EC cell lines KLE, ECC-1 and HEC-1A were originally obtained from the American Type Culture Collection (ATCC), and the Ishikawa cells were purchased from Sigma-Aldrich (St. Louis, MO). The KLE cells were cultured in DMEM/F12 medium with 10% fetal bovine serum (FBS). The HEC-1A cells were maintained in McCoy’s 5A medium with 10% FBS. The ECC-1 cells were cultured in 1,640 medium with 5% FBS. The Ishikawa cells were maintained in MEM with 10% FBS at 37°C under 5% CO₂. All cell lines are authenticated annually by LabCorp (Burlington, NC) using short tandem repeat (STR) profiling, and mycoplasma tests were performed in these cell lines every 6 months. All antibodies used for in vitro studies were purchased from Cell Signaling Technology (Danvers, MA). All chemicals were from Thermo Fisher Scientific (Waltham, MA).

The ASP extract was obtained from Shandong Juxinyuan Asparagus Industry Development Research Institute, P.R. China. Asparagus stems and shoots were used to prepare ASP extracts following the protocol of our previous publication (Xu et al., 2021). Briefly, the crushed stems and shoots of ASP were rinsed in distilled water at 95°C before being extracted using squeezer equipment. After centrifuging to remove insoluble solids, the crude extract was concentrated in vacuum to produce a concentrated solution containing 20% soluble solids. This concentrated extract is further concentrated through a water bath. Finally, the concentrated extract includes 70% soluble components and is sterilized through pasteurization (Supplementary Table S1). After the ASP extraction was completed, the sample of ASP was sent to local government agencies to examine 11 pesticide components. There was no pesticide ingredients found in the ASP extract used in this study (Xu et al., 2021).

The HPLC analysis system (Shimadzu LC 20AT, Columbia, MD) equipped with a vacuum degasser, a quaternary pump, an autosampler, and a diode array detector or ELSD (for flavonoids) was used to qualitatively detect flavonoids, saponins, phenolic compounds and other active metabolites in the ASP extracts. All samples were filtered through 0.45 µm PTFE filters before HPLC analysis. Chromatographic separations were performed on an Agilent ZORBAXSB-C18 column (4.6 × 250 mm, 5 µm). The mobile phase was a mixture of methanol and water (10%) or a mixture of formic acid and water (2%, for flavonoids). The temperature of the column was 25°C. The detection wavelengths range were from 210–500 nm. The flow rate was 1 mL/min. All measurements were performed in triplicate. Data acquisition was performed using the Shimadzu LCsolution software program. All measurements of ASP extracts were performed in Institute of Oceanology, Chinese Academy of Science (Qingdao, China, Supplementary Table S2). The major bioactive metabolites were shown in Table 1.

The KLE, ECC-1 and HEC-1A and Ishikawa Cells were seeded in 96-well plates, with 4,000–6,000 cells per well for 24 h at 37°C. The cells were then treated with the indicated doses (0.001, 0.01.0.1, 0.5, 0.75.1, 5 and 10 mg/mL) of ASP for 72 h. Each treatment was tested in quadruplicate replicates. 5 μL MTT (5 mg/mL) was added to each well for 1 hour at 37°C. After incubation, the formazan crystals are dissolved in 100 µL dimethyl sulfoxide (DMSO)/well. Absorbance at 575 nm on 96-well plates was measured using a microplate reader (Tecan, Durham, NC). The IC₅₀ value for the KLE, ECC-1 and HEC-1A and Ishikawa cells was calculated using the IC₅₀ Calculator (AAT Bioquest, Sunnyvale, CA). Bliss independence model was used to evaluate the synergistic effect of ASP combined with rapamycin on cell proliferation (Foucquier and Guedj, 2015). CI = 1 (additive), CI < 1 (synergistic), or CI > 1 (antagonistic).

The KLE and Ishikawa cells were treated with 0.01, 0.1, and 1 mg/mL ASP extracts for 36 h in 5% CO₂ at 37°C. The cells were harvested with 0.5% trypsin, fixed with 90% ice cold methanol for 1 hour, and then incubated in a Propidium Iodide/RNAse staining solution (Nexcelom Bioscience, Lawrence, MA) for 20 min. Cell cycles were detected by Cellometer (Nexcelom Bioscience). The data were analyzed by FCS4 Express (De Novo Software, Pasadena, CA).

The KLE and Ishikawa cells were plated in 6-well plates at the concentration of 2.5 × 10⁵ cells/well overnight. The cells were exposed to 0.01,0.1 and 1 mg/mL ASP for 12 h and lysed with 1X caspase lysis buffer. BCA assay (Thermo Fisher) was used to detect protein concentrations. 150 μL of cell lysate was added to each well in a black 96-well plate, and reaction buffers with cleaved caspase 3, 8 and 9 substrates were mixed with the cell lysate at 37°C in the dark for 20 min. The fluorescence intensity for cleaved caspase 3 (Ex/Em = 400/505), cleaved caspase 8 (Ex/Em = 376/482), and cleaved caspase 9 (Ex/Em = 341/441) were recorded using a Tecan microplate reader.

Intracellular reactive oxygen species (ROS) production was determined using the DCFH-DA assay. The KLE and Ishikawa cells were cultured in black 96-well plates overnight and treated with ASP extracts for 12 h 200 μM ROS inducer (pyocyanin) was used as a positive control. The cells were incubated with 20 µM DCFH-DA solution for 30 min. Cellular ROS generation was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a Tecan plate reader. Cells were incubated with 20 µM DCFH-DA solution for 30 min.

Mitochondrial membrane potential was assayed using the TMRE assay (AAT Bioquest, Pleasanton, CA). The KLE and Ishikawa cells were seeded in a 96-well plate (10,000/well) overnight and incubated with 0.01, 0.1, and 1 mg/mL ASP extracts for 12 h. For positive control, we treated the cells with 20 µM FCCP for 30 min. After 100 µL of culture medium with 500 µM TMRE was added to each well and then incubated at 37°C for 30 min. TMRE fluorescence was measured using a Tecan plate reader at an excitation wavelength of 549 nm and an emission wavelength of 575 nm.

The KLE and Ishikawa cells were plated into black 96-well plates at 6,000 cells/well overnight and treated with different concentrations of ASP extracts for 12 h. Each well on the 96-well plate was replaced with 100 µL glucose-free medium containing 100 ug/mL 2-NBDG for 20 min. The four wells without 2NBDG were used as negative controls. After washing each well with HBSS twice, fluorescence intensity (Ex/Em = 465/540) was measured via the Tecan plate reader.

The content of L-lactate in culture medium in the KLE and Ishikawa cells was measured using the L-Lactate Assay Kit (Eton Bio, San Diego, CA) following the manufacturer’s instruction. In brief, the cells were treated with different concentrations of ASP extracts (0.01, 0.1, and 1 mg/mL) for 24 h. Subsequently, 25 µL of the sample medium was transferred into a 96-well plate and then 50 µL of distilled water and 25 µL of lactate assay solution were added to each well for 60 min at room temperature in the dark. The absorbance at 490 nm was measured using a Tecan microplate reader.

Cellular ATP production was assessed using the Luminometric ATP Assay Kit (Fisher Scientific) following the manufacturer’s instruction. After the KLE and Ishikawa cells were treated with ASP extracts (0.01, 0.1, and 1 mg/mL) for 24 h in a 96-well plate, the ATP reaction mix was added to each well and incubated for 10–20 min in the dark. Cellular ATP production was measured with a Tecan plate reader. ATP levels were normalized based on the viable cell counts measured by the MTT assay.

The KLE and Ishikawa cells were seeded in plates pre-coated with laminin-1 (Sigma-Aldrich) at 6,000 cells/well and treated with 0.01, 0.1, and 1 mg/mL ASP extracts for 2 h. After aspirating the supernatant, the cell lines were fixed with 5% glutaraldehyde for 30 min and then the plate was stained with 0.1% crystal violet solution for 15 min at room temperature. 100 μL of 10% acetic acid was added to each well to solubilize the dye. The plate was measured at a wavelength of 575 nm by a Tecan plate reader.

Transwell assay was conducted in 96-well plates pre-coated with 0.5-1 x BME (Trevigen, Gaithersburg, MD). The KLE and Ishikawa cells were starved in an FBS-free medium for 12 h and then plated in the upper chamber with a density of 3 × 10⁴. ASP extracts (0.01, 0.1, and 1 mg/mL) were added to the upper chambers and then the lower chambers were filled with regular medium. The plates were incubated for 6 h for cell invasion into the lower chambers at 37°C. 100 μL of calcein AM solution (Invitrogen, Carlsbad, CA) was added to the lower chambers and incubated for another 30 min. The plate was measured using a Tecan plate reader with an excitation/emission wavelength of 485/520 nm.

After KLE and Ishikawa cells were treated with 0.01, 0.1, and 1 mg/mL ASP extracts for 24 h, the cells were lysed in RIPA buffer supplemented with protease inhibitors (Roche, Seattle, WA). Equal amounts of protein were separated by electrophoresis through 10%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred into 0.2 µm PVDF membranes (Bio-Rad, Hercules, CA). After blocking with 5% non-fat milk at room temperature for 1 hour, the membranes were incubated with various primary antibodies overnight at 4°C. The primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies. Immunoblotting signals were visualized by Super Signal WestPico™ (Thermo Scientific) and analyzed using the Bio-Rad ChemiDoc™ image system.

The Lkb1 ^fl/fl p53 ^fl/fl mouse model of EC has been described previously (Guo et al., 2019). Our animal protocol (19–141) for this study was approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill (UNC-CH). To induce endometrial tumors, 5 µL of 1 × 10¹⁰ pfu/mL Ad5-CMV-Cre (Transfer Vector Core, University of Iowa, IA) was injected into the left uterine horn of female Lkb1 ^fl/fl p53 ^fl/fl mice at 6–8 weeks of age. To compare the effect of concentrations of ASP extracts on tumor growth, the mice were initially divided into three groups (18 mice per group) and treated with 200 or 800 mg/kg ASP or vehicle orally (daily) for 4 weeks. Given that obesity is one of the leading risk factors for carcinogenesis and progression of EC, we explored the effect of ASP on tumor growth in Lkb1 ^fl/fl p53 ^fl/fl mice under obese and lean conditions. The mice were fed a high-fat diet (HFD) or a low-fat diet (LFD) starting at 3 weeks of age. Approximately 8 weeks after Ad5-CMV-Cre injection for tumor induction, mice were then divided into four groups (18 mice/group) and treated with 200 mg/kg ASP extract daily for 4 weeks: HFD control, HFD + ASP, LFD control, and LFD + ASP. Endometrial tumors and blood samples were collected and stored at −80°C until use. The control mice received the same volume of concentrated extract without ASP.

Bip, VEGF, phosphorylated-S6, and Ki67 antibodies were used for IHC staining on 4 mm paraffin sections of formalin-fixed tissue from the LKB1 ^fl/fl p53 ^fl/fl mice at the UNC-CH animal facility. All slides were scanned by Motic and analyzed by ImagePro software (Rockville, MD).

Data are presented as a mean ± the standard error of the mean. All experiments were repeated three times except for animal experiments. Graphs and statistical analysis were performed by GraphPad Prism 8 software. The unpaired Student’s t-test and analysis of variance (ANOVA) were performed for comparison between treatment and control groups in this study. p values < 0.05 were considered statistically significant.

The anti-proliferative effects of different concentrations of ASP on the ECC-1, HEC-1A, KLE, and Ishikawa were evaluated using MTT assay. The results showed that ASP extracts significantly inhibited cell proliferation in a dose-dependent manner in all 4 cell lines after treatment with ASP for 72 h. The IC₅₀ values of ASP in ECC-1, HEC-1A, KLE, and Ishikawa cells were 1.83, 2.91, 0.64, and 2.14 mg/mL, respectively (Figure 1A). To investigate whether ASP inhibits tumor growth in vivo, we treated Lkb1 ^fl/fl p53 ^fl/fl mice with 200 or 800 mg/kg ASP extract (oral gavage, daily) for 4 weeks. Compared with the vehicle group, the tumor weight of mice in the 200 mg/kg group was significantly reduced by 41.6% ± 6.2%, and the tumor weight of mice in the 800 mg/kg group was significantly reduced by 51.8% ± 7.1%. However, there was no statistical difference in tumor weight between the two treatment groups (Figure 1B). During ASP treatment, none of the mice showed abnormal activity, and no abnormalities were found in weekly body weight or blood glucose assessments (Supplementary Figure S1).

Given that obesity is an important contributor to carcinogenesis in EC and diet-induced obesity effectively promotes tumor growth in Lkb1 ^fl/fl p53 ^fl/fl mice (Guo et al., 2019; Pierce et al., 2021), the effect of ASP on tumor growth under obese and lean conditions was investigated in Lkb1 ^fl/fl p53 ^fl/fl mice. Before the treatment, the average body weight of the mice in the HFD was 35.5 g, and the average body weight in the LFD was 27.9 g. The obese and lean mice were treated with ASP extract (200 mg/kg, daily, oral gavage) for 4 weeks. Compared with the LFD group, the tumor weight in the HFD group increased significantly by 36.4%. In the HFD group, ASP effectively decreased tumor growth by 48.9%, while in the LFD group, ASP reduced tumor weight by 42.4% (Figure 1C). ASP exhibited greater anti-tumorigenic activity in obese mice compared with lean mice (p < 0.05). Similarly, further analysis of the expression of Ki-67 in EC tissues revealed that ASP was able to reduce the expression of Ki-67 by 47.2% in obese mice and 23.5% in lean mice (Figure 1D).

The KLE and Ishikawa were treated with 0.01, 0.1, and 1 mg/mL ASP extracts for 36 h, and an analysis of cell cycle prolife showed that ASP extract induced cell cycle G1 phase arrest in both cell lines (Figure 2A). Treatment of cells with 1 mg/mL ASP significantly increased G1 phase by 11.36% in the KLE cells and 12.06% in the Ishikawa cells, respectively. To understand the molecular mechanism underlying cell cycle G1 arrest induced by ASP, the expression of cell cycle-related proteins was detected by Western blotting assay after the KLE and Ishikawa cells were treated with ASP extract for 24 h. The results demonstrated that ASP extract reduced the expression of CDK4 and CDK6 in a concentration-dependent manner in both cell lines (Figure 2B). These results suggest that cell cycle G1 arrest is involved in ASP-induced inhibition of cell proliferation in EC cells.

To investigate the effect of ASP on apoptosis, the KLE and Ishikawa cells were treated with varying concentrations of ASP for 16 h and then stained with Annexin V and PI. As shown in Figure 3A, a significant increase in early apoptotic cells was observed after treatment with 1 mg/mL ASP extract in both cell lines. These results were confirmed by Western blotting results. ASP extract reduced the expression of Bcl-2 and Mcl-1 in a concentration-dependent manner in both cell lines (Figure 3B). To examine whether ASP induced apoptosis through the extrinsic/intrinsic apoptotic pathway, the activities of cleaved caspase 3, 8, and 9 were assessed by ELISA assays. As shown in Figures 3C–E, the activities of cleaved caspase 3, 8, and 9 were increased in a dose-dependent manner by treatment with increasing concentrations of ASP for 12 h in both cell lines. 1 mg/mL ASP extract increased the activities of caspase 3, 8, and 9 by 120%, 78%, and 77% in the KLE cells and by 98%, 89%, and 94% in the Ishikawa cells, respectively. These results suggest that inhibition of cell proliferation by ASP depends on the induction of extrinsic and intrinsic apoptotic pathways in EC cells.

Intercellular ROS production was measured after treatment of the KLE and Ishikawa cells with ASP extract for 12 h. ASP-treated cells showed a significant increase in ROS production in a concentration-dependent manner in both cell lines (Figure 4A). TMRE assay also showed that ASP extract reduced the mitochondrial membrane potential in both cell lines after 12 h of treatment. 1 mg/mL ASP effectively reduced mitochondrial membrane potential by 26% in the KLE cells and 29% in the Ishikawa cells compared to control cells, respectively (Figure 4B). Furthermore, after treatment of both cells with ASP for 24 h, Western blotting results showed that ASP extract increased the expression of cellular stress-related proteins PERK and Bip (Figure 4C). IHC results showed that there was no difference in Bip expression between obese and lean mice, but 4 weeks of ASP treatment increased Bip expression in both obese and lean mice (Figure 4D), indicating that ASP treatment significantly increased cellular stress in endometrial tumors.

Since ASP induced oxidant stress in vitro and in vivo, N-Acetylcysteine (NAC) was used to block ASP-induced cellular stress pathway to observe whether blocking the cellular stress pathway would reverse the inhibitory function of ASP on cell growth. The KLE and Ishikawa cells were pretreated with NAC (1 mM) for 4 h and then treated with ASP (0.1 mg/mL) for 12 h, Western blotting results showed that NAC significantly revised the ASP-induced PERK expression in both cells compared with control, NAC-treated, and ASP-treated groups (Figure 4E). The ROS assay confirmed that NAC partially reversed increased ROS production induced by 1 mg/mL ASP (Figure 4F). Similar results were found in cleaved caspase 3 assay. Pretreated with NAC effectively reduced cleaved caspase 3 activity by 82.1% in the KLE cells and 58% in the Ishikawa cells, respectively (Figure 4G). Importantly, the combination of NAC and ASP treatment partially reversed the inhibitory effect of 1 mg/mL ASP on cell proliferation, from 52.5% to 26.8% in the KLE cells, and 56.3%–29.6% in the Ishikawa cells, respectively (Figure 4H). These results suggest that cellular stress is associated with ASP-induced inhibition of cell proliferation and tumor growth in EC.

Our previous study found that ASP extract effectively inhibited cell adhesion and invasion and reduced VEGF expression in ovarian cancer cell lines and in a transgenic mouse model of ovarian cancer (Xu et al., 2021). To investigate the role of ASP in cellular adhesion and invasion in EC cells, the KLE and Ishikawa cells were cultured in laminin-1-coated 96-well plates and treated with 0.01, 0.1, and 1 mg/mL ASP extracts for 2 hours. The results showed that ASP significantly inhibited cell adhesion ability in a concentration-dependent manner in both cell lines (Figure 5A). Similarly, ASP extract decreased the invasive ability of the KLE and Ishikawa cells using a transwell migration assay. Cell invasion was reduced by 41.5% and 28.3% in the KLE and Ishikawa cells, respectively, after treatment with 1 mg/mL ASP for 6 h (Figure 5B). To evaluate the effect of ASP on epithelial-to-mesenchymal transition (EMT) and angiogenesis in EC cells, both cell lines were treated with 0.01, 0.1, and 1 mg/mL ASP extracts for 18 h. Western blotting results demonstrated that ASP decreased the expression of VEGF, β-catenin, Slug and vimentin and increased the expression of E-Cadherin in the KLE and Ishikawa cells (Figure 5C). More importantly, in the EC tissues of Lkb1 ^fl/fl p53 ^fl/fl mice, IHC staining results showed that HFD significantly increased the expression of VEGF compared with LFD mice, and ASP extract effectively reduced VEGF expression in obese and lean conditions (Figure 5D). These results confirm that ASP may reduce adhesion and invasion in EC cells and Lkb1 ^fl/fl p53 ^fl/fl mice.

Given that AKT/mTOR and MAPK pathways are involved in the anti-tumorigenic activity of ASP in cancer (Zhang et al., 2023; Li et al., 2023; Liang et al., 2022), the effect of ASP on the AKT/mTOR and MAPK pathways in EC cells was detected by Western blotting using phosphorylated AMPK, AKT, p42/44, and S6 antibodies. As shown in Figure 6A, ASP extracts decreased the expression of phosphorylated AKT, p42/44, and S6 and increased the expression of AMPK after the KLE and Ishikawa cells were treated with ASP extracts for 18 h. IHC results demonstrated that HFD significantly promoted the expression of phosphorylated S6 compared with the LFD group. ASP effectively reduced the expression of phosphorylated-S6 in obese and lean mice after 4 weeks of treatment (Figure 6B).

To further investigate the regulatory role of AKT/mTOR/S6 pathway on ASP-induced cell proliferation, the KLE and Ishikawa cells were treated with rapamycin (a mTOR inhibitor), ASP extract, and the combination of ASP and rapamycin overnight. The results of Western blotting confirmed that rapamycin (0.5 nM) significantly reduced phosphorylated S6 expression compared to untreated cells, and the combination of ASP (0.01 mg/mL) and rapamycin showed a more potent inhibitory effect on the expression of phosphorylation of S6 in both cells (Figure 6C). When compared to ASP or rapamycin treatment alone, the combined treatment in both cell lines showed more pronounced inhibitory effects on cell proliferation after 72 h of treatment (Figure 6D). Similar results were found for the effect of combination treatment on cleaved caspase 3 activity in both cells, where ASP combined rapamycin increased more activity of cleaved caspase 3 in both cells compared to single agent after 12 h of treatment (Figure 6E). Overall, these results suggest that the ability of ASP to inhibit cell proliferation is dependent on the PTEN/AKT/S6 pathway in EC.

Platinum-based chemotherapy is the standard therapeutic strategy for patients with advanced EC. To evaluate the synergistic effect of ASP on cisplatin in EC cells, the KLE and Ishikawa cells were treated with varying concentrations of cisplatin (0.001–250 µM) and then in combination with 0.01, 0.1 and 1 mg/mL ASP for 72 h. MTT assay was used to evaluate cell viability. Relative cell proliferation was reduced in a dose dependent manner following cisplatin treatment. Treatment of both cell lines with 0.01, 0.1 and 1 mg/mL ASP in combination with cisplatin (0.01–50 uM) significantly enhanced the inhibitory effect on cell proliferation compared with ASP or cisplatin alone (Figures 7A, B). The combination index (CI) calculated by CompuSyn software also confirmed that the combination of low-dose ASP and low-dose cisplatin produced a synergistic cytotoxic effect (CI < 1) in the 2 cell lines (Figure 7C). To investigate the effect of the combination of ASP and cisplatin on cleaved caspase 3 activity, ELISA assay showed that the combination of 0.1 mg/mL ASP and 10 uM cisplatin elevated cleaved caspase 3 activity to 1.83-fold compared with 1.55-fold and 1.41 of ASP and cisplatin, respectively, in the KLE cells, and to 2.16-fold compared with 1.37-fold and 1.48-fold of ASP and cisplatin in the Ishikawa cells, respectively (Figure 7D). These results indicate that the combination of ASP and cisplatin produces a synergistic effect in inhibiting cell proliferation in EC cells.

Given the hypoglycemic effect of ASP in diabetic rats (Zhao et al., 2011; Hafizur et al., 2012), the effect of ASP extract on glycolysis was investigated in the KLE and Ishikawa cells. 1 mg/mL ASP significantly reduced glucose uptake by nearly 17.8% in the KLE cells and by 22.1% in the Ishikawa cells after 12 h of treatment compared with control cells (Figure 8A). Western blotting results showed that ASP decreased the expression of GLUT 1 and GLUT 4 in both cell lines after 12 h of treatment (Figure 8B). Finally, we measured the production of lactate in the culture media and the cellular ATP levels by ELISA assays. ASP decreased lactate production by 21.2% in the KLE cells and 26.4% in the Ishikawa cells in cell culture media compared to untreated cells, respectively (Figure 8C). In addition, 1 mg/mL ASP significantly reduced cellular ATP level by 23.3% in the KLE and 25.6% in the Ishikawa cells, respectively, after 24 h of treatment (Figure 8D, p < 0.01). These results support the ability of ASP to reduce glycolytic activity in EC cells.’