AhR was proven to participate

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) irak inhibitor (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. 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-α, 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 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 (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 10nmol/l for 24h 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 (10nmol/l for 24h and 48h) 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).