ONC201 inhibited cell viability in OC cell lines
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 of ONC201, 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. Subsequently, because colony formation assay is a well-established in vitro technique for testing the proliferative capability of treated cells (25), we investigated the long-term effect of ONC201 on OC cell growth. The OVCAR5 and SKOV3 cells were seeded in same density and incubated 10 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 58.3% in OVCAR5 and 57.5% in SKOV3, respectively (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 (5), 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 ONC201 treatment significantly increased the expression of the DR5 protein and decreased the expression of DRD2 in a dose-dependent manner in both cell lines (Figure 1C).
ONC201 caused cell cycle G1 arrest in OC cells
We next investigated whether ONC201 modulates cell cycle progression in OC cells. As illustrated in Figure 2A and 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 100uM 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). 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 level of cyclin D1 expression 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 30 hours (Figure 2C). These data suggest that ONC201 induces cell cycle G1 arrest through cyclin D1 degradation in OC cells.
ONC201 induced apoptosis 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 significant increases 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. 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 (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.
ONC201 inhibited tumor growth in a transgenic mouse model of OC
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 (24). The HFD-fed or LFD-fed KpB mice were divided into four groups: HFD, HFD+ONC201, LFD and LFD+ONC201. 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), 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 (Figure 4A-C). ONC201 caused a greater anti-tumor effect in obese mice compared with control mice and, in non-obese mice. These findings imply that ONC201 caused a greater anti-tumor effect in obese mice compared to non-obese KpB mice although gene expression and metabolomic profiling showed statistically significant differences between the ovarian tumors from the obese versus lean mice(26). 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. Consistent with our results in vitro, ONC201 inhibited Ki-67 expression in the ONC201-treated obese mice by 33.6% and the ONC201-treated lean mice by 26.1%. We also found that the expression of DRD5 was reduced in the obese and lean mice treated with ONC201 but not in the placebo-treated mice (Figure 4D), suggesting that ONC201 inhibits ovarian tumor growth through the DRD5 pathway.
ONC201 induced cellular stress in OC cells
ROS have been implicated as mediators of TRAIL-induced apoptosis in cancer cells via different pathways (27). 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 a dose-dependent 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 (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 showed 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 mitochondrial transmembrane potential by 20.8% and 23.5 % in the OVCAR5 and SKOV3 cells (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, the protein expression of ClpP was downregulated significantly by ClpP siRNA in both cells when compared to the cells transfected with mock siRNA, which was accompanied by a decrease in the expression of PERK and phosphorylation S6, indicating mTOR/S6 signaling pathway might be involved in ClpP induced stress response. The siRNA-mediated knockdown of ClpP effectively inhibited the expression of ATF4 and PERK induced by ONC201 (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 the anti-tumorigenic effects of ONC201 are in part depend on ClpP activated the integrated stress response in OC.
ONC201 inhibited adhesion and invasion in OC cells and ovarian tumors
Given that ONC201 exhibited anti-invasive ability in uterine serous carcinoma 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. 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% (Figure 6B).
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 ONC 201 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(28), 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(29). 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 (6D).
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.
Inhibition of cellular stress reduced the effects of ONC201 on cell proliferation and invasion.
Girven that 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 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 (Figure 7A and B). 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 (Figure 7B and C). These results suggest that cellular stress is essential for the anti-proliferative activity of ONC201 in OC cells.
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.
ONC201 inhibited P13K/AKT/mTOR and MAPK pathways in vitro and in vivo
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 (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.