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The role of aryl hydrocarbon receptor (AhR) in the pathology of pleomorphic adenoma in parotid gland
Archives of Oral Biology 61 (2016) 53–59
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The role of aryl hydrocarbon receptor (AhR) in the pathology of pleomorphic adenoma in parotid gland Agnieszka Drozdzika,* , Robert Kowalczykb , Mariusz Lipskic, Joanna Łapczukd , Elzbieta Urasinskae, Mateusz Kurzawskid a
Department of General Dentistry, Pomeranian Medical University, Powstancow Wlkp 72, 70-111 Szczecin, Poland Department of Maxillofacial Surgery, Pomeranian Medical University, Powstancow Wlk 72, 70-111 Szczecin, Poland Department of Preclinical Conservative and Endodontic Dentistry, Powstancow Wlkp 72, 70-111 Szczecin, Poland d Department of Pharmacology, Pomeranian Medical University, Powstancow Wlkp 72, 70-111 Szczecin, Poland e Department of Pathomorphology, Pomeranian Medical University, Unii Lubelskiej 1, 71-252 Szczecin, Poland b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 October 2014 Received in revised form 4 September 2015 Accepted 12 October 2015
Objectives: Pleomorphic adenoma (benign mixed tumor) is one of the most common salivary gland tumors. However, molecular mechanisms implicated in its development are not entirely defined. Therefore, the study aimed at definition of aryl hydrocarbon receptor (AhR) involvement in pleomorphic adenoma pathology, as the AhR controlled gene system was documented to play a role in development of various human tumors. Design: The study was carried out in pleomorphic adenoma and control parotid gland tissues where gene expression of AHR, AhR nuclear translocator (ARNT), AhR repressor (AHRR), as well as AhR controlled genes: CYP1A1 and CYP1B1, at mRNA and protein (immunohistochemistry) levels were studied. Functional evaluation of AhR system was evaluated in HSY cells (human parotid gland adenocarcinoma cells) using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as AhR specific inducer. Results: Pleomorphic adenoma specimens showed cytoplasmic and nuclear AhR expression in epithelial cells as well as in mesenchymal cells. In parotid gland AhR was expressed in cytoplasm of duct cells. Quantitative expression at mRNA level showed significantly higher expression of AHR, ARNT and CYP1B1, and comparable levels of CYP1A1 in pleomorphic adenoma tissue in comparison to healthy parotid gland. The HSY cell study revealed significantly higher expression level of AHRR in HSY as compared with MCF7 cells (human breast adenocarcinoma cell line used as reference). Upon TCDD stimulation a drop in AHRR level in HSY cells and an increase in MCF-7 cells were observed. The HSY and MCF-7 cell proliferation rate (measured by WST-1 test) was not affected by TCDD. Conclusions: Summarizing both in vitro and in vivo observations it can be stated that AhR system may play a role in the pathology of pleomorphic adenoma. ã 2015 Published by Elsevier Ltd.
Keywords: Salivary gland tumor Pleomorphic adenoma AhR
1. Introduction Pleomorphic adenoma (benign mixed tumor) is one of the most frequent salivary gland tumors. Histologically, within pleomorphic adenoma tumor mass both epithelial and mesenchymal elements with a marked morphological diversity are found. It is generally accepted that the adenomas arise from intercalated duct cells with myoepithelial cell differentiation into epithelial and connectivetissue structures (Batsakis, Sneige, & El-Naggar, 1992).
* Corresponding author at: Department of General Dentistry, Pomeranian Medical University, Powstancow Wlkp 72, 70-111 Szczecin, Poland. Fax: +48 91 4661600. E-mail address: [email protected] (A. Drozdzik). http://dx.doi.org/10.1016/j.archoralbio.2015.10.016 0003-9969/ ã 2015 Published by Elsevier Ltd.
Despite numerous studies, pathophysiology of pleomorphic adenomas is still not well defined. Therefore, there is a need for studies on mechanisms associated with tumorigenesis in order to better characterize factors contributing to the development and progression of these tumors. AhR is a transcription factor, which coordinates expression of genes involved mainly in xenobiotics' metabolism (including carcinogens), in response to endo- and exogenous compounds. Ligand binding to AhR triggers its translocation into the nucleus, combined with subsequent heterodimerisation to AhR nuclear translocator (ARNT). AhR/ARNT heterodimer recognizes response elements in regulatory sequences (promoter or enhancer regions) of target genes, and modulates transcription. AhR signaling is regulated by AhR repressor (AHRR) by its competition with AhR for
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ARNT dimerization and binding to AhR-responsive elements (AHREs) (Beischlag, Luis Morales, Hollingshead, & Perdew, 2008; Harper, Riddick, & Okey, 2006; Hahn, Allan, & Sherr, 2009; Murray, Patterson, & Perdew, 2014). The available data, both experimental and clinical, supports an important role of AhR in carcinogenesis. Ahr-D (defective) mouse Hepa1c1c7 cells are not well differentiated and functionally defective. Loss of AhR results also in decreased rate of cell proliferation and an increased number of cells in G0/G1 phase of the cell cycle (Ma & Whitlock, 1996). Characteristics of 967 cancer cell lines for AHR mRNA expression revealed that esophageal, upper respiratory and digestive, pancreatic, and liver cancer cell lines were characterized by relatively high AHR levels, whereas many subtypes of leukemia cells expressed low mRNA levels (Safe, Lee, & Jin, 2013). Therefore, contribution of AhR to carcinogenesis may involve various mechanisms, which are cell type specific. Likewise, clinical studies provide observations that AhR protein expression in pancreatic, prostate, urinary tract, lung, esophageal tumors and papillary thyroid carcinoma (especially with BRAF mutations) is relatively high but not in pituitary tumors. The cellular location of the receptor, which defines its functional state, i.e., cytosolic and/or nuclear, was variable (Safe et al., 2013; Mian et al., 2014). Our previous study revealed expression of AhR in human parotid gland in cytoplasm of striated duct cells (Drozdzik, ska, & Kurzawski, 2013). In a further study we Kowalczyk, Urasin observed regulation of AhR expression and function by its specific inducer, i.e. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat parotid gland (Drozdzik, Wajda, Łapczuk, & Laszczynska, 2014). The AhR expression in human parotid gland as well as its response to ligands suggest its role in the physiology and pathology of the gland. However, there is no information on AhR role (expression, regulation) in pleomorphic adenoma in parotid gland. The aforementioned findings suggesting AhR involvement in tumorigenesis focused our present study on evaluation of AhR involvement in pleomorphic adenoma pathology. 2. Materials and methods 2.1. Tissue specimens Tissue specimens were from 14 patients, aged 49–66 years (8 females, 6 males) diagnosed with pleomorphic adenoma. From each patient a neoplastic tissue as well as tissue from healthy part of the parotid gland were dissected. A part of each specimen sampled was immediately preserved in RNAlater (Applied Biosystems, USA) for RNA expression analysis and the adjacent healthy and neoplastic tissues were embedded in formalin for immunohistochemistry. The study protocol was approved by local ethics committee, and all patients gave informed consent. 2.2. Cell culture A human parotid gland adenocarcinoma cells (HSY) (provided by Dr. M. Sato, Tokushima University, Japan) and human breast adenocarcinoma cell line (MCF-7, reference cell line) cells were seeded in 24-well tissue culture plates, 5 104 per well into DMEM medium (Sigma, Germany), supplemented with 10% FBS (Invitrogen, USA) and 0.4% streptomycin/penicillin (Sigma, Germany), and incubated at 37 C in a humidified incubator supplied with 5% CO2. After 24 h, the medium was replaced with DMEM medium without FBS, containing 0.5% BSA and 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD)—final concentration 10 nmol/l, as well as in control cells a respective medium with DMSO (without TCDD). After the subsequent 24 or 48 h of incubation, the medium was decanted and RNA immediately extracted from cells for
subsequent qRT-PCR analysis, using RNAqueous Micro Kit (Ambion, USA). The experiments were performed in triplicate. 2.3. Cell proliferation/viability assay Cell proliferation was evaluated using water-soluble tetrazolium salt WST-1 assay (Roche Applied Science, Mannheim, Germany). The cell proliferation WST-1 test is based on the reduction of the tetrazolium salt WST-1 to a soluble red-colored formazan by mitochondrial dehydrogenase of metabolically active cells. The amount of formazan dye formed directly correlates with the number of metabolically active cells. For the present study HSY and MCF-7 cells were seeded into a 96-well plate (at a density of 8 103/well and 4 103/well respectively) in DMEM medium (Sigma, Germany) supplemented with 10% FBS (Sigma, Germany), 0.4% penicillin-streptomycin (Sigma, Germany) and L-glutamine (2 mM, Sigma, Germany). Following 24 h of incubation period, cells were treated with TCDD (10 nmol/l) or vehiculum (0.1% DMSO) in DMEM medium for 24 and 48 h. After the respective incubation time, WST-1 reagent was added to each well for 0.5 h. Subsequently, the plate was shaken gently for 1 min, and the absorbance was measured at 450 nm and 620 nm wavelength (reference wavelength, background correction) using a multifunctional microplate reader (Infinite 200 PRO, Tecan, Switzerland). Cell proliferation/viability was calculated using the following equation: % of cell number = [(Atest Ablank)/(Acontrol Ablank)] 100%. 2.4. Quantitative real-time PCR analysis Total RNA was extracted from 20 mg tissue specimen by means of Direct-zol RNA MiniPrep Kit (Zymo Research Corporation, USA). Subsequently cDNA was prepared from 500 ng of total RNA in 20 ml of reaction volume, using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Lithuania) with oligo-dT primers, according to the manufacturer’s instructions. Quantitative expression of the following genes was measured using two-step reverse transcription PCR, using pre-validated Taqman Gene Expression Assays (Life Technologies, USA): AHR (assay ID: Hs00169233_m1), ARNT (Hs00231048_m1), AHRR (Hs00324967_m1) CYP1A1 (Hs00153120_m1) and CYP1B1 (Hs00164383_m1), together with house-keeping endogenous control genes: GAPDH (glyceraldehyde-3-phosphate dehydrogenase, Hs99999905_m1), PPIA (cyclophilin A, Hs99999904_m1) and GUSB (beta-glucuronidase, Hs99999908_m1). qRT-PCR was performed in ViiA7 Real Time PCR System (Life Technologies, USA), with TaqMan Fast Advanced Master Mix (Life Technologies, USA) and 1.5 ml of cDNA for each reaction mix of 15 ml. Each sample was analyzed simultaneously in two technical replicates, and mean CT values were used for further analysis. Calculations were performed using the DDCt relative quantification method, using integrated instrument software (Life Technologies, USA). The thresholds were set manually to compare data between runs, and CT values were extracted. All CT values for each sample were normalized to the geometric mean value obtained for three control genes, processed in the same run. In the present study, each tumor sample was compared to the adjacent normal tissue. The results of relative gene expression for each patient (tumor and normal tissue) were treated as repeated measurements. Fold change between groups was calculated from the means of the logarithmic expression values. 2.5. Immunohistochemical staining Formalin-fixed, paraffin-embedded 5 mm sections from the specimens from healthy parotid gland tissue as well as from pleomorphic adenoma were deparaffinized, rehydrated and immersed in pH 9.0 buffer. Heat-induced antigen retrieval was
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performed in a pressure cooker (Pascal, Dako, Denmark) at 120 C for 3 minutes. Slides were incubated with primary mouse monoclonal anti-AhR antibody (antigen used: synthetic peptide corresponding to amino acid residues 12–31 from the mouse AhR protein with amino acids 18- being omitted; SM5031, Acris Antibodies GmbH, Germany) at 1:200 dilution for 30 minutes at room temperature and immunostained with a Dako Envision+ kit for 30 minutes, AEC+ as a chromogen and hematoxylin as counterstain. Normal mouse immunoglobulins were substituted for primary antibodies as negative controls. A semi-quantitative analysis was performed, with the following grading system: (+++) very strong expression, (++) strong expression, (+) weak expression, () lack expression. 2.6. Statistical analysis Data are shown as mean value SEM. Differences in gene expression between tumorous and normal samples as well as differences in proliferation rate were determined using nonparametric Wilcoxon signed-rank test. Relative expression of the studied genes in cells incubated with TCDD and untreated control cells was compared using Student’s t-test. The level of statistical significance was set at p < 0.05. All calculations were performed using Statistica 10.0 Software Package (Statsoft, Poland). 3. Results Expression of AhR was observed in human parotid salivary gland and pleomorphic adenoma tissue, both at mRNA and protein level (evaluated by immunohistochemistry). In parotid gland AhR was expressed mainly in duct cells, especially in the cytoplasm. Nuclear expression was scarcely seen duct cells. The serous cells were mostly negative for AhR expression (Table 1 and Fig. 2). In pleomorphic adenoma both myoepithelial/epithelial cells as well as mesenchymal cells demonstrated AhR expression, both in cytoplasm and nucleus (Table 1 and Fig. 2). Quantitative expression analysis at mRNA level showed significantly higher expression of AHR, ARNT and CYP1B1, and comparable levels of CYP1A1 in pleomorphic adenoma tissue in comparison to healthy parotid gland (Fig. 1). For AHR, in 50% of tumors (n = 7) mRNA level was elevated (>90th percentile of values obtained in control samples), while it was comparable to normal tissue in the remaining tumors (between 10th and 90th percentile for the control samples). In the case of ARNT, 43% of tumors showed gene overexpression (n = 6), and only 14% (n = 2) in the case of CYP1A1 (respectively, in 58% and 86% of tumor samples AHR and CYP1A1 gene expression was comparable to the control tissues). For CYP1B1, gene overexpression was observed in 79% of tumors (n = 11), 7% (n = 1) showed mRNA levels similar to normal tissue and 14% of tumors (n = 2) presented decreased gene expression, compared to normal tissue (below 10th percentile for control samples). Expressions of AHRR in both pleomorphic adenoma and healthy parotid gland were below quantification limit (Fig. 1).
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The HSY cell study revealed an effect of specific AhR inducer, i.e. TCDD. Significantly higher expression level of AHRR was observed in HSY as compared with MCF-7 cells. Upon TCDD stimulation a drop in AHRR level in HSY cells and an increase in MCF-7 cells were observed. The expression of AHR and ARNT did not differentiated HSY and MCF-7 cells, and were not changed significantly under TCDD treatment (Fig. 3). The effects of TCDD on HSY and MCF-7 cell proliferation/ viability did not reveal an impact of the dioxin on the studied parameters (using WST-1 test). Mitochondrial activity of HSY cells after 24 h and 48 h from initiation of TCDD treatment did not differ statistically from cells incubated with vehiculum-DMSO (the control), and yielded 110% and 101% at the respective time points. Likewise, no statistically significant changes in the number/ viability of MCF-7 cells under TCDD induction were observed. The number of cells treated with TCDD vs. the controls at 24 h and 48 h was as follows: 109% and 120%, respectively (Fig. 4). 4. Discussion The pathology of pleomorphic adenoma is not well defined. It is accepted that the tumor originates from stem cells or a reserve cells of intercalated ducts with further epithelial and mesenchymal cell differentiation. This pool of stem cell or a reserve cell population is a reservoir of cells to maintain morphological and functional integrity or may give an origin for neoplasia. The semipleuripotential bicellular hypothesis for tumor induction explains morphological diversity observed in pleomorphic adenoma (Batsakis et al., 1992). However, the trigger mechanisms and other factors implicated in the tumor progression and development still require definition. AhR was proven to participate in carcinogenesis. Its high expression was demonstrated in a variety of tumors, i.e. pancreatic, prostate, urinary tract, lung and papillary thyroid carcinoma (Safe et al., 2013; Mian et al., 2014). However, cell line studies showed variable AhR levels/responses. AhR knockdown studied revealed that loss of AHR was associated with decreased rate of Ahr-D (defective) mouse Hepa1c1c7 cell proliferation (Ma & Whitlock, 1996), as well as in rat hepatoma cell line (Weiss, Kolluri, Kiefer, & Göttlicher, 1996) or in human HepG2 (Puga et al., 2000) cells (where growth inhibition was recorded). AhR silencing in HN30 head and neck cancer cells decreased IL-6 production, cell migration and proliferation (DiNatale et al., 2012), and reduced T24 urothelial cancer cell invasion (Portal-Nuñez et al., 2012). AHR overexpression studies revealed also in some models that high AhR levels promote carcinogenesis. Mice constitutively expressing AHR (Ca-AHR) rapidly develop stomach cancer (Andersson et al., 2002) and liver tumors (Moennikes et al., 2004). However, AHR overexpression in MCF-7 breast cancer cells inhibited cell growth (Köhle et al., 2002). Those in vitro and in vivo findings point out the need of further definition of the AhR role in carcinogenesis in different tumors. The is no available data on AhR expression in pleomorphic adenoma in salivary glands.
Table 1 Immunolocalization and immunoexpression of AhR in pleomorphic adenoma and parotid gland. Tissue
AhR expression
Parotid gland
Cytosolic nuclear
Pleomorphic adenoma
Cytosolic nuclear
Serous cells
Duct cells
Myoepithelial/epithelial cells +++ Positive
+++/++ /+ (Scarce cells positive) Mesenchymal cells +++ Positive
(+++)—very strong expression, (++)—strong expression, (+)—weak expression, ()—lack of expression.
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Fig. 1. Relative expression of AhR pathway genes in pleomorphic adenoma as compared with healthy salivary gland tissue. Mean values and standard errors are presented, *p < 0.05 for differences between tumor and salivary gland, as evaluated by means of Wilcoxon signed-rank test. AHR—AhR receptor; ARNT—AhR nuclear translocator; CYP1A1—cytochrome P450 1A1; CYP1B1—cytochrome P450 1B1. The AHRR gene expression is not shown (below quantification).
Fig. 2. Immunohistochemical localization and immunoexpression of AhR in human parotid gland (A) and pleomorphic adenoma (B) (magnification 200). (A) Parotid gland: very strong cytosolic and scarce nuclear expression of AhR in duct cells; in serous cells AhR staining is negative. (B) Pleomorphic adenoma: strong positive nuclear and cytosolic AhR expression in moepithelial/epithelial cells as well as mesenchymal cells.
The present study demonstrate an increased expression of AhR in pleomorphic adenoma in comparison to normal parotid gland tissue. Within normal salivary gland high AhR expression levels
were seen in duct cells, that according to Batsakis et al. (1992) gave origin for pleomorphic adenoma. Comparing healthy parotid gland tissue with pleomorphic adenoma, an elevated levels of AHR and ARNT were observed, and they were accompanied by significantly higher expression of AhR-controlled gene CYP1B1 (CYP1A1 expression was similar, but this enzyme is typical for hepatocytes, where it is functionally responsive to AhR ligands). This findings suggest that pleomorphic adenoma is characterized with increased activity of AhR, as it was documented for pancreatic, prostate, urinary tract, lung cancers, papillary thyroid carcinoma, which in turn activates processes involved in carcinogenesis (Safe et al., 2013; Mian et al., 2014). Our previous study has also revealed that AhR in HSY cells responds to TNF-a, which may point out on the potential role of inflammation in recruitment of AhR system (Drozdzik, Dziedziejko, & Kurzawski, 2014). In in vitro studies HSY cells (human parotid gland adenocarcinoma cell line), that have an ultrastructure similar to human salivary intercalated duct cells, were used (Nagamine et al., 1990). As one of hypotheses of pleomorphic adenoma pathology points out on reserve cells of intercalated duct as origin cells of the neoplasia, HSY cells might be used as model cells to study salivary cells pathophysiology, and its role in pleomorphic adenoma development. However, it should also be stated that HSY cells are adenocarcinoma cells, not benign ones. In vitro studies in HSY parotid gland adenocarcinoma cells and in MCF-7 breast cancer cells (characterized by high AhR as well as CYP1A1 and CYP1B1 activity, and used as model cells to study AhR system function, especially dioxin induction) (Taylor, Wang, Hsu, & Hankinson, 2009) demonstrated that the main difference between these cell lines is seen in basal AHRR expression. This finding is in keeping with our previous observations on CYP1A1 and CYP1B1 activities in both cell lines. Higher AhR repressor level
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Fig. 3. Relative expression of AHR,AHRR and ARNT genes in HSY cells as compared with MCF7 breast cancer cell line, incubated in growth medium and treated with dioxin (TCDD, 10 nmol/l, 24 and 48 h). Expression level in non-treated MCF7 cells after 24 hours of incubation was used as reference (100%). *Significant differences (p < 0.05, Student t-test): AHRR expression between HSY and MCF cells in the same time point, HSY TCDD-treated and not-treated cells, and MCF7 TCDD-treated and not-treated cells (only after 24 h of incubation).
in HSY cells was associated with lower CYP1A1 and CYP1B1 activity (Drozdzik, Dziedziejko et al., 2014).
There is general consensus that TCDD is a carcinogen, which is evidenced by experimental findings and clinical observations
Fig. 4. HSY and MCF-7 viability under TCDD exposure for 24 h and 48 h. Viability of the cells cultivated with vehiculum (used to dissolve TCDD) at respective time-points was used as reference (100%). No significant changes were seen.
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(TCDD is classified as a Group I human carcinogen—IARC, 1997. The mechanisms triggered by AhR induction by TCDD depend on cell type, concentration and time of exposure, and are variable in different cells (Safe et al., 2013). Analyzing the TCDD induction results in the studied neoplastic cell lines, HSY and MCF-7, it is evident that dioxin stimulation of AhR system is associated with suppression of AHRR levels in HSY cells, whereas MCF-7 cells responded with AHRR expression increase. So, HSY model, due to a high constitutive expression of AHRR not seen in both normal salivary gland and pleomorphic adenoma tissues, may not reflect clinical situation, and activation of AhR in pleomorphic adenoma seems to be related to the increase in AHR and ARNT levels. But both mechanisms, i.e. decrease in AHRR level (observed in TCDD stimulated HSY cells) and increase in AHR and ARNT levels (observed in pleomorphic adenoma tissue) may lead to activation of AhR system, which in turn can promote cell proliferation and cancer development. However, in vitro observations of Barhoover et al. carried out in MCF-7 cells demonstrated that TCDD at concentration of 10 nmol/l for 24 h disrupted interaction of AhR with cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1), and thus lead to G1 cell cycle arrest (Barhoover, Hall, Greenlee, & Thomas, 2010). The observations of our studies do not support the latter observations as no significant influence of TCDD (10 nmol/l for 24 h and 48 h) was seen on proliferation of both MCF-7 and HSY cells. It should be stated that we used the test based on mitochondrial activity measurements, which indirectly can provide information on the number of cells (directly informs on total mitochondrial activity). The functional observations from the present study are based on neoplastic cells (HSY), and comparative expressions between normal salivary and pleomorphic adenoma tissues. However, there is a paucity of information on AhR expression and function in normal salivary gland, not neoplastic, cells. mRNA expression of AhR in human salivary gland was reported by Nishimura, Naito, Yokoi, and Drozdzik (2004) and Drozdzik et al. (2013), and in rats by Drozdzik, Wajda et al. (2014). Cellular localization in rat and human parotid glands was found to be similar, i.e. predominant AhR expression (defined by immunohistochemistry) in striated duct cells. Functional studies in rats under TCDD stimulation revealed a shift of AhR from cytoplasm to nucleus, accompanied by activation of AhR controlled genes, i.e. CYP1A1 and CYP1B1. These findings may suggest that AhR in salivary gland is functional, and may participate in the gland physiology and pathology. However, AHR expression was significantly lower in rat parotid gland in comparison to liver, most probably due to relatively high expression level of AHRR (Drozdzik, Wajda et al., 2014; Drozdzik, Dziedziejko et al., 2014), as demonstrated in the present study (comparison of HSY and MCF-7 cells). Summarizing both in vitro and in vivo observations from the present study it can be stated that AhR system can play a role in neoplastic transformation in human parotid gland, giving an origin to pleomorphic adenoma. However, other detailed studies are required to verify findings of the present study. Competing interests The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article. Ethical approval The study protocol was approved by local ethics committee at Pomeranian Medical University (reference number KB-0080/22/ 09).
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Archives of Oral Biology journal homepage: www.elsevier.com/locate/aob
The role of aryl hydrocarbon receptor (AhR) in the pathology of pleomorphic adenoma in parotid gland Agnieszka Drozdzika,* , Robert Kowalczykb , Mariusz Lipskic, Joanna Łapczukd , Elzbieta Urasinskae, Mateusz Kurzawskid a
Department of General Dentistry, Pomeranian Medical University, Powstancow Wlkp 72, 70-111 Szczecin, Poland Department of Maxillofacial Surgery, Pomeranian Medical University, Powstancow Wlk 72, 70-111 Szczecin, Poland Department of Preclinical Conservative and Endodontic Dentistry, Powstancow Wlkp 72, 70-111 Szczecin, Poland d Department of Pharmacology, Pomeranian Medical University, Powstancow Wlkp 72, 70-111 Szczecin, Poland e Department of Pathomorphology, Pomeranian Medical University, Unii Lubelskiej 1, 71-252 Szczecin, Poland b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 October 2014 Received in revised form 4 September 2015 Accepted 12 October 2015
Objectives: Pleomorphic adenoma (benign mixed tumor) is one of the most common salivary gland tumors. However, molecular mechanisms implicated in its development are not entirely defined. Therefore, the study aimed at definition of aryl hydrocarbon receptor (AhR) involvement in pleomorphic adenoma pathology, as the AhR controlled gene system was documented to play a role in development of various human tumors. Design: The study was carried out in pleomorphic adenoma and control parotid gland tissues where gene expression of AHR, AhR nuclear translocator (ARNT), AhR repressor (AHRR), as well as AhR controlled genes: CYP1A1 and CYP1B1, at mRNA and protein (immunohistochemistry) levels were studied. Functional evaluation of AhR system was evaluated in HSY cells (human parotid gland adenocarcinoma cells) using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as AhR specific inducer. Results: Pleomorphic adenoma specimens showed cytoplasmic and nuclear AhR expression in epithelial cells as well as in mesenchymal cells. In parotid gland AhR was expressed in cytoplasm of duct cells. Quantitative expression at mRNA level showed significantly higher expression of AHR, ARNT and CYP1B1, and comparable levels of CYP1A1 in pleomorphic adenoma tissue in comparison to healthy parotid gland. The HSY cell study revealed significantly higher expression level of AHRR in HSY as compared with MCF7 cells (human breast adenocarcinoma cell line used as reference). Upon TCDD stimulation a drop in AHRR level in HSY cells and an increase in MCF-7 cells were observed. The HSY and MCF-7 cell proliferation rate (measured by WST-1 test) was not affected by TCDD. Conclusions: Summarizing both in vitro and in vivo observations it can be stated that AhR system may play a role in the pathology of pleomorphic adenoma. ã 2015 Published by Elsevier Ltd.
Keywords: Salivary gland tumor Pleomorphic adenoma AhR
1. Introduction Pleomorphic adenoma (benign mixed tumor) is one of the most frequent salivary gland tumors. Histologically, within pleomorphic adenoma tumor mass both epithelial and mesenchymal elements with a marked morphological diversity are found. It is generally accepted that the adenomas arise from intercalated duct cells with myoepithelial cell differentiation into epithelial and connectivetissue structures (Batsakis, Sneige, & El-Naggar, 1992).
* Corresponding author at: Department of General Dentistry, Pomeranian Medical University, Powstancow Wlkp 72, 70-111 Szczecin, Poland. Fax: +48 91 4661600. E-mail address: [email protected] (A. Drozdzik). http://dx.doi.org/10.1016/j.archoralbio.2015.10.016 0003-9969/ ã 2015 Published by Elsevier Ltd.
Despite numerous studies, pathophysiology of pleomorphic adenomas is still not well defined. Therefore, there is a need for studies on mechanisms associated with tumorigenesis in order to better characterize factors contributing to the development and progression of these tumors. AhR is a transcription factor, which coordinates expression of genes involved mainly in xenobiotics' metabolism (including carcinogens), in response to endo- and exogenous compounds. Ligand binding to AhR triggers its translocation into the nucleus, combined with subsequent heterodimerisation to AhR nuclear translocator (ARNT). AhR/ARNT heterodimer recognizes response elements in regulatory sequences (promoter or enhancer regions) of target genes, and modulates transcription. AhR signaling is regulated by AhR repressor (AHRR) by its competition with AhR for
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ARNT dimerization and binding to AhR-responsive elements (AHREs) (Beischlag, Luis Morales, Hollingshead, & Perdew, 2008; Harper, Riddick, & Okey, 2006; Hahn, Allan, & Sherr, 2009; Murray, Patterson, & Perdew, 2014). The available data, both experimental and clinical, supports an important role of AhR in carcinogenesis. Ahr-D (defective) mouse Hepa1c1c7 cells are not well differentiated and functionally defective. Loss of AhR results also in decreased rate of cell proliferation and an increased number of cells in G0/G1 phase of the cell cycle (Ma & Whitlock, 1996). Characteristics of 967 cancer cell lines for AHR mRNA expression revealed that esophageal, upper respiratory and digestive, pancreatic, and liver cancer cell lines were characterized by relatively high AHR levels, whereas many subtypes of leukemia cells expressed low mRNA levels (Safe, Lee, & Jin, 2013). Therefore, contribution of AhR to carcinogenesis may involve various mechanisms, which are cell type specific. Likewise, clinical studies provide observations that AhR protein expression in pancreatic, prostate, urinary tract, lung, esophageal tumors and papillary thyroid carcinoma (especially with BRAF mutations) is relatively high but not in pituitary tumors. The cellular location of the receptor, which defines its functional state, i.e., cytosolic and/or nuclear, was variable (Safe et al., 2013; Mian et al., 2014). Our previous study revealed expression of AhR in human parotid gland in cytoplasm of striated duct cells (Drozdzik, ska, & Kurzawski, 2013). In a further study we Kowalczyk, Urasin observed regulation of AhR expression and function by its specific inducer, i.e. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat parotid gland (Drozdzik, Wajda, Łapczuk, & Laszczynska, 2014). The AhR expression in human parotid gland as well as its response to ligands suggest its role in the physiology and pathology of the gland. However, there is no information on AhR role (expression, regulation) in pleomorphic adenoma in parotid gland. The aforementioned findings suggesting AhR involvement in tumorigenesis focused our present study on evaluation of AhR involvement in pleomorphic adenoma pathology. 2. Materials and methods 2.1. Tissue specimens Tissue specimens were from 14 patients, aged 49–66 years (8 females, 6 males) diagnosed with pleomorphic adenoma. From each patient a neoplastic tissue as well as tissue from healthy part of the parotid gland were dissected. A part of each specimen sampled was immediately preserved in RNAlater (Applied Biosystems, USA) for RNA expression analysis and the adjacent healthy and neoplastic tissues were embedded in formalin for immunohistochemistry. The study protocol was approved by local ethics committee, and all patients gave informed consent. 2.2. Cell culture A human parotid gland adenocarcinoma cells (HSY) (provided by Dr. M. Sato, Tokushima University, Japan) and human breast adenocarcinoma cell line (MCF-7, reference cell line) cells were seeded in 24-well tissue culture plates, 5 104 per well into DMEM medium (Sigma, Germany), supplemented with 10% FBS (Invitrogen, USA) and 0.4% streptomycin/penicillin (Sigma, Germany), and incubated at 37 C in a humidified incubator supplied with 5% CO2. After 24 h, the medium was replaced with DMEM medium without FBS, containing 0.5% BSA and 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD)—final concentration 10 nmol/l, as well as in control cells a respective medium with DMSO (without TCDD). After the subsequent 24 or 48 h of incubation, the medium was decanted and RNA immediately extracted from cells for
subsequent qRT-PCR analysis, using RNAqueous Micro Kit (Ambion, USA). The experiments were performed in triplicate. 2.3. Cell proliferation/viability assay Cell proliferation was evaluated using water-soluble tetrazolium salt WST-1 assay (Roche Applied Science, Mannheim, Germany). The cell proliferation WST-1 test is based on the reduction of the tetrazolium salt WST-1 to a soluble red-colored formazan by mitochondrial dehydrogenase of metabolically active cells. The amount of formazan dye formed directly correlates with the number of metabolically active cells. For the present study HSY and MCF-7 cells were seeded into a 96-well plate (at a density of 8 103/well and 4 103/well respectively) in DMEM medium (Sigma, Germany) supplemented with 10% FBS (Sigma, Germany), 0.4% penicillin-streptomycin (Sigma, Germany) and L-glutamine (2 mM, Sigma, Germany). Following 24 h of incubation period, cells were treated with TCDD (10 nmol/l) or vehiculum (0.1% DMSO) in DMEM medium for 24 and 48 h. After the respective incubation time, WST-1 reagent was added to each well for 0.5 h. Subsequently, the plate was shaken gently for 1 min, and the absorbance was measured at 450 nm and 620 nm wavelength (reference wavelength, background correction) using a multifunctional microplate reader (Infinite 200 PRO, Tecan, Switzerland). Cell proliferation/viability was calculated using the following equation: % of cell number = [(Atest Ablank)/(Acontrol Ablank)] 100%. 2.4. Quantitative real-time PCR analysis Total RNA was extracted from 20 mg tissue specimen by means of Direct-zol RNA MiniPrep Kit (Zymo Research Corporation, USA). Subsequently cDNA was prepared from 500 ng of total RNA in 20 ml of reaction volume, using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Lithuania) with oligo-dT primers, according to the manufacturer’s instructions. Quantitative expression of the following genes was measured using two-step reverse transcription PCR, using pre-validated Taqman Gene Expression Assays (Life Technologies, USA): AHR (assay ID: Hs00169233_m1), ARNT (Hs00231048_m1), AHRR (Hs00324967_m1) CYP1A1 (Hs00153120_m1) and CYP1B1 (Hs00164383_m1), together with house-keeping endogenous control genes: GAPDH (glyceraldehyde-3-phosphate dehydrogenase, Hs99999905_m1), PPIA (cyclophilin A, Hs99999904_m1) and GUSB (beta-glucuronidase, Hs99999908_m1). qRT-PCR was performed in ViiA7 Real Time PCR System (Life Technologies, USA), with TaqMan Fast Advanced Master Mix (Life Technologies, USA) and 1.5 ml of cDNA for each reaction mix of 15 ml. Each sample was analyzed simultaneously in two technical replicates, and mean CT values were used for further analysis. Calculations were performed using the DDCt relative quantification method, using integrated instrument software (Life Technologies, USA). The thresholds were set manually to compare data between runs, and CT values were extracted. All CT values for each sample were normalized to the geometric mean value obtained for three control genes, processed in the same run. In the present study, each tumor sample was compared to the adjacent normal tissue. The results of relative gene expression for each patient (tumor and normal tissue) were treated as repeated measurements. Fold change between groups was calculated from the means of the logarithmic expression values. 2.5. Immunohistochemical staining Formalin-fixed, paraffin-embedded 5 mm sections from the specimens from healthy parotid gland tissue as well as from pleomorphic adenoma were deparaffinized, rehydrated and immersed in pH 9.0 buffer. Heat-induced antigen retrieval was
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performed in a pressure cooker (Pascal, Dako, Denmark) at 120 C for 3 minutes. Slides were incubated with primary mouse monoclonal anti-AhR antibody (antigen used: synthetic peptide corresponding to amino acid residues 12–31 from the mouse AhR protein with amino acids 18- being omitted; SM5031, Acris Antibodies GmbH, Germany) at 1:200 dilution for 30 minutes at room temperature and immunostained with a Dako Envision+ kit for 30 minutes, AEC+ as a chromogen and hematoxylin as counterstain. Normal mouse immunoglobulins were substituted for primary antibodies as negative controls. A semi-quantitative analysis was performed, with the following grading system: (+++) very strong expression, (++) strong expression, (+) weak expression, () lack expression. 2.6. Statistical analysis Data are shown as mean value SEM. Differences in gene expression between tumorous and normal samples as well as differences in proliferation rate were determined using nonparametric Wilcoxon signed-rank test. Relative expression of the studied genes in cells incubated with TCDD and untreated control cells was compared using Student’s t-test. The level of statistical significance was set at p < 0.05. All calculations were performed using Statistica 10.0 Software Package (Statsoft, Poland). 3. Results Expression of AhR was observed in human parotid salivary gland and pleomorphic adenoma tissue, both at mRNA and protein level (evaluated by immunohistochemistry). In parotid gland AhR was expressed mainly in duct cells, especially in the cytoplasm. Nuclear expression was scarcely seen duct cells. The serous cells were mostly negative for AhR expression (Table 1 and Fig. 2). In pleomorphic adenoma both myoepithelial/epithelial cells as well as mesenchymal cells demonstrated AhR expression, both in cytoplasm and nucleus (Table 1 and Fig. 2). Quantitative expression analysis at mRNA level showed significantly higher expression of AHR, ARNT and CYP1B1, and comparable levels of CYP1A1 in pleomorphic adenoma tissue in comparison to healthy parotid gland (Fig. 1). For AHR, in 50% of tumors (n = 7) mRNA level was elevated (>90th percentile of values obtained in control samples), while it was comparable to normal tissue in the remaining tumors (between 10th and 90th percentile for the control samples). In the case of ARNT, 43% of tumors showed gene overexpression (n = 6), and only 14% (n = 2) in the case of CYP1A1 (respectively, in 58% and 86% of tumor samples AHR and CYP1A1 gene expression was comparable to the control tissues). For CYP1B1, gene overexpression was observed in 79% of tumors (n = 11), 7% (n = 1) showed mRNA levels similar to normal tissue and 14% of tumors (n = 2) presented decreased gene expression, compared to normal tissue (below 10th percentile for control samples). Expressions of AHRR in both pleomorphic adenoma and healthy parotid gland were below quantification limit (Fig. 1).
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The HSY cell study revealed an effect of specific AhR inducer, i.e. TCDD. Significantly higher expression level of AHRR was observed in HSY as compared with MCF-7 cells. Upon TCDD stimulation a drop in AHRR level in HSY cells and an increase in MCF-7 cells were observed. The expression of AHR and ARNT did not differentiated HSY and MCF-7 cells, and were not changed significantly under TCDD treatment (Fig. 3). The effects of TCDD on HSY and MCF-7 cell proliferation/ viability did not reveal an impact of the dioxin on the studied parameters (using WST-1 test). Mitochondrial activity of HSY cells after 24 h and 48 h from initiation of TCDD treatment did not differ statistically from cells incubated with vehiculum-DMSO (the control), and yielded 110% and 101% at the respective time points. Likewise, no statistically significant changes in the number/ viability of MCF-7 cells under TCDD induction were observed. The number of cells treated with TCDD vs. the controls at 24 h and 48 h was as follows: 109% and 120%, respectively (Fig. 4). 4. Discussion The pathology of pleomorphic adenoma is not well defined. It is accepted that the tumor originates from stem cells or a reserve cells of intercalated ducts with further epithelial and mesenchymal cell differentiation. This pool of stem cell or a reserve cell population is a reservoir of cells to maintain morphological and functional integrity or may give an origin for neoplasia. The semipleuripotential bicellular hypothesis for tumor induction explains morphological diversity observed in pleomorphic adenoma (Batsakis et al., 1992). However, the trigger mechanisms and other factors implicated in the tumor progression and development still require definition. AhR was proven to participate in carcinogenesis. Its high expression was demonstrated in a variety of tumors, i.e. pancreatic, prostate, urinary tract, lung and papillary thyroid carcinoma (Safe et al., 2013; Mian et al., 2014). However, cell line studies showed variable AhR levels/responses. AhR knockdown studied revealed that loss of AHR was associated with decreased rate of Ahr-D (defective) mouse Hepa1c1c7 cell proliferation (Ma & Whitlock, 1996), as well as in rat hepatoma cell line (Weiss, Kolluri, Kiefer, & Göttlicher, 1996) or in human HepG2 (Puga et al., 2000) cells (where growth inhibition was recorded). AhR silencing in HN30 head and neck cancer cells decreased IL-6 production, cell migration and proliferation (DiNatale et al., 2012), and reduced T24 urothelial cancer cell invasion (Portal-Nuñez et al., 2012). AHR overexpression studies revealed also in some models that high AhR levels promote carcinogenesis. Mice constitutively expressing AHR (Ca-AHR) rapidly develop stomach cancer (Andersson et al., 2002) and liver tumors (Moennikes et al., 2004). However, AHR overexpression in MCF-7 breast cancer cells inhibited cell growth (Köhle et al., 2002). Those in vitro and in vivo findings point out the need of further definition of the AhR role in carcinogenesis in different tumors. The is no available data on AhR expression in pleomorphic adenoma in salivary glands.
Table 1 Immunolocalization and immunoexpression of AhR in pleomorphic adenoma and parotid gland. Tissue
AhR expression
Parotid gland
Cytosolic nuclear
Pleomorphic adenoma
Cytosolic nuclear
Serous cells
Duct cells
Myoepithelial/epithelial cells +++ Positive
+++/++ /+ (Scarce cells positive) Mesenchymal cells +++ Positive
(+++)—very strong expression, (++)—strong expression, (+)—weak expression, ()—lack of expression.
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Fig. 1. Relative expression of AhR pathway genes in pleomorphic adenoma as compared with healthy salivary gland tissue. Mean values and standard errors are presented, *p < 0.05 for differences between tumor and salivary gland, as evaluated by means of Wilcoxon signed-rank test. AHR—AhR receptor; ARNT—AhR nuclear translocator; CYP1A1—cytochrome P450 1A1; CYP1B1—cytochrome P450 1B1. The AHRR gene expression is not shown (below quantification).
Fig. 2. Immunohistochemical localization and immunoexpression of AhR in human parotid gland (A) and pleomorphic adenoma (B) (magnification 200). (A) Parotid gland: very strong cytosolic and scarce nuclear expression of AhR in duct cells; in serous cells AhR staining is negative. (B) Pleomorphic adenoma: strong positive nuclear and cytosolic AhR expression in moepithelial/epithelial cells as well as mesenchymal cells.
The present study demonstrate an increased expression of AhR in pleomorphic adenoma in comparison to normal parotid gland tissue. Within normal salivary gland high AhR expression levels
were seen in duct cells, that according to Batsakis et al. (1992) gave origin for pleomorphic adenoma. Comparing healthy parotid gland tissue with pleomorphic adenoma, an elevated levels of AHR and ARNT were observed, and they were accompanied by significantly higher expression of AhR-controlled gene CYP1B1 (CYP1A1 expression was similar, but this enzyme is typical for hepatocytes, where it is functionally responsive to AhR ligands). This findings suggest that pleomorphic adenoma is characterized with increased activity of AhR, as it was documented for pancreatic, prostate, urinary tract, lung cancers, papillary thyroid carcinoma, which in turn activates processes involved in carcinogenesis (Safe et al., 2013; Mian et al., 2014). Our previous study has also revealed that AhR in HSY cells responds to TNF-a, which may point out on the potential role of inflammation in recruitment of AhR system (Drozdzik, Dziedziejko, & Kurzawski, 2014). In in vitro studies HSY cells (human parotid gland adenocarcinoma cell line), that have an ultrastructure similar to human salivary intercalated duct cells, were used (Nagamine et al., 1990). As one of hypotheses of pleomorphic adenoma pathology points out on reserve cells of intercalated duct as origin cells of the neoplasia, HSY cells might be used as model cells to study salivary cells pathophysiology, and its role in pleomorphic adenoma development. However, it should also be stated that HSY cells are adenocarcinoma cells, not benign ones. In vitro studies in HSY parotid gland adenocarcinoma cells and in MCF-7 breast cancer cells (characterized by high AhR as well as CYP1A1 and CYP1B1 activity, and used as model cells to study AhR system function, especially dioxin induction) (Taylor, Wang, Hsu, & Hankinson, 2009) demonstrated that the main difference between these cell lines is seen in basal AHRR expression. This finding is in keeping with our previous observations on CYP1A1 and CYP1B1 activities in both cell lines. Higher AhR repressor level
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Fig. 3. Relative expression of AHR,AHRR and ARNT genes in HSY cells as compared with MCF7 breast cancer cell line, incubated in growth medium and treated with dioxin (TCDD, 10 nmol/l, 24 and 48 h). Expression level in non-treated MCF7 cells after 24 hours of incubation was used as reference (100%). *Significant differences (p < 0.05, Student t-test): AHRR expression between HSY and MCF cells in the same time point, HSY TCDD-treated and not-treated cells, and MCF7 TCDD-treated and not-treated cells (only after 24 h of incubation).
in HSY cells was associated with lower CYP1A1 and CYP1B1 activity (Drozdzik, Dziedziejko et al., 2014).
There is general consensus that TCDD is a carcinogen, which is evidenced by experimental findings and clinical observations
Fig. 4. HSY and MCF-7 viability under TCDD exposure for 24 h and 48 h. Viability of the cells cultivated with vehiculum (used to dissolve TCDD) at respective time-points was used as reference (100%). No significant changes were seen.
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(TCDD is classified as a Group I human carcinogen—IARC, 1997. The mechanisms triggered by AhR induction by TCDD depend on cell type, concentration and time of exposure, and are variable in different cells (Safe et al., 2013). Analyzing the TCDD induction results in the studied neoplastic cell lines, HSY and MCF-7, it is evident that dioxin stimulation of AhR system is associated with suppression of AHRR levels in HSY cells, whereas MCF-7 cells responded with AHRR expression increase. So, HSY model, due to a high constitutive expression of AHRR not seen in both normal salivary gland and pleomorphic adenoma tissues, may not reflect clinical situation, and activation of AhR in pleomorphic adenoma seems to be related to the increase in AHR and ARNT levels. But both mechanisms, i.e. decrease in AHRR level (observed in TCDD stimulated HSY cells) and increase in AHR and ARNT levels (observed in pleomorphic adenoma tissue) may lead to activation of AhR system, which in turn can promote cell proliferation and cancer development. However, in vitro observations of Barhoover et al. carried out in MCF-7 cells demonstrated that TCDD at concentration of 10 nmol/l for 24 h disrupted interaction of AhR with cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1), and thus lead to G1 cell cycle arrest (Barhoover, Hall, Greenlee, & Thomas, 2010). The observations of our studies do not support the latter observations as no significant influence of TCDD (10 nmol/l for 24 h and 48 h) was seen on proliferation of both MCF-7 and HSY cells. It should be stated that we used the test based on mitochondrial activity measurements, which indirectly can provide information on the number of cells (directly informs on total mitochondrial activity). The functional observations from the present study are based on neoplastic cells (HSY), and comparative expressions between normal salivary and pleomorphic adenoma tissues. However, there is a paucity of information on AhR expression and function in normal salivary gland, not neoplastic, cells. mRNA expression of AhR in human salivary gland was reported by Nishimura, Naito, Yokoi, and Drozdzik (2004) and Drozdzik et al. (2013), and in rats by Drozdzik, Wajda et al. (2014). Cellular localization in rat and human parotid glands was found to be similar, i.e. predominant AhR expression (defined by immunohistochemistry) in striated duct cells. Functional studies in rats under TCDD stimulation revealed a shift of AhR from cytoplasm to nucleus, accompanied by activation of AhR controlled genes, i.e. CYP1A1 and CYP1B1. These findings may suggest that AhR in salivary gland is functional, and may participate in the gland physiology and pathology. However, AHR expression was significantly lower in rat parotid gland in comparison to liver, most probably due to relatively high expression level of AHRR (Drozdzik, Wajda et al., 2014; Drozdzik, Dziedziejko et al., 2014), as demonstrated in the present study (comparison of HSY and MCF-7 cells). Summarizing both in vitro and in vivo observations from the present study it can be stated that AhR system can play a role in neoplastic transformation in human parotid gland, giving an origin to pleomorphic adenoma. However, other detailed studies are required to verify findings of the present study. Competing interests The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article. Ethical approval The study protocol was approved by local ethics committee at Pomeranian Medical University (reference number KB-0080/22/ 09).
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