EZH2 Contributes To Ovarian Cancer Anoikis Resistance

Objective We have found that EZH2 could promote the implantation of ovarian cancer in peritoneal cavity. However, the underlying mechanism remains unclear. The aim of the present study was to investigate whether EZH2 can contribute to ovarian cancer anoikis resistance.Materials and methods 1. To establish anoikis models, ovarian cancer cells SKOV3 and A2780 were cultured in suspension in poly-HEMA coated plates. The morphological changes were observed under an inverted microscope. 2. To identify the anoikis models of ovarian cancer cells, the apoptosis rates in ovarian cancer cells cultured in suspension for different time periods ranging from 0h to 96 h was detected using Annexin V Apoptosis Detection Kit. 3. The changes in migration property and proliferation of ovarian cancer cells resulted from anoikis were evaluated by scratch healing assays, Transwell migration assays, and Ed U assays. 4. The EZH2 levels in ovarian cancer cells that had been cultured in suspension for 0h to 96 h were detected by western blot.5. Transfecting a lentivirus with short hairpin EZH2 silencing RNA(sh RNA) of SKOV3, we measured the protein level of EZH2 by Western Blot. 6. The SKOV3 cells were treated with DMSO(1 ?g/ml), DZNEP(1 ?g/ml), and GSK126(1 ?g/ml), cultured in suspension for 48 h, the cells were stained using APC Annexin V Apoptosis Detection Kit and the apoptosis rates were assessed by flow cytometry. In addition the apoptosis rates in SKOV3 sh EZH2 and SKOV3 sh NC were compared. 7. The tissues obtained from intraperitoneal xenografts of SKOV3 sh EZH2 and SKOV3 sh NC that had been established in our previous study were used to investigate the effects of EZH2 on anoikis in vivo. The levels of EZH2, Ki67 and cleaved-caspase-3 were detected by IHC, and TUNEL assays were performed to evaluate the apoptosis in xenografts.Results 1. In adherent culture, SKOV3 cells were irregular polygon, while the A2780 cells were long fusiform. After being cultured in suspension for 24 h, obvious morphological changes, including smaller size and spherical retraction, were observed in both SKOV3 and A2780 cells. After being cultured in suspension for 48 h, large and evacuated cell dumps of SKOV3 were formed, whereas A2780 cells were in small and dense cell mass. 2. The apoptosis of ovarian cancer cells SKOV3 was stable after they had been suspended for 48 h, while the apoptosis of A2780 was increasing depending on the time of suspension. 3. Both SKOV3 and A2780 showed no significant change(P>0.05) in proliferation between the adherent cells and the suspended cells. Suspension culture could significantly promote the migration of SKOV3 cells(P<0.05). However, the migration of A2780 cells were not affected by suspension culture(P>0.05). 4. The EZH2 level in SKOV3 cells was decreased gradually along with the increase of time in suspension as revealed by western blot, and the expression of EZH2 tended tobe stable afer 48 h in suspension. However, the expression of EZH2 in A2780 cells was not changed after suspension. In the SKOV3 cells tranfected with sh EZH2(SKOV3 sh EZH2), the expression of EZH2 was significantly reduced and the apoptosis rate was increased compared with the vehicle controls(SKOV3 sh NC) after being cultured for 48 h in suspension. 5. The apoptosis rates in SKOV3 cells treated with DZNEP(1 ?g/ml) and GSK126(1 ?g/ml) were significantly increased compared with the DMSO control. 6. In the SKOV3 sh EZH2 xenografts, TUNEL-positive tumor cells and cleaved-caspase3 expression were increased, and a decrease in Ki67-positive cells was observed.Conclusion EZH2 can enhance anoikis resistance in ovarian cancer, which may be one of the important mechanisms for the metastasis of ovarian cancer.