants generate a wide range of substances, including secondary metabolites, which have been utilised for over 5000 years as antibiotics, pain relievers, anticancer, and cardioprotective medications. This vast potential of plant components, which has now been refined by scientific advancement and the capacity to precisely identify plant-based bioactive compounds, has aided in the creation of new health-related technology. Many plants are being studied for their anticancer qualities, and the results are highly encouraging. Approximately 80–90% of the population in underdeveloped nations still consumes plant-based medications. The separation and identification of physiologically active chemicals derived from plants has resulted in the discovery of novel medicinal treatments, hence boosting the growth of the health and pharmaceutical industries [3].

Silibinin is the main active component of silymarin, a blend of flavonolignans obtained from milk thistle (Silybum marianum) [4]. Silibinin exhibits anticancer [5], neuroprotective [6], and anti-diabetic [7], properties, according to earlier investigations. Cell death through a mechanism known as apoptosis is crucial for development. Cell shrinkage, DNA breakage, chromatin condensation, and the creation of apoptotic bodies are all signs of apoptosis, which is controlled genetically. Nowadays, a number of anti-cancer therapies rely on chemically inducing apoptosis, however doing so runs the risk of unintended side effects and recurrence [8].

Several investigations show that silibinin's anti-cancer effects stem from a range of biological mechanisms, such as metastasis, apoptosis promotion, cell cycle arrest, and suppression of angiogenesis [9]. Pro- and anti-apoptotic B-cell lymphoma 2 (Bcl-2) family proteins are known to control apoptosis. The death receptor-mediated extrinsic route and the mitochondria-dependent intrinsic system are the two primary pathways via which the caspase cascade is activated [10]. Human oral squamous carcinoma (SCC-25) cells were used to determine the cytotoxic impact of silibinin. A potential therapeutic option for the treatment of oral squamous cell carcinoma, according to the study's findings, is silibinin [11]. In a different study, silibinin was employed as a nanoparticle encapsulated in a polymersome to induce apoptosis and lower the expression level of miR-125b/miR-182 in human breast cancer cells [12]. Human breast cancer cells MCF-7 and MDA-MB-231 undergo apoptosis in the presence of silibinin [13]. The downregulation of p73 was implicated in the cytoprotective action of silibinin, as evidenced by the reduction of UVB-induced apoptosis when p73 was inhibited by siRNA [14]. TGF- βs act as tumor suppressors in premalignant tumors and tumor promoters in advanced tumors in relation to carcinoma, as is well acknowledged [15]. In a dose-dependent way, silibinin reduced the viability of HT-29 cancer cells. The Smad2 signaling pathway may have been involved in silibinin's inhibition of TGF-β-stimulated MMP-2 and MMP-9 production in HT-29 cells [16].

Bcl-2 is a member of the protein family that controls programmed cell death. i.e. apoptosis via an intricate intracellular process [17]. Apoptosis is inhibited by the proto-oncogene bcl-2, which expression increases cell survival by making tumor cells resistant to apoptosis. Previous studies have identified the expression of bcl-2 in human lung cancer, osteosarcoma, and metastases, in addition to its function in the growth of tumors [18].

Several studies shows that the silibinin have exhibited its anti-tumor effects in breast cancer by targeting the stem cells reducing their tumorgenicity and metastasis [19], and also exhibits its anti- cancer activity on human ovarian cancer by increasing apoptosis and inhibiting mesenchymal transition [20]. Silibinin also shows its effect in prostate cancer and shows its migration and invasion [21]; in addition, it overcomes EMT driven lung cancer which were resistance to new generation ALK inhibitors [22]. The silibinin were also used as gold nanoparticles to treat lung cancer in a conjugated therapy [23]. We investigated the anticancer effect of silibinin on human osteosarcoma (MG-63) cell lines and hence its effect on Cell Migration by Targeting the TGF-β Signaling Pathway in cells.

2.1 Cell line maintenance

The NCCS in Pune provided human osteosarcomacell lines (MG-63) for this study. The cells were grown in T25 culture flasks supplemented with DMEM and RPMI, along with 10% FBS and 1% antibiotics. The cells were maintained in a humid atmosphere with 5% CO2 at 37°C. After being trypsinized, confluent cells were passaged.

2.2 Cell viability (MTT) assay

The osteosarcoma cell line treated with silibinin was tested for cell viability using the MTT assay. The test depends on metabolically active cells converting soluble yellow tetrazolium salt into insoluble purple formazan crystals. The osteosarcoma cell line was plated in 96-well plates with a 5x103 cell density per well. Cells were starved by being incubated in serum-free media for 3 hours at 37oC, two times, 24 hours after plating. After fasting, cells were exposed to Silibinin at varying doses (10–100 M/ml) for 24 hours. At the end of the treatment, 100 l of MTT-containing DMEM (0.5 mg/ml) was added to each well, replacing the media from the control and Silibinin-treated cells. The cells were then placed in a CO2 incubator and kept at 37°C for 4 hours. After that, the MTT-containing media was removed, and 1x PBS was used to wash the cells. The created formazan crystals were dissolved in 100 l of dimethyl sulfoxide, and the mixture was then allowed to settle for an hour in the dark. The color's intensity was then measured at 570 nm using a Micro ELISA plate reader. To express the amount of viable cells, the percentage of control cells grown in serum-free medium is utilized. The cell viability in the control medium without any treatment was indicated as 100%. The formula is used to calculate the cell viability.

% cell viability = [A570 nm of treated cells/A570 nm of control cells] ×100.

2.3 Morphology study

Based on the results of the MTT experiment, we chose the best doses of silibinin (IC-50: 60 mM and 100 mM for osteosarcoma cell line and 100 mM and 150 mM for MG-63 cell line) for further research. By using a phase contrast microscope, changes in cell morphology are examined. In 6-well plates, 2 105 cells were plated and given a 24-hour Silibinin treatment. At the end of the incubation period, the medium was removed from the cells and they were given a single wash in phosphate buffer saline (PBS, pH 7.4). A phase contrast microscope was used to view the plates [24].

2.4 Determination of mode of cell death by acridine orange (AO)/ethidium bromide (EtBr) dual staining

The AO/EtBr dual staining method previously described was used to assess the effects of Silibinin on osteosarcoma cell death. Silibinin was applied to the cells for 24 hours, after which the cells were collected and rinsed with ice-cold PBS. The pellets were redissolved in 5 milliliters of acridine orange (1 mg/mL) and 5 milliliters of EtBr (1 mg/mL). The apoptotic alterations in the tagged cells were then examined using a fluorescence microscope [25].

2.5 Cell cycle analyses by flow cytometer

The MG-63 cells were cultivated on 100-mm culture plates with growth media, with 1 × 106 cells per plate. The cells were allowed to fast for a while before being treated for 24 hours with 60 M/ml silibinin, collected with 0.25% trypsin, and centrifuged for five minutes at 3000xg. After that, PBS was used to wash the cells. After centrifugation, the cells were frozen at -20°C for an entire night in 70% ice-cold ethanol. Following that, the cells were incubated for 30 minutes in PBS with 1 mg/ml ribonuclease and 50 g/ml propidium iodide [26]. The data were analyzed using BD FACSC anto clinical software, and cell cycle studies were carried out using a BD FACSCantoTMII (Becton and Dickinson Biosciences, Mountain View, CA, USA).

2.6 Real Time PCR

The gene expression of the apoptotic signaling molecules was examined using real-time PCR. The total RNA was extracted using a standardized procedure using the Trizol Reagent (Sigma). Reverse transcription-based cDNA synthesis was performed using 2µg of RNA and a PrimeScript, first strand cDNA synthesis kit (TakaRa, Japan). Targeted gene amplification was accomplished with specific primers. The PCR reaction was carried out using the GoTaq® qPCR Master Mix (Promega), which contained SYBR green dye and other required PCR components. Real-time PCR was conducted on a Biorad CFX96 PCR apparatus. 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 [27].

2.7 LDH assay

LDH is a stable cytosolic enzyme present in the cytosol with intact cell membrane but it released once the cell losses membrane integrity or damage and it can be measured quantitatively by LDH assay. For LDH leakage assay, the same MTT assay treatment protocol used and the conditioned medium alone were taken for this assay. 0.1 ml of condition media was added to 1 ml of buffered substrate, and kept in water bath at 37oC. Then NAD+ (0.2 ml) solution was added and mixed, after 15 min incubation at 37 ◦C 1ml of DNPH solution was added and incubated for 15 min. lastly, 0.4N of sodium hydroxide (10ml) was added and incubated for 1–5 min and the absorbance was measured at 440 nm. Sodium pyruvate used as standards to prepare the standard graph. LDH activity = OD of unknown/OD of known ×standard concentration = µg of Lactate liberated/ml of conditioned media.

2.8 Scratch wound healing assay

MG-63 cells (2×105 cells/well) were seeded onto six-well culture plates. The cell monolayer was scratched using 200µl tip to create wound, washed with PBS and photographed in inverted microscope. Silibinin treated for 24 h and control cells received with vehicle DMSO (0.01%) in culture medium and without Silibinin, after the treatment period, the wounded area was photographed using the same microscope. And the experiments were repeated in triplicate for each treatment group.

2.9 Protein Preparation

The crystal structures of B-cell lymphoma 2 (BCL-2) (PDB ID: 1R2D) [28] and Tumour Necrosis Factor-Beta (TGF- β) (PDB ID: 1TGJ) [29] were acquired from the RCSB database. The resolution of the BCL-2 structure is 1.95Å, while the TGF- β structure has a resolution of 2.00Å. Upon retrieving them, the structures went through preparation procedures, which involved eliminating complexes that were attached to the protein receptor molecules and determining the protonation statuses of ionizable residues to ensure precise electrostatic interactions during docking. To optimize the system, water molecules and redundant ligands were removed from the protein structures. Subsequently, force field parameters were utilized to replicate the protein's behavior in docking simulations. Afterward, optimization and refinement processes were performed on the protein structures before beginning molecular docking studies.

2.10 Ligand preparation

The chemical structures of the silibinin molecule were obtained from PubChem [30]. Using Chimera [31] the molecular structure of silibinin was converted from the SDF to the PDB format, facilitating a thorough examination of their three-dimensional arrangements. For precise target protein interaction analysis, silibinin's structural data must be accurately represented during ligand preparation in molecular docking studies.

2.11 Molecular Docking

The molecular docking analysis is carried out using AutoDock [32], leveraging the robustness of the Lamarckian Genetic Algorithm. The algorithm iteratively refines the positions and orientations of the ligand within the protein's binding site, effectively optimizing the ligand's conformation to achieve the highest binding affinity.

2.12 Statistical analysis

All of the data were examined using SPSS and were expressed as mean ± SD for triplicates. A One-way ANOVA was followed by a student’s-t-test. The level of statistical significance was set at p < 0.05.

3.1 Silibinin inhibits cell proliferation and induces cytotoxicity in osteosarcoma cells:

The MTT test was utilized to determine the cytotoxic effect of silibinin on the MG-63 cells after they were exposed to varying doses of silibinin for a whole day in order to evaluate the inhibition of human osteosarcoma cell growth. In a concentration-dependent manner, the treatment of silibinin resulted in a significant reduction in cell proliferation as depicted in Fig. 1.

Figure 1. The cytotoxic effects of Silibinin extract on osteosarcoam cells. After 24 hours of treatment with silibinin extract (10–100 µM/ml), the MTT test was used to assess the vitality of the cells. Data are shown as means ± SD (n = 3). * Compared with the control blank group, p < 0.05.

3.2 Silibinin alters the cell morphology of osteosarcoma cells.

After cell proliferation, the MG-63 cells treated with silibinin of two concentrations (40 &60µM/ml) for 24 hrs, were undergone morphology studies to determine the alteration of cell morphology. Following silibinin treatment, it was found that the cells showed cytoplasmic membrane blebbing, shrinkage, and a reduction in overall cell count.

3.3 silibinin induces apoptotic cells in osteosarcoma cells.

The induction of apoptosis by silibinin on osteosarcoma cell line MG-63 was assessed with AO/EtBr staining and the results were showed in Fig. 2. a. AO/EtBr staining was used to distinguish the live and dead cells. The green cells are alive ones, whereas ethidium bromide can only reach dead cells. Acrylin orange stain can penetrate both living and dead cells. MG-63 cells treated with silibinin showed increased number of dead cells in our AO/EtBr staining.

3.4 Silibinin suppresses cell growth and causes cell cycle arrest in osteosarcoma cells.

Apoptosis is directly related to cell cycle inhibition. So, employing a flow cytometry test, silibinin (60µM/ml) -induced apoptosis was further studied. Apoptosis was analyzed by Propidium Iodide double staining method. After treatment with various concentrations of silibinin for 24 hrs, the percentage of apoptosis was G0-G1:92.20%, S: 5.44%, and G2/M: 2.22% for control whereas the cells which were treated with silibinin shows G0-G1: 81.61%, S: 8.08% and G2/M: 10.19% in MG-63 cells were elucidated in Fig. 2.b. These results show that MG-63 cells treated with silibinin had higher percentages of S phase and G2/M phase cells than control. These findings revealed that silibinin suppressed proliferation and induced cell cycle blockage by interfering with cell cycle and proliferation-related proteins.

3.5 Silibinin modulates the mRNA expression of apoptotic genes in osteosarcoma cells.

Programmed cell death, or apoptosis, plays a role in a number of physiological and pathological processes, such as body homeostasis, cell elimination, and embryonic development. Dysregulation can trigger diseases like malignant tumors, and alterations in apoptosis pathways can lead to cancer recurrence and metastasis. Numerous studies aim to understand the relationship between apoptosis and cancer development to improve patient prognosis. In this present study focused on inducing apoptosis in the osteosarcoma cells by silibinin via intrinsic apoptotic pathway. To evaluate the effect of silibinin on apoptotic genes (p53, Bcl-2, Bax and Caspase-3) in the MG-63 cells were quantified. Figure 5. shows that the MG-63 cell line's mRNA expression of apoptotic genes was significantly elevated by silibinin (100 and 150µM/ml) in comparison to the untreated control cells. In parallel, silibinin increased the expression of pro-apoptotic genes such as p53, Bax, and caspase-3 and decreased the expression of the anti-apoptotic gene Bcl-2 in this MG-63 osteosarcoma cell line.

3.6 Lactate dehydrogenase (LDH) assay

The LDH cytotoxicity test was used to assess the impact of silibinin on bone osteosarcoma cells, revealing the release of LDH enzyme and increased absorbance. treatment of silibinin with osteosarcoma cell line showed a dose dependently increase in LDH release. As seen in Fig. 1, after 24-hour treatment of MG-63 cancer cells of silibinin, we have found a significant release. In this study first, we evaluated the viability of the cells at different concentrations (10–100 µM) using the LDH assay. It was discovered that, in comparison to other concentrations, 60µM/ml was the most effective.

3.7 Scratch Wound healing assay_ MG-63

In this assay, MG-63 cells were migrated, and the effects of silibinin were assessed using an in vitro wound healing assay. After starting medication, photos were taken to determine how many cells had moved into the area that had been scratched, and the number of cells was converted into a percentage of migration.A wound-healing assay was used to test the migration capacity of MG-63 cells (Fig. 5). After a 24 hours incubation period for wounded cells, the percentage wound width increased in the groups treated with 40 and 60 µM silibinin, respectively (Fig. 5). The results of this study revealed that significantly and independently reduced the migration of MG-63 cells.

3.8 Molecular Docking

The molecular docking study of silibinin against TGF-β and BCL2 proteins reveals significant binding affinities and specific interactions, suggesting its potential therapeutic applications. Silibinin binds to TGF-β with a binding energy of -6.99 kJ/mol, through multiple hydrogen bonds with CYS48 (2.93935 Å), ASN66 (3.18625 Å), CYS77 (3.23595 Å), CYS78 (3.07785 Å) and SER45 (3.52791 Å). It also forms a hydrophobic Pi-Sigma interaction with SER45 (3.08159 Å), indicating strong affinity and potential to modulate TGF-β activity, crucial in cellular proliferation and differentiation processes. Against BCL2, silibinin demonstrates a binding energy of -6.02 kJ/mol. It forms conventional hydrogen bonds with ARG139 (3.03178 Å), carbon-hydrogen bonds with GLY138 (3.72456 Å) and ARG139 (3.12654 Å). It engages in hydrophobic interactions including Pi-Sigma with ALA142 (3.91996 Å), Pi-Pi T-shaped with PHE97 (4.84123 Å) and TYR195 (4.96485 Å), and Pi-Alkyl with TYR101 (4.01496 Å). These interactions suggest silibinin's potential as a pro-apoptotic agent by interfering with BCL2's anti-apoptotic function.

Table 1

Interaction of silibinin with TGF-β and BCL2 protein
Compound Name	Protein name	Binding energy (kJ/mol)	Amino acid	Distance	Interaction type	Interaction bond
Silibinin	TGF-β	-6.99	CYS48	2.93935	Hydrogen Bond	Conventional Hydrogen Bond
			ASN66	3.18625	Hydrogen Bond	Conventional Hydrogen Bond
			CYS77	3.23595	Hydrogen Bond	Conventional Hydrogen Bond
			CYS78	3.07785	Hydrogen Bond	Conventional Hydrogen Bond
			SER45	3.08159	Hydrophobic	Pi-Sigma
			SER45	3.52791	Hydrogen Bond	Conventional Hydrogen Bond
			CYS48	2.93935	Hydrogen Bond	Conventional Hydrogen Bond
			ASN66	3.18625	Hydrogen Bond	Conventional Hydrogen Bond
			CYS77	3.23595	Hydrogen Bond	Conventional Hydrogen Bond
			CYS78	3.07785	Hydrogen Bond	Conventional Hydrogen Bond
	BCL2	-6.02	ARG139	3.03178	Hydrogen Bond	Conventional Hydrogen Bond
			GLY138	3.72456	Hydrogen Bond	Carbon Hydrogen Bond
			ARG139	3.12654	Hydrogen Bond	Carbon Hydrogen Bond
			ALA142	3.91996	Hydrophobic	Pi-Sigma
			PHE97	4.84123	Hydrophobic	Pi-Pi T-shaped
			TYR195	4.96485	Hydrophobic	Pi-Pi T-shaped
			TYR101	4.01496	Hydrophobic	Pi-Alkyl

Osteosarcoma is the most frequent orthopaedic tumour that affects teenagers. Osteosarcoma spreads quickly to other organs, including the lungs. Patients with osteosarcoma will have a much lower 5-year survival rate after metastasis begins [33].

In this study, firstly we performed MTT assay to evaluate the viability of cells under various concentrations (10–100 µM/ml). From that 60µM/ml were found to be effective when compared to other concentrations. Salmani et al., (2022). have performed MTT assay for various concentrations and studies the morphological changes where the cell membrane density whereas at 48 hours there is a change in features of apoptotic cells and membrane blebs were reduced in MG- 63 human osteosarcoma cells [34].

The study found that increased silibinin levels increased early apoptotic cell proportion, leading to cell death. Morphological analyses revealed membrane blebs, chromatin and nuclear condensation, DNA fragmentation, and apoptotic bodies. Shafiei et al. (2023) have synthesized silibinin loaded in MNNPs and studied the MTT assay along with its morphological characteristics. They concluded that the silibinin loaded in MNNPs showed a greater number of cell death when compared to pure silibinin [35].

In this present study we have performed AO/EB staining to detect the apoptosis for the 40 and 60µM/ml. In this study, we found that MG-63 cells treated with silibinin had higher levels of apoptotic cell death and nuclear damage, which was shown by AO/EB staining. Reduced osteosarcoma cell adhesion and migration were also seen after silibinin therapy. Si et al., (2023) have enhanced apoptosis in PINK1-depleted U2OS cells and confirmed these findings using AO/EB staining. In addition, they have also found out PINK1 inhibition increased the expression of proapoptotic proteins such as p53, Bad, and Bax in U2OS cells. These findings show that suppressing PINK1 causes U2OS cells to die [36].

The study utilized flow cytometry to analyze cell apoptosis in osteo sarcoma MG-63 cells, examining the impact of silibinin on initiating apoptosis and determining its cause.Silibinin induced a considerable amount of apoptosis in comparison to the control; in the control, the percentage of apoptosis was G0-G1:92.20%, S: 5.44%, and G2/M: 2.22%; in the MG-63 cells treated with silibinin, the percentage was G0-G1: 81.61%, S: 8.08%, and G2/M: 10.19%. The study reveals that MG-63 cells treated with silibinin showed higher S phase and G2/M phase cell percentages, suggesting that silibinin suppressed proliferation and induced cell cycle blockage.Pagano et al., (2022) have examined the Silibinin's effect on DC antigen-presenting ability, as determined by flow cytometry, which measures CD4 + T-cell responsiveness to alloantigens. While treated with Silibinin, the population hardly grew and the percentage of IFN-γ and IL-17 CD4 + T-cells was much reduced. The results demonstrated that LPS-matured DC promoted high proliferation and elevated IFN-γ and IL-17 production [37].

In this present study, we have performed real time PCR analysis expression for genes (p53, Bcl-2, Bax and Caspase-3). Among the four genes Bcl2 showed decreased apoptotic activity in compare to other three genes. Results showed that expression of p53 with 2.1 and 3.9-fold, Bax with 2.9 and 6-fold and Caspase-3 with 4.2 and 6.2-fold were significantly increased and for Bcl-2 with 0.7- and 0.3-fold decreased insigificantlty in silibinin over-expressed osteosarcoma cell line MG-63. Pourgholi et al. (2021) have examined the expression, by real-time PCR, of P53, caspase-3, 7, cyclin D1, survivin, Bax, Bcl-2, and hTERT in MCF-7 and MDA-MB-231 cancer cell lines. Reduced anti-apoptotic hTERT, Cyclin D1, Survivin, and Bcl-2 gene transcription levels were observed in nanoformulated SIL, while increased caspase-3, P53, and Bax mRNA levels were observed [38].

In this study first, we evaluated the viability of the cells at different concentrations (10–100 µM) using the LDH assay. It was discovered that, in comparison to other concentrations, 60µM/ml was the most effective. Gîrd et al., (2021) They have studied found that osteosarcoma cells treated with dry extracts showed a dose-dependent increase in LDH release. After 24-hour treatment with 700 µg/mL OE, MG-63 cancer cells showed a significant release of LDH. However, no significant differences were observed for other OE concentrations. When treated with RE, significant LDH release was observed [39].

The study tested MG-63 cell migration capacity using a wound-healing assay. Results showed that silibinin treatment significantly reduced the migration of MG-63 cells after 24 hours, increasing wound width and reducing migration. Du et al., (2018) Have evaluated the effects of hesperidin on MG-63 cell migration using an in vitro wound healing assay. Results showed that increasing doses of hesperidin led to cell migration inhibition in a dose-dependent manner. The untreated group showed no inhibition, but treatment with 5, 50, and 150 µM hesperidin reduced the percentage of migrated cells from 94.5% to 24.5 [40].

Silibinin decreased the TGF-β (40µM/ml-fold 0.7, 60µM/ml-fold 0.6) and SMAD-2 (100µM/ml-fold 0.9, 150µM/ml-fold 0.7) expression in osteosarcoma cell line. Chen et al., (2020) They have found that silibinin treatment significantly decreased infarct size compared to vehicle treatment, and inhibited myocardial apoptosis, caspase-3 activity, and down-regulation of Bcl-2 and Bax. It also restored the viability of H9C2 cells in hypoxia/reperfusion and increased CCK-8 value. In vitro, silibinin treatment reduced the number of TUNEL-positive H9C2 cells, lowered LDH and caspase-3 activity, and restored dysregulated expression of Bcl-2 and Bax in H9C2 cells under hypoxia/reperfusion [41].

Silibinin, although an "old drug" with a long history of clinical research and use, is regarded as a novel treatment for osteosarcoma, with the goal of decreasing tumour development and postponing the course of the condition. Future research should focus more on this topic because the intrinsic process is still unclear and complex. Finally, our results demonstrate that silibinin can kill cells in the MG-63 human osteosarcoma cell line. By turning on the cell migration and apoptosis pathway, silibinin induced apoptosis. Therefore, our current research may contribute to the development of silibinin as a possible therapy option for osteosarcoma and provide new insights into the molecular mechanisms behind its potential anticancer actions.

Acknowledgments:

None

Funding

None

Data availability statement

None

Author contribution

Nancy sheela. S: Original draft – writing and editing; Gnanamathy. G – Original draft – editing; Jeevitha. R – Methodology; Elumalai. P – Supervision, Validation; Sridevi. M: Supervision, Validation.

Disclosure of Interest:

The authors declare that they have no potential conflict of interest relevant to this article.

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No competing interests reported.

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Silibinin Induces Apoptosis and Suppresses Cell Migration by Targeting the Transforming growth factor-β Signaling Pathway in Osteosarcoma Cells

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Abstract

Figures

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 Cell line maintenance

2.2 Cell viability (MTT) assay

2.3 Morphology study

2.4 Determination of mode of cell death by acridine orange (AO)/ethidium bromide (EtBr) dual staining

2.5 Cell cycle analyses by flow cytometer

2.6 Real Time PCR

2.7 LDH assay

2.8 Scratch wound healing assay

2.9 Protein Preparation

2.10 Ligand preparation

2.11 Molecular Docking

2.12 Statistical analysis

3. RESULTS

3.1 Silibinin inhibits cell proliferation and induces cytotoxicity in osteosarcoma cells:

3.2 Silibinin alters the cell morphology of osteosarcoma cells.

3.3 silibinin induces apoptotic cells in osteosarcoma cells.

3.4 Silibinin suppresses cell growth and causes cell cycle arrest in osteosarcoma cells.

3.5 Silibinin modulates the mRNA expression of apoptotic genes in osteosarcoma cells.

3.6 Lactate dehydrogenase (LDH) assay

3.7 Scratch Wound healing assay_ MG-63

3.8 Molecular Docking

4. DISCUSSION

5. CONCLUSION

Declarations

References

Additional Declarations

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