Advertisement

Molecular Biology Reports

, Volume 46, Issue 1, pp 1275–1284 | Cite as

The unfolded protein response controls endoplasmic reticulum stress-induced apoptosis of MCF-7 cells via a high dose of vitamin C treatment

  • Peter BoberEmail author
  • Zuzana Tomková
  • Michal Alexovič
  • Ivan Ropovik
  • Ján Sabo
Original Article
  • 168 Downloads

Abstract

Recent in vitro studies have shown that vitamin C (Vit C) with pro-oxidative properties causes cytotoxicity of breast cancer cells by selective oxidative stress. However, the effect of Vit C in itself at different concentration levels on MCF-7 breast cancer cell line after 24 h, has not yet been described. We aimed to examine the effect of Vit C on the viability and signalling response of MCF-7/WT (MCF-7 wild-type) cells that were exposed to various concentrations (0.125–4 mM) of Vit C during 24 h. The cytotoxic effect of Vit C on MCF-7/VitC (MCF-7/WT after added 2 mM Vit C) was observed, resulting in a decrease of cell index after 12 h. Also, the cytotoxicity of Vit C (2 mM) after 24 h was confirmed by flow cytometry, i.e., increase of dead, late apoptotic, and depolarized dead MCF-7/VitC cells compared to MCF-7/WT cells. Moreover, changes in proteomic profile of MCF-7/VitC cells compared to the control group were investigated via label-free quantitative mass spectrometry and post-translational modification. Using bioinformatics assessment (i.e., iPathwayGuide and SPIA R packages), a significantly impacted pathway in MCF-7/VitC was identified, namely the protein processing in endoplasmic reticulum. The semi-quantitative change (log2fold change = 4.5) and autophosphorylation at Thr-446 of protein kinase (PKR) (involved in this pathway) indicates that PKR protein could be responsible for the unfolded protein response and inhibition of the cell translation during endoplasmic reticulum stress, and eventually, for cell apoptosis. These results suggest that increased activity of PKR (Thr-446 autophosphorylation) related to cytotoxic effect of Vit C (2 mM) may cause the MCF-7 cells death.

Keywords

MCF-7 cells Vitamin C Unfolded protein response Endoplasmic reticulum stress PKR protein 

Notes

Acknowledgements

This work was supported by the Agency of the Slovak Ministry of Education for the Structural Funds of the EU, under project ITMS: 26220220143.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants and/or animals performed by any of the authors.

Supplementary material

11033_2019_4598_MOESM1_ESM.xls (42 kb)
Supplementary material 1 (XLS 42 KB)
11033_2019_4598_MOESM2_ESM.xlsx (184 kb)
Supplementary material 2 (XLSX 184 KB)

References

  1. 1.
    da Mata AMOF, de Carvalho RM, de Alencar MVOB et al (2016) Ascorbic acid in the prevention and treatment of cancer. Rev da Assoc Med Bras 62:680–686CrossRefGoogle Scholar
  2. 2.
    Park S (2013) The effects of high concentrations of vitamin C on cancer cells. Nutrients 5:3496–3505CrossRefGoogle Scholar
  3. 3.
    Takemura Y, Satoh M, Satoh K et al (2010) High dose of ascorbic acid induces cell death in mesothelioma cells. Biochem Biophys Res Commun 394:249–253CrossRefGoogle Scholar
  4. 4.
    Chen Q, Espey MG, Krishna MC et al (2005) Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl Acad Sci 102:13604–13609CrossRefGoogle Scholar
  5. 5.
    Creagan ET, Moertel CG, Fallon JR et al (1979) Failure of high-dose vitamin C (ascorbic acid) therapy to benefit patients with advanced cancer. N Engl J Med 301:687–690CrossRefGoogle Scholar
  6. 6.
    Moertel CG, Fleming TR, Creagan ET et al (1985) High-dose vitamin C versus placebo in the treatment of patients with advanced cancer who have had no prior chemotherapy. N Engl J Med 312:137–141CrossRefGoogle Scholar
  7. 7.
    Benade L, Howard T, Burk D (1969) Synergistic killing of ehrlich ascites carcinoma cells by ascorbate and 3-amino-1,2,4,-triazole. Oncology 23:33–43CrossRefGoogle Scholar
  8. 8.
    Koch CJ, Biaglow JE (1978) Toxicity, radiation sensitivity modification, and metabolic effects of dehydroascorbate and ascorbate in mammalian cells. J Cell Physiol 94:299–306CrossRefGoogle Scholar
  9. 9.
    McQuiston A, Diehl JA (2017) Recent insights into PERK-dependent signaling from the stressed endoplasmic reticulum. F1000Research 6:1897CrossRefGoogle Scholar
  10. 10.
    Boyce M, Yuan J (2006) Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ 13:363–373CrossRefGoogle Scholar
  11. 11.
    Clemens MJ, Elia A (1997) The double-stranded RNA-dependent protein kinase PKR: structure and function. J Interfaces Cytokine Res 17:503–524CrossRefGoogle Scholar
  12. 12.
    Gil J, Esteban M (2000) Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis 5:107–114CrossRefGoogle Scholar
  13. 13.
    Ung TL (2001) Heterologous dimerization domains functionally substitute for the double-stranded RNA binding domains of the kinase PKR. EMBO J 20:3728–3737CrossRefGoogle Scholar
  14. 14.
    Nanduri S (2000) A dynamically tuned double-stranded RNA binding mechanism for the activation of antiviral kinase PKR. EMBO J 19:5567–5574CrossRefGoogle Scholar
  15. 15.
    DuRose JB, Scheuner D, Kaufman RJ et al (2009) Phosphorylation of eukaryotic translation initiation factor 2 ~ coordinates rRNA transcription and translation inhibition during endoplasmic reticulum stress. Mol Cell Biol 29:4295–4307CrossRefGoogle Scholar
  16. 16.
    Kustermann S, Boess F, Buness A et al (2013) A label-free, impedance-based real time assay to identify drug-induced toxicities and differentiate cytostatic from cytotoxic effects. Toxicol Vitr 27:1589–1595CrossRefGoogle Scholar
  17. 17.
    Sandin M, Teleman J, Malmström J, Levander F (2014) Data processing methods and quality control strategies for label-free LC/MS protein quantification. Biochim Biophys Acta-Protein Proteom 1844:29–41CrossRefGoogle Scholar
  18. 18.
    Dar AC, Dever TE, Sicheri F (2005) Higher-order substrate recognition of eIF2 alpha by the RNA-dependent protein kinase PKR. Cell 122:887–900CrossRefGoogle Scholar
  19. 19.
    Buettner GR (1993) The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate. Arch Biochem Biophys 300:535–543CrossRefGoogle Scholar
  20. 20.
    Samuni A, Aronovitch J, Godinger D et al (1983) On the cytotoxicity of vitamin C and metal ions. A site- specific fenton mechanism. Eur J Biochem 137:119–124CrossRefGoogle Scholar
  21. 21.
    Sakagami H, Satoh K, Hakeda Y et al (2000) Apoptosis-inducing activity of vitamin C and vitamin K. Cell Mol Biol 46:129–143Google Scholar
  22. 22.
    Clément MV, Ramalingam J, Long LH et al (2001) The in vitro cytotoxicity of ascorbate depends on the culture medium used to perform the assay and involves hydrogen peroxide. Antioxid Redox Signal 3:157–163CrossRefGoogle Scholar
  23. 23.
    Chen Q, Espey MG, Sun AY (2008) Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc Natl Acad Sci 105:11105–11109CrossRefGoogle Scholar
  24. 24.
    Michels AJ, Frei B (2013) Myths, artifacts, and fatal flaws: identifying limitations and opportunities in vitamin C research. Nutrients 5:5161–5192CrossRefGoogle Scholar
  25. 25.
    Golubitskii GB, Budko EV, Basova EM et al (2007) Stability of ascorbic acid in aqueous and aqueous–organic solutions for quantitative determination. J Anal Chem 62:742–747CrossRefGoogle Scholar
  26. 26.
    Gonzalez MJ, Miranda-Massari JR (2014) New insights on vitamin C and cancer, springerbriefs in cancer research. Springer, New York, pp 17–22Google Scholar
  27. 27.
    Wang C, Youle RJ (2009) The role of mitochondria in apoptosis. Annu Rev Genet 43:95–118CrossRefGoogle Scholar
  28. 28.
    Ly JD, Grubb DR, Lawen A (2003) The mitochondrial membrane potential (∆ψm) in apoptosis; an update. Apoptosis 8:115–128CrossRefGoogle Scholar
  29. 29.
    Hanks SK, Hunter T (1995) Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J 9:576–596CrossRefGoogle Scholar
  30. 30.
    Nanduri S, Carpick BW, Yang Y et al (1998) Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation. EMBO J 17:5458–5465CrossRefGoogle Scholar
  31. 31.
    Dey M, Mann BR, Anshu A, Mannan MA (2013) Activation of protein kinase PKR requires dimerization-inducedcis-phosphorylation within the activation loop. J Biol Chem 289:5747–5757CrossRefGoogle Scholar
  32. 32.
    Faitova J, Krekac D, Hrstka R, Vojtesek B (2006) Endoplasmic reticulum stress and apoptosis. Cell Mol Biol Lett 11:488–505CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Medical and Clinical Biophysics, Faculty of MedicineUniversity of P.J. Šafárik in KošiceKošiceSlovakia
  2. 2.Department of Pre-school and Elementary Education and Psychology, Faculty of Education, University of PresovPresovSlovakia

Personalised recommendations