Bangladesh Journal of Pharmacology Research Article BJP Introduction Osteosarcoma, a highly aggressive malignancy with a pronounced incidence among children and adolescents under the age of 20, is characterized by its high metas- tatic potential (Xu et al., 2020), poor prognosis (Li et al., 2020), and currently limited five-year survival rates (Lei et al., 2021). Despite extensive research over the past three decades, there has been no substantial improve- ment in the efficacy of pharmacological treatments for osteosarcoma. Consequently, pursuing safer and more effective novel therapeutic agents against osteosarcoma holds paramount importance and urgency. Glucocorticoids, vital small-molecule substances within the body, play pivotal roles in regulating growth (Zhao et al., 2024), development (Taves and Ashwell, 2021), metabolism (Felten and Arnaud, 2020), and immunity (Shimba and Ikuta, 2020). They exhibit extensive and significant physiological and pharmacological effects and are among the most widely used and effective anti- inflammatory and immunosuppressive agents in clini- cal practice. Commonly prescribed glucocorticoids include prednisone, hydrocortisone and dexametha- sone, which also have demonstrated important roles in the treatment of breast cancer (Pang et al., 2020) and hematological malignancies (Ahmad, et al., 2019; Gebru et al., 2020). Prednisone, a synthetic glucocorticoid with an elimination half-life of 3-4 hours in adults and 1-2 hours in children (Puckett et al., 2024), is employed in the management of asthma (Abaya et al., 2018), Abstract Prednisone, a synthetic glucocorticoid, possesses a moderate duration of action in the human body. To investigate the effect of prednisone on osteosarcoma cells in vitro, CCK8, wound healing, Transwell assays, flow- cytometry, western blot, and ELISA were used to measure cell proliferation, cell migration, cell invasion, cell cycle, cell apoptosis, and the key proteins in Wnt/β-catenin pathway. The results showed prednisone could inhibit the proliferation in osteosarcoma cells and exhibit lower proliferation toxicity towards normal bone cells compared to cisplatin by 17.5% (p<0.001). Cell apoptosis was increased by 6.6 and 7.4%, cell migration was decreased to 62.6 and –23.9%, G0/G1 phase cells were increased by 33.0 and 17.9%, and S phase cells were reduced by 29.6 and 18.0% (all p<0.001) in Mg63 and Saos-2 cells respectively after prednisone treatment. The expressions of β-catenin, cMYC, cyclin D1, and MMP7 were also reduced. In conclusion, prednisone inhibits osteosarcoma cells by regulating the Wnt/β-catenin signaling pathway. Article Info Received: 18 July 2024 Accepted: 31 October 2024 Available Online: 13 December 2024 DOI: 10.3329/bjp.v19i3.76379 Cite this article: Zhang Y, Zhang H, Yang S, Gao X， Guo W. Prednisone inhibits osteosar- coma cell by regulating the Wnt/β- catenin signaling pathway. Bangla- desh J Pharmacol. 2024; 19: 73-80. Prednisone inhibits osteosarcoma cell by regulating the Wnt/β- catenin signaling pathway You Zhang1, Hongmei Zhang1, Shanshan Yang1, Xiufeng Gao2，and Wenhao Guo3 1Clinical Translational Innovation Center/Molecular Medicine Research Center, West China Hospital, Sichuan University, Chengdu, Sichuan Province, China; 2West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, Sichuan Province, China; 3Department of Abdominal Oncology, Cancer Center and State Key Laboratory of Biotherapy, Medical School, West China Hospital, Sichuan University, Sichuan Province, China. This work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2024; 19: 73-80 Journal homepage: www.bdpsjournal.org; www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 myeloma (Kaedbey et al., 2024), and prostate cancer (Duarte et al., 2019). Although the long-acting gluco- corticoid dexamethasone (Kudawara et al., 2001; Yama- moto, et al., 2002) and the short-acting glucocorticoid hydrocortisone (Hindmarsh et al., 2015; Zhang et al., 2023) have been proven to have some effects on osteo- sarcoma inhibition, the effect of the medium-acting glucocorticoid prednisone on osteosarcoma remains unexplored. Studying the inhibitory effect of predni- sone on osteosarcoma may help to fully understand the role of glucocorticoid in anti-tumor therapy and pro- vide more comprehensive treatment options and theo- retical basis for clinical practice. Materials and Methods Cells The human osteosarcoma cell line Mg63, human osteo- sarcoma cell line Saos-2, and human normal osteocyte hFOB1.19 were all purchased from the Chinese Aca- demy of Sciences Cell Bank in Shanghai. Drugs and reagents Cell counting Kit-8 (CCK8), Bradford protein quantifi- cation Kit, myelocytomatosis viral oncogene homolog (cMYC) antibody, cyclin D1 antibody and glyceralde- hyde-3-phosphate dehydrogenase (GAPDH) antibody were purchased from Beijing Biyuntian Biotechnology Co., Ltd. Secondary antibodies were purchased from Beijing Zhongshan Jinqiao Technology Co., Ltd. High- sensitivity enhanced chemiluminescence substrate, propidium iodide cell cycle detection kit, and apoptosis detection kit were purchased from Shanghai Sizhengbai Biotechnology Co., Ltd. Prednisone was purchased from Selleck Biotechnology Co., Ltd. and dissolved in dimethyl sulfoxide (DMSO) before use. Human matrix metalloproteinase 7 (MMP7) enzyme-linked immuno- sorbent assay kit and crystal violet were purchased from Shanghai Biological Engineering Co., Ltd. Dulbecco's Modified Eagle Medium (DMEM) culture medium and fetal bovine serum were purchased from GIBCO Medical and Biotechnology Co., Ltd.. Cell culture Mg63 and Saos-2 human osteosarcoma cells are cul- tured in a 5% CO2-saturated incubator at 37°C, using DMEM cell culture medium supplemented with 100 IU/mL penicillin, 15% fetal bovine serum, and 100 μg/ mL streptomycin. The medium is replaced every 48 hours. The human fetal osteoblastic cell line hFOB1.19 was maintained in F-12 Dulbecco's Modified Eagle Medium (GIBCO, USA) supplemented with 10% fetal bovine serum and 0.3 mg/mL of neomycin (Biyuntian Biotech, China) at 33.5℃. CCK8 assay for cell proliferation To assess cell proliferation, the cell suspensions were seeded into 96-well plates at 100 μL per well, containing approximately 5,000 cells per well. Different concentra- tions (0-1000 μM) of prednisone were added to the treatment group wells, and the plates were incubated continuously for 48 hours. CCK8 (10 μL CCK8 per 100 μL DMEM) was added to each well, and the absorbance at 450 nm was measured (Multiskan, USA). The optical density (OD) values obtained reflect the number of viable cells in each well (Jiang, et al., 2024). Wound healing assay MG63 and Saos-2 cells were inoculated into a 6-well plate at 5 × 105 cells/well at 37°C and 5% CO2 for 24 hours to form a monolayer of cells. After 24 hours, a scratch was subsequently performed in the cell mono- layer. Carefully wash three times with PBS to remove residual cell debris, and then add drug-free and predni- sone-containing media to incubate without serum to avoid the effect of cell proliferation. Then the plate was incubated at 37°C and 5% CO2 for 48 hours, and subsequently photographed (Yang et al., 2024). 74 Bangladesh J Pharmacol 2024; 19: 73-80 Box 1: Western Blot Principle Western blot is based on the principle of immunochromato- graphy where proteins are separated into polyacrylamide gel according to their molecular weight. Requirements BCA protein assay kit (Beyotime, China, P0010), High sensi- tivity enhanced chemiluminescence substrate (Beyotime, China, P0018M), Cells pellet, RIPA buffer, Protease cocktail (Beyotime, China, P1005), Blocking buffer, Primary antibodies, Secondary antibodies, Phosphate saline buffer, PVDF membrane, 1×Tris-buffered saline, 0.1%Tween, Sodium polyacrylamide gel, Transfer buffer. Procedure Step 1: The proteins were separated using a 1% sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS- PAGE) gel and electrotransferred onto a PVDF membrane. Step 2: The membrane was washed, blocked, and then a primary antibody at a 1:2500 dilution was added. Step 3: It was incubated at room temperature for 1 to 2 hours or overnight at 4°C. Step 4: After the membrane was washed, a secondary antibody at an appropriate dilution of 1:4000 was added, and it was incubated at room temperature for 1 hour with gentle agitation. Step 5: Following another wash, the target protein was detected, and the grayscale values were analyzed using ImageJ software. Reference Zheng et al., 2024 https://www.thermofisher.cn/cn/zh/home/life-science/lab-equipment/microplate-instruments/plate-readers/models/multiskan-fc.html Transwell assay The incubated upper chamber of the Transwell was placed in the 24-well plate with 5×104 cells, and 600 µL culture medium containing 20% fetal bovine serum was added to the bottom of the well, which was the lower chamber. After incubation at 5% CO2, cells were placed in 0.1% 600 µL crystal violet staining solution for 30 min at room temperature. Upon completion of staining, the crystal violet was eluted with 30% acetic acid, and the optical density values were measured at a wavelength of 570 nm for statistical analysis of the results (Yang et al., 2024). Cell apoptosis and cell cycle detection The cell cycle and apoptosis experiments were performed as described previously (Li et al., 2019; Le et al., 2024). Cells were harvested with trypsin without ethylenediamine-tetraacetic acid (EDTA), washed with phosphate- buffered saline (PBS), and then resuspended in binding buffer (0.1 M HEPES/NaOH, 1.4M NaCl, 25 mM CaCl2) to adjust the cell concentration to between 1 × 106/mL and 5 × 106/mL. 100 µL of the suspension was aspirated into a 1.5 mL tube, mixed with 5 µL of annexin V/PE (Sizhengbai Technology, China), incubi- ted at room temperature for 5 min in the dark, and then added with 10 µL of 20 µg/mL 7AAD+ (Sizhengbai Technology, China) and 400 µL of PBS before imme- diate flow cytometry analysis (Flowy XP, 0211, China). Cells were digested with EDTA-free trypsin, washed with cold PBS, and fixed with precooled 75% ethanol overnight at 4°C. The cells were washed twice with PBS, centrifuged, and treated with RNase A and propi- dium iodide (PI) for 30 min at 37°C in the dark. Finally, the stained cells were analyzed by a flow cytometer (BD -cytometer, LSRII, USA). Statistical analysis All data are presented as statistical graphs generated using GraphPad Prism 5. Differences between the two groups were determined using a two-tailed t-test. Differences between three or more groups were determined using a two-way analysis of variance. Data were measured using the mean ± SEM. P values less than 0.05 were considered statistically significant. Results Proliferation of osteosarcoma cells and toxicity towards normal bone cells compared to cisplatin Human normal osteocyte hFOB1.19 cells, human osteosarcoma cells Mg63, and human osteosarcoma cells Saos-2 were cultured in an incubator at 37°C and then seeded into 96-well plates. They were treated with different concentrations of prednisone (ranging from 0 to 1000 μM) or cisplatin (ranging from 0 to 40 μM), respectively, and further cultured for 48 hours. Cell viability was then measured by CCK8 assay, with the results presented in Figures 1A and 1B. At cisplatin concentrations ranging from 0 to 5 μg/mL, no significant effect on the proliferation of either normal osteocyte or osteosarcoma cells was observed. However, when cisplatin concentrations reached 10-40 μg/mL, an inhibition of proliferation was observed in three types of cells. At a cisplatin concentration of 20 μg/mL, the proliferation rate of normal osteocytes was significantly lower than that of the osteosarcoma cells (Figure 1A), suggesting a higher proliferation toxicity of cisplatin towards normal bone cells compared to osteosarcoma cells. At lower concentrations of prednisone (0-62.5 μM), no significant inhibition of proliferation was observed in any of the three cells (Figure 1B). Nevertheless, as the concentration of prednisone increased (125-1000 μM), prednisone suppressed the proliferation of both normal osteocyte and osteosarcoma cells, leading to a signifi- cant decrease in survival rates. This finding underscores A B C Figure 1: Prednisone inhibits osteosarcoma cells and is less toxic to the proliferation of normal osteocyte than cisplatin. Effects of cis- platin on normal bone cells hFOB1.19, osteosarcoma cells Mg63 and osteosarcoma cells Saos-2 (A). Effects of prednisone on hFOB1.19, Mg63 and Saos-2 (B). The inhibition on the proliferation of prednisone to normal bone cells was lower than that of cisplatin (C). aIndicates statistical results compared with the control group; dindicates statistical results compared with normal bone cell hFOB1.19 with p<0.05; eindicates statistical results compared with normal bone cell hFOB1.19 with p<0.001; ap<0.05, bp<0.01， cp<0.001 100 50 0 Saos - 2 Mg63 a Saos - 2 Mg63 Prednisone Cisplatin a b c c c c c c c c c c c c c c c a a b c c c c c c c e b 1 0 0 0 5 0 0 2 5 0 1 2 5 6 2 .5 3 1 .2 5 1 5 .6 2 0 4 0 2 0 1 0 5 0 2 .5 V ia b ili ty ( % C o n tr o l) R e la ti v e v ia b ili ty ( % C o n tr o l) d Concentration (µM) Concentration (µM) Bangladesh J Pharmacol 2024; 19: 73-80 75 the ability of prednisone to inhibit the proliferation of osteosarcoma cells. To compare the differences in the inhibitory effects of prednisone and cisplatin on cell proliferation, three types of cells were treated with 20 μg/mL cisplatin or 250 μM prednisone for 48 hours (Figure 1D). The results showed that there was no significant difference in the survival rate of the two osteosarcoma cells treated with 20 μg/mL cisplatin and 250 μM prednisone (both p>0.05), while the survival rate of the normal osteocyte hFOB1.19 treated with prednisone was significantly higher than that of cisplatin by 17.5% (p<0.05). This indicates that compared to cisplatin, which has a similar inhibitory effect on osteosarcoma cell proliferation, prednisone has significantly lower toxicity in inhibiting the proliferation of normal osteocytes than cisplatin. Migration of osteosarcoma cells To investigate the effect of prednisone on the migration of osteosarcoma cells, human Mg63 osteosarcoma cells and human Saos-2 osteosarcoma cells were cultured in 6-well plates, scratched, and cultured in serum-free medium with prednisone treatment for 48 hours (Figure 2A). The migration of Mg63 and Saos-2 cells decreased to 62.6% (p<0.001) and -23.9% (p<0.001), respectively, compared to the control group, indicating that prednisone treatment significantly inhibited the migration of osteosarcoma cells. Invasion of osteosarcoma cells MG63 and Saos-2 osteosarcoma cells were grown in the chamber and treated with prednisone for 48 hours. The results showed that after 48 hours of treatment with prednisone on Mg63 and Saos-2 cells, the number of cells that had crossed the chamber membrane decreased to 54.3% (p<0.001) and 50.7% (p<0.001), respectively, compared to the control group, indicating that predni- sone treatment significantly inhibited the invasion of osteosarcoma cell (Figure 2B). Apoptosis of osteosarcoma cells Apoptosis of osteosarcoma cells treated with predni- sone for 48 hours was analyzed by flow cytometry (Figure 3A). Compared with the control group, the average apoptosis rate increased by 6.6% (p<0.001) and 7.4% (p<0.001) after treatment with prednisone in Mg63 and Saos-2 osteosarcoma cells, respectively. The results suggested that prednisone treatment significantly increased the apoptosis in osteosarcoma cells. Osteosarcoma cell cycle Flow cytometry was used to analyze the cell cycle in Mg63 and Saos-2 cells after treatment with 250 µM prednisone for 48 hours. As shown in Figure 3B, com- pared with Mg63 and Saos2 cells without any drug treatment, the number of cells in the G0/G1 phase significantly increased by 33.0% (p<0.001) and 17.9% (p<0.001), respectively, while the number of cells in the S phase decreased by 29.6% (p<0.001) and 18.0% (p<0.001), respectively. This indicated that prednisone significantly causes cell cycle arrest in osteosarcoma cells. Control Prednisone Mg63 Saos-2 100 50 0 R e la ti v e m ig ra ti o n (% C o n tr o l) Mg63 Saos-2 100 50 0 R e la ti v e m ig ra ti o n (% C o n tr o l) a a Control Prednisone 100 50 0 % W o u n d c lo s e r 100 50 0 % W o u n d c lo s e r a a A B Figure 2: Effects and statistical chart of pred- nisone on the migration and invasion of oste- osarcoma cells. The invasion number of cells was calculated by the average of 3 random horizons and analyzed in the histogram, with a scale of 5 µm (A). The effects and statistics of the migration of MG63 and Saos-2 cells. The unhealed area was measured, and the values were organized and shown in the histogram. The initial wound area was used as a 100% control (B). Scale bars: 100 µm. Data are the mean ± SEM. (n=3) and repre- sented as the fold change. ap<0.001 76 Bangladesh J Pharmacol 2024; 19: 73-80 Osteosarcoma cells by regulating the Wnt/β-catenin pathway To explore the molecular mechanism of prednisone in inhibiting osteosarcoma cells, the key proteins of the Wnt/β-catenin signaling pathway were investigated. Osteosarcoma cells Mg63 and Saos-2 were divided into control- and prednisone- (250 µM) treated groups. After treating the cells for 48 hours, total protein was collected for western blot experiment to detect the expression of key proteins β-catenin and proteins rela- ted to tumor cell proliferation, migration, and invasion in the Wnt/β-catenin pathway, and statistics were performed on protein bands. Then, the content of MMP7 in the protein lysate was detected using an ELISA kit. As shown in Figure 4, compared with cells without any drug treatment, the protein expression/ concentration of cMYC, cyclin D1, β-catenin, and MMP7 significantly decreased in Mg63 and Saos-2 osteosarcoma cells after treating with 250 µM predni- sone, indicating that prednisone inhibited the prolifera- tion, migration, and invasion of osteosarcoma cells by Control Prednisone S G2/M G0/G1 a a Saos-2 C o u n t Propidium iodide 100 80 60 40 20 0 % P h a s e 80 60 40 20 0 % P h a s e a a Annexin FITC-A 7 a a d Mg63 Control Prednisone % C e ll a p o p to s is % C e ll a p o p to s is 15 10 5 0 15 10 5 0 a a Mg63 Saos-2 Control Prednisone Saos-2 Mg63 A B Figure 3: The treatment of prednisone increased apoptosis and cell cycle in osteosarcoma cells. Apoptosis and cell cycle of osteosar- coma cells were detected by flow cytometry (A). Cell cycle of osteosarcoma cells were detected by flow cytometry (B). Data are the mean ± SEM. n=3, ap<0.001 Bangladesh J Pharmacol 2024; 19: 73-80 77 inhibiting the Wnt/β-catenin signaling pathway. Discussion Literature had documented the inhibitory effects of dexamethasone and hydrocortisone on osteosarcoma cells. For instance, the proliferation of osteosarcoma cells was suppressed to varying degrees upon adminis- tering different doses of dexamethasone to Dunn cells (Yamamoto et al., 2002). A significant correlation between the inhibition of osteosarcoma proliferation and the dosage of dexamethasone in male C3H/He mice after 14 days of treatment (Kudawara et al., 2001). Hydrocortisone inhibits osteosarcoma both in vivo and in vitro (Zhang et al., 2023). These findings suggest that glucocorticoids may serve as a novel therapeutic agent for osteosarcoma. While hydrocortisone is prone to causing water-sodium retention and edema, dexame- thasone are associated with adverse effects such as central obesity, hypertension, and diabetes. Interme- diate-acting glucocorticoids (e.g., prednisone) offer a balanced anti-inflammatory and immuno-suppressive effect, making them suitable treating various auto- immune and inflammatory diseases. However, effects of prednisone on osteosarcoma remain unexplored. To find a new drug for treating osteosarcoma with low side effects, the inhibitory effect of prednisone on osteosarcoma and its molecular mechanism was explor- ed. First, normal bone cells hFOB1.19, osteosarcoma cells Mg63, and osteosarcoma cells Saos-2 were treated with cisplatin or prednisone. The results showed that prednisone successfully inhibited the proliferation of two types of osteosarcoma cells, with similar inhibitory effects to cisplatin but the survival rate of prednisone- treated normal bone cells hFOB1.19 was significantly higher than that of cisplatin. This indicated that prednisone has less impact on the proliferation of normal bone cells than cisplatin, which may help to reduce bone-related complications such as osteoporosis and fractures caused by drug side effects during the treatment of osteosarcoma. In addition, cell experi- ments also found that prednisone can inhibit the inva- sion and migration of osteosarcoma cells, which means that it can reduce the spread of tumor cells to tissues and distant organs and reduce the risk of tumor metas- tasis. Through flow cytometry experiments, it was found that prednisone can induce cell cycle arrest and apoptosis in osteosarcoma cells, which means that prednisone can affect the proliferation rate of tumor cells, thereby limiting tumor volume and disease pro- gression. These results have not been reported before, providing a basis for the pharmacological effects of prednisone in inhibiting osteosarcoma. Moreover, the drug concentration at which prednisone exerts its inhibitory effect on osteosarcoma is lower than that of hydrocortisone (Zhang et al., 2023), potentially provi- ding a broader therapeutic window, reducing treatment costs, and having higher application value. To date, this is the first report to show that prednisone also inhibits osteosarcoma. The Wnt/β-catenin signaling pathway is a highly conserved pathway in biological evolution, which plays an important role in cell proliferation, differentiation, migration, and apoptosis. β-catenin is an important regulatory protein in the Wnt/β-catenin signaling path- way. Mutations at Ser33, Ser37, and Thr41 sites of β- catenin protein led to higher levels of β-catenin protein in tumor cells than in normal cells, which is observed in many tumor cell lines (Gui et al., 2021; Zhang et al., 2019; Zhang et al., 2024). β-catenin can enter the nucleus B Control Mg63 Saos-2 Control beta-Catenin cMYC Cyclin D1 GAPDH b c Control PDN Control PDN 40 30 20 10 0 M M P 7 c o n c e n tr a ti o n (n g /m L ) 1.5 1.0 0.5 0.0 R e la ti v e e x p re s s io n (F o ld c h a n g e ) Control Mg63 Mg63 Control Control Saos-2 Control Saos-2 Control Saos-2 Mg63 Control beta-Catenin beta-Catenin cMYC cMYC Cyclin D1 Cyclin D1 b b b a a a Prednisone Prednisone Figure 4: Effects of prednisone (PDN) on proteins of Wnt/β-catenin pathway in Saos-2 and Mg63 cells. The histograms show the quantitative analysis of the samples. Data were measured using the mean ± SEM. The statistical significance of differences between two groups was analyzed using the two-tailed unpaired t test. n=3, ap<0.05, bp<0.01, cp<0.001 78 Bangladesh J Pharmacol 2024; 19: 73-80 from the cytoplasm to become a transcription factor (Nusse and Clevers, 2017), activating the transcription and expression of several downstream target proteins, such as cMYC, cyclin D1, and MMP7. cMYC is a gene that promote tumor cell proliferation, apoptosis, and division, which is associated with the occurrence and development of various tumors (McMahon, 2014). Cyclin D1 is a protein encoded by the human CCND1 gene that is involved in regulating cycle-dependent kinase (CDKs). Throughout the cell cycle, the abun- dance of cyclin D1 protein changes periodically (Mon- talto and De Amicis, 2020; Tchakarska and Sola, 2020). Matrix metalloproteinase 7 is a member of the matrix metalloproteinase (MMPs) family, which is involved in the degradation of extracellular matrix and is associated with tumor cell migration and invasion. This gene exhibits high expression levels in various human can- cers (Liu et al., 2020; Nomden et al., 2020). Through western blot experiments, it was found that the expression of β-catenin, cMYC, MMP7, and cyclin D1 proteins in the Wnt/β-catenin signaling pathway in osteosarcoma cells was significantly down-regulated after prednisone treatment, indicating that prednisone can regulate the proliferation, apoptosis, cell cycle, and migration of osteosarcoma cells by regulating the Wnt/ β-catenin signaling pathway, providing new ideas for novel drugs with low side effects on osteosarcoma. Conclusion Prednisone inhibits osteosarcoma cells by regulating β- catenin, cMYC, MMP7, and cyclin D1, proteins in the Wnt/β-catenin signaling pathway, thereby inhibiting the proliferation, apoptosis, cell cycle, migration, and invasion of osteosarcoma cells. Financial Support This work was supported by the National Natural Science Foundation of China (No. 82070299, 81870221, and 81670249). Ethical Issue The immortalized cell lines used in the experiments are not relevant materials, as they have been created outside of the human body. For research purposes, the storage and use of immortalized human cell lines do not require ethical approval. Conflict of Interest Authors declare no conflict of interest Acknowledgements Gratitude is extended to Mr. Youwei Huang for conducting part of the cell culture and western blotting experiments. Special thanks also go to Prof. Wei Jiang for providing guidance throughout the experiments. References Abaya R, Jones L, Zorc JJ. Dexamethasone compared to prednisone for the treatment of children with acute asthma exacerbations. Pediatr Emerg Care. 2018; 34: 53-58. Ahmad N, Abosoudah IF, Sobaihi MM, Algiraigri AH, Roujouleh F, Ghurab F, Chanoine JP. Adrenal function following acute discontinuation of glucocorticoids in children with acute lymphocytic leukemia: A prospective study. Pediatr Hemat Oncol. 2019; 36: 422-31. Duarte C, Jimeno A, Kessler ER. Abiraterone acetate to treat metastatic castration-resistant prostate cancer in combina- tion with prednisone. Drug Today. 2019; 55: 5-15. Felten R, Arnaud L. Is it possible to stop glucocorticoids in systemic lupus? Joint Bone Spine. 2020; 87: 528-30. Gebru MT, Wang HG, Xavier J. Glucocorticoids act synergis- tically with flt3 inhibitors to enhance cell death in flt3 mutant acute myeloid leukemia. Cancer Res. 2020; 80: 630- 36. Gui ZH, Yan H, Wu JF, Zhang MX, Wu AR, He J. Aberrant activation of Wnt/catenin signaling and overexpression of ABCG2 contributes to apoptosis down regulation and tumor progression of high grade ovarian cancer. Acta Biochim Pol. 2021; 68: 127-33. Hindmarsh PC, Charmandari E. Variation in absorption and half-life of hydrocortisone influence plasma cortisol concen- trations. Clin Endocrinol. 2015; 82: 557-61. Jiang L, Yang DD, Zhang ZY, Xu LY, Jiang QY, Tong YX , Zheng LZ. Elucidating the role of Rhodiola rosea L in sepsis-induced acute lung injury via network pharma- cology: Emphasis on inflammatory response, oxidative stress, and the PI3K-AKT pathway. Pharm Biol. 2024, 10: 2319117. Kaedbey R, Reece D, Venner CP, Mccurdy A, Su JD, Chu MC, Louzada M, Jimenez-Zepeda VH, Mian H, Song KV, Sebag M, Stakiw J, White D, Reiman A, Aslam M, Kotb R, Bergstrom D, Gul E, Leblanc R. Long-term follow-up of outcomes including progression-free survival 2 in patients with transplant-ineligible multiple myeloma in the real- world practice: A multi-institutional report from the Cana- dian myeloma research group (CMRG) database. Ejhaem 2024; 5: 474-84. Kaplan D. Regulatory T cells mediate the anti-inflammatory function of glucocorticoids. J Invest Dermatol. 2020; 140: 2098-98. Kudawara I, Ueda T, Yoshikawa H, Miyama T, Yamamoto T, Nishizawa Y. Inhibition of tumour growth by dexametha- sone in murine osteosarcomas. Eur J Cancer. 2001; 37: 1703- 08. Le LHM, Elqamoudi B, Colon N, Cramond A, Poly F, Ying L, Korolik V, Ferrero RL. Campylobacter jejuni extracellular vesicles harboring cytolethal distending toxin bind host cell glycans and induce cell cycle arrest in host cells. Microbiol Spectrum. 2024; 12: e0323223. Lei L, Zhang YC, Jian Q, Lei L, Lv N, Williamson RA, Chen P, Bangladesh J Pharmacol 2024; 19: 73-80 79 Author Info Xiufeng Gao，Wenhao Guo (Principal contact and contributed equally) e-mail: wenhaoguo2011@163.com Zhang D, Hu JS. Resistance of osteosarcoma cells to the proapoptotic effects of carfilzomib involves activation of mitogen activated protein kinase pathways. Exp Physiol. 2021; 106: 438-49. Li H, Chen HY, Li RL, Xin JJ, Wu SS, Lan J, Xue KY, Li X, Zuo CL, Jiang W, Zhu, L. Cucurbitacin I induces cancer cell death through the endoplasmic reticulum stress pathway. J Cell Biochem. 2019; 120: 2391-03. Li JP, Zhang F, Li HH, Peng F, Wang Z, Peng H, He JX, Li Y, He L, Wei LQ. Circ_0010220-mediated mir-503-5p/cdca4 axis contributes to osteosarcoma progression tumorigenesis. Gene 2020; 763: 145068. Liu Z, Tan RJ, Liu YH. The many faces of matrix metallo- proteinase-7 in kidney diseases. Biomolecules 2020; 10: 960. McMahon SB. Myc and the control of apoptosis. Csh Perspect Med. 2014; 4: a014407. Montalto FI, De Amicis F. Cyclin D1 in cancer: A molecular connection for cell cycle control, adhesion and invasion in tumor and stroma. Cells-Basel. 2020; 9: 9122648. Nomden M, Beljaars L, Verkade HJ, Hulscher JBF, Olinga P. Current concepts of biliary atresia and matrix metallo- proteinase-7: A review of literature. Front Med-Lausanne. 2020; 7: 221-31. Nusse R, Clevers H. Wnt/β-catenin signaling, disease, and emerging therapeutic modalities. Cell 2017; 169: 985-99. Pang JM, Huang YC, Sun SP, Pan YR, Shen CY, Kao MC, Wang RH, Wang LH, Lin KT. Effects of synthetic glucocor- ticoids on breast cancer progression. Steroids 2020; 164: 108738. Puckett Y, Gabbar A, Bokhari AA. Prednisone. San Fran -cisco, StatPearls, 2024. Shimba A, Ikuta K. Control of immunity by glucocorticoids in health and disease. Semin Immunopathol. 2020; 42: 669-80. Taves MD, Ashwell JD. Glucocorticoids in T cell develop- ment, differentiation and function. Nat Rev Immunol. 2021; 21: 233-43. Tchakarska G, Sola B. The double dealing of cyclin D1. Cell Cycle. 2020; 19: 163-78. Xu QX, He LM, Ma L, Fan L, Yan LH, Zhao XL, Li YY. Linc01410 accelerated the invasion and proliferation of osteosarcoma by sponging mir-3128. Aging-Us. 2020; 12: 24957-66. Yamamoto T, Nishiguchi M, Inoue N, Goto HG, Kudawara I, Ueda T, Yoshikawa H, Tanigaki Y, Nishizawa Y. Inhibition of murine osteosarcoma cell proliferation by glucocorticoid. Anticancer Res. 2002; 22: 4151-56. Yang RY, Gao WH, Wang ZB, Jian HY, Peng L, Yu XP, Xue PS, Peng W, Li KX, Zeng PH. Polyphyllin I induced ferroptosis to suppress the progression of hepatocellular carcinoma through activation of the mitochondrial dysfunc- tion via Nrf2/HO-1/GPX4 axis. Phytomedicine 2024; 122: 155135. Zhang LY, Chen Y, Jia J, Zhu X, He Y, Wu LM. Mir-27a promotes emt in ovarian cancer through active Wnt/β- catenin signalling by targeting foxo1. Cancer Biomark. 2019; 24: 31-42. Zhang RT, Shi HR, Ren F, Feng W, Cao Y, Li GL, Liu ZY, Ji PC, and Zhang MH. Microrna-338-3p suppresses ovarian cancer cells growth and metastasis: Implication of Wnt/catenin beta and Mek/ Erk signaling pathways (Retraction of vol 38, art No. 494, 2019). J Exp Clin Canc Res. 2024; 43: 168-82. Zhang Y, Li NJ, Li H, Chen MJ, Jiang W, Guo WH. Thiram, an inhibitor of 11β-hydroxysteroid dehydrogenase type 2, enhances the inhibitory effects of hydrocortisone in the treatment of osteosarcoma through Wnt/β-catenin pathway. BMC Pharmacol Toxicol. 2023; 24: 258-67. Zhao CX, He L, Li LJ, Deng FY, Zhang MH, Wang CH, Qiu JL, Gao QQ. Prenatal glucocorticoids exposure and adverse cardiovascular effects in offspring. Front Endocrinol. 2024; 15: 1430334. Zheng F, Zhong JM, Chen KL, Shi Y, Wang F, Wang SC, Tang S, Yuan XY, Shen ZJ, Tang SS, Xia DJ, Wu YH, Lu WG. PINK1-PTEN axis promotes metastasis and chemoresis- tance in ovarian cancer via non-canonical pathway. 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