Retracted Article: FOXO4 overexpression suppresses hypoxia-induced-MCF-7 cell survival and promotes apoptosis through the HIF-2α/Bnip3 signal pathway

Abstract

Transcriptional regulator forkhead box O (FOXO) has implications in many diverse carcinomas and often acts as a tumour suppressor. Evidence suggests that FOXO4 may play a role in cancer cell proliferation and apoptosis; however, the function and mechanism of FOXO4 on breast cancer cell growth are still unknown. FOXO4 can respond to hypoxia and in the current study, our aim is to investigate the function and molecular mechanism of FOXO4 in hypoxia-induced MCF-7 cells. We first observed that hypoxia treatment reduced FOXO4 mRNA and protein expression in MCF-7 cells. Moreover, FOXO4 overexpression reversed hypoxia-induced MCF-7 cell survival. Hypoxia treatment markedly impeded MCF-7 cell apoptosis and inhibited caspase-3 activity, whereas FOXO4 overexpression promoted apoptosis and increased caspase-3 activity in hypoxia-induced MCF-7 cells. Further studies indicated that FOXO4 overexpression inhibited hypoxia-induced HIF-2α and Bnip3 expression in MCF-7 cells; moreover, FOXO4 suppressed Bnip3 expression, which is dependent on the low level of HIF-2α. Finally, we demonstrated that Bnip3 overexpression reversed the effects of FOXO4 overexpression on cell survival and apoptosis in hypoxia-induced MCF-7 cells. In conclusion, the present study suggests that FOXO4 overexpression mediated the HIF-2α/Bnip3 signal pathway, which has implications in cell survival and apoptosis in hypoxia-induced MCF-7 cells.

Fig. 1. Hypoxia reduced FOXO4 expression in MCF-7 cells. (A) RT-PCR was used to measure FOXO4 mRNA levels; (B) FOXO4 protein expression was assayed by Western blot. MCF-7 cells were transfected with pcDNA3.1-FOXO4 for 48 h and then cultured under normoxic (21% O₂; 5% CO₂) or hypoxic (1% O₂; 5% CO₂) conditions for 24 h. Data are expressed as means ± SD from three independent experiments. *p < 0.05 compared with the control group.

Fig. 2. FOXO4 overexpression inhibited hypoxia-induced MCF-7 cell survival. (A) RT-PCR was used to measure FOXO4 mRNA levels; (B) FOXO4 protein expression was assayed by Western blot; (C) MCF-7 cell survival was determined by the CCK-8 assay. MCF-7 cells were transfected with pcDNA3.1-FOXO4 for 48 h and then cultured under normoxic (21% O₂; 5% CO₂) or hypoxic (1% O₂; 5% CO₂) conditions for 24 h. Data are expressed as means ± SD from three independent experiments. *p < 0.05 compared with the control group, #p < 0.05 compared with the hypoxia group.

Fig. 3. FOXO4 overexpression promoted apoptosis in hypoxia-induced MCF-7 cells. (A) Cell death detection ELISA kit was used to detect cell apoptosis; (B) caspase-3 activity was detected using an APOPCYTO Caspase-3 Colorimetric Assay Kit. MCF-7 cells were transfected with pcDNA3.1-FOXO4 for 48 h and then cultured under normoxic (21% O₂; 5% CO₂) or hypoxic (1% O₂; 5% CO₂) conditions for 24 h. Data are expressed as means ± SD from three independent experiments. *p < 0.05 compared with the control group, #p < 0.05 compared with the hypoxia group.

Fig. 4. FOXO4 overexpression suppressed HIF-2α and Bnip3 expression in hypoxia-induced MCF-7 cells. Western blot was used to determine the expression of HIF-2α and Bnip3. MCF-7 cells were transfected with pcDNA3.1-FOXO4 or co-transfected with pcDNA3.1-FOXO4 and pcDNA3.1-HIF-2α for 48 h and then cultured under normoxic (21% O₂; 5% CO₂) or hypoxic (1% O₂; 5% CO₂) conditions for 24 h. Data are expressed as means ± SD from three independent experiments. *p < 0.05 compared with the control group, #p < 0.05 compared with the hypoxia group, and p < 0.05 compared with the pcDNA3.1-FOXO4 group.

Fig. 5. FOXO4 plays roles in hypoxia-induced MCF-7 cells dependent on Bnip3 signal. (A) Western blot was used to determine the expression of Bnip3; (B) MCF-7 cell survival was determined using a CCK-8 assay; (C) cell death detection ELISA kit was used to detect cell apoptosis; (D) caspase-3 activity was detected using an APOPCYTO Caspase-3 Colorimetric Assay Kit. MCF-7 cells were transfected with pcDNA3.1-FOXO4 or co-transfected with pcDNA3.1-FOXO4 and pcDNA3.1-Bnip3 for 48 h and then cultured under normoxic (21% O₂; 5% CO₂) or under hypoxic (1% O₂; 5% CO₂) conditions for 24 h. Data are expressed as means ± SD from three independent experiments. *p < 0.05 compared with the hypoxia + pcDNA3.1 group, #p < 0.05 compared with the hypoxia + pcDNA3.1-FOXO4 group.