In vitro evaluation of the pogostone effects on the expression of PTEN and DACT1 tumor suppressor genes, cell cycle, and apoptosis in ovarian cancer cell line

Abstract

Background and purpose: Ovarian cancer is one of the most dangerous cancers among women. Pogostone has anticancer effects and is rich in polyphenol compounds. In the present study, we investigated the effects of pogostone on ovarian cancer cell lines (OVCAR-3).

Experimental approach: OVCAR-3 cells were treated with pogostone at IC₅₀(90 μg/mL) for 24 and 48 h. Cell viability and apoptotic rate in the cells were measured using MTT assay and flow cytometry. Real-time PCR was used to determine the expression of genes involved in the cell cycle and apoptosis. The expression of caspase-3 (CASP3) protein was evaluated by the CASP3 assay.

Findings/results: Treatment of OVCAR-3 cells with pogostone increased the expression levels of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and Dapper antagonist of catenin-1 (DACT1) tumor suppressor genes, as well as the apoptotic genes CASPs3, 8, and 9. Moreover, the ratio of the expressed BCL2 associated X (BAX)/BCl2 genes, as pro- and anti-apoptotic genes, was increased. The expression levels of the genes related to the cell cycle progression including cyclin D1 (CCND1) and cyclin- dependent kinase 4 (CDK4) were inhibited. The data obtained from flow cytometry indicated that pogostone induced cell apoptosis in 24 and 48 pogostone groups. The CASP3 colorimetric assay revealed that pogostone increased the expression of CASP3 protein in the treated groups.

Conclusion and implication: Pogostone, by inducing the expression of PTEN and DACT1 tumor suppressor genes and regulation of downstream genes may decrease cell proliferation and increase the rate of apoptosis in OVCAR-3.

Keywords: Apoptosis; Cell cycle; DACT1; Ovarian cancer; PTEN; Pogostone.

Fig. 1

Effect of pogostone on the viability of OVCAR-3 cells. The cells were incubated with different concentrations (10-250 ug/mL) of pogostone for 24 h. Cell viability was measured with an MTT assay. Based on the results, IC₅₀ of pogostone was in the range of 90 μg/mL.

Fig. 2

Comparison of mean cell viability between groups. ***P < 0.001 Indicates significant differences compared to DMSO 24 and ^###P < 0.001 versus DMSO 48.

Fig. 3

The effects of pogostone on cell apoptosis which was determined by flow cytometry. Treatment with 90 μg/mL pogostone for 24 and 48 h, significantly induced apoptosis in OVCAR-3 cells compared with the DMSO treated cells. (A, B) Cells without treatment with any substance in 24 and 48 h, respectively; (C, D) cells treated with DMSO, as the solvent of pogostone, for 24 and 48, respectively; (E, F) cells treated with 90 μg/mL pogostone for 24 and 48 h, respectively.

Fig. 4

The effect of pogostone on the apoptotic cell in different groups evaluated by flow cytometry. ***P < 0.001 Indicates significant differences compared to DMSO 24 and ^###P < 0.001 versus DMSO 48.

Fig. 5

The effects of pogostone on the expression level of genes involved in the signaling pathway, cell cycle, and apoptosis process evaluated by real-time polymerase chain reaction. (A) The expression level of genes involved in phosphoinositide 3-kinases (PI3K)/AKT/mTOR signaling pathway (PTEN, AKT, and mTOR) in different groups; (B) the expression level of genes involved in Wnt/β-catenin signaling pathway (DACT1, GSK3B, and C-MYC) in different groups; (C) the expression level of genes involved in the cell cycle (CDK4 and CCND1) in different groups; (D) the expression level of pro- and anti-apoptotic genes (BAX and BCL2) in different groups; (E) the expression level of genes involved in the apoptotic process (CASP3, CASP8, and CASP9) in different groups. ***P < 0.001 Indicates significant differences compared to DMSO 24 group and ^###P < 0.001 versus DMSO 48 group. PTEN, phosphatase, and tensin homologue deleted on chromosome ten; AKT, protein kinase B; mTOR, mammalian target of rapamycin; DACT1, Dapper antagonist of catenin-1; CDK4, cyclin-dependent kinase 4; CCND1, cyclin D1; CASP, caspase.

Fig. 6

Protein expression of caspase3 in different groups evaluated by caspase colorimetric. ***P < 0.001 Indicates significant differences compared to DMSO 24 and ^###P < 0.001 versus DMSO 48.