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Apoptosis

, Volume 22, Issue 9, pp 1079–1097 | Cite as

Fluoxetine induces apoptosis through endoplasmic reticulum stress via mitogen-activated protein kinase activation and histone hyperacetylation in SK-N-BE(2)-M17 human neuroblastoma cells

  • Ji Hyun Choi
  • Yeon Ju Jeong
  • Ah-Ran Yu
  • Kyung-Sik Yoon
  • Wonchae Choe
  • Joohun Ha
  • Sung Soo Kim
  • Eui-Ju YeoEmail author
  • Insug KangEmail author
Article

Abstract

Fluoxetine (FLX) is an antidepressant drug that belongs to the class of selective serotonin reuptake inhibitors. FLX is known to induce apoptosis in multiple types of cancer cells. In this study, the molecular mechanisms underlying the anti-cancer effects of FLX were investigated in SK-N-BE(2)-M17 human neuroblastoma cells. FLX induced apoptotic cell death, activation of caspase-4, -9, and -3, and expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitors, salubrinal and 4-phenylbutyric acid or CHOP siRNA transfection reduced FLX-induced cell death. FLX induced phosphorylation of mitogen-activated protein kinases (MAPKs) family, p38, JNK, and ERK, and an upstream kinase apoptosis signal kinase 1 (ASK1). Inhibition of MAPKs and ASK1 reduced FLX-induced cell death and CHOP expression. We then showed that FLX reduced mitochondrial membrane potential (MMP) and ER stress inhibitors as well as MAPK inhibitors ameliorated FLX-induced loss of MMP. Interestingly, FLX induced hyperacetylation of histone H3 and H4, upregulation of p300 histone acetyltransferase (HAT), and downregulation of histone deacetylases (HDACs). Treatment with a HAT inhibitor anacardic acid or p300 HAT siRNA transfection blocked FLX-induced apoptosis in SK-N-BE(2)-M17 cells. However, FLX did not induce histone acetylation and anacardic acid had no protective effect on FLX-induced cell death and CHOP expression in MYCN non-amplified SH-SY5Y human neuroblastoma and MYCN knockdowned SK-N-BE(2)-M17 cells. These findings suggest that FLX induces apoptosis in neuroblastoma through ER stress and mitochondrial dysfunction via the ASK1 and MAPK pathways and through histone hyperacetylation in a MYCN-dependent manner.

Keywords

Apoptosis ER stress SK-N-BE(2)-M17 cells Fluoxetine MAPKs Histone hyperacetylation 

Notes

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (Nos. 2011-0030721; 2015R1D1A1A01057508) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (No. HI14C2700).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10495_2017_1390_MOESM1_ESM.jpg (73 kb)
Fig. S1 Effects of FLX on ER stress and histone acetylation in U251MG human glioblastoma cells. a Cells were treated with vehicle or 25 μM FLX for the indicated times. Cell lysates were analyzed by Western blot with antibodies against CHOP, sXBP-1, ATF6α (p90), cleaved caspase-4(C), and β-actin. b, c cells were preincubated with 10 μM salubrinal or 1 mM 4-PBA for 1 h and then treated with FLX for 24 h. Cell viability was determined by MTT assay and the percent viabilities are plotted as the mean ± standard deviation of at least three experiments (b). The protein levels of CHOP and β-actin were detected by Western blot analysis (c). d, e Cells were pretreated with 5 μM anacardic acid (AA) for 1 h or transfected with Scr control or p300 siRNA for 24 h and then followed by FLX for 24 h. Cell viability was determined by MTT assay and the percent viabilities are plotted as the mean ± standard deviation of at least three experiments (d). Cell lysates were analyzed by Western blot with antibodies against CHOP, cleaved caspase-4(C), Ac-H3, Ac-H4, p300, and β-actin (e). f Cells were treated with FLX for the indicated times. Cell lysates were analyzed by Western blot with antibodies against HDACs 1-6, Ac-H3, Ac-H4, and β-actin. *P < 0.01 and **P < 0.01 compared with vehicle- or Scr siRNA-treated cells. #P < 0.01 and ##P < 0.01 compared with FLX-treated cells with vehicle (b) or Scr siRNA transfection (d) (JPG 72 kb)
10495_2017_1390_MOESM2_ESM.jpg (43 kb)
Fig. S2 Effects of MYCN on FLX-induced histone hyperacetylation in U251MG cells. a Cells were transfected with Scr control or MYCN siRNA for 24 h. Cells were then treated with vehicle or 5 μM anacardic acid (AA) for 1 h and followed by 15 μM FLX for 24 h. Cell viability was determined by MTT assay. *P < 0.01 compared with vehicle-treated cells with Scr siRNA transfection. #P < 0.01 compared with FLX-treated cells with Scr siRNA transfection. b After Scr or MYCN siRNA transfection for 24 h, cells were treated with 15 μM FLX for 12 or 24 h. Cell lysates were subjected to Western blot with antibodies against CHOP, cleaved caspase-4(C), Ac-H3, Ac-H4, HDACs 1-4, p300, N-Myc, and β-actin (JPG 42 kb)

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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Ji Hyun Choi
    • 1
  • Yeon Ju Jeong
    • 2
  • Ah-Ran Yu
    • 2
  • Kyung-Sik Yoon
    • 1
  • Wonchae Choe
    • 1
  • Joohun Ha
    • 1
  • Sung Soo Kim
    • 1
  • Eui-Ju Yeo
    • 3
    Email author
  • Insug Kang
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular Biology, School of Medicine, Medical Research Center for Bioreaction to Reactive Oxygen Species, Biomedical Science InstituteKyung Hee UniversitySeoulRepublic of Korea
  2. 2.Department of Biomedical Sciences, Graduate SchoolKyung Hee UniversitySeoulRepublic of Korea
  3. 3.Department of Biochemistry, College of MedicineGachon UniversityIncheonRepublic of Korea

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