Suppressed epithelial-mesenchymal transition and cancer stem cell properties mediate the anti-cancer effects of ethyl pyruvate via regulation of the AKT/nuclear factor-κB pathway in prostate cancer cells

Abstract

Castration-resistant prostate cancer (CRPC) is a leading cause of mortality among cases of prostate cancer (PCa). Current treatment options for CRPC are limited. Ethyl pyruvate (EP), a lipophilic derivative of pyruvic acid, has been reported to have antitumor activities. In the present study, the efficacy of EP against PCa was investigated using two human PCa cell lines and a mouse xenograft tumor model. PC3 and CWR22RV1 cells were treated with EP, and cytotoxicity was evaluated via Cell Counting Kit-8 and colony formation assays, while cell cycle distribution was assessed by flow cytometry. Changes in cell migration and invasion caused by EP treatment were also evaluated with Transwell and wound healing assays, and changes in the expression of intracellular signaling pathway components were detected by western blotting. EP treatment reduced cell viability, induced G1 arrest, and activated the intrinsic apoptosis pathway. Additionally, the in vivo experiments revealed that EP administration markedly inhibited tumor growth. EP also reversed epithelial-mesenchymal transition and suppressed cancer stem cell properties in part through negative regulation of AKT/nuclear factor-κB signaling. These results indicate that EP has anticancer activity in vitro and in vivo, and is therefore a promising therapeutic agent for the treatment of PCa.

Keywords: AKT/nuclear factor-κB; cancer stem cells; epithelial-mesenchymal transition; ethyl pyruvate; prostate cancer.

Figure 1.

Effect of EP on PCa cell viability, cell colony formation and tumourigenesis. (A) EP show cytotoxic potential against the PC3 and 22Rv1 with IC50 values of 11.56 mM and 10.93 mM, respectively. (B) The kinetics of cell viability in PC3 and CWR22RV1 cells treated with EP. Cells were treated with EP (10 mmol/l) at the indicated time. (C) The dosage effect of EP on the cell viability in PC3 and CWR22RV1 cells. (D) The tumor cell colony formation was analyzed, and (E) the colony formation rate was calculated. The data represented means ± SEM (*P<0.05, **P<0.01, ***P<0.001 n=3) as the percentage of viable cells normalized to percentage of viable cells in saline-treated (control) cells. (F) Gross observation of xenograft tumour size. (G) Plot of tumour volume over time. (H) Body weight of tumor-bearing mice. Significant differences are between EP treatment groups and Saline vehicle control groups. EP, ethyl pyruvate; PCa, prostate cancer; means ± SEM, mean ± standard error of mean.

Figure 2.

EP causes cell cycle arrest and induces apoptosis. (A) EP causes cell cycle arrest of different PCa cells in G0/G1 phases of the cell cycle. The data represented the mean ± SEM (*P<0.05, n=3). Comparisons shown: *significant differences between EP treatment groups and Saline vehicle control groups. (B) The expression of P21, CDK4, Cyclin D1 was detected by western blot in vitro. (C) The expression of PARP and caspase-3, cleaved PARP and caspase-3 on protein level was detected by western blot in vitro. (D) TUNNEL staining showed the apoptotic cells in two groups. Magnification, ×200 (*P<0.05 for Saline vs. Saline + EP (50 mg/kg)). EP, ethyl pyruvate; PCa, prostate cancer; means ± SEM, mean ± standard error of mean.

Figure 3.

EP inhibited PCa cells invasion and migration. (A) EP reduced migration ability in PCa cells, as revealed by the cell wound healing assay. (B) Quantification. (C) EP Inhibition of cell invasion of CWR22RV1 and PC3 cells after EP treatment for 24 h (magnification, ×200). (D) Dose-dependent inhibition of cell invasion of CWR22RV1 and PC3 cells treated with EP. Data represented means ± SEM of three independent experiments. (E) Total cell lysates were extracted from CWR22RV1 and PC3 cells after EP treatment, and the expressions of E-cadherin, Vimentin protein were detected by western blot. β-actin was used as a loading control. The data represented the mean ± SEM (**P<0.01, ***P<0.001, n=3). Comparisons shown: *significant differences between EP treatment groups and Saline vehicle control groups. EP, ethyl pyruvate; PCa, prostate cancer; means ± SEM, mean ± standard error of mean.

Figure 4.

EP suppressed CSC properties and CSC markers expression and AKT/NF-κB signaling pathway. (A) The representative pictures of tumorspheres of PC3 and CWR22Rv1 cells with or without EP treatment (magnification, ×400). Quantification of the suppressive effect of EP on sphere formation of PC3 and CWR22Rv1 cells. The data represented means ± SEM (*P<0.05, n=3). *Significant differences between EP treatment groups and Saline vehicle control groups. (B) PC3 and CWR22Rv1 cells were treated with EP, and then the level of NANOG and SOX2 were determined by western blotting assay as described in the Materials and methods section. Results (mean ± SEM) were obtained from three independent experiments. *P<0.05 between indicated groups. (C) PCa cells were treated with EP (10 and 15 mM) for 48 h. Cell lysates were immunoblotted with specific antibodies to P-AKT, AKT, P65, P-P65. β-actin was used for normalization. (D) PCa cells were stimulated by 10 ng/ml TNF-α for 2 h after treatment with EP (15 mM) for 10 h. The amount of nuclear NF-κB/p65 level was analyzed by fractionation and western blotting assay. Histone 3 was used for normalization. EP, ethyl pyruvate; PCa, prostate cancer; means ± SEM, mean ± standard error of mean.