Gambogic acid suppresses osteosarcoma progression through upregulation of FOXO3a

Abstract

Background

Osteosarcoma is a prevalent bone cancer in children and adolescents, posing significant treatment challenges due to its high metastatic potential and resistance to traditional chemotherapy. Gambogic Acid (GA), a natural compound, has demonstrated promising anticancer properties, including inhibition of cell proliferation and induction of apoptosis.

Methods

This study evaluated the anticancer effects of GA on osteosarcoma cell lines 143B and U2OS through cell viability assays, proliferation tests, wound healing assays, and flow cytometry to assess migration and apoptosis. RNA sequencing and RT-qPCR analyses were conducted to identify molecular mechanisms, with a focus on the tumor suppressor transcription factor FOXO3a. siRNA-mediated knockdown of FOXO3a was performed to determine its role in GA’s mechanism of action.

Results

GA treatment significantly reduced cell viability and proliferation in a dose- and time-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and CCK-8 assays. Wound healing assays revealed a marked inhibition of cell migration, while flow cytometry confirmed a significant increase in apoptosis rates following GA treatment. RNA sequencing identified FOXO3a as a key upregulated gene in both cell lines after GA exposure, which was validated by RT-qPCR. Importantly, FOXO3a knockdown diminished GA's effects on cell viability, migration, and apoptosis, underscoring its pivotal role in mediating GA's anticancer activity.

Conclusions

The findings suggest that GA exerts potent anticancer effects on osteosarcoma cells through the upregulation of FOXO3a, a critical tumor suppressor. These results provide a foundation for further exploration of GA as a novel therapeutic agent for osteosarcoma, potentially offering a safer alternative to conventional chemotherapy. Future research will aim to elucidate the detailed mechanisms underlying the interaction between GA and FOXO3a and to optimize GA-based therapeutic strategies.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-025-02776-w.

Introduction

Osteosarcoma is the most common malignant bone tumor in children and adolescents, presenting significant treatment challenges due to its high metastatic potential and resistance to conventional therapies [1, 2]. Current therapeutic approaches typically employ a multimodal strategy, including preoperative systemic chemotherapy, surgical intervention, and postoperative chemotherapy. Despite advancements in treatment modalities, survival rates have plateaued over the past decades, largely due to the aggressive nature of osteosarcoma and the development of resistance to standard chemotherapeutic agents [3, 4]. Furthermore, the severe side effects of traditional chemotherapy, such as myelosuppression, hearing loss, renal dysfunction, and gonadal toxicity, underscore the pressing need for new therapeutic strategies that are both effective and safer [5, 6].

Natural compounds have long been recognized for their therapeutic potential in treating various diseases, including cancer. These compounds, often derived from plants, possess diverse pharmacological properties, such as antiviral, anti-inflammatory, and antitumor effects [7]. Among them, flavonoids, polyphenols, and phytochemicals have gained particular attention for their ability to selectively target cancer cells while sparing normal tissues [8]. Gambogic Acid (GA), a flavonoid extracted from the resin of the Garcinia tree, has demonstrated significant anticancer activity in preclinical studies. Its mechanisms of action include inducing apoptosis, inhibiting proliferation, suppressing angiogenesis, and overcoming chemoresistance in various cancer types, including liver, gastric, and breast cancers [9–12]. Importantly, GA has shown low toxicity to normal tissues in animal models, further supporting its potential as a promising anticancer agent [13, 14]. However, despite its preclinical success, GA remains in the experimental stage, and no large-scale clinical trials have yet validated its efficacy and safety in humans.

FOXO3a, a key member of the Forkhead Box O (FOXO) transcription factor family, plays a pivotal role in regulating cellular processes critical for tumor suppression, including apoptosis, cell cycle arrest, and oxidative stress response [15–18]. Through the upregulation of pro-apoptotic genes (e.g., Bim, Puma) and cyclin-dependent kinase inhibitors (e.g., p21, p27), FOXO3a serves as a critical regulator of cell proliferation and survival [19, 20]. However, in many cancers, FOXO3a activity is downregulated due to mutations, epigenetic alterations, or oncogenic signaling pathways such as PI3 K/Akt, which lead to its nuclear exclusion and functional inactivation [21, 22]. This loss of FOXO3a activity is closely associated with cancer progression, therapy resistance, and poor prognosis. Consequently, reactivating FOXO3a has emerged as a promising therapeutic strategy to restore its tumor-suppressive functions [23, 24].

In osteosarcoma, FOXO3a plays a critical role in regulating apoptosis. Recent studies have shown that in cisplatin-resistant osteosarcoma sublines, FOXO3a expression is decreased, and autophagy is increased. Restoring FOXO3a expression has been found to enhance sensitivity to cisplatin by activating the intrinsic apoptosis pathway, suggesting that reactivating FOXO3a could be a potential therapeutic strategy for overcoming cisplatin resistance [25]. Natural compounds like Gambogic Acid have shown potential to reactivate FOXO3a and restore its regulatory functions. By inducing FOXO3a expression, GA could promote apoptosis and cell cycle arrest, offering a novel therapeutic approach for treating osteosarcoma.

In this study, we explored the anticancer effects of GA on osteosarcoma cells, with a particular focus on its ability to upregulate FOXO3a and modulate its downstream pathways. Using RNA sequencing (RNA-seq), we identified FOXO3a as a key gene differentially expressed in response to GA treatment. Functional assays demonstrated that GA effectively inhibited cell proliferation, migration, and induced apoptosis in osteosarcoma cells. Additionally, siRNA-mediated knockdown of FOXO3a significantly attenuated these effects, highlighting its central role in GA's mechanism of action. These findings suggest that GA may exert its anticancer effects by restoring FOXO3a activity, thereby disrupting key oncogenic pathways and reinstating tumor suppressive mechanisms.

Our study underscores the therapeutic potential of natural compounds like GA in the treatment of osteosarcoma. By elucidating the molecular mechanisms underlying GA’s effects, particularly its interaction with FOXO3a, we aim to provide a foundation for future development of GA-based therapeutic strategies for this aggressive cancer.

Methods

Cell culture and GA treatment protocol

Osteosarcoma cell lines 143B and U2OS were sourced from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 μg/ml streptomycin at 37 °C in a 5% CO₂ humidified atmosphere. Gambogic Acid (GA; Sigma-Aldrich, PHL80455, Darmstadt, Germany) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. For experiments, cells were treated with final concentrations of 0, 200, 400, and 600 nM GA, based on preliminary dose–response results. The chosen concentrations enabled us to assess both dose-dependent effects and cytotoxicity levels of GA, with cells treated for time points of 24, 48, and 72 h.

Transfection protocol

For FOXO3a knockdown, osteosarcoma cells were transfected with small interfering RNA (siRNA) targeting FOXO3a (GenePharma, Suzhou, China) or a scrambled control siRNA. Transfections were performed using Lipofectamine 3000 reagent (Invitrogen, L3000150, Californiam, USA), following the manufacturer’s protocol. Cells were seeded at 70–80% confluence and transfected with 50 nM siRNA using Lipofectamine 3000, at a reagent-to-siRNA ratio of 1:1 (µl reagent: pmol siRNA), in serum-free medium. The volume of Lipofectamine 3000 was calculated based on the final siRNA concentration and the manufacturer's protocol for optimal transfection efficiency. After 6 h, the transfection medium was replaced with DMEM containing 10% FBS, and cells were incubated for an additional 24 h before GA treatment or further analysis.

Sample size and power analysis

Each experiment was conducted with a minimum of three independent replicates per condition to ensure robust statistical analysis. Although a formal power analysis was not conducted, sample sizes were selected based on prior literature demonstrating that three or more replicates typically provide adequate power in in vitro studies. This approach ensured sufficient statistical sensitivity to detect significant effects of GA treatment on osteosarcoma cell behavior.

MTT assay

Osteosarcoma cells were seeded in 96-well plates at a density of 10,000 cells per well in 100 μl of culture medium and incubated overnight under standard conditions (37 °C, 5% CO₂). Cells were treated with GA for 24 h, 48 h, and 72 h, after which 20 μl of MTT solution (5 mg/ml in PBS) was added to each well and incubated at 37 °C for 4 h. The medium was then carefully aspirated, and 200 μl of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The plates were gently shaken for 10 min, and absorbance was measured at 570 nm and 630 nm using a PerkinElmer microplate reader. Results were calculated as a percentage of cell viability relative to untreated controls. Each condition was tested in triplicate, and experiments were repeated at least three times.

Cell proliferation assay

Cell proliferation was assessed using the CCK-8 assay kit (Transgen, FC101-01,Beijing, China). Osteosarcoma cells were seeded in 96-well plates at a density of 3000 cells per well in 100 μl of culture medium. The cells were cultured under standard conditions (37 °C, 5% CO₂) for 24, 48 and 72 h. At each specified time point, 10 μl of CCK-8 solution was added directly to each well, followed by gentle mixing. The plates were incubated at 37 °C for 3 h to allow for the enzymatic conversion of the reagent into a colored formazan product.

After incubation, the optical density (OD) of the resulting solution was measured at 450 nm using a microplate reader. The OD values, reflecting the number of viable cells, were normalized to the control group and used to evaluate cell proliferation rates. Each condition was tested in triplicate, and the experiment was performed independently at least three times to ensure accuracy and reproducibility.

Wound healing migration assays

Wound healing migration assays were conducted to assess the migration ability of osteosarcoma cells following GA treatment. Cells were seeded in six-well plates at a density of 500,000 cells per well and allowed to reach full confluence. A scratch was created down the center of each well using a sterile 10 μl pipette tip. The wells were washed with phosphate-buffered saline (PBS) to remove detached cells, and fresh medium with or without GA was added. Images of the wound area were captured immediately (0 h) and after 48 h using the Olympus IX73 inverted microscope (Olympus Corporation, Tokyo, Japan).Image analysis was performed using ImageJ software (National Institutes of Health, USA), following standard protocols for wound healing assays. The wound area was measured at each time point, and the percentage of wound closure was calculated using the formula:

$$\text{Wound}\text{Closure}\left(\%\right)=\left(\text{Width}\text{of}\text{Initial}\text{Gap}-\text{Width}\text{of}\text{Final}\text{Gap}\right)/\left(\text{Width}\text{of}\text{Initial}\text{Gap}\right)\times 100\%$$

This allowed for accurate quantification of cell migration and wound closure in response to GA treatment.

Annexin V-FITC staining and flow cytometry

Cell apoptosis was evaluated using the Annexin V-FITC/PI Apoptosis Detection Kit (Yeasen, 40305ES20, Shanghai, China), following the manufacturer's instructions. Osteosarcoma cells were pre-treated with Gambogic Acid for the specified duration and then harvested by centrifugation at 300g for 5 min at 4 °C. The cell pellets were washed once with cold PBS and resuspended in 100 μl of 1 × binding buffer to a final concentration of 1 × 10⁶ cells/ml.

Subsequently, 5 μl of Annexin V-FITC and 10 μl of propidium iodide (PI) staining solution were added to each sample. The cells were gently mixed and incubated for 10 min in the dark at room temperature to prevent light-induced degradation of the fluorophores. After incubation, an additional 400 μl of 1 × binding buffer was added to each sample before analysis.

Flow cytometry was conducted using a BD Biosciences flow cytometer, with fluorescence signals collected in the FITC (Annexin V) and PE (PI) channels. Data analysis distinguished early apoptotic (Annexin V⁺/PI⁻) and late apoptotic/necrotic cells (Annexin V⁺/PI⁺). All experiments were performed in triplicate, and representative data were presented to ensure reproducibility.

Quantitative real-time PCR

Quantitative real-time PCR (qRT-PCR) was conducted to measure the expression levels of FOXO3a in osteosarcoma cells following GA treatment. Total RNA was extracted using the RNAeasy™ RNA Isolation Kit (Beyotime Biotechnology, R0026, Shanghai, China), and cDNA synthesis was performed with the BeyoRT™ II cDNA Synthesis Kit (Beyotime Biotechnology, D7168M, Shanghai, China). FOXO3a expression was quantified using SYBR Green qPCR Master Mix (Takara, 640,022, Dalian, China) with GAPDH as the internal control. Primers used for FOXO3a were as follows: forward, 5'-CGGACAAACGGCTCACTCT-3'; reverse, 5'-GGACCCGCATGAATCGACTAT-3'. Primers used for GAPDH were as follows: forward, 5'-ACAACTTTGGTATCGTGGAAGG-3'; reverse, 5'-GCCATCACGCCACAGTTTC-3'. qRT-PCR analysis confirmed that GA treatment significantly upregulated FOXO3a expression.

RNA-Seq analysis

To explore GA’s effects on gene expression at a broader level, RNA sequencing (RNA-seq) analysis was performed on GA-treated osteosarcoma cells. Cells treated with GA for 48 h were harvested, and total RNA was extracted using the RNAeasy™ RNA Isolation Kit (Beyotime Biotechnology, R0026, Shanghai, China). RNA quality and integrity were assessed using an Agilent Bioanalyzer 2100. Library preparation was conducted with the Illumina TruSeq RNA Library Preparation Kit (Illumina, RS-122–2001, USA), and sequencing was performed on an Illumina platform, yielding paired-end reads.

Bioinformatic analysis was conducted to identify differentially expressed genes (DEGs) between GA-treated and control samples. Reads were mapped to the reference genome using HISAT2, and differential expression analysis was performed using DESeq2. Genes with an adjusted p-value < 0.05 and |log2(fold change) |> 1 were considered significantly differentially expressed. Among the DEGs, FOXO3a was identified as a significantly upregulated gene in both cell lines. This finding directed our focus on FOXO3a as a potential mediator of GA’s antitumor effects, further validated through qRT-PCR.

Statistical analysis

All data are presented as mean ± standard deviation (SD). Each experiment included biological replicates (n ≥ 3) and was performed with at least three technical repeats to ensure reproducibility. Statistical analyses were conducted using GraphPad Prism (version 8.0, GraphPad Software, San Diego, CA, USA) and SPSS (version 20.0, IBM, Chicago, IL, USA). For comparisons between two groups, we used either the two-sided Student's t-test or the two-tailed Mann–Whitney U-test, depending on data distribution. For multiple group comparisons, one-way analysis of variance (ANOVA) was performed, followed by Bonferroni post hoc tests for pairwise comparisons. A p-value < 0.05 was considered statistically significant. In figures, significance levels are denoted as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

Results

Gambogic Acid decreases osteosarcoma cell growth

The chemical structure of Gambogic Acid (GA) is shown in Fig. 1A, highlighting its potential as a natural anticancer compound. The concentrations of GA (0, 200, 400, and 600 nM) were selected based on previous literature reporting its cytotoxic effects in various cancer models [26], as well as preliminary dose–response experiments conducted in our study to identify effective yet non-lethal concentrations for osteosarcoma cells.

Fig. 1.

Impact of GA on the growth of osteosarcoma cells. A Chemical structure of GA. B, C Cell viability of U2OS and 143B osteosarcoma cell lines was measured using the MTT assay after 48 h of treatment with increasing concentrations of GA (0, 200, 400, and 600 nM). Data represent mean ± SD from three independent experiments, with statistical significance indicated as follows: *p < 0.05 and **p < 0.01, comparing each treatment group to the control. D, E Cell proliferation in U2OS and 143B cells was assessed using the CCK-8 assay at 24, 48, and 72 h following GA treatment with 600 nM GA. A significant reduction in proliferative capacity was observed in a dose- and time-dependent manner, as indicated by **p < 0.01. Error bars represent mean ± SD from three independent experiments, highlighting the inhibitory effects of GA on osteosarcoma cell growth

This study evaluated GA’s effects on cell viability and proliferation in osteosarcoma cell lines 143B and U2OS. The results demonstrated a dose-dependent reduction in cell viability, as shown in Fig. 1B, C and Supplementary Fig. 2 A–D, confirming the cytotoxic efficacy of GA over 24 h, 48 h, and 72 h. The CCK-8 assay, performed to measure cell proliferation over time, further validated GA's ability to significantly inhibit the proliferative capacity of these cells in a concentration- and time-dependent manner (Fig. 1D, E).

Notably, at 72 h, 400 nM GA exhibited stronger cytotoxic effects, while 200 nM GA showed moderate but significant inhibition of cell viability, supporting the potential of lower doses for clinical applications. As expected, 600 µM GA led to extensive cell death, highlighting the importance of optimizing GA concentration. These findings suggest that GA limits osteosarcoma cell growth, potentially through mechanisms involving cell cycle arrest or the induction of apoptosis. These findings suggest that GA limits osteosarcoma cell growth, potentially through mechanisms involving cell cycle arrest or the induction of apoptosis.

To assess the selective toxicity of GA, we also tested its effects on the human osteoblastic cell line hFOB 1.19, a normal bone cell line (Supplementary Fig. 2E). At 200 nM and 400 nM concentrations, GA exhibited minimal cytotoxicity toward hFOB 1.19 cells. However, at 600 µM, some cell death was observed, although the cytotoxicity was lower compared to the osteosarcoma cell lines. These results suggest that GA selectively targets osteosarcoma cells at therapeutic concentrations while exerting less toxicity on normal bone cells, thereby supporting its potential for clinical use.

Gambogic acid increases osteosarcoma cell apoptosis

Following the initial assessment of Gambogic Acid's (GA) anticancer efficacy, we extended our investigation to examine its impact on cancer cell migration, a key factor in the spread of cancer. Utilizing the osteosarcoma cell lines U2OS and 143B, we applied GA and conducted a wound healing assay to assess migration inhibition. The results unambiguously demonstrated GA's effectiveness in significantly reducing cell migration in both cell lines, aligning with our hypothesis (Fig. 2A–C). In addition, Supplementary Fig. 1 A, B shows that GA treatment significantly increased the protein level of E-cadherin, while markedly reducing MMP9 levels in both U2OS and 143B cell lines. These results suggest that GA may inhibit migration by promoting epithelial characteristics and suppressing extracellular matrix degradation.

Fig. 2.

Effect of GA on osteosarcoma cell apoptosis and migration. A–C Migration abilities of U2OS and 143B cells were evaluated using wound healing assays after treatment with 600 nM GA for 48 h. Representative images and quantitative analysis show a significant dose-dependent inhibition of wound closure, indicating reduced migratory capacity. Data represent mean ± SD from three independent experiments, with statistical significance indicated as follows: **p < 0.01 compared to the control group. D, E Apoptosis of U2OS and 143B cells was assessed by flow cytometry after 48-h GA treatment with 600 nM GA. Cells were stained with Annexin V-FITC and PI, allowing discrimination between live, early apoptotic, and late apoptotic/necrotic cells. Quantitative results show a significant, dose-dependent increase in apoptotic cell populations, confirming the pro-apoptotic effects of GA. Data represent mean ± SD from three independent experiments, with **p < 0.01 considered statistically significant

In addition to inhibiting osteosarcoma cell migration, GA may also promote apoptosis, another key mechanism in tumor suppression. To further investigate whether GA exerts its anticancer effects through apoptosis induction, we performed Annexin V-FITC/PI staining followed by flow cytometry analysis. GA treatment significantly increased apoptosis in both U2OS and 143B osteosarcoma cell lines, as assessed by Annexin V-FITC and PI staining. Flow cytometry analysis revealed a marked rise in the proportion of apoptotic cells following treatment with GA for 48 h compared to untreated control cells.

In Fig. 2D, representative scatter plots for U2OS and 143B cells illustrate the distribution of cells in different stages of apoptosis. The lower left quadrant represents live cells (Annexin V-negative, PI-negative), the lower right quadrant represents early apoptotic cells (Annexin V-positive, PI-negative), and the upper right quadrant represents late apoptotic or necrotic cells (Annexin V-positive, PI-positive). GA-treated cells displayed a significant increase in both early and late apoptotic populations compared to the control.

Figure 2E quantitatively summarizes these findings, showing the percentage of apoptotic cells in each group. GA treatment led to a nearly threefold increase in the apoptotic rate for both cell lines, with U2OS and 143B cells exhibiting approximately 15% apoptosis in the GA-treated group compared to less than 5% in controls. Statistical significance is denoted by **p < 0.01, indicating a highly significant increase in apoptosis for GA-treated cells relative to controls. Data are shown as mean ± SD from three independent experiments.

These results indicate that GA exerts a potent pro-apoptotic effect on osteosarcoma cells, which may contribute to its overall antitumor efficacy.

FOXO3a expression is upregulated by Gambogic Acid in osteosarcoma cells

To investigate the molecular mechanisms underlying Gambogic Acid’s (GA) anticancer effects, we performed RNA sequencing (RNA-seq) analysis on U2OS and 143B osteosarcoma cells treated with 600 nM GA for 48 h. Differentially expressed genes (DEGs) were identified using DESeq2, with thresholds set at adjusted p-value < 0.05 and |log2 fold-change|≥ 1. The volcano plots in Fig. 3A, B illustrate the distribution of DEGs in U2OS and 143B cell lines, respectively. In U2OS cells, 372 genes were upregulated and 13 genes were downregulated following GA treatment, while in 143B cells, 514 genes were upregulated and 6 genes were downregulated. Among these, 87 genes were commonly upregulated in both cell lines (Fig. 3C). Importantly, FOXO3a, a known tumor suppressor, was identified as one of the significantly upregulated genes in both cell lines.

Fig. 3.

Regulation of FOXO3a expression by GA in osteosarcoma cells. A, B Differential gene expression analysis was performed on U2OS and 143B cells using RNA sequencing after 48-h treatment with 600 nM GA. Red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent non-differentially expressed genes (Nodiff). Thresholds for differential expression were set at |log2 fold change|≥ 1 and adjusted p-value < 0.05. C Venn diagram showing the overlap of upregulated genes in both U2OS and 143B cells, identifying FOXO3a as a commonly upregulated gene following GA treatment. D, E Validation of 10 selected upregulated genes by RT-qPCR in U2OS and 143B cells, respectively. GA treatment significantly increased the expression levels of these genes compared to control, with FOXO3a showing the highest upregulation. Data represent mean ± SD from three independent experiments, with statistical significance indicated as follows: *p < 0.05, **p < 0.01 compared to the control group

To validate the RNA-seq findings, we selected 10 genes from the upregulated genes for further validation in both cell lines. RT-qPCR results demonstrated that these genes were significantly upregulated following GA treatment, among which FOXO3a exhibited the highest increase in expression (Fig. 3D, E). These findings further corroborate the RNA-seq results and highlight FOXO3a as a key mediator of GA’s anticancer effects.

Knockdown of FOXO3a promotes cell proliferation in GA-treated osteosarcoma cells

In our study aimed at understanding the mechanisms behind Gambogic Acid (GA) induced tumor suppression, we specifically targeted FOXO3a, a key player in cellular apoptosis and proliferation, using siRNA to knock down its expression in osteosarcoma cell lines U2OS and 143B. Following the silencing of FOXO3a, we treated these cell lines with GA at a concentration of 600 nM, known for its anticancer properties, to observe the compound's effect in the absence of this transcription factor. The knockdown efficiency was validated through RT-qPCR, showing a significant decrease in FOXO3a expression, depicted in Fig. 4A and Supplementary Fig. 3 A. Subsequent analyses of cell viability and proliferation through MTT and CCK-8 assays, respectively, revealed that the suppression of FOXO3a negated the GA-induced decrease in both cell viability and proliferation (Fig. 4B, C and Supplementary Fig. 3B, C). In addition, the MTT assay also revealed no significant differences in cell viability between the Control, DMSO, and Lipo groups, indicating that Lipofectamine 3000 did not impact cell viability under the experimental conditions tested (Supplementary Fig. 1 C). These results suggest that FOXO3a is a critical mediator of GA's antitumor effects, implicating the transcription factor's restoration as a potential strategy for enhancing the efficacy of GA and similar agents in osteosarcoma treatment.

Fig. 4.

Role of FOXO3a knockdown in enhancing GA-induced proliferation in osteosarcoma cells. A FOXO3a expression levels were quantified using RT-qPCR in U2OS cells following siRNA-mediated knockdown of FOXO3a and treatment with 600 nM GA for 48 h. Knockdown efficiency was confirmed, with significant reductions in FOXO3a expression observed. Data represent mean ± SD from three independent experiments, with **p < 0.01 compared to the control group. B Cell viability of U2OS cells was assessed using the MTT assay after FOXO3a knockdown and 48-h treatment with 600 nM GA. Results indicate that FOXO3a depletion attenuates the GA-induced reduction in cell viability. Data represent mean ± SD from three independent experiments, with *p < 0.05 compared to the control group. C Cell proliferation was evaluated using the CCK-8 assay in FOXO3a-depleted U2OS cells treated with 600 nM GA for 24, 48, and 72 h. Proliferation rates were significantly higher in FOXO3a-knockdown cells compared to controls, highlighting the critical role of FOXO3a in mediating GA’s antitumor effects. Data represent mean ± SD from three independent experiments, with **p < 0.01 compared to the control group

FOXO3a mediates the antitumor effects of Gambogic Acid in osteosarcoma cells

In our investigation into the effects of FOXO3a on the migratory and apoptotic behaviors of osteosarcoma cells, we conducted wound healing assays and flow cytometry analysis post siRNA-mediated FOXO3a knockdown, followed by treatment with Gambogic Acid (GA). Our findings provide compelling evidence of FOXO3a's pivotal role in regulating osteosarcoma cell dynamics. Specifically, the wound healing assays revealed that FOXO3a depletion nullified the migration-suppressing effect of GA, as osteosarcoma cells continued to migrate at rates comparable to untreated controls, showcased in Fig. 5A, B and Supplementary Fig. 3D, E. This indicates that FOXO3a is integral to GA's mechanism of impeding cell migration, suggesting that its expression levels directly influence osteosarcoma cells'ability to spread. Additionally, flow cytometry results further elucidated the role of FOXO3a in cell survival, demonstrating that its absence abrogated the apoptotic increase typically induced by GA treatment in osteosarcoma cells, as observed in Fig. 5C, D and Supplementary Fig. 3 F–G. This dual functionality of FOXO3a, influencing both cell migration and apoptosis, underlines its significance in cancer cell biology and GA's targeted anticancer effects. These findings underscore the importance of FOXO3a as a regulatory factor in osteosarcoma progression and its potential as a therapeutic target.

Fig. 5.

Contribution of FOXO3a to GA's antitumor effects in osteosarcoma cells. A, B Migration of U2OS cells was assessed using wound healing assays after FOXO3a knockdown and 48-h treatment with 600 nM GA. Representative images and quantitative analysis demonstrate that FOXO3a knockdown significantly attenuated the inhibitory effect of GA on cell migration, highlighting FOXO3a's role in regulating migratory capacity. Data are presented as mean ± SD from three independent experiments, with *p < 0.05 considered statistically significant. C, D Apoptosis of U2OS cells was analyzed by flow cytometry following FOXO3a knockdown and 48-h GA treatment. Annexin V-FITC/PI staining results indicate a marked reduction in GA-induced apoptosis rates in FOXO3a-depleted cells compared to controls, underscoring the critical role of FOXO3a in mediating GA's pro-apoptotic effects. Data are presented as mean ± SD from three independent experiments, with *p < 0.05 considered statistically significant.

Discussion

This study explored the potential application of Gambogic Acid (GA) as an anticancer agent, particularly focusing on its effects against osteosarcoma cell lines 143B and U2OS. The findings reveal that GA significantly inhibits osteosarcoma cell survival, suppresses proliferation, impedes migration, and promotes apoptosis. Mechanistically, FOXO3a upregulation is likely to play a significant role in mediating these antitumor effects. These results suggest that GA holds significant potential as a novel therapeutic agent for treating osteosarcoma, a malignancy that remains challenging to manage with current therapeutic strategies.

Osteosarcoma treatment has long relied on a combination of surgery and chemotherapy, yet survival rates for metastatic or recurrent cases remain dismal [5]. Resistance to conventional chemotherapeutics and severe treatment-related side effects, such as myelosuppression, nephrotoxicity, and neurotoxicity, further complicate patient outcomes [27]. These limitations underscore the urgent need for alternative therapies that are both effective and less toxic. Natural compounds, including flavonoids such as GA, have emerged as promising candidates due to their selective cytotoxicity, targeting tumor cells while sparing normal tissues [28]. GA, derived from the Garcinia tree, has demonstrated potent anticancer effects in preclinical models of various cancers, including liver, breast, and gastric cancers [10]. Despite these promising preclinical findings, GA remains in the experimental stage, with no clinical trials to date validating its efficacy or safety in human patients. This study advances the understanding of GA’s potential by elucidating its molecular mechanisms in osteosarcoma cells.

The concentration of 600 nM GA was selected based on preliminary dose–response experiments that demonstrated a significant inhibition of cell proliferation and induction of apoptosis. However, at this concentration, GA also induced notable cytotoxicity in vitro, which highlights the challenges of using such high concentrations. While it effectively inhibited cell proliferation and induced apoptosis, the associated cytotoxicity suggests the need for careful consideration of concentration in therapeutic applications. However, the applicability of this concentration in vivo is subject to limitations. Achieving such high concentrations in human tissues is often restricted by factors such as bioavailability, solubility, and rapid clearance. GA’s pharmacokinetic profile has been reported to include low water solubility and rapid elimination, which could limit its therapeutic efficacy [29–31]. To overcome these challenges, future research should explore advanced delivery systems, such as nanoparticle encapsulation or liposomal formulations, to enhance GA's solubility, stability, and tissue penetration. These approaches could potentially allow lower doses of GA to achieve therapeutic effects in vivo while minimizing systemic toxicity.

In addition to its effects on various apoptotic pathways, GA has been reported to act as an inhibitor of Bcl-2, an anti-apoptotic protein [32]. By inhibiting Bcl-2, GA promotes the activation of pro-apoptotic signaling, thereby enhancing apoptosis in cancer cells. This mechanism likely contributes to GA's pro-apoptotic effects observed in osteosarcoma cells, further supporting its potential as a therapeutic agent in cancer treatment.

FOXO3a emerged as a pivotal mediator of GA's antitumor effects in this study. GA treatment led to a significant upregulation of FOXO3a expression, which orchestrates various downstream pathways critical for tumor suppression. FOXO3a activation is known to induce cell cycle arrest through the upregulation of cyclin-dependent kinase inhibitors, such as p21 and p27, which impede the activity of cyclin-CDK complexes required for cell cycle progression [33]. This mechanism likely contributed to the reduced proliferation observed in osteosarcoma cells following GA treatment. Additionally, FOXO3a promotes apoptosis by activating pro-apoptotic genes such as BIM, PUMA, and FasL [34]. These factors are essential for initiating apoptotic pathways and disrupting mitochondrial integrity, resulting in programmed cell death. Furthermore, FOXO3a enhances oxidative stress responses by upregulating antioxidant enzymes, including catalase and MnSOD, which maintain reactive oxygen species (ROS) balance and mitigate oxidative damage [35]. This dual role of FOXO3a in regulating apoptosis and oxidative stress underscores its critical function in tumor suppression.

Interestingly, FOXO3a knockdown significantly attenuated the antitumor effects of GA, confirming its central role in mediating GA’s activity. This finding aligns with previous reports that FOXO3a is frequently inactivated in cancer cells due to mutations, epigenetic modifications, or oncogenic signaling pathways such as PI3 K/Akt. These pathways promote FOXO3a cytoplasmic sequestration, thereby impairing its tumor-suppressive functions. By reactivating FOXO3a, GA disrupts these oncogenic processes, offering a multifaceted approach to counteract tumor progression and therapy resistance.

The therapeutic implications of GA’s effects on FOXO3a extend beyond its activation of pro-apoptotic and anti-proliferative pathways. FOXO3a activation may also inhibit key oncogenic pathways, including Wnt/β-catenin, NF-κB, and mTOR, which are associated with tumor growth, metastasis, and chemoresistance [36]. By targeting these pathways, GA could potentially overcome the limitations of current treatments for osteosarcoma, providing a comprehensive strategy for managing this aggressive cancer. Additionally, FOXO3a activation enhances antioxidant defenses, which may reduce oxidative stress-induced DNA damage and mutagenesis, further supporting its role in maintaining genomic stability.

While these findings highlight GA’s therapeutic potential, several limitations must be acknowledged. First, the high in vitro concentration of GA required for cytotoxic effects may not be directly translatable to clinical applications. Future studies should investigate alternative delivery systems to improve GA's bioavailability and pharmacokinetics. Second, the absence of in vivo data limits our ability to evaluate GA’s therapeutic efficacy and safety under physiological conditions. Animal studies are necessary to determine its pharmacodynamic properties, systemic toxicity, and impact on tumor growth in osteosarcoma models. Third, the possibility of drug resistance to prolonged GA treatment warrants investigation to identify potential resistance mechanisms and develop combination therapies to mitigate this risk. Lastly, tumor heterogeneity, including variations in genetic mutations, metastatic potential, and cellular phenotypes, may influence responses to GA. For example, the differential genetic backgrounds of 143B and U2OS cells may account for variations in sensitivity to GA, suggesting that personalized treatment approaches may be required.

Our findings underscore the importance of targeting FOXO3a as a therapeutic strategy for osteosarcoma. FOXO3a’s ability to modulate multiple pathways critical for tumor growth and survival makes it a promising target for cancer therapies. GA’s ability to upregulate FOXO3a provides a foundation for developing novel treatments that exploit FOXO3a's tumor-suppressive functions. Moreover, combining GA with other therapies that modulate the PI3 K/Akt or mTOR pathways could enhance its efficacy and overcome resistance in osteosarcoma.

In conclusion, this study demonstrates that GA exerts significant antitumor effects on osteosarcoma cells by upregulating FOXO3a and modulating its downstream pathways. These findings provide valuable insights into the molecular mechanisms of GA’s action and support its potential as an alternative therapeutic agent for osteosarcoma. However, further studies are required to address the challenges of translating these findings into clinical applications. Future research should prioritize in vivo studies to evaluate GA’s pharmacokinetics, long-term safety, and therapeutic efficacy, as well as investigate additional molecular targets influenced by GA. By addressing these challenges, GA could be developed into a viable and effective option for osteosarcoma treatment, offering hope for improved patient outcomes in this challenging malignancy.

Author contributions

Study conception and design: Yawei Hu, Songqing Ye, Zengfeng Guo; draft manuscript preparation: Yawei Hu, Songqing Ye, Hao Zhang, Zengfeng Guo, Xiaohong Han; review and editing: Jiawen Wu; visualization: Jianhua Zhou; supervision: Yawei Hu, Jianhua Zhou. All authors reviewed the results and approved the final version of the manuscript.

Funding

This work was supported by grants from the Scientific Research Projects of Medical and Health Institutions of Longhua District, Shenzhen:2023041, and Shenzhen Science and Technology Program (JCYJ20230807145002004).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.