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The bacterial protein azurin impairs invasion and FAK/Src signaling in P-cadherin-overexpressing breast cancer cell models.

Bernardes N, Ribeiro AS, Abreu S, Mota B, Matos RG, Arraiano CM, Seruca R, Paredes J, Fialho AM - PLoS ONE (2013)

Bottom Line: The invasive phenotype of these breast cancer cells was significantly reduced by azurin.More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins.Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biotechnology and Bioengineering, Center for Biological and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal.

ABSTRACT
P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by azurin. Azurin (50-100 µM) also caused a specific decrease on P-cadherin protein levels from 30-50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.

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Azurin impairs invasion of breast cancer cells over-expressing P-cadherin.a) Schematic representation of the invasive profile of cell lines used in this work (upper panel) and E- and P-cadherin protein expression levels for each cell line (lower panel) b) Azurin decreases invasion of MCF-7/AZ.Pcad and SUM149 cells. Matrigel Invasion Assays showed that one single treatment of azurin at 50 µM for 48 h (MCF-7/AZ.Mock and MCF-7/AZ.Pcad) or 24 h (SUM149) significantly reduced the invasive behaviour of breast cancer cells.MCF-7/AZ.Mock cell line was used as a control and the invasion of this cell line was used to normalize the levels of invasion. c) Cell viability assessed with MTT assay of MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 cell lines in the presence of azurin. Cells were plated in 96-well plates in the presence of 50 and 100 µM of azurin for 24 h (SUM149) or 48 h (MCF-7/AZ.Mock and MCF-7/AZ.Pcad) to match the time course of invasion assays for each cell line. Control cells received complete media without azurin.
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pone-0069023-g001: Azurin impairs invasion of breast cancer cells over-expressing P-cadherin.a) Schematic representation of the invasive profile of cell lines used in this work (upper panel) and E- and P-cadherin protein expression levels for each cell line (lower panel) b) Azurin decreases invasion of MCF-7/AZ.Pcad and SUM149 cells. Matrigel Invasion Assays showed that one single treatment of azurin at 50 µM for 48 h (MCF-7/AZ.Mock and MCF-7/AZ.Pcad) or 24 h (SUM149) significantly reduced the invasive behaviour of breast cancer cells.MCF-7/AZ.Mock cell line was used as a control and the invasion of this cell line was used to normalize the levels of invasion. c) Cell viability assessed with MTT assay of MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 cell lines in the presence of azurin. Cells were plated in 96-well plates in the presence of 50 and 100 µM of azurin for 24 h (SUM149) or 48 h (MCF-7/AZ.Mock and MCF-7/AZ.Pcad) to match the time course of invasion assays for each cell line. Control cells received complete media without azurin.

Mentions: Previous work from our group has demonstrated that invasion of breast cancer cells can be induced by P-cadherin overexpression in a wild-type E-cadherin context [6]. In fact, we showed that expression of P-cadherin in MCF-7/AZ cells induces an increase in cell invasion [6], whereas the knocking-down of P-cadherin by siRNA causes a decrease in the invasive behavior of SUM149 breast cancer cells [21]. Thus, in order to study if azurin could impair the invasion mediated by P-cadherin, these same human breast cancer cell models were used (Figure 1a). Using Matrigel Invasion Assays, we observed that a sub-killing dose of azurin (one single addition at 50 µM) reduces invasion of both MCF-7/AZ.Pcad and SUM149 breast cancer cells lines, 66% and 44%, respectively (Figure 1b). MCF-7/AZ.Mock cells did not change its non-invasive behavior after azurin treatment (Figure 1b).


The bacterial protein azurin impairs invasion and FAK/Src signaling in P-cadherin-overexpressing breast cancer cell models.

Bernardes N, Ribeiro AS, Abreu S, Mota B, Matos RG, Arraiano CM, Seruca R, Paredes J, Fialho AM - PLoS ONE (2013)

Azurin impairs invasion of breast cancer cells over-expressing P-cadherin.a) Schematic representation of the invasive profile of cell lines used in this work (upper panel) and E- and P-cadherin protein expression levels for each cell line (lower panel) b) Azurin decreases invasion of MCF-7/AZ.Pcad and SUM149 cells. Matrigel Invasion Assays showed that one single treatment of azurin at 50 µM for 48 h (MCF-7/AZ.Mock and MCF-7/AZ.Pcad) or 24 h (SUM149) significantly reduced the invasive behaviour of breast cancer cells.MCF-7/AZ.Mock cell line was used as a control and the invasion of this cell line was used to normalize the levels of invasion. c) Cell viability assessed with MTT assay of MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 cell lines in the presence of azurin. Cells were plated in 96-well plates in the presence of 50 and 100 µM of azurin for 24 h (SUM149) or 48 h (MCF-7/AZ.Mock and MCF-7/AZ.Pcad) to match the time course of invasion assays for each cell line. Control cells received complete media without azurin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3716805&req=5

pone-0069023-g001: Azurin impairs invasion of breast cancer cells over-expressing P-cadherin.a) Schematic representation of the invasive profile of cell lines used in this work (upper panel) and E- and P-cadherin protein expression levels for each cell line (lower panel) b) Azurin decreases invasion of MCF-7/AZ.Pcad and SUM149 cells. Matrigel Invasion Assays showed that one single treatment of azurin at 50 µM for 48 h (MCF-7/AZ.Mock and MCF-7/AZ.Pcad) or 24 h (SUM149) significantly reduced the invasive behaviour of breast cancer cells.MCF-7/AZ.Mock cell line was used as a control and the invasion of this cell line was used to normalize the levels of invasion. c) Cell viability assessed with MTT assay of MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 cell lines in the presence of azurin. Cells were plated in 96-well plates in the presence of 50 and 100 µM of azurin for 24 h (SUM149) or 48 h (MCF-7/AZ.Mock and MCF-7/AZ.Pcad) to match the time course of invasion assays for each cell line. Control cells received complete media without azurin.
Mentions: Previous work from our group has demonstrated that invasion of breast cancer cells can be induced by P-cadherin overexpression in a wild-type E-cadherin context [6]. In fact, we showed that expression of P-cadherin in MCF-7/AZ cells induces an increase in cell invasion [6], whereas the knocking-down of P-cadherin by siRNA causes a decrease in the invasive behavior of SUM149 breast cancer cells [21]. Thus, in order to study if azurin could impair the invasion mediated by P-cadherin, these same human breast cancer cell models were used (Figure 1a). Using Matrigel Invasion Assays, we observed that a sub-killing dose of azurin (one single addition at 50 µM) reduces invasion of both MCF-7/AZ.Pcad and SUM149 breast cancer cells lines, 66% and 44%, respectively (Figure 1b). MCF-7/AZ.Mock cells did not change its non-invasive behavior after azurin treatment (Figure 1b).

Bottom Line: The invasive phenotype of these breast cancer cells was significantly reduced by azurin.More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins.Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biotechnology and Bioengineering, Center for Biological and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal.

ABSTRACT
P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by azurin. Azurin (50-100 µM) also caused a specific decrease on P-cadherin protein levels from 30-50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.

Show MeSH
Related in: MedlinePlus