Salivary cytokines in cell proliferation and cancer

Clinica Chimica Acta 412 (2011) 1740–1748

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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

Invited critical review

Salivary cytokines in cell proliferation and cancer Mirco Schapher a,⁎, Olaf Wendler a, Michael Gröschl b a b

University of Erlangen-Nuremberg, Department of Otorhinolaryngology, Head and Neck Surgery, Waldstrasse 1, 91054 Erlangen, Germany University of Erlangen-Nuremberg, Department of Pediatrics, Loschgestrasse 15, 91054 Erlangen, Germany

a r t i c l e

i n f o

Article history: Received 11 February 2011 Received in revised form 19 June 2011 Accepted 21 June 2011 Available online 27 June 2011 Keywords: Saliva Cytokines Salivary cytokines Cell proliferation Tumor marker Leptin

a b s t r a c t While the presence of multiple systemic steroids, amines and peptides in saliva has been reported, other hormones of the circulation do not appear in saliva. Substances present within saliva may be classified in different groups: first, those which passively display blood plasma concentrations and constitute a promising alternative to evaluate certain systemic parameters. Second, molecules which seem to play a more active, regulatory role within the upper gastrointestinal tract. Concerning the latter, a growing awareness, especially with regards to salivary peptides has been established. Up to now, understanding the distinct effects of salivary peptides known so far is in its infancy. Various publications, however, emphasize important effects of their presence. Salivary peptides can influence inflammatory processes and cell proliferation in epithelia of the upper digestive tract. These include transforming growth factors (TGFs), epidermal growth factors (EGFs), vascular endothelial growth factors (VEGFs) as well as amines such as melatonin. Of those, candidate cytokines like interleukin 8, tumor necrosis factors (TNFs) and leptin are involved in neoplastic activities of salivary glands and the oral cavity. The exact mechanisms of action are not yet completely understood, but their presence can be utilized for diagnostic purposes. Salivary gland tumors in patients may, in certain circumstances, be identified by saliva diagnostics. Saliva samples of the concerned patients, for instance, reveal significantly higher leptin concentrations than those of healthy individuals. Numerous studies postulate that, beside single indicators, the establishment of salivary hormone profiles may assist clinicians and researchers in detecting tumors and other pathologies of the oral cavity, including adjacent tissues, with high sensitivity and specificity. © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hormones and peptides in human saliva . . . . . . . . . . . . . . . . . . 2.1. Salivary peptides and hormones: origins and influences on concentration 2.2. Physiologic functions of salivary growth factors . . . . . . . . . . . . 2.3. Saliva, ontogeny and cell proliferation . . . . . . . . . . . . . . . . 3. Salivary cytokines in different diseases and tumorigenesis . . . . . . . . . . 3.1. Salivary markers: selected pathologies . . . . . . . . . . . . . . . . 3.2. Tumor marker candidates . . . . . . . . . . . . . . . . . . . . . . 3.3. Salivary leptin is a promising marker in salivary gland tumors . . . . . 3.4. Additional findings and possible explanations . . . . . . . . . . . . . 4. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ⁎ Corresponding author at: University of Erlangen-Nuremberg, Department of Internal Medicine 4, Nephrology, Loschgestrasse 8, 91054 Erlangen, Germany. Tel.: + 49 9131 8539205; fax: + 49 9131 8539202. E-mail address: [email protected] (M. Schapher). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.06.026

While the determination of plasma parameters represents a fundamental method in clinical routine diagnostics, other body fluids are used as auxiliary sources to a much lesser extent. During recent

M. Schapher et al. / Clinica Chimica Acta 412 (2011) 1740–1748

years, the validity of saliva analysis has gained growing attention among basic researchers. Saliva specimen is easy to obtain: fast, noninvasive, stress-free and relatively cheap, compared with blood samples requiring sterile provisions. In particular clinical endocrinologists have been investigating salivary hormones for decades [1–5], especially with regard to their suitability as substitutes for evaluating blood plasma concentrations. Meanwhile, the salivary glands have been proven to be a site of active hormone synthesis with endocrine and exocrine release [6– 21]. The interest in salivary hormones, especially peptides, has shifted to the yearning for understanding their active and diverse mechanisms of action. For some molecules, the impacts within the gastrointestinal tract might be comparable with those already known from the systemic circulation, however, for others they might be different or unique. Conversely, some of the effects discovered in cells exposed to distinct salivary peptides may also prove to be true for other parenchymes or cells exposed to the same peptide within the systemic circulation. Nevertheless, it cannot be denied that current knowledge about the effects of salivary peptides is still fragmentary. This review attempts to provide insights into the function of selected salivary hormones and growth factors released into the oral cavity and the upper gastrointestinal tract. In the first part the available knowledge about the role of the salivary glands as an autonomous endocrine and exocrine tissue is compiled, thus providing an overview on known functions of salivary hormones and growth factors. The second part illustrates recent findings about the contribution of salivary hormones and growth factors to cell proliferation and tumorigenesis. 2. Hormones and peptides in human saliva 2.1. Salivary peptides and hormones: origins and influences on concentration It is well known that salivary steroid hormones, which enter the duct system by diffusion, reflect the non-protein-bound levels of the systemic circulation [1–4]. Several peptides, in contrast, are synthesized autonomously by the salivary glands and reach the glandular lumen by exocytosis [5]. The latter process is both active and selective; consequently, salivary peptide concentrations do not necessarily mirror plasma levels. In addition, several of those peptides which are expressed and secreted by the salivary glands appear in saliva in high concentrations, while others just appear in traces, related to their synthesized amount. Nevertheless, not every peptide detected in saliva is necessarily synthesized by the salivary glands. Peptides such as insulin can also be imported from the blood into salivary gland epithelium cells, followed by secretion into saliva [5,22]. To reveal an autonomous production, appropriate methods are needed to verify mRNA and peptide levels within the gland tissue. Often, but not always, a correlation exists between the expression of specific mRNAs within the salivary glands and a release of the corresponding peptide into saliva. In contrast, insinuations exist that some proteins produced by salivary glands are subject not to exocrine, but to endocrine secretion — as it may apply to leptin produced by white adipocytes of the parotid gland [23]. Furthermore, the individual glands differentially participate in peptide secretion [21], in so far as different parts of the glands or sections of the duct system are responsible for production, secretion and modification of salivary hormones [7,9,10,13–19,21,24–30] (Fig. 1). This secretion and selectivity in hormone modification partially affects the saliva to plasma ratio. EGF, for instance, is present in equal to higher levels in mixed saliva compared with plasma levels [24], while tumor necrosis factor α (TNFα) and leptin appear in lower concentrations [31,32], though all of the aforementioned are produced autonomously by the salivary glands.

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The concentrations of many salivary hormones and peptides follow a circadian rhythm, which influences secretion rates and the composition of saliva. This rhythm is itself controlled by systemic hormones. Moreover, salivary hormone levels underlie external influences like food intake, diet, smoking, alcohol, other drugs affecting salivation, gender, and age [33–43]. The complex salivary hormone framework is consequently susceptible to even small ascendancies, which may result in formidable alterations in saliva composition. To obtain reliable, comparable and reproducible data when analyzing saliva, it is essential to comply with certain standards [44,45] and to take into account at least the listed parameters. 2.2. Physiologic functions of salivary growth factors Little is known about physiologic functions of salivary peptides under healthy conditions — most data is obtained from observations of altered saliva composition under pathological circumstances. Consequently, this chapter briefly describes some selected physiologic properties of salivary peptides; for further reading and overview, please confer to recommended articles [46–50]. First, salivary growth factors and cytokines support the maintenance of mucosal integrity within the gastrointestinal and upper respiratory tract. In particular, the EGF content of saliva has a significant impact on mucosal cell renewal. This impact extends from the oral cavity to the esophagus [51,52], the stomach – where salivary EGF additionally inhibits gastric acid secretion, while its deficiency promotes the development of gastric peptic ulcers [53] – , the small intestine [54] and perhaps even to the colon [55–57]. Olsen and coworkers described an increased incidence of gastric and duodenal ulcers in rats after removal of the submandibular gland, a measure which decreased salivary EGF levels [58]. A mere substitution of salivary EGF clearly reduced lesion incidence and severity. These findings were confirmed in other contexts by Sarosiek, Skov and Kiluk, respectively [59–61]. Moreover, the inactivation of the EGF receptor in mice results in a haemorrhagic enteritis that resembles necrotizing enterocolitis (NEC) [62]. Noteworthy, salivary glands even contribute to the maintenance of systemic EGF levels [19, 21], with the parotid gland being responsible for the largest part of salivary gland derived EGF production. Other salivary gland derived peptides are responsible for mucosal homeostasis [63, 64] in a similar way. Leptin increases the expression and secretion of growth factors in oral epithelia [65] and may act as a kind of upstream regulator for additional growth factors which sustain cell renewal. Salivary VEGF, a multifunctional cytokine, is a useful indicator of oral inflammatory processes [66,67]— it controls reactions in periodontial disease, both during inflammatory states and the healing processes which occur simultaneously. Its concentration is significantly elevated in saliva during inflammatory processes or injuries within the oral cavity, compared with a healthy state [68–70]. Second, many salivary cytokines are capable of controlling and directing immune responses within the oral cavity and adjacent epithelia. Leptin promotes the secretion of defensins [71], which directly act as host defense mechanisms against infective pathogens, and upholds salivary gland mucin synthesis, otherwise impaired by bacterial lipopolysaccharides [72]. Thus, the antibacterial effect of numerous other molecules contained in saliva is preserved. Furthermore, the protective features of leptin seem important for other mucosae, as a study revealed additional antiinfective actions in the respiratory system of mice [64]. A growing number of other small proteins in saliva were shown to assist antimicrobial actions, which is subject to further study. Third, salivary peptides like leptin and ghrelin, which are thought to act partly as antagonists, are part of a control circuit reactive to and presumably influencing carbohydrate uptake. Salivary ghrelin secretion is rapidly suppressed by sugar loads, followed by much slower

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BV WA

SD SA, MEC BV

BV ID Saliva flow

BV

Blood vessel

WA

White adipocyte

SA, MEC

Serous acinus, myoepithelial cell

SD

Striated duct

ID

Intercalated duct

Cross section Passive diffusion Enzymatic steroid conversion

Peptide, produced by salivary glands Peptide, imported from blood plasma Steroid, unconjugated Steroid, conjugated Steroid, after conversion Expression and secretion Active transport

Modification of concentrations Endocrine secretion Fig. 1. Transport mechanisms of hormones within the parotid gland. Salivary peptides are either synthesized and secreted by the salivary glands (e. g. EGF, leptin) or imported from blood plasma through active transport mechanisms (e. g. insulin). Modification of peptide concentrations occurs along the duct system. Unconjugated steroids enter the glands by passive diffusion, where some of them are enzymatically converted before they appear in saliva. Indications exist that some of the cells contribute to endocrine secretion (see text for further details).

rising salivary leptin levels, indicating satiety [73]. It is not yet clear to which extent the salivary concentrations of these hormones influence food intake behavior in comparison with systemic levels. Their autonomous expression and release from the salivary glands as well as the tight regulation of both processes may, however, be an indication that they do have a controlling activity. As a fourth example, structure and remodeling processes in jaws and bones connected to the oral cavity are influenced by molecules present in saliva [74–76]. Melatonin reduces damage due to oxidative stress and stimulates osteoblast activity as well as extracellular matrix synthesis of alveolar bone structures [77–79]. Conversely, systemic bone structure alterations, as in osteoporosis or in juvenile idiopathic arthritis, affect saliva composition [80,81]. 2.3. Saliva, ontogeny and cell proliferation During ontogeny, the emergence and formation of acini [82] as well as the branching of salivary ducts [83,84] is controlled by fibroblastgrowth-factors (FGFs) and members of the bone-morphogenic-proteinfamily, in particular TGF-β3. Although FGFs are expressed within the glands [85–88], most family members could not, or hardly, be detected

in saliva, with the exception of FGF-2. However, receptors for FGF-family members are present in all salivary glands [82,83,86–88] and oral epithelia [5]. The involved interactions between mesenchymal and developing epithelial structures are complex. Certain parallels between salivary gland, kidney or pancreas ontogeny are expected to exist, but knowledge of these alignments is still in its infancy. To what extent salivary peptides are responsible for the development and maintenance of the gland structures is also not yet clear. In adults, some salivary peptides can significantly enhance proliferation of oral mucosal cells, especially EGF, ghrelin [65,89] and leptin [65,84,90–92], the latter being a further stimulus for the secretion of fibroblast-growth-factor-7 (FGF-7). EGF and leptin are expressed and released into saliva by salivary gland tissues [8,93] in conspicuous amounts [31,34,36]. FGF-7 was found to expand the pool of progenitor cells in salivary glands [94]— therefore leptin may also play a role during the development or homeostatic maintenance of salivary gland tissue and its functions. EGF-levels in saliva are higher than those of the systemical blood circulation, and its concentration increases significantly after injury [95,96] or after stimulation with other cytokines [65]. The proliferation stimulating effects [92,97,98] of salivary EGF are clearly dependent on its concentration: a loss of such

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leads to impairment of mucosal homeostasis as described in gastric ulcers, whereas an excess manifests as increased proliferation, visible in gingival overgrowth during ciclosporin medication [99,100]. Wounds within the oral cavity heal much faster than external wounds of the keratinized squamous epithelium [101]. Within mammals, wound licking behavior is known in many species. Tissue healing effects of saliva, even in cutaneous defects [95], have been known since antiquity [102,103] and are now mainly attributed to its peptidergic content. Oudgoff and colleagues published that histatins function as major wound-closing factors in human saliva, augmenting cell division and migration [104]. NGF as with EGF participates in the healing process [94–96,101,105–109]. Numerous other salivary growth factors are involved in the interaction of cytokines during oral mucosal proliferation. Indirectly, the decrease of salivary cytokines during and after radiotherapy of head and neck cancer patients could be the cause for impaired wound healing in the oral cavity and adjacent tissues, due to the fact that the salivary glands are often located within the beam projection [29,68,69,94,105,106,110]. 3. Salivary cytokines in different diseases and tumorigenesis 3.1. Salivary markers: selected pathologies An increasing number of different diseases, including systemic pathologies, can be diagnosed or their development monitored by saliva analysis (Table 1). Deep vein thrombosis [111], chronic heart failure [112] and necrotising enterocolitis [55] as well as the patient's response in multiple sclerosis [113] or hepatitis C therapy [114] belong to this category. Analysis is mostly based on measurement of peptide concentrations, but nucleid acid levels were shown to serve as auxiliary sources for analysis [114,115]. 3.2. Tumor marker candidates Distinct salivary gland derived cytokines display suppressing effects on tumor growth in vitro. Numbers of dysplastic cells in Ehrlich-tumors (a spontaneous breast carcinoma in rodents) were reported to decrease when incubated with salivary gland tissue extracts. Fractions of necrotic or apoptotic tumor cells increased by 30%, leaving healthy cells almost unaffected. However, not all tumor cells were impaired: those expressing EGF or NGF were found to be enriched, suggesting a potential selective anti-tumorous effect or, in all probability, a protective one for cells expressing certain markers [116], conceivably by survival advantage. A promising candidate molecule for this supposed antitumorous effect of saliva has not yet been detected, and further details remain unknown. Although most studies mention elevated concentrations of candidate tumor markers in saliva, this is not true for all of them, as reductions in peptide expression have also been reported. Ghrelin,

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which is presumably involved in oral cell proliferation, was found to be significantly expressed by healthy salivary glands [89], but was absent in salivary gland tumor tissues [117]. To date, there is no satisfying explanation for this discovery. Nonetheless, the majority of studies investigating salivary peptides in tumor patients describe elevated levels of certain candidates. Interestingly, evidence exists that not only local, but also systemic neoplasias can be diagnosed by altered saliva composition. In breast cancer patients, Brooks and coworkers reported that VEGF, EGF and CEA (carcinoembryonic antigen) were significantly elevated in saliva compared with healthy individuals [118]. The combined analysis of salivary VEGF and EGF yielded a sensitivity of 83% and a specificity of 74% to detect the tumor. A similar result was obtained by Navarro et al. [119]. In mice models of melanoma and non-small cell lung cancer (NSCLC), Gao and colleagues recently found that the model tumors changed salivary transcriptomes and consequently salivary biomarker profiles [120]. Likewise findings were published by Michiels and colleagues [121]. In tumors of the salivary glands, the oral cavity or the head and neck, numerous studies addressed altered expression profiles, altered functions and other possibly tumor specific properties within the affected parenchyme, including genetic instability, DNA repair mechanisms, cell cycle progression, apoptosis, levels of extracellular matrix cleaving proteins, intracellular or membrane bound proteins and mRNA expression patterns. Unfortunately, most of these changes require a cytological specimen or biopsy of the diseased organ for diagnostic analysis. All the more important for diagnostic routines, some of the aforementioned cytological modifications result in altered saliva compositions. A great deal of effort is put into the analysis of the salivary proteome. Recently, Scarano and colleagues reported the identification of more than 1400 nonredundant salivary proteins [122], the group of Wong even a number of 1939 [123]. Since 2008, the Sys-BodyFluid database has provided information about proteomes of various body fluids, including saliva [124]. Miller [125], Bandhakavi [126] and many other scientists emphasize the potency of salivary biomarker profiles in identifying and managing various diseases. Especially cytokine levels have been repeatedly proven to be increased in saliva of tumor patients. The best investigated candidates are EGF, interleukin 6 and 8, and to a lesser extent other NFκB-derived members such as TNFα or interleukin 1, further VEGF, bFGF, interleukin 4 and 10, TNFβ, and endothelin [32,110,127–141]. The majority of investigations have targeted EGF. This growth factor not only positively influences cell proliferation, but its levels also positively correlate with the invasive behavior of oral cancer cells [98,142–144]. Overexpression of this cytokine was found to be linked to the development, growth advantage and metastasis of numerous different neoplasias, including the oral cavity and upper digestive tract. Consequently, EGF and its receptors have become important targets in anti tumor therapy, as in breast cancer [145], malignancies

Table 1 Salivary markers for detection and monitoring of selected pathologies. Associated pathology

Salivary marker

Comment

Reference

Chronic heart failure

Endothelin

[112]

Deep vein thrombosis (DVT)

B-thromboglobulin

Necrotizing enterocolitis (NEC)

EGF

Multiple sclerosis (MS)

Soluble Human Leukocyte Antigen II (sHLAII) Hepatitis C Virus RNA

Interleukin 8 (IL-8)

Elevated salivary levels correspond to disease severity according to New York Heart Association Classes. Sensitivity N 88% in diagnosing DVT postoperatively after total hip replacement arthroplasty. Salivary EGF levels are significantly lower in NEC infants. Low EGF in saliva may predict risk for NEC. Significantly higher salivary sHLA-ll levels in MS patients than in controls. Potential marker of therapeutic response to IFN beta-1a. Monitoring and follow up of chronic hepatitis C infection in patients obtaining Interferon alpha 2a treatment. Salivary virus RNA levels decreased during and after successful therapy independently from serum levels. Significantly higher saliva levels in patients with OSCC.

Leptin

Significantly increased saliva levels in patients with parotid gland tumors.

[23]

Chronic Hepatitis C

Oropharyngeal squamous cell carcinoma (OSCC) Salivary gland tumors

[111] [55] [113] [114]

[141]

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of the head and neck [146] and many others [147–149]. Although most of these findings are proven for EGF of the systemic circulation or locally increased EGF levels, elevated salivary EGF concentrations are believed to play a role in the development of oral cancer [127,150]. With regard to salivary glands, levels of EGF receptors are increased in adenoid-cystic-carcinoma (ACC) tissues [151]. Studies related to the use of EGFR targeting drugs in ACC as suitable therapeutics, especially during relapse or metastatic disease, are in progress. In 2004, St. John and colleagues demonstrated salivary IL-8 to be a promising tumor marker, being elevated in saliva samples of individuals suffering from oropharyngeal squamous-cell-carcinoma [141]. IL-8 has repeatedly been linked to tumor growth and metastasis [152]. Recently, we reported salivary leptin as being significantly increased in patients with neoplasms of salivary glands [23]. It is autonomously expressed and secreted by the tumors [8,153] and was shown to enhance expression of EGF and FGF-7 in oral epithelia in vitro[65]. The following section provides further information about leptin's role in salivary gland tumors. 3.3. Salivary leptin is a promising marker in salivary gland tumors Leptin, the product of the ob-gene, is a 146 amino acid comprising 16 kDa type I cytokine. Initially described as an adipocyte derived hormone influencing food intake [154,155] and body weight, there is an increasing awareness that it has an additional influence on reproduction, immunity and carcinogenesis. Leptin receptors, members of the gp130 cytokine receptor family, exist in several isoforms, namely OB-Ra, OB-Rb, OB-Rc, OB-Rd and OB-Rf, which share a common extracellular domain, albeit there exist different cytoplasmatic domains. Signals are intracellularly transduced through the JAK/ STAT pathway. OB-Re is a soluble isoform which contains only the extracellular domain and appears in blood plasma [156]. It is of statistical relevance that obese people more often suffer from malignancies than individuals with a lower body-mass-index (BMI). Consequently, future investigations will have to show whether mechanisms of the leptin/leptin receptor axis play a key role in the development of specific tumors. Moreover, these studies will also have to rule out other confounding factors on tumorigenesis mostly coinciding in obese patients. Many different neoplasias were shown to express or overexpress either leptin and/or its different receptors, which have been related to the development and metastasis in breast, colorectal and endometrial cancer [157–163]. For a growing number of malignancies, leptin and leptin receptors have recently started to be accepted as markers whose expression or presence can be utilized to predict clinical outcomes or therapeutic options [164–167]. Within the gastrointestinal tract, including salivary glands, leptin meets criteria of endocrine and exocrine secretion [168]. Saliva of healthy subjects contains leptin [8,169], which is produced autonomously by the salivary glands in acinar and intralobular duct cells [8,153,170]. Most tumors affecting the salivary glands arise from the parotid gland (Fig. 2). Immunohistochemical and immunofluorescence staining of parotid gland tumors revealed that in all entities studied, both benign and malignant, leptin and its receptors are strongly overexpressed in nearly all areas of the neoplastic tissue. In contrast, their expression is limited to basal parts of the acini and defined ductal structures in healthy glands [23]. Intracellular leptin is located in cytoplasmatic granules, and in polarized tumor cells as in adenolymphomas, shows the highest density in areas between the nucleus and the apical cell pole, presumably depicting its secretion pathway from the endoplasmatic reticulum to the cell surface. In addition to the cytokine itself, the leptin receptor-mRNAsynthesis is likewise strongly enhanced in the tumors. Overexpression ranges from more than 70-fold to 125-fold for Ob-Ra and 40-fold to 70-fold for Ob-Rb. Though mRNA-expression is not equatable with protein expression, immunohistochemical analyses substantiated

Parotid gland

Sublingual gland

Submandibular gland

Fig. 2. Location of the major human salivary glands. Human mixed saliva is mainly produced by the three indicated paired major salivary glands, supplemented by several hundreds of smaller gland structures within the oral cavity. Secretion rates and peptide synthesis of the individual glands are influenced by numerous parameters and to different extents. As a consequence, the flow rate and the composition of saliva are affected by even small conditional changes.

the trend of these results. It is unclear whether an auto- and paracrine mechanism exists in cells overexpressing both the cytokine and its receptor. Due to the fact that salivary gland tumor cell lines are rare and largely inadequately characterized, experiments to investigate the role of leptin and its receptors in salivary glands in vitro are difficult to perform, furthermore the establishment of primary cultures of these tissues is complicated. For cell lines of other origins, proliferation enhancing effects of leptin have been observed [65]. Interestingly, immunoblots revealed that leptin is not only enriched in parotid gland tumor tissues in comparison with healthy glands, but appears to show up as oligomers, which have not been reported for plasma leptin. Within the parenchyme of healthy and tumor salivary gland tissues, the predominant form of the hormone emerges as a dimeric one, but higher oligomers are also present. In contrast, leptin immunoblots obtained from saliva samples revealed that monomers are the most abundant secreted variant, whereas the relative amount of oligomers decreases. It is of note that Cammisotto and colleagues also reported leptin dimers secreted within the gastrointestinal tract [171]. It is unclear whether these oligomers have different physiological functions compared with monomers, or if they constitute a precursor form. One result of augmented leptin production in salivary gland tumor cells is a significantly elevated concentration of leptin in saliva specimen. While healthy patients were found with average salivary leptin concentrations of 125 pg/mL (±36, SD), mean levels rose to 676 pg/mL (±474, SD) in benign neoplasias and even to 880 pg/mL (±618, SD) in carcinomas, implicating a strong p-value of 0.001 on a 5% significance level comparing healthy with overall tumor samples. Unfortunately, this test did not allow significant distinction between benign and malignant tumors. However, although data is limited due to the rarity of this process, there might be an important trend: salivary leptin levels in patients suffering from pleomorphic adenomas are settled in dimensions mentioned for overall benignomas, but the levels further increase in patients in which the tumor processes to malignancy, the so called carcinoma ex pleomorphic adenoma. These patients then exhibit levels reflecting values more associated with overall malignomas. This observation clearly demands more evidence and theories are as yet speculative, but tendentially salivary leptin levels could display proliferation activity, malignant potency and aggressiveness of these tumors.

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Intriguingly, the sizes of the tumors investigated – from small extents in cases of early diagnosis to overwhelming magnitude in advanced stages – did not have a significant impact on salivary leptin levels, an effect which was not expected. As mentioned before, this also may be due to the fact that most tumor entities arise from the duct lining epithelium, and consequently newly originated leptin producing tumor cells could start directly after their emergence with leptin secretion into the lumen. In the growing tumor, additional tumor cells are translocated into the deeper parenchyma more distant from the lumen. As the number of ducts and their surface area does not seem to be enlarged in the tumors, the number of duct lining cells being capable of secreting leptin seems to be limited. This may explain that there is no direct correlation between salivary leptin levels and tumor size, although this underlying theory is speculative. Furthermore, serum leptin concentrations did not significantly influence salivary leptin levels, and therefore did not interfere with the significance in increased salivary leptin concentrations in tumor patients. Correlation of salivary leptin to patients' Body Mass Index (BMI) did not affect the results in tumor or healthy patients, except the finding that it might be possible to distinguish between other nontumorous pathologies. Other parameters such as age, gender or smoking behavior were matched in tumor and control groups of the referred study and did not seem to compromise the significance of increased salivary leptin levels in salivary gland tumor patients. 3.4. Additional findings and possible explanations When leptin mRNA-levels within tumorous salivary glands are analyzed by quantitative realtime-PCR and compared with nontumorous glands, it is surprising that the difference in leptin mRNAexpression is not as noticable as expected after immunohistochemical staining, western blot analysis and measurement of salivary leptin concentrations. A possible explanation might be that the healthy parotid gland in particular contains a lot of white adipocytes. However, these cells do not or rarely appear within the tumor tissue, but are still present within the nontumorous areas of the diseased gland. Interestingly, although these cells express leptin-mRNA in considerable amounts, the number of white fat cells does not or scarcely influence the salivary levels of leptin. In the healthy gland, these adipocytes do not have direct contact to the lumen. Apparently these adipocytes do not excrete leptin into the ductal system and consequently into saliva, but rather contribute to the endocrine secretion of the cytokine — as it may apply to tumor cells which are translocated to the deeper parenchyma within the growing tumor, as described above. Conversely, leptin production of tumor cells is mirrored by an elevated leptin concentration in saliva, presumably because some of these cells are connected to the ductal system. This additional leptin secretion leads to a tremendous rise in saliva levels, whereas leptin produced by adipocytes or deeper parenchymal tumor cells might be subject to endocrine secretion, but in an insufficient quantity to alter systemic leptin concentrations. By quantitative realtime-PCR, no statement can be made about the mRNA-turnover within the cell, which may be very different between adipocytes and tumor cells. Moreover adipocytes are much larger than neoplastic cells of nearly all salivary gland tumor entities. By normalization to housekeeping genes, the difference in cell size and therefore different ratios in mRNA-levels might be lost. As a consequence therefore, a high leptin mRNA production within a limited number of adipocytes could result in an overestimation of the healthy gland's leptin mRNA production. 4. Conclusions and perspectives Finally, more questions remain open than have been answered. Knowledge about the role of salivary hormones concerning their physiological function and their impact on the development of oral

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tumors is fragmentary. Some salivary hormones enter saliva by diffusion, and it is not clear whether or which task they fulfill in the oral cavity and the digestive tract. Others, such as numerous peptides found in saliva, are produced and secreted by salivary glands, and assumptions that they serve distinct functions are conceivable. Several cytokines, in particular members of the EGF-family, interleukines and leptin contribute to oral cell proliferation and enhance the wound healing features of saliva. The effects on cell proliferation have recently been further clarified, albeit incomplete, with regard to possible impacts on tumorigenesis. It has, however, been repeatedly stated that the analysis of salivary cytokine profiles may offer screening potential in the diagnosis of diseases, including cancer. Numerous publications suggest the use of salivary cytokine profiles for diagnosis of oral tumors, and several promising candidates have been reported [23,141,172,173]. One of the most encouraging markers is leptin, and its function is linked with an abundance of relevant metabolic events. The question if leptin actually performs a function or is involved in tumor growth still remains unanswered. However, the mitogenic effect of leptin on oral epithelia [65] allied with additional studies reporting proliferative effects as well as the increased expression of leptin in many different tumor tissues support the hypothesis that its role is important. Salivary leptin elevation is found in patients with salivary gland tumors, the determination of its salivary concentration can assist clinical diagnosis and could therefore enable clinicians to distinguish tumors from other room occupying processes [23]— a finding that needs additional elucidation and confirmation. Further analysis of salivary glands, their metabolism and saliva may provide useful information about mechanisms in immune system modulation, cell metabolism and cell proliferation behavior, including tumorigenesis. Especially during the process of carcinogenesis in vivo, saliva analysis could help researchers to gain new insights. Changes in cell metabolism or expression patterns due to neoplastic activities – which may be missed easily within the systemical circulation due to dilution – could possibly reveal themselves in saliva much earlier, with salivary gland tumors and saliva as a reporting fluid being a possible model for studies about physiologic and pathologic functions of hormones and peptides.

Acknowledgments We thank Stefano Celotti for excellent graphical illustration as well as E. Schoenwasser and C. Telford for linguistic revision.

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