Arsenic trioxide-induced growth arrest of breast cancer MCF-7 cells involving FOXO3a and IκB kinase β expression and localization

Abstract

Currently, arsenic has been clinically investigated as a therapeutic agent for a variety of solid malignancies, including breast cancer. However, the exact underlying molecular mechanisms through which arsenic trioxide (As(2)O(3)) induces cell growth arrest and apoptosis in solid tumors have not been clearly understood. The aim of our study was to gain an insight into the effect of As(2)O(3) on the human breast cancer MCF-7 cell line and investigate cell growth inhibition, apoptosis, and the molecular mechanism after As(2)O(3) treatment in MCF-7 cells. Expression of FOXO3a, nuclear-FOXO3a, caspase-3, and IκB kinase β (IKKβ) mRNA levels in MCF-7 cells was determined by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression was examined by the Western blot analysis and immunocytochemical staining. The distribution of apoptotic cells was assessed by flow cytometry, and the morphology of the apoptotic cells was investigated by Hoechest33258 staining. Our results showed that As(2)O(3) significantly induced the apoptosis of MCF-7 cells tested in this study in a dose-dependent manner. As(2)O(3) induced the decrease of IKKβ expression and the increase of total as well as nuclear FOXO3a expression, which triggered the phosphorylation of cytoplasmic FOXO3a at the Thr32 residue decrease. RT-PCR, Western blot analysis, and immunocytochemistry revealed that the expression of IKKβ in MCF-7 cells was upregulated when As(2)O(3) was combined with tumor necrosis factor-α (TNF-α), whereas the expression of FOXO3a was downregulated in comparison with the As(2)O(3)-alone group. These findings indicated a specific molecular mechanism by which MCF-7 cell lines were susceptible to the As(2)O(3) therapy through FOXO3a expression and localization. This FOXO3a accumulation may be well correlated with the As(2)O(3)-induced reduction of active IKKβ, which may provide new insights into As(2)O(3)-related signaling activities.

FIG. 1.

As₂O₃-induced apoptosis of breast cancer MCF-7 cells. The human breast cancer MCF-7 cells were treated with As₂O₃ (2.0, 4.0, and 8.0 μM), or PBS for 24 hours followed by the flow cytometric analysis. (A, B) Apoptosis analysis was conducted using Annexin V-FITC and PI double staining. The Annexin V-positive cells in the total cell population represented apoptotic cells. The percentage of apoptotic cells was significantly higher in 2.0, 4.0, and 8.0 μM groups than that in the PBS group (*p<0.05, compared with the PBS groups, respectively). Results are representative of three independent experiments. As₂O₃, arsenic trioxide; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PI, propidium iodide.

FIG. 2.

Microscopic examination of Hoechst-stained nuclei of breast cancer MCF-7 cells in the untreated group (A) and groups treated with As₂O₃ 2.0 μM (B), As₂O₃ 4.0 μM (C), and As₂O₃ 8.0 μM (D). After treatment with As₂O₃ for 24 hours, MCF-7 cells of visible apoptosis were detected by Hoechst33258 staining (arrows: the apoptotic cells).

FIG. 3.

mRNA expression of FOXO3a, caspase-3, nuclear FOXO3a, and IKKβ in MCF-7 cells after As₂O₃ treatment. Total RNA was isolated, transferred to cDNA, and amplified with FOXO3a, caspase-3, nuclear FOXO3a, and IKKβ primers. (A) RT-PCR analysis of FOXO3a (369 bp) and caspase-3 (243 bp) mRNA levels in MCF-7 cancer cells treated with 2.0, 4.0, and 8.0 μM As₂O₃ for 24 hours, β-actin (205 bp) was used as an internal control for normalization. (B) Densitometric analysis of the amplified FOXO3a and caspase-3 PCR products after normalization with β-actin (*p<0.05, compared with the PBS group cells). (C) RT-PCR analysis of nuclear FOXO3a (369 bp) mRNA level in MCF-7 cancer cells treated with 4.0 μM As₂O₃ at different time points. (D) Densitometric analysis of the amplified nuclear FOXO3a PCR products after normalization with β-actin (*p<0.05, compared with control group cells). (E) RT-PCR analysis of FOXO3a (369 bp) and IKKβ (435 bp) mRNA levels in MCF-7 cancer cells treated with 4.0 μM As₂O₃ for 24 hours and with TNF-α for 2 hours. (F) Densitometric analysis of the amplified FOXO3a and IKKβ PCR product after normalization with β-actin (*p<0.05, compared with control group cells; **p<0.01, compared with 4.0 μM As₂O₃ group). Data were representative of three independent experiments. IKKβ, IκB kinase β; RT-PCR, reverse transcription–polymerase chain reaction; TNF-α, tumor necrosis factor-α.

FIG. 4.

Protein expression of FOXO3a, nuclear FOXO3a, P-FOXO3a, caspase-3, and IKKβ in MCF-7 cells after As₂O₃ treatment was analyzed by Western blot. (A) The expression of phosphorylated and total FOXO3a (91 and 79 kDa) and active caspase-3 (17 kDa) protein levels was analyzed in MCF-7 cells treated with 2.0, 4.0, and 8.0 μM As₂O₃ for 24 hours, respectively; also shown is a blot for β-actin as a loading control, with statistically significant differences in each group (*p<0.05; **p<0.01, compared with the PBS group). (B) The expression of nuclear and cytoplasmic p-FOXO3a protein levels was evaluated in MCF-7 cells treated with 4.0 μM As₂O₃ at different time points, with statistically significant differences in each group (*p<0.05; **p<0.01, compared with the untreated group). (C) The expression of FOXO3a and IKKβ (89 kDa) protein levels was examined in MCF-7 cancer cells treated with 4.0 μM As₂O₃ for 24 hours, followed by combination with TNF-α for 2 hours, with statistically significant differences in each group (*p<0.05; **p<0.01, compared with the 4.0 μM As₂O₃ group). Data were representative of three independent experiments.

FIG. 5.

Expression levels of FOXO3a and IKKβ proteins in MCF-7 cells after As₂O₃ and TNF-α treatment were analyzed by immunocytochemical staining (A, D). Immunocytochemical analyses of IKKβ and FOXO3a expression and cytoplasmic and nuclear staining in normal MCF-7 cells (B, E). Cytoplasmic staining of IKKβ and nuclear staining of FOXO3a in MCF-7 cancer cells were performed following As₂O₃ treatment (4 μM) for 24 hours (p<0.01, compared with the PBS group) (C, F). The expression variations of nuclear FOXO3a and cytoplasmic IKKβ in MCF-7 cells were evaluated after 24 hours of As₂O₃ treatment (4 μM) and subsequent 2 hours of combination with TNF-α (p<0.05, compared with the As₂O₃ [4 μM] group). Data were representative of three independent experiments.