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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.

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Related in: MedlinePlus

α1A-AR and cav-1 differential expressions in normal, cancerous and hyperplastic human prostate.(A)(a) Western blot showing the expression of α1A-AR in DU145 cells transfected with two siRNA against α1A-AR (siADR1A-1 and siADR1A-2) and control siRNA (siCTL). Numbers above gels indicate relative densitometry assessed on actin expression. (b) Representative immunofluorescence of normal prostate acinus showing the presence of the apical epithelial marker cytokeratin 18 (cytk 18, green) and α1A-AR or cav-1 fluorescence (red); Bar, 50 µm. Star indicates the acinus lumen and white arrowhead fibromuscular stroma. The middle left panel shows representative stage III cancerous acinus. Typically, cancerous apical epithelial cells detected with cytokeratin 18 (green) have completely invaded the lumen and express both α1A-AR and cav-1 (red); Bar, 50 µm. The middle right panel shows a section of a representative hyperplastic acinus where the lumen (star) is enlarged; Bar, 20 µm. (B)(a) Agarose gel showing the expression of α1A-AR (178 pb), cav-1 (220 pb), cytokeratin 18 (241 pb) and cytokeratin 14 (210 pb) amplicons in six different prostatic androgen-independent carcinoma tissues and six different benign hyperplasia tissues. A no-template control was also run with the PCR samples where cDNA was replaced with water (H2O). A 1-kilobase DNA ladder (MW (bp)) was used as a DNA size marker. GAPDH (236 pb) was used as an internal control. (b) Ratios of mean mRNA quantities of α1A-AR, cav-1, cytokeratin 18 (cytk 18) and 14 (cytk 14) as compared to GAPDH in androgen-independent (AI) cancer (black columns) and hyperplastic samples (grey hatched columns). Plots are the average cumulative data (mean ± SEM) of six samples by condition. Statistical analysis used the t test; *, p<0.05, **, p<0.01.
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pone-0007068-g006: α1A-AR and cav-1 differential expressions in normal, cancerous and hyperplastic human prostate.(A)(a) Western blot showing the expression of α1A-AR in DU145 cells transfected with two siRNA against α1A-AR (siADR1A-1 and siADR1A-2) and control siRNA (siCTL). Numbers above gels indicate relative densitometry assessed on actin expression. (b) Representative immunofluorescence of normal prostate acinus showing the presence of the apical epithelial marker cytokeratin 18 (cytk 18, green) and α1A-AR or cav-1 fluorescence (red); Bar, 50 µm. Star indicates the acinus lumen and white arrowhead fibromuscular stroma. The middle left panel shows representative stage III cancerous acinus. Typically, cancerous apical epithelial cells detected with cytokeratin 18 (green) have completely invaded the lumen and express both α1A-AR and cav-1 (red); Bar, 50 µm. The middle right panel shows a section of a representative hyperplastic acinus where the lumen (star) is enlarged; Bar, 20 µm. (B)(a) Agarose gel showing the expression of α1A-AR (178 pb), cav-1 (220 pb), cytokeratin 18 (241 pb) and cytokeratin 14 (210 pb) amplicons in six different prostatic androgen-independent carcinoma tissues and six different benign hyperplasia tissues. A no-template control was also run with the PCR samples where cDNA was replaced with water (H2O). A 1-kilobase DNA ladder (MW (bp)) was used as a DNA size marker. GAPDH (236 pb) was used as an internal control. (b) Ratios of mean mRNA quantities of α1A-AR, cav-1, cytokeratin 18 (cytk 18) and 14 (cytk 14) as compared to GAPDH in androgen-independent (AI) cancer (black columns) and hyperplastic samples (grey hatched columns). Plots are the average cumulative data (mean ± SEM) of six samples by condition. Statistical analysis used the t test; *, p<0.05, **, p<0.01.

Mentions: The α1A-AR is abundant in the fibromuscular tissue in normal and hyperplastic prostates [45], [46] however, its role in prostate epithelial cells is still not well defined. Moreover, cav-1 has been described to play an important role in the survival/growth of PCa cells contributing to their metastatic activities [47], [48]. To determine the association of α1A-AR and cav-1 expression with established features of PCa as well as BPH in comparison to normal prostate, we performed α1A-AR and cav-1 immunostaining on tissue microarrays containing specimen cores from 40 patients. Cytokeratin 18 immunostaining was used as a label of apical epithelial cells present in acini. Normal tissue regions from 9 of these patients were present on these slides. The figures shown here are representative of all samples observed. We first confirmed the specificity of the α1A-AR antibody by the use of two siRNA against α1A-AR (siADR1A-1 and siADR1A-2) (Figure 6A, a). The use of these siRNA induced a 40% and 60% decrease of α1A-AR as compared to DU145 cells transfected with the control siRNA. Previous studies revealed that cav-1 overexpression is associated with aggressive PCa [49] but little is known about α1A-AR expression in PCa progression. Here, we found that cav-1 and α1A-AR were strongly expressed in luminal, invasive epithelial cells in stage III PCa (Figure 6A, b, cancerous acinus). Furthermore, in hyperplastic tissues cav-1 and α1A-AR immunostaining was present in the simple lining of cytokeratin 18-positive-acinar epithelium and in the fibromuscular stroma (Figure 6A, b, hyperplastic acinus). The majority of the normal regions of the samples expressed low levels of cav-1 and α1A-AR in apical epithelial cells and the presence of the two proteins was mostly observed in the stroma (Figure 6A, b, normal acinus). Overall, these results demonstrated elevated expressions of cav-1 and α1A-AR in advanced PCa epithelial cells, more numerous and invasive in these conditions than in BPH.


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)

α1A-AR and cav-1 differential expressions in normal, cancerous and hyperplastic human prostate.(A)(a) Western blot showing the expression of α1A-AR in DU145 cells transfected with two siRNA against α1A-AR (siADR1A-1 and siADR1A-2) and control siRNA (siCTL). Numbers above gels indicate relative densitometry assessed on actin expression. (b) Representative immunofluorescence of normal prostate acinus showing the presence of the apical epithelial marker cytokeratin 18 (cytk 18, green) and α1A-AR or cav-1 fluorescence (red); Bar, 50 µm. Star indicates the acinus lumen and white arrowhead fibromuscular stroma. The middle left panel shows representative stage III cancerous acinus. Typically, cancerous apical epithelial cells detected with cytokeratin 18 (green) have completely invaded the lumen and express both α1A-AR and cav-1 (red); Bar, 50 µm. The middle right panel shows a section of a representative hyperplastic acinus where the lumen (star) is enlarged; Bar, 20 µm. (B)(a) Agarose gel showing the expression of α1A-AR (178 pb), cav-1 (220 pb), cytokeratin 18 (241 pb) and cytokeratin 14 (210 pb) amplicons in six different prostatic androgen-independent carcinoma tissues and six different benign hyperplasia tissues. A no-template control was also run with the PCR samples where cDNA was replaced with water (H2O). A 1-kilobase DNA ladder (MW (bp)) was used as a DNA size marker. GAPDH (236 pb) was used as an internal control. (b) Ratios of mean mRNA quantities of α1A-AR, cav-1, cytokeratin 18 (cytk 18) and 14 (cytk 14) as compared to GAPDH in androgen-independent (AI) cancer (black columns) and hyperplastic samples (grey hatched columns). Plots are the average cumulative data (mean ± SEM) of six samples by condition. Statistical analysis used the t test; *, p<0.05, **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007068-g006: α1A-AR and cav-1 differential expressions in normal, cancerous and hyperplastic human prostate.(A)(a) Western blot showing the expression of α1A-AR in DU145 cells transfected with two siRNA against α1A-AR (siADR1A-1 and siADR1A-2) and control siRNA (siCTL). Numbers above gels indicate relative densitometry assessed on actin expression. (b) Representative immunofluorescence of normal prostate acinus showing the presence of the apical epithelial marker cytokeratin 18 (cytk 18, green) and α1A-AR or cav-1 fluorescence (red); Bar, 50 µm. Star indicates the acinus lumen and white arrowhead fibromuscular stroma. The middle left panel shows representative stage III cancerous acinus. Typically, cancerous apical epithelial cells detected with cytokeratin 18 (green) have completely invaded the lumen and express both α1A-AR and cav-1 (red); Bar, 50 µm. The middle right panel shows a section of a representative hyperplastic acinus where the lumen (star) is enlarged; Bar, 20 µm. (B)(a) Agarose gel showing the expression of α1A-AR (178 pb), cav-1 (220 pb), cytokeratin 18 (241 pb) and cytokeratin 14 (210 pb) amplicons in six different prostatic androgen-independent carcinoma tissues and six different benign hyperplasia tissues. A no-template control was also run with the PCR samples where cDNA was replaced with water (H2O). A 1-kilobase DNA ladder (MW (bp)) was used as a DNA size marker. GAPDH (236 pb) was used as an internal control. (b) Ratios of mean mRNA quantities of α1A-AR, cav-1, cytokeratin 18 (cytk 18) and 14 (cytk 14) as compared to GAPDH in androgen-independent (AI) cancer (black columns) and hyperplastic samples (grey hatched columns). Plots are the average cumulative data (mean ± SEM) of six samples by condition. Statistical analysis used the t test; *, p<0.05, **, p<0.01.
Mentions: The α1A-AR is abundant in the fibromuscular tissue in normal and hyperplastic prostates [45], [46] however, its role in prostate epithelial cells is still not well defined. Moreover, cav-1 has been described to play an important role in the survival/growth of PCa cells contributing to their metastatic activities [47], [48]. To determine the association of α1A-AR and cav-1 expression with established features of PCa as well as BPH in comparison to normal prostate, we performed α1A-AR and cav-1 immunostaining on tissue microarrays containing specimen cores from 40 patients. Cytokeratin 18 immunostaining was used as a label of apical epithelial cells present in acini. Normal tissue regions from 9 of these patients were present on these slides. The figures shown here are representative of all samples observed. We first confirmed the specificity of the α1A-AR antibody by the use of two siRNA against α1A-AR (siADR1A-1 and siADR1A-2) (Figure 6A, a). The use of these siRNA induced a 40% and 60% decrease of α1A-AR as compared to DU145 cells transfected with the control siRNA. Previous studies revealed that cav-1 overexpression is associated with aggressive PCa [49] but little is known about α1A-AR expression in PCa progression. Here, we found that cav-1 and α1A-AR were strongly expressed in luminal, invasive epithelial cells in stage III PCa (Figure 6A, b, cancerous acinus). Furthermore, in hyperplastic tissues cav-1 and α1A-AR immunostaining was present in the simple lining of cytokeratin 18-positive-acinar epithelium and in the fibromuscular stroma (Figure 6A, b, hyperplastic acinus). The majority of the normal regions of the samples expressed low levels of cav-1 and α1A-AR in apical epithelial cells and the presence of the two proteins was mostly observed in the stroma (Figure 6A, b, normal acinus). Overall, these results demonstrated elevated expressions of cav-1 and α1A-AR in advanced PCa epithelial cells, more numerous and invasive in these conditions than in BPH.

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