Apoptosis

, Volume 18, Issue 3, pp 324–336

TRAIL promotes membrane blebbing, detachment and migration of cells displaying a dysfunctional intrinsic pathway of apoptosis

Authors

  • Syam Prakash Somasekharan
    • Department of Cell BiologyUniversity of Geneva
    • Department of Pathology and Laboratory MedicineUniversity of British Columbia
    • Department of Molecular OncologyBritish Columbia Cancer Research Centre
  • Michal Koc
    • Institute of Molecular GeneticsCzech Academy of Sciences
  • Alexandre Morizot
    • INSERM, UMR866
  • Olivier Micheau
    • INSERM, UMR866
  • Poul H. B. Sorensen
    • Department of Pathology and Laboratory MedicineUniversity of British Columbia
    • Department of Molecular OncologyBritish Columbia Cancer Research Centre
  • Olivier Gaide
    • Department of Dermatology-VenereologyGeneva University Hospital
  • Ladislav Andera
    • Institute of Molecular GeneticsCzech Academy of Sciences
    • Department of Cell BiologyUniversity of Geneva
Original Paper

DOI: 10.1007/s10495-012-0782-6

Cite this article as:
Somasekharan, S.P., Koc, M., Morizot, A. et al. Apoptosis (2013) 18: 324. doi:10.1007/s10495-012-0782-6
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Abstract

Recently, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) has been shown to be a potential candidate for cancer therapy. TRAIL induces apoptosis in various cancer cells but not in normal tissues. Here we show that HCT116 and SW480 cells with a deficient mitochondrial apoptotic pathway were resistant to TRAIL-induced apoptosis, whereas HCT116 and SW480 cells with a functional mitochondrial apoptotic pathway underwent apoptosis upon exposure to TRAIL. Surprisingly, TRAIL induced phenotypic changes in cells with a dysfunctional mitochondrial apoptotic pathway, including membrane blebbing and a transient loss of adhesion properties to the substratum. Accordingly, TRAIL stimulated the ability of these cells to migrate. This behavior was the consequence of a transient TRAIL-induced ROCK1 cleavage. In addition, we report that Bax-deficient HCT116 cells exposed to TRAIL for a prolonged period lost their sensitivity to TRAIL as a result of downregulation of TRAIL receptor expression, and became resistant to combination of TRAIL and other drugs such as MG-132 and bortezomib. These findings may have important consequences for TRAIL anti-cancer therapy.

Keywords

TRAILMembrane blebbingROCK1HCT116 Bax−/−Cancer cell migrationDrug resistanceBortezomibProteasome

Abbreviations

TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand

TNFR1

Tumor necrosis factor receptor-1

DISC

Death inducing signaling complex

TRAIL-R1

TRAIL receptor-1

TRAIL-R2

TRAIL receptor-2

MLC

Myosin light chain

ROCK1

Rho-associated, coiled-coil containing protein kinase 1

IAPs

Inhibitors of apoptosis

XIAP

X-linked inhibitor of apoptosis

Supplementary material

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Supplementary material Movie 1 HCT116 Bax−/+ cells treated with TRAIL (100 ng/ml) for 30 h as described in the Materials and methods. Movie was created using ImageJ software using time-lapse images captured every 7 min. (AVI 2565 kb)

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Supplementary material Movie 2 HCT116 Bax−/− cells treated with TRAIL (100 ng/ml) for 30 h as described in the ‘Materials and methods’. Movie was created using ImageJ software using time-lapse images captured every 7 min. (AVI 2753 kb)

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Supplementary material Fig. 1 HCT116 Bax−/+ cells overexpressing Bcl-2 or Bcl-xL behave like Bax−/−HCT116 cells in response to TRAIL treatment. a HCT116 Bax−/+ cells stably expressing a control or Bcl-2 or Bcl-xL expression vector were treated with TRAIL (100 ng/ml) and photographed at the indicated time points using a phase contrast microscope. b The number of blebbing cells in each condition was counted in three different fields. Mean values ± SD are shown for three independent experiments with *p < 0.05, **p < 0.01 (JPG 518 kb)
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Supplementary material Fig. 2 Detachment and blebbing of SW480 cells overexpressing Bcl-xL upon TRAIL treatment. a SW480 cells transfected with a control or Bcl-xL expression DNA vector were treated with TRAIL (100 ng/ml), proteosome inhibitor MG-132 (10 μM) or TRAIL (100 ng/ml) and MG-132 (10 μM) for 5 h. Cells were harvested, stained with propidium iodide and analyzed by FACS. Note that Bcl-xL overexpressing cells were found to be resistant to TRAIL while they were sensitized by a co-treatment with TRAIL and MG-132. b Control or Bcl-xL overexpressing SW480 cells were treated with TRAIL (100 ng/ml) or TRAIL (100 ng/ml) and ROCK1 inhibitor Y-27632 (10 μM) for the indicated time points and imaged. c SW480 cells overexpressing Bcl-xL were treated with TRAIL (100 ng/ml) for 5 h and the detached cells were collected, counted and expressed as percentage of the total number of cells in the well. Mean values ± SD are shown for three independent experiments with *p < 0.05. d SW480 cells overexpressing Bcl-xL or control were treated with TRAIL (100 ng/ml), the detached cells were collected and seeded in a 3.5 cm petriplate at a concentration of 1 × 105 cells/ml. The reattached cells were counted after 24 h of incubation. Results are an average of three independent experiments. e Control or Bcl-xL overexpressing SW480 cells were cultured in the presence or absence of TRAIL (100 ng/ml) for 5 h. Cell lysates were prepared and analyzed by Western blotting using antibodies as indicated. Note that caspase-8, caspase-3, PARP and ROCK1 were efficiently cleaved in control cells. In Bcl-xL overexpressing cells, caspase-8 and ROCK1 were efficiently processed while caspase-3 was partially processed. (TIFF 11804 kb)
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Supplementary material Fig. 3 Cleavage of ROCK1 is sufficient to induce membrane blebbing. a HCT116 Bax–/– cells were transiently transfected (Neon Transfection System, Invitrogen) with a plasmid encoding GFP alone (upper panel) or GFP and ROCK1 (G1114opa) (at a ratio of 5:1) (middle panels) and imaged after 24 h. Cells showing blebbing phenotype are indicated by arroheads. A single cell transfected with both GFP and ROCK1 (G1114opa) is enlarged and shown in the bottom panel. b Green cells showing blebbing morphology were counted and expressed as percentage of the total number of green cells. Results are an average of three independent experiments. Mean values ± SD are shown for three independent experiments with **p < 0.01. (JPG 2079 kb)

Copyright information

© Springer Science+Business Media New York 2012