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.

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% CO₂ 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).

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)".

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 1×10⁷ 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 1×10⁴ 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% CO₂ 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 × 10⁴ 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 × 10⁵/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 1×10⁵ 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.

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(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 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(Figs. 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.

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(Figs. 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(Figs. 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(Figs. 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(Figs. 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(Figs. 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(Figs. 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).

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(Figs. 5a-h).

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 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.^[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.

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