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Inhibition of autophagy enhances DENSpm-induced apoptosis in human colon cancer cells in a p53 independent manner

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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Fig. 1: DENSpm-induced apoptotic cell death is increased by 3-MA pre-treatment in colon cancer cells.
Fig. 2: Effect of 3-MA on DENSpm-induced cell viability, cell death and mitochondrial membrane potential.
Fig. 3: Increased effect of 3-MA pre-treatment on DENSpm-induced cell cycle arrest and apoptotic cell death.
Fig. 5
Fig. 6: Involvement of PA in DENSpm-induced apoptotic cell death under Beclin-1 silencing conditions in colon cancer cells.
Fig. 7: Role of p53 in DENSpm-induced autophagy under autophagy inhibition conditions.
Fig. 8: Modulation of DENSpm-induced cell viability loss, cell cycle arrest and apoptotic cell death under autophagy inhibition conditions in p53 deficient HCT116 and p53 null HT29 cells.
Fig. 9: Autophagy inhibition triggers DENSpm-induced PA depletion by activating PA catabolic enzymes in a p53 dependent manner in colon cancer cells.

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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Correspondence to Ajda Coker Gurkan.

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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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Keywords

  • Colon cancer
  • Apoptosis
  • Autophagy
  • Polyamine analogue
  • DENSpm