Sulindac exhibits anti-proliferative and anti-invasive effects and enhances the sensitivity to paclitaxel in ovarian cancer

Abstract

Objective: Chronic inflammation is a key contributor to carcinogenesis, progression, and chemoresistance in ovarian cancer, making inflammatory pathways a logical therapeutic target for the treatment of this disease. Sulindac, a commonly used non-steroidal anti-inflammatory drug, has demonstrated anti-proliferative and anti-invasive effects on several preclinical models of cancer. In this study, we investigated the antitumorigenic effects of sulindac in human ovarian cancer cell lines and a transgenic mouse model of ovarian cancer (KpB).

Methods: MTT and colony formation assays were used to evaluate cell proliferation. Cell cycle was detected by Cellometer. ELISA assays were conducted to evaluate the changes of cellular stress, apoptosis and adhesion, while invasion was determined by wound healing assay. Protein expression was examined through Western blotting and immunohistochemistry.

Results: Our results demonstrated that sulindac significantly inhibited cell proliferation, induced cellular stress and apoptosis, caused G1 phase cell cycle arrest, and reduced cell invasion, and suppressed Cox-2 and NF-κB pathways in the MES and OVCAR5 cell lines. Inhibition of cellular stress by N-acetylcysteine partially reversed the anti-proliferative and anti-invasive effects of sulindac. The combination of sulindac and paclitaxel produced synergistic effects in inhibiting cell growth in both paclitaxel sensitive and resistant MES cells. Treatment with sulindac for 4 weeks effectively reduced tumor growth, improved serum levels of inflammatory cytokines and chemokines, and reduced the expression of Cox-2 of ovarian tumors in KpB mice compared with untreated mice.

Conclusions: These findings provide support for the development of clinical trials repurposing sulindac in the treatment of OC, possibly in combination with paclitaxel.

Keywords: cell proliferation; invasion; ovarian cancer; paclitaxel; sulindac; synergy.

FIGURE 1

Sulindac inhibited cell proliferation and tumor growth in OC cell lines and KpB mice. MTT assay demonstrated that sulindac inhibited cell proliferation in a dose-dependent manner in OV433, OVCAR5, MES, and OVCAR3 cells after 72 h of treatment (A). The MES and OVCAR5 cells were treated with sulindac for 72 h at 25, 75, and 100 μM, and then cultured for an additional 14 days. Colony assay showed that sulindac inhibited colony formation (B). Sulindac reduced the expression of phosphorylated Akt and phosphorylated S6 in MES and OVCAR5 cells after 16 h of treatment (C). The KpB mice were treated with sulindac (7.5 mg/kg, daily) or vehicle for 4 weeks. Sulindac effectively reduced tumor volume and tumor weight compared with control mice (n = 15) (D,E). IHC results demonstrated that sulindac treatment decreased the expression of Ki-67 in OC tissues from KpB mice (n = 6) (F). Data are presented as mean ± SD. Statistical significance was assessed using two-sided unpaired Student’s t-test (two groups) or one-way ANOVA with Tukey’s post hoc test (multiple groups). *p < 0.05, **p < 0.01, compared with control.

FIGURE 2

Sulindac inhibited inflammatory responses in OC cell lines and the KpB mice. Sulindac decreased the expression of Cox-2 in MES and OVCAR5 cells after 16 h of treatment (A). Pretreatment of OVCAR5 cells with 75 µM sulindac for 3 h followed by treatment with 10 ng/mL TNF-α for 15 and 30 min resulted in a decrease in TNF-α-induced expression of NF-κB phosphorylation (B). Sulindac treatment for 4 weeks affected serum concentrations of IL-10, INF-γ, CCL7, CXCL16, CCL24, and CCL11 in KpB mice compared with control mice (n = 15) (C). IHC results showed that sulindac inhibited the expression of Cox-2 in OC tissues of KpB mice (n = 6) (D). Data are presented as mean ± SD. Statistical significance was assessed using two-sided unpaired Student’s t-test (two groups) or one-way ANOVA with Tukey’s post hoc test (multiple groups). *p < 0.05, **p < 0.01, compared with control. #p < 0.05, ##p < 0.01, between groups.

FIGURE 3

Sulindac induced cellular stress in OC cells. Sulindac significantly increased ROS levels in the MES and OVCAR5 cells after 8 h of treatment (A). The JC-1 and TMRE assays demonstrated that sulindac decreased mitochondria membrane potential in the MES and OVCAR5 cells (B,C). Western blotting results showed that sulindac increased expression of cell stress-related proteins such as BiP, ATF-4, and PDI in both cell lines after 8 h of treatment (D). Data are presented as mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01 compared with control.

FIGURE 4

Sulindac induced cell cycle G1 arrest in OC cells. Sulindac treatment resulted in cell cycle G1 phase arrest in the MES and OVCAR5 cells after 24 h of treatment (A). Western blotting results showed that sulindac inhibited the expression of CDK4, CDK6 and cyclin D1 after 24 h of treatment in both cell lines (B). Data are presented as mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01 compared with control.

FIGURE 5

Sulindac induced apoptosis in OC cells and KpB mice. The results of ELISA assay showed that treatment with 75 and 100 µM sulindac for 14 h significantly increased the activities of cleaved caspase 3, 8, and 9 in the MES and OVCAR5 cells (A–C). Sulindac increased the expression of Bax and decreased the expression of Mcl-1 and Bcl-xL in both cell lines (D). IHC results revealed that sulindac inhibited the expression of Bcl-xL in OC tissues of KpB mice (n = 6) (E). Z-VAD-FMK (10 µM) was pretreated with both cell lines for 2 h followed by treatment with sulindac for 14 and 72 h. Pretreatment with Z-VAD-FMK effectively blocked the 75 µM sulindac-induced cleaved caspase 3 activity (F). The MTT assay showed that pretreatment with Z-VAD-FMK significantly reversed sulindac (75 µM)-induced inhibition of cell proliferation in both cell lines (G). Data are presented as mean ± SD. Statistical significance was assessed using two-sided unpaired Student’s t-test (two groups) or one-way ANOVA with Tukey’s post hoc test (multiple groups). *p < 0.05, **p < 0.01, compared with control. #p < 0.05, ##p < 0.01, between groups.

FIGURE 6

Sulindac inhibited cell adhesion and invasion in OC cells. The laminin-1 assay showed that treatment with 75 and 100 µM sulindac significantly inhibited cell adhesion in the MES and OVCAR5 cells (A). The wound healing assay demonstrated that 10 and 25 µM sulindac inhibited cell migration after 24 h of treatment in the MES and OVCAR5 cells (B). Western blotting showed that sulindac reduced the expression of Slug and β-Catenin after 24 h of treatment in both cell lines (C). Data are presented as mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, compared with control.

FIGURE 7

Anti-proliferative and anti-invasion activities of sulindac depends on cell stress pathway in OC cells. The MES and OVCAR5 cells were pre-treated with N-acetylcysteine (NAC, 1 mM) for 2 h and then treated with 75 µM sulindac for 8 h. ROS assays revealed that pretreatment with NAC completely reversed the sulindac-induced increase in ROS levels in both cell lines (A). Pretreatment with NAC for 4 h partially reversed 75 µM sulindac-induced cleaved caspase 3 activities in the MES and OVCAR5 (B). The MTT assay demonstrated that NAC partially reversed the inhibition of proliferation induced by 75 µM sulindac for 72 h in both cell lines (C). Pretreatment with NAC effectively reversed the inhibition of cell migration induced by 10 µM sulindac for 24 h in the MES and OVCAR5 cells (D). Western blot analysis showed that the expression of BiP, ATF-4, Mcl-1, and Bcl-xL was altered after treatment with sulindac, NAC, or the combination of sulindac and NAC (E). Data are shown as mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, compared with control. #p < 0.05, ##p < 0.01, between groups.

FIGURE 8

The combination of sulindac and PTX synergistically enhances cell growth inhibition in PTX-sensitive and -resistant OC cells The MES and MES-TP cells were treated with sulindac (1, 10, 25, 50, 75, 100, 150 and 250 µM), PTX (0.1, 1, 10, 25, 50 and 100 nM), and the combination for 72 h. MTT assay revealed the inhibitory effects of sulindac and PTX on cell proliferation (A). The CI value for the combination of sulindac (25, 50, and 75 µM) and PTX (1, 10, 25 and 50 µM) was calculated by CompuSyn for each combination group (B). MTT assay revealed changes in cell proliferation after treatment with sulindac (75 µM) and PTX (1, 10, 25, and 50 nM) in both cells (C). In both cell lines, the combination of sulindac (75 µM) and PTX (5 nM) significantly increased cleaved caspace3 levels after 14 h of treatment compared with either agent alone (D). Western blotting demonstrated the changes in the expression of phosphorylated H2A.X, Rad51, and Bcl-xL after treatment with sulindac, PTX or their combination in both cell lines (E). Data are presented as mean ± SD from three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, compared with control. #p < 0.05, ##p < 0.01, between groups.