Expression of FOXO4 Inhibits Cholangiocarcinoma Cell Proliferation In Vitro via Induction of G0/G1 Arrest

Abstract

Background/aim: Forkhead box O4 (FOXO4) has been demonstrated to be a tumor suppressor and proposed as target for treatment of a variety of cancer types. However, the role of FOXO4 in cholangiocarcinoma (CCA), a dangerous cancer of bile-duct epithelium, has rarely been explored.

Materials and methods: The proliferative rate of CCA cell lines KKU-213B, KKU-055 and KKK-D068 was investigated using the sulforhodamine B (SRB) assay. Levels of FOXO4, cyclin E1 (CCNE1), CCNE2, cyclin-dependent kinase 2 (CDK2) and cell division cycle 25A (CDC25A) expression were measured using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The cell-cycle profile was explored using flow cytometry.

Results: The SRB assay demonstrated that KKU-213B expressed low levels of FOXO4 but its proliferative rate was highest of all cell lines tested. Interestingly, ectopic expression of FOXO4 significantly suppressed proliferation of KKU-213B cells. Cell-cycle analysis revealed that the cell population in the G₀/G₁ phase was significantly higher in FOXO4-transfected KKU-213B cells than in controls. RT-qPCR analysis demonstrated that the levels of expression of genes that play a role in the G₁/S transition, namely CCNE1, CCNE2, CDK2 and CDC25A, were significantly lower in FOXO4-transfected KKU-213B cells compared to controls.

Conclusion: FOXO4 suppressed CCA cell proliferation partly via down-regulating the expression of genes involved in the G₁/S transition, leading to G₀/G₁ arrest. Our findings suggest that induction of FOXO4 expression might be an alternative approach for the treatment of CCA.

Keywords: FOXO4; G0/G1 arrest; cell cycle; cholangiocarcinoma.