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, 42 (12), 884-892

Increased Expression of FosB Through Reactive Oxygen Species Accumulation Functions as Pro-Apoptotic Protein in Piperlongumine Treated MCF7 Breast Cancer Cells

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Increased Expression of FosB Through Reactive Oxygen Species Accumulation Functions as Pro-Apoptotic Protein in Piperlongumine Treated MCF7 Breast Cancer Cells

Jin-Ah Park et al. Mol Cells.

Abstract

Piperlongumine (PL), a natural alkaloid compound isolated from long pepper (Piper longum), can selectively kill cancer cells, but not normal cells, by accumulation of reactive oxygen species (ROS). The objective of this study was to investigate functional roles of expression of SETDB1 and FosB during PL treatment in MCF7 breast cancer cells. PL downregulates SETDB1 expression, and decreased SETDB1 expression enhanced caspase 9 dependent-PARP cleavage during PL-induced cell death. PL treatment generated ROS. ROS inhibitor NAC (N-acetyl cysteine) recovered SETDB1 expression decreased by PL. Decreased SETDB1 expression induced transcriptional activity of FosB during PL treatment. PARP cleavage and positive annexin V level were increased during PL treatment with FosB overexpression whereas PARP cleavage and positive annexin V level were decreased during PL treatment with siFosB transfection, implying that FosB might be a pro-apoptotic protein for induction of cell death in PL-treated MCF7 breast cancer cells. PL induced cell death in A549 lung cancer cells, but molecular changes involved in the induction of these cell deaths might be different. These results suggest that SETDB1 mediated FosB expression may induce cell death in PL-treated MCF7 breast cancer cells.

Keywords: FosB; SETDB1; breast cancer; cell death; piperlongumine; reactive oxygen species.

Conflict of interest statement

Disclosure

The authors have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. PL treatment downregulates SETDB1 expression
(A) MCF7 cells were treated with PL at indicated concentration for 24 h. mRNA and protein levels were estimated by RT-PCR and western blot, respectively. (B) MCF7 cells were immuno-stained with specific antibodies to SETDB1 (green). DAPI was used as a counter staining for nucleus. Scale bars = 50 μm. (C) MCF7 cells were transiently transfected with SETDB1 siRNA for 16 h followed by treatment with 10 μM PL for 24 h. Protein levels of SETDB1 and PARP cleavage were examined by western blot analyses. (D) Cell viability was assessed by MTT assay. Statistical significance is indicated as ***P < 0.001.
Fig. 2
Fig. 2. Decreased SETDB1 expression is associated with ROS
(A) MCF7 cells were treated with 10 mM NAC for 1 h followed by treatment with 10 μM of PL for 6 h. ROS were stained with H2DCFDA and observed under a fluorescence microscope. Scale bars = 50 μm. (B) MCF7 cells were immune-stained with SETDB1 antibody (red). DAPI was used for nuclear staining. Scale bars = 50 μm. (C) mRNA and protein levels were analyzed after treatment with NAC or PL. Left, RT-PCR; right, Western blot.
Fig. 3
Fig. 3. SETDB1 mediated FosB expression is regulated by ROS during PL treatment
(A) Luciferase assay for FosB promoter was performed after siSETDB1 transfection and PL treatment. FosB promoter activity was increased in the presence of siSETDB1. It was increased after siSETDB1 transfection along with PL treatment. *P < 0.05, **P < 0.01, ***P < 0.001. (B) MCF7 cells were transiently transfected with siSETDB1 for 16 h followed by treatment with 10 μM PL for 24 h. mRNA and protein expression levels were analyzed by RT-PCR and western blot, respectively. (C) MCF7 cells were treated with 10 mM NAC for 1 h followed by treatment with 10 μM PL for 24 h. Protein level of FosB was measured by Western blot.
Fig. 4
Fig. 4. FosB expression triggers death of PL-treated MCF7 cells
(A) MCF7 cells were transfected with FosB plasmid for 16 h followed by treatment with 10 μM PL for 24 h. MTT assay was performed to evaluate death of MCF7 cells. (B) PARP cleavage was analyzed by Western blot after FosB transfection along with PL treatment. (C) Annexin V positive cells were observed under a confocal microscope after FosB transfection along with PL treatment. Scale bars = 50 μm. (D) Annexin V/PI positive cells were sorted by flow cytometry. Negative stained cells for both annexin V and PI are viable cells (lower left). Cells stained positive for annexin V and negative for PI mean early apoptotic stage (lower right). Positively stained cells for both annexin V and PI are at the end stage of apoptosis (upper right). In the graph, percentages of dead or dying cells were significantly increased after FosB transfection along with PL treatment (***P < 0.001).
Fig. 5
Fig. 5. PL-induced cell death is decreased by siFosB transfection
(A) MCF7 cells were transfected with siFosB followed by PL treatment. MTT assay was performed to evaluate death of MCF7 cells (*P < 0.05). (B) PARP cleavage was analyzed by Western blot after siFosB transfection along with PL treatment. (C) Annexin V positive cells were observed under a confocal microscope after siFosB transfection along with PL treatment. Scale bars = 50 μm. (D) Annexin V/PI positive cells were sorted by flow cytometry after siFosB transfection along with PL treatment. In the graph, percentages of cell death were significantly decreased after siFosB transfection along with PL treatment (***P < 0.001).

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References

    1. Al Emran A., Marzese D.M., Menon D.R., Stark M.S., Torrano J., Hammerlindl H., Zhang G., Brafford P., Salomon M.P., Nelson N., et al. Distinct histone modifications denote early stress-induced drug tolerance in cancer. Oncotarget. 2018;9:8206–8222. doi: 10.18632/oncotarget.23654. - DOI - PMC - PubMed
    1. Barrett C.S., Millena A.C., Khan S.A. TGF-beta effects on prostate cancer cell migration and invasion require FosB. Prostate. 2017;77:72–81. doi: 10.1002/pros.23250. - DOI - PMC - PubMed
    1. Dhillon A.S., Hagan S., Rath O., Kolch W. MAP kinase signalling pathways in cancer. Oncogene. 2007;26:3279–3290. doi: 10.1038/sj.onc.1210421. - DOI - PubMed
    1. Du D., Katsuno Y., Meyer D., Budi E.H., Chen S.H., Koeppen H., Wang H., Akhurst R.J., Derynck R. Smad3-mediated recruitment of the methyltransferase SETDB1/ESET controls Snail1 expression and epithelial-mesenchymal transition. EMBO Rep. 2018;19:135–155. doi: 10.15252/embr.201744250. - DOI - PMC - PubMed
    1. Gajewski P.A., Turecki G., Robison A.J. Differential expression of FosB proteins and potential target genes in select brain regions of addiction and depression patients. PLoS One. 2016;11:e0160355. doi: 10.1371/journal.pone.0160355. - DOI - PMC - PubMed
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