Anti-ovarian cancer effects of forsythiaside A: insights from in vitro and in vivo studies

Introduction

Ovarian cancer is a highly lethal gynecological malignancy with a progressively increasing global incidence, ranking as the eighth most common cancer in women worldwide and accounting for an estimated 3.7% of all cancer cases and 4.7% of cancer-related deaths (1,2). Epithelial carcinoma is the predominant form, constituting approximately 80% of ovarian malignancies (3,4). Despite an overall 5-year survival rate of 46%, prognosis is critically dependent on the stage at diagnosis: the 5-year relative survival rate is 92% for early-stage disease but drops sharply to 29% for advanced disease (5,6). Compounding this challenge, the often asymptomatic nature of ovarian cancer means that significant majority of patients (approximately 58–75%) are diagnosed at an advanced stage (III or IV), where 5-year survival rates are starkly low at 27% for stage III and 13% for stage IV (7,8). The high mortality in these advanced stages is also attributable to the development of resistance to standard therapies (9). Given these significant challenges, exploring new therapeutic candidates is essential, with natural products representing a particularly promising area of interest (10-12).

Forsythiaside A (FSA, Figure 1A) is a key bioactive compound isolated from Forsythia suspensa (Thunb.) Vahl (Lianqiao), a plant widely used in traditional Chinese medicine for its diverse pharmacological properties (13,14). With the chemical formula C₂₉H₃₆O₁₅ and a molecular mass of 624.59, FSA has demonstrated anti-inflammatory, antibacterial, antioxidant, antiviral, and antipyretic effects. Mechanistic studies indicate that FSA can modulate key signaling pathways, such as PPAR-γ/RXR-α, TLR4/MAPK/NF-κB, and MLCK/MLC2, thereby suppressing inflammation in animal models (15). FSA has also shown protective effects in models of brain injury associated with severe acute pancreatitis, and by activating Nrf2, it protects against bile duct ligation-induced liver fibrosis (16). Furthermore, it has been found to mitigate pulmonary fibrosis and ischemia-reperfusion injury through the modulation of oxidative stress and related pathways (17,18).

Figure 1 Anti-proliferative activity of FSA against SK-OV-3 and OVCAR-3 cells. The viability of cells treated with FSA for 24 or 48 hours was measured by CCK-8 assay. (A) Chemical structure of forsythoside A (CAS: 79916-77-1). (B) Dose-response curves illustrating the effect of FSA on SK-OV-3 cell viability after 24 and 48 hours of treatment. (C) Dose-response curves depicting the viability of OVCAR-3 cells following 24 and 48 hours of FSA treatment. Data are shown as mean ± standard deviation. Number of replicates: n=6. CAS, Chemical Abstracts Service; CCK-8, Cell Counting Kit-8; FSA, forsythiaside A.

Beyond these broad activities, FSA exhibits notable anti-tumor effects in various cancers. It has been shown to reduce tumor growth in esophageal squamous cell carcinoma by influencing BCL2, BAX, and p21 expression (19). FSA can also target CD44 to alleviate liver fibrosis by regulating NLRP3-mediated pyroptosis (20,21). Notably, FSA inhibits the KLRB1-CLEC2D immune checkpoint, a pathway relevant to tumor progression and immune evasion (22). Despite these promising anti-cancer effects in other contexts, the potential of FSA against ovarian cancer remains largely unexplored. Therefore, this study aims to investigate the inhibitory effects of FSA on human ovarian cancer cells in vitro and in vivo, providing a data-driven rationale for its potential as a novel therapeutic agent for this challenging disease. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-980/rc).

Methods

Reagents and cell culture

FSA (Cat No. SM1030-100mg) was purchased from Beyotime Biotechnology (Shanghai, China) and stored at −20 ℃. SK-OV-3 cells (Cat No. CL-0215) were cultured in McCoy’s 5A medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). OVCAR3 cells (Cat No. CL-0178) were cultured in RPMI-1640 medium containing 20% FBS and 1% PS. Cells were maintained at 37 ℃ in a humidified atmosphere with 5% CO₂. FBS (Cat No. 164210), PS (Cat No. PB180120), McCoy’s 5A (Cat No. PM150710), and RPMI-1640 (Cat No. PM150110) were obtained from Wuhan Procell Life Science & Technology (Wuhan, China).

Cell proliferation assay

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. SK-OV-3 and OVCAR3 cells (3×10⁴ cells/well) were seeded into 96-well plates and treated with various concentrations of FSA in their respective culture media for 24 and 48 hours. Medium was then discarded, and cells were incubated with 100 µL of medium containing 10% CCK-8 solution (Cat No. CA1210; Solarbio, Beijing, China) for 1.5–2.0 hours. Absorbance was measured at 450 nm using a microplate reader. Half maximal inhibitory concentration (IC₅₀) values were calculated using GraphPad Prism 9.5 software.

Cell cycle analysis

SK-OV-3 and OVCAR3 cells were seeded into 6-well plates at 2×10⁵ cells/mL. Cells were treated with FSA at their respective IC₅₀ concentrations (43.88 µM for SK-OV-3, 108.6 µM for OVCAR3) or dimethyl sulfoxide (DMSO) vehicle for 48 hours. Cells were harvested by trypsinization, fixed in 70% ice-cold ethanol overnight at −20 ℃, washed with phosphate buffered saline (PBS), and stained with 2 µL RNase A (1 mg/mL) (Cat No. R1030; Solarbio, Beijing, China) and 50 µL propidium iodide (PI) (100 µg/mL) (Cat No. C0080; Solarbio, Beijing, China) in the dark for 20 minutes. Cell cycle distribution was analyzed by flow cytometry (BD Biosciences, San Jose, USA), and the percentage of cells in each phase (G1, S, G2/M) was quantified using FlowJo_V10 software.

Cell apoptosis and necrosis detection

Cell apoptosis and necrosis were detected using the YO-PRO-1 iodide (YO-PRO-1)/PI cell apoptosis and necrosis detection kit (Cat No. C1075S; Beyotime Biotechnology). SK-OV-3 and OVCAR3 cells were seeded in 6-well plates and incubated for 24 hours at 37 ℃ in a humidified 5% CO₂ atmosphere. The SK-OV-3 cells were treated with FSA at 61.87 µM (IC₅₀), and the OVCAR3 cells were treated with FSA at 141.6 µM (IC₅₀). Control cells received only the vehicle. Following incubation, the medium was aspirated, and the cells were washed once with PBS. The cells were then incubated with 1 mL of YO-PRO-1/PI staining solution per well at 37 ℃ in the dark for 20 minutes. Fluorescence staining was observed using a fluorescence microscope.

Cell migration assay

Migration capacity was evaluated using a wound-healing assay. SK-OV-3 and OVCAR3 cells were cultured in 6-well plates until achieving confluence. A linear scratch wound was generated in the cell monolayer using a 200 µL pipette tip. Detached cells were removed by washing the monolayers two to three times with PBS. Subsequently, cells were cultured in serum-free McCoy’s 5A (for SK-OV-3) or RPMI-1640 (for OVCAR3) medium. Monitoring and photographic documentation of cell migration occurred at various time intervals. Measurement of the wound area was conducted using ImageJ software. The migratory rate was calculated using the following formula: migration rate (%) = (initial wound width − wound width at the end of experiment)/initial wound width ×100%.

Cell invasion assay

Cell invasion was assessed using a Transwell assay. Transwell inserts (8-µm pores) were coated with Matrigel (Cat No. 356234; Solarbio, Beijing, China) and incubated at 37 ℃ for 1–2 hours. Serum-free medium was added to the upper chamber for equilibration (30 min). Cells (1.5×10⁵) in 100–200 µL serum-free medium were seeded in the upper chamber. The lower chamber contained 650 µL medium with 20% FBS. Cells were incubated at 37 ℃ for 24–48 hours. Non-invading cells on the upper membrane were removed. Invaded cells on the lower surface were fixed in methanol or paraformaldehyde (15 min), stained with 0.1% crystal violet (30 min) (Cat No. Y268091; Beyotime Biotechnology), and washed. Membranes were air-dried. Invaded cells were visualized and photographed under an inverted microscope (random fields). For quantification, cells were destained in anhydrous ethanol, and absorbance was measured at 595 nm using a microplate reader. Data analysis was performed using GraphPad Prism 9.5.

Western blot analysis

Total protein was extracted from SK-OV-3 and OVCAR3 cells, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked (Epizyme, Cat. No. PS108, Shanghai, China) for 15 min, then incubated overnight at 4 ℃ with primary antibodies: BCL2-associated X protein (BAX) (Cat. No. 50599-2-Ig), Caspase-3 (CASP3) (Cat. No. 19677-1-AP), beta-actin (β-actin) (Cat. No. 81115-1-RR), BH3 interacting domain death agonist (BID) (Cat. No. 10988-1-AP), and Cytochrome C (CYCS) (Cat. No. 10993-1-AP) from Proteintech (Wuhan, China), and B-cell lymphoma 2 (BCL2) (Cat. No. bs-0032R; Bioss, Beijing, China). After three washes with 1× Tris-Buffered Saline with Tween 20 (TBST), membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (Cat. No. bs-0295G-HRP; Bioss) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (SQ101; Epizyme) after washing and quantified with ImageJ. β-actin was the loading control.

RNA sequencing and analysis

SK-OV-3 cells were cultured in McCoy’s 5A medium with 10% FBS and 1% PS. At 70–80% confluence, cells were treated with FSA (IC₅₀) or DMSO control for 48 h. Cells were collected, washed, detached, and centrifuged (500 ×g, 5 min, 4 ℃). Cell pellets were lysed in 1 mL TRIzol (≤10⁷ cells) (Cat No. R0016; Beyotime Biotechnology) and stored at −80 ℃ before shipment to Qingke Biotechnology Co., Ltd. (Beijing, China) for RNA sequencing. Data was processed for gene expression quantification. Differentially expressed genes (DEGs) were identified using DESeq2 [adjusted P<0.01, |log₂ fold change (FC)| >1]. Pathway enrichment analysis used Qingke Biotechnology’s platform.

Animal experiments

A protocol was prepared before the study without registration. Six-week-old male BALB/c nude mice (19–21 g) from Sipeifu Biotechnology Co., Ltd. (Beijing, China) were used to establish subcutaneous xenograft models. SK-OV-3 cells (5×10⁶) were injected subcutaneously into the right axilla. When tumors reached ~50 mm³, mice were randomized into two groups (n=5/group): FSA (50 mg/kg) and control (PBS with 5% DMSO). FSA (50 mg/kg in 200 µL vehicle) was given daily by gavage; control mice received vehicle (200 µL) daily. Tumor size was measured with calipers, and volume was calculated: volume = (length × width²)/2. When tumors reached size endpoint, mice were euthanized, and tumors were excised, weighed, measured, and photographed.

Statistical analysis

Data were presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was performed using GraphPad Prism 9.5. Differences were analyzed using one-way analysis of variance (ANOVA) or Student’s t-test. P<0.05 was considered statistically significant.

Ethical statement

Animal experiments were performed under a project license (No. PB20250418003) granted by the Science and Technology Ethics Committee of Tarim University, in compliance with the institutional animal welfare guidelines of Tarim University.

Results

Anti-proliferative activity of FSA against human ovarian cancer cells SK-OV-3 and OVCAR-3 in vitro

The anti-proliferative effects of FSA on human ovarian cancer cell lines SK-OV-3 and OVCAR-3 were assessed via CCK-8 assays to determine IC₅₀ values. SK-OV-3 and OVCAR-3 cells were treated with graded concentrations of FSA for 24 and 48 hours. A dose- and time-dependent decrease in the count of viable SK-OV-3 and OVCAR-3 cells was evident. For the 24-hour treatment period, IC₅₀ values were calculated as 61.87 µM for SK-OV-3 cells and 141.6 µM for OVCAR-3 cells. At 48 hours post-treatment, the IC₅₀ values were lower, reaching 43.8 µM for SK-OV-3 and 108.6 µM for OVCAR-3 (refer to Figure 1B,1C). These data demonstrate FSA’s capability to effectively inhibit the in vitro proliferation of both SK-OV-3 and OVCAR-3 cell lines.

G1 phase cell cycle arrest in SK-OV-3 and OVCAR-3 cells induced by FSA

Analysis by flow cytometry indicated that FSA treatment resulted in G1 phase cell cycle arrest in both SK-OV-3 and OVCAR-3 cell lines (Figure 2). In SK-OV-3 cells, the G1 phase percentage increased significantly from a baseline of 63.97% to 75.45% upon treatment (P<0.01). This corresponded with a decline in the S phase population (from 25.2% to 21.21%) and the G2 phase population (from 10.83% to 3.34%) (Figure 2A,2B). OVCAR-3 cells displayed a comparable effect; the G1 population significantly expanded from 73.03% to 94.63% (P<0.01), while percentages in S phase decreased from 23.01% to 4.07%, and G2 phase from 3.96% to 1.30% after FSA treatment (Figure 2C,2D). These results suggest that FSA effectively causes G1 phase cell cycle blockage in human ovarian cancer cells.

Figure 2 FSA-induced G1 arrest in SK-OV-3 and OVCAR-3 cells. Cell cycle status was assessed via flow cytometry after 48 hours of FSA exposure. (A) Flow cytometry histograms representing the cell cycle distribution of SK-OV-3 cells (control and FSA-treated). (B) Bar graph summary showing the percentage of SK-OV-3 cells in G1, S, and G2 phases. (C) Flow cytometry histograms illustrating the cell cycle distribution of OVCAR-3 cells (control and FSA-treated). (D) Bar graph summary showing the percentage of OVCAR-3 cells in G1, S, and G2 phases. Data represent mean ± standard deviation from three independent experiments (n=3). Statistical significance was determined by Student’s t-test. Statistical significance: **, P<0.01; ****, P<0.0001. FSA, forsythiaside A; NC, negative control; PE, phycoerythrin.

FSA inhibits migration and invasion of human ovarian cancer cells

Wound-healing assays were conducted to assess the migratory impact of FSA on ovarian cancer cells. Specifically, SK-OV-3 cell migration rate significantly decreased from 61.3% in the control group to 18.4% in the FSA-treated group following 24 hours of exposure (P<0.001) (Figure 3A,3B). OVCAR-3 cells showed a comparable reduction, with the migration rate falling from 12.3% in the control group to 4.87% in the FSA-treated group within the same period (P<0.001) (Figure 3C,3D). This highlights a significant inhibitory effect of FSA on their motility.

Figure 3 Anti-migratory effects of FSA on SK-OV-3 and OVCAR-3 cells. Wound healing assays were performed to assess cell migration. (A) Representative images showing wound closure over 24 hours in SK-OV-3 cells treated with NC or FSA (0- and 24-hour views). Scale bar: 100 µm. (B) Bar graph illustrating the migration rate (%) of SK-OV-3 cells in the control and FSA-treated groups. (C) Representative images of wound closure in OVCAR-3 cells at 0 and 24 hours, comparing NC and FSA treatment. Scale bar: 100 µm. (D) Bar graph quantifying the migration rate (%) of OVCAR-3 cells exposed to NC or FSA. Data represent mean ± standard deviation from three independent experiments (n=3). Statistical significance was determined by Student’s t-test. Statistical significance: ****, P<0.0001. FSA, forsythiaside A; NC, negative control.

Transwell assays demonstrated that FSA inhibits the invasion of ovarian cancer cells (Figure 4). Microscopic observation showed fewer cells invading through the Matrigel-coated membrane in FSA-treated wells compared to controls (Figure 4A,4C), a finding supported by quantitative analysis of eluted crystal violet absorbance at 595nm, which revealed a significant decrease in invasion rate (P<0.001) (Figure 4B,4D). These results, combined with the migration assay results, indicate that FSA suppresses the migration and invasion of ovarian cancer cells.

Figure 4 FSA inhibits invasion of SK-OV-3 and OVCAR-3 ovarian cancer cells. Cell invasion was assessed using Transwell assays. (A) Representative images of SK-OV-3 cells that invaded through the Matrigel-coated membrane in the NC and FSA-treated groups. Scale bar: 100 µm. (B) Quantitative analysis of invaded SK-OV-3 cells based on OD595 measurement of eluted crystal violet. (C) Representative images showing invaded OVCAR-3 cells in the NC and FSA-treated groups. Scale bar: 100 µm. (D) Quantitative analysis of invaded OVCAR-3 cells based on OD595 measurement of eluted crystal violet. Data represent mean ± standard deviation from three independent experiments (n=3). Statistical significance was determined by Student’s t-test. Statistical significance: ***, P<0.001; ****, P<0.0001. FSA, forsythiaside A; NC, negative control.

FSA promotes apoptosis and necrosis in ovarian cancer cells in vitro

To evaluate FSA’s ability to induce apoptosis and necrosis in SK-OV-3 and OVCAR-3 ovarian cancer cells, cells were treated for 48 hours and analyzed using the YO-PRO-1/PI kit. Microscopic observation revealed increased cell death after treatment compared to control groups. Compared to the control group, FSA-treated SK-OV-3 and OVCAR-3 cells displayed a higher number of cells positive for YO-PRO-1 (indicating early apoptosis) and PI (indicating late apoptosis/necrosis) under microscopic observation (Figure 5). These findings suggest that FSA promotes cell death in ovarian cancer cells, likely involving both apoptotic and necrotic pathways (Figure 5).

Figure 5 YO-PRO-1/PI staining reveals FSA-induced apoptosis and necrosis in SK-OV-3 and OVCAR-3 cells. Cells were stained with YO-PRO-1 (green) and PI (red) following 48-hour control or FSA treatment to visualize cell death stages. Representative fluorescence microscopy images of SK-OV-3 and OVCAR-3 cells in the control and treated conditions are shown, displaying merged, YO-PRO-1, and PI signals. Scale bar: 50 µm. The number of replicates (n=3). FSA, forsythiaside A; NC, negative control; PI, propidium iodide.

FSA modulates apoptosis-related protein expression in SK-OV-3 and OVCAR-3 cells

Expression levels of apoptosis-related proteins in SK-OV-3 and OVCAR-3 cells were examined using Western blot following 48-hour FSA treatment, revealing altered expression (Figure 6). Analysis showed a significant increase in the expression of pro-apoptotic proteins BAX, CASP3, BID, and CYCS in SK-OV-3 and OVCAR-3 cells upon treatment (P<0.05) (Figure 6A,6C), which was quantitatively confirmed (Figure 6B,6D). Significantly reduced expression of the anti-apoptotic protein BCL2 was observed in both SK-OV-3 and OVCAR-3 cell lines following 48 hours of FSA treatment (P<0.05) (Figure 5A,5C), consistent with quantitative findings. In summary, FSA modulates the expression of apoptosis-related proteins in ovarian cancer cells, increasing pro-apoptotic factors (BAX, CASP3, BID, CYCS) while decreasing the anti-apoptotic factor BCL2.

Figure 6 FSA alters apoptosis protein profiles in SK-OV-3 and OVCAR-3 cells. Expression of BAX, BID, CASP3, CYCS, and BCL2 was determined by Western blot following 48-h treatment with control or FSA. β-actin served as a loading control. (A) Representative Western blot images for BAX, BID, CASP3, CYCS, BCL2, and β-actin in SK-OV-3 cells (NC and FSA). (B) Bar graph analysis of protein expression levels relative to β-actin in SK-OV-3 cells. (C) Representative Western blot images for BAX, BID, CASP3, CYCS, BCL2, and β-actin in OVCAR-3 cells (NC and FSA). (D) Bar graph analysis of protein expression levels relative to β-actin in OVCAR-3 cells. Data represent mean ± standard deviation from three independent experiments (n=3). Statistical significance was determined by Student’s t-test. Statistical significance markers: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. FSA, forsythiaside A; NC, negative control.

Transcriptome analysis of FSA-treated SK-OV-3 ovarian cancer cells

Significant changes in the gene expression landscape of SK-OV-3 cells following FSA treatment were identified by transcriptome analysis. Principal component analysis (PCA) visualization (Figure 7A) illustrated clear differentiation between the control and treated groups, primarily along PC1 (95.83%) and PC2 (2.20%). Analysis presented in the volcano plot (Figure 7B) revealed 9,394 DEGs (Padj <0.01), comprising 4,401 genes whose expression was elevated and 4,993 genes whose expression was reduced in response to FSA. Enrichment analysis (Figure 7C) identified 15 pathways with significant enrichment, notably involving endocytosis, protein processing in endoplasmic reticulum, cell senescence, nucleocytoplasmic transport, hepatocellular carcinoma, hepatitis b, ribosome, ubiquitin mediated proteolysis, cell cycle, small cell lung cancer, colorectal cancer, ErbB signaling pathway, ribosome biogenesis in eukaryotes, chronic myeloid leukemia, mitophagy-animal, and an ovarian cancer related pathway. In summary, transcriptome analysis reveals that FSA modulates diverse cellular pathways in SK-OV-3 ovarian cancer cells, suggesting its influence on processes like cell cycle regulation, protein metabolism, and pathways specifically relevant to ovarian cancer.

Figure 7 RNA sequencing analysis of FSA’s impact on SK-OV-3 cells. (A) PCA plot showing separation of the control and FSA-treated samples, with PC1 and PC2 variance percentages indicated. (B) Volcano plot visualizing gene expression changes in SK-OV-3 cells treated with FSA. Red dots represent upregulated genes, blue dots downregulated, and grey dots non-significant genes. Differential expression thresholds (Padj <0.01, |log₂FC| >1) are displayed. (C) Enriched pathways from DEG analysis. Bubble size indicates gene count, color shows adjusted P value, and the x-axis shows gene ratio. Number of independent experiments for each group: n=3. DEG, differentially expressed gene; FC, fold change; FSA, forsythiaside A; PC, principal component; PCA, principal component analysis.

Anti-tumorigenic effects of FSA on SK-OV-3 cells in vivo

Investigation into the in vivo effects of FSA on SK-OV-3 tumor growth was conducted using a nude mouse xenograft model (Figure 8). Analysis of tumor volumes (Figure 8B) showed a significant reduction in the FSA-treated group’s tumor size compared to controls (P<0.01). This quantitative suppression was consistent with the visual observation that tumors from mice receiving FSA treatment were smaller (Figure 8A). These findings suggest that FSA successfully inhibits SK-OV-3 xenograft tumor growth in nude mice.

Figure 8 FSA suppresses SK-OV-3 xenograft tumor growth in vivo. (A) Representative images of tumors excised from nude mice treated with vehicle control (top) or FSA (bottom). (B) Tumor volumes measured throughout the study for each group (n=5). The control group received PBS + 5% DMSO via gavage, while the FSA-treated group received 50 mg/kg FSA via gavage. Data represent mean ± standard deviation. Student’s t-test was used for volume comparison. Tumor volume was significantly reduced in the FSA group compared to the control group (***, P<0.001). DMSO, dimethyl sulfoxide; FSA, forsythiaside A; NC, negative control; PBS, phosphate buffered saline.

Discussion

This study provides robust evidence for FSA’s anti-ovarian cancer efficacy, demonstrated through both in vitro and in vivo models. Our key findings reveal that FSA inhibits the proliferation, migration, and invasion of SK-OV-3 and OVCAR3 ovarian cancer cells. Furthermore, FSA induces G1 phase cell cycle arrest and promotes apoptosis. These effects are closely associated with FSA’s modulation of apoptosis-related proteins, specifically upregulating BAX, CASP3, BID, and CYCS, while downregulating BCL2. Transcriptome analysis corroborated these findings, confirming significant alterations in gene expression within pathways critical for cell cycle regulation and apoptosis. In vivo, FSA significantly retarded the growth of SK-OV-3 xenograft tumors.

Previous research has established the broader anti-neoplastic potential of FSA across various cancer types, highlighting its versatile anti-cancer activity. Consistent with our observations, studies have shown FSA’s ability to modulate specific molecular targets in cancer, such as BCL2, BAX, and p21 in esophageal squamous cell carcinoma (19), and CD44 in liver fibrosis, where it regulates NLRP3-mediated pyroptosis (20). Moreover, FSA inhibits the KLRB1-CLEC2D immune checkpoint, a pathway implicated in tumor progression and immune evasion (22). Our study builds upon these findings by demonstrating similar effects in ovarian cancer, with a specific emphasis on the role of G1 arrest and apoptosis mediated through the modulation of BAX, CASP3, BID, CYCS, and BCL2.

This study’s strengths include in vitro and in vivo validation of FSA’s effects, with analysis of cell cycle, apoptosis, and gene expression. Limitations are the use of only two ovarian cancer cell lines, suggesting broader studies are needed. The subcutaneous xenograft model is also a limitation; future in vivo studies should use more clinically relevant models.

Conclusions

FSA demonstrates significant anti-ovarian cancer activity in both in vitro and in vivo models. FSA effectively inhibits the proliferation, migration, and invasion of ovarian cancer cells by inducing G1 phase cell cycle arrest and promoting apoptosis. These findings suggest that FSA holds potential as a novel therapeutic agent for ovarian cancer and warrants further investigation in clinical settings to evaluate its therapeutic efficacy.

Acknowledgments

The authors acknowledge the assistance of Instrumental Analysis Center of Tarim University.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-980/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-980/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-980/prf

Funding: This study was supported by the Xinjiang Uyghur Autonomous Region Department of Science and Technology and Xinjiang Production and Construction Corps Science and Technology Bureau, which jointly fund the “Tianshan Talents”—Science and Technology Innovation Leading Talents Training Project (No. 2023TSYCLJ0057), Guidance Science and Technology Program of the Corps (No. 2023ZD115), Tianshan Talent Introduction Program for Young Doctors (No. 524307007) and Tarim University graduate students creation project (No. TDBSCX202209).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-980/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Animal experiments were performed under a project license (No. PB20250418003) granted by the Science and Technology Ethics Committee of Tarim University, in compliance with the institutional animal welfare guidelines of Tarim University.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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