Limits...
Caveolae contribute to the apoptosis resistance induced by the alpha(1A)-adrenoceptor in androgen-independent prostate cancer cells.

Katsogiannou M, El Boustany C, Gackiere F, Delcourt P, Athias A, Mariot P, Dewailly E, Jouy N, Lamaze C, Bidaux G, Mauroy B, Prevarskaya N, Slomianny C - PLoS ONE (2009)

Bottom Line: In addition, we showed that agonist stimulation of the alpha(1A)-adrenoceptor induced resistance to thapsigargin-induced apoptosis and that caveolin-1 was necessary for this process.We also show by immunoblotting that the TG-induced apoptosis resistance described in DU145 cells is mediated by extracellular signal-regulated kinases (ERK).In conclusion, we propose that alpha(1A)-adrenoceptor stimulation in androgen-independent prostate cancer cells via caveolae constitutes one of the mechanisms contributing to their protection from TG-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Inserm U800, Université Lille 1 Sciences et Technologies, Villeneuve d'Ascq, France.

ABSTRACT

Background: During androgen ablation prostate cancer cells' growth and survival become independent of normal regulatory mechanisms. These androgen-independent cells acquire the remarkable ability to adapt to the surrounding microenvironment whose factors, such as neurotransmitters, influence their survival. Although findings are becoming evident about the expression of alpha(1A)-adrenoceptors in prostate cancer epithelial cells, their exact functional role in androgen-independent cells has yet to be established. Previous work has demonstrated that membrane lipid rafts associated with key signalling proteins mediate growth and survival signalling pathways in prostate cancer cells.

Methodology/principal findings: In order to analyze the membrane topology of the alpha(1A)-adrenoceptor we explored its presence by a biochemical approach in purified detergent resistant membrane fractions of the androgen-independent prostate cancer cell line DU145. Electron microscopy observations demonstrated the colocalization of the alpha(1A)-adrenoceptor with caveolin-1, the major protein component of caveolae. In addition, we showed that agonist stimulation of the alpha(1A)-adrenoceptor induced resistance to thapsigargin-induced apoptosis and that caveolin-1 was necessary for this process. Further, immunohistofluorescence revealed the relation between high levels of alpha(1A)-adrenoceptor and caveolin-1 expression with advanced stage prostate cancer. We also show by immunoblotting that the TG-induced apoptosis resistance described in DU145 cells is mediated by extracellular signal-regulated kinases (ERK).

Conclusions/significance: In conclusion, we propose that alpha(1A)-adrenoceptor stimulation in androgen-independent prostate cancer cells via caveolae constitutes one of the mechanisms contributing to their protection from TG-induced apoptosis.

Show MeSH

Related in: MedlinePlus

PHE enhances TG-induced apoptosis resistance in DU145 cells also via bax activation.Representative immunofluorescence of (A) DU145, (B) DushCTL and (C) Dushcav-1 cells in (a) non-treated conditions, (b) 10 µM TG for 48 h, (c) 10 µM PHE 3-day pretreatment (d) with the presence of 1 µM PRA, followed by 10 µM TG for 48 h showing the presence of activated Bax recognised by the anti-bax (6A7) antibody. The presence of cells in the observation field is indicated by DAPI-stained nuclei. Bar, 50 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2742726&req=5

pone-0007068-g005: PHE enhances TG-induced apoptosis resistance in DU145 cells also via bax activation.Representative immunofluorescence of (A) DU145, (B) DushCTL and (C) Dushcav-1 cells in (a) non-treated conditions, (b) 10 µM TG for 48 h, (c) 10 µM PHE 3-day pretreatment (d) with the presence of 1 µM PRA, followed by 10 µM TG for 48 h showing the presence of activated Bax recognised by the anti-bax (6A7) antibody. The presence of cells in the observation field is indicated by DAPI-stained nuclei. Bar, 50 µm.

Mentions: Bax is a 23 kD pro-apoptotic protein, member of the Bcl-2 family. The critical events in the activation process of Bax are: its translocation to mitochondria and its N-terminal conformational change closely coupled to mitochondrial membrane insertion and oligomerisation [42], [43]. The insertion of Bax into the mitochondrial outer membrane is closely associated with the release into the cytosol of several proteins such as cytochrome c and procaspase-3 which are essential to the execution of the apoptotic program [44]. We investigated Bax activation by immunofluorescence using an antibody which specifically recognizes the activated conformation of Bax (see “Methods” section). The figures shown here are representative of all slides observed. TG treatment of DU145 WT (Figure 5A, b), DUshCTL cells (Figure 5B, b) and Dushcav-1 cells (Figure 5C, b) induced an increase in activated-Bax staining as compared to non-treated conditions (Figure 5A, B and C, a). In DU145 and DushCTL cells, a three-day treatment by 10 µM PHE preceding TG reduced activated-Bax expression (Figure 5A and B, c) as compared to TG treatment alone (Figure 5A and B, b) suggesting decreased apoptosis in these conditions. Once more, 1 µM PRA pretreatment opposed the PHE-induced decrease in activated-Bax staining (Figure 5A and B, d). No significant consequence of PHE pretreatment (with or without PRA) in the resistance to TG-induced apoptosis was observed in DUshcav-1 cells (Figure 5C, b, c and d) once again strongly suggesting the involvement of caveolae in this function of α1-AR. DAPI-stained nuclei are indicative of the number of cells present in the observation field. The absence of apoptotic figures (chromatin condensation and nuclear fragmentation) in cells expressing activated Bax is expectable as Bax activation is an early-stage apoptotic process as opposed to nuclear fragmentation.


Caveolae contribute to the apoptosis resistance induced by the alpha(1A)-adrenoceptor in androgen-independent prostate cancer cells.

Katsogiannou M, El Boustany C, Gackiere F, Delcourt P, Athias A, Mariot P, Dewailly E, Jouy N, Lamaze C, Bidaux G, Mauroy B, Prevarskaya N, Slomianny C - PLoS ONE (2009)

PHE enhances TG-induced apoptosis resistance in DU145 cells also via bax activation.Representative immunofluorescence of (A) DU145, (B) DushCTL and (C) Dushcav-1 cells in (a) non-treated conditions, (b) 10 µM TG for 48 h, (c) 10 µM PHE 3-day pretreatment (d) with the presence of 1 µM PRA, followed by 10 µM TG for 48 h showing the presence of activated Bax recognised by the anti-bax (6A7) antibody. The presence of cells in the observation field is indicated by DAPI-stained nuclei. Bar, 50 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742726&req=5

pone-0007068-g005: PHE enhances TG-induced apoptosis resistance in DU145 cells also via bax activation.Representative immunofluorescence of (A) DU145, (B) DushCTL and (C) Dushcav-1 cells in (a) non-treated conditions, (b) 10 µM TG for 48 h, (c) 10 µM PHE 3-day pretreatment (d) with the presence of 1 µM PRA, followed by 10 µM TG for 48 h showing the presence of activated Bax recognised by the anti-bax (6A7) antibody. The presence of cells in the observation field is indicated by DAPI-stained nuclei. Bar, 50 µm.
Mentions: Bax is a 23 kD pro-apoptotic protein, member of the Bcl-2 family. The critical events in the activation process of Bax are: its translocation to mitochondria and its N-terminal conformational change closely coupled to mitochondrial membrane insertion and oligomerisation [42], [43]. The insertion of Bax into the mitochondrial outer membrane is closely associated with the release into the cytosol of several proteins such as cytochrome c and procaspase-3 which are essential to the execution of the apoptotic program [44]. We investigated Bax activation by immunofluorescence using an antibody which specifically recognizes the activated conformation of Bax (see “Methods” section). The figures shown here are representative of all slides observed. TG treatment of DU145 WT (Figure 5A, b), DUshCTL cells (Figure 5B, b) and Dushcav-1 cells (Figure 5C, b) induced an increase in activated-Bax staining as compared to non-treated conditions (Figure 5A, B and C, a). In DU145 and DushCTL cells, a three-day treatment by 10 µM PHE preceding TG reduced activated-Bax expression (Figure 5A and B, c) as compared to TG treatment alone (Figure 5A and B, b) suggesting decreased apoptosis in these conditions. Once more, 1 µM PRA pretreatment opposed the PHE-induced decrease in activated-Bax staining (Figure 5A and B, d). No significant consequence of PHE pretreatment (with or without PRA) in the resistance to TG-induced apoptosis was observed in DUshcav-1 cells (Figure 5C, b, c and d) once again strongly suggesting the involvement of caveolae in this function of α1-AR. DAPI-stained nuclei are indicative of the number of cells present in the observation field. The absence of apoptotic figures (chromatin condensation and nuclear fragmentation) in cells expressing activated Bax is expectable as Bax activation is an early-stage apoptotic process as opposed to nuclear fragmentation.

Bottom Line: In addition, we showed that agonist stimulation of the alpha(1A)-adrenoceptor induced resistance to thapsigargin-induced apoptosis and that caveolin-1 was necessary for this process.We also show by immunoblotting that the TG-induced apoptosis resistance described in DU145 cells is mediated by extracellular signal-regulated kinases (ERK).In conclusion, we propose that alpha(1A)-adrenoceptor stimulation in androgen-independent prostate cancer cells via caveolae constitutes one of the mechanisms contributing to their protection from TG-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Inserm U800, Université Lille 1 Sciences et Technologies, Villeneuve d'Ascq, France.

ABSTRACT

Background: During androgen ablation prostate cancer cells' growth and survival become independent of normal regulatory mechanisms. These androgen-independent cells acquire the remarkable ability to adapt to the surrounding microenvironment whose factors, such as neurotransmitters, influence their survival. Although findings are becoming evident about the expression of alpha(1A)-adrenoceptors in prostate cancer epithelial cells, their exact functional role in androgen-independent cells has yet to be established. Previous work has demonstrated that membrane lipid rafts associated with key signalling proteins mediate growth and survival signalling pathways in prostate cancer cells.

Methodology/principal findings: In order to analyze the membrane topology of the alpha(1A)-adrenoceptor we explored its presence by a biochemical approach in purified detergent resistant membrane fractions of the androgen-independent prostate cancer cell line DU145. Electron microscopy observations demonstrated the colocalization of the alpha(1A)-adrenoceptor with caveolin-1, the major protein component of caveolae. In addition, we showed that agonist stimulation of the alpha(1A)-adrenoceptor induced resistance to thapsigargin-induced apoptosis and that caveolin-1 was necessary for this process. Further, immunohistofluorescence revealed the relation between high levels of alpha(1A)-adrenoceptor and caveolin-1 expression with advanced stage prostate cancer. We also show by immunoblotting that the TG-induced apoptosis resistance described in DU145 cells is mediated by extracellular signal-regulated kinases (ERK).

Conclusions/significance: In conclusion, we propose that alpha(1A)-adrenoceptor stimulation in androgen-independent prostate cancer cells via caveolae constitutes one of the mechanisms contributing to their protection from TG-induced apoptosis.

Show MeSH
Related in: MedlinePlus