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. 2018 Feb 28;41(3):297–317. doi: 10.1007/s13402-017-0369-x

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

Ajda Coker Gurkan 1,✉, Elif Damla Arisan 1, Pinar Obakan Yerlikaya 1, Halime Ilhan 1, Narcin Palavan Unsal 1
PMCID: PMC12995235  PMID: 29492901

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.

Keywords: Colon cancer, Apoptosis, Autophagy, Polyamine analogue, DENSpm

Introduction

Putrescine (Put), spermidine (Spd) and spermine (Spm) are natural polyamines (PAs) with essential roles in cell proliferation, growth and differentiation. Increased intracellular PA levels have, however, been found to be associated with cancer initiation and progression [1]. Consequently, agents that activate PA catabolism have gained interest as potential new anticancer drugs [2]. This has resulted in the synthesis of various PA analogues, such as N1,N11-bis(ethyl)norspermine (BENSpm), N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm) and N1,N11-diethylnorspermine (DENSpm), to decrease the intracellular PA pool [3] and to assess their apoptotic cell death potential in various cancer cell types [4].

Recently, DENSpm, which is one of the most studied PA analogues, has been shown to induce G1 cell cycle arrest in MALME-3 M melanoma cells via targeting the p53-p21-Rb signalling axis [5]. Other effects of DENSpm include apoptotic cell death through spermidine/spermine acetyl transferase (SSAT) activation and induction of caspase-3 cleavage and cytochrome c release in SK-MEL-28 melanoma [6], prostate [7], breast [8] and non-small cell lung [9] cancer cells. Conversely, in phase I and II breast and prostate cancer trials, the efficacy of DENSpm was found to be relatively low compared to its in vitro effects [10, 11]. As a consequence, attempts were made to enhance the apoptotic potential of DENSpm by combination treatment with DNA damaging agents (oxaliplatin, cisplatin) in ovarian cancer cells [12] and 5-fluorouracil (5-FU) in colon cancer cells [13]. It was also found that DENSpm and paclitaxel co-treatment may overcome Bcl-2 mediated paclitaxel resistance in MCF-7 breast cancer cells [14]. Besides its apoptosis inducing potential, we recently reported DENSpm-induced autophagy in HCT116, SW480 and HCT116 p53−/− colon cancer cells [15].

Autophagy is a self-degradation process of cellular components that involves elimination of misfolded or aggregated proteins and damaged organelles, such as mitochondria, endoplasmic reticulum and peroxisomes, as well as intracellular pathogens [16–18]. The autophagy process is initiated by sequestering intracellular components within crescent-shaped membranes (phagophores), which are maintained to form closed double-membranes (autophagosomes), ultimately leading to fusion with lysosomes to form autolysosomes [19]. One of the key molecules of autophagy, Beclin-1, exhibits a structural resemblance to the Bcl-2 homology (BH) 3 domain present in Bcl-2 family members. Consequently, it has been proposed that Beclin-1 may act as a linker protein between apoptosis and autophagy [20]. Although DENSpm has been shown to induce both apoptosis and autophagy through acting on key molecular players and triggering PA depletion, the apoptotic cell death potential of DENSpm under autophagy inhibition conditions and the role of PAs therein have so far not been investigated.

The aim of this study was to assess the potential apoptotic effect of DENSpm on PA metabolism in HCT116 p53 wild-type, SW480 p53 mutant and HT29 p53 null colon cancer cells under autophagy inhibition conditions imposed by 3-methyladenine (3-MA) treatment or siRNA-mediated Beclin-1 silencing. We found that a combined treatment of 3-MA with DENSpm caused a significant increase in cell viability loss and apoptotic cell death through cleavage of poly(ADP-ribose) polymerase (PARP), caspase-3 and caspase-9 in HCT16 and SW480 cells. In addition, we found that autophagy inhibition by Beclin-1 silencing increased cell viability loss and apoptotic cell death through Atg5 expression down-regulation, LC-3 cleavage inhibition (lipidation) and p62 degradation in HCT116 and SW480 cells. PA pool depletion, ornithine decarboxylase (ODC) down-regulation and spermidine/spermine N1-acetyltransferase (SSAT) up-regulation were observed after DENSpm apoptosis induction and autophagosome formation inhibition by both 3-MA treatment and Beclin-1 silencing. Although DENSpm-induced apoptotic cell death was observed in p53 deficient colon cancer cells [pifithrin pre-treated HCT116 and HT29 (p53 null) cells], no additional apoptotic effect was observed after 3-MA treatment or Beclin-1 silencing. This information may be instrumental for the development of autophagy-based therapeutic interventions in colon cancer.

Materials and methods

Drugs, chemical and antibodies

DENSpm was purchased from TOCRIS (Tocris Bioscience, Bristol, UK), dissolved in water to prepare a 10 mM stock solution of which aliquots were kept at −20 °C. 3-Methyl adenine (3-MA), Acridine Orange (AO), Monodansyl Cadaverin (MDC) and Pifithrin were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). 3-MA was dissolved in Dimethylformamide (DMF) to prepare a 5 mM stock solution, whereas the other drugs were dissolved in DMSO to prepare 10 mM stock solutions. All aliquots were kept at −20 °C. Rabbit anti-β-actin, anti-PARP, anti-caspase-9, anti-caspase-3, anti-PI3K, anti-Akt, anti-Atg5, anti-Atg12 and anti-LC3 antibodies (each 1:1000 dilution) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA), whereas rabbit anti-SSAT and anti-PAO antibodies (each 1:1000 dilution) were purchased from Santa Cruz (Santa Cruz, CA, USA). ODC and AZI were kindly gifted by Prof. Chaim Kahana from The Weizmann Institute, Israel. Mouse anti-Beclin-1 and anti-p62 antibodies (each 1:1000 dilution) were purchased from Becton Dickinson Biosciences (BD Biosciences, Bedford, MA, USA), whereas HRP-conjugated secondary anti-rabbit and anti-mouse antibodies (1:5000) were purchased from CST (Danvers, MA, USA).

Cell lines and culture conditions

HCT116 (CCL-247), SW480 (CCL-228) and HT29 (HTB-38) colon cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, USA). The cells were maintained in McCoy’s medium (PAN Biotech, Aidenbach, Germany) and MEM (PAN Biotech, Aidenbach, Germany) supplemented with 2 mM L-glutamine, 10% fetal calf serum (PAN Biotech, Aidenbach, Germany), 1% non-essential amino acids (Biological Industries, Kibbutz Beit-Haemek, Israel) and 100 U/100 mg/ml penicillin/streptomycin (Biological Industries, Kibbutz Beit-Haemek, Israel) in the presence of 5% CO2 in a humidified atmosphere at 37 °C.

MTT cell viability assay

The dose-dependent effect of DENSpm and/or autophagy inhibition (3-MA treatment or siRNA-mediated Beclin-1 silencing) on cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (Roche, Indianapolis, IN, USA) in HCT116 (p53 wild-type), SW480 (p53 mutant) and HT29 (p53 null) cells. Each cell line was seeded at a density of 1 × 104 cells/well in 96-well plates and treated with various concentrations of DENSpm (0–50 μM) for 24, 48 and 72 h. 3-MA (10 μM) was applied as a 2 h pre-treatment, whereas Beclin-1 siRNA was co-treated with DENSpm (10 μM). Pifithrin (2 μM) pre-treated (2 h) HCT116 cells were used as a p53 inhibited model. After exposure of cells to DENSpm and autophagy inhibition, 10 μl MTT dye (5 mg/ml in 1× PBS) was added and incubated at 37 °C for another 4 h to allow MTT conversion to formazan crystals. Following aspiration of the medium, 200 μl DMSO (Sigma-Aldrich, St. Louis, MO, USA) was added and the absorbance was measured at 570 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

Fluorescence staining

HCT116 and SW480 cells were seeded at a density of 1 × 105 cells/well in 6-well plates. Following treatment of the cells (see above) they were exposed to 1 mg/ml Propidium Iodide (PI) for 30 min, to 1 μl/ml DAPI (4′,6-diamidino-2-phenylindole) for 5 min, to 4 nM DiOC6 (3,3′-dihexyloxacarbocyanine iodide) for 15 min and to 5 μg/ml Acridine Orange (AO) solution for 15 min at 37 °C. After dye incubation, the cells were carefully re-suspended in 1× PBS and visualized by fluorescence microscopy (Olympus, Japan).

GFP-tagged LC3 localization

HCT116, SW480 and HT29 cells were seeded in 6-well plates and transfected with a green fluorescent protein (GFP)-tagged LC3 expression vector (0.5 μg/ml) using a FuGENE6 (Promega, Sunnyvale, CA, USA) transfection reagent. After a 24 h transfection period, the cells were exposed to DENSpm (10 μM) with or without 3-MA for 0–72 h, after which the media were carefully discarded. P53 activity in HCT116 cells was inhibited by pifithrin (2 μM) pre-treatment, after which these cells were exposed to DENSpm (10 μM) with or without 3-MA for 0–48 h. Finally, HCT116 and SW480 cells were collected in 1× PBS and evaluated using fluorescence microscopy (IX70, Olympus, Japan). HT29 and pifithrin pre-treated HCT116 cells were analyzed using a BD Accuri C6 Flow Cytometer (BD Biosciences, Bedford, MA, USA).

siRNA-mediated Beclin-1 silencing

HCT116 and SW480 cells were seeded at a density of 4 × 105 in 60 mm petri dishes and allowed to adhere overnight at 37 °C. Next, 1 μg Beclin-1 siRNA (Santa Cruz Biotechnology, CA, USA) in 1:6 siRNA transfection reagent (Santa Cruz Biotechnology, CA, USA) was prepared in 200 μl serum-free culture medium and incubated for 15 min at room temperature. The mixture was gently added drop-wise to the cells in 800 μl culture medium with 10% FBS and incubated for 0–72 h at 37 °C in a CO2 incubator. Following transient silencing for 0–72 h, the cells were treated with 10 μM DENSpm for 0–72 h after which total proteins were extracted.

Cell cycle analysis by PI staining

HCT116 and SW480 cells were seeded at a density 2 × 105 cells/well in 6-well plates and treated with DENSpm (10 μM) alone or with autophagy inhibitors [3-MA (10 μM), Beclin-1 siRNA (1 μg)] for 24 and 48 h. HT29 and pifithrin (2 μM) pre-treated HCT116 cells were exposed to DENSpm (10 μM) alone or with 3-MA for 48 h. Both floating and adherent cells were collected and fixed with 70% ethanol. Following incubation on ice for 30 min, the samples were centrifuged at 1200 rpm for 5 min, after which the pellets were re-suspended in 1× PBS, RNase (100 μg/ml) and PI solution (40 μg/ml). The resulting samples were kept for 30 min at 37 °C in the dark and analyzed using a BD Accuri C6 Flow Cytometer (BD Biosciences, Bedford, MA, USA) equipped with a 15-mW, 488-nm, air-cooled argon ion laser. Fluorescence emission was collected through a 570-nm band-pass filter. Cell cycle distribution analyses were performed using C6 software (BD Biosciences, Bedford, MA, USA).

AnnexinV/PI double staining

HCT116 and SW480 cells were seeded at a density of 2 × 105 cells/well in 6-well plates and treated with DENSpm alone or with autophagy inhibitors [3-MA (10 μM), Beclin-1 siRNA (1 μg)] for 48 h. P53 deficient cells [pifithrin (2 μM) pre-treated HCT116 and HT29 cells] were treated with DENSpm (10 μM) alone or with 3-MA (10 μM) for 48 h. Both floating and adherent cells were collected and, after centrifugation, the cell pellets were re-suspended in 1× PBS, RNase (100 μg/ml) and Annexin V (10 μg/ml), incubated for 10 min, followed by the addition of PI (40 μg/ml), kept in the dark for 5 min and analyzed using a BD Accuri C6 Flow Cytometer (BD Biosciences, Bedford, MA, USA) equipped with a 15-mW, 488-nm, air-cooled argon ion laser. Fluorescence emission was collected through a 570-nm band-pass filter. Cell cycle distribution analyses were performed using C6 software (BD Biosciences, Bedford, MA, USA).

PA analysis by HPLC

Intracellular PA contents were determined by HPLC analysis following a benzoylation procedure. HCT116, SW480 and p53 deficient [pifithrin (2 μM) pre-treated HCT116 and HT29] cells were seeded at a density of 1.2 × 106 in 100 mm petri dishes and allowed to adhere overnight. Next, the cells were treated with DENSpm alone or with 3-MA for 0–72 h. Following washing in 1× PBS the cells were collected by scraping and transferred to a new microfuge tube, after which 50% trichloroacetic acid was added to each sample (1:10, v/v). The samples were kept at −20 °C until benzoylation. Following benzoylation, the samples were immediately run on a HPLC system (Agilent, Santa Clara, CA, USA) using a UV detector set at 226 mV. The results obtained were evaluated against the internal standard 1,7-diaminoheptane, and Put, Spd, and Spm standard curves (each 10 mM stock concentration).

Protein extraction and Western blotting

The effects of DENSpm on autophagy key players and on apoptotic markers were assessed by Western blotting after 2 h pre-treatment with 3-MA (10 μM) and 48 h siRNA-mediated Beclin-1 silencing (1 μg/ml) in HCT116 and SW480 cells. The role of p53 in DENSpm induced apoptotic cell death and autophagy was evaluated in p53 inhibited cells (pifithrin pre-treated HCT116 and p53 null HT29 cells). All samples were washed with ice-cold 1× PBS and lysed on ice in a solution containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, Nonidet P-40 0.5% (v/v), 1 mM EDTA, 0.5 mM PMSF, 1 mM DTT and a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). After lysis, cell debris was removed by centrifugation for 15 min at 13200 rpm, and protein concentrations were determined using a Bradford protein assay (Bio-Rad, Hercules, CA, USA). Next, total protein lysates (30 μg) were separated by 12% SDS-PAGE and transferred to PVDF membranes (Roche, Indianapolis, IN, USA). After this, the membranes were blocked with 5% milk blocking solution in Tris buffered saline (TBS)-Tween20 (Sigma-Aldrich, St. Louis, MO, USA) and incubated with appropriate primary and horseradish peroxidase (HRP)-conjugated secondary antibodies (CST, Danvers, MA, USA) in antibody buffer containing 5% (v/v) milk blocking solution. Following gentle washing with 1× TBS-Tween20, protein bands were analyzed using an enhanced chemiluminescence detection system (ECL).

Statistical analysis

All the experiments were statistically analyzed by two-way ANOVA using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). Statistically significant results by ANOVA were further analyzed by Bonferroni post-hoc analysis (where indicated). A p-value < 0.05 was considered statistically significant. Error bars in the graphs were generated using ± standard deviation (SD) values. Western blot experiments were repeated at least twice and Image J was applied to calculate band intensities.

Results

DENSpm inhibits proliferation and induces autophagy in HCT116 and SW480 cells in a time-dependent manner

In order to assess the cytotoxic effects of DENSpm on both p53 wild-type (HCT116) and p53 mutant (SW480) colon cancer cells, MTT cell viability assays were carried out. We found that DENSpm treatment (0−50 μM) induced loss of cell viability in HCT116 and SW480 cells at the different time points measured (within 72 h) (Fig. 1a, b). Specifically, we found that 10 μM DENSpm treatment decreased the viability of HCT116 cells by 16, 37 and 52% compared to untreated cells at 24, 48 and 72 h, respectively (Fig. 1a; ***p < 0.001). We found that the cell viability loss after DENSpm treatment for 24 h was less in SW480 cells (12%; *p < 0.05), whereas after a longer exposure (48 and 72 h) the cell viability decreased further by 23 and 33%, respectively (Fig. 1b). In addition, we observed significant vacuole formation in a time-dependent manner in SW480 cells (Fig. 1c). According to Acridine Orange (AO) and Monodansyl Cadaverin (MDC) staining and a GFP-LC3 puncta assay we found that, although DENSpm-induced vacuole formation was present in both cell lines, DENSpm-induced autophagy was more overtly induced in SW480 cells in a time-dependent manner (Fig. 1d).

Fig. 1. DENSpm-induced apoptotic cell death is increased by 3-MA pre-treatment in colon cancer cells.

Fig. 1

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The effect of DENSpm on cell viability was determined by MTT assay after DENSpm (0–50 μM) treatment in a time-dependent manner (24, 48, 72 h) in a. HCT116 and b. SW480 cells. Columns represent mean ± S.D. of two independent experiments and at least 3 replicates. c. Time-dependent effect of DENSpm (10 μM) on the morphology of colon cancer cells visualized by light microscopy. d. HCT116 and SW480 cells were seeded in 6-well plates, treated with DENSpm (10 μM) for 0–72 h, stained with AO or MDC and visualized by immunofluorescence microscopy. DENSpm-induced LC3 localization was determined after GFP-LC3 plasmid transfection. Statistical differences were analyzed using two-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001

Autophagy inhibition by 3-MA increases DENSpm-induced apoptosis in HCT116 and SW480 cells

The potential apoptotic effect of DENSpm on colon cancer cells after autophagy inhibition was evaluated using a MTT cell viability assay, PI and DiOC6 staining, and flow cytometry (FACS) in conjunction with PI and AnnexinV/PI staining. We found that, based on the MTT cell viability data, DENSpm and 3-MA pre-treatment significantly increased cell viability loss compared to DENSpm treatment alone, in a time-dependent manner in HCT116 and SW480 cells, respectively (Fig. 2a, b). In order to assess a potential effect of 3-MA on DENSpm-induced colony formation, we performed a soft agar assay. We found that inhibition of DENSpm-induced autophagy by 3-MA significantly reduced the colony sizes in both cell lines compared to DENSpm treatment alone (Fig. 2c). In addition, we found that autophagy inhibition by 3-MA pre-treatment inhibited DENSpm-induced colony formation and rapid mitochondrial membrane potential loss (DiOC6 staining) as well as cell death (PI staining) in a time-dependent manner (Fig. 2d).

Fig. 2. Effect of 3-MA on DENSpm-induced cell viability, cell death and mitochondrial membrane potential.

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The effect of 3-MA on DENSpm-induced cell viability loss was determined by MTT assay after DENSpm (10 μM) treatment with/without 3-MA in a time-dependent manner (24, 48, 72 h) in a. HCT116 and b. SW480 cells. The columns represent mean ± S.D. of two independent experiments and at least 3 replicates. c. Effect of DENSpm under 3-MA mediated autophagosome inhibition conditions on soft agar colony formation in HCT116 and SW480 cells. Time-dependent effects of 3-MA pre-treatment on DENSpm-induced d. cell death and mitochondrial membrane potential loss determined by PI and DiOC6 staining, respectively

We also found that 3-MA pre-treatment increased DENSpm-induced apoptotic cell death in HCT116 and SW480 cells compared to DENSpm treatment alone (24 and 48 h; Fig. 3a, b). Based on FACS analysis after PI staining, we found that combined 3-MA and DENSpm treatment resulted in an accumulation of SW480 cells in the G2/M phase of the cell cycle compared to DENSpm treatment alone for 24 and 48 h. Although a significant difference in G1 arrest in HCT116 cells was observed after DENSpm treatment alone compared to DENSpm plus 3-MA pre-treatment for 24 h, the subG1 population only increased from 2.0% to 3.3% after DENSpm treatment alone compared to 3-MA and DENSpm treatment for 48 h (Fig. 3a). Concomitantly, we found that 3-MA pre-treatment triggered apoptotic cell death in a time-dependent manner in both cell lines, as assessed by AnnexinV/PI FACS analysis (Fig. 3b). Finally, we found that autophagy inhibition triggered DENSpm-induced apoptotic cell death through preventing LC3 cleavage and p62 degradation, through inducing Beclin-1 expression and through inducing caspase-3 and caspase-9 cleavage in both HCT116 and SW480 cells (Fig. 3c).

Fig. 3. Increased effect of 3-MA pre-treatment on DENSpm-induced cell cycle arrest and apoptotic cell death.

Fig. 3

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a. HCT116 and SW480 cells were treated with 10 μM DENSpm and/or 3-MA for 48 or 72 h, harvested, fixed, stained with PI and subjected to flow cytometry to assess cell cycle distribution. The results shown represent two independent experiments. b. HCT116 and SW480 cells were treated with 10 μM DENSpm and/or 3-MA for 48 or 72 h, harvested, stained with AnnexinV/PI and subjected to flow cytometry. c. Expression of PI3K, Akt, p62, Beclin-1, LC3, caspase-3, caspase-9 and PARP in HCT116 and SW480 cells treated with 10 μM DENSpm and/or 3-MA for 48 or 72 h

Role of PA metabolism in DENSpm-induced apoptosis under autophagy inhibition conditions

In order to reveal a possible role of intracellular PA on apoptosis induction by DENSpm under 3-MA mediated autophagy inhibition conditions in HCT116 and SW480 cells, we performed HPLC and Western blot analyses (Fig. 4a-c). The HPLC analysis revealed that DENSpm treatment in combination with 3-MA pre-treatment decreased the Put, Spd and Spm levels compared to DENSpm treatment alone in HCT116 cells at 48 and 72 h. In contrast, no significant differences were found in the Put and Spd levels in SW480 cells after DENSpm treatment alone or combined 3-MA and DENSpm treatment at 48 h. Conversely, a significant decrease in Spm level was observed after DENSpm treatment with or without 3-MA treatment in a time-dependent manner in SW480 cells (Fig. 4c). Next, we set out to evaluate potential effects on the expression of PA metabolic enzymes after DENSpm-induced apoptotic cell death under 3-MA mediated autophagy inhibition conditions in both cell lines by Western blotting (Fig. 4d). We found that autophagy inhibition resulted in significantly downregulated ODC and Antizyme Inhibitor (AZI) expression and upregulated polyamine catabolic enzyme SSAT expression levels compared to DENSpm treatment alone in a time-dependent manner in both cell lines.

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Beclin-1 silencing increases DENSpm-induced cell viability loss and apoptosis in HCT116 and SW480 cells

Next, we set out to silence the expression of one of the key autophagy players, Beclin-1, in HCT116 and SW480 cells to validate the increased apoptotic induction potential of DENSpm under autophagy inhibition conditions. The efficiency of siRNA-mediated Beclin-1 silencing was assessed using qRT-PCR and Western blotting (Fig. 5a, b). We found that Beclin-1 was efficiently silenced 48 h after siRNA transfection in both cell lines. Following Beclin-1 silencing, both cell lines were treated with DENSpm during different time intervals. Using a MTT assay, we observed a significant cell viability loss compared to parental HCT116 and SW480 cells after 48 and 72 h treatment (***p < 0.001) (Fig. 5c, d). Subsequent PI staining, performed to assess apoptotic cell death under Beclin-1 silencing conditions, revealed that when Beclin-1 was silenced, the DENSpm-induced apoptotic cell death rate increased from 4.3% to 8.2% in HCT116 cells and from 1.6% to 4.9% in SW480 cells (Fig. 5e), This result was confirmed by AnnexinV/PI staining for 48 h (Fig. 5f).

Fig. 5.

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DENSpm-induced cell viability loss, cell cycle arrest and apoptosis after Beclin-1 silencing in colon cancer cells. siRNA-mediated Beclin-1 silencing in a time-dependent manner in a. HCT116 and b. SW480 cells. The effect of DENSpm on cell viability loss was assessed by MTT assay in a time-dependent manner after Beclin-1 silencing in c. HCT116 and d. SW480 cells. DENSpm-induced cell cycle arrest and apoptotic cell death after Beclin-1 silencing in e. HCT116 and f. SW480 cells determined by PI and Annexin V/PI staining and flow cytometry, respectively. g. Expression of Beclin-1, Atg5, p62, LC3, PARP, caspase-9 and caspase-3 under Beclin-1 silencing conditions determined by Western blotting. β-actin was used as a loading control. Statistical differences were analyzed using two-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, D: DENSpm

Next, we checked the expression profiles of apoptotic markers and key autophagy players under Beclin-1 silencing conditions in HCT116 and SW480 cells using Western blotting. We found that siRNA-mediated Beclin-1 silencing led to an increased apoptotic effect of DENSpm via increased caspase-3, caspase-9 and PARP cleavage in HCT116 and SW480 cells (Fig. 5g). Since an increase in DENSpm-induced apoptotic cell death was assumed to occur through expression suppression of key autophagy players under autophagy-inhibition conditions, we next assessed the expression of Beclin-1, Atg5, p62 and LC3 by Western blotting. We found that, next to Beclin-1, the expression of Atg5 was downregulated, and that p62 degradation and LC3 cleavage were inhibited by DENSpm treatment following Beclin-1 silencing compared to DENSpm treatment alone in both cell lines.

Beclin-1 silencing increases DENSpm-induced polyamine depletion in HCT116 and SW480 cells

Although starvation triggers autophagy induction and DENSpm depletes intracellular PA levels, the association between DENSpm-induced PA starvation and autophagy induction has not yet been fully elucidated. Consistent with previous data, we found that DENSpm treatment resulted in decreased Put, Spd and Spm levels, and that Beclin-1 silencing led to increased DENSpm-induced PA depletion compared to DENSpm treatment alone in HCT116 cells (Fig. 6a). Also, although no significant effect was observed in Put levels with or without Beclin-1 silencing in SW480 cells, significant increases were observed in Spd and Spm levels in Beclin-1 silenced SW480 cells (Fig. 6b). When we assessed the expression profiles of PA metabolic enzymes in cells with or without Beclin-1 silencing, we found by Western blotting that ODC was downregulated and SSAT was upregulated more significantly by DENSpm treatment in Beclin-1 silenced cells than in non-silenced cells. This change in expression was more significant in HCT116 (p53 wild-type) cells than in SW480 (p53 mutant) cells (Fig. 6c).

Fig. 6. Involvement of PA in DENSpm-induced apoptotic cell death under Beclin-1 silencing conditions in colon cancer cells.

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Effect of DENSpm on intracellular a, b. Put, Spd and Spm levels determined by HPLC in HCT116 (left) and SW480 (right) cells. c. PA metabolic enzyme ODC, SSAT and AZI expression after DENSpm treatment determined by Western blotting. β-actin was used as a loading control. Statistical differences were analyzed using two-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001

Autophagy inhibition prevents DENSpm-induced autophagy in p53 deficeint colon cancer cells

In order to further unravel the role of p53 in DENSpm-induced autophagy, we performed AO staining and FACS analysis after GFP-LC3 plasmid transfection. We also assessed the expression profiles of Atg5, Atg12 and Beclin-1 by Western blotting in pifithrin (2 μM) pre-treated HCT116 and HT29 (p53 null) cells. We found that AO staining due to DENSpm-induced vacuole formation was prevented by 3-MA pre-treatment in both cell lines. Using GFP-LC3 FACS analysis, we found that DENSpm mediated GFP-LC3 expression was inhibited by 3-MA pre-treatment in both cell lines (Fig. 7a, b). In addition, we found that DENSpm-induced Atg5 and Beclin-1 upregulation were prevented by 3-MA pre-treatment in both p53 deficient cell lines (Fig. 7c).

Fig. 7. Role of p53 in DENSpm-induced autophagy under autophagy inhibition conditions.

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DENSpm-induced autophagy vacuole formation visualized by AO staining and flow cytometry after GFP-LC3 plasmid transfection in a. HCT116 and b. HT29 cells. c. Expression of Atg5, Atg12 and Beclin-1 under 3-MA mediated autophagy inhibition conditions in functionally inactive p53 HCT116 (pifithrin pre-treated) and HT29 cells determined by Western blotting. β-actin was used as a loading control. P: Pifithrin, D: DENSpm

Autophagy inhibition increases DENSpm-induced apoptotic cell death in p53 deficient colon cancer cells

Next, the role of p53 in DENSpm-induced apoptotic cell death under autophagy inhibition conditions was investigated by MTT and FACS analysis after PI and AnnexinV/PI staining. Based on the MTT results, we found that DENSpm and 3-MA pre-treatment led to a significant increase in loss of cell viability compared to DENSpm treatment alone, in both pifithrin (2 μM) pre-treated HCT116 and HT29 cells, respectively (***p < 0.001) (Fig. 8a, b). Based on FACS analysis following PI staining, we found that inhibition of autophagy by 3-MA had no significant effect on DENSpm-induced subG1accumulation of p53 deficient cells for 48 h (Fig. 8c). Concomitantly, we found that 3-MA pre-treatment did not alter the DENSpm-induced apoptotic cell death in either of the two cell lines as demonstrated by AnnexinV/PI analysis (Fig. 8d). Finally, we found that autophagy inhibition triggered DENSpm-induced apoptotic cell death through caspase-3, -7 and -9 activation and Bcl-2 downregulation in both pifithrin (2 μM) pre-treated HCT116 and HT29 (p53 null) cells (Fig. 8e).

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. 8

Fig. 8

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DENSpm induced cell viability loss determined by MTT assay in a. HCT116 (pifithrin pre-treated) and b. HT29 cells. c. HCT116 (pifithrin pre-treated) and HT29 cells were treated with 10 μM DENSpm and/or 3-MA for 48 h, harvested, fixed, stained with PI and subjected to flow cytometry to assess cell cycle distribution. The results shown represent two independent experiments. d. HCT116 (pifithrin pre-treated) and HT29 cells were treated with 10 μM DENSpm and/or 3-MA for 48 h, harvested, stained with Annexin V/PI and subjected to flow cytometry. e. Expression of pro-caspase-3, -7, -9 and Bcl-2 in HCT116 and HT29 cells treated with 10 μM DENSpm and/or 3-MA for 48 h determined by Western blotting. β-actin was used as a loading control. Statistical differences were analyzed using two-way ANOVA; *p < 0.05, ** p < 0.01, *** p < 0.001. P: Pifithrin, D: DENSpm

Role of PA metabolism in the apoptotic effect of DENSpm under autophagy inhibition conditions in p53 deficient colon cancer cells

In order to reveal the role of intracellular PA levels on increased DENSpm-induced apoptosis under autophagy inhibition conditions in pifithrin (2 μM) pre-treated HCT116 and HT29 (p53 null) cells, we performed HPLC and Western blot analyses. Through HPLC analysis we found that DENSpm treatment combined with 3-MA pre-treatment led to decreased Put and Spd levels compared to DENSpm only treatment for 48 h in the p53 deficient cells. However, significant increases were observed in Spm levels after DENSpm treatment alone and 3-MA plus DENSpm co-treatment after 48 h in both cell lines (Fig. 9a, b). We also evaluated the potential effect of the expression levels of PA metabolic enzymes after DENSpm-induced apoptosis under autophagy inhibition conditions in p53 deficient cells by Western blotting (Fig. 9c). We found that autophagy inhibition did not alter the DENSpm effect on SSAT and PAO expression 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.

Fig. 9

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The role of p53 in DENSpm-induced intracellular PA levels with/without 3-MA treatment were determined by HPLC in a. pifithrin pre-treated HCT116 and b. HT29 cells. c. PA metabolic enzymes SSAT, PAO and ODC expression after 10 μM DENSpm treatment and/or 3-MA pre-treatment determined by Western blotting. β-actin was used as a loading control. P: Pifithrin, D: DENSpm

Discussion

Since elevated intracellular PA levels have been associated with cancer initiation and progression, PA catabolism activating chemotherapeutics have gained interest [2]. Previous studies have shown that DENSpm may, via triggering intracellular PA pool depletion and activation of the PA catabolic enzyme SSAT, inhibit cell proliferation, induce cell cycle arrest and cause apoptotic cell death [3, 4] in melanoma [6], breast [8], non-small cell lung [9], prostate [11] and colon [13] cancer cells. Next to these in vitro results, DENSpm has been found to act as a candidate cancer drug in both in vivo studies and in clinical trials [10, 11]. But, although the clinical trials revealed a promising efficacy of DENSpm as a single therapeutic agent in prostate, breast and colon cancer, co-treatment with conventional therapeutic agents, such as 5-FU, cisplatin and paclitaxel has been found to enhance its potential [14, 21, 22].

Previously, we found that DENSpm may induce autophagy in the colon cancer-derived HCT116 and SW480 cells [15]. Here, we found that DENSpm decreased the viability and induced autophagy in a time-dependent manner in both cell lines, although more significantly in SW480 than in HCT116 cells. Previously, we found that the expression of autophagy key players increased in a time-dependent manner in both cell lines [15]. Autophagy is a multi-step process involving autophagosome and autolysosome formation, and increased autophagy has been found to result from amino acid and energy starvation, unfolded protein and degenerated organelle accumulation and pathogen endocytosis [23]. Recently, it has been reported that some therapeutic drugs may trigger autophagy, which is thought to act as a drug resistance strategy of cancer cells. In support of this notion, it has been found that autophagy inhibition may increase the apoptotic cell death potential of therapeutic drugs [24]. Therefore, we set out to investigate alterations in the apoptotic efficacy of DENSpm under autophagy inhibition conditions (either via 3-MA pre-treatment or Beclin-1 silencing) in HCT116 and SW480 cells and in p53 deficient (pifithrin pre-treated HCT116 and p53 null HT29 cells) colon cancer cells. We found that 3-MA mediated autophagy inhibition increased the DENSpm-induced proliferation inhibition and apoptotic death in a time-dependent manner in HCT116 and SW480 cells, although a more significant apoptotic effect was observed in p53 wild-type HCT116 cells than in p53 mutant SW480 cells. No significant effects on DENSpm-induced apoptosis under autophagy inhibition conditions were observed in p53 null HT29 and p53 inhibited (pifithrin) HCT116 cells. These results are in accordance with the biological potential of p53 in autophagy regulation through alteration of its migration to the nucleus [24], which determines the cell death decision. Previously, we found that different biologically active p53 proteins may alter DENSpm-induced autophagy by enhancing Beclin-1 expression, p62 degradation and LC3 cleavage in colon cancer cells [15]. In addition, it is well known that p53 inactivation may lead to the development of resistance to therapeutic drugs in cancer cells [25]. Thus, the role of p53 in DENSpm-induced apoptotic cell death during autophagy inhibition may be essential in understanding the therapeutic potential of DENSpm in colon cancer cells. Previously, it has been reported that the apoptotic efficacy of DENSpm in breast cancer cells may be p53 independent [8]. It has also been reported that DENSpm may induce apoptotic cell death without cell cycle arrest in p53 mutant SK-MEL-28 melanoma cells, and induce cytochrome c release and caspase activation in a time-dependent manner [6]. Conversely, we previously found that HCT116 p53−/− colon cancer cells showed resistance to DENSpm treatment in a time- and dose-dependent manner compared to HCT116 p53 wild-type cells [15]. Similarly, it has been shown that, although DENSpm can induce G1 cell cycle arrest in MALME-3 M melanoma cells that exhibit functional p53 expression, DENSpm failed to arrest p53 mutant SK-MEL-28 melanoma cells at the G1 phase [5]. Thus, autophagy may prevent DENSpm-induced apoptotic cell death, regardless of p53, in colon cancer cells. Accordingly we found that, although DENSpm activated key autophagy players in a time-dependent manner, 3-MA inhibited DENSpm-induced autophagy in both cell lines, regardless p53 status.

Previously, it has been shown that some chemotherapeutics may inhibit the apoptotic efficacy of DENSpm in melanoma [6], breast [14, 26] and colon [13, 21] cancer cells. Here, we found that autophagy inhibition synergises with the apoptotic effect of DENSpm in colon cancer cells. To underscore this notion, we found that autophagy inhibition enhanced the apoptotic potential of DENSpm in these cells through triggering PARP cleavage and caspase activation. Based on these results, we conclude that autophagy may serve as an obstacle to DENSpm-induced apoptosis in colon cancer cells. In addition, we found that no significant additional effect was observed on DENSpm-mediated apoptotic cell death under autophagy inhibition conditions in p53 inhibited (pifithrin) HCT116 and p53 null HT29 cells. Thus, as a key transcription factor, p53 does not only play a role in cell cycle control, but also in directing the autophagy process, which is assumed to play an essential role in the apoptotic-autophagy decision of cancer cells upon drug exposure.

The PA catabolic enzyme SSAT can transfer acetyl groups to Spd and Spm. Once acetylated, these molecules may be excreted from the cell or converted to Spd or Put by another PA catabolic enzyme, PAO [27]. Previously, DENSpm has been shown to induce PA depletion in MALME-3 M and SK-MEL-28 melanoma cells [28] and in HCT116 and SW480 colon cancer cells [15]. In NCI-H157 non-small cell lung cancer cells DENSpm has been shown to cause a dramatic depletion of the intracellular PA pool within 24 h [29]. Similarly, DENSpm has been found to cause a reduction in PA levels in various breast cancer cell lines, including MCF-7, SK-BR-3 and HCC1937. These cells underwent DENSpm-induced apoptosis after S-phase cell cycle arrest [30]. Based on these results, chemotherapeutic drugs inducing PA depletion through activating the expressions of PA catabolic enzymes (SSAT or PAO) in cancer cells were anticipated to suit clinical application. As an effective PA analogue, DENSpm has been found to induce apoptotic cell death via PA metabolism modulation in T24 and J82 bladder cancer cells [31]. Since it has been found that DENSpm can increase SSAT activity up to 1000-fold, this was suggested to be the major reason for its anti-proliferative effect in cancer cells [32]. Interestingly, Chinese hamster ovary cells were found to be resistant to DENSpm treatment due to a point mutation in the SSAT gene [33]. Moreover, it has been found that SSAT silencing prevents DENSpm-induced apoptotic cell death and caspase activation in SK-MEL-28 melanoma cells [28]. Within this context, we decided to evaluate the role of PA metabolism in DENSpm-induced apoptotic cell death during autophagy inhibition by 3-MA. Although no significant differences in Spd levels in SW480 cells were observed, the intracellular Put and Spm levels were found to be significantly decreased after 3-MA and DENSpm co-treatment compared to DENSpm treatment alone in HCT116 cells in a time-dependent manner. The increased apoptotic potential of DENSpm under autophagy inhibition conditions may be due to ODC downregulation and SSAT upregulation in these cells. Thus, as an autophagy induction agent, increased DENSpm-induced apoptotic cell death when autophagy is prevented by 3-MA pre-treatment may be due to PA depletion via ODC suppression and SSAT upregulation in biologically active p53 colon cancer cells.

The major autophagy player Beclin-1 has a BH domain, and its cross-talk with Bcl-2 emphasises its essential role in linking autophagy with apoptotic cell death. Overexpression of Bcl-2 increases cell survival and triggers resistance against chemotherapeutic drugs and prevents autophagy via binding to Beclin-1. In addition, it has been found that Beclin-1 silencing prevents drug-induced apoptotic cell death in cancer cells [34]. We found that Beclin-1 silencing increased DENSpm-induced cell viability loss and increased apoptotic cell death through PARP cleavage, caspase-9 activation and Atg5 downregulation, while p62 accumulation was observed in a p53-dependent manner in colon cancer cells. In order to assess the role of PA and metabolic enzyme expression levels in DENSpm-induced apoptotic cell death under Beclin-1 silencing conditions, we performed HPLC and Western blot analyses. We found that DENSpm-induced Put and Spd depletion was significantly enhanced after Beclin-1 silencing in HCT116 cells. In SW480 cells, Beclin-1 silencing led to increased DENSpm-induced Spd and Spm levels. It has been reported that DENSpm-induced PA depletion may serve as a molecular mechanism underlying DENSpm-induced apoptotic cell death in prostate [7], breast [8] and non-small cell lung [9] cancer cells. We found that Beclin-1 silencing increased DENSpm-induced cell viability loss, cell cycle arrest and apoptotic cell death via intracellular PA depletion through upregulation of SSAT expression in colon cancer cells. This is the first report indicating increased DENSpm-induced apoptotic cell death in Beclin-1 silenced colon cancer cells. Intracellular PA depletion may stabilize DENSpm-induced apoptotic cell death in p53 wild-type colon cancer cells.

Conclusions

Although we found that DENSpm may induce autophagy in both p53 wild-type and p53 mutant/deficient colon cancer cells, autophagy inhibition by 3-MA treatment or Beclin-1 silencing led to an increased DENSpm-induced apoptotic cell death via the suppression of key autophagy players and depletion of intracellular PA levels through SSAT activation. Although we found that functional p53 activates DENSpm-induced apoptosis via autophagy inhibition, mutant p53 may be an obstacle to overcome DENSpm-induced drug resistance. As such, autophagy inhibition may increase the efficacy of DENSpm-induced apoptotic cell death in p53 mutant colon cancer cells. We found that p53 deficiency did not incline the DENSpm-induced apoptotic cell death under autophagy inhibition conditions in the colon cancer cells tested. This study is the first to show that, next to apoptosis, DENSpm induces autophagy in colon cancer cells, and that p53 may play a role in autophagy/apoptosis decision making in response to DENSpm treatment of these cells. In order to increase the therapeutic efficacy of DENSpm in colon cancer and to overcome resistance, autophagy inhibition may represent an effective step.

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.

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

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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