Ephrin type‑A receptor 2 regulates sensitivity to paclitaxel in nasopharyngeal carcinoma via the phosphoinositide 3‑kinase/Akt signalling pathway

Abstract

Ephrin type‑A receptor 2 (EphA2) is a receptor tyrosine kinase that is associated with cancer cell metastasis. There has been little investigation into its impact on the regulation of sensitivity to paclitaxel in nasopharyngeal carcinoma (NPC). In the present study, upregulation of EphA2 expression enhanced the survival of NPC 5‑8F cells, compared with control cells exposed to the same concentrations of paclitaxel. Flow cytometry and western blot analysis demonstrated that over‑expression of EphA2 decreased NPC cancer cell sensitivity to paclitaxel by regulating paclitaxel‑mediated cell cycle progression but not apoptosis in vitro. This was accompanied by alterations in the expression of cyclin‑dependent kinase inhibitors, p21 and p27, and of inactive phosphorylated‑retinoblastoma protein. Furthermore, paclitaxel stimulation and EphA2 over‑expression resulted in activation of the phosphoinositide 3‑kinase (PI3K)/Akt signalling pathway in NPC cells. Inhibition of the PI3K/Akt signalling pathway restored sensitivity to paclitaxel in 5‑8F cells over‑expressing EphA2, which indicated that the PI3K/Akt pathway is involved in EphA2‑mediated paclitaxel sensitivity. The current study demonstrated that EphA2 mediates sensitivity to paclitaxel via the regulation of the PI3K/Akt signalling pathway in NPC.

Figure 1

Effects of EphA2 on paclitaxel sensitivity of nasopharyngeal carcinoma 5-8F cells in vitro. (A) Western blot analysis was conducted to confirm the efficiency of EphA2 over-expression. (B) Proliferation assay curves of each group. EphA2, ephrin type-A receptor 2.

Figure 2

Effect of EphA2 over-expression on cell-cycle distribution and apoptosis following exposure to paclitaxel. (A) Graphs of cell cycle distribution in each group. (B) Percentages of cells in different cell cycle phases in each group. (C) Graphs showing apoptosis in each group. (D) Percentage of apoptotic cells in each group. Results are presented as the mean ± standard deviation of at least three independent experiments. ^*P<0.05. FITC, fluorescein isothiocyanate; EphA2, ephrin type-A receptor 2.

Figure 3

Effect of EphA2 over-expression on cyclin-dependent kinase inhibitors, p21Cip1 and p27Kip1, in NPC 5-8F cells. Western blot analysis was used to detect the expression of p21Cip1, p27Kip1 CDK2, Cyclin E and p-Rb in NPC 5-8F and CNE-2 nasopharyngeal carcinoma cells. All data were obtained by three independent experiments, which produced similar results. EphA2, ephrin type-A receptor 2; CDK, cyclin-dependent kinase; Rb, retinoblastoma protein; p-Rb, phosphorylated Rb; NPC, nasopharyngeal carcinoma.

Figure 4

Effect of paclitaxel stimulation and EphA2 overexpression on activation of the PI3K/Akt signalling pathway in NPC 5-8F cells. (A) NPC 5-8F cells were treated with varying concentrations of paclitaxel and PI3K/Akt pathway signalling molecules (total Akt, p-Akt) were measured by western blot analysis. (B) EphA2 over-expression stimulates the PI3K/Akt pathway. The total quantity Akt, p-Akt, GSK-3β and p-GSK-3β was determined by western blot analysis. EphA2, ephrin type-A receptor 2; PI3K, phosphoinositide 3-kinase; p-Akt, phosphorylated Akt; GSK-3β, glycogen synthase kinase-3β; p-GSK-3β, phosphorylated-GSK-3β.

Figure 5

EphA2 mediated paclitaxel sensitivity in NPC 5–8 cells via modulation of the PI3K/Akt signalling pathway. (A) PI3K/Akt signalling pathway inhibitor, LY294002, reverses paclitaxel resistance caused by EphA2 over-expression. (B) Effect of LY294002 on the cell-cycle distribution in EphA2 over-expressing NPC cells pre-treated with paclitaxel. (C) Effect of LY294002 on the apoptotic rate in NPC cells over-expressing EphA2, pretreated with paclitaxel. (D) LY294002 restores the changes in expression of cyclin-dependent kinase inhibitors, p21Cip1 and p27Kip1, caused by EphA2 over-expression. ^*P<0.05. EphA2, ephrin type-A receptor 2; NPC, nasopharyngeal carcinoma; PI3K, phosphoinositide 3-kinase; p-Akt, phsophorylated Akt; Rb, retinoblastoma protein; p-Rb, phosphorylated Rb.