Curcumol Targeting PAX8 Inhibits Ovarian Cancer Cell Migration and Invasion and Increases Chemotherapy Sensitivity of Niraparib

Abstract

Objective: To investigate the effects of Curcumol on invasion, migration and epithelial-mesenchymal transformation of IGROV-1 and OVCAR-3 cells in ovarian cancer and its potential mechanism. Meanwhile, the effect of Curcumol on the antitumor activity of Niraparib was analyzed.

Methods: Cell Counting Kit 8 (CCK-8) was used to detect the effects of Curcumol on the activity of IGROV-1 and OVCAR 3 cells. In vitro invasion assay (Transwell) was used to test the invasiveness of cells. Cell migration was detected by scratch assay. The inhibitory effect of Curcumol on PAX8 was detected by QRT-PCR. To evaluate the antitumor effect of Curcumol in subcutaneous tumor-bearing animal model.

Results: Knockdown of PAX8 could inhibit the proliferation, invasion and migration of ovarian cancer cells. After Curcumol treated IGROV-1 and OVCAR-3 cells, the cell proliferation ability was decreased, the number of invasive cells was significantly reduced, and the scratch closure rate was significantly reduced, in a dose-dependent manner. Mechanism studies showed that Curcumol increased the antitumor activity of Niraparib by inhibiting PAX8.

Conclusion: Curcumol can inhibit the invasion, migration and epithelial-mesenchymal transformation of IGROV-1 and OVCAR-3 cells in ovarian cancer, and its mechanism is related to the targeted inhibition of PAX8. Curcumol also increased the sensitivity of Niraparib chemotherapy by inhibiting PAX8.

Figure 1

PAX8 is up-regulated in ovarian cancer. A. Bioinformatics analysis PAX8 is up-regulated in ovarian cancer. Data were obtained from GEPIA database (http://gepia.cancer-pku.cn/index.html).B. Immunohistochemistry verified that PAX8 is highly expressed in ovarian cancer. Data is obtained from the human protein atlas database (https://www.proteinatlas.org/).∗P <0.05,Tumor (n =426) vs Normal (n =88) tissues group.

Figure 2

PAX8 is highly expressed in ovarian cancer. (a). The expression level of PAX8 in ovarian cancer tissues is higher than that in adjacent control tissues. N =16. (b). The expression level of PAX8 is positively correlated with the co-expression of Snail. (c). The expression level of PAX8 is positively correlated with the co-expression of Twsit1. (d). PAX8 expression is positively correlated with Zeb2 co-expression. (e). The expression of PAX8 is positively correlated with the co-expression of MMP13. (f). The expression of PAX8 is negatively correlated with the co-expression of E-cadherin. (g). PAX8 expression is positively correlated with Vimentin co-expression.

Figure 3

Knockdown of PAX8 inhibits the proliferation, invasion and EMT of ovarian cancer. (a). Verification of the efficiency of knocking down shRNA PAX8 in IGROV-1 and OVCAR-3 cells. (b). CCK-8 experiment verified that knocking down PAX8 inhibits the proliferation of ovarian cancer cells IGROV-1 and OVCAR-3. (c). Knockdown of PAX8 inhibits the migration ability of ovarian cancer cells IGROV-1 and OVCAR-3. (d). Knockdown of PAX8 inhibits the invasion of ovarian cancer cells IGROV-1 and OVCAR-3 by transwell assay. (e). After knocking down PAX8, detection of Snail1 expression in IGROV-1 and OVCAR-3 cells. (f). After knocking down PAX8, detection of Twist1 expression in IGROV-1 and OVCAR-3 cells. (g). Detection of Zeb2 expression in IGROV-1 and OVCAR-3 cells after knocking down PAX8. (h). After knocking down PAX8, detection of MMP13 expression in IGROV-1 and OVCAR-3 cells. (i). After knocking down PAX8, detection of E-cadherin expression in IGROV-1 and OVCAR-3 cells. (j). After knocking down PAX8, detection of Vimentin expression in IGROV-1 and OVCAR-3 cells. ∗P <0.05, ∗∗P <0.01, vs sh-NC group.

Figure 4

Curcumol inhibits the expression of PAX8 and inhibits the malignant progression of ovarian cancer cells. (a). Detection of PAX8 expression in IGROV-1 and OVCAR-3 cells after treatment with different concentrations of Curcumol (10, 20, 40 μM). (b). After treatment with different concentrations of Curcumol, the proliferation ability of IGROV-1 and OVCAR-3 cells was detected by CCK-8. (c). After treatment with different concentrations of Curcumol, the invasion ability of IGROV-1 and OVCAR-3 cells was detected by Transwell assay. ∗P <0.05, ∗∗P <0.01, ∗∗∗P <0.001, vs Control group.

Figure 5

Subcutaneous tumor-bearing experiments in nude mice prove that Curcumol inhibits the proliferation and metastasis of ovarian cancer cells. (a). Tumor growth curve of nude mice (Curcumol: 100 mg/kg). (b). Gross picture of nude mouse tumor. (c). Tumor weight of nude mice. (d). Detection of the expression level of PAX8 in two groups of tumor tissues. (e). The expression of E-cadherin in the control and Curcumol treated tissue was detected. (f). Vimentin expression in the control and Curcumol treated tissue was detected. N =6. ∗P <0.05, ∗∗P <0.01, ∗∗∗P <0.001, vs Control group.

Figure 6

Knockdown of PAX8 or combination of Curcumol can increase the sensitivity of ovarian cancer chemotherapy. (a). After different treatments, cell proliferation detection. (b). After different treatments, cell Transwell detection. (c). After treatment with different concentrations of Curcumol, the expression of E-cadherin in OVCAR-3 cells was detected. (d). Detection of Vimentin expression in OVCAR-3 cells after treatment with different concentrations of Curcumol. ∗P <0.05, ∗∗P <0.01.