Erianin induces apoptosis of osteosarcoma cells through ferroptosis signaling pathway, and inhibits proliferation and migration of osteosarcoma cells

Abstract

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. 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. In vitro, Erianin inhibits proliferation and migration of OS cells by regulating apoptotic proteins (Bcl-2, Bax and Cleaved Caspase3), migration proteins(MMP-9, N-cadherin, Vimentin and E-cadherin) and cyclin proteins (CyclinB1 and CDK1), leading to ferroptosis; In vivo, Erianin inhibits tumor growth(Volume inhibition rate: 81.10%, Weight inhibition rate: 53.25%) and causes ferroptosis. 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.

Introduction

Osteosarcoma (OS) is a malignant tumor with clinical manifestations of bone and joint pain and local mass (Chen et al. 2021). Research shows that the average age of osteosarcoma is 17.1 years old, and the peak of incidence rate of primary osteosarcoma in children and adolescents is 10–24 years old. At present, the standard treatment for osteosarcoma is chemotherapy and surgical resection. However, in the past few decades, the 5-year survival rate of osteosarcoma patients has not significantly improved, indicating that existing treatment strategies are inappropriate (Shoaib et al. 2021; Tian et al. 2023). 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 (Li et al. 2023). This natural compound is similar to the active ingredients of various traditional medicinal plants. For example, in bone disease research, traditional Chinese medicine extracts can improve bone density in ovariectomized rats (Zhang et al. 2016). In the field of anti-cancer, natural toxins such as paclitaxel exert their effects by regulating ion channels (Liang et al. 2023). Hypericum perforatum in blood can treat major depressive disorder (Xu et al. 2024). The water extract of Hedyotis diffusa contain a novel and potent anti Helicobacter pylori adhesive (Ou et al. 2024), which shows the therapeutic potential of natural products in infectious diseases. The efficacy of different traditional Chinese medicine therapies in treating age-related osteoporosis has been validated by a network meta-analysis. 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 (Duan et al. 2022). 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 (Yang et al. 2023). The present research shows that Erianin has a certain role in the treatment of tumor, inflammation, diabetic nephropathy, retinopathy and other diseases (Sun et al. 2020; Dou et al. 2020; Zhang et al. 2019). However, the effect of Erianin on OS and its mechanism are not clear.

The accumulation of iron in cells leads to high expression of unsaturated fatty acids and lipid peroxidation on the cell membrane, which is the main mechanism of iron deposition (Dixon et al. 2012). Iron deposition is a novel programmed cell death that relies on iron, different from apoptosis, cell necrosis, and autophagy that cause cell death. Its core features are the accumulation of lipid reactive oxygen species (ROS) and the lipid peroxidation cascade reaction triggered by the inhibition of glutathione peroxidase 4 (GPX4) activity (Park and Chung 2019). When GPX4 inhibition or GPX4 deficiency occurs, it can directly lead to the accumulation of lipid peroxides, thereby activating ferroptosis (Xie et al. 2023). TRIM3 facilitates ferroptosis in non-small cell lung cancer through promoting SLC7A11/xCT K11-linked ubiquitination and degradation (Wang et al. 2024a). For example, inhibiting DDR1 can reverse gefitinib resistance in non-small cell lung cancer by activating desferriosis (Wang et al. 2024b). In recent years, newly discovered death pathways such as copper poisoning and disulfide poisoning, although sharing oxidative stress characteristics with iron deposition, have significant differences in their core regulatory genes (such as ATP7B, SLC31A1 copper metabolism genes) and triggering mechanisms (abnormal disulfide bond accumulation), suggesting that different cell death pathways may have interactive regulatory networks in the tumor microenvironment (Liu 2023; Liu and Tang 2022, 2023; Liu et al. 2023). Earlier research has confirmed that GPX4 inhibitor RAS selective lethal compound 3 (RSL3) can induce ferroptosis in cancer cells (Li et al. 2021) Ferroptosis has been linked to the genesis of various diseases, such as tumors, ischemia–reperfusion harm, degenerative diseases, and stroke (Stockwell et al. 2017). 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 (Xiang et al. 2021) and it has been demonstrated that Erianin inhibits the growth and metastasis through autophagy-dependent ferroptosis in KRAS^G13D colorectal cancer (Miao et al. 2023). 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

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, Shandong First Medical University. Throughout the experiment, these mice were housed in the SPF grade animal facility of Shandong First Medical University. They are maintained in a 12 h 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 °C incubator with 100% humidity and 5% CO₂ concentration. The medium was supplemented with 10% fetal bovine serum (Cat.No. C04001-050; VivaCell Biosciences; Shanghai), 100U/mL penicillin and 100 µg/mL streptomycin (Cat.No. P1400; Solarbio; Beijing).

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)”.

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 h, and the model group was treated with 80 nM of Erianin (Cat.No. HY-N0517; MCE; Shanghai) for 24 h. Then the cells were digested with pancreatic enzyme, cleaned with PBS, and the concentration of cells was adjusted to 1 × 10⁷ cells/mL. Respectively inoculate cells into the subcutaneous axillary area of 8 mice, 0.2 mL/mouse. Inject Erianin (10 mg/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 (Rudolph et al. 2023) (0.2 mL/10 g) (Cat.No. C804539; MACKLIN; Shanghai), followed by subcutaneous tumor extraction, weighing, and recording.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay

MG-63 cells were inoculated with 1 × 10⁴ cells/well into 96-well plates for normal culture, and 5 compound pores were set up. After 4 h 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 °C for 4 h. Subsequently, the supernatant was discarded and 100 μL DMSO (Cat.No. D8370; Solarbio; Beijing) was added in per well. After 10 min in a shaker, the absorbance was measured by spectrophotometer at 490 nm 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 h for further experiments.

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% CO₂ at 37 °C 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 15 min and stained with 0.1% crystal violet (Cat.No. C8407; Solarbio; Beijing) for 30 min. The number of colonies was counted using ImageJ.

Cell viability staining

Inoculate 4 × 10⁴ cells per milliliter on 6-well plates for each group of cells. Cultivate for 4 h. When the cells were attached, they were treated with different concentrations of Erianin for 24 h. 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).

Flow cytometry

Collect cells from each group, adjust them to a cell concentration of 1 × 10⁵/mL, and fix overnight with 75% cold alcohol at 4 °C. 24 h later, clean the cells with PBS, then add RNase and stain with 50 μg/mL propidium iodide (Cat.No. C0080; Solaibio), and keep for 30 min in dark. Test samples on NovoCyte (Novo Express; 1.3.0; ACEA Biosciences; Beijing).

Cell migration assay

After collecting cells from each group, they were suspended in a basic medium with a density of 1 × 10⁵ cells per milliliter. To the upper chamber of Transwell chamber (Cat.No. TCS003024; BIOFIL; Guangzhou), 200 μL cell suspension was added, and 500 μL complete culture medium was added to the lower chamber. After 24 h in a cell incubator at 37 °C, 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 min, away from light. Stain the wells with 0.1% crystal violet for 15 min, then measure cell migration number under an optical microscope.

Wound healing assay

Each group of cells was seeded on a six well plate with 1 × 10⁵ cells per well. 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 20 μL pipette gun tip. Then cleaned with PBS, and treated with different concentrations of Erianin or RSL3 (Cat.No. HY-100218A; MCE; Shanghai) for 24 h. Observe the relative distance of healed scratches using microscope (Nikon; 80i; Japan) and Image J (Image J; 1.53t; National Institutes of Health; USA).

Determination of glutathione (GSH)

Measure the intracellular total glutathione level according to the operating instructions of the glutathione assay kit (Cat.No.S0055; Beyotime; Shanghai). The detection range of this kit is 0.1-10U/mL, with a sensitivity of 0.05 U/mL. Reduced GSH reacts with DTNB to produce a yellow product. Firstly, prepare standard samples, including 1 mg/mL GSH standard and 200 μg/mL GSH standard. Further dilute 200 μg/mL GSH standard to achieve a final concentration of 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL, 10 μg/mL, 5 μg/mL, 2 μg/mL, 0 μg/mL. Before each experiment, prepare a new standard and preferably use the prepared standard within 4 h. Then prepare the samples, collect 2 × 10⁶ cells from each group, wash the cells twice with PBS, centrifuge at 12000 rpm and 4 °C for 5 min. Resuspend cells in 200 μL Extraction Buffer, repeat freezing and thawing twice, centrifuge at 12,000 rpm and 4 °C for 10 min, and take the supernatant for the next step of analysis. Preheat the enzyme-linked immunosorbent assay (ELISA) reader for at least 30 min and adjust the wavelength to 412 nm. Add samples to a 96 well plate with blank wells of 0 μL sample volume, 20 μL ultrapure water, 0 μL standard, 140 μL Assay Buffer, and 4 μL Chromogen; Standard wells: 0 μL sample size, 0 μL ultrapure water, 20 μL standard, 140 μL Sassay Buffer, and 40 μL Chromogen; Measurement wells: 20 μL sample size, 0 μL ultrapure water, 0 μL standard, 140 μL AssayBuffer, and 40 μL Chromogen. The liquid is thoroughly mixed and incubated for 2 min at room temperature and in the dark, and the absorbance value at a wavelength of 412 nm is recorded and analyzed. Finally, calculate the content of GSH.

Western blot

After drug treatment, cells in each group were collected and placed in RIPA lysta(cat.no. G4535; Servicebio; Wuhan). After 30 min at low temperature, the supernatant solution was centrifuged at 4 °C 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(concentration of concentrated gel is 5%, concentration of separation gel is 10%), 30 μg protein samples were transferred to PVDF (Polyvinylidene Difluoride) membranes. 5% skimmed milk was blocked for 2 h 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), Cleaved Caspase3(1: 500; cat.no. ab32042; Abcam; Britain), 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 (HRP labeled goat anti mouse IgG: 1:100,000; cat.no. GB23301; Servicebio; Wuhan; HRP labeled goat anti rabbit IgG: 1:100,000; cat.no. GB23303; Servicebio; Wuhan). Incubated for 1 h 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 min 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.

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

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 (Fig. 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 h 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 (Fig. 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 (Fig. 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 (Fig. 1e). It is suggested that Erianin can induce G2/M phase arrest in OS cells.

Fig. 1.

Erianin 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. *P < 0.05 versus the Control(Con) group; **P < 0.01 versus the Con group; ***P < 0.001 versus the Con group

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 (Fig. 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 (Fig. 2d, e).

Fig. 2.

Erianin 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. *P < 0.05 versus the Control(Con) group; **P < 0.01 versus the Con group; ***P < 0.001 versus the Con group

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 Cleaved Caspase3 were augmented (Fig. 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 (Fig. 3a, e–h). Erianin decreased the expression of CyclinB1 and CDK1 in a dose-dependent manner (Fig. 3i–k).

Fig. 3.

Erianin 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 < 0.05 versus the Control(Con) group; **P < 0.01 versus the Con group; ***P < 0.001 versus the Con group

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 (Fig. 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 (Fig. 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 (Fig. 4h). Western Blot revealed the expression of a related protein to ferroptosis, with the results being consistent (Figs. 4i–k).

Fig. 4.

Erianin 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. *P < 0.05 versus the Control(Con) group; **P < 0.01 versus the Con group; ***P < 0.001 versus the Con group

Erianin inhibits the proliferation and migration of OS cells through ferroptosis signaling pathway

We also conducted clone formation experiments, scratch experiments, migration experiments, and flow cytometry using the GPX4 inhibitor RSL3 to verify its inhibitory effects on the proliferation, migration, and cell cycle of MG-63 cells. In addition, we also verified the effect of RSL3 on ferroptosis expression in OS cells and found that RSL3 can reduce the expression of GPX4 and SLC7A11 in cells. These effects are consistent with the effects of Erianin. Therefore, we conclude that Erianin inhibits the proliferation and migration of OS cells through the iron ferroptosis signaling pathway (Fig. 5a–h).

Fig. 5.

Erianin 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, f 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. *P < 0.05 versus the Control(Con) group; **P < 0.01 versus the Con group; ***P < 0.001 versus the Con group

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 (Wang et al. 2020; Pan et al. 2021; Qiu et al. 2020). 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 (Mondal et al. 2020). 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 (Liu et al. 2024). 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 (Balamurugan et al. 2023). 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 (Na et al. 2020). 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 (Loon et al. 2023). 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 (Wood and Endicott 2018). 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 (Progression et al. 2022). CyclinB-CDK1 mainly plays a role at the end of G2 and can guide cells into the M phase of mitosis (Linder et al. 2017). 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 (Li et al. 2023). 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 Ca²⁺/CaM signaling, resulting in the increase of Ca²⁺ and Fe²⁺ levels. CaM can adjust voltage dependence l-type Ca²⁺ channel (L-typevoltagptedependentCa²⁺ channel, LVDCC), not only is the key to Ca²⁺ transport, this also is the key to iron absorption. After treated with Erianin, CaM of lung cancer cells are activated, Ca²⁺/CaM signal pathway is activated, and increased Ca²⁺ uptake induces ROS accumulation and elevated Fe²⁺ levels, which in turn induces the occurrence of cell ferroptosis (Chen et al. 2020). 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. Recent studies have emphasized the progress of liquid biopsy for cancer diagnosis and monitoring. Research has demonstrated the practicality of detecting circulating tumor DNA (ctDNA) through liquid biopsy technology (Jahangiri 2024). In addition, emerging sequencing technologies have improved the sensitivity and specificity of DNA analysis, and methylation is also used for detection (Ohyama et al. 2024; Gonzalez et al. 2024). The mechanism in this study may also be applicable for identification and diagnosis using this technology in the near future. In addition, there are some limitations to this study, such as the small sample size of animal experiments, which may result in certain errors. It is recommended to repeat animal experiments for validation before conducting clinical studies.

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.

Author contributions

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.

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).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical 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)”.