Graphical abstract [41]graphic file with name ga1.jpg [42]Open in a new tab Keywords: Demethylzeylasteral, Osteosarcoma, PI3K/AKT pathway, Autophagy Highlights * • DEM potently inhibits osteosarcoma cell proliferation via G2/M arrest and apoptosis induction. * • DEM suppresses metastasis by reversing EMT-related protein expression. * • DEM modulates autophagy and PI3K/AKT signaling pathways, as revealed by RNA sequencing. * • DEM activates autophagy through PI3K/AKT inhibition, accompanied by ROS elevation. * • DEM demonstrates robust anti-tumor efficacy in vivo with minimal toxicity. * • First evidence of DEM as a multi-target therapeutic candidate for osteosarcoma. Abstract Background Osteosarcoma (OS) remains a highly aggressive malignancy with limited treatment options, necessitating the discovery of novel therapeutic agents. Demethylzeylasteral (DEM), a compound previously shown to exert anti-tumor properties in several malignancies, has not been sufficiently explored for its potential in OS treatment. Purpose This study focused on the anti-tumor properties of DEM on OS cells as well as the potential mechanisms. Methods OS cell lines (MG63 and 143B) were exposed to varying concentrations of DEM, followed by assessment of diverse cell functions. RNA sequencing was implemented to identify the molecular pathways affected by DEM exposure. The mechanistic underpinnings of DEM’s action were also studied via a series of assays. Additionally, the therapeutic potential was validated utilizing xenograft models. Results DEM evidently repressed OS cell proliferation in a dose- and time-dependent fashion, arrested cells in G2/M phase, and facilitated apoptosis through the modulation of the BCL2/BAX ratio. Furthermore, DEM suppressed cell migration and invasion by reversing EMT-related protein expression. RNA sequencing revealed that DEM primarily affected autophagy-related pathways, particularly through the PI3K/AKT signaling. DEM treatment led to an elevation in ROS generation and enhanced autophagic activity, as demonstrated by elevated LC3B puncta formation and autophagy-related protein expression. In vivo, DEM effectively suppressed tumor growth while showing a favorable safety profile. Conclusion This study provides comprehensive evidence that DEM exerts potent anti-tumor properties in OS via the PI3K/AKT pathway, highlighting the significance of DEM as a therapeutic candidate for OS. 1. Introduction Osteosarcoma (OS) is an exceptionally aggressive and malignant bone tumor that predominantly affecting children and adolescents. This type of cancer is known for its high propensity for metastasis and recurrence[[43][1], [44][2], [45][3], [46][4]]. Despite considerable advancements in modern medical approaches to tumor treatment, the prognosis for OS patients remains challenging, especially in cases of late-stage metastasis[[47]5]. Conventional treatment modalities, including surgical resection, chemotherapy, and radiotherapy, have shown limited success in improving survival outcomes. These approaches are often hindered by challenges such as tumor resistance to drugs, severe adverse effects, and the inability to completely eradicate tumor cells. Hence, it is crucial to explore innovative therapeutic strategies that are more effective, less toxic, and capable of overcoming the limitations of current treatments[[48]6,[49]7]. Over the past several decades, natural product compounds have played an irreplaceable role in drug discovery, owing to their unique and complex chemical structures as well as their remarkable biological activities. These compounds have provided an invaluable library of molecules for combating human diseases[[50][8], [51][9], [52][10]]. Historically, natural compounds derived from plants, microorganisms, and marine organisms have been crucial sources for the development of drug discovery. [[53]11]Their structural diversity and distinct pharmacological mechanisms make them ideal candidates for new drug development[[54]12,[55]13], particularly in the fields of anticancer and cardiovascular therapies. Modern pharmaceutical research has consistently demonstrated that natural products offer significant advantages in drug development. These advantages include higher bioavailability and lower toxicity, attributes closely related to their evolution through natural selection[[56]14]. Furthermore, research data highlights that a substantial proportion of clinical drugs in use today are either directly derived from or inspired by natural products, underscoring their critical role in drug discovery and development[[57]11]. Demethylzeylasteral (DEM) is a pentacyclic triterpene compound derived from Tripterygium wilfordii Hook. f., garnering significant attention for its diverse pharmacological actions and broad therapeutic potential[[58]15]. As a natural product from traditional Chinese medicine, DEM demonstrates a broad spectrum of biological activities, including anticancer, anti-inflammatory, immunosuppressive, anti-fertility, antibacterial, and antiviral properties. In oncological studies, DEM has demonstrated potent antitumor activities against multiple malignancies, including glioma, breast cancer, hepatocellular carcinoma (HCC), gastric cancer, pancreatic cancer, colorectal cancer (CRC), and melanoma [[59]16]. Its anticancer mechanisms involve diverse pathways, including cell cycle arrest, apoptosis induction, autophagy regulation, and inhibition of tumor cell migration and metastasis[[60]17]. For instance, in CRC, DEM induces G0/G1 phase arrest and caspase-3-mediated apoptosis while enhancing chemosensitivity to 5-fluorouracil. In breast cancer models, it suppresses metastasis by modulating the TGF-β pathway and epithelial-mesenchymal transition (EMT)[[61]18]. Similarly, in glioma cells, it reverses EMT by upregulating E-cadherin and downregulating Vimentin and MYBL2[[62]19]. Beyond oncology, DEM’s immunosuppressive properties make it a promising candidate for treating immune-related disorders such as rheumatoid arthritis, systemic lupus erythematosus, and organ transplant rejection. It has shown significant efficacy in alleviating vascular inflammation and atherosclerosis by modulating immune cell activity and reducing lipid deposition in arterial walls[[63]20,[64]21]. Recent studies have also revealed DEM’s antiviral potential, particularly against SARS-CoV-2. By binding strongly to the viral spike protein receptor-binding domain (RBD) and the ACE2 receptor, DEM interferes with viral host cell invasion, suggesting its potential for treating emerging infectious diseases[[65]22]. Although DEM has demonstrated potent antitumor activities in diverse cancers, its specific effects and potential mechanisms in OS remain largely unexplored. Particularly, whether DEM can inhibit malignant progression of OS by modulating key pathways or metabolic processes warrants further investigation. Therefore, this study focuses on the antitumor effects of DEM on OS and its potential molecular mechanisms. We propose to conduct comprehensive in vitro and in vivo experiments to evaluate the modulatory effects of DEM on OS cell biological capabilities. Through RNA sequencing technology, we will analyze gene expression changes in OS cells following DEM treatment, explore its potential influence on tumor cell metabolism, and validate its antitumor efficacy and safety in xenograft models. 2. Materials and methods 2.1. Cell culture and drug treatment MG63 and 143B human osteosarcoma lines, sourced from the Cell Bank, Chinese Academy of Sciences (Shanghai, China), served as the experimental models. Standard propagation involved high-glucose DMEM (Gibco, Grand Island, NY, USA) as the basal medium, augmented with 10 % FBS (Gibco) and 1 % penicillin/streptomycin solution (Solarbio, Beijing, China). The cells were housed under standard conditions (37°C, 5 % CO2, humidified atmosphere). Demethylzeylasteral (DEM), the compound under investigation, was obtained from MedChemExpress (MCE, Shanghai, China). For experimental use, DEM was dissolved in DMSO (Solarbio, Beijing, China) to generate a 10 mM stock, which was then diluted in culture medium to final working concentrations (10, 20, or 40 μM). Vehicle control groups received DMSO at a final concentration maintained at ≤ 0.5 %. 2.2. Cell viability and proliferation assessment The impact of DEM on cellular survival was determined using the Cell Counting Kit-8 (CCK-8) assay (Yeasen, Shanghai, China). Cells were initially plated in 96-well formats (3 × 10^4 cells/well) and permitted to adhere overnight. Subsequently, treatments involving various DEM concentrations or vehicle control were applied for durations of 24, 48, or 72 h. Post-treatment, CCK-8 reagent (10 µL/well) was introduced, followed by a 1–2 h incubation at 37°C. A BioTek Instruments microplate reader was employed to capture absorbance readings at 450 nm. Survival rates were calculated relative to the vehicle-treated controls. GraphPad Prism (v9.0) facilitated the calculation of IC50 values. 2.3. Colony formation analysis To evaluate DEM's effect on sustained proliferative capacity, colony formation assays were conducted. Cells were sparsely seeded (500 cells/well) into 6-well plates and allowed to attach overnight. The medium was then replaced with formulations containing the designated DEM concentrations or vehicle. Cultures were maintained for 10–14 days, receiving fresh medium every 2–3 days, until visible colonies formed. Fixation of colonies was achieved using 4 % paraformaldehyde (Solarbio, 30 min, room temp), followed by staining with 0.1 % crystal violet (30 min). After thorough rinsing with water and air-drying, colonies were imaged and counted microscopically. The rate of colony formation was expressed as a percentage relative to the control group. 2.4. Cell migration and invasion assays Cellular migration and invasion assays utilized Transwell chambers featuring 8 µm pores (Corning, NY, USA). For migration assessment, cells (2 × 10^5) underwent 24-hour serum starvation prior to seeding into the upper chambers containing serum-free medium with varying DEM levels. Complete medium with 10 % FBS in the lower chamber acted as the migration stimulus. Invasion assays required the upper membranes to be pre-coated with Matrigel (BD Biosciences; 1:8 dilution in serum-free medium), polymerized for 2 h at 37°C before cell seeding. After a 24-hour incubation, non-migrated/non-invaded cells on the membrane's upper side were mechanically removed via cotton swabs. Cells successfully traversing to the lower membrane surface were fixed (4 % paraformaldehyde) and stained (0.1 % crystal violet). Five random fields per membrane were imaged (200 × magnification), and cell counts were performed using ImageJ (NIH, USA) to quantify migration/invasion. 2.5. Flow cytometric analysis Cell Cycle Profiling: DEM's influence on cell cycle distribution was investigated. Cells seeded in 6-well plates (3 × 10^5 cells/well) were exposed to DEM for 48 h. Post-treatment, cell fixation occurred in 70 % ice-cold ethanol (−20°C, overnight). Fixed cells were subsequently stained using a PI (50 µg/mL) and RNase A (100 µg/mL) solution (37°C, 30 min, dark). Cell cycle phase percentages were determined using a BD FACSCanto^TM II instrument, with subsequent analysis via FlowJo software. Apoptosis Measurement: Apoptosis levels were quantified following identical treatment protocols. The Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) was employed according to the provided instructions. In brief, harvested cells were washed, resuspended in 1 × binding buffer, and stained with Annexin V-FITC and PI (15 min, room temp, dark). The apoptotic cell fraction was measured by flow cytometry (BD FACSCanto^TM II), followed by data analysis in FlowJo software. 2.6. Immunoblotting Western blotting was performed to probe protein level changes. Cell disruption was achieved using RIPA buffer (Solarbio) containing protease/phosphatase inhibitors. Cleared lysates (12,000 rpm, 10 min, 4°C) were quantified for protein content using the BCA method (Thermo Fisher Scientific). Standardized protein amounts were subjected to SDS-PAGE separation and subsequently transferred onto PVDF membranes. Blocking utilized 5 % non-fat milk in TBST (1 h, room temp). Membranes underwent overnight incubation (4°C) with primary antibodies targeting: Cyclin B1(ImmunoWay, 1:1000), CDK2(ImmunoWay, 1:1000), BAX (ImmunoWay, 1:1000), BCL2(ImmunoWay, 1:1000), Vimentin (ImmunoWay, 1:1000), E-Cadherin, N-Cadherin (ImmunoWay, 1:1000); PI3K(CST, 1:1000), p-PI3K (CST, 1:1000); AKT (CST, 1:1000), p-AKT (Abcam, 1:1000); MEK (CST, 1:1000), p-MEK (Servicebio, 1:1000); ERK(CST, 1:1000), p-ERK(CST, 1:1000), β-actin (Proteintech, 1:10000). Following TBST washes, membranes were incubated (1 h, room temp) with HRP-linked secondary antibodies. Signal detection relied on enhanced chemiluminescence (Millipore), captured using a Bio-Rad ChemiDoc system. Densitometric analysis of bands was done with ImageJ, normalizing to β-actin signals. 2.7. RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis Gene expression analysis involved total RNA isolation using TRIzol (Invitrogen). Spectrophotometry (NanoDrop 2000) confirmed RNA purity and concentration, while integrity was verified by agarose gel electrophoresis. Reverse transcription of 1 µg total RNA into cDNA was accomplished using HiScript® II Q RT SuperMix (Vazyme, Nanjing, China). Quantitative PCR was executed on a LightCycler® 480 (Roche) with ChamQ™ SYBR® qPCR Master Mix (Vazyme). Each 20 µL reaction contained master mix (10 µL), forward/reverse primers (1 µL each), cDNA (1 µL), and nuclease-free water (8 µL). Thermal cycling involved 95°C for 30 s, then 40 cycles of (95 °C, 10 s; 60°C, 30 s). Relative transcript abundance was determined using the 2^-ΔΔCt methodology, with GAPDH serving as the reference gene. All primers were synthesized by Sangon Biotech (Shanghai, China), and their sequences are listed in [66]Table 1. Table 1. The Primers used in this study. MAP1LC3B-F GCCGCACCTTCGAACAAA MAP1LC3B-R TCGTTCTATTATCACCGGGATTTT ATG7-F CGTTGCCCACAGCATCATCTTC ATG7-R CACTGAGGTTCACCATCCTTGG BECN1-F CTGGACACTCAGCTCAACGTCA BECN1-R CTCTAGTGCCAGCTCCTTTAGC ULK1-F TACACAGCAAGGGCATCATTCACC ULK1-R CGGGCAAATCCAAAGTCAGCAATC ATG5-F TCTGGATGGGATTGCAAAATG ATG5-R TTTCTTCTGCAGGATATTCCATGA GAPDH-F CCTTCATTGACCTCAACTACATGG GAPDH-R CTCGCTCCTGGAAGATGGTG [67]Open in a new tab 2.8. RNA sequencing and bioinformatics analysis To profile genome-wide expression changes, MG63 cells received 48-hour treatment with either 25 µM DEM or DMSO. Extraction of total RNA followed the procedure outlined previously. Guangzhou Gene Denovo Biotechnology Co., Ltd., performed library preparation and sequencing using the Illumina NovaSeq 6000 platform. Differentially expressed genes (DEGs) were defined by |log2(fold change)| ≥1.5 coupled with a Q-value < 0.05. Bioinformatic interpretation included GO term enrichment and KEGG pathway mapping for the identified DEGs. GSEA was additionally performed to identify significantly enriched functional gene sets. 2.9. Immunofluorescence Immunofluorescence staining visualized protein localization. Cells cultured on appropriate coverslips were treated with DEM (0–40 µM) for 48 h. Post-treatment processing involved fixation (4 % PFA, 20 min), permeabilization (0.1 % Triton X-100, 10 min), and blocking (5 % BSA, 1 h) at ambient temperature. Overnight incubation (4°C) with anti-LC3B primary antibody (CST, 1:200) was followed by PBST washes and incubation (1 h, room temp, dark) with Alexa Fluor 488-conjugated secondary antibody (1:500). Nuclei were visualized using DAPI (5 min). A Leica SP8 confocal system was used for image capture. 2.10. Detection of cellular ROS levels Flow Cytometry: Cells plated in 6-wells (2 × 10^5/well) and treated for 48 h with DEM (0–40 µM) were collected. They were then loaded with 10 µM DCFH-DA (serum-free medium, 30 min, 37°C). After PBS washing, fluorescence was read on a BD FACSCalibur (488 nm excitation). Analysis used FlowJo software (triplicate assays). Microscopy: Visually, cells on coverslips treated identically were loaded with DCFH-DA (10 µM, 30 min, 37°C), washed (PBS), and immediately observed under fluorescence illumination (Ex/Em: 488/525 nm). Relative fluorescence quantification used ImageJ (triplicate experiments). 2.11. Animal studies and in vivo efficacy All procedures involving animals received ethical approval from Jinan University's Animal Ethics Committee and adhered to institutional guidelines. Male BALB/c-nu/nu mice (6–8 weeks, Vital River, Beijing) were group-housed and acclimatized for 7 days. Osteosarcoma xenografts were established by subcutaneous injection of 1 × 10^6 MG63 cells (suspended in 100 µL PBS) into the right axillary area. Once tumors became palpable and reached an average volume near 100 mm^3, mice were assigned randomly to treatment cohorts (n = 5 per cohort): vehicle (saline i.p.), DEM 5 mg/kg (i.p.), or DEM 10 mg/kg (i.p.). Treatments occurred daily for four weeks. Tumor size (length, width) was measured with calipers every seven days, and volume calculated [(length × width^2)/2]. Body weights were monitored concurrently. Upon study completion, mice were euthanized, tumors harvested, weighed, and processed for further examination. 2.12. Histological and immunological analysis of tumor tissues For histological and immunological analysis of tumor tissues following euthanasia, a portion of each tumor was fixed in 4 % paraformaldehyde and embedded in paraffin, while the remainder was snap-frozen and stored at −80 °C. Paraffin-embedded tissues were sectioned at 4 µm. Standard Hematoxylin and Eosin (H&E) staining was performed on designated sections to evaluate histopathological changes. For Immunohistochemistry (IHC), sections underwent deparaffinization, rehydration, antigen retrieval, quenching of endogenous peroxidase, and blocking before incubation with an anti-Ki67 primary antibody ([68]Proteintech 1:400), followed by HRP-linked secondary antibody and DAB visualization, with hematoxylin counterstaining, to assess cell proliferation. For Immunofluorescence (IF), sections were similarly processed, including permeabilization, and incubated with an anti-LC3B primary antibody ([69]Proteintech 1:300) followed by an Alexa Fluor 488-conjugated secondary antibody and DAPI counterstaining; images were captured via confocal microscopy to evaluate autophagy. For Western Blotting, frozen tissues were homogenized in RIPA buffer, and total protein was extracted to detect the expression and phosphorylation levels of PI3K, p-PI3K, AKT, and p-AKT via BCA quantification, SDS-PAGE, PVDF transfer, incubation with primary and HRP-linked secondary antibodies, and Enhanced Chemiluminescence (ECL) detection. 2.13. Statistical analysis Quantitative data, derived from a minimum of three independent biological replicates, are expressed as mean ± SD. GraphPad Prism 9.0 handled all statistical computations. Direct comparisons between two groups employed the unpaired Student's t-test. Multi-group comparisons utilized one-way ANOVA followed by Bonferroni's correction. A p-value less than 0.05 was deemed statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). 3. Results 3.1. DEM exhibits potent anti-proliferative effects on OS cells The chemical structure of DEM is illustrated in [70]Fig. 1A. To investigate the anti-tumor potential of DEM on OS cells, we first evaluated its cytotoxicity using CCK8 assays. Both MG63 and 143B cells were exposed to varying concentrations of DEM for 24, 48, and 72 h. The results demonstrated that DEM distinctly inhibited cell viability in a dose-and time-dependent fashion ([71]Fig. 1B-C). The IC[50] values were calculated and are summarized in [72]Fig. 1D, further highlighting its potent cytotoxic effects. To further validate the anti-proliferative properties of DEM, colony formation assays were implemented and revealed that DEM treatment (10, 20, and 40 μm) markedly suppressed colony formation in both cell lines relative to the DMSO control group, with a clear dose-dependent reduction in colony numbers ([73]Fig. 1E-H). Fig. 1. [74]Fig. 1 [75]Open in a new tab Demethylzeylasteral (DEM) inhibits osteosarcoma cell proliferation and colony formation in a dose- and time-dependent manner. (A) Chemical structure of Demethylzeylasteral. (B, C) Viability of MG63 and 143B osteosarcoma cells treated with varying concentrations of DEM (0, 10, 20, and 40 uM) for 24, 48, and 72 h, assessed using a CCK8 assay. (D) Summary of IC[50] values calculated for MG63 and 143B cells at 24, 48, and 72 h. (E-H) Colony formation assays revealing a significant suppression of colony-forming ability in MG63 and 143B cells following DEM treatment at different concentrations (0, 10, 20, and 40 uM) in comparison to DMSO controls. *p < 0.05, **p < 0.01, ***p < 0.001. DEM, Demethylzeylasteral. 3.2. DEM induces cell cycle arrest and apoptosis in OS cells To investigate the mechanisms underlying the anti-tumor effects of DEM, we probed into its potential functions in cell cycle progression and apoptosis. Flow cytometry analysis revealed that DEM treatment caused significant cell cycle arrest in both MG63 and 143B cells ([76]Fig. 2A). A quantitative analysis demonstrated a dose-dependent accumulation of cells in G2/M phase following DEM treatment ([77]Fig. 2B). Moreover, immunoblotting confirmed that DEM treatment resulted in a dose-dependent reduction in CDK2 and Cyclin B1 expression ([78]Fig. 2C), with densitometric analysis confirming the statistical significance of these changes ([79]Fig. 2D). Fig. 2. [80]Fig. 2 [81]Open in a new tab DEM induces G2/M phase cell cycle arrest and promotes apoptosis in osteosarcoma cells. (A, B) Flow cytometric analysis demonstrating cell cycle alterations in MG63 and 143B cells after treatment with DMSO or DEM (0,10, 20, and 40 uM) for 48 h. (C, D) Western blot analysis showing the expression levels of cell cycle regulators (CDK2 and Cyclin B1) in MG63 and 143B cells following DEM treatment for 48 h. (E, F) Flow cytometric assessment of apoptosis in cells treated with DMSO or DEM for 48 h using Annexin V/PI staining. (G, H) Western blot analysis for apoptosis-related proteins (BCL2 and BAX), confirming the induction of apoptosis by DEM. *p < 0.05, **p < 0.01, ***p < 0.001. Furthermore, flow cytometry utilizing Annexin V/PI staining revealed a notable increase in apoptotic cell populations following DEM treatment ([82]Fig. 2E). Statistical analysis confirmed that apoptosis induction was dose-dependent ([83]Fig. 2F). Further examination of the expression of key apoptosis-related proteins utilizing immunoblotting revealed that DEM treatment led to a marked decrease in the BCL2 expression (anti-apoptotic protein), while increasing the levels of BAX (pro-apoptotic protein) ([84]Fig. 2G). Quantitative analysis of protein expression confirmed these changes occurred in a dose-dependent fashion ([85]Fig. 2H). 3.3. DEM suppresses malignant properties of OS cells To further explore the anti-metastatic potential of DEM in OS, we evaluated its impact on cell migration and invasion capabilities. Transwell assays demonstrated that DEM treatment notably reduced both migration and invasion in the 143B and MG63 cells in a concentration-dependent fashion ([86]Fig. 3A, 3C). Quantitative analysis revealed a marked reduction in the number of migrated and invaded cells following DEM treatment, with significant differences confirmed ([87]Fig. 3B, 3D). Fig. 3. [88]Fig. 3 [89]Open in a new tab DEM suppresses migration, invasion, and epithelial-mesenchymal transition (EMT) in osteosarcoma cells. (A, B) Transwell migration assays for assessing migration of 143B and MG63 cells treated with DMSO or DEM (0, 10, 20, and 40 uM) for 48 h. (C, D) Transwell invasion assays demonstrating decreased invasion potential of DEM-treated cells compared to controls. (E-H) Western blot analysis of EMT-related markers in 143B and MG63 cells, showing downregulation of mesenchymal markers (Vimentin and N-cadherin) and upregulation of the epithelial marker (E-cadherin) upon DEM treatment. *p < 0.05, **p < 0.01, ***p < 0.001. EMT, epithelial-mesenchymal transition. Since EMT is a critical process in cancer metastasis, we next examined whether DEM influenced its related proteins. Immunoblotting validated that DEM treatment led to significant alterations in the levels of EMT markers in both cell cells. Specifically, DEM diminished the expression of mesenchymal markers (Vimentin and N-cadherin) while increasing that of the epithelial marker E-cadherin ([90]Fig. 3E, 3G). Densitometric analysis confirmed that these changes were dose-dependent ([91]Fig. 3F, 3H). 3.4. DEM induces transcriptomic changes and pathway enrichment in OS cells To systematically investigate the molecular mechanisms underlying the anti-tumor properties of DEM, we performed RNA sequencing on MG63 cells treated with DEM (25 μM) for 48 h. The transcriptome analysis revealed significant changes in gene expression profiles, with 1048 genes upregulated and 285 genes downregulated (|log2(fold change)| ≥ 1 and Q value < 0.01; [92]Fig. 4A-C). Pathway enrichment analysis using the KEGG demonstrated that DEGs were predominantly involved in autophagy-related pathways, particularly those associated with the PI3K/AKT signaling cascade and cellular autophagy regulation ([93]Fig. 4D-F). These findings were further validated through GSEA, which confirmed significant enrichment of autophagy-associated gene sets ([94]Fig. 4F). Subsequently, we verified five upregulated autophagy-related genes (ATG5, BECN1, MAP1LC3B, ATG7, and ULK1) utilizing RT-qPCR, and the results were consistent with the RNA sequencing data ([95]Fig. 4G). Fig. 4. [96]Fig. 4 [97]Open in a new tab DEM modulates gene expression in osteosarcoma cells, affecting autophagy and the PI3K/AKT signaling. (A) Overview of differentially expressed genes in MG63 cells after 48-hour exposure to 25 uM DEM, with 723 genes upregulated and 1,356 downregulated (|log2(fold change)| ≥ 1 and Q value < 0.01). (B) Volcano plot illustrating the differentially expressed genes from RNA sequencing analysis. (C) Heatmap showing clustering of the differentially expressed genes. (D, E) KEGG pathway analysis revealing significant enrichment in autophagy-related and PI3K/AKT signaling. (F) Gene set enrichment analysis (GSEA) confirming the activation of autophagy-associated pathways. (G) RT-qPCR validation of five upregulated autophagy-related genes (ATG5, BECN1, MAP1LC3B, ATG7, and ULK1). **p < 0.01, ***p < 0.001. 3.5. DEM promotes autophagy in OS cells through the PI3K/AKT pathway Transcriptome analysis revealed that DEM notably affected autophagy-related genes ([98]Fig. 4B), and GSEA further confirmed the enrichment of DEM-regulated DEGs in autophagy-related pathways ([99]Fig. 4F). To validate these findings, we first evaluated oxidative stress levels, which are often linked to autophagy induction. Flow cytometry analysis revealed that DEM treatment evidently elevated ROS levels in OS cells in a dose-dependent fashion ([100]Fig. 5A, B). Immunofluorescence staining demonstrated a marked increase in LC3B puncta formation following DEM treatment, indicating the induction of autophagosome formation ([101]Fig. 5C). These observations were further supported by Immunoblotting ([102]Fig. 5D), which demonstrated substantial upregulation of key autophagy markers, including LC3B-II conversion, as well as dencreased expression of p62. Transcriptomic data also suggested that DEM’s effect could be mediated through the MAPK and PI3K/AKT pathways. To verify this, we used immunoblotting to investigate the involvement of these pathways. As depicted in ([103]Fig. 5D, G), DEM predominantly suppressed the PI3K/AKT pathway, rather than the MAPK pathway. Therefore, DEM appears exert its anti-tumor effects by facilitating autophagy through modulation of the PI3K/AKT signaling cascade. Fig. 5. [104]Fig. 5 [105]Open in a new tab DEM promotes autophagy in osteosarcoma cells through the PI3K/AKT pathway. (A, B) Flow cytometric analysis of ROS levels in osteosarcoma cells treated with increasing concentrations of DEM. (C) Immunofluorescence imaging showing enhanced LC3B puncta formation in DEM-treated cells. (D-G) Western blot analysis demonstrating increases in autophagy markers (LC3B-II conversion, decreased p62 expression) and suppression of PI3K/AKT signaling (p-PI3K and p-AKT) in DEM-treated cells. *p < 0.05, **p < 0.01, ***p < 0.001. 3.6. DEM suppresses OS growth in xenograft models To validate the therapeutic potential of DEM in vivo, the xenograft models were established using MG63 cells, followed by a systematic drug administration protocol. The experimental results demonstrated that DEM treatment effectively inhibited tumor development, with treated animals exhibiting reducted tumor volumes relative to the controls ([106]Fig. 6A, C). Continuous monitoring revealed slower tumor growth and lower final tumor weights in DEM-treated mice ([107]Fig. 6B), further confirming its anti-tumor efficacy. Importantly, throughout the treatment period, the DEM-treated mice maintained stable body weights, suggesting minimal systemic toxicity ([108]Fig. 6D). Fig. 6. [109]Fig. 6 [110]Open in a new tab In vivo efficacy of DEM in osteosarcoma xenograft models. (A) Representative tumor images showing the effect of DEM treatment on MG63 xenograft mouse models. (B) Analysis of tumor growth rates and final tumor weights, demonstrating the anti-tumor effects of DEM. (C) Tumor volume measurements showing significant reductions in the DEM-treated group compared to the vehicle control.(D) Monitoring of body weight throughout the treatment period, indicating that DEM treatment causes minimal systemic toxicity. (E) Representative images of Hematoxylin and Eosin (HE) staining (top) and Ki67 immunohistochemistry (IHC) (bottom) of tumor tissues. (F) Representative images of LC3-B immunofluorescence (IF) staining (Green: LC3-B; Blue: DAPI) in tumor tissues, showing autophagy levels. (G) Western blot analysis of p-PI3K, PI3K, p-AKT, and AKT expression in tumor tissues from each group. (H) Schematic diagram illustrating the proposed molecular mechanism of DEM in osteosarcoma cells. Data are presented as mean ± SD (n = 5 per group). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the DMSO control group. (For interpretation of the references to colour in this figure legend, the