The human ovarian cancer cell lines OV433, OVCAR5, OVCAR3, MES, and paclitaxel-resistant MES-TP were utilized in this study. The MES-TP cell line was kindly provided by Dr. Sikic (Stanford University School of Medicine) and was established by stepwise exposure of MES cells to increasing concentrations of paclitaxel in combination with the P-glycoprotein inhibitor PSC833 (Moisan et al., 2014). This cell line is at least 10-fold more resistant to paclitaxel than its parental cell line (MES), with increased expression of P-glycoprotein (Zhang et al., 2023). Both MES and MES-TP were grown in McCoy’s 5A medium. OV433 cells were cultured in the RPMI 1640 medium, while OVCAR5 and OVCAR3 cells were maintained in the DMEM/F12 medium. All mediums contained 10% fetal bovine serum (FBS) (Thermo Fisher Scientific; Waltham, MA), 1% penicillin/streptomycin, and 1% L-Glutamine (Gibco Cell Culture, CA). All cell lines were cultured under humidified conditions with 5% CO₂ at 37°C. Sulindac sulfide was purchased from MedChemExpress (Newark, NJ). MTT, N-acetylcysteine (NAC), Z-VAD-FMK, and paclitaxel (PTX) were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies used were from Cell Signaling Technology (Beverly, MA) and ABclonal (Woburn, MA). The Western Lightning™ Plus Chemiluminescence Reagent was purchased from PerkinElmer (Waltham, MA).

4,000–8,000 cells per well were incubated in 96-well plates overnight and treated with various doses of sulindac (ranging from 10 to 500 μM) for 72 h 5 μL of MTT solution (5 mg/mL) was added to each well, and the plate was incubated for another 1 h at 37°C. The absorbance of MTT was measured at 562 nm using a microplate reader (Tecan; Morrisville, NC) after adding 100 μL DMSO to each well. The effect of sulindac on OC cell proliferation was evaluated as a percentage of control cell growth, and then IC50 analysis was determined using the AAT Bioquest calculator (Sunnyvale, CA).

The MES cells and OVCAR5 cell lines were plated at a density of 200 and 400 cells/well in 6-well plates respectively, overnight. Subsequently, the plates were treated with varying concentrations of sulindac (25, 75, and 100 μM) for 72 h. The cells were cultured continuously for 14 days, and the medium was changed every 3 days. Cells were stained with 0.5% crystal violet, and colonies were counted under a microscope for analysis.

Intracellular ROS levels were quantified by DCFH-DA assay. The MES and OVCAR5 cells were seeded at a density of 8,000 cells/well in 96-well plates overnight, and then treated with 25, 75, and 100 μM sulindac for 8 h at 37°C. After treatment, the supernatants were removed, and phenol red-free medium containing 15 µM DCFH-CA (Sigma-Aldrich) was added to each well for an additional 30-minute incubation. The fluorescence intensity was then measured at Ex/Em 485/525 nm using a Tecan microplate reader.

The MES and OVCAR5 cells were cultured at 1.5 × 10⁴ cells/well and 2.5 × 10⁴ cells/well, respectively, in 96-well plates overnight. The cells were treated with sulindac at doses of 25, 75, and 100 μM for 6 h and then incubated with 200 µM JC-1 dye (Sigma-Aldrich) for 30 min at 37°C in the dark. After washing each well with warm PBS, the green JC-1 signal was detected at Ex/Em 485/535 nm, and the red JC-1 signal was measured at Ex/Em 535/590 nm using a Tecan microplate reader.

The MES and OVCAR5 cells, plated at 1 × 10⁵ and 1.5 × 10⁵ cells/well, respectively, were incubated in black clear-bottom 96-well plates overnight. The cells were then exposed to 25, 75, and 100 μM sulindac for 6 h. After removing the supernatants, 100 µL of 1 mM TMRE (Sigma-Aldrich) was added to each well and incubated for an additional 30 min at 37°C in the dark. After washing each well with PBS, the plate was measured at Ex/Em 549/575 nm using a Tecan microplate reader.

The MES and OVCAR5 cells were plated in 6-well plates at a density of 2.5 × 10⁵ cells overnight, and then treated with 25, 75, and 100 µM sulindac for 24 h. After treatment, the cells were harvested using 0.25% trypsin (Thermo Fisher Scientific) and fixed in 100% methanol for 30 min. The cells were then resuspended in a solution containing 10 mM propidium iodide (PI), 50 ug RNase A, and 0.05% Triton X-100 and then incubated at 37°C for 30 min in the dark. Cell cycle progression was measured by Cellometer (Nexcelom, Lawrence, MA), and the results were analyzed using FCS7 Express software (Molecular Devices, Sunnyvale, CA).

The MES and OVCAR5 cells were cultured at a concentration of 2.5 × 10⁵ cells/mL in 6-well plates overnight, followed by treatment with sulindac at 25, 75, and 100 μM for 14 h. The cells were then lysed using 150 µL/well of 1X caspase lysis buffer, and the protein concentration was determined using the BCA assay (Thermo Fisher Scientific). Lysates (30–40 µg) were added to a black clear bottom 96-well plate and incubated with reaction buffer containing 200 µM caspase substrates for 30 min. Specifically, the substrates Ac-DEVD-AMC, Ac-IETD-AFC, and Ac-LEHD-AMC (AAT Bioquest) were used to detect cleaved caspase 3, 8, and 9 activities, respectively. The fluorescence intensity of cleaved caspase 3 and 9 was measured at an excitation/emission (Ex/Em) wavelengths of 341/441 nm, while the fluorescence intensity of cleaved caspase 8 was measured at Ex/Em 376/482 nm using a Tecan microplate reader.

96-well plates were coated with 80 μL of laminin-1 (10 μg/mL) (Sigma-Aldrich) and incubated overnight at 4°C before use. After each well was treated with a blocking buffer (0.5% BSA in PBS) for 30 min, 1 × 10⁵/well of MES and OVACR5 cells, and varying concentration of sulindac (25, 75, and 100 μM) were added, followed by incubation for 90 min. After aspirating the supernatants, the adhered cells were fixed with 5% glutaraldehyde for 30 min and stained with 0.1% crystal violet for 15 min at room temperature. After washing twice with PBS, 100 µL of 10% acetic acid was added to each well to dissolve the dye. The absorbance was read at 575 nm using a Tecan microplate reader.

The MES and OVCAR5 cells were seeded in 6-well plates at a density of 5 × 10⁵/well and cultured overnight. After replacing the culture medium to containing 1% FBS, three uniform wounds were scratched across the cell monolayer using a 200 μL pipette tip. The cells were then gently washed twice with PBS and treated with sulindac (5, 10, and 25 μM) for 24 h. Images of each well were taken using a microscope at 24 h after treatment. Wound width was measured and calculated using the ImageJ software (National Institutes of Health; Bethesda, MD).

The MES and OVCAR5 cells were seeded at a density of 2.5 × 10⁵ cells/well in 6-well plates and cultured overnight. Then cells were treated with sulindac at doses of 25, 75, and 100 μM for 8–24h. Total cell lysate was prepared using pre-cold RIPA lysis buffer, and the protein concentration was determined by BCA assay (Thermo Fisher Scientific). Equal amounts of 20–25 µg protein were electrophoresed on 10% or 12% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad; Hercules, CA) at 4°C for 90–120 min. After blocking with 5% fat-free milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with appropriate primary antibodies. The detailed information of primary antibodies was listed in (Supplementary Table S1). The next day, membranes were washed with TBS-T buffer and then incubated with secondary antibodies for 1 h at room temperature. Membranes were developed using the Western Lightning™ Plus-ECL (PerkinElmer) and visualized using the ChemiDoc Image System (Bio-Rad, Hercules, CA). The quantitative assessment of protein expression was analyzed by ImageJ software.

The K18−gT₁₂₁ ^+/−; p53^fl/fl; Brca1^fl/fl (KpB) mouse model of high-grade serous epithelial OC has been described previously (Makowski et al., 2014; Szabova et al., 2012). Our animal protocol (21–229) was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina at Chapel Hill. For the induction of ovarian tumors, 5 ul of 2.5 × 10⁷ P.F.U of recombinant adenovirus Ad5-CMV-Cre (AdCre, Transfer Vector Core, University of Iowa) was injected into the left ovarian bursa cavity of KpB mice at 6–8 weeks of age. Mice were examined weekly by abdominal palpation for the appearance of ovarian tumors. When ovarian tumors reached 0.1–0.2 cm in diameter, the mice were randomly divided into two groups (15 mice per group): a control group and a sulindac-treated group. Mice in the treatment group were treated with 7.5 mg/kg of sulindac daily via oral gavage for 4 weeks. Throughout the treatment period, all animals were checked daily for any signs of toxicity and weighed weekly. The size of ovarian tumors was checked twice a week by palpation. Mice were euthanized by CO₂ asphyxiation after finishing the treatment. Ovarian tumors and blood samples were weighted and stored at −80°C until further use. Ovarian tumor volumes were calculated using the formula: (width² × length)/2.

The mouse ovarian tumor tissues were fixed in formalin, then paraffin-embedded and sectioned into 4 μm slides at the Animal Histopathology Core Facility at UNC-CH. The slides were deparaffinized with xylene and hydrated with ethanol. Subsequently, fresh antigen retrieval buffer was used to boil the slides for 3 min in a pressure cooker, followed by soaking in cold water for 10 min. To block nonspecific binding, the slides were treated with a protein block solution (Dako, Agilent Technologies, Santa Clara, CA) for 1 h at room temperature and then incubated with primary antibodies for Ki-67 (1:400), Bcl-xL (1: 1,200), and Cox-2 (1:200) overnight at 4°C. Slides were washed three times with TBST buffer and then incubated with secondary antibodies (Biotinylated goat anti-rabbit, Vector Labs, Burlingame, CA, United States) at room temperature for 1 h. Afterward, the slides were subjected to an ABC Substrate System (Vector Labs, Burlingame, CA) for color development and counterstained with Mayer’s hematoxylin. All IHC slides were scanned using Motic (Houston, TX) and scored by ImagePro 10 software (Vista, CA).

Serum inflammatory cytokines and chemokines were measured using the mouse multiplexed Luminex assays (Bio-Rad, Hercules, CA) according to the manufacturer’s protocols in the Animal Core Facility at UNC-CH. The plate was measured using the Luminex-200 system (Austin, TX).

All experiments were repeated at least three times for consistency of results. Data were presented as mean ± SD. Statistical analysis and graphical representation were generated using GraphPad Prism eight software (La Jolla, CA). A two-sided unpaired Student’s t-test was used for comparisons between two groups with at least three replicates. One-way ANOVA with Tukey’s multiple comparison test was used when comparing multiple groups. The combination index of sulindac and PTX were calculated by CompuSym software (Biosoft, MO, United States). Statistically significant was set at P values <0.05. All in vitro experiments were repeated at least 3 times.

To investigate the effect of sulindac on cell proliferation, the OV433, MES, OVCAR5, and OVCAR3 cell lines were treated with sulindac at concentrations ranging from 10 to 500 μM for 72 h. The results of the MTT assay revealed that sulindac reduced cell proliferation in a dose-dependent manner in four OC cell lines. The mean IC50 values of sulindac were 90.5 ± 2.4 µM, 76.9 ± 1.7 µM, 80.2 ± 1.3 µM and 52.7 ± 3.7 µM for OV433, OVCAR5, MES and OVCAR3 cells, respectively (Figure 1A). Colony formation assay was used to assess the long-term effect of sulindac on cell proliferative capability. The MES and OVCAR5 cells were exposed to 25, 75, and 100 µM sulindac for 72 h and then cultured for 14 days. The results showed that 100 µM sulindac was able to reduce the colony formation of both cell lines, with the colony formation reduced by 95.0% and 81.9% in MES and OVCAR5 cells, respectively, compared to untreated cells (Figure 1B). Western blotting results demonstrated that sulindac effectively decreased the expression of phosphorylated Akt and phosphorylated S6 in both cell lines after 16 h of treatment (Figure 1C).

To investigate the effect of sulindac on tumor growth, the KpB transgenic mice of OC were treated with sulindac for 4 weeks (7.5 mg/kg, daily, oral garage) when ovarian tumors reached approximately 0.1–0.2 cm in diameter by palpation. During the treatment period, mice exhibited tolerance to sulindac treatment without any discernible changes in behavior and body weight. Sulindac-treated mice demonstrated a significant 73.7% reduction in tumor volume and 67.1% reduction in tumor weight compared with the control mice at the end of the treatment (Figures 1D,E). IHC staining showed that Ki-67 expression in sulindac-treated tumors was significantly decreased by 48.6% compared with untreated tumors (Figure 1F). These findings demonstrated sulindac is effective in inhibiting cell proliferation and tumor growth in vitro and in vivo.

To determine the effect of sulindac on Cox-2 pathway in OC cells, the MES and OVCAR5 cells were treated with sulindac at 25, 75, and 100 µM for 16 h. Western blotting results showed that sulindac at 75 and 100 µM significantly reduced the expression of Cox-2 in both cell lines (Figure 2A). Since NF-κB is closely involved in regulating the biological effects of sulindac and TNF-α is commonly used to induce NF-κB activity (Yi et al., 2021), OVCAR5 cells were pretreated with 75 µM sulindac for 3 h and then treated with 10 ng/mL of TNF-α for 15 and 30 min. Western blotting showed that sulindac treatment dramatically reduced the expression of phosphorylated NF-κB induced by TNF-α (Figure 2B). These results suggest that sulindac is capable of inhibiting the Cox-2 pathway and TNF-α induced NF-κB activity in OC cells.

To further investigate the effect of sulindac on inflammatory response in vivo, the serum levels of inflammatory cytokines and chemokines from KpB mice were measured by ELISA assay. Compared to the control group, the serum interleukin-10 (IL-10) level was significantly decreased, and the serum interferon-γ (INF-γ), chemokine (C-C motif) ligand 7 (CCL7), chemokine (C-X-C motif) ligand 16 (CXCL 16), chemokine (C-C motif) ligand 24 (CCL24), and chemokine (C-C motif) ligand 11 (CCL11) levels were decreased in the sulindac-treated group (Figure 2C). Meanwhile, the expression of Cox-2 of OC tissues in sulindac-treated mice was reduced by 66.9% compared to control mice (Figure 2D). Altogether, these results suggest that sulindac treatment for 4 weeks significantly improves the inflammatory environment in vivo and reduces Cox-2 activity in ovarian tumors.

To determine the effects of sulindac on cellular stress in OC cells, the MES and OVCAR5 cells were treated with 25, 75, and 100 µM sulindac for 8 h. ROS levels were quantified using the DCFH-DA assay. Treatment with 75 and 100 μM sulindac significantly increased cellular ROS production in the MES and OVCAR5 cells. Sulindac at 100 μM increased the level of ROS by 49.3% in MES cells and 31.3% in OVCAR5 cells, respectively (Figure 3A). Next, JC-1 and TMRE assays were used to detect the effect of sulindac on mitochondrial membrane potential. Compared with control cells, sulindac at doses of 75 and 100 µM effectively reduced mitochondrial membrane potential in both cells after 6 h of treatment, with 100 µM sulindac reducing JC-1 levels by 46.7% and 20.0% in MES and OVCAR5 cells, and TMRE levels by 21.5% and 22.2% in MES and OVCAR5 cells, respectively (Figures 3B,C). Western blotting results revealed that sulindac unregulated the expression of cellular stress-related proteins BiP, ATF-4, and PDI in both MES and OVCAR5 cells following 8 h of treatment (Figure 3D). These results confirmed that sulindac exerts a pro-stress role in inhibiting cell proliferation in OC cells.

To investigate the potential modulation of cell cycle progression by sulindac, cell cycle profiles were measured via Cellometer after treatment of the MES and OVCAR5 cells with 25, 75 and 100 μM sulindac for 24 h 75 and 100 μM sulindac significantly induced cell cycle G1 phase arrest and decreased G2 phase in both the MES and OVCAR5 cells. Sulindac at a dose of 100 µM increased the G1 arrest phase from 46.74% in untreated cells to 62.8% in MES cells, and from 50.0% to 65.6% in OVCAR5 cells, respectively (Figure 4A). Western blotting results confirmed that after 24 h of sulindac treatment, the expression of cell cycle-related proteins such as CDK4, CDK6, and cyclin D1 were decreased in both the MES and OVCAR5 cells (Figure 4B). These data confirm that sulindac induces cell cycle G1 arrest in OC cells.

To evaluate whether sulindac induces apoptosis in OC cells, the levels of cleaved caspase 3, 8, and 9 in the MES and OVCAR5 cells were measured by ELISA assays after 14 h of treatment. Sulindac (100 µM) significantly increased the production of cleaved caspase 3, 8, and 9 by 1.86-fold, 1.26-fold, and 1.28-fold in the MES cells, respectively, and 1.78-fold, 1.20-fold, and 1.74-fold in the OVCAR5 cells, respectively, compared with untreated cells (Figures 5A–C). Meanwhile, Western blotting showed that sulindac treatment for 8 h reduced the expression of Mcl-1 and Bcl-xl, while increasing the expression of Bax in both cell lines (Figure 5D). IHC staining further confirmed that the expression of Bcl-xL was reduced by 66.0% in ovarian tumors of KpB mice compared with the control group (Figure 5E).

To further explore the role of mitochondrial apoptotic pathway in sulindac-induced apoptosis, the MES and OVCAR5 cells were pretreated with a pan-caspase inhibitor Z-VAD-FMK (10 µM) for 2 h, followed by treatment with 75 µM sulindac. Pre-treatment with 10 μM of Z-VAD-FMK effectively blocked the increase in cleaved caspase 3 level induced by 14 h of sulindac treatment in both cell lines (Figure 5F). Similar results were observed via MTT assay. Pre-treatment with Z-VAD-FMK greatly reduced the inhibition of cell proliferation induced by 72 h of sulindac treatment in both cell lines, with inhibition decreasing from 28.9% to 7.2% in MES cells and from 43.9% to 18.3% in OVCAR5 cells, respectively (Figure 5G).

To assess the effect of sulindac on cell adhesion and migration, laminin-1 adhesion and wound healing assays were conducted in the MES and OVCAR5 cells. 75 and 100 µM sulindac significantly suppressed the cell adhesion in MES and OVCAR5 cells. Specifically, sulindac at a dose of 100 µM inhibited adhesion by 31.4% in MES cells and 22.7% in OVCAR5 cells, respectively, compared with control cells (Figure 6A). The wound healing assay demonstrated that sulindac inhibited cell migration in both cell lines. Treatment with 25 µM sulindac for 24 h significantly increased wound healing width by 2.0-fold and 2.13-fold in MES and OVCAR5 cells, respectively, compared with untreated cells (Figure 6B). Meanwhile, sulindac effectively decreased the expression of EMT-related proteins including Slug and β-Catenin after 24 h of treatment in both cells (Figure 6C). These findings indicate that sulindac has the ability to inhibit adhesion and invasion in OC cells.

Given that sulindac induces cellular stress in OC cells, which is a trigger for the induction of apoptosis and cell cycle arrest, the role of cellular stress pathways in sulindac-induced cell growth inhibition was investigated. The MES and OVCAR5 cells were pretreated with the antioxidant N-acetylcysteine (1 mM) for 2 h, followed by treatment with 75 µM sulindac for 8 h. ROS assay revealed that pre-treatment with NAC totally reversed the increase in ROS levels induced by 75 µM sulindac treatment in both cell lines (Figure 7A). Pretreatment with NAC (1 mM for 4 h) also partially reversed sulindac-induced (75 µM) cleaved caspase 3 activities from 34.8% to 13.6% in MES cells and from 45.1% to 15.9% in OVCAR5 cells (Figure 7B). Similar to caspase 3 results, NAC partially reversed the inhibition of proliferation induced by 75 µM sulindac for 72 h in both cells, reducing the proliferation inhibition from 31.0% to 9.4% in MES cells and from 42.9% to 21.1% in OVCAR5 cells, respectively (Figure 7C). Importantly, blocking cell stress by NAC also partially reversed the inhibition of cell migration induced by 10 µM sulindac for 24 h, as the wound width was reduced from 40.8% to 14.4% in MES cells, and from 89.3% to 13.9% in OVCAR5 cells (Figure 7D). Western blotting confirmed that combination pretreatment with NAC and sulindac decreased the sulindac-stimulated BiP and ATF-4 expression, and increased the levels of Mcl-1, Bcl-xL, and Slug compared with monotherapy in both cell lines (Figure 7E). All these results indicate that anti-proliferative and anti-metastatic effects of sulindac are partially dependent on cellular stress pathways in OC cells.

In order to explore whether sulindac and paclitaxel have synergistic effects in OC cells, the effects of sulindac combined with PTX on the cell proliferation of MES and its PTX-resistant cell line MES-TP were studied. After treatment of the two cell lines with of PTX (0.1, 1, 10, 25, 50 and 100 nM) and sulindac (1, 10, 25, 50, 75, 100, 150 and 250 µM) for 72 h, the IC50 values of PTX were different in both cell lines, with an IC50 value of 15.4 ± 0.7 nM in MES cells and an IC50 value of 126.0 ± 13.0 μM in MES-TP cells. However, they showed similar inhibitory responses to sulindac treatment (Figure 8A). According to the inhibitory response of the two cell lines to sulindac and PTX, three doses of sulindac (25, 50, and 75 µM) and four doses of PTX (1, 10, 25, and 50 nM) were chosen to co-treat the MES and MES-TP cells for 72 h. The combination index (CI) values were calculated using CompuSyn Software based on the Chou-Talalay model (Figure 8B). Sulindac at a dose of 75 µM synergistically increased the sensitivity of both cell lines to PTX (Figure 8C, CI < 1). Furthermore, 75 µM sulindac combined with 5 nM PTX produced more potent in inducing cleaved caspase 3 activity compared with either agent alone in both cell lines (Figure 8D). Western blot results showed that the combination of 75 µM sulindac and 5 nM PTX produced greater effects on decreasing expression of DNA damage markers, including phosphorylated H2A.X and Rad51, and increasing expression of the apoptotic marker Bcl-xL, than sulindac or PTX alone in both cells (Figure 8E). These results indicate that sulindac significantly increases the sensitivity of PTX both in PTX sensitive or resistant OC cells; and thus, may be useful in overcoming resistance to PTX.