Mitochondrial Inside-Out Signalling During Alkylating Agent-Induced Anoikis
Matthieu Sourdeval1, Emmanuelle Boisvieux-Ulrich2, Marie-Claude Gendron3, Francelyne Marano2
1
Laboratoire de Genetique et Biologie Cellulaire, UMR 8159, Universite de Versailles St Quentin en Yvelines, Batiment Buffon, 45 Avenue des Etats-Unis, 78035 Versailles Cedex, France, 2Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Universite Paris 7 Denis Diderot,3Plate-forme de Cytometrie en flux, Institut Jacques Monod CNRS, Universites Paris 6 et 7, 2 Place Jussieu, 75251 Paris Cedex 05, France
TABLE OF CONTENTS
- 1. Abstract
- 2. Introduction
- 3. Material and methods
- 3.1. Cells and cell culture
- 3.2. Chemical treatment
- 3.3. Immunofluorescence
- 3.4. Flow cytometry analysis
- 3.5. Western blot analysis
- 3.6. Cell counts
- 3.7. Cell attachment assays
- 3.8. Discrimination between live, apoptotic and necrotic cells
- 3.9. Statistical analysis
- 4. Results
- 4.1. Localization and expression of integrins and E-cadherin are not affected in adherent cells by HN2 treatment
- 4.2. Integrins and E-cadherin cell surface expression is reduced in HN2 treated detached cells
- 4.3. HN2 treatment induces the disorganization of F-actin in both adherent and detached cells
- 4.4. Effect of caspase inhibitors
- 4.5. Effect of Inhibitors of mitochondrial potential disruption
- 4.6. Effect of inhibitors of matrix metalloproteinase
- 4.7. Cell attachment assays
- 5. Discussion
- 5.1. Disruption of actin-F and subsequent cell rounding may be closely related to onset of cell detachment
- 5.2. Loss of adherent proteins follows cell detachment
- 5.3. The role of caspases in down-regulation of F-actin and adherent proteins
- 5.4. The role of mitochondria in anoikis
- 5.5. Mitochondria depolarization correlates with down regulation of F-actin and adherent proteins
- 5.6. Effect of matrix metalloproteinase.
- 5.7. Maintain of cell adhesiveness and survival is actively sustained by inhibition of mitochondrial depolarization
- 6. References
1. ABSTRACT
Exposure of epithelial respiratory cells to the alkylating agent, mechlorethamine (HN2), induces anoikis initiated by mitochondrial depolarization and caspase-2 activation. The mechanisms of disruption of cell interactions were investigated and expression of integrins, E-cadherin, and actin were therefore studied after HN2 treatment. In the adherent cells, an early disruption of F-actin occurred associated with cell rounding. Inhibitors of caspase-2 resulted in attenuating of the decline of adhesion proteins and microfilaments. HN2-induced down-regulation of beta1 integrin, E-cadherin expression and F-actin pattern occurred in detached cells but were efficiently prevented by inhibitors of mitochondrial permeabilization. Moreover, inhibiting mitochondrial depolarization improved significantly both cell survival and capacity of detached cells to re-adhere. These findings confirm the pro-survival integrins and E-cadherin mediated signalling pathway. The central role of mitochondria in HN2-induced cell detachment is reinforced, suggesting that mitochondria acts as a key executor of reduced cell adherence during anoikis and could be responsible of an inside-out signalling. Present data support the potential of these therapeutics, generated via the inhibition of mitochondrial depolarization, as protectors against the alkylating agent lesions.
