Four ovarian cancer cell lines, OVCAR3, OVCAR5, IGROV-1 and SKOV3, were used in this study. The cells were grown in DMEM/F12(1:1) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100U/ml penicillin and 100 ug/ml streptomycin under 5% CO₂. ONC201 was kindly provided by Oncoceutics, Inc. MTT, propidium iodide and RIPA buffer were purchased from Sigma (St. Louis, MO). Antibodies to PERK (#5683), ATF4 (#11815), CHOP (#2895), BCL-XL (#2764), MCL-1 (#5453), PARP (#9542), DR5 (#8074), β-actin (#3722), VEGF-C (#2445), Slug (#9585), Snail (#3879), CyclinD1 (#2978), CDK4 (#12790), and CDK6 (#3136) were obtained from Cell Signaling Technology (Beverly, MA). DRD2 (B-10, sc-5303) and DRD5 (E-12, sc-376088) were purchased from Santa Cruz Biotechnology. The TMRE (#22220) and JC-1 (#22200) probes were purchased from AAT bioquest (Sunnyvale, CA).

The four OC cell lines were incubated in 96-well plates (4000 cells/well) in the presence of varying concentrations of ONC201 for 72 hours. The control group received vehicle only (0.1% DMSO). 5ul MTT (5 mg/mL) was added in each well following by incubation for 1-2 hours at 37°C. MTT absorbance was measured at 575 nm after 100 ul DMSO was added to the plates. Effects of ONC201 on cell proliferation were assessed as a percentage of control cell proliferation obtained from 0.1% DMSO treated cells grown in the same 96-well plates followed by IC50 analysis.

The OVCAR5 and SKOV3 cells were plated at a density of 200 cells in 6-well plates in triplicated for each treatment group for 24 hours. The cells were treated with indicated concentrations of ONC201 or vehicle controls for 48 hours and then incubated at 37°C for 10-14 days. The culture medium was changed every third or fourth day. The cells were fixed in methanol and stained with 0.5% crystal violet. The colonies were counted utilizing Photoshop software.

The cells were grown in in 6-well plates overnight and then incubated with varying concentrations of ONC201 or vehicle control for 36 hours. Cells were harvested, washed with PBS, fixed in 2 ml of ice-cold 90% ethanol and stored overnight at -20°C until cell cycle analysis was performed. On the day of analysis, the cells were resuspended in 100 ul RNase A solution containing propidium iodide (2 mg/ml) for 30 min at room temperature in the dark. DNA content was determined by Cellometer (Nexcelom, Lawrence, MA). Cell cycle was analyzed using FCS4 express software (Molecular Devices, Sunnyvale, CA).

Cell apoptosis was determined using Annexin-V FITC kit (BioVision, Mountain View, CA) following manufacture’ protocol. The cells were seeded into 6-well plates at a density of 2.5 × 10⁵ cells/well and then treated with vehicle or varying concentrations of ONC201 for 30 hours. The cells were harvested and stained with 100 ul of Annexin-V and PI dual-stain solution for 15 min in the dark. Annexin V expression was determined by a Cellometer. Apoptosis cells were analyzed by FCS4 express software.

Caspase activity assays were performed with modifications as previously described (18). In brief, the OVCAR5 and SKOV3 cells were seeded in 6-well plates at a density of 2.5 × 10⁵ cells/well overnight. The cells were treated with ONC201 at different concentrations for 8-12 hours. The controls cells are treated with cell culture media at DMSO concentration of 0.1%. 150-180 ul 1X caspase lysis buffer was added to each well. BCA protein assay was used for quantitation of protein concentration. 10-15 ug lysates in a black clear bottom 96-well plate were incubated with reaction buffer and 200 uM of caspase substrates for 30 min. The fluorescence of each well was determined using a microplate reader (Tecan, Morrisville, NC). The selective substrates Ac-DEVD-AMC, Ac-IETD-AFC and Z-IETD-AFC (AAT Bioquest) were used for caspase 3, caspase 8 and caspase 9, respectively. Each experiment was repeated three times to assess for consistency of results.

ROS production was determined using the DCFH-DA assay, as previously described (19). The OVCAR5 and SKOV3 cells (8000 cells/well) were cultured in a black 96-well plate overnight followed by treatment with indicated doses of ONC201 or vehicle for 12 hours. Cells were then incubated with 20 uM DCFH-DA in regular growth medium for 30 minutes. ROS accumulation was measured by a plate reader (Tecan) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Mitochondrial membrane potential was analyzed using the specific fluorescent probes JC-1 and TMRE, respectively (20, 21). The cells were cultured overnight in a 96 well plate and then treated with different concentrations of ONC201 or vehicle control for 8 hours. Treated cells were incubated with 2 uM JC-1 or 1 uM TMRE for 30 minutes at 37°C. The levels of the fluorescent probes were measured using a Tecan plate reader at Ex/Em= 549/575 nm for TMRE. For JC-1, green JC-1 signals were measured at Ex/Em 485/535 nm and red signals were measured at 535/590 nm. Each experiment was repeated three times to assess for consistency of results.

Cell adhesion was measured by using laminin adhesion assay as previously described (11). Briefly, a 96-well plate was coated with 100 ul laminin-1 (10 ug/ml) for 1 hour and was blocked by 3% BSA for 30 min at 37°C. The cells were pre-treated with ONC201 for 24 hours and then added to the laminin coated wells (25,000 cells). The plate was incubated at 37°C for 2 hour in serum-free medium. The cells were washed twice with PBS to remove nonadherent cells. Attached cells were fixed by adding 100 ul of 5% glutaraldehyde for 20-30 min and stained with 0.1% crystal violet for 30 min. Next, the cells were lysed with 10% acetic acid and the absorbance of the solution was measured at 570 nm in a plate reader (Tecan).

Cell invasion assays were performed using a 96-well plate coated with 0.5-1 x BME. The OVCAR5 and SKOV3 cells were starved for 12 hours and then seeded in the upper chambers of the wells and the lower chambers were filled with regular medium and differing concentrations of ONC201. The media contained 0.1% DMSO as a vehicle control. The plates were incubated for 4 hours to allow invasion into the lower chambers. After washing the upper and lower chambers with PBS, 100 ul of calcein AM solution (Invitrogen, Carlsbad, CA) was added to the lower chambers and incubated for 30-60 minutes. The lower chamber plate was measured by plate reader for reading fluorescence at EX/EM 485/520 nm. Each experiment was repeated twice.

The cells were plated in 6-well plates at 3.5 × 10⁵ cells/well for 24 hours and then replaced with media with 0.5% charcoal stripped FBS for 12 hours. A sterilized 200 ul pipette tip was used to draw a straight line across the plate in one direction. The cells were then washed with fresh media to remove the detached cells, and cells were then treated with different concentrations of ONC201 in the media supplemented with 0.5% charcoal stripped serum for 24 to 48 hours. The images were acquired at different timing points (24, 36 and 48 hours). Measurements of the width of the wound were performed at random intervals with the Adobe Photoshop CS6.

The organotypic culture was performed as previously described (22). 1 × 10⁶ mouse stromal cells transfected with hTERT were mixed with 3.5 volumes of Matrigel^®, 1 volume of 10X DMEM, 1 volume of FCS, and 1 volume of DMEM/F12, and then were pipetted into each well of a 24-well plate. After 18 hours, 5 × 10⁵ OVCAR5 cells, suspended in 1 ml regular media supplemented with 10% FCS, were added to each well. The OVCAR5 cells were treated with 10 uM ONC201 or vehicle control for 36 hours. Then the gel with stromal and cancer cells was lifted and placed onto a stainless steel grid in a 6 well plate. DMEM/F12 media was added to the well so that the gel plug was exposed to the air from above and to the media from below. The media was changed every 3 days maintaining the air-liquid interface. The organotypic gels were cultured for 12 to 14 days and then fixed in 4% formaldehyde for 24 hours. The gels were bisected and processed to paraffin blocks. Each slide was stained using standard haematoxylin and eosin (H&E), and then scanned by Motic. The “Invasion Index” was calculated by ImagePro as MCY × N × A (MCY: Mean Cord Y, N: the number of cancer islands, A: sum of areas of the cancer islands). The gels not treated with ONC201 were used as control.

The OVCAR5 and SKOV3 cells were transiently transfected with either siRNA targeting ClpP (15 nM) and scrambled control siRNA (15 nM) using Mission SiRNA Transfection Reagent (Sigma, St. Louis, MO), according to manufacturer’s protocol. MTT assay were performed at 48 hours post-transfection. Western blotting were performed at 24 hours after transfection.

The OVCAR5 and SKOV3 cells were treated with ONC201 or vehicle for 30 hours. Total cell lysates were prepared in RIPA buffer plus PhosStop. Protein concentration was quantified using BCA assay (Sigma, St. Louis, MO). Equal amounts of lysates were electrophoresed on 10-12% SDS-PAGE and transferred onto a PVDF membrane. The membranes were probed at 4°C overnight with appropriate primary antibodies. Proteins were visualized using SuperSignal West Pico Substrate (Thermo Scientific, Waltham, MA) by the ChemiDoc image system (Bio Rad, Hercules, CA).

The KpB ( K 18 -gT 121 + / − ;p53^fl/fl;Brca1^fl/fl; KpB) transgenic mouse model of high grade serous epithelial OC has been described previously in detail (23–25). Animal protocol was approved by the UNC-CH Institutional Animal Care and Use Committee (IACUC). Mice were housed on a 12 hours light, 12 hours dark cycle, with free access to food and water The KpB female mice were fed a high fat diet (HFD, 60% calories from fat) or a low fat diet (LFD, 10% calories from fat, Research Diets) at 3 weeks of age. 5 ul of 2.5 x10⁷ P.F.U of recombinant adenovirus Ad5-CMV-Cre (AdCre, Transfer Vector Core, University of Iowa) was injected into the left ovarian bursa cavity at 6–8 weeks age (24). The mice were checked weekly by abdominal palpation for the appearance of ovarian tumors. Once tumors had reached an average size of 0.1x0.1 cm in diameter by palpation, the mice fed with HFD or LFD were assigned into the four groups: HFD control, LFD control, HFD+ONC201 and LFD+ONC201 (N=15/group). ONC201 was given weekly in 0.1 ml containing either 130 mg/kg or placebo in oral gavage for 4 weeks. The size of ovarian tumors were measured twice a week using palpation. The mice were weighted weekly and observed daily any signs of toxicity or distress. No mice died during the treatment. After 4 weeks of treatment, mice were sacrificed by CO2 asphyxiation and tumors were weighted. Ovarian tumor volumes were calculated as following: (width2 × length)/2.

Five micrometer paraffin sections from the KpB mice tumors were processed for IHC analysis at IHC Mice Core Facility at UNC. Briefly, the slides were incubated with Ki-67, phosphorylated p42/44, phosphorylated-S6, and VEGF at 4°C overnight, respectively, and then treated with HRP-conjugated antibody for 1 hour. The sections were further applied with ABC-Staining Kits (Vector Labs, Burlingame, CA) for color reaction and hematoxylin for counterstaining. All IHC slides were scanned by Motic and scored by ImagePro software (Rockville, MD).

The VEGF productions of mice serum after treatment with ONC201 were detected using a VEGF ELISA Kit (#MMV00, R&D Systems, Minneapolis, MN), according to the manufacturer’s directions. Each sample from ONC201 and control groups was measured in duplicate. Plates were read at 570 nm using a Tecan plate reader.

Data are given as the mean ± SD. Statistical significance was analyzed by the two-sided unpaired Student’s t-test from at least three replicates. Tumor growth in different treatment arms was analyzed by One-way & Two-way ANOVA test. GraphPad Prism 5 (La Jolla, CA USA) was used for all graphs and significance tests. P values of <0.05 were considered to have significant group differences.

ONC201−mediated inhibition of OC cell viability was assessed using MTT assay. The OVCAR3, IGROV-1, OVCAR5 and SKOV3 were cultured in media with various concentrations of ONC201 for 72 hours. The MTT results showed that with increasing ONC201 concentrations, a dose-dependent growth inhibition was observed in four OC cell lines compared to the control cells ( Figure 1A ). The mean IC50 values of ONC201 were 4.2 uM for OVCAR3, 3.1 uM for IGROV-1, 3.2 uM for OVCAR5 and 2.1 uM for SKOV3, respectively. Subsequently, because colony formation assay is a well-established in vitro technique for testing the proliferative capability of treated cells (26), we investigated the long-term effect of ONC201 on OC cell growth. OVCAR5 and SKOV3 cells were seeded in same density in six-well plates and incubated 1, 10 and 100 uM of ONC201 for 2 days and subsequent culture of the cells for 12 days. The results showed that the colony-forming ability was reduced by 4.8%, 58.3% and 79.75% in OVCAR5, and 22.2%, 57.5% and 86.1% in SKOV3, respectively, after the cells were treated with 1-100 uM ONC201 compared with vehicle control ( Figure 1B ). These results suggest that OC cells are sensitive to the anti-proliferative effects of ONC201.

Because ONC201 is a selective antagonist of the G protein-coupled receptor DRD2 that causes p53-independent apoptosis through upregulation of TRAIL and DR5 in tumor cells (4), we next detected the effect of ONC201 on DRD2, DRD5 and DR5 in the OVCAR5 and SKOV3 cells. The cells were treated with ONC201 at 1, 10 and 100 uM for 24 hours. Western blotting results showed that Treatment with OVCAR5 and SKOV3 cells with 10 or 100 uM ONC201 reduced the protein levels of DRD2 and DRD5, with DR5 expression being increased compared with vehicle control ( Figure 1C ).

We next investigated whether ONC201 modulates cell cycle progression in OC cells. As illustrated in Figures 2A, B , treatment with 1, 10 and 100 uM ONC201 for 36 hours caused significant increases in the G1 phase and decreases S phase in a dose-dependent manner in the OVCAR5 and SKOV3 cells. G1 arrest phase increased from 44.69% in control cells to 60.12% in the 100 uM ONC201-treated OVCAR5 cells and 56.89 to 71.55% in the SKOV3 cells; in parallel, the S phase cell population decreased from 25.41 to 15.62% with increasing concentrations of ONC201 in the OVCAR5 and 21.2 to 13.93% in the SKOV3 cells (p<0.05), respectively. To further understanding the mechanism underlying the cell cycle arrest, cell cycle-related proteins were analyzed by western blotting in the ONC201-treated OVCAR5 and SKOV3 cells. The results showed that ONC201 resulted in reduced expression of cyclin D1 in both cell lines after 36 hours of treatment. Moreover, ONC201 also inhibited the expression of the cyclin D1 regulatory partners, CDK4 and CDK6, following ONC201 treatment for 36 hours compared with vehicle control ( Figure 2C ). These data suggest that ONC201 induces cell cycle G1 arrest through cyclin D1 degradation in OC cells.

To characterize the underlying mechanism of growth inhibition by ONC201, the apoptotic cells were analyzed by performing an Annexin-V assay after treating the OVCAR5 and SKOV3 cell lines with ONC201 (1-100 uM). Annexin-V analysis showed a meaningful increase in apoptotic cells in a dose-dependent manner after 30 hours of ONC201 treatment, along with decreasing expression of MCL-1 and BCL-XL protein ( Figure 3A ). Annexin V expression increased from 6.61% in control cells to 19.15% in the 100uM ONC201-treated OVCAR5 cells and 6.68 to 16.12% in the SKOV3 cells, respectively (p<0.01). To examine whether ONC201 induces apoptosis through the mitochondrial apoptosis pathways in the OC cells, western blotting results showed that ONC201 significantly induced cleaved PARP and caspase 9 protein expressions in both cell lines compared with vehicle control ( Figure 3B ). Furthermore, a dose-dependent increase in the activity of cleaved caspase 3, 8 and 9 was found by ELISA assays in OVCAR5 and SKOV3 cells in response to ONC201 treatment ( Figure 3C ). These results indicated that apoptosis induced by ONC201 was executed through activation of either the extrinsic pathway (death receptor) or the intrinsic pathway in OC cells.

Given that obesity is associated with worse outcomes for ovarian cancer and dopamine signaling pathway contributes to the distinct metabolic profiles of obese and non-obese patients, we sought to evaluate whether ONC201 was able to inhibit tumor growth in a transgenic mouse model of high grade serous OC (KpB) under obese and non-obese conditions. Immune competent mice carrying Cre-inducible oncogenic Brca1 in combination with deletion of p53 and Rb in the ovaries develop high grade serous OC about 4-6 months after injection with AdCre (25). The HFD-fed or LFD-fed KpB mice were divided into four groups (15 mice/group): HFD, HFD+ONC201, LFD and LFD+ONC201, respectively. The mice were treated once a week by oral gavage with either ONC201 (130mg/kg, 4 weeks) or placebo after tumor induction. The tumor size was monitored twice a week by palpation. The mice showed tolerance to ONC201 treatment and did not show any obvious changes in behavior and body weight. After 4 weeks of treatment, the tumors were excised, weighed, and examined histologically. ONC201 effectively inhibited tumor growth and reduced tumor weight in both HFD and LFD groups compared to control groups. Ovarian tumor weights in obese KpB mice were significantly greater than that in the non-obese control mice (2.92 g versus 1.80 g, p<0.05), suggesting that obesity promoted ovarian tumor growth. ONC201 treatment decreased tumor weight by 75.5% in obese mice and 65.2% in non-obese mice compared to their control groups ( Figures 4A, B , p<0.01). These findings imply that ONC201 caused similar anti-tumor effects in obese mice compared to non-obese KpB mice although gene expression and metabolomics profiling showed statistically significant differences between the ovarian tumors from the obese versus lean mice (27). Collectively, these data demonstrate that treatment with ONC201 significantly suppressed OC growth in a genetically engineered mouse model of OC under obese and non-obese conditions.

To determine the anti-tumorigenic mechanisms of ONC201 in vivo, the expression of Ki-67 and DRD5 in ovarian tumors was evaluated by IHC in obese and non-obese KpB mice after 4 weeks of treatment. The tumors from obese mice displayed increased expression of the Ki67 and DRD5 compared to non-obese mice (p<0.05). Consistent with our results in vitro, ONC201 inhibited Ki-67 expression in the ONC201-treated obese mice by 33.6% compared with obese control mice and the ONC201-treated lean mice by 26.1% compared with lean control mice (p<0.01). In addition, we found that high expression of DRD5 was identified in ovarian tumors of obese mice, and the expression of DRD5 was reduced in the obese and lean mice treated with ONC201 compared with the control mice ( Figure 4C , p<0.05), suggesting that ONC201 inhibits ovarian tumor growth through the DRD2/DRD5 pathway.

ROS have been implicated as mediators of TRAIL-induced apoptosis in cancer cells via different pathways (28). To examine the involvement of oxidative stress in the anti-tumorigenic effect of ONC201 in OC cells, intracellular ROS levels were detected using the DCFH-DA assay. The results showed an increase in ROS production when OVCAR5 and SKOV3 cells were treated with ONC201 at different concentrations for 12 hours. At a concentration of 100 uM, ONC201 significantly increased DCFH-DA fluorescence 1.49 and 1.30- fold in OVCAR5 and SKOV3 cells compared with vehicle control cells (p<0.01), respectively ( Figure 5A ).

To further evaluate the underlying mechanism of ROS effect in association with mitochondrial function, we next set out to assess the ability of ONC201 to depolarize mitochondrial membranes by JC-1 and TMRE ELISA assays. JC-1 assay indicated that ONC201 induced the loss of mitochondrial transmembrane potential (ΔΨm) in both cell lines after 8 hours of treatment compared to control cells. ONC201 at 10 uM significantly reduced ΔΨm by 20.8% and 23.5% in the OVCAR5 and SKOV3 cells compared with vehicle control (p<0.01), respectively ( Figure 5B ). Similarly, these changes of ΔΨm in response to treatment were also observed in a TMRE assay in both cell lines ( Figure 5C ), which further strengthens the reliability of our results. Moreover, western blotting analysis showed that ONC201 significantly increased expression of mitochondrial protease, ClpP, and endoplasmic reticulum (ER) stress-related markers including ATF4, PERK, CHOP and IRE-1a in a dose-dependent manner in both cell lines after 24 hours of treatment ( Figure 5D ).

ClpP is essential for the oxidative stress response, and ONC201 is an allosteric agonist of ClpP. To understand the effect of ClpP on ONC201 mediated cell proliferation and cell stress, OVCAR5 and SKOV3 cells were transfected with ClpP siRNA or scramble RNA, respectively. As shown in Figure 5E , ClpP siRNA significantly downregulated the protein expression of ClpP in both cells compared with cells transfected with mock siRNA, which was accompanied by a decrease in PERK expression in both cells. Knockdown of ClpP resulted in decreased expression of phosphorylated S6 in SKOV3 cells. However, in OVCAR5 cells, this effect on phosphorylated S6 was not as pronounced as in SKOV3 cells. The siRNA-mediated knockdown of ClpP effectively inhibited the expression of ATF4 and PERK induced by ONC201 in OVCAR5 cells ( Figure 5F ). ONC201 mediated inhibition of cell proliferation in OVCAR5 and SKOV3 cells partially recovered by transfection with ClpP siRNA ( Figure 5G ). Importantly, IHC results confirmed ONC201 significantly increased the expression of ClpP in obese and lean mice after 4 weeks of treatment ( Figure 5H ). These results suggest that activation of ClpP through ONC201 is involved in its anti-tumorigenic activity in OC.

Given that ONC201 exhibited anti-invasive ability in USC cells (11), we investigated the impact of ONC201 on cell adhesion, migration and invasiveness in the OVCAR5 and SKOV3 cell lines. In the assessment of cell adhesion, both cell lines were incubated in laminin-coated 96 well plates and treated with ONC201 for 2 hours. As shown in Figure 6A , cellular adhesion was decreased by 61.6% to 35.1% in the SKOV3 and OVCAR5 cells, respectively, at a dose of 100 uM compared with control cells (p<0.01). Cell invasion was measured using a transwell invasion assay with a Matrigel-coated filter. Both cell lines were seeded in the upper chambers of the transwell and treated with ONC201 (1-100 uM) for 4 hours. The invasive capacity of the OVCAR5 and SKOV3 cell lines was reduced by ONC201 treatment in a dose-dependent manner. ONC201 (100 uM) significantly reduced the invasive ability of the OVCAR5 and SKOV3 cell lines by 23.3% and 36.0% compared with vehicle control cells ( Figure 6B , p<0.05).

To evaluate the effect of ONC201 on cell migration, a wound-healing assay was performed in OVCAR5 and SKOV3 cells. The cells were treated with different concentrations of ONC201 and evaluated the cell migration capacity at 24, 48 and 72 hours. ONC201 had a significant inhibitory effect on cell migration of OVCAR5 and SKOV3 cells at all times compared with vehicle-treated control cells (p<0.05), which was most pronounced at 48 hours of treatment as shown in Figure 6C .

Given that stromal cells have an active role in inducing EMT and enhancing invasive potential, and the pattern of invasion produced in organotypic cultures displays a similar invasion patterns observed in human (29), we used organotypic cultures containing stromal cells to measure the invasion capacity of OC cells after treatment with ONC201. The organotypic gels stained with H&E were analyzed to generate an invasion index (30). Similar to wound healing and transwell assays, 14 days after treatment, addition of 10 uM ONC201 for 24 hours significantly reduced the invasion index, and OVCAR5 cells invasion was inhibited by 39.3% in organotypic cultures compared to control ( Figure 6D , p<0.01).

We next examined the effect of ONC201 on the EMT and angiogenesis in the OC cells. Treatment with ONC201 for 24 hours significantly decreased the expression of VEGF-C, Slug and Snail in both cell lines ( Figure 6E ). IHC results showed that obesity increased VEGF expression in the ovarian tumors, when comparing tumors from obese versus lean mice. However, ONC201 reduced VEGF expression by 42.5% in the obese mice and by 42.2% in the lean mice as compared to controls ( Figure 6F , p<0.05). In addition, we found that the production of serum VEGF decreased by 14.0% in obese mice and 19.4% in lean mice in comparison to placebo-treated mice ( Figure 6G ). Together, these findings support the contention that ONC201 has an ability to inhibit adhesion, invasion and angiogenesis in OC cells and ovarian tumors in KpB mice.

Because ONC201 is a potent activator of the ClpP-induced integrated stress response, we investigated the role of cellular stress in ONC201’s anti-proliferative and anti-invasive effects. The OVCAR5 and SKOV3 cells were treated with vehicle control or ONC201 for 72 hours in the presence and absence of 1 mM of the antioxidant, N-acetylcysteine (NAC). The results showed that NAC partially reversed the cytotoxic effects of ONC201 in both cell lines compared to the control cells ( Figures 7A, B , p<0.05). Similarly, pre-treatment of NAC for 6 hours effectively reversed ONC201-induced decreases in mitochondrial membrane potential and inhibited ONC201-induced increases in intracellular ROS levels ( Figures 7B, C , p<0.05).

Because NAC significantly inhibited ONC201-induced oxidative stress, efforts were made to explore whether ONC201 exerted anti-invasive effects through oxidative stress pathways. The wound healing assay was used to detect the ability of invasion after ONC201 treatment in both cell lines. In the presence of 10 μM or 100 μM ONC201, pre-treatment with NAC for 6 hours partially prevented ONC201-induced anti-invasive activity by 43.5% and 35.0% in the OVCAR5 cells, and 29.2% and 36.2% in the SKOV3 cells, respectively ( Figure 7D , p<0.01). Western blotting results indicated that 1 mM NAC treatment did not change the expression of CLpP induced by ONC201. However, in NAC-treated groups, NAC partially blocked 10 μM ONC201-evoked decreases in VEGF and Snail expression ( Figure 7E ). Therefore, it appears that ONC201 exerted its anti-proliferative and anti-invasive effects partially through the integrated stress response in OC cells.

To gain insight into the role of ONC201 in TRAIL-mediated signaling, we evaluated whether P13K/AKT/mTOR and MAPK pathways were involved in the anti-proliferative effects of ONC201 in OC cells. OVCAR5 and SKOV3 cells were treated with ONC201 (1, 10 and 100 uM) for 24 hours. mTOR activity was determined by phosphorylation of S6 (Ser235/236) and MAPK activation by detecting P42/44 phosphorylated on Thr202 and Tyr204. ONC201 inhibited AKT phosphorylation and activity of mTOR and MAPK pathways without changing the total levels of S6, p42/44 and AKT proteins in the both cells compared with control cells ( Figure 8A ). The effects of ONC201 on the phosphorylation-dependent activation of p42/44 and S6 in the KpB serous OC mouse model are shown in Figure 8B . ONC201 reduced p42/44 phosphorylation by 24.3% in obese mice and 38.8% in lean mice, respectively (p<0.05). Similarly, ONC201 also reduced phosphorylation of S6 by 46.1% and 37.3% in obese and lean mice, respectively, compared to untreated mice. Overall, these data confirm that ONC201 reduces cell growth via inhibition of the AKT/mTOR and MAPK signaling pathways in OC cells and tumors.