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WIN induces apoptotic cell death in human colon cancer cells through a block of autophagic flux dependent on PPARγ down-regulation

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Abstract

Cannabinoids have been reported to possess anti-tumorigenic activity in cancer models although their mechanism of action is not well understood. Here, we show that the synthetic cannabinoid WIN55,212-2 (WIN)-induced apoptosis in colon cancer cell lines is accompanied by endoplasmic reticulum stress induction. The formation of acidic vacuoles and the increase in LC3-II protein indicated the involvement of autophagic process which seemed to play a pro-survival role against the cytotoxic effects of the drug. However, the enhanced lysosomal membrane permeabilization (LMP) blocked the autophagic flux after the formation of autophagosomes as demonstrated by the accumulation of p62 and LC3, two markers of autophagic degradation. Data also provided evidence for a role for nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in cannabinoid signalling. PPARγ expression, at both protein and mRNA levels, was significantly down-regulated after WIN treatment and its inhibition, either by specific antagonists or by down-regulation via gene silencing, induced effects on cell viability as well as on ER stress and autophagic markers similar to those obtained in the presence of WIN. Moreover, the observation that the increase in p62 level and the induction of LMP were also modified by PPARγ antagonists seemed to indicate that PPARγ down-regulation was crucial to determinate the block of autophagic flux, thus confirming the critical role of PPARγ in WIN action. In conclusion, at our knowledge, our results are the first to show that the reduction of PPARγ levels contributes to WIN-induced colon carcinoma cell death by blocking the pro-survival autophagic response of cells.

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Abbreviations

ER:

Endoplasmic reticulum

PPARγ:

Peroxisome proliferator-activated receptor γ

CHOP:

CCAAT/enhancer binding protein homologous protein

GRP78:

Glucose-regulated protein 78/Binding immunoglobulin protein

TRB3:

Tribbles-related protein 3

LC3 (MAP1LC3):

Microtubule-associated protein 1 light chain 3

PARP:

Poly-(ADP-ribose) polymerase

WIN:

(WIN55,212-2) R-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl] pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl methanone mesylate

MTT:

3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide

MDC:

Monodansylcadaverine

PI:

Propidium iodide

3-MA:

3-Methyladenine

AO:

Acridine orange

CCCP:

Carbonylcyanide m-chlorophenylhydrazone

LMP:

Lysosomal membrane permeabilization

Baf A1:

Bafilomycin A1

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Acknowledgments

This work was supported by University of Palermo [Grant ORPA07EZ5Z and ORPA06F3TB].

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Laboratory of Cellular and Developmental Genetics, Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, PROTEO and IBIS, Université Laval, Quebec, QC, Canada

    Ornella Pellerito

  2. Dipartimento STEBICEF Scienze e Tecnologie biologiche, chimiche e farmaceutiche, Laboratorio di Biochimica, Università di Palermo, Policlinico Universitario, Via del Vespro 129, 90127, Palermo, Italy

    Antonietta Notaro, Selenia Sabella, Anna De Blasio, Renza Vento, Giuseppe Calvaruso & Michela Giuliano

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  1. Ornella Pellerito
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  2. Antonietta Notaro
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  4. Anna De Blasio
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  5. Renza Vento
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  6. Giuseppe Calvaruso
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Corresponding author

Correspondence to Michela Giuliano.

Additional information

O. Pellerito and A. Notaro have contributed equally to this study.

G. Calvaruso and M. Giuliano share senior co-authorship.

Electronic supplementary material

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10495_2014_985_MOESM1_ESM.jpg

Supplementary Fig. 1. Complete depolarization of mitochondria was evaluated by means of carbonyl cyanide 3-chlorophenylhydrazone (CCCP). 50 μM CCCP was added to HT29 cells for 10 min at room temperature. Then, mitochondrial transmembrane potential (Δψm) dissipation was quantified by flow cytometry in the presence of the lipophilic dye DiOC6, as reported in “Materials and methods” section. Filled profile: untreated cells; continuous line: 50 μM CCCP (JPEG 106 kb)

10495_2014_985_MOESM2_ESM.jpg

Supplementary Fig. 2. Uptake of propidium iodide and cell cycle profile of WIN-treated HT29 cells. a Cell were treated for 36 h with 10 μM WIN. The percentage of dead cells was evaluated by cytofluorimetric analysis of the uptake of propidium iodide (PI), indicative of a loss of plasma membrane integrity, as reported in “Materials and methods” section. b Effects of WIN and/or GW9662 on cell cycle distribution of HT29 cells. Flow cytometric analysis was carried out on propidium iodide-stained cells treated for 16 h with 10 μM WIN or 36 h with 10 μM WIN and/or 60 μM GW9662. The percentage of cells in each phase of the cell cycle was calculated as reported in “Materials and methods” section using Expo32 software. **p<0.001 (JPEG 465 kb)

10495_2014_985_MOESM3_ESM.jpg

Supplementary Fig. 3. Representative western blots of different transcription factors involved in PPARγ expression in HT29 cells treated for 36 h in the presence of 10 μM WIN. Cell lysates were analysed via immunoblotting using specific antibodies as reported in “Materials and methods” section. Actin blots were included as a loading control (JPEG 216 kb)

10495_2014_985_MOESM4_ESM.jpg

Supplementary Fig. 4. Effects of PPARγ antagonist T007 in HT29 cells. a Table 1 Determination of mitochondrial transmembrane potential (Δψm) dissipation and DNA fragmentation in HT29 cells treated with 10 μM WIN and/or 25 μM T007. Δψm and DNA fragmentation were evalualted by means of flow cytometry after 16 h or 36 h of treatment, respectively, as reported in “Materials and methods” section. b Effects of WIN and/or T007 on HT29 cell viability. Cells were incubated for the indicated times with 10 μM WIN and/or 25 μM T007. Cell survival was estimated by MTT assay, as reported in “Materials and methods” section, and expressed as the percentage of control cells. c-d Representative western blots of PPARγ (c), CHOP, Beclin-1, p62 and LC3 (d) levels. After treatment for 36 h with 10 μM WIN and/or 25 μM T007, cell lysates were analysed via immunoblotting using specific antibodies as reported in “Materials and methods” section. Actin blots were included as a loading control. e Effects of T007 on WIN-induced lysosomal destabilization. HT29 cells were loaded with 5 μg/ml AO for 15 min before treatment with 10 μM WIN and/or 25 μM T007 for 5 h. Induction of lysosomal destabilization was evaluated under fluorescence microscopy. Control cells show punctate red staining of acidic organelles red fluorescence while in WIN and/or T007-treated cells a diffuse green fluorescence is observed. The data shown are from one of three experiments giving essentially the same results. **p<0.001 (JPEG 619 kb)

10495_2014_985_MOESM5_ESM.jpg

Supplementary Fig. 5. Effects of anti-Beclin-1 siRNA on autophagic markers and on HT29 cell viability after WIN treatment. a Cells were transfected with siRNA control (siScr) or specific anti-Beclin-1 siRNA (siBeclin-1) as reported in “Materials and methods” section and treated with 10 μM WIN for 16 h. Then, the level of Beclin-1 and LC3 were analysed by western blot. Actin blots were included as a loading control. b Effects of siBeclin-1 on viability of WIN-treated HT29 cells. Silenced cells were incubated for 16 h with 10 μM WIN, cell survival was estimated by MTT assay, as reported in “Materials and methods” section, and expressed as the percentage of control cells. *p<0.01; **p<0.001 (JPEG 304 kb)

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Pellerito, O., Notaro, A., Sabella, S. et al. WIN induces apoptotic cell death in human colon cancer cells through a block of autophagic flux dependent on PPARγ down-regulation. Apoptosis 19, 1029–1042 (2014). https://doi.org/10.1007/s10495-014-0985-0

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