Radiosensitization of human glioma cells by tamoxifen is associated with the inhibition of PKC-ι activity in vitro

Abstract

The present study aimed to investigate the radiosensitizing effects of tamoxifen (TAM), a non-steroidal anti-estrogen drug, in human glioma A172 and U251 cells in vitro. A colony-forming assay revealed that TAM enhances radiosensitivity in A172 and U251 cells. Treatment with TAM also increased the percentage of apoptotic cells subsequent to ionizing radiation, and increased the expression of apoptotic markers, including cleaved caspase-3 and poly(ADP-ribose) polymerase. Ionizing radiation induced G2/M phase arrest, which was alleviated within 24 h when the radiation-induced DNA damage was repaired. However, flow cytometry analysis revealed that TAM treatment delayed the recovery of cell cycle progression. Additional examination demonstrated that TAM-mediated protein kinase C-ι (PKC-ι) inhibition may lead to the activation of pro-apoptotic B-cell lymphoma 2-associated death promoter, and the dephosphorylation of cyclin-dependent kinase 7, resulting in increased cell apoptosis and sustained G2/M phase arrest following exposure to radiation. The present data indicate that the radiosensitizing effects of TAM on glioma cells are partly due to the inhibition of PKC-ι activity in vitro.

Keywords: Bad; cyclin-dependent kinase 7; glioma; protein kinase C-ι; radiosensitivity; tamoxifen.

Figure 1.

TAM enhances the radiosensitivity of the glioma A172 and U251 cell lines. The A172 and U251 cells were treated with various doses of X-ray irradiation and 10 µM TAM for 48 h. A clonogenic survival assay was then performed. *P<0.05 vs control (n=3). TAM, tamoxifen.

Figure 2.

TAM enhances radiation-induced apoptosis in A172 and U251 cells. (A) Flow cytometry was used to determine A172 and U251 cell apoptosis following treatment with either 4-Gy X-rays or 10 µM TAM alone or in combination for 24 h or 48 h. *P<0.05 and **P<0.01 vs. IR group. (B) Western blot analysis was performed to evaluate the expression of activated apoptotic markers. β-actin was used as the loading control. TAM, tamoxifen; IR, irradiation; PARP, poly(ADP-ribose) polymerase.

Figure 3.

TAM alters radiation-induced cell cycle arrest. A172 and U251 cells were treated with either 4-Gy X-rays or 10 µM TAM alone or in combination for 12 h, 24 h and 48 h. Flow cytometry was employed to investigate the cell cycle distribution of glioma cells. TAM, tamoxifen

Figure 4.

Inhibition of PKC-ι signaling by treatment with TAM. A172 or U251 cells were treated with either 4 Gy X-ray or 10 µM TAM alone or in combination for 24 h. Western blot analysis was performed to detect the expression of p-PKC-ι, Bad and p-cdk7. β-actin was used as the loading control. TAM, tamoxifen; IR, irradiation; PKC-ι, protein kinase C-ι; p-PKC-ι, phosphorylated PKC-ι; Bad, B-cell lymphoma 2-associated death promoter; cdk7, cyclin-dependent kinase 7; p-cdk7, phosphorylated cdk7.