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Inflammation and cancer: role of annexin A1 and FPR2/ALX in proliferation and metastasis in human laryngeal squamous cell carcinoma.

Gastardelo TS, Cunha BR, Raposo LS, Maniglia JV, Cury PM, Lisoni FC, Tajara EH, Oliani SM - PLoS ONE (2014)

Bottom Line: ANXA1(2-26) treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression.ANXA1(2-26) treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling.These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.

View Article: PubMed Central - PubMed

Affiliation: From the Post-graduation in Structural and Functional Biology, Federal University of São Paulo (UNIFESP), Paulista School of Medicine (EPM), São Paulo, SP, Brazil.

ABSTRACT
The anti-inflammatory protein annexin A1 (ANXA1) has been associated with cancer progression and metastasis, suggesting its role in regulating tumor cell proliferation. We investigated the mechanism of ANXA1 interaction with formylated peptide receptor 2 (FPR2/ALX) in control, peritumoral and tumor larynx tissue samples from 20 patients, to quantitate the neutrophils and mast cells, and to evaluate the protein expression and co-localization of ANXA1/FPR2 in these inflammatory cells and laryngeal squamous cells by immunocytochemistry. In addition, we performed in vitro experiments to further investigate the functional role of ANXA1/FPR2 in the proliferation and metastasis of Hep-2 cells, a cell line from larynx epidermoid carcinoma, after treatment with ANXA1(2-26) (annexin A1 N-terminal-derived peptide), Boc2 (antagonist of FPR) and/or dexamethasone. Under these treatments, the level of Hep-2 cell proliferation, pro-inflammatory cytokines, ANXA1/FPR2 co-localization, and the prostaglandin signalling were analyzed using ELISA, immunocytochemistry and real-time PCR. An influx of neutrophils and degranulated mast cells was detected in tumor samples. In these inflammatory cells of peritumoral and tumor samples, ANXA1/FPR2 expression was markedly exacerbated, however, in laryngeal carcinoma cells, this expression was down-regulated. ANXA1(2-26) treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA1(2-26) treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling. Overall, this study identified potential roles for the molecular mechanism of the ANXA1/FPR2 interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing promising starting points for future research. ANXA1 may contribute to the regulation of tumor growth and metastasis through paracrine mechanisms that are mediated by FPR2/ALX. These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.

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Gene expression in Hep-2 cells using real-time PCR.(A) Down- and up-regulated expression of MMP2 and MMP9, respectively, after ANXA12–26 treatment. (B) Overexpression of EP4, MMP2 and MMP9, and down-regulation of EP3 after ANXA12–26+Boc. Hep-2 cells were seeded in MEM-Earle medium at a density of 2×106 cells in 75-cm2 culture flasks, and then were incubated with serum-free medium, 24 hours prior to the addition of ANXA12–26 (1 µM) and ANXA12–26 (1 µM)+Boc2 (10 µM). All of the experiments are representative of three separate experiments. GAPDH was used as the internal reference and the control sample was used as the calibrator. Values were Log2 transformed (y-axis) so that all values below −1 indicated down-regulation in gene expression while values above 1 represented up-regulation compared with control cells.
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pone-0111317-g006: Gene expression in Hep-2 cells using real-time PCR.(A) Down- and up-regulated expression of MMP2 and MMP9, respectively, after ANXA12–26 treatment. (B) Overexpression of EP4, MMP2 and MMP9, and down-regulation of EP3 after ANXA12–26+Boc. Hep-2 cells were seeded in MEM-Earle medium at a density of 2×106 cells in 75-cm2 culture flasks, and then were incubated with serum-free medium, 24 hours prior to the addition of ANXA12–26 (1 µM) and ANXA12–26 (1 µM)+Boc2 (10 µM). All of the experiments are representative of three separate experiments. GAPDH was used as the internal reference and the control sample was used as the calibrator. Values were Log2 transformed (y-axis) so that all values below −1 indicated down-regulation in gene expression while values above 1 represented up-regulation compared with control cells.

Mentions: Hep-2 cells that were treated with ANXA12–26 showed a significant decrease (Log2≤−1.0) in MMP2 expression and an increase (Log2≥1.0) in MMP9 expression compared with control cells (Fig. 6). However, incubation of Hep-2 cells with ANXA12–26 plus Boc mediated up-regulation (Log2≥1.0) of EP4, MMP2 and MMP9 gene expression and down-regulation (Log2≤−1.0) of EP3 expression compared with control cells (Fig. 6).


Inflammation and cancer: role of annexin A1 and FPR2/ALX in proliferation and metastasis in human laryngeal squamous cell carcinoma.

Gastardelo TS, Cunha BR, Raposo LS, Maniglia JV, Cury PM, Lisoni FC, Tajara EH, Oliani SM - PLoS ONE (2014)

Gene expression in Hep-2 cells using real-time PCR.(A) Down- and up-regulated expression of MMP2 and MMP9, respectively, after ANXA12–26 treatment. (B) Overexpression of EP4, MMP2 and MMP9, and down-regulation of EP3 after ANXA12–26+Boc. Hep-2 cells were seeded in MEM-Earle medium at a density of 2×106 cells in 75-cm2 culture flasks, and then were incubated with serum-free medium, 24 hours prior to the addition of ANXA12–26 (1 µM) and ANXA12–26 (1 µM)+Boc2 (10 µM). All of the experiments are representative of three separate experiments. GAPDH was used as the internal reference and the control sample was used as the calibrator. Values were Log2 transformed (y-axis) so that all values below −1 indicated down-regulation in gene expression while values above 1 represented up-regulation compared with control cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260827&req=5

pone-0111317-g006: Gene expression in Hep-2 cells using real-time PCR.(A) Down- and up-regulated expression of MMP2 and MMP9, respectively, after ANXA12–26 treatment. (B) Overexpression of EP4, MMP2 and MMP9, and down-regulation of EP3 after ANXA12–26+Boc. Hep-2 cells were seeded in MEM-Earle medium at a density of 2×106 cells in 75-cm2 culture flasks, and then were incubated with serum-free medium, 24 hours prior to the addition of ANXA12–26 (1 µM) and ANXA12–26 (1 µM)+Boc2 (10 µM). All of the experiments are representative of three separate experiments. GAPDH was used as the internal reference and the control sample was used as the calibrator. Values were Log2 transformed (y-axis) so that all values below −1 indicated down-regulation in gene expression while values above 1 represented up-regulation compared with control cells.
Mentions: Hep-2 cells that were treated with ANXA12–26 showed a significant decrease (Log2≤−1.0) in MMP2 expression and an increase (Log2≥1.0) in MMP9 expression compared with control cells (Fig. 6). However, incubation of Hep-2 cells with ANXA12–26 plus Boc mediated up-regulation (Log2≥1.0) of EP4, MMP2 and MMP9 gene expression and down-regulation (Log2≤−1.0) of EP3 expression compared with control cells (Fig. 6).

Bottom Line: ANXA1(2-26) treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression.ANXA1(2-26) treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling.These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.

View Article: PubMed Central - PubMed

Affiliation: From the Post-graduation in Structural and Functional Biology, Federal University of São Paulo (UNIFESP), Paulista School of Medicine (EPM), São Paulo, SP, Brazil.

ABSTRACT
The anti-inflammatory protein annexin A1 (ANXA1) has been associated with cancer progression and metastasis, suggesting its role in regulating tumor cell proliferation. We investigated the mechanism of ANXA1 interaction with formylated peptide receptor 2 (FPR2/ALX) in control, peritumoral and tumor larynx tissue samples from 20 patients, to quantitate the neutrophils and mast cells, and to evaluate the protein expression and co-localization of ANXA1/FPR2 in these inflammatory cells and laryngeal squamous cells by immunocytochemistry. In addition, we performed in vitro experiments to further investigate the functional role of ANXA1/FPR2 in the proliferation and metastasis of Hep-2 cells, a cell line from larynx epidermoid carcinoma, after treatment with ANXA1(2-26) (annexin A1 N-terminal-derived peptide), Boc2 (antagonist of FPR) and/or dexamethasone. Under these treatments, the level of Hep-2 cell proliferation, pro-inflammatory cytokines, ANXA1/FPR2 co-localization, and the prostaglandin signalling were analyzed using ELISA, immunocytochemistry and real-time PCR. An influx of neutrophils and degranulated mast cells was detected in tumor samples. In these inflammatory cells of peritumoral and tumor samples, ANXA1/FPR2 expression was markedly exacerbated, however, in laryngeal carcinoma cells, this expression was down-regulated. ANXA1(2-26) treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA1(2-26) treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling. Overall, this study identified potential roles for the molecular mechanism of the ANXA1/FPR2 interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing promising starting points for future research. ANXA1 may contribute to the regulation of tumor growth and metastasis through paracrine mechanisms that are mediated by FPR2/ALX. These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.

Show MeSH
Related in: MedlinePlus