The authors have declared that no competing interests exist.
Conceived and designed the experiments: SMO EHT. Performed the experiments: TSG BRC. Analyzed the data: TSG SMO EHT FCRL. Contributed reagents/materials/analysis tools: SMO EHT. Wrote the paper: TSG SMO EHT. Collected the surgical samples: LSR JVM. Performed the pathological analyses: PMC.
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 ANXA12–26 (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. ANXA12–26 treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA12–26 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.
Laryngeal cancer is one of the most common types of head and neck tumors that has a high mortality rate and a poor prognosis
Only 5% to 10% of all cancers are caused by the inheritance of mutated genes, whereas the remaining 90% to 95% of cases have been linked to genetic and epigenetic alterations caused by lifestyle and environmental factors, such as cigarette smoking and alcohol use
Inflammatory cells secrete numerous cytokines, chemokines and growth factors that can stimulate proliferation, inhibit apoptosis, induce morphogenesis and generate DNA-damaging reactive oxygen species
It has been demonstrated that mast cells and neutrophils can be recruited by tumor cells. Increased numbers of mast cells have been reported in mammary
From these studies, it is clear that various inflammatory mediators are differentially expressed in several cancers and can stimulate disease progression
Dysregulation of ANXA1 has been reported in multiple neoplasms, suggesting that this protein may play important roles in tumor development and progression
Advances in this area have shown a functional link between the anti-inflammatory properties of ANXA1 and receptor for formyl-Met-Leu-Phe (fMLP) FPR
Several signaling pathways mediate inflammation-associated tumorigenesis. For example, cyclooxygenase-2 (COX-2), an enzyme involved in the conversion of arachidonic acid to prostaglandins, is considered to play important roles in cancer development by modulating cell proliferation and other important biological processes via their metabolites, G protein-coupled receptors and downstream effectors
Given the potential role for ANXA1 in multiple neoplasms such as the regulation of cell proliferation, differentiation and metastasis, we focused on the molecular mechanisms by which ANXA1 modulates these cellular responses. In this report, we showed that ANXA1 protein is down-regulated in human laryngeal carcinoma and can regulate tumor growth in a paracrine manner that is mediated by the receptor FPR2/ALX. This investigation demonstrates the potential significance of this ANXA1/FPR2 interaction in tumorigenesis. Overall, this study identified potential roles for the molecular mechanism of this protein interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing evidence for the potential of ANXA1 as a therapeutic target and promising starting points for future research.
Human invasive laryngeal cancer tissues were obtained from 20 patients with laryngeal squamous cell carcinoma who were treated at the Hospital de Base in São José do Rio Preto, Brazil. The patients were male, alcoholic smokers, their age ranged from 50 to 80 years, and none of them had received radiotherapy or chemotherapy before intervention. In each patient (n = 20), the surgical tumor samples were collected from the center of the laryngeal tumor, the peritumoral samples were collected from the tumor periphery, and the control samples were collected from the region free of tumor cells. Because of the high heterogeneity of laryngeal carcinoma, the tumor tissues were dissected and reviewed by a senior pathologist and were characterized as moderately differentiated, invasive carcinomas. These tissue samples were used in a previous study that was conducted in our laboratory, and the number of patients was sufficient to obtain statistically significant data
The Hep-2 cell line, which was originally established from an epidermoid carcinoma of the larynx, was used in the present study (ATCC, Rockville, Maryland, USA). Cell line authentication was performed using the AmpFLSTR Identifiler PCR Amplification Kit (Life Technologies) at the Special Techniques Laboratory, Hospital Israelita Albert Einstein (LATE -HIAE), Sao Paulo. We found 100% identity of our cell line compared with the American Type Culture Collection (ATCC) database. The cells were seeded at a density of 2×106 cells in a 75-cm2 culture flask (Corning, NY, USA) and incubated in MEM-Earle medium (Cultilab, Campinas, SP, Brazil), pH 7.5, supplemented with 20% fetal calf serum (Cultilab), 1% non-essential amino acids, and 0.1% antibiotic/antimycotic solution (Invitrogen Corporation, Carlsbad, CA, USA), at 37°C in a humid atmosphere of 5% CO2
For the pharmacological experiments, Hep-2 cells were seeded as previously described. Twenty-four hours later, after the cells had already adhered, they were incubated in serum-free medium to synchronize the cell cycle. After an additional 24 hours, the medium was replaced with complete growth medium containing the following: (a) 1 µM of ANXA12–26 peptide (Ac-AMVSEFLKQAWFIENEEQEYVQTVK) (Invitrogen); (b) 1 µM of ANXA12–26 peptide and 10 µM Boc2, a nonselective FPR antagonist (N-t-BOC-MET-LEU-PHE) (MP Biomedicals, Aurora, OH); (c) 0.01 µM dexamethasone (a glucocorticoid; Sigma-Aldrich, St. Louis, MO, USA); (d) 0.01 µM dexamethasone and 10 µM Boc2; or (e) 10 µM Boc2 alone. The drugs were first dissolved in small amounts of dimethyl sulfoxide (DMSO) and then diluted in medium (the final concentration of DMSO never exceeded 1%). The concentrations of the drugs were selected based on preliminary experiments and literature data
Laryngeal samples and Hep-2 cells were fixed in 4% paraformaldehyde, 0.5% glutaraldehyde, and 0.1 mol/L sodium cacodylate buffer (pH 7.4) for 24 hours at 4°C, washed in sodium cacodylate, dehydrated through graded percentages of ethanol, and embedded in LRGold (London Resin Co., Reading, UK). For histopathological and morphological analyses, tissue sections (0.5-µm thick) were cut on an ultramicrotome (Reichert Ultracut; Leica, Austria), stained with 1% toluidine blue in 1% borax solution (TAAB Laboratories, Aldermaston, UK) and examined using an AXIOSKOP 2-Mot Plus ZEISS microscope (Carl Zeiss, Jena, Germany). For electron microscopy, sections (∼90 nm) were cut on an ultramicrotome and placed on nickel grids for immunogold labeling
Immunocytochemistry reaction (double labeling) was used to detect the expression and co-localization of ANXA1 and FPR2/ALX in mast cells, neutrophils, and control and tumor cells from laryngeal tissues and Hep-2 cells.
The Hep-2 cells were incubated with control medium, ANXA12–26 and ANXA12–26+Boc2. After 48 hours, the culture was harvested, and the cells were embedded in LRGold resin for immunocytochemical analysis. Ultrathin sections of laryngeal tissues and Hep-2 cells were incubated in a step-by-step manner using the following reagents and/or conditions at room temperature: 1) distilled water; 2) 0.1 mol/L phosphate buffer containing 1% egg albumin (PBEA); 3) 0.1 mol/L PBS containing 5% PBEA for 30 minutes; 4) sheep polyclonal antibody LCPS1, raised against the N-terminal segment of ANXA1 (1∶500 in PBEA) and rabbit polyclonal antibody FPR2/ALX (Abcam, Cambridge, UK) (1∶500 in PBEA) for 2 hours; normal sheep and rabbit sera were used as the control (1∶500); 5) three washes in PBEA containing 0.01% Tween 20; 6), donkey anti-sheep IgG (Fc fragment-specific) antibody (1∶100 in PBEA) conjugated to 15-nm colloidal gold (British Biocell, UK) was added to detect ANXA1, and goat anti-rabbit IgG (Fc fragment-specific) antibody (1∶100 in PBEA) conjugated to 10-nm colloidal gold (British Biocell) was added to detect FPR2/ALX). After 1 hour, the sections were washed in PBEA containing 0.01% Tween 20, and then in distilled water
To quantify the pro-inflammatory cytokines interleukins 6 (IL-6) and IL-8 as well as monocyte chemotactic protein-1 (MCP-1), in the Hep-2 cell culture supernatants, we used the multiplex instrument LUMINEX xMAP MAGPIX (Millipore Corporation, Billerica, MA, USA). The cells were incubated with control medium, ANXA12–26, ANXA12–26+Boc2, Dexa, Dexa+Boc2 or Boc2. After 48 hours of treatment, the culture supernatants were removed, centrifuged and stored at −20°C. Antibody beads, controls, wash buffer, serum matrix and standards were prepared following the manufacturer's instructions (MILLIPLEX HCYTOMAG-60K kit). Next, 200 µL of wash buffer was added to each well of a magnetic 96-well plate and mixed on a shaker for 10 min. The wash buffer was decanted, and 25 µl of standards, controls and samples were added to the wells. Next, 25 µl of assay buffer was added to the samples, and 25 µl of serum matrix was added to the standards. Finally, 25 µl of magnetic beads (coated with a specific capture antibody) was added to all wells and incubated overnight at 4°C on a shaker. The next day, the plate was washed with wash buffer and incubated with 25 µl of detection antibodies for 1 hour on a shaker. Next, 25 µL of streptavidin-phycoerythrin was added to each well and incubated for 30 minutes on a shaker. The plate was washed and incubated with 150 µl of drive fluid for 5 minutes on a shaker. Finally, the plate was analyzed using MAGPIX with xPONENT software
The Hep-2 cells were incubated with control medium, ANXA12–26 or ANXA12–26+Boc2 and harvested after 48 hours. Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer's protocol. The genomic DNA was removed by DNase treatment according to the manufacturer's description (Promega). Aliquots (2 µg) of total RNA from control and treated cells were used for double-stranded cDNA synthesis using a High Capacity cDNA Archive kit (Applied Biosystems) according to the manufacturer's instructions. Four differentially expressed genes (
The values concerning the quantification of mast cells and neutrophils of the in vivo tissue samples were expressed as the mean ± SEM of the number of cells per mm2 in three sections of 0.5 µm (leaving a space of 40 µm between each section) for each patient (n = 20 patients).
Hep-2 cells were counted using the Countess Automated Cell Counter (Invitrogen). The concentration of the cytokines was determined using MAGPIX Xponent software (Millipore Corporation). The in vitro analyses were repeated a minimum of three times.
The ultrastructural and immunocytochemistry expression of ANXA1 and FPR2 was determined using ten cells for each group investigated. The area of each cell compartment was determined using Axiovision imaging software (Zeiss). In the nucleus and cytoplasm, the density of colloidal gold particles was calculated as the mean ± SEM of the number of particles per µm2. In the plasma membrane, the density of colloidal gold particles was calculated as the mean ± SEM of the number of particles per µm.
The significant differences between the means were determined using analysis of variance. This test was followed by the Bonferroni post-hoc test on select experimental groups using Graph-Pad Prism 4.0 (Graph-Pad, San Diego, CA, USA). A probability value less than 0.05 was considered to be statistically significant.
To verify the interaction of inflammatory cells with laryngeal tumor cells, we performed histological analyses in larynx tissue samples to quantify mast cells and neutrophils, important cells of the inflammatory response. In control samples, we observed intact mast cells with metachromatic granules in close vicinity to blood vessels (
(
Statistical analysis showed that there were high numbers of mast cells in the control sections of the larynx and significantly fewer mast cells in the peritumoral and tumor regions (
Ultrastructural immunocytochemistry showed for the first time the expression of FPR2/ALX and its co-localization with ANXA1 in mast cells (
Immunolabeling with 10-nm (FPR2/ALX) and 15-nm (ANXA1) colloidal gold particles. Mast cells (
Quantitative analysis revealed that there is higher expression of ANXA1 and its receptor in the cytoplasm and nucleus compared with that in the plasma membrane (
Approximately 87% to 97% of the Hep-2 cells were viable under the experimental conditions (control, ANXA12–26, ANXA12–26+Boc2, Dexa, Dexa+Boc2 and Boc2) (
Treatment with ANXA12–26 reduced the cellular growth. The antagonist Boc2 partially inhibited the anti-proliferative effect of ANXA12–26. The cells treated with Boc2 alone showed a level of proliferation similar to that of the control. The Hep-2 cells were seeded in MEM-Earle medium at a density of 2×106 cells in 75-cm2 culture flask, and then were incubated with serum-free medium 24 hours prior to the addition of ANXA12–26 (1 µM), ANXA12–26 (1 µM)+Boc (10 µM) or Boc (10 µM) alone. All of the experiments were performed in triplicate to confirm the results. Data are expressed as the mean ± SEM of the cell number ×106. **
Treatment with the peptide ANXA12–26 significantly reduced the proliferation of Hep-2 laryngeal cancer cells compared with the control cells (
The epithelial cells of control, peritumoral and tumor laryngeal tissues displayed immunoreactivity to ANXA1 and FPR2/ALX, as well as co-localizations of the proteins, in the plasma membrane, cytoplasm and nucleus (
Immunolabeling with 10-nm (FPR2/ALX) and 15-nm (ANXA1) colloidal gold particles. (
Hep-2 cells that were treated with ANXA12–26 or ANXA12–26 plus Boc2 showed immunoreactivity for ANXA1 and FPR2/ALX (
The expression pattern of IL-6, IL-8 and MCP-1 was similar among the experimental conditions (
Low expression of IL-6 (
To investigate the possible effect of ANXA1 on the expression of genes that are involved in the prostaglandin pathway and, therefore, are related to tumorigenesis and inflammation, we carried out real time PCR in Hep-2 cells that were incubated with ANXA12–26 and Boc2. Prior to RT-PCR, we evaluated the quality of mRNA and cDNA obtained from the samples (
Hep-2 cells that were treated with ANXA12–26 showed a significant decrease (Log2≤−1.0) in
(
Although advances in new diagnostic tools and treatments have led to declining mortality rates, cancer remains a leading cause of death in industrialized countries. A better understanding of the interactions between tumors and inflammation may lead to new biological targets for therapeutic intervention in different neoplasms within the context of chronic inflammation, such as laryngeal carcinoma. We showed that ANXA1 protein may control tumor growth in a paracrine manner that is mediated by the receptor FPR2/ALX. In the inflammatory cells of human laryngeal carcinoma tissue samples, ANXA1/FPR2 expression was markedly exacerbated; however, in laryngeal carcinoma cells, this expression was down-regulated. Treatment with ANXA12–26 reduced the proliferation of Hep-2 cells and up-regulated ANXA1/FPR2 expression in these cells. Our results established, for the first time, the role of FPR2/ALX in laryngeal carcinoma cells as demonstrated by ultrastructural analyses showing the co-localization of ANXA1/FPR2.
The inflammatory components in a developing neoplasm may include several types of immune cells, including mast cells and neutrophils, which can produce an array of cytokines, such as the cell-killing mediators TNF-α and interleukins (ILs)
Mast cells may be effective in inducing neutrophil migration to inflammatory sites
It is now evident that inflammatory cells have powerful effects on tumor development. Early in the neoplastic process, these cells are malignant promoters, producing an attractive environment for tumor growth, facilitating genomic instability and promoting angiogenesis
In this anti-inflammatory and anti-proliferative study, we have unveiled whether ANXA1 protein could be mediated by receptors for formylated peptides (FPRs) in larynx cancer. The functions of FPRs in cancer are still not well established.
We analyzed the role of ANXA1/FPR2 interactions in the proliferation of Hep-2 cells after treatment with ANXA12–26, dexamethasone and/or Boc2. The addition of ANXA12–26 and Dexa to the cell culture reduced cell growth, showing the antiproliferative effects of these anti-inflammatory drugs. However, the pharmacological antagonist Boc2 attenuated the antiproliferative activity of ANXA1, suggesting that this receptor plays central roles in the proliferation response in larynx cancer. These data corroborate the results of a recent study
To uncover the subcellular localization and possible interaction of ANXA1/FPR2, we performed immunocytochemistry reactions in the inflammatory and epithelial cells of laryngeal tissue samples and in Hep-2 cells. Co-localization of ANXA1/FPR2 was detected in the mast cells, neutrophils, and tumor cells. These data suggest that the anti-inflammatory/antiproliferative activity of ANXA1 may be mediated by FPR2/ALX and show, for the first time, the presence of the receptor FPR2/ALX and its interaction with ANXA1 in mast cells and laryngeal tumor cells. Recently, we also detected the co-localization of ANXA1/Fpr2 in murine neutrophils, suggesting that the anti-inflammatory ANXA1 effects may be mediated through this receptor
The mast cells and neutrophils in the peritumoral and tumor sections revealed high expression of ANXA1/FPR2. The tumor-activated inflammatory cells appear to increase the synthesis of ANXA1/FPR2 proteins as an anti-inflammatory response mechanism to resolve the inflammation and proliferation in laryngeal cancer. The cellular differentiation and, in some cases, activation are stimuli for the synthesis of ANXA1, although the molecular processes are not yet fully understood
Loss of ANXA1 and FPR2/ALX expression was detected in laryngeal carcinoma cells. However, Hep-2 cells had up-regulated expression of ANXA1, FPR2/ALX and co-localization after treatment with ANXA12–26 peptide. These results could signify the regulatory role of the ANXA1/FPR2 interaction in cancer development and, corroborate the previous investigation performed in our laboratory
The electron microscopy data demonstrated that ANXA1/FPR2 expression was high in the cytoplasm and nucleus and low in the plasma membrane. ANXA1, which is usually located in the cytoplasm, can translocate to the nucleus after stimulation. Recently, Lin and colleagues
Because of the importance of cytokine control of the inflammatory microenvironment favoring or inhibiting tumor progression
Additionally, we have begun to outline the anti-inflammatory pathway of ANXA1 in Hep-2 cells which may result in the inhibition of angiogenic and invasive phenotypes. Thus, we investigated the effect of ANXA1 on the expression of genes that are involved in prostaglandin signaling and whose role in head and neck cancer deserves evaluation
Prostaglandin E2 is the most important COX metabolite present in human tumors and that bind to its receptors (EP1–4) on the plasma membrane of neoplastic, stromal or immune cells. The EP receptors may have distinct functions depending on the cell and tissue type
Overall, our in vivo data indicated that mast cells and neutrophils, under activation by the tumor microenvironment, demonstrate up-regulation of ANXA1/FPR2 as an anti-inflammatory response mechanism to resolve inflammation and proliferation in laryngeal cancer. However, in vitro results have indicated that treatment with the peptide mimetic ANXA12–26 promotes the reduction of Hep-2 cell proliferation, up-regulation of ANXA1/FPR2 expression, down-regulation of inflammatory cytokines (IL-6, IL-8 and MCP-1) and MMP2.
Integration of the in vivo and in vitro data showed that ANXA1 may be effective in the regulation of tumor growth and metastasis through paracrine mechanisms mediated by FPR2/ALX. Detailed studies of mast cell and neutrophil function in different types of cancer should aid the development of effective antitumor strategies. In light of the potential importance ascribed to ANXA1, our study not only has generated novel and important information about ANXA1 mechanisms but also may justify further investigation of the roles of the complex FPR receptor family. In addition, a combination of anti-inflammatory approaches that target the tumor microenvironment with more sophisticated and selective tumoricidal drugs may lead to future therapeutic intervention in human laryngeal cancer.
Analysis of Hep-2 cell viability. Treatment with ANXA12–26, ANXA12–26+Boc2, Dexa, Dexa+Boc2 or Boc2 did not affect the percentage of cellular viability during growth curve analysis. The 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), ANXA12–26 (1 µM)+Boc2 (10 µM), Dexa (0.01 µM), Dexa (0.01 µM)+Boc2 (10 µM) or Boc2 (10 µM) alone. All experiments were performed in triplicate to confirm the results. Data are expressed as the mean ± SEM of the cell percentage number.
(TIF)
Effect of dexamethasone on the proliferation of Hep-2 cells. Treatment with dexamethasone (Dexa) reduced the cellular growth. The antagonist Boc2 had no effect on Dexa. 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 Dexa (0.01 µM) and Dexa (0.01 µM)+Boc2 (10 µM). All of the experiments were performed in triplicate to confirm the results. Data are expressed as the mean ± SEM of the cell number ×106. **
(TIF)
Validation of mRNA and cDNA integrity. Agarose gels showing the quality of mRNA (A) and cDNA (B) from Hep-2 cells after treatment. 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 were performed in triplicate to confirm the results.
(TIF)
We thank the Special Techniques Laboratory, Hospital Israelita Albert Einstein (LATE-HIAE) for providing and performing the STR analysis. We thank Elena Outon Alonso, for performing the assay and Dr. Roberta Sitnik for data analysis.