PKR-Mediated Phosphorylation of eIF2a and CHK1 Is Associated with Doxorubicin-Mediated Apoptosis in HCC1143 Triple-Negative Breast Cancer Cells

Abstract

Triple-negative breast cancer is more aggressive than other types of breast cancer. Protein kinase R (PKR), which is activated by dsRNA, is known to play a role in doxorubicin-mediated apoptosis; however, its role in DNA damage-mediated apoptosis is not well understood. In this study, we investigated the roles of PKR and its downstream players in doxorubicin-treated HCC1143 triple-negative breast cancer cells. Doxorubicin treatment induces DNA damage and apoptosis. Interestingly, doxorubicin treatment induced the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) via PKR, whereas the inhibition of PKR with inhibitor C16 reduced eIF2α phosphorylation. Under these conditions, doxorubicin-mediated DNA fragmentation, cell death, and poly(ADP ribose) polymerase and caspase 7 levels were recovered. In addition, phosphorylation of checkpoint kinase 1 (CHK1), which is known to be involved in doxorubicin-mediated DNA damage, was increased by doxorubicin treatment, but blocked by PKR inhibition. Protein translation was downregulated by doxorubicin treatment and upregulated by blocking PKR phosphorylation. These results suggest that PKR activation induces apoptosis by increasing the phosphorylation of eIF2α and CHK1 and decreasing the global protein translation in doxorubicin-treated HCC1143 triple-negative breast cancer cells.

Keywords: CHK1; ER; PKR; apoptosis; doxorubicin; eIF2α; triple-negative breast cancer.

Figure 1

Doxorubicin induces DNA damage and apoptosis in HCC1143 triple-negative breast cancer cells. (A,B) Cells were treated with doxorubicin for the indicated time period or dose. DNA was extracted from cells and electrophoresed on agarose gels. Cisplatin (cis) was used as a positive control. (C,D) Cells were treated with the indicated concentrations of doxorubicin for 48 h and stained with Hoechst 33342, at room temperature, for 10 min. Nuclear morphological changes were observed using a confocal laser scanning microscope (magnification 100×). Fragmented nuclei are indicated by arrows, and the lower left squares are enlarged representative images (C). Scale bar is 100 μm. Fragmented nuclei were counted using a confocal laser scanning microscope (D). (E). HCC1143 cells were treated with doxorubicin at various concentrations for 48 h and MTT assay was performed to measure viability of cells. (F,G) Cells were treated with 1.5 μM doxorubicin for the indicated time period (F) or with increasing concentrations of doxorubicin for 48 h. (G) Immunoblotting analyses were conducted using specific antibodies against PARP, cleaved form of caspase-7 (c-Cas-7), and β-actin. Arrowhead indicates cleaved form of PARP-1.

Figure 2

Doxorubicin-mediated activation of protein kinase R (PKR), but not PKR-like ER kinase (PERK), is responsible for eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation in HCC1143 cells. (A,B) Cells were treated with 2 μM doxorubicin for the indicated time period and analyzed via immunoblotting using antibodies against phosphorylated form of eIF2α (p-eIF2α), eIF2α, phosphorylated form of PKR (p-PKR), PKR, PERK, and β-Actin. Tg was used as a positive control. Fold change is the ratios of p-PKR to PKR and p-eIF2α to eIF2α. (C) Cells were treated with the indicated concentrations of doxorubicin for 32 h and analyzed via immunoblotting using specific antibodies against p-PKR, PKR, p-eIF2α, eIF2α, and β-Actin. (D) Cells pretreated with or without 0.5 μM C16 for 1 h were treated with the indicated concentrations of doxorubicin for 32 h and analyzed via immunoblotting using specific antibodies against p-PKR, PKR, p-eIF2α, eIF2α, and β-Actin. Fold change is the ratios of p-PKR to PKR and p-eIF2α to eIF2α.

Figure 3

Inhibition of PKR reduces the doxorubicin-mediated apoptosis of HCC1143 cells. (A) Cells pretreated with or without 0.5 μM C16 for 1 h were treated with the indicated concentrations of doxorubicin for 48 h. DNA extracted from the cells was electrophoresed on agarose gels. (B,C) Cells pretreated with or without 0.5 μM C16 for 1 h were treated with 0.5 μM doxorubicin for 48 h and stained with Hoechst 33342, at room temperature, for 10 min. Nuclear morphological changes were observed using a confocal laser scanning microscope (magnification 100×). Scale bar is 100 μm. Lower-left squares are enlarged representative images (B). In each group, fragmented nuclei were counted using a confocal laser scanning microscope. Statistical significance was analyzed via the Student’s t-test (*** p < 0.001) (C). (D) Cells pretreated with or without 0.5 μM C16 for 1 h were treated with the indicated concentrations of doxorubicin for 32 h and analyzed via immunoblotting using specific antibodies against PARP, c-Cas-7, and β-actin. Arrowhead indicates cleaved form of PARP-1.

Figure 4

Doxorubicin-induced PKR-mediated apoptosis is caused by eIF2α/CHOP signaling and checkpoint kinase 1 (CHK1) phosphorylation in HCC1143 cells. (A,B) Cells pretreated with or without 0.5 μM C16 for 1 h were treated with 2 μM doxorubicin for the indicated time period. RT-PCR was performed using specific primers for C/EBP homologous protein (CHOP) and β-actin (A). Cells were treated with 10 μg/mL puromycin for 10 min. Cell lysates were subjected to immunoblotting analysis using specific antibodies against puromycin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (B). (C) Cells were treated with 2 μM doxorubicin for the indicated time period and analyzed via immunoblotting using specific antibodies against CHK1, phosphorylated form of CHK1 (p-CHK1), and β-Actin. (D,E) Cells pretreated with or without 0.5 μM C16 for 1 h were treated with the indicated concentrations of doxorubicin for 32 h (D) or 2 μM doxorubicin for the indicated time period (E) and analyzed via immunoblotting using specific antibodies against p-CHK1, CHK1, p-p53, p53, and β-Actin. Fold change is the ratio of p-CHK1 to CHK1 (D) and the ratios of p-CHK1 to CHK1 and p-p53 to p53 (E).