The human normal ovarian epithelial cell line IOSE-80 and human GCT-derived cell lines KGN and COV434 were used for investigation in this study. The KGN cells are derived from an adult-type GCT. The COV434 cells are derived from a juvenile-type GCT (Jamieson and Fuller, 2012). All cell lines were purchased from Shanghai iCell Bioscience Inc and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin (Gibco), and 1% streptomycin (Gibco), at 37°C in a 5% CO₂ humidified atmosphere.

The SIRT3 selective inhibitor 77-39 was provided by Professor Xiaoyu Li (The University of Hong Kong, China). The p53 inhibitor PFTα (S2929) and NF-κB inhibitor BAY 11-7082 (S2913) were purchased from Selleck (USA). Anti-p-IKKα/β (2697), anti-p65 (8242), anti-p-p65 (3033), anti-IκBα (4812), anti-p-IκBα (2859), anti-p53 (2524), anti-p21 (2947), anti-p18 (2896), anti-p16 (18769), anti-β-actin (4970), anti-mouse secondary antibody (7076) and anti-rabbit secondary antibody (7074) were purchased from Cell Signaling Technology (USA). Anti-IKKα/β (PA5-105292) antibody was purchased from Invitrogen (USA). Anti-ac-p53 (HW137) antibody was purchased from Signalway (China). Anti-SIRT3 (ab217319) antibody was from Abcam (USA).

SiRNA targeting SIRT3 (siSIRT3) and siRNA control (siNC) were synthesized by GenePharma Co., Ltd. (China). The indicated siRNA was transfected into KGN and COV434 cells using the RNAiMAX transfection reagent (Invitrogen). The siSIRT3 sequence was listed as follows: 5′-CCA​GCA​UGA​AAU​ACA​UUU​ATT-3’. The SIRT3 overexpression plasmid was synthesized by and purchased from GenePharma Co., Ltd. The plasmid was transfected into KGN and COV434 cells using the Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s instructions.

The KGN and COV434 cells were treated with the indicated treatments. Then, cells were lysed using RIPA buffer (Beyotime, China) containing phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor cocktail (Sigma-Aldrich, USA). The protein concentration of samples was quantified using a BCA Protein Assay Kit (Beyotime). Proteins were separated by 8%–12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis and subsequently transferred to polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% Skim milk, the membrane was incubated with primary antibodies at 4°C overnight. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 h. Finally, we used Clarity Western ECL Substrate (Bio-rad, USA) for imaging. The uncropped blots are available in the Supplementary Material.

Cell senescence was assessed by the detection of SA-β-gal activity using a senescence β-galactosidase staining kit (Beyotime). The cells were first fixed with the fixative solution at room temperature for 15 min. Then the cells were stained with the staining buffer at 37°C overnight. The stained cells were photographed and counted randomly from 4 to 9 fields per well under a microscope.

A total number of 300 KGN or 500 COV434 cells were seeded into a six-well plate. Indicated concentrations of SIRT3 inhibitor 77-39 were added to the medium. The cells were allowed to grow for 2 weeks. When the colonies were visible, cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed.

DNA synthesis was assessed by EdU assay using an EdU cell proliferation kit (Beyotime) according to the manufacturer’s instructions. As previously described (Zeng et al., 2024a), after treatment with 77–39, cells were incubated with the EdU staining buffer for 2.5 h and then fixed with 4% paraformaldehyde. Subsequently, the cell nuclei were stained with Hoechst and photographed under a fluorescence microscope.

Cell viability was evaluated by CCK8 assay. Briefly, cells were seeded at a density of 3000 cells per well in 96-well plates. Then, cells were treated with the indicated concentrations of 77–39 for 24 h. A volume of 10 μL CCK8 solution (Beyotime) was added to 100 μL culture medium and incubated with cells at 37°C for 1 h. The optical density (OD) at 450 nm was measured using a microplate reader.

TRIzol reagent (Invitrogen) was used to extract total RNA of cells, followed by the synthesis of complementary DNA (cDNA) using a PrimeScript RT reagent Kit (Takara, Japan). qPCR was performed using a TB Green Premix Ex Taq Kit (Takara) on a CFX96 real-time system (Bio-rad, USA). GAPDH was used as a reference control for normalization. The primer sequences were listed as follows: IL6 forward: 5′-AGA​CAG​CCA​CTC​ACC​TCT​TCA​G-3′ and reverse: 5′-TTC​TGC​CAG​TGC​CTC​TTT​GCT​G-3’; IL-1B forward: 5′-CCA​CAG​ACC​TTC​CAG​GAG​AAT​G-3′ and reverse: 5′-GTG​CAG​TTC​AGT​GAT​CGT​ACA​GG-3’; CXCL2 forward: 5′-GGC​AGA​AAG​CTT​GTC​TCA​ACC​C-3′ and reverse: 5′-CTC​CTT​CAG​GAA​CAG​CCA​CCA​A-3’; GAPDH forward: 5′-GGG​AAA​CTG​TGG​CGT​GAT-3′ and reverse: 5′-GAG​TGG​GTG​TCG​CTG​TTG​A-3’.

KGN and COV434 cells seeded in 10 cm culture dishes were lysed using IP lysis buffer (Beyotime, China) with protease inhibitors. Primary antibody was added to and incubated with the cleared lysate at 4°C overnight with gentle rotation. Then protein A/G beads (Invitrogen, USA) were added to and incubated with the lysate-antibody mixture at 4°C for 2 h with gentle rotation, followed by washing 3-4 times. The immunoprecipitate was eluted with the addition of elution buffer. The immunoprecipitated proteins were subsequently subjected to WB analysis.

Transcriptome sequencing and differential expression analysis were performed by Novogene Inc. (Tianjin, China). As previously reported (Zeng et al., 2024b), the total RNA of 77–39 treated and non-treated KGN or Cov434 cells was extracted with TRIzol reagent. Total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, USA). When the libraries were constructed and qualified, they were subsequently subjected to RNA sequencing with Illumina NovaSeq 6000.

GSEA was performed using GSEA software (https://www.gsea-msigdb.org/gsea/index.jsp) as previously described (Zeng et al., 2024a). The RNA sequencing expression profiles of 77–39 non-treated group and treated group were imported into the GSEA software. When the false discovery rate (FDR) was <0.25 and the nominal p-value was <0.05, the enriched gene sets were considered statistically significant.

Four-week-old female BALB/c nude mice (six mice per group) were purchased from Charles River Laboratories and maintained under specific pathogen-free conditions. KGN cells (1 × 10⁶ cells in the mixture of 0.1 mL PBS and 0.1 mL Matrigel (Corning, USA) per mouse) were injected subcutaneously into the right dorsal flank (designated as day 1). Fourteen days after injection, mice were randomly assigned to 5 groups (6 mice per group). 77-39 was dissolved in normal saline. One hundred microliters of indicated concentrations of 77–39 were administered by gavage every day. Cisplatin (CDDP) was injected intraperitoneally into mice every other day. Tumor volume was measured every other day, and tumor volume was calculated as volume (mm³) = length × width². Mice were euthanized on day 31. Tumors were isolated and harvested from indicated mice and subsequently were fixed, embedded with paraffin, and examined by hematoxylin and eosin (H&E), SA-β-gal, and immunohistochemical (IHC) staining. All experimental procedures and protocols were reviewed and approved by Shenzhen TopBiotech Co., Ltd. Animal Center (Shenzhen, China). The H-score scoring system was employed to assess the differences in IHC staining, with the scoring formula defined as H-score = ∑ (percentage of positive cells × intensity). The percentage of positive cells was expressed as a fraction of 100 (e.g., 20% = 20). The intensity of staining was graded on a scale from 0 to 3, with 0 representing no staining, 1 representing weak staining, 2 representing moderate staining, and 3 representing strong staining.

Data shown in this study were representative of the statistics [mean value ±standard deviation (SD)] of the results from at least three independent experiments. The students’ two-tailed t-test was used for all statistical analyses, with the level of significance set at (***) p < 0.005, (**) p < 0.01, and (*) p < 0.05. All the statistical analyses were conducted by GraphPad Prism 7 software.

The chemical structural formula of 77–39 is shown in Figure 1A. To evaluate the effect of 77–39 on GCT cells, we first examined the cell viability of normal ovarian epithelial cell line IOSE-80 and two GCT cell lines KGN and CO434 with the treatment of indicated 77-39 concentration. The results showed that 77–39 significantly suppressed the viability of KGN and COV434 cells in a concentration-dependent manner, while it did not inhibit the normal cell line IOSE-80 as effectively (Figure 1B). We further performed EdU assay and colony formation assay to assess the impact of 77–39 on GCT cell proliferation. As shown in Figures 1C,D, the proportion of EdU-positive cells and the number of colonies were significantly higher in the control group, which were remarkably reduced in a concentration-dependent manner in the presence of 77–39, indicating that 77-39 inhibited the proliferative activity of GCT cells. As SIRT3 is a protein related to aging and senescence (Zhou et al., 2022), we then tested whether 77-39 induces GCT senescence. By SA-β-gal staining assay, we found that 77–39 significantly induced cellular senescence compared with the control group (Figure 1E). Cellular senescence results in the secretion of a wide range of cytokines, chemokines, growth factors, and proteases/inhibitors, collectively known as the senescence-associated secretory phenotype (SASP) (Lasry and Ben-Neriah, 2015). We detected several SASP-associated proteins, such as IL-6, IL-1B, and CXCL2, by qPCR. As expected, treatment with 77-39 significantly elevated the mRNA expression levels of all cytokines (Figure 1F). In summary, the results demonstrate that 77-39 inhibits GCT cell growth by inducing senescence.

To elucidate the potential mechanisms by which 77-39 inhibits the proliferation of GCT cells, we performed RNA sequencing on KGN and COV434 cells treated with or without 77-39. As summarized in Figure 2A, following treatment of 77–39, 2898 genes were significantly upregulated while 3067 genes were significantly downregulated in COV434 cells, 633 genes were significantly upregulated while 640 genes were significantly downregulated in KGN cells. Subsequently, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using the differentially expressed genes (DEGs), and we noticed that the term “p53 signaling pathway” was significantly enriched in both COV434 and KGN cells (Figure 2B), indicating that 77–39 might regulate p53 pathway in GCT. Indeed, p53 has been reported to be a downstream factor of SIRT3, and deacetylated and suppressed by SIRT3 (Li et al., 2010; Chen et al., 2017; Xiong et al., 2018). We further performed GSEA and found that gene sets including “FRIDMAN_SENESCENCE_UP” and “WANG_NFKB_TARGETS” were significantly enriched in the 77-39 treated group, indicating 77–39 might activate the NF-κB pathway and induce senescence (Figure 3A). By WB analyses, we found that, following treatment of 73–39, the protein expression of cell cycle-related proteins like p16, p18, p21, p53, and acetylated p53 were elevated in a dose-dependent manner. The key proteins in the NF-κB pathway, such as p65, IκBα, and IKKα/β, were also activated (Figure 3B). These results indicate that 77-39 inhibited cell growth and induced senescence through the p53 and NF-κB signaling in GCT cells.

To verify that 77-39 functioned through targeting SIRT3, we knocked down the expression of SIRT3 in KGN and COV434 cells by siRNA (Figure 4A). By EdU assay and SA-β-gal staining, we found that the silence of SIRT3 significantly inhibited cell proliferation and promoted senescence in GCT cells (Figures 4B,C). The protein expression of cell cycle-related proteins and the phosphorylation levels of key proteins in the NF-κB pathway also underwent significant changes (Figure 4D), which were consistent with the results observed upon treatment with 77–39.

Correspondingly, we overexpressed SIRT3 in KGN and COV434 cells (Figure 5A). The inhibitory effect of 77–39 on cell proliferation and senescence in GCT cells was reversed by overexpression of SIRT3 (Figures 5B,C). The expression of proteins related to the p53 and NF-κB pathways was also suppressed by the overexpression of SIRT3 (Figure 5D). Overall, the above results indicated that SIRT3 is the target through which 77-39 exerts its effects.

To verify that p53 is a downstream effector molecule of SIRT3, we performed co-IP experiments and found that p53 interacted with SIRT3 in both KGN and COV434 cells (Figure 6A). To determine whether p53 mediated the inhibitory effect of 77–39 on GCT cells, we inhibited p53 using the inhibitor PFTα. The results showed that inhibiting p53 led to a significant recovery in cell proliferation and a reduced rate of senescence in GCT cells treated with 77–39 (Figures 6B,C). The proteins that inhibited the cell cycle process were also downregulated (Figure 6D). Intriguingly, we found that the inhibition of p53 significantly affected the phosphorylated level of NF-κB-related proteins, which indicated that p53 might regulate NF-κB signaling in GCT (Figure 6D).

The NF-κB pathway was reported to be activated in senescent cells and implicated in regulating the expression of the inflammatory factors (Chen et al., 2018; Wang et al., 2022). Similarly, we also inhibited the activity of NF-κB in GCT cells using the inhibitor BAY 11-7082. The proliferative capacity of GCT cells and the expression of related proteins in GCT cells treated with 77–39 were restored upon inhibition of NF-κB. (Figures 7A–C). In summary, these results demonstrated that p53 and NF-κB are downstream effectors of SIRT3.

To evaluate the efficacy and safety of 77–39 in inhibiting the tumorigenicity of GCT in vivo, we established a subcutaneous tumor model in nude mice. KGN cells were injected subcutaneously into the right dorsal flank of nude mice. Fourteen days after injection, mice were randomly assigned to 5 groups (6 mice per group). One hundred microliters of indicated concentrations of 77–39 were administered by gavage every day. CDDP, a chemotherapeutic agent used clinically for the treatment of GCT, was injected intraperitoneally into mice every other day. The results showed that both CDDP and 77-39 could effectively inhibit the tumor growth of GCT in vivo (Figures 8A–C). CDDP demonstrated the best tumor-inhibitory capacity (Figure 8C). However, the body weight of mice in the CDDP-treated group significantly decreased (Figure 8D), while the 77-39-treated groups did not show significant changes in body weight, indicating that, compared with CDDP, 77-39 exhibited lower drug toxicity. In addition, xenograft tumors excised from nude mice were further subjected to HE staining, SA-β-gal staining, and IHC staining. Consistent with our in vitro experimental results, 77-39 was capable of inducing tumor senescence, inhibiting tumor proliferation, and regulating the expression of cell cycle-related proteins (Figure 8E). At the endpoint of the animal experiment, we collected blood from the nude mice and measured the indicators of liver and kidney function. The results showed that compound 77–39 did not exhibit significant toxicity to the liver and kidneys, indicating its safety profile (Figure 8F).