Abstract
Purpose
One of the recently developed polyamine (PA) analogues, N1,N11-diethylnorspermine (DENSpm), has been found to act as an apoptotic inducer in melanoma, breast, prostate and colon cancer cells. Also, its potential to induce autophagy has been established. Unfolded protein responses and starvation of amino acids are known to trigger autophagy. As yet, however, the molecular mechanism underlying PA deficiency-induced autophagy is not fully clarified. Here, we aimed to determine the apoptotic effect of DENSpm after autophagy inhibition by 3-methyladenine (3-MA) or siRNA-mediated Beclin-1 silencing in colon cancer cells.
Methods
The apoptotic effects of DENSpm after 3-MA treatment or Beclin-1 silencing were determined by PI and AnnexinV/PI staining in conjunction with flow cytometry. Intracellular PA levels were measured by HPLC, whereas autophagy and the expression profiles of PA key players were determined in HCT116, SW480 and HT29 colon cancer cells by Western blotting.
Results
We found that DENSpm-induced autophagy was inhibited by 3-MA treatment and Beclin-1 silencing, and that apoptotic cell death was increased by PA depletion and spermidine/spermine N1-acetyltransferase (SSAT) upregulation. We also found that autophagy inhibition led to DENSpm-induced apoptosis through Atg5 down-regulation, p62 degradation and LC3 lipidation in both HCT116 and SW480 cells. p53 deficiency did not alter the response of the colon cancer cells to DENSpm-induced apoptotic cell death under autophagy suppression conditions.
Conclusions
From our results we conclude that DENSpm-induced apoptotic cell death is increased when autophagy is inhibited by 3-MA or Beclin-1 siRNA through PA depletion and PA catabolic activation in colon cancer cells, regardless p53 mutation status.
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Abbreviations
- AO:
-
Acridine Orange
- AZI:
-
Antizyme Inhibitor
- BENSpm:
-
N1,N11-bis (ethyl) norspermine
- BH:
-
Bcl-2 homology
- CHENSpm:
-
N1-ethyl-N11-((cycloheptyl) methyl)-4,8-diazaundecane
- DiOC6 :
-
3,3′-Dihexyloxacarbocyanine Iodide
- DENSpm:
-
N1,N11-diethylnorspermine
- DMSO:
-
Dimethylsulfoxide
- GFP:
-
Green Fluorescence Protein
- MDC:
-
Monodansyl Cadaverin
- MTT:
-
3–4,5-Dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide
- ODC:
-
Ornithine Decarboxylase
- PAO:
-
Polyamine Oxidase
- PBS:
-
Phosphate-buffered saline
- PI:
-
Propidium Iodide
- Put:
-
Putrescine
- PVDF:
-
Polyvinyldifluoride
- SDS-PAGE:
-
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
- Spd:
-
Spermidine
- Spm:
-
Spermine
- SSAT:
-
Spermidine/spermine N1-acetyltransferase
- TBS:
-
Tris-buffered saline
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Acknowledgements
This work was supported by The Scientific and Technological Research Council of TURKEY [(TUBITAK), Grant Number; 212T227] and the Istanbul Kultur University Scientific Projects Support Center. We are thankful to Esin Guvenir, Merve Karatas and Derya Bulut for their technical assistance.
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Gurkan, A.C., Arisan, E.D., Yerlikaya, P.O. et al. Inhibition of autophagy enhances DENSpm-induced apoptosis in human colon cancer cells in a p53 independent manner. Cell Oncol. 41, 297–317 (2018). https://doi.org/10.1007/s13402-017-0369-x
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DOI: https://doi.org/10.1007/s13402-017-0369-x