Erianin Induces Apoptosis of Osteosarcoma Cells Through Ferroptosis Signaling Pathway, and Inhibits Proliferation and Migration of Osteosarcoma Cells

Erianin Induces Apoptosis of Osteosarcoma Cells Through Ferroptosis Signaling Pathway, and Inhibits Proliferation and Migration of Osteosarcoma Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Erianin Induces Apoptosis of Osteosarcoma Cells Through Ferroptosis Signaling Pathway, and Inhibits Proliferation and Migration of Osteosarcoma Cells Beilei Yu, Shuyuan Feng, Zhongzhe Li, Bin Wang, Kaige Gao, Lu Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6119041/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Aug, 2025 Read the published version in Cytotechnology → Version 1 posted 7 You are reading this latest preprint version Abstract Purpose Erianin plays a certain role in the treatment of tumors, inflammation, diabetes nephropathy, retinopathy and other diseases. However, the impact and mechanism of Erianin on osteosarcoma(OS) are still unclear. This article aims to investigate the mechanism of action of Erianin in OS. Methods Animal experiments were conducted using nude mice to investigate the in vivo effects of Erianin on OS. Investigations into the in vitro effects of Erianin on OS were conducted through cell experiments utilizing MG-63 and U-2 OS human OS cell lines. Firstly, use the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to detect cell viability and calculate IC50. Using colony-formation assay to detect the inhibitory effect of Erianin on cell proliferation. Use wound healing assay and cell migration assay to detect the effect of Erianin on the migration of OS cells. Use flow cytometry to detect cell apoptosis. Observe the effect of Erianin on the survival of OS cells under a microscope using Acridine Orange/Propidium Iodide(AO/PI)‌ staining. Use glutathione detection kit to detect the effect of Erianin on the ferroptosis signaling pathway in OS cells. Verify protein expression using western blot. Results In vitro, Erianin inhibits proliferation and migration of OS cells by regulating cyclin and apoptotic proteins, leading to ferroptosis of OS cells; In vivo, Erianin can also inhibits tumor growth and cause ferroptosis of OS. Conclusion The findings of Erianin's induction of apoptosis and ferroptosis, as well as its inhibition of proliferation and migration of OS cells, point to it as a potential therapeutic agent for OS that warrants further investigation. Erianin Osteosarcoma Ferroptosis Cell Proliferation Migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Osteosarcoma(OS) is a malignant tumor with clinical manifestations of bone and joint pain and local mass. [ 1 ] The incidence is highest among adolescents. The current treatment methods include preoperative chemotherapy, focal resection and postoperative chemotherapy. [ 2 ] The etiology and pathogenesis of OS remain unclear, so clarification is imperative in order to improve long-term survival in patients with OS and facilitate the development of targeted anti-cancer therapies. Isolated from Dendrobium officinale, Erianin is a natural product of low molecular weight. [ 3 ] Dendrobium is a valuable traditional Chinese medicinal material in China. It has many pharmacological effects, including polysaccharides, bibenzyl, alkaloids, sesquiterpenoids, fluorenones, coumarins, triterpenoids and volatile oils. [ 4 ] Pharmacological studies have found that Erianin has anti-bacterial, anti-viral, anti-oxidation, anti-angiogenesis and other effects, and can regulate a variety of signaling pathways, which is a promising new drug. [ 5 ] The present research shows that Erianin has a certain role in the treatment of tumor, inflammation, diabetic nephropathy, retinopathy and other diseases. [ 6 – 8 ] However, the effect of Erianin on OS and its mechanism are not clear. The accumulation of iron in cells, resulting in the high expression of unsaturated fatty acids on the cell membrane and lipid peroxidation, is the primary mechanism of ferroptosis - a novel type of programmed cell death dependent on iron, distinct from apoptosis, cell necrosis and autophagy, which leads to cell death. [ 9 ] Moreover, it also manifests as a decrease in the expression of glutathione peroxidase 4(GPX4), the core regulatory enzyme of the intracellular antioxidant system - glutathione system. [ 10 ] When GPX4 inhibition or GPX4 deficiency occurs, it can directly lead to the accumulation of lipid peroxides, thereby activating ferroptosis. [ 11 ] Earlier research has confirmed that GPX4 inhibitor RAS selective lethal compound 3 (RSL3) can induce ferroptosis in cancer cells [ 12 ] Ferroptosis has been linked to the genesis of various diseases, such as tumors, ischemia-reperfusion harm, degenerative diseases, and stroke. [ 13 ] However, the relationship between OS and Ferroptosis requires further study. In recent years, it has been found that Erianin may be a new regulator of ferroptosis. The inhibition of the NF-E2-related factor 2(Nrf2)/Heme Oxygenase-1(HO-1)/GPX4 pathway by Erianin may lead to ferroptosis in cancer cells,, [ 14 ] and it has been demonstrated that Erianin inhibits the growth and metastasis through autophagy-dependent ferroptosis in KRAS G13D colorectal cancer. [ 15 ] However, whether Erianin exerts its effect through ferroptosis in OS still needs further research. The purpose of this research is to investigate Erianin's part in OS and its potential molecular processes, thereby furnishing a novel theoretical basis for clinical treatment of OS. Materials and Methods 1. Animals and Cells. A total of 8 SPF healthy Balb/c nude mice, all male, aged 8w and weighing (15±2)g, were provided by the College of Laboratory Animals, ****. Throughout the experiment, these mice were housed in the SPF grade animal facility of *****. They are maintained in a 12 hour light dark cycle, requiring ventilation 10-15 times per hour, with a temperature of 25 ± 1 ℃ and a relative humidity of 55% ± 10%. Provide sufficient specialized food and sterile water for mice to use freely, and replace sterile padding every three days. Human osteosarcoma cell line MG-63 and U-2 OS were purchased from Pricella(cat.no. CL-0157; CL-0236; Wuhan). All cells were identified by Short Tandem Repeat(STR), and the matching degree was ≥ 80%. Samples did not show mycoplasma contamination. The cells were cultured in DMEM medium(cat.no. KGM12800; KeyGEN BioTECH; Jiangsu) in a 37℃ incubator with 100% humidity and 5% CO 2 concentration. The medium was supplemented with 10% fetal bovine serum(cat.no. C04001-050; VivaCell Biosciences; Shanghai), 100U/ml penicillin and 100µg/ml streptomycin(cat.no. P1400; Solarbio; Beijing). 2. Ethical Review Ethics Review Committee of Shandong First Medical University gave the experiment their approval, with the ethical code of W202312220339. This experiment follows the "Regulations on the Administration of Experimental Animals of the People's Republic of China (Revised Draft for Comments)". 3. Model Construction and Grouping. Randomization's principle was adhered to, with the nude mice split into two groups - the control and the model - each containing four. First, the logarithmic growth of MG-63 cells was divided into two groups, the control group was cultured for 24 hours, and the model group was treated with 80nM of Erianin(cat.no. HY-N0517; MCE; Shanghai) for 24 hours. Then the cells were digested with pancreatic enzyme, cleaned with PBS, and the concentration of cells was adjusted to 1×10 7 cells/mL. Respectively inoculate cells into the subcutaneous axillary area of 8 mice, 0.2mL/mouse. Inject Erianin(10mg/kg) or an equal volume of physiological saline into the peritoneum of mice every three days. Following 14 days of treatment, all mice were euthanized by intraperitoneal injection of 10% chloral hydrate [16] (0.2ml/10g)(cat.no. C804539; MACKLIN; Shanghai), followed by subcutaneous tumor extraction, weighing, and recording. 4. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) Assay. MG-63 cells were inoculated with 1×10 4 cells/well into 96-well plates for normal culture, and 5 compound pores were set up. After 4h of culture, the cells were attached to the wall and cultured with 0, 10, 20, 40, 80, 160 nM of Erianin for 24 h. The MTT solution(cat.no. M1020; Solarbio; Beijing), at a concentration of 0.5 mg/ml, was added to the plate and incubated at 37℃ for 4h. Subsequently, the supernatant was discarded and 100μl DMSO(cat.no. D8370; Solarbio; Beijing) was added in per well. After 10 minutes in a shaker, the absorbance was measured by spectrophotometer at 490nm and the cell inhibition rate was calculated. The experimental results divided the cells into four groups, each of which were exposed to 0 nM, 20 nM, 40 nM and 80 nM for 24 hours for further experiments. 5. Colony-formation Assay. Infecting each group's cells with 500 cells per well into a 6-well plate, the culture medium was incubated in an incubator with 5% CO 2 at 37℃ for two weeks. The fresh culture medium was replaced every three days. Finally, the cells were fixed with 4% paraformaldehyde(cat.no. G1101; Servicebio; Wuhan) for 15min and stained with 0.1% crystal violet(cat.no. C8407; Solarbio; Beijing) for 30min. The number of colonies was counted using ImageJ. 6. Cell Viability Staining. Inoculate 4 × 10 4 cells per milliliter on 6-well plates for each group of cells. Cultivate for 4 hours. When the cells were attached, they were treated with different concentrations of Erianin for 24h. After washing with PBS buffer, Acridine Orange/Propidium Iodide(AO/PI) staining solution(cat.no. CA1143; Solarbio; Beijing; &cat.no. G1021; Servicebio; Wuhan) was added and the cells were observed under fluorescence microscope(Nikon; 80i; Japan). 7. Flow cytometry Collect cells from each group, adjust them to a cell concentration of 1 × 10 5 /ml, and fix overnight with 75% cold alcohol at 4℃. 24 hours later, clean the cells with PBS, then add RNase and stain with 50μg/mL propidium iodide(cat.no. C0080; Solaibio), and keep for 30 minutes in dark. Test samples on NovoCyte(Novo Express; 1.3.0; ACEA Biosciences; Beijing). 8. Cell Migration Assay. After collecting cells from each group, they were suspended in a basic medium with a density of 1×10 5 cells per milliliter. To the upper chamber of Transwell chamber(cat.no. TCS003024; BIOFIL; Guangzhou), 200μL cell suspension was added, and 500μL complete culture medium was added to the lower chamber. After 24 hours in a cell incubator at 37℃, the chamber was removed and the side of it was cleaned with a cotton swab. They were placed in 4% paraformaldehyde at room temperature for 10 minutes, away from light. Stain the wells with 0.1% crystal violet for 15 minutes, then measure cell migration number under an optical microscope. 9. Wound Healing Assay. The cells of each group were inoculated on a six-well plate. Wait for the cells to fill the bottom and scratches were made on single cells along the bottom of the culture plate with the tip of a sterilized 20μl pipette gun tip. Then cleaned with PBS, and treated with different concentrations of Erianin or RSL3(cat.no. HY-100218A; MCE; Shanghai) for 24h. Observe the relative distance of healed scratches using microscope(Nikon; 80i; Japan) and Image J(Image J; 1.53t; ‌National Institutes of Health; USA). 10. Determination of Glutathione(GSH) . Collect cells by centrifuge, freeze-thawed repeatedly, and GSH was determined by supernatant. Measure total intracellular glutathione levels according to the operating instructions of the glutathione assay kit(cat.no. S0055; Beyotime; Shanghai). 11. Western Blot. After drug treatment, cells in each group were collected and placed in RIPA lysta(cat.no. G4535; Servicebio; Wuhan). After 30min at low temperature, the supernatant solution was centrifuged at 4℃ and the total protein concentration was determined using Coomassie Brilliant Blue(cat.no. G250; Servicebio; Wuhan), and SDS-PAGE electrophoresis was performed. After polyacrylamide gel electrophoresis, 30μg protein samples were transferred to PVDF (Polyvinylidene Difluoride) membranes. 5% skimmed milk was blocked for 2h at room temperature, incubated overnight with primary antibodies for CyclinB1 (1 : 500; cat.no. GB11255; Servicebio; Wuhan), CDK1(1 : 1000; cat.no. GB11634; Servicebio; Wuhan), GAPDH(1 : 1000; cat.no. AF1186; Beyotime; Shanghai), Bcl-2(1 : 1000; cat.no. T40056; Abmart; Shanghai), Bax(1 : 1000; cat.no. GB11690; Servicebio; Wuhan), Caspase3(1 : 2000; cat.no. TA6311; Abmart; Shanghai), Mmp9(1 : 1000; cat.no. AF5234; Beyotime; Shanghai), N-cadherin(1 : 1000; cat.no. CY5015; Abways; Beijing), E-cadherin(1 : 1000; cat.no. AB3386; Abways; Beijing), Vimentin(1 : 1000; cat.no. AF1975; Beyotime; Shanghai), β-Actin(1 : 5000; cat.no. AB0035; Abways; Beijing), GPX4(1 : 1000; cat.no. CY6959; Abways; Beijing), SLC7A11(1 : 1000; cat.no. AF7992; Beyotime; Shanghai). Next day, secondary antibody dilution labeled by HRP was added and incubated for 1h at room temperature for development. β-Actin and GAPDH were used as references. Then the color reaction was carried out according to the instructions of ECL kit(cat.no. BL520A; Biosharp; Beijing). Incubate the PVDF membrane in a cassette containing developer for 2 minutes and gently shake the cassette. Then place it in the instrument for exposure and photography. Use Image J 2.3.0 to calculate the grayscale values of the stripes. 12. Statistical Analysis. All the above tests were repeated three times. Using GraphPad Prism 9.5.1(GraphPad Software; USA) software, a statistical analysis of the data was conducted. Independent sample t-test was employed to contrast the mean values of two samples, which were displayed as mean ± standard deviation. ANOVA was then utilized to contrast the mean values in multiple groups, with P < 0.05 being deemed statistically significant. Results 1. Erianin Inhibits the Proliferation of OS Cells by Inducing G2/M Phase Arrest. According to GraphPad Prism analysis, the IC50 of Erianin inhibiting the activity of MG-63 cells in vitro was 41.32 nM(Figure 1a). The IC50 revealed that the cells were split into 4 distinct groups: control group, 20 nM Erianin treatment group, 40 nM Erianin treatment group, and 80 nM Erianin treatment group. These were then cultured for 24 hours and utilized for subsequent experiments. Compared with the control group(Con), cloning experiments showed that Erianin inhibits MG-63 and U-2 OS cell proliferation(Figures 1b-c). The staining of AO/PI revealed a dose-dependent increase in the apoptosis of MG-63 cells in 20, 40 and 80 nM Erianin groups when compared to the Con group(Figure 1d). The results of Flow cytometry, which was employed to detect the cell cycle, revealed that after Erianin was administered, the amount of G0/G1 phase cells diminished, while the amount of S phase and G2/M phase cells augmented(Figure 1e). It is suggested that Erianin can induce G2/M phase arrest in OS cells. 2. Erianin Inhibits the Migration of OS Cells. The results of Transwell experiment showed that the number of cells migrating to the lower chamber gradually decreased with the increase of the concentration of Erianin, indicating that Erianin could inhibit the migration of OS cells(Figures 2a-c). Compared with the Control(Con) group, wound healing assays results showed that the migration distance of OS cells in the 20, 40, 80 nM Erianin treatment groups gradually decreased in a dose-dependent manner(Figures 2d-e). 3. Erianin Regulates Protein Expression of Apoptosis, Migration, and Cyclin in OS cells. Results of Western Blot analysis indicated a decrease in the expression of apoptosis protein Bcl-2, yet Bax and Caspase3 were augmented(Figures 3a-d). In a dose-dependent fashion, MMP-9, N-cadherin and Vimentin were all expressed less, whereas E-cadherin was seen to be more (Figure3A, e-h). Erianin decreased the expression of CyclinB1 and CDK1 in a dose-dependent manner(Figures 3i-k). 4. Erianin Induced Ferroptosis in MG-63 Cells in Vivo and Vitro. Compared with the control group, the subcutaneous tumor volume and weight of MG-63 cells in 80 nM Erianin group of mice were significantly smaller than that in the control group(Figures 4a-d). Western Blot was employed to identify the ferroptosis protein expression in the tumor tissues, and the results revealed a decrease in GPX4 and SLC7A11 expression in the 80 nM Erianin treatment group compared to the control(Con) group, implying that Erianin can induce ferroptosis in tumor cells in vivo(Figures 4e-g). The dose-dependent decrease in GSH levels in MG-63 cells caused by Erianin treatment was observed to be distinct from that of the Con group, suggesting that the ferroptosis of MG-63 cells was induced by the treatment(Figure 4h). Western Blot revealed the expression of a related protein to ferroptosis, with the results being consistent(Figures 4i-k). 5. Erianin Inhibits the Proliferation and Migration of OS Cells through Ferroptosis Signaling Pathway . We also used the GPX4 inhibitor RSL3 and found a inhibition of the proliferation, migration and cell cycle of OS cells, proving that Erianin inhibits the proliferation and migration of OS cells through ferroptosis signaling pathway(Figures 5a-h). Discussion Our experiments revealed that Erianin can impede the proliferation and expansion of OS cells, and MTT results indicated that Erianin could suppress the endurance of tumor cells in a dose-dependent way. According to GraphPrism analysis, the IC50 of Erianin inhibiting the activity of MG-63 cells in vitro was 41.32 nM. Then divided the cells into four groups for follow-up verification. Cloning, wound healing, and Transwell assays were used to measure the proliferation and migration capacity of OS cells when exposed to various concentrations of Erianin. The results revealed that with a rise in Erianin concentration, the proliferation and migration of OS cells were progressively inhibited, implying that Erianin has an inhibitory effect on the proliferation and migration of OS cells. Subsequently, Western Blot assay was used to verify the changes of cell migration proteins. Results indicated a dose-dependent decrease in the expression of MMP-9, N-cadherin, and Vimentin, while E-cadherin was observed to be amplified. MMP-9 is an enzyme that mainly degrades type IV collagen and elastin. MMP-9 has been demonstrated to be significantly elevated in the tissues of malignant tumors, such as colon cancer, stomach cancer, lung cancer and cervical cancer, and has become the focus of anti-tumor drugs [ 17 – 19 ] . This is due to its ability to both promote the growth of tumor cells by forming new blood vessels and to induce infiltration and invasion of tumor cells to the basement membrane by degrading and destroying it. [ 20 ] N-cadherin and E-cadherin belong to the cadherin family and are related to intercellular adhesion, differentiation and embryogenesis. In epithelial cells, N-cadherin promotes the development of cell morphology into fibroblasts, making the cells more aggressive. [ 21 ] E-cadherin is a calcium-dependent adhesion molecule, and its intracellular structure can connect with other proteins to form complexes, which play an important role in maintaining normal epithelial cell morphology, cell polarity, intercellular adhesion and tissue integrity. [ 22 ] The expression of E-cadherin, when either deleted or down-regulated, results in a loss of polarity between cells, a decrease in adhesion, and a heightened susceptibility to invasion and metastasis. [ 23 ] Vimentin, a type of intermediate filament protein, is implicated in the formation of cytoskeletal and cell membrane structures, and plays a role in many physiological processes, such as embryo development, wound healing, and tumor invasion. [ 24 ] The expression of MMP-9, N-cadherin, and vimentin in tumor was observed to be reduced after Erianin treatment, while E-cadherin expression was augmented, thus demonstrating that Erianin could impede the migration and invasion of tumor cells and impede tumor metastasis. Flow cytometry and Western Blot were employed to further explore the inhibition of tumor cell proliferation by Erianin. The results indicated that the proportion of cells in G2/M phase augmented with the concentration of Erianin. Moreover, Western Blot revealed that, in comparison to the control group, CyclinB1 and CDK1 expressions decreased after treatment with Erianin. Cyclins are A, B, D, E, G, and H, which bind to a key protein kinase, the cyclin-dependent kinase CDK, and regulate its enzyme activity to help, drive, and coordinate the cell cycle. [ 25 ] M-phase cyclins mainly consist of CyclinB, which begins to be synthesized in late G1 phase, increases in expression in S phase, peaks in late G2 phase and M phase, and enters the nucleus to bind to CDK1 to form a complex, thereby activating the kinase activity of CDK1. [ 26 ] CyclinB-CDK1 mainly plays a role at the end of G2 and can guide cells into the M phase of mitosis. [ 27 ] When the cell exited the M phase, CyclinB degraded, CDK1 kinase activity inactivated, substrate dephosphorylation promoted chromosome agglutination, nucleolar reconstruction, and guided the cell into the next cell cycle. [ 28 ] The experiment revealed a decrease in CyclinB1 and CDK1 expression after Erianin was administered, implying that the treatment of Erianin could impede the cell cycle, postpone its progression, and thus impede cell proliferation. We conclude that inhibition of proliferation and migration of OS cells by Erianin is related to the physiological process of ferroptosis, which is consistent with the results of previous papers. A recent paper found that Erianin may be involved in the ferroptosis process. Despite the utilization of Caspase inhibitor Z-VAD-FMK, Necrostatin 1 and autophagy inhibitor chloroquine to impede apoptosis, necrosis and autophagy in lung cancer cells, Erianin was still able to cause the death of these cells. It was discovered that ROS accumulation, GSH consumption and lipid peroxidation had significantly risen in lung cancer cells. The use of ferroptosis inhibitors Ferrostatin-1 and Liproxstatin-1 could reduce the Erianin-induced cell death, indicating that ferroptosis was involved in the Erianin-induced cell death process. Transcriptomic analysis also found that the expression of GPX4 was down-regulated, and the enrichment of KEGG pathway showed that information related to ferroptosis pathway was enriched. It was found that Erianin could induce ferroptosis in lung cancer cells by activating Ca 2+ /CaM signaling, resulting in the increase of Ca 2+ and Fe 2+ levels. CaM can adjust voltage dependence l-type Ca 2+ channel (L-typevoltagptedependentCa2 + channel, LVDCC), not only is the key to Ca 2+ transport, this also is the key to iron absorption. After treated with Erianin, CaM of lung cancer cells are activated, Ca 2+ / CaM signal pathway is activated, and increased Ca 2+ uptake induces ROS accumulation and elevated Fe2 + levels, which in turn induces the occurrence of cell ferroptosis. [ 29 ] In this study, we detected the levels of GSH and ROS in MG-63 cells after treatment with Erianin. The Erianin treatment caused a decrease in GSH levels in cells, as compared to the control group, as evidenced by the results. Western Blot also detected reduced expression of GPX4 and SLC7A11 proteins, indicating ferroptosis in MG-63 cells. These results suggest that Erianin can induce apoptosis of OS cells by inducing ferroptosis, and inhibit proliferation and migration of OS cells. Subsequently, we further validated and treated the cells with GPX4 inhibitors(RSL3), and found that both cell proliferation and migration were inhibited. This study demonstrates that Erianin can control the growth and tumorigenesis of OS in nude mice, as well as the proliferation, migration, and apoptosis of OS cells in vitro. It has the potential to become a new candidate target for OS and provide new theoretical basis for clinical treatment of OS. Conclusion OS cells can be inhibited in their proliferation and migration by Erianin, G2/M phase arrest is brought about, and apoptosis is induced. This may be related to the ferroptosis signaling pathway Declarations Competing Interests The authors declare that there are no competing interests regarding the publication of this paper. Ethics approval Ethics Review Committee of Shandong First Medical University gave the experiment their approval, with the ethical code of W202312220339. This experiment follows the "Regulations on the Administration of Experimental Animals of the People's Republic of China (Revised Draft for Comments)". Funding Many thanks are given to the teachers and professors for providing us with technical support. This work was supported by Projects of Medical and Health Technology Development Program in Shandong Province under Grant (number 202002020809) and Shandong First Medical University College Students Innovation and Entrepreneurship Training Program(2022104391661) . Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Beilei Yu and Shuyuan Feng. The first draft of the manuscript was written by Zhongzhe Li, Bin Wang and Kaige Gao and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Beilei Yu and Shuyuan Feng made equal contributions to this article. Lu Zhou is the corresponding author. Acknowledgement Many thanks are given to the teachers and professors for providing us with technical support. Data Availability The datasets during the current study are available from the corresponding authors on reasonable request. 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Proc Natl Acad Sci U S A, 2020. 117(11): p. 5931-5937. van Loon, K., M.E. van Breest Smallenburg, E.J.M. Huijbers, A.W. Griffioen and J.R. van Beijnum, Extracellular vimentin as a versatile immune suppressive protein in cancer. Biochim Biophys Acta Rev Cancer, 2023. 1878(6): p. 188985. Wood, D.J. and J.A. Endicott, Structural insights into the functional diversity of the CDK-cyclin family. Open Biol, 2018. 8(9). . Linder, M.I., M. Köhler, P. Boersema, M. Weberruss, C. Wandke, J. Marino, et al., Mitotic Disassembly of Nuclear Pore Complexes Involves CDK1- and PLK1-Mediated Phosphorylation of Key Interconnecting Nucleoporins. Developmental Cell, 2017. 43(2): p. 141-156.e7. . Chen, P., Q. Wu, J. Feng, L. Yan, Y. Sun, S. Liu, et al., Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits lung cancer cell growth and migration via calcium/calmodulin-dependent ferroptosis. Signal Transduct Target Ther, 2020. 5(1): p. 51. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 01 Aug, 2025 Read the published version in Cytotechnology → Version 1 posted Editorial decision: Revision requested 12 Apr, 2025 Reviews received at journal 12 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers invited by journal 30 Mar, 2025 Editor assigned by journal 28 Feb, 2025 Submission checks completed at journal 28 Feb, 2025 First submitted to journal 27 Feb, 2025 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. 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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-6119041","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":422174628,"identity":"b744c0c0-d80e-412a-acc2-2cb998896f26","order_by":0,"name":"Beilei Yu","email":"","orcid":"","institution":"Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Beilei","middleName":"","lastName":"Yu","suffix":""},{"id":422174629,"identity":"c4070268-75ee-4916-a9af-2a1f4df7824c","order_by":1,"name":"Shuyuan Feng","email":"","orcid":"","institution":"Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuyuan","middleName":"","lastName":"Feng","suffix":""},{"id":422174630,"identity":"fbabd89e-be6e-47db-8652-99d2702966dc","order_by":2,"name":"Zhongzhe Li","email":"","orcid":"","institution":"Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhongzhe","middleName":"","lastName":"Li","suffix":""},{"id":422174631,"identity":"ce4bca3b-4e94-449a-b829-9220a2ab85be","order_by":3,"name":"Bin Wang","email":"","orcid":"","institution":"Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Wang","suffix":""},{"id":422174632,"identity":"a05a9a45-053a-4933-99e8-c70dec7a6193","order_by":4,"name":"Kaige Gao","email":"","orcid":"","institution":"Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kaige","middleName":"","lastName":"Gao","suffix":""},{"id":422174633,"identity":"d04b55a9-72fd-4a59-9692-be67be5117aa","order_by":5,"name":"Lu Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYHACxgOMDQwMfAzMBw58+EGkHrAWNga2xIMze0jTwmN8mIONCOX87c0HDv7cYZPYJpHz4TADD4M8v9gB/FokzhxLOCB5Jg2oJXfD4QILBsOZsxPwazGQyDE4YNh2GKJlBg9DgsFtYrQkgrXkPDjMw0asloMQLQzEaQH55WBjW5pxG88zA2AgSxD2CzDEDj782WYj28+e/PjDhx828vzSBLRg2Eqa8lEwCkbBKBgF2AEA/DxJNRqHjt4AAAAASUVORK5CYII=","orcid":"","institution":"Shandong First Medical University","correspondingAuthor":true,"prefix":"","firstName":"Lu","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-02-27 08:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6119041/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6119041/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10616-025-00792-w","type":"published","date":"2025-08-01T16:13:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77613532,"identity":"b6d1101b-245d-4d24-80bd-532ba8b299e4","added_by":"auto","created_at":"2025-03-03 14:44:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":432389,"visible":true,"origin":"","legend":"\u003cp\u003eErianin inhibits the proliferation of osteosarcoma(OS) cells by inducing G2/M phase arrest. (a)The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay for testing tumor OS cell activity. (b-c) Cell cloning experiments to evaluate cell proliferation activity. (d)Acridine Orange/Propidium Iodide(AO/PI)‌ staining of MG-63 cells. Live cells emit green fluorescence after AO staining, while dead cells emit red fluorescence after PI staining. (e) Detection of cell cycle by flow cytometry in OS cells. Each assay was replicated three times. Data are shown as mean ± standard deviation. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus the Control(Con) group; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus the Con group; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the Con group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6119041/v1/fd82e1094cd9e9b1287b704d.png"},{"id":77612155,"identity":"929088e0-12ae-4032-91c4-ac157d1ce71b","added_by":"auto","created_at":"2025-03-03 14:36:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":736464,"visible":true,"origin":"","legend":"\u003cp\u003eErianin inhibits the migration of osteosarcoma(OS) cells. (a-c)Transwell experiment to test OS cell migration activity. (d-e)Wound healing assays to test cell migration activity. Each assay was replicated three times. Data are shown as mean ± standard deviation. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus the Control(Con) group; *\u003cem\u003e*P\u003c/em\u003e\u0026lt;0.01 versus the Con group; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the Con group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6119041/v1/a604bcefb60e6b23e85b777e.png"},{"id":77613533,"identity":"d502d1ff-0fb3-4d34-a498-a47fba127669","added_by":"auto","created_at":"2025-03-03 14:44:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":232044,"visible":true,"origin":"","legend":"\u003cp\u003eErianin regulates the expression of apoptosis, migration, and cyclin in osteosarcoma(OS) cells. (a-h)The effect of Erianin on apoptosis and migration proteins in OS cells. (i-k)The effect of Erianin on cyclin in OS cells. Each assay was replicated three times. Data are shown as mean ±standard deviation. *P\u0026lt;0.05 versus the Control(Con) group; **P\u0026lt;0.01 versus the Con group; ***P\u0026lt;0.001 versus the Con group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6119041/v1/815d895cd3fe6d6d4c1d0702.png"},{"id":77612156,"identity":"d59853eb-a616-4851-bbc0-3a6f579df08d","added_by":"auto","created_at":"2025-03-03 14:36:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":197313,"visible":true,"origin":"","legend":"\u003cp\u003eErianin induces ferroptosis in MG-63 cells in vivo and vitro. (a-d) Subcutaneous tumor volume and weight in nude mice. (e-g)Expression of ferroptosis related protein in vivo. (h)Determination of Glutathione(GSH) in vitro. (i-k)Expression of ferroptosis related protein in vitro. Each assay was replicated three times. Data are shown as mean ± standard deviation. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus the Control(Con) group; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus the Con group; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the Con group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6119041/v1/9f11ac9b269d860424cc0846.png"},{"id":77612159,"identity":"428de0d6-9a66-4a5b-b963-e8bc0282aedb","added_by":"auto","created_at":"2025-03-03 14:36:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":522966,"visible":true,"origin":"","legend":"\u003cp\u003eErianin inhibits the proliferation and migration of osteosarcoma(OS) cells through ferroptosis signaling pathway . (a-b) Cell cloning experiments to evaluate OS cell proliferation activity. (c-d) Wound healing assays to test cell migration activity. (e) Transwell experiment to test cell migration activity. (g) Detection of cell cycle by flow cytometry in MG-63. (h) Expression of ferroptosis related protein. Each assay was replicated three times. Data are shown as mean ± standard deviation. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 versus the Control(Con) group; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus the Con group; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the Con group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6119041/v1/9502aa8a8c43eba12e18d069.png"},{"id":88268273,"identity":"1c984e09-9480-44ab-b4cf-a80c4c307930","added_by":"auto","created_at":"2025-08-04 16:50:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2439054,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6119041/v1/1327c935-3147-44b3-95f5-74fc8d7c9772.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Erianin Induces Apoptosis of Osteosarcoma Cells Through Ferroptosis Signaling Pathway, and Inhibits Proliferation and Migration of Osteosarcoma Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteosarcoma(OS) is a malignant tumor with clinical manifestations of bone and joint pain and local mass.\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e The incidence is highest among adolescents. The current treatment methods include preoperative chemotherapy, focal resection and postoperative chemotherapy.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e The etiology and pathogenesis of OS remain unclear, so clarification is imperative in order to improve long-term survival in patients with OS and facilitate the development of targeted anti-cancer therapies.\u003c/p\u003e \u003cp\u003eIsolated from Dendrobium officinale, Erianin is a natural product of low molecular weight.\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e Dendrobium is a valuable traditional Chinese medicinal material in China. It has many pharmacological effects, including polysaccharides, bibenzyl, alkaloids, sesquiterpenoids, fluorenones, coumarins, triterpenoids and volatile oils.\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e Pharmacological studies have found that Erianin has anti-bacterial, anti-viral, anti-oxidation, anti-angiogenesis and other effects, and can regulate a variety of signaling pathways, which is a promising new drug.\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e The present research shows that Erianin has a certain role in the treatment of tumor, inflammation, diabetic nephropathy, retinopathy and other diseases.\u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e However, the effect of Erianin on OS and its mechanism are not clear.\u003c/p\u003e \u003cp\u003eThe accumulation of iron in cells, resulting in the high expression of unsaturated fatty acids on the cell membrane and lipid peroxidation, is the primary mechanism of ferroptosis - a novel type of programmed cell death dependent on iron, distinct from apoptosis, cell necrosis and autophagy, which leads to cell death.\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e Moreover, it also manifests as a decrease in the expression of glutathione peroxidase 4(GPX4), the core regulatory enzyme of the intracellular antioxidant system - glutathione system.\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e When GPX4 inhibition or GPX4 deficiency occurs, it can directly lead to the accumulation of lipid peroxides, thereby activating ferroptosis.\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e Earlier research has confirmed that GPX4 inhibitor RAS selective lethal compound 3 (RSL3) can induce ferroptosis in cancer cells\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e Ferroptosis has been linked to the genesis of various diseases, such as tumors, ischemia-reperfusion harm, degenerative diseases, and stroke.\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e However, the relationship between OS and Ferroptosis requires further study.\u003c/p\u003e \u003cp\u003eIn recent years, it has been found that Erianin may be a new regulator of ferroptosis. The inhibition of the NF-E2-related factor 2(Nrf2)/Heme Oxygenase-1(HO-1)/GPX4 pathway by Erianin may lead to ferroptosis in cancer cells,,\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e and it has been demonstrated that Erianin inhibits the growth and metastasis through autophagy-dependent ferroptosis in KRAS\u003csup\u003eG13D\u003c/sup\u003e colorectal cancer.\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e However, whether Erianin exerts its effect through ferroptosis in OS still needs further research. The purpose of this research is to investigate Erianin's part in OS and its potential molecular processes, thereby furnishing a novel theoretical basis for clinical treatment of OS.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e1. Animals and Cells.\u003c/p\u003e\n\u003cp\u003eA total of 8 SPF healthy Balb/c nude mice, all male, aged 8w and weighing (15±2)g, were provided by the College of Laboratory Animals, ****. Throughout the experiment, these mice were housed in the SPF grade animal facility of *****. They are maintained in a 12 hour light dark cycle, requiring ventilation 10-15 times per hour, with a temperature of 25\u0026nbsp;±\u0026nbsp;1\u0026nbsp;℃\u0026nbsp;and a relative humidity of 55%\u0026nbsp;±\u0026nbsp;10%. Provide sufficient specialized food and sterile water for mice to use freely, and replace sterile padding every three days.\u003c/p\u003e\n\u003cp\u003eHuman osteosarcoma cell line MG-63 and U-2 OS were purchased from Pricella(cat.no. CL-0157; CL-0236; Wuhan). All cells were identified by Short Tandem Repeat(STR), and the matching degree was ≥ 80%. Samples did not show mycoplasma contamination. The cells were cultured in DMEM medium(cat.no. KGM12800; KeyGEN BioTECH; Jiangsu) in a 37℃ incubator with 100% humidity and 5% CO\u003csub\u003e2\u003c/sub\u003e concentration. The medium was supplemented with 10% fetal bovine serum(cat.no. C04001-050; VivaCell Biosciences; Shanghai), 100U/ml penicillin and 100µg/ml streptomycin(cat.no. P1400; Solarbio; Beijing).\u003c/p\u003e\n\u003cp\u003e2. Ethical Review\u003c/p\u003e\n\u003cp\u003eEthics Review Committee of Shandong First Medical University gave the experiment their approval, with the ethical code of W202312220339. This experiment follows the \"Regulations on the Administration of Experimental Animals of the People's Republic of China (Revised Draft for Comments)\".\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. Model Construction and Grouping.\u003c/p\u003e\n\u003cp\u003eRandomization's principle was adhered to, with the nude mice split into two groups - the control and the model - each containing four. First, the logarithmic growth of MG-63 cells was divided into two groups, the control group was cultured for 24 hours, and the model group was treated with 80nM of Erianin(cat.no. HY-N0517; MCE; Shanghai) \u0026nbsp;for 24 hours. Then the cells were digested with pancreatic enzyme, cleaned with PBS, and the concentration of cells was adjusted to 1×10\u003csup\u003e7\u003c/sup\u003e cells/mL. Respectively inoculate cells into the subcutaneous axillary area of 8 mice, 0.2mL/mouse. Inject Erianin(10mg/kg) or an equal volume of physiological saline into the peritoneum of mice every three days. Following 14 days of treatment, all mice were euthanized by intraperitoneal injection of 10% chloral hydrate\u003csup\u003e[16]\u003c/sup\u003e (0.2ml/10g)(cat.no. C804539; MACKLIN; Shanghai), followed by subcutaneous tumor extraction, weighing, and recording.\u003c/p\u003e\n\u003cp\u003e4. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) \u0026nbsp;Assay.\u003c/p\u003e\n\u003cp\u003eMG-63 cells were inoculated with 1×10\u003csup\u003e4\u003c/sup\u003e cells/well into 96-well plates for normal culture, and 5 compound pores were set up. After 4h of culture, the cells were attached to the wall and cultured with 0, 10, 20, 40, 80, 160 nM of Erianin for 24 h. The MTT solution(cat.no. M1020; Solarbio; Beijing), at a concentration of 0.5 mg/ml, was added to the plate and incubated at 37℃ for 4h. Subsequently, the supernatant was discarded and 100μl DMSO(cat.no. D8370; Solarbio; Beijing) was added in per well. After 10 minutes in a shaker, the absorbance was measured by spectrophotometer at 490nm and the cell inhibition rate was calculated. The experimental results divided the cells into four groups, each of which were exposed to 0 nM, 20 nM, 40 nM and 80 nM for 24 hours for further experiments.\u003c/p\u003e\n\u003cp\u003e5. Colony-formation Assay.\u003c/p\u003e\n\u003cp\u003eInfecting each group's cells with 500 cells per well into a 6-well plate, the culture medium was incubated in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃ for two weeks. The fresh culture medium was replaced every three days. Finally, the cells were fixed with 4% paraformaldehyde(cat.no. G1101; Servicebio; Wuhan) for 15min and stained with 0.1% crystal violet(cat.no. C8407; Solarbio; Beijing) for 30min. The number of colonies was counted using ImageJ.\u003c/p\u003e\n\u003cp\u003e6. Cell Viability Staining.\u003c/p\u003e\n\u003cp\u003eInoculate 4 × 10\u003csup\u003e4\u003c/sup\u003e cells per milliliter on 6-well plates for each group of cells. Cultivate for 4 hours. When the cells were attached, they were treated with different concentrations of Erianin for 24h. After washing with PBS buffer, Acridine Orange/Propidium Iodide(AO/PI) staining solution(cat.no. CA1143; Solarbio; Beijing; \u0026amp;cat.no. G1021; Servicebio; Wuhan) was added and the cells were observed under fluorescence microscope(Nikon; 80i; Japan).\u003c/p\u003e\n\u003cp\u003e7. Flow cytometry\u003c/p\u003e\n\u003cp\u003eCollect cells from each group, adjust them to a cell concentration of 1 × 10\u003csup\u003e5\u003c/sup\u003e/ml, and fix overnight with 75% cold alcohol at 4℃. 24 hours later, clean the cells with PBS, then add RNase and stain with 50μg/mL propidium iodide(cat.no. C0080; Solaibio), and keep for 30 minutes in dark. Test samples on NovoCyte(Novo Express; 1.3.0; ACEA Biosciences; Beijing).\u003c/p\u003e\n\u003cp\u003e8. Cell Migration Assay.\u003c/p\u003e\n\u003cp\u003eAfter collecting cells from each group, they were suspended in a basic medium with a density of 1×10\u003csup\u003e5\u003c/sup\u003e cells per milliliter. To the upper chamber of Transwell chamber(cat.no. TCS003024; BIOFIL; Guangzhou), 200μL cell suspension was added, and 500μL complete culture medium was added to the lower chamber. After 24 hours in a cell incubator at 37℃, the chamber was removed and the side of it was cleaned with a cotton swab. They were placed in 4% paraformaldehyde at room temperature for 10 minutes, away from light. Stain the wells with 0.1% crystal violet for 15 minutes, then measure cell migration number under an optical microscope.\u003c/p\u003e\n\u003cp\u003e9. Wound Healing Assay.\u003c/p\u003e\n\u003cp\u003eThe cells of each group were inoculated on a six-well plate. Wait for the cells to fill the bottom and scratches were made on single cells along the bottom of the culture plate with the tip of a sterilized 20μl pipette gun tip. Then cleaned with PBS, and treated with different concentrations of Erianin or RSL3(cat.no. HY-100218A; MCE; Shanghai) for 24h. Observe the relative distance of healed scratches using microscope(Nikon; 80i; Japan) and Image J(Image J; 1.53t; ‌National Institutes of Health; USA).\u003c/p\u003e\n\u003cp\u003e10. Determination of Glutathione(GSH) .\u003c/p\u003e\n\u003cp\u003eCollect cells by centrifuge, freeze-thawed repeatedly, and GSH was determined by supernatant. Measure total intracellular glutathione levels according to the operating instructions of the glutathione assay kit(cat.no. S0055; Beyotime; Shanghai).\u003c/p\u003e\n\u003cp\u003e11. Western Blot.\u003c/p\u003e\n\u003cp\u003eAfter drug treatment, cells in each group were collected and placed in RIPA lysta(cat.no. G4535; Servicebio; Wuhan). After 30min at low temperature, the supernatant solution was centrifuged at 4℃\u0026nbsp;and the total protein concentration was determined using Coomassie Brilliant Blue(cat.no. G250; Servicebio; Wuhan), and SDS-PAGE electrophoresis was performed. After polyacrylamide gel electrophoresis, 30μg protein samples were transferred to PVDF (Polyvinylidene Difluoride) membranes. 5% skimmed milk was blocked for 2h at room temperature, incubated overnight with primary antibodies for CyclinB1 (1 : 500; cat.no. GB11255; Servicebio; Wuhan), CDK1(1 : 1000; cat.no. GB11634; Servicebio; Wuhan), GAPDH(1 : 1000; cat.no. AF1186; Beyotime; Shanghai), Bcl-2(1 : 1000; cat.no. T40056; Abmart; Shanghai), Bax(1 : 1000; cat.no. GB11690; Servicebio; Wuhan), Caspase3(1 : 2000; cat.no. TA6311; Abmart; Shanghai), Mmp9(1 : 1000; cat.no. AF5234; Beyotime; Shanghai), N-cadherin(1 : 1000; cat.no. CY5015; Abways; Beijing), E-cadherin(1 : 1000; cat.no. AB3386; Abways; Beijing), Vimentin(1 : 1000; cat.no. AF1975; Beyotime; Shanghai),\u0026nbsp;β-Actin(1 : 5000; cat.no. AB0035; Abways; Beijing), GPX4(1 : 1000; cat.no. CY6959; Abways; Beijing), SLC7A11(1 : 1000; cat.no. AF7992; Beyotime; Shanghai). Next day, secondary antibody dilution labeled by HRP was added and incubated for 1h at room temperature for development.\u0026nbsp;β-Actin and GAPDH were used as references. Then the color reaction was carried out according to the instructions of ECL kit(cat.no. BL520A; Biosharp; Beijing). Incubate the PVDF membrane in a cassette containing developer for 2 minutes and gently shake the cassette. Then place it in the instrument for exposure and photography. Use Image J 2.3.0 to calculate the grayscale values of the stripes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e12. Statistical Analysis.\u003c/p\u003e\n\u003cp\u003eAll the above tests were repeated three times. Using GraphPad Prism 9.5.1(GraphPad Software; USA) software, a statistical analysis of the data was conducted. Independent sample t-test was employed to contrast the mean values of two samples, which were displayed as mean ± standard deviation. ANOVA was then utilized to contrast the mean values in multiple groups, with \u003cem\u003eP \u0026lt;\u003c/em\u003e0.05 being deemed statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e1. Erianin Inhibits the Proliferation of OS Cells by Inducing G2/M Phase Arrest.\u003c/p\u003e\n\u003cp\u003eAccording to GraphPad Prism analysis, the IC50 of Erianin inhibiting the activity of MG-63 cells in vitro was 41.32 nM(Figure 1a). The IC50 revealed that the cells were split into 4 distinct groups: control group, 20 nM Erianin treatment group, 40 nM Erianin treatment group, and 80 nM Erianin treatment group. These were then cultured for 24 hours and utilized for subsequent experiments. Compared with the control group(Con), cloning experiments showed that Erianin inhibits MG-63 and U-2 OS cell proliferation(Figures 1b-c). The staining of AO/PI revealed a dose-dependent increase in the apoptosis of MG-63 cells in 20, 40 and 80 nM Erianin groups when compared to the Con group(Figure 1d). The results of Flow cytometry, which was employed to detect the cell cycle, revealed that after Erianin was administered, the amount of G0/G1 phase cells diminished, while the amount of S phase and G2/M phase cells augmented(Figure 1e). It is suggested that Erianin can induce G2/M phase arrest in OS cells.\u003c/p\u003e\n\u003cp\u003e2. Erianin Inhibits the Migration of OS Cells.\u003c/p\u003e\n\u003cp\u003eThe results of Transwell experiment showed that the number of cells migrating to the lower chamber gradually decreased with the increase of the concentration of Erianin, indicating that Erianin could inhibit the migration of OS cells(Figures 2a-c). Compared with the Control(Con) group, wound healing assays results showed that the migration distance of OS cells in the 20, 40, 80 nM Erianin treatment groups gradually decreased in a dose-dependent manner(Figures 2d-e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. Erianin Regulates Protein Expression of Apoptosis, Migration, and Cyclin in OS cells.\u003c/p\u003e\n\u003cp\u003eResults of Western Blot analysis indicated a decrease in the expression of apoptosis protein Bcl-2, yet Bax and Caspase3 were augmented(Figures 3a-d). In a dose-dependent fashion, MMP-9, N-cadherin and Vimentin were all expressed less, whereas E-cadherin was seen to be more (Figure3A, e-h). Erianin decreased the expression of CyclinB1 and CDK1 in a dose-dependent manner(Figures 3i-k).\u003c/p\u003e\n\u003cp\u003e4. Erianin Induced Ferroptosis in MG-63 Cells in Vivo and Vitro.\u003c/p\u003e\n\u003cp\u003eCompared with the control group, the subcutaneous tumor volume and weight of MG-63 cells in 80 nM Erianin group of mice were significantly smaller than that in the control group(Figures 4a-d). Western Blot was employed to identify the ferroptosis protein expression in the tumor tissues, and the results revealed a decrease in GPX4 and SLC7A11 expression in the 80 nM Erianin treatment group compared to the control(Con) group, implying that Erianin can induce ferroptosis in tumor cells in vivo(Figures 4e-g). The dose-dependent decrease in GSH levels in MG-63 cells caused by Erianin treatment was observed to be distinct from that of the Con group, suggesting that the ferroptosis of MG-63 cells was induced by the treatment(Figure 4h). Western Blot revealed the expression of a related protein to ferroptosis, with the results being consistent(Figures 4i-k).\u003c/p\u003e\n\u003cp\u003e5. Erianin Inhibits the Proliferation and Migration of OS Cells through Ferroptosis Signaling Pathway .\u003c/p\u003e\n\u003cp\u003eWe also used the GPX4 inhibitor RSL3 and found a inhibition of the proliferation, migration and cell cycle of OS cells, proving that Erianin inhibits the proliferation and migration of OS cells through ferroptosis signaling pathway(Figures 5a-h).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur experiments revealed that Erianin can impede the proliferation and expansion of OS cells, and MTT results indicated that Erianin could suppress the endurance of tumor cells in a dose-dependent way. According to GraphPrism analysis, the IC50 of Erianin inhibiting the activity of MG-63 cells in vitro was 41.32 nM. Then divided the cells into four groups for follow-up verification. Cloning, wound healing, and Transwell assays were used to measure the proliferation and migration capacity of OS cells when exposed to various concentrations of Erianin. The results revealed that with a rise in Erianin concentration, the proliferation and migration of OS cells were progressively inhibited, implying that Erianin has an inhibitory effect on the proliferation and migration of OS cells.\u003c/p\u003e \u003cp\u003eSubsequently, Western Blot assay was used to verify the changes of cell migration proteins. Results indicated a dose-dependent decrease in the expression of MMP-9, N-cadherin, and Vimentin, while E-cadherin was observed to be amplified. MMP-9 is an enzyme that mainly degrades type IV collagen and elastin. MMP-9 has been demonstrated to be significantly elevated in the tissues of malignant tumors, such as colon cancer, stomach cancer, lung cancer and cervical cancer, and has become the focus of anti-tumor drugs\u003csup\u003e[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. This is due to its ability to both promote the growth of tumor cells by forming new blood vessels and to induce infiltration and invasion of tumor cells to the basement membrane by degrading and destroying it.\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e N-cadherin and E-cadherin belong to the cadherin family and are related to intercellular adhesion, differentiation and embryogenesis. In epithelial cells, N-cadherin promotes the development of cell morphology into fibroblasts, making the cells more aggressive.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e E-cadherin is a calcium-dependent adhesion molecule, and its intracellular structure can connect with other proteins to form complexes, which play an important role in maintaining normal epithelial cell morphology, cell polarity, intercellular adhesion and tissue integrity.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e The expression of E-cadherin, when either deleted or down-regulated, results in a loss of polarity between cells, a decrease in adhesion, and a heightened susceptibility to invasion and metastasis.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e Vimentin, a type of intermediate filament protein, is implicated in the formation of cytoskeletal and cell membrane structures, and plays a role in many physiological processes, such as embryo development, wound healing, and tumor invasion.\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e The expression of MMP-9, N-cadherin, and vimentin in tumor was observed to be reduced after Erianin treatment, while E-cadherin expression was augmented, thus demonstrating that Erianin could impede the migration and invasion of tumor cells and impede tumor metastasis.\u003c/p\u003e \u003cp\u003eFlow cytometry and Western Blot were employed to further explore the inhibition of tumor cell proliferation by Erianin. The results indicated that the proportion of cells in G2/M phase augmented with the concentration of Erianin. Moreover, Western Blot revealed that, in comparison to the control group, CyclinB1 and CDK1 expressions decreased after treatment with Erianin. Cyclins are A, B, D, E, G, and H, which bind to a key protein kinase, the cyclin-dependent kinase CDK, and regulate its enzyme activity to help, drive, and coordinate the cell cycle.\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e M-phase cyclins mainly consist of CyclinB, which begins to be synthesized in late G1 phase, increases in expression in S phase, peaks in late G2 phase and M phase, and enters the nucleus to bind to CDK1 to form a complex, thereby activating the kinase activity of CDK1.\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e CyclinB-CDK1 mainly plays a role at the end of G2 and can guide cells into the M phase of mitosis.\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e When the cell exited the M phase, CyclinB degraded, CDK1 kinase activity inactivated, substrate dephosphorylation promoted chromosome agglutination, nucleolar reconstruction, and guided the cell into the next cell cycle.\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e The experiment revealed a decrease in CyclinB1 and CDK1 expression after Erianin was administered, implying that the treatment of Erianin could impede the cell cycle, postpone its progression, and thus impede cell proliferation.\u003c/p\u003e \u003cp\u003eWe conclude that inhibition of proliferation and migration of OS cells by Erianin is related to the physiological process of ferroptosis, which is consistent with the results of previous papers. A recent paper found that Erianin may be involved in the ferroptosis process. Despite the utilization of Caspase inhibitor Z-VAD-FMK, Necrostatin 1 and autophagy inhibitor chloroquine to impede apoptosis, necrosis and autophagy in lung cancer cells, Erianin was still able to cause the death of these cells. It was discovered that ROS accumulation, GSH consumption and lipid peroxidation had significantly risen in lung cancer cells. The use of ferroptosis inhibitors Ferrostatin-1 and Liproxstatin-1 could reduce the Erianin-induced cell death, indicating that ferroptosis was involved in the Erianin-induced cell death process. Transcriptomic analysis also found that the expression of GPX4 was down-regulated, and the enrichment of KEGG pathway showed that information related to ferroptosis pathway was enriched. It was found that Erianin could induce ferroptosis in lung cancer cells by activating Ca\u003csup\u003e2+\u003c/sup\u003e/CaM signaling, resulting in the increase of Ca\u003csup\u003e2+\u003c/sup\u003e and Fe\u003csup\u003e2+\u003c/sup\u003e levels. CaM can adjust voltage dependence l-type Ca\u003csup\u003e2+\u003c/sup\u003e channel (L-typevoltagptedependentCa2\u0026thinsp;+\u0026thinsp;channel, LVDCC), not only is the key to Ca\u003csup\u003e2+\u003c/sup\u003e transport, this also is the key to iron absorption. After treated with Erianin, CaM of lung cancer cells are activated, Ca\u003csup\u003e2+\u003c/sup\u003e / CaM signal pathway is activated, and increased Ca\u003csup\u003e2+\u003c/sup\u003e uptake induces ROS accumulation and elevated Fe2\u0026thinsp;+\u0026thinsp;levels, which in turn induces the occurrence of cell ferroptosis.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e In this study, we detected the levels of GSH and ROS in MG-63 cells after treatment with Erianin. The Erianin treatment caused a decrease in GSH levels in cells, as compared to the control group, as evidenced by the results. Western Blot also detected reduced expression of GPX4 and SLC7A11 proteins, indicating ferroptosis in MG-63 cells. These results suggest that Erianin can induce apoptosis of OS cells by inducing ferroptosis, and inhibit proliferation and migration of OS cells. Subsequently, we further validated and treated the cells with GPX4 inhibitors(RSL3), and found that both cell proliferation and migration were inhibited.\u003c/p\u003e \u003cp\u003eThis study demonstrates that Erianin can control the growth and tumorigenesis of OS in nude mice, as well as the proliferation, migration, and apoptosis of OS cells in vitro. It has the potential to become a new candidate target for OS and provide new theoretical basis for clinical treatment of OS.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOS cells can be inhibited in their proliferation and migration by Erianin, G2/M phase arrest is brought about, and apoptosis is induced. This may be related to the ferroptosis signaling pathway\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare that there are no competing interests regarding the publication of this paper.\u003c/p\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003eEthics Review Committee of Shandong First Medical University gave the experiment their approval, with the ethical code of W202312220339. This experiment follows the \"Regulations on the Administration of Experimental Animals of the People's Republic of China (Revised Draft for Comments)\".\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eMany thanks are given to the teachers and professors for providing us with technical support. This work was supported by Projects of Medical and Health Technology Development Program in Shandong Province under Grant (number 202002020809) and Shandong First Medical University College Students Innovation and Entrepreneurship Training Program(2022104391661) .\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Beilei Yu and Shuyuan Feng. The first draft of the manuscript was written by Zhongzhe Li, Bin Wang and Kaige Gao and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Beilei Yu and Shuyuan Feng made equal contributions to this article. Lu Zhou is the corresponding author.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eMany thanks are given to the teachers and professors for providing us with technical support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets during the current study are available from the corresponding authors on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChen, C., L. Xie, T. Ren, Y. Huang, J. Xu, and W. Guo, Immunotherapy for osteosarcoma: Fundamental mechanism, rationale, and recent breakthroughs. Cancer Letters, 2021. 500: p. 1-10.\u003c/li\u003e\n\u003cli\u003eShoaib, Z., T.M. Fan and J.M.K. Irudayaraj, Osteosarcoma mechanobiology and therapeutic targets. British Journal of Pharmacology, 2021. 179(2): p. 201-217.\u003c/li\u003e\n\u003cli\u003eLi, G., H. Zhang, H. Lai, G. Liang, J. Huang, F. Zhao, et al., Erianin: A phytoestrogen with therapeutic potential. 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Gumbiner, The functional activity of E-cadherin controls tumor cell metastasis at multiple steps. Proc Natl Acad Sci U S A, 2020. 117(11): p. 5931-5937.\u003c/li\u003e\n\u003cli\u003evan Loon, K., M.E. van Breest Smallenburg, E.J.M. Huijbers, A.W. Griffioen and J.R. van Beijnum, Extracellular vimentin as a versatile immune suppressive protein in cancer. Biochim Biophys Acta Rev Cancer, 2023. 1878(6): p. 188985.\u003c/li\u003e\n\u003cli\u003eWood, D.J. and J.A. Endicott, Structural insights into the functional diversity of the CDK-cyclin family. Open Biol, 2018. 8(9).\u003c/li\u003e\n\u003cli\u003e\u0026lt;Cell Cycle Progression and Synchroni Source Methods Mol Biol SO 2022.pdf\u0026gt;.\u003c/li\u003e\n\u003cli\u003eLinder, M.I., M. K\u0026ouml;hler, P. Boersema, M. Weberruss, C. Wandke, J. Marino, et al., Mitotic Disassembly of Nuclear Pore Complexes Involves CDK1- and PLK1-Mediated Phosphorylation of Key Interconnecting Nucleoporins. Developmental Cell, 2017. 43(2): p. 141-156.e7.\u003c/li\u003e\n\u003cli\u003e\u0026lt;Mechanisms and regulation of the deg Source Philos Trans R Soc Lond B Biol Sci SO 1999 Sep 29 354 1389 1571 5 discussion 1575 6.PDF\u0026gt;.\u003c/li\u003e\n\u003cli\u003eChen, P., Q. Wu, J. Feng, L. Yan, Y. Sun, S. Liu, et al., Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits lung cancer cell growth and migration via calcium/calmodulin-dependent ferroptosis. Signal Transduct Target Ther, 2020. 5(1): p. 51.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cytotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cyto","sideBox":"Learn more about [Cytotechnology](http://link.springer.com/journal/10616)","snPcode":"10616","submissionUrl":"https://submission.nature.com/new-submission/10616/3","title":"Cytotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Erianin, Osteosarcoma, Ferroptosis, Cell Proliferation, Migration","lastPublishedDoi":"10.21203/rs.3.rs-6119041/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6119041/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eErianin plays a certain role in the treatment of tumors, inflammation, diabetes nephropathy, retinopathy and other diseases. However, the impact and mechanism of Erianin on osteosarcoma(OS) are still unclear. This article aims to investigate the mechanism of action of Erianin in OS.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAnimal experiments were conducted using nude mice to investigate the in vivo effects of Erianin on OS. Investigations into the in vitro effects of Erianin on OS were conducted through cell experiments utilizing MG-63 and U-2 OS human OS cell lines. Firstly, use the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to detect cell viability and calculate IC50. Using colony-formation assay to detect the inhibitory effect of Erianin on cell proliferation. Use wound healing assay and cell migration assay to detect the effect of Erianin on the migration of OS cells. Use flow cytometry to detect cell apoptosis. Observe the effect of Erianin on the survival of OS cells under a microscope using Acridine Orange/Propidium Iodide(AO/PI)\u0026zwnj; staining. Use glutathione detection kit to detect the effect of Erianin on the ferroptosis signaling pathway in OS cells. Verify protein expression using western blot.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn vitro, Erianin inhibits proliferation and migration of OS cells by regulating cyclin and apoptotic proteins, leading to ferroptosis of OS cells; In vivo, Erianin can also inhibits tumor growth and cause ferroptosis of OS.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe findings of Erianin's induction of apoptosis and ferroptosis, as well as its inhibition of proliferation and migration of OS cells, point to it as a potential therapeutic agent for OS that warrants further investigation.\u003c/p\u003e","manuscriptTitle":"Erianin Induces Apoptosis of Osteosarcoma Cells Through Ferroptosis Signaling Pathway, and Inhibits Proliferation and Migration of Osteosarcoma Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 14:36:46","doi":"10.21203/rs.3.rs-6119041/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-13T02:49:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-12T21:34:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154069887869640573326166051981607826029","date":"2025-04-01T11:19:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T02:17:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-28T08:15:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-28T08:11:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cytotechnology","date":"2025-02-27T08:29:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cytotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cyto","sideBox":"Learn more about [Cytotechnology](http://link.springer.com/journal/10616)","snPcode":"10616","submissionUrl":"https://submission.nature.com/new-submission/10616/3","title":"Cytotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"29ab1741-2a11-42ac-95e0-6d3a6f8f1842","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:45:31+00:00","versionOfRecord":{"articleIdentity":"rs-6119041","link":"https://doi.org/10.1007/s10616-025-00792-w","journal":{"identity":"cytotechnology","isVorOnly":false,"title":"Cytotechnology"},"publishedOn":"2025-08-01 16:13:37","publishedOnDateReadable":"August 1st, 2025"},"versionCreatedAt":"2025-03-03 14:36:46","video":"","vorDoi":"10.1007/s10616-025-00792-w","vorDoiUrl":"https://doi.org/10.1007/s10616-025-00792-w","workflowStages":[]},"version":"v1","identity":"rs-6119041","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6119041","identity":"rs-6119041","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}