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Increased risk of oral cancer in diabetic animals is not associated with c-jun activation pathway
ARTICLE IN PRESS Journal of Cranio-Maxillofacial Surgery (2007) 35, 382–387 r 2007 European Association for Cranio-Maxillofacial Surgery doi:10.1016/j.jcms.2007.05.006, available online at http://www.sciencedirect.com
Increased risk of oral cancer in diabetic animals is not associated with c-jun activation pathway Eleftherios VAIRAKTARIS1, Lambros GOUTZANIS1, Georgios KALOKERINOS1, Stavros VASSILIOU1, Sofia SPYRIDONIDOU1, Dimitris AVGOUSTIDIS1, Pashalis STRANTZIAS1, Andreas LAZARIS2, Georgios PAPAGEORGIOU1, Vassilis RAGOS1, Christos YAPIJAKIS1, Efstratios PATSOURIS2 1
Department of Oral and Maxillofacial Surgery (Head: Prof. E. Vairaktaris, MD, PhD, PhD), University of Athens Medical School, Athens, Greece; 2Department of Pathology (Head: Prof. E. Patsouris, MD, PhD), University of Athens Medical School, Athens, Greece
SUMMARY. Purpose: The expression of oncogenic protein c-jun was investigated in an experimental model of chemically induced carcinogenesis in normal and diabetic (type I) Sprague-Dawley rats. Material and methods: Thirteen diabetic and twelve normal rats developed cancer after 4-nitroquinoline-N-oxide treatment, while six diabetic and six normal animals were used as controls. The biopsies were classified pathologically from oral mucosal dysplasia to moderately differentiated oral squamous cell carcinoma (OSCC) and studied immunohistochemically using monoclonal antibody against c-jun protein. Results: Higher c-jun levels were observed in non-cancerous and precancerous stages of normal rats compared with diabetic rats, while in different tumour stages, the expression of c-jun was practically identical for both groups. Conclusion: It seems that diabetes does not affect the c-jun N-terminal kinase (JNK)/c-jun pathway. r 2007 European Association for Cranio-Maxillofacial Surgery
Keywords: c-jun, oral squamous cell carcinoma, immunohistochemistry, histological grade, animal/rats, diabetes
involves insulin receptor substrate-1 (IRS-1) and focal adhesion kinase pp125 (FAK) (Goutzanis et al., 2007). IRS-1 inactivation, which contributes to insulin resistance in diabetic states, is promoted by c-jun N-terminal kinase (JNK) through Ser307 phosphorylation of IRS-1 (Moxham et al., 1996; Miller et al., 1996; Virkamaki et al., 1999; Aguirre et al., 2000). Under diabetic conditions, the JNK activity is abnormally elevated in various tissues due to high glucose levels (Fernyhough et al., 1999; Liu et al., 2000; Purves et al., 2001; Hirosumi et al., 2002). In addition, JNK is stimulated by multiple factors including cytokines (tumour necrosis factor and several interleukins), DNA-damaging agents (drugs and radiation) and environmental stresses (hypoxia, glucose deprivation) (Zayzafoon et al., 2000; Weston and Davis, 2002). The activity of JNK may be monitored by c-jun expression, since the oncogenic protein c-jun is activated directly and exclusively by JNK (Vasilevskaya and O’Dwyer, 2003). c-jun is the main component of the AP-1 transcription factor complex, which regulates the expression of many genes required for the establishment of the malignant phenotype (Angel and Karin, 1991; Vogt, 2001; Hess et al., 2004). Although other Mitogen Activated Protein Kinases (MAPKs) activate various AP-1 components and thus exert their effects on the multitude of genes regulated by this transcription
INTRODUCTION Oral cancer is a common malignancy in the Western world and one of the leading cancers in developing countries (Bsoul et al., 2005). Despite progress in surgical, radiological and chemotherapeutical approaches in the past 20 years, the survival rate as well as the economic and psychological burden of oral cancer has not been improved (Wolff et al., 2004; Hassanein et al., 2005; Kovacs, 2006). Smoking, heavy alcohol consumption and some viruses have been implicated as risk factors for oral squamous cell carcinoma (OSCC), which develops through successive and/or cumulative gene alterations involving the activation of oncogenes and inactivation of tumour suppressor genes (Scully et al., 2000; Llewellyn et al., 2001; Kozomara et al., 2005; Pyo et al., 2007). Research in recent years has been focused on molecular mechanisms of oral carcinogenesis and their clinical relevance as well as prognostic value (Schimming et al., 2004; Hundsdorfer et al., 2005; Derka et al., 2006). Diabetes has been linked with development of premalignant and malignant oral disease in a few epidemiological studies (Dietrich et al., 2004; Ujpal et al., 2004). Recently, a molecular basis for this association was demonstrated by our group in an experimental animal model, indicating that a possible mechanism linked to both diabetes and cancer 382
ARTICLE IN PRESS Increased risk of oral cancer in diabetic animals is not associated with c-jun activation pathway 383
factor, the only MAPK that specifically phosphorylates c-jun is JNK (Vasilevskaya and O’Dwyer, 2003). c-jun is generally considered as an early marker of differentiation but there are very few studies regarding its role in early carcinogenesis (Szabo et al., 1996). Our study, which established involvement of IRS-1 in the molecular basis for the association between oral cancer and type I diabetes, used a rat animal model of sequential stages of oral oncogenesis (Goutzanis et al., 2007). By using the same experimental material, the purpose of the present study was the investigation of c-jun expression in normal oral mucosa tissues and in sequential stages of tumour formation, as a marker of the JNK involvement further delineating the effect of diabetes in oral cancer.
MATERIAL AND METHODS Animal model of induced diabetes and oral cancer The experimental animal model of oral carcinogenesis in diabetic and normal control rats has been previously described in detail (Goutzanis et al., 2007). Briefly, it involved 37 female Sprague-Dawley rats purchased from the Hellenic Pasteur Institute (Athens) at the age of 6 weeks and weighting approximately 135 g each. The rats were handled in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). The animals were randomly allocated to four groups. Group D (n ¼ 6): Diabetic rats without carcinogenesis, Group Dc (n ¼ 13): Diabetic rats used for induced carcinogenesis, Group N (n ¼ 6): Normal rats without carcinogenesis, Group Nc (n ¼ 12): Normal rats used for induced carcinogenesis. The induction of diabetes was performed in 19 rats previously overnight fasted animals by a single intraperitoneal injection of streptozotocin (STZ) dissolved in saline buffer at a dose of 70 mg/kg of weight (ZANOSAR, Pharmacia & Upjohn Co., USA) and determined by blood glucose levels after 3 weeks, as previously described (Goutzanis et al., 2007). After confirmation of diabetes, oral cancer was induced in Dc and Nc animals by application of the carcinogen 4-nitroquinoline N-oxide (4NQO) at a concentration of 5% in propylene glycol 3 times per week for 5 months to the rats’ hard palate (Fluca AG Chemische Fabrik, Switzerland), as previously described (Goutzanis et al., 2007). Clinical signs of oral lesions putatively tumour-related were observed within 6 months after the last application of carcinogen. After sacrifice of the animal using ether, the oral regions with cancer (mainly palate and tongue) of Dc and Nc rats and the respective regions of D and N rats were excised for immunohistochemical analysis (Goutzanis et al., 2007).
Pathological evaluation The histological status of the lesions was defined after examination of the complete section under light microscopy and the tissue profiles were classified in the following categories: normal mucosa, hyperplasia, dysplasia, early invasion, well- and moderately differentiated carcinoma. In every sample all possible different lesions were evaluated. Immunohistochemical analysis Surgical specimens were fixed in 10% neutralized formaldehyde solution and embedded in paraffin. Three sections of 4 mm were prepared from each specimen and were mounted on Super Frost Pluscoated glass slides (Menzel and Co., Braunschweig, Germany). One section was stained with haematoxylin and eosin for routine histological evaluation, while the other two were used for immunohistochemical detection of c-jun protein as previously described (Derka et al., 2006) with monoclonal primary antibodies against c-jun (H-79: sc-1694, Santa Cruz Biotechnology, Inc., diluted 1:100). Prostate carcinoma which strongly expresses c-jun was used as a positive control. Negative controls for each biopsy were processed in the same manner, using PBS instead of the primary antibody. All slides were independently and blindly reviewed by two investigators. The consecutive haematoxylin–eosinstained slides were evaluated by a pathologist experienced in oral pathology, without knowing the c-jun staining patterns. Statistical analysis Statistical analyses were performed using the twotailed Student’s t-test for each group of animals and each histological category. The percentage of positively stained cells from each non-cancerous or precancerous condition (hyperplasia, dysplasia) were compared with normal tissue, while the percentage of positively stained cells from each tumour (early invasion, well- and moderately differentiated carcinoma) were compared with the mean value of percentages of the precancerous conditions. In all cases with no normal distribution, the results of both the Wilcoxon test and two-tailed student’s t-test provided the same level of significance.
RESULTS The histological status of biopsies observed from various regions of rats of all the groups (N, Nc, D, Dc) after induced oral oncogenesis is summarized in Table 1. As previously described, there were no significant differences in histological status of oral cancer biopsies obtained from diabetic and normal rats after carcinogen treatment for a prolonged period of 5 months (Goutzanis et al., 2007).
ARTICLE IN PRESS 384 Journal of Cranio-Maxillofacial Surgery
The experimental model allowed the collection of several normal, non-cancerous, precancerous and cancerous regions in the various tissue biopsies and further analysis of immunostaining data was implemented (Fig. 1). In all cases with no normal distribution, the results of both the Wilcoxon test and two-tailed student’s t-test provided the same level of significance. The percentages of positive expression of c-jun, in the various histological categories for non-diabetic animals from groups N and Nc are shown in Table 2. Statistical analysis revealed a significant increase in c-jun levels in oral mucosal dysplasia when compared with normal mucosa (Po0.05). No statistically significant differences were observed when c-jun expression in early invasive and well-differentiated carcinomas was compared with the mean value of percentages of non-cancerous and precancerous conditions, indicating an almost stable pattern of expression of c-jun in initial stages of oral oncogenesis. Nevertheless, in moderately differentiated OSCC a significant decrease in c-jun levels was noticed (Po0.05). Table 3 summarizes the percentages of positively stained cells for c-jun in the various categories of
histological status for diabetic animals from groups D and Dc. During the non-cancerous and precancerous stages, c-jun was elevated significantly compared with normal oral mucosa. In addition, the comparison of c-jun percentages between the tumour stages and the mean value of percentages of non-cancerous and precancerous conditions revealed a statistically significant increase for early invasion and welldifferentiated OSCC, as well as a non-significant decrease in the case of moderately differentiated OSCC. Fig. 2 illustrates the compared expression of c-jun in the different tissue categories in normal and diabetic rats. From normal mucosa to dysplasia, c-jun expression displays higher levels in normal rats when compared with diabetic rats, while in different tumour stages, surprisingly, the expression of c-jun is practically identical.
DISCUSSION In accordance with previous epidemiological studies in humans which implicated diabetes mellitus as a
Table 1 – Histological status of biopsies in groups N, D, Nc and Dc
Normal tissue Hyperplasia Dysplasia Early invasion Well-differentiated carcinoma Moderately differentiated carcinoma
Control group (N)
Control group (D)
3 3
5 1
Experimental group (Nc)
Experimental group (Dc)
1 1 6 4
1 4 1 4 3
Fig. 1 – (A) Minimal c-jun immunoreactivity in normal oral mucosa of normal rats ( 400), (B) Intense nuclear immunostaining in dysplastic nuclei in normal rats ( 400), (C) Evident c-jun immunoreaction in an area of early invasion in diabetic rats ( 200), (D) Weak c-jun immunostaining in a moderately differentiated oral carcinoma in diabetic rats ( 400).
ARTICLE IN PRESS Increased risk of oral cancer in diabetic animals is not associated with c-jun activation pathway 385 Table 2 – Percentages of c-jun positive cells in the various categories of tissue status for normal rats (N and Nc) Non-cancerous and precancerous
c-jun Mean of percentages Probability of ttest
Tumour
Normal oral mucosa
Oral mucosal hyperplasia
Oral mucosal dysplasia
Early invasion
Welldifferentiated OSCC
Moderately differentiated OSCC
2.25 (N ¼ 4)
4 (N ¼ 9)
5 (N ¼ 4)
4.25 (N ¼ 4)
3.67 (N ¼ 6)
2.25 (N ¼ 4)
N.S.a
Po0.05
N.S.a
N.S.a
Po0.05
Comparisons of percentages were made between each non- cancerous or precancerous condition with normal tissue, and from each tumour stage with the mean value of percentages of the non-cancerous and precancerous conditions. a N.S: No statistical difference. Table 3 – Percentages of c-jun positive cells in the various categories of tissue status for diabetic rats (D and Dc) Non-cancerous and precancerous
c-jun Mean of percentages Probability of ttest
Tumor
Normal oral mucosa
Oral mucosal hyperplasia
Oral mucosal dysplasia
Early invasion
Well differentiated OSCC
Moderately differentiated OSCC
0.27 (N ¼ 22)
1.15 (N ¼ 33)
2.8 (N ¼ 21)
4.25 (N ¼ 4)
3.65 (N ¼ 23)
2.18 (N ¼ 11)
Po0.01
Po0.001
Po0.05
Po0.01
N.S.a
Comparisons of percentages were made between each non- cancerous or precancerous condition with normal tissue, and from each tumour stage with the mean value of percentages of the non-cancerous and precancerous conditions. a N.S: No statistical difference.
6 c-jun D 5
c-jun N
4 3 2 1 0 Normal
Hyperplasia
Dysplasia
Early invasion
Well Moderately differentiated differentiated
Fig. 2 – Expression of c-jun in the different tissue categories in normal (N) and diabetic (D) rats.
risk factor for the development of OSCC, a previous experimental study by our group established that association through the IRS-1 pathway (Dietrich et al., 2004; Ujpal et al., 2004; Goutzanis et al., 2007). In the present study, c-jun expression was investigated using the same experimental system of chemically induced oral cancer in diabetic and normal rats, in order to further delineate the molecular pathway linking diabetes and oral cancer. The present study is, to our knowledge, the first one investigating the expression of c-jun in an
experimental animal system of chemically induced oral carcinogenesis both in non-diabetic and diabetic rats. The c-jun expression was investigated in sequential stages of oral oncogenesis, varying from normal oral mucosa to moderately differentiated oral carcinomas, obtained from normal and diabetic rats. A similar pattern of c-jun expression was observed for both diabetic and non-diabetic rats, which consists of increased levels of c-jun during the initial stages of oral oncogenesis, and decreased levels in moderately differentiated OSCC. Higher c-jun levels
ARTICLE IN PRESS 386 Journal of Cranio-Maxillofacial Surgery
were observed in non-cancerous and precancerous stages of normal rats compared with diabetic rats, while in various tumour stages, the expression of c-jun was practically identical for both animal groups. It has been reported that the activity of c-jun N-terminal kinase (JNK) and subsequently the activity of c-jun, is abnormally elevated in various tissues under diabetic conditions (Fernyhough et al., 1999; Liu et al., 2000; Purves et al., 2001; Hirosumi et al., 2002). It has been documented that JNK is activated in sensory neurons and pulmonary artery endothelium, due to high glucose levels observed in type I diabetes (Liu et al., 2000; Purves et al., 2001). In these tissues, activation of the JNK pathway as seen under diabetic conditions increases insulin resistance, and in contrast, suppression of the JNK pathway decreases insulin resistance and markedly improves glucose tolerance in diabetic animal models (Purves et al., 2001). No previous data exists in the literature concerning c-jun expression in oral tissues under diabetic conditions. In the light of this, the present study resulted in unexpectedly diverse observations. The observed increase of c-jun expression in non-cancerous and precancerous stages of non-diabetic rats compared with the equivalent ones in diabetic animals may be attributed to physiological differences of oral tissues in comparison with previously studied tissues. Insulin-deficient states (such as diabetes type I) result in increased numbers of insulin receptors, increased phosphorylation of IRS-1 and increased IRS-1-associated PI 3-kinase activity (Kondo and Kahn, 2004). On the other hand, IRS-1 inactivation, which contributes to insulin resistance in diabetes II, is promoted by c-jun N-terminal kinase (JNK) through Ser307 phosphorylation of IRS-1 (Moxham et al., 1996; Miller et al., 1996; Virkamaki et al., 1999; Aguirre et al., 2000). A previous study by our group using the same experimental system observed that the expression of IRS-1 was significantly higher in diabetic than in normal animals (Goutzanis et al., 2007). This significant elevation of IRS-1 may be attributed to lack of IRS-1 inactivation by reduced activity of JNK. Decreased JNK protein levels are also responsible for the reduction of c-jun protein levels in diabetic animals (Vasilevskaya and O’Dwyer, 2003). The latter was observed in the present study, in which lower c-jun levels were detected in diabetic compared with normal animals during early stages of oral oncogenesis. Insulin signal transduction is mediated by a series of phosphorylation cascades linking initial activation of the insulin receptor (IR) to downstream substrates. Activation of the receptor for insulin results in tyrosine phosphorylation of IRS-1, which binds Grb2/SOS complex mediating the activation of N-ras, thereby activating the ras/raf/MAP kinase cascade (White, 2002). These data were linked with a comparison between the findings of the present study and those of a previous report by our group
investigating N-ras/Ets-1 expression in the same experimental system (Vairaktaris et al., 2007). In diabetic animals, N-ras was found to be significantly increased in the last stages of oral cancer in contrast to c-jun expression which was found to be decreased in the same stages. It seems that the ras/raf/ MAP kinase plays an important role in diabetic states while the JNK/c-jun pathway may not be affected by diabetes.
CONCLUSIONS The findings of the present study suggest that diabetes does not affect the c-jun N-terminal kinase (JNK)/c-jun pathway during early and advanced tumour stages of oral carcinogenesis. The rat model of oral oncogenesis is a very useful experimental animal model for studying the effect of diabetes on various mechanisms and stages towards establishment of oral squamous cell carcinoma. ACKNOWLEDGEMENTS
Source of support: This work was co-funded by the European Social Fund and National Resources (EPEAEK II ‘‘Pythagoras’’ 70/3/7391) grant to E.V.
References Aguirre V, Uchida T, Yenush L, Davis R, Whitei MF: The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J Biol Chem 275: 9047–9054, 2000 Angel P, Karin M: The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1072: 129–157, 1991 Bsoul SA, Huber MA, Terezhalmy GT: Squamous cell carcinoma of the oral tissues: A comprehensive review for oral healthcare providers. J Contemp Dent Pract (6): 1–16, 2005 Derka S, Vairaktaris E, Papakosta V, Vassiliou S, Acil Y, Vylliotis A, Spyridonidou S, Lazaris AC, Mourouzis C, Kokkori A, Moulavasili P, Perrea D, Donta I, Yapijakis C, Patsouris E: Cell proliferation and apoptosis culminate in early stages of oral oncogenesis. Oral Oncol 42 (5): 540–550, 2006 Dietrich T, Reichart PA, Scheifele C: Clinical risk factors of oral leukoplakia in a representative sample of the US population. Oral Oncol 40: 158–163, 2004 Fernyhough P, Gallagher A, Averill SA, Priestley JV, Hounsom L, Patel J, Tomlinson DR: Aberrant neurofilament phosphorylation in sensory neurons of rats with diabetic neuropathy. Diabetes 48: 881–889, 1999 Goutzanis L, Vairaktaris E, Yapijakis C, Kavantzas N, Nkenke E, Derka S, Vassiliou S, Acil Y, Kessler P, Stavrianeas N, Perrea D, Donta I, Patsouris E: Diabetes may increase risk for oral cancer through the insulin receptor substrate-1 and focal adhesion kinase pathway. Oral Oncol 43: 165–173, 2007 Hassanein KA, Musgrove BT, Bradbury E: Psychological outcome of patients following treatment of oral cancer and its relation with functional status and coping mechanisms. J Craniomaxillofac Surg 33: 404–409, 2005 Hess J, Angel P, Schorpp-Kistner M: AP-1 subunits: quarrel and harmony among siblings. J Cell Sci 117: 5965–5973, 2004 Hirosumi J, Tuncman G, Chang L, Gorgun CZ, Uysal KT, Maeda K, Karin M, Hotamisligil GS: A central role for JNK in obesity and insulin resistance. Nature 420: 333–336, 2002
ARTICLE IN PRESS Increased risk of oral cancer in diabetic animals is not associated with c-jun activation pathway 387 Hundsdorfer B, Zeilhofer HF, Bock KP, Dettmar P, Schmitt M, Kolk A, Pautke C, Horch HH: Tumour-associated urokinasetype plasminogen activator (uPA) and its inhibitor PAI-1 in normal and neoplastic tissues of patients with squamous cell cancer of the oral cavity – clinical relevance and prognostic value. J Craniomaxillofac Surg 33: 191–196, 2005 Kondo T, Kahn CR: Altered insulin signaling in retinal tissue in diabetic states. J Biol Chem 279: 37997–38006, 2004 Kovacs AF: Maximized combined modality treatment of an unselected population of oral and oropharyngeal cancer patients. Final results of a pilot study compared with a treatment-dependent prognosis index. J Craniomaxillofac Surg 34: 74–84, 2006 Kozomara R, Jovic N, Magic Z, Brankovic-Magic M, Minic V: p53 mutations and human papillomavirus infection in oral squamous cell carcinomas: correlation with overall survival. J Craniomaxillofac Surg 33: 342–348, 2005 Liu W, Schoenkerman A, Lowe Jr. WL: Activation of members of the mitogen-activated protein kinase family by glucose in endothelial cells. Am J Physiol 279: 782–790, 2000 Llewellyn CD, Johnson NW, Warnakulasuriya KAAS: Risk factors for squamous cell carcinoma of the oral cavity in young people – a comprehensive literature review. Oral Oncol 37: 401–418, 2001 Miller BS, Shankavaram UT, Horney MJ, Gore ACS, Kurtz DT, Rosenzweig SA: Activation of cJun NH2-terminal kinase/stressactivated protein kinase by insulin. Biochemistry 35: 8769–8775, 1996 Moxham CM, Tabrizchi A, Davis RJ, Malbon CC: Jun N-terminal kinase mediates activation of skeletal muscle glycogen synthase by insulin in vivo. J Biol Chem 271: 30765–30773, 1996 Purves T, Middlemas A, Agthong S, Jude EB, Boulton AJ, Fernyhough P, Tomlinson DR: A role for mitogen-activated protein kinases in the etiology of diabetic neuropathy. FASEB J 15: 2508–2514, 2001 Pyo SW, Hashimoto M, Kim YS, Kim CH, Lee SH, Johnson KR, Wheelock MJ, Park JU: Expression of E-cadherin, P-cadherin and N-cadherin in oral squamous cell carcinoma: correlation with the clinicopathologic features and patient outcome. J Craniomaxillofac Surg 35: 1–9, 2007 Schimming R, Reusch P, Kuschnierz J, Schmelzeisen R: Angiogenic factors in squamous cell carcinoma of the oral cavity: do they have prognostic relevance? J Craniomaxillofac Surg 32: 176–181, 2004 Scully C, Field JK, Tanzawa H: Genetic aberrations in oral and head and neck squamous cell carcinoma (SCCHN) 1: carcinogen metabolism, DNA repair and cell cycle control. Oral Oncol 36: 256–263, 2000
Szabo E, Riffe ME, Steinberg SM, Birrer MJ, Linnoila RI: Altered c-jun expression:an early event in human lung carcinogenesis. Cancer Res 56: 305–315, 1996 Ujpal M, Matos O, Bibok G, Somogyi A, Szabo G, Suba Z: Diabetes and oral tumors in Hungary. Epidemiological correlations. Diabetes Care 27: 770–774, 2004 Vairaktaris E, Goutzanis L, Kalokerinos G, Yannopoulos A, Yapijakis C, Derka S, Vassiliou S, Spyridonidou S, Vylliotis A, Nkenke E, Papageorgiou G, Lazaris AC, Tesseromatis C, Tsigris C, Patsouris E: Diabetes increases both N-ras and ets-1 expression during oral oncogenesis resulting in enhanced cell proliferation and metastatic potential. In Vivo 21: 615–621, 2007 Vasilevskaya I, O’Dwyer PJ: Role of Jun and Jun kinase in resistance of cancer cells to therapy. Drug Resist Updat 6: 147–156, 2003 Virkamaki A, Ueki K, Kahn CR: Protein–protein interaction in insulin signalling and the molecular mechanisms of insulin resistance. J Clin Invest 103: 931–943, 1999 Vogt PK: Jun, the oncoprotein. Oncogene 20: 2365–2377, 2001 Weston CR, Davis RJ: The JNK signal transduction pathway. Curr Opin Genet Dev 12: 14–21, 2002 White MF: IRS proteins and the common path to diabetes. Am J Physiol Endocrinol Metab 283: 413–422, 2002 Wolff D, Hassfeld S, Hofele C: Influence of marginal and segmental mandibular resection on the survival rate in patients with squamous cell carcinoma of the inferior parts of the oral cavity. J Craniomaxillofac Surg 32: 318–323, 2004 Zayzafoon M, Stell C, Irwin R, McCabe LR: Extracellular glucose influences osteoblast differentiation and c-jun expression. J Cell Biochem 79: 301–310, 2000
Prof. Dr. Eleftherios VAIRAKTARIS, MD, DDS, PhD, PhD. Department of Oral and Maxillofacial Surgery University of Athens Medical School ‘‘Attikon’’ Hospital Rimini 1 Athens 12462 Greece Tel.: +30 210 6443035 Fax: +30 210 6443803 E-mail: [email protected] Paper received 7 September 2006 Accepted 18 May 2007
Increased risk of oral cancer in diabetic animals is not associated with c-jun activation pathway Eleftherios VAIRAKTARIS1, Lambros GOUTZANIS1, Georgios KALOKERINOS1, Stavros VASSILIOU1, Sofia SPYRIDONIDOU1, Dimitris AVGOUSTIDIS1, Pashalis STRANTZIAS1, Andreas LAZARIS2, Georgios PAPAGEORGIOU1, Vassilis RAGOS1, Christos YAPIJAKIS1, Efstratios PATSOURIS2 1
Department of Oral and Maxillofacial Surgery (Head: Prof. E. Vairaktaris, MD, PhD, PhD), University of Athens Medical School, Athens, Greece; 2Department of Pathology (Head: Prof. E. Patsouris, MD, PhD), University of Athens Medical School, Athens, Greece
SUMMARY. Purpose: The expression of oncogenic protein c-jun was investigated in an experimental model of chemically induced carcinogenesis in normal and diabetic (type I) Sprague-Dawley rats. Material and methods: Thirteen diabetic and twelve normal rats developed cancer after 4-nitroquinoline-N-oxide treatment, while six diabetic and six normal animals were used as controls. The biopsies were classified pathologically from oral mucosal dysplasia to moderately differentiated oral squamous cell carcinoma (OSCC) and studied immunohistochemically using monoclonal antibody against c-jun protein. Results: Higher c-jun levels were observed in non-cancerous and precancerous stages of normal rats compared with diabetic rats, while in different tumour stages, the expression of c-jun was practically identical for both groups. Conclusion: It seems that diabetes does not affect the c-jun N-terminal kinase (JNK)/c-jun pathway. r 2007 European Association for Cranio-Maxillofacial Surgery
Keywords: c-jun, oral squamous cell carcinoma, immunohistochemistry, histological grade, animal/rats, diabetes
involves insulin receptor substrate-1 (IRS-1) and focal adhesion kinase pp125 (FAK) (Goutzanis et al., 2007). IRS-1 inactivation, which contributes to insulin resistance in diabetic states, is promoted by c-jun N-terminal kinase (JNK) through Ser307 phosphorylation of IRS-1 (Moxham et al., 1996; Miller et al., 1996; Virkamaki et al., 1999; Aguirre et al., 2000). Under diabetic conditions, the JNK activity is abnormally elevated in various tissues due to high glucose levels (Fernyhough et al., 1999; Liu et al., 2000; Purves et al., 2001; Hirosumi et al., 2002). In addition, JNK is stimulated by multiple factors including cytokines (tumour necrosis factor and several interleukins), DNA-damaging agents (drugs and radiation) and environmental stresses (hypoxia, glucose deprivation) (Zayzafoon et al., 2000; Weston and Davis, 2002). The activity of JNK may be monitored by c-jun expression, since the oncogenic protein c-jun is activated directly and exclusively by JNK (Vasilevskaya and O’Dwyer, 2003). c-jun is the main component of the AP-1 transcription factor complex, which regulates the expression of many genes required for the establishment of the malignant phenotype (Angel and Karin, 1991; Vogt, 2001; Hess et al., 2004). Although other Mitogen Activated Protein Kinases (MAPKs) activate various AP-1 components and thus exert their effects on the multitude of genes regulated by this transcription
INTRODUCTION Oral cancer is a common malignancy in the Western world and one of the leading cancers in developing countries (Bsoul et al., 2005). Despite progress in surgical, radiological and chemotherapeutical approaches in the past 20 years, the survival rate as well as the economic and psychological burden of oral cancer has not been improved (Wolff et al., 2004; Hassanein et al., 2005; Kovacs, 2006). Smoking, heavy alcohol consumption and some viruses have been implicated as risk factors for oral squamous cell carcinoma (OSCC), which develops through successive and/or cumulative gene alterations involving the activation of oncogenes and inactivation of tumour suppressor genes (Scully et al., 2000; Llewellyn et al., 2001; Kozomara et al., 2005; Pyo et al., 2007). Research in recent years has been focused on molecular mechanisms of oral carcinogenesis and their clinical relevance as well as prognostic value (Schimming et al., 2004; Hundsdorfer et al., 2005; Derka et al., 2006). Diabetes has been linked with development of premalignant and malignant oral disease in a few epidemiological studies (Dietrich et al., 2004; Ujpal et al., 2004). Recently, a molecular basis for this association was demonstrated by our group in an experimental animal model, indicating that a possible mechanism linked to both diabetes and cancer 382
ARTICLE IN PRESS Increased risk of oral cancer in diabetic animals is not associated with c-jun activation pathway 383
factor, the only MAPK that specifically phosphorylates c-jun is JNK (Vasilevskaya and O’Dwyer, 2003). c-jun is generally considered as an early marker of differentiation but there are very few studies regarding its role in early carcinogenesis (Szabo et al., 1996). Our study, which established involvement of IRS-1 in the molecular basis for the association between oral cancer and type I diabetes, used a rat animal model of sequential stages of oral oncogenesis (Goutzanis et al., 2007). By using the same experimental material, the purpose of the present study was the investigation of c-jun expression in normal oral mucosa tissues and in sequential stages of tumour formation, as a marker of the JNK involvement further delineating the effect of diabetes in oral cancer.
MATERIAL AND METHODS Animal model of induced diabetes and oral cancer The experimental animal model of oral carcinogenesis in diabetic and normal control rats has been previously described in detail (Goutzanis et al., 2007). Briefly, it involved 37 female Sprague-Dawley rats purchased from the Hellenic Pasteur Institute (Athens) at the age of 6 weeks and weighting approximately 135 g each. The rats were handled in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). The animals were randomly allocated to four groups. Group D (n ¼ 6): Diabetic rats without carcinogenesis, Group Dc (n ¼ 13): Diabetic rats used for induced carcinogenesis, Group N (n ¼ 6): Normal rats without carcinogenesis, Group Nc (n ¼ 12): Normal rats used for induced carcinogenesis. The induction of diabetes was performed in 19 rats previously overnight fasted animals by a single intraperitoneal injection of streptozotocin (STZ) dissolved in saline buffer at a dose of 70 mg/kg of weight (ZANOSAR, Pharmacia & Upjohn Co., USA) and determined by blood glucose levels after 3 weeks, as previously described (Goutzanis et al., 2007). After confirmation of diabetes, oral cancer was induced in Dc and Nc animals by application of the carcinogen 4-nitroquinoline N-oxide (4NQO) at a concentration of 5% in propylene glycol 3 times per week for 5 months to the rats’ hard palate (Fluca AG Chemische Fabrik, Switzerland), as previously described (Goutzanis et al., 2007). Clinical signs of oral lesions putatively tumour-related were observed within 6 months after the last application of carcinogen. After sacrifice of the animal using ether, the oral regions with cancer (mainly palate and tongue) of Dc and Nc rats and the respective regions of D and N rats were excised for immunohistochemical analysis (Goutzanis et al., 2007).
Pathological evaluation The histological status of the lesions was defined after examination of the complete section under light microscopy and the tissue profiles were classified in the following categories: normal mucosa, hyperplasia, dysplasia, early invasion, well- and moderately differentiated carcinoma. In every sample all possible different lesions were evaluated. Immunohistochemical analysis Surgical specimens were fixed in 10% neutralized formaldehyde solution and embedded in paraffin. Three sections of 4 mm were prepared from each specimen and were mounted on Super Frost Pluscoated glass slides (Menzel and Co., Braunschweig, Germany). One section was stained with haematoxylin and eosin for routine histological evaluation, while the other two were used for immunohistochemical detection of c-jun protein as previously described (Derka et al., 2006) with monoclonal primary antibodies against c-jun (H-79: sc-1694, Santa Cruz Biotechnology, Inc., diluted 1:100). Prostate carcinoma which strongly expresses c-jun was used as a positive control. Negative controls for each biopsy were processed in the same manner, using PBS instead of the primary antibody. All slides were independently and blindly reviewed by two investigators. The consecutive haematoxylin–eosinstained slides were evaluated by a pathologist experienced in oral pathology, without knowing the c-jun staining patterns. Statistical analysis Statistical analyses were performed using the twotailed Student’s t-test for each group of animals and each histological category. The percentage of positively stained cells from each non-cancerous or precancerous condition (hyperplasia, dysplasia) were compared with normal tissue, while the percentage of positively stained cells from each tumour (early invasion, well- and moderately differentiated carcinoma) were compared with the mean value of percentages of the precancerous conditions. In all cases with no normal distribution, the results of both the Wilcoxon test and two-tailed student’s t-test provided the same level of significance.
RESULTS The histological status of biopsies observed from various regions of rats of all the groups (N, Nc, D, Dc) after induced oral oncogenesis is summarized in Table 1. As previously described, there were no significant differences in histological status of oral cancer biopsies obtained from diabetic and normal rats after carcinogen treatment for a prolonged period of 5 months (Goutzanis et al., 2007).
ARTICLE IN PRESS 384 Journal of Cranio-Maxillofacial Surgery
The experimental model allowed the collection of several normal, non-cancerous, precancerous and cancerous regions in the various tissue biopsies and further analysis of immunostaining data was implemented (Fig. 1). In all cases with no normal distribution, the results of both the Wilcoxon test and two-tailed student’s t-test provided the same level of significance. The percentages of positive expression of c-jun, in the various histological categories for non-diabetic animals from groups N and Nc are shown in Table 2. Statistical analysis revealed a significant increase in c-jun levels in oral mucosal dysplasia when compared with normal mucosa (Po0.05). No statistically significant differences were observed when c-jun expression in early invasive and well-differentiated carcinomas was compared with the mean value of percentages of non-cancerous and precancerous conditions, indicating an almost stable pattern of expression of c-jun in initial stages of oral oncogenesis. Nevertheless, in moderately differentiated OSCC a significant decrease in c-jun levels was noticed (Po0.05). Table 3 summarizes the percentages of positively stained cells for c-jun in the various categories of
histological status for diabetic animals from groups D and Dc. During the non-cancerous and precancerous stages, c-jun was elevated significantly compared with normal oral mucosa. In addition, the comparison of c-jun percentages between the tumour stages and the mean value of percentages of non-cancerous and precancerous conditions revealed a statistically significant increase for early invasion and welldifferentiated OSCC, as well as a non-significant decrease in the case of moderately differentiated OSCC. Fig. 2 illustrates the compared expression of c-jun in the different tissue categories in normal and diabetic rats. From normal mucosa to dysplasia, c-jun expression displays higher levels in normal rats when compared with diabetic rats, while in different tumour stages, surprisingly, the expression of c-jun is practically identical.
DISCUSSION In accordance with previous epidemiological studies in humans which implicated diabetes mellitus as a
Table 1 – Histological status of biopsies in groups N, D, Nc and Dc
Normal tissue Hyperplasia Dysplasia Early invasion Well-differentiated carcinoma Moderately differentiated carcinoma
Control group (N)
Control group (D)
3 3
5 1
Experimental group (Nc)
Experimental group (Dc)
1 1 6 4
1 4 1 4 3
Fig. 1 – (A) Minimal c-jun immunoreactivity in normal oral mucosa of normal rats ( 400), (B) Intense nuclear immunostaining in dysplastic nuclei in normal rats ( 400), (C) Evident c-jun immunoreaction in an area of early invasion in diabetic rats ( 200), (D) Weak c-jun immunostaining in a moderately differentiated oral carcinoma in diabetic rats ( 400).
ARTICLE IN PRESS Increased risk of oral cancer in diabetic animals is not associated with c-jun activation pathway 385 Table 2 – Percentages of c-jun positive cells in the various categories of tissue status for normal rats (N and Nc) Non-cancerous and precancerous
c-jun Mean of percentages Probability of ttest
Tumour
Normal oral mucosa
Oral mucosal hyperplasia
Oral mucosal dysplasia
Early invasion
Welldifferentiated OSCC
Moderately differentiated OSCC
2.25 (N ¼ 4)
4 (N ¼ 9)
5 (N ¼ 4)
4.25 (N ¼ 4)
3.67 (N ¼ 6)
2.25 (N ¼ 4)
N.S.a
Po0.05
N.S.a
N.S.a
Po0.05
Comparisons of percentages were made between each non- cancerous or precancerous condition with normal tissue, and from each tumour stage with the mean value of percentages of the non-cancerous and precancerous conditions. a N.S: No statistical difference. Table 3 – Percentages of c-jun positive cells in the various categories of tissue status for diabetic rats (D and Dc) Non-cancerous and precancerous
c-jun Mean of percentages Probability of ttest
Tumor
Normal oral mucosa
Oral mucosal hyperplasia
Oral mucosal dysplasia
Early invasion
Well differentiated OSCC
Moderately differentiated OSCC
0.27 (N ¼ 22)
1.15 (N ¼ 33)
2.8 (N ¼ 21)
4.25 (N ¼ 4)
3.65 (N ¼ 23)
2.18 (N ¼ 11)
Po0.01
Po0.001
Po0.05
Po0.01
N.S.a
Comparisons of percentages were made between each non- cancerous or precancerous condition with normal tissue, and from each tumour stage with the mean value of percentages of the non-cancerous and precancerous conditions. a N.S: No statistical difference.
6 c-jun D 5
c-jun N
4 3 2 1 0 Normal
Hyperplasia
Dysplasia
Early invasion
Well Moderately differentiated differentiated
Fig. 2 – Expression of c-jun in the different tissue categories in normal (N) and diabetic (D) rats.
risk factor for the development of OSCC, a previous experimental study by our group established that association through the IRS-1 pathway (Dietrich et al., 2004; Ujpal et al., 2004; Goutzanis et al., 2007). In the present study, c-jun expression was investigated using the same experimental system of chemically induced oral cancer in diabetic and normal rats, in order to further delineate the molecular pathway linking diabetes and oral cancer. The present study is, to our knowledge, the first one investigating the expression of c-jun in an
experimental animal system of chemically induced oral carcinogenesis both in non-diabetic and diabetic rats. The c-jun expression was investigated in sequential stages of oral oncogenesis, varying from normal oral mucosa to moderately differentiated oral carcinomas, obtained from normal and diabetic rats. A similar pattern of c-jun expression was observed for both diabetic and non-diabetic rats, which consists of increased levels of c-jun during the initial stages of oral oncogenesis, and decreased levels in moderately differentiated OSCC. Higher c-jun levels
ARTICLE IN PRESS 386 Journal of Cranio-Maxillofacial Surgery
were observed in non-cancerous and precancerous stages of normal rats compared with diabetic rats, while in various tumour stages, the expression of c-jun was practically identical for both animal groups. It has been reported that the activity of c-jun N-terminal kinase (JNK) and subsequently the activity of c-jun, is abnormally elevated in various tissues under diabetic conditions (Fernyhough et al., 1999; Liu et al., 2000; Purves et al., 2001; Hirosumi et al., 2002). It has been documented that JNK is activated in sensory neurons and pulmonary artery endothelium, due to high glucose levels observed in type I diabetes (Liu et al., 2000; Purves et al., 2001). In these tissues, activation of the JNK pathway as seen under diabetic conditions increases insulin resistance, and in contrast, suppression of the JNK pathway decreases insulin resistance and markedly improves glucose tolerance in diabetic animal models (Purves et al., 2001). No previous data exists in the literature concerning c-jun expression in oral tissues under diabetic conditions. In the light of this, the present study resulted in unexpectedly diverse observations. The observed increase of c-jun expression in non-cancerous and precancerous stages of non-diabetic rats compared with the equivalent ones in diabetic animals may be attributed to physiological differences of oral tissues in comparison with previously studied tissues. Insulin-deficient states (such as diabetes type I) result in increased numbers of insulin receptors, increased phosphorylation of IRS-1 and increased IRS-1-associated PI 3-kinase activity (Kondo and Kahn, 2004). On the other hand, IRS-1 inactivation, which contributes to insulin resistance in diabetes II, is promoted by c-jun N-terminal kinase (JNK) through Ser307 phosphorylation of IRS-1 (Moxham et al., 1996; Miller et al., 1996; Virkamaki et al., 1999; Aguirre et al., 2000). A previous study by our group using the same experimental system observed that the expression of IRS-1 was significantly higher in diabetic than in normal animals (Goutzanis et al., 2007). This significant elevation of IRS-1 may be attributed to lack of IRS-1 inactivation by reduced activity of JNK. Decreased JNK protein levels are also responsible for the reduction of c-jun protein levels in diabetic animals (Vasilevskaya and O’Dwyer, 2003). The latter was observed in the present study, in which lower c-jun levels were detected in diabetic compared with normal animals during early stages of oral oncogenesis. Insulin signal transduction is mediated by a series of phosphorylation cascades linking initial activation of the insulin receptor (IR) to downstream substrates. Activation of the receptor for insulin results in tyrosine phosphorylation of IRS-1, which binds Grb2/SOS complex mediating the activation of N-ras, thereby activating the ras/raf/MAP kinase cascade (White, 2002). These data were linked with a comparison between the findings of the present study and those of a previous report by our group
investigating N-ras/Ets-1 expression in the same experimental system (Vairaktaris et al., 2007). In diabetic animals, N-ras was found to be significantly increased in the last stages of oral cancer in contrast to c-jun expression which was found to be decreased in the same stages. It seems that the ras/raf/ MAP kinase plays an important role in diabetic states while the JNK/c-jun pathway may not be affected by diabetes.
CONCLUSIONS The findings of the present study suggest that diabetes does not affect the c-jun N-terminal kinase (JNK)/c-jun pathway during early and advanced tumour stages of oral carcinogenesis. The rat model of oral oncogenesis is a very useful experimental animal model for studying the effect of diabetes on various mechanisms and stages towards establishment of oral squamous cell carcinoma. ACKNOWLEDGEMENTS
Source of support: This work was co-funded by the European Social Fund and National Resources (EPEAEK II ‘‘Pythagoras’’ 70/3/7391) grant to E.V.
References Aguirre V, Uchida T, Yenush L, Davis R, Whitei MF: The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J Biol Chem 275: 9047–9054, 2000 Angel P, Karin M: The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1072: 129–157, 1991 Bsoul SA, Huber MA, Terezhalmy GT: Squamous cell carcinoma of the oral tissues: A comprehensive review for oral healthcare providers. J Contemp Dent Pract (6): 1–16, 2005 Derka S, Vairaktaris E, Papakosta V, Vassiliou S, Acil Y, Vylliotis A, Spyridonidou S, Lazaris AC, Mourouzis C, Kokkori A, Moulavasili P, Perrea D, Donta I, Yapijakis C, Patsouris E: Cell proliferation and apoptosis culminate in early stages of oral oncogenesis. Oral Oncol 42 (5): 540–550, 2006 Dietrich T, Reichart PA, Scheifele C: Clinical risk factors of oral leukoplakia in a representative sample of the US population. Oral Oncol 40: 158–163, 2004 Fernyhough P, Gallagher A, Averill SA, Priestley JV, Hounsom L, Patel J, Tomlinson DR: Aberrant neurofilament phosphorylation in sensory neurons of rats with diabetic neuropathy. Diabetes 48: 881–889, 1999 Goutzanis L, Vairaktaris E, Yapijakis C, Kavantzas N, Nkenke E, Derka S, Vassiliou S, Acil Y, Kessler P, Stavrianeas N, Perrea D, Donta I, Patsouris E: Diabetes may increase risk for oral cancer through the insulin receptor substrate-1 and focal adhesion kinase pathway. Oral Oncol 43: 165–173, 2007 Hassanein KA, Musgrove BT, Bradbury E: Psychological outcome of patients following treatment of oral cancer and its relation with functional status and coping mechanisms. J Craniomaxillofac Surg 33: 404–409, 2005 Hess J, Angel P, Schorpp-Kistner M: AP-1 subunits: quarrel and harmony among siblings. J Cell Sci 117: 5965–5973, 2004 Hirosumi J, Tuncman G, Chang L, Gorgun CZ, Uysal KT, Maeda K, Karin M, Hotamisligil GS: A central role for JNK in obesity and insulin resistance. Nature 420: 333–336, 2002
ARTICLE IN PRESS Increased risk of oral cancer in diabetic animals is not associated with c-jun activation pathway 387 Hundsdorfer B, Zeilhofer HF, Bock KP, Dettmar P, Schmitt M, Kolk A, Pautke C, Horch HH: Tumour-associated urokinasetype plasminogen activator (uPA) and its inhibitor PAI-1 in normal and neoplastic tissues of patients with squamous cell cancer of the oral cavity – clinical relevance and prognostic value. J Craniomaxillofac Surg 33: 191–196, 2005 Kondo T, Kahn CR: Altered insulin signaling in retinal tissue in diabetic states. J Biol Chem 279: 37997–38006, 2004 Kovacs AF: Maximized combined modality treatment of an unselected population of oral and oropharyngeal cancer patients. Final results of a pilot study compared with a treatment-dependent prognosis index. J Craniomaxillofac Surg 34: 74–84, 2006 Kozomara R, Jovic N, Magic Z, Brankovic-Magic M, Minic V: p53 mutations and human papillomavirus infection in oral squamous cell carcinomas: correlation with overall survival. J Craniomaxillofac Surg 33: 342–348, 2005 Liu W, Schoenkerman A, Lowe Jr. WL: Activation of members of the mitogen-activated protein kinase family by glucose in endothelial cells. Am J Physiol 279: 782–790, 2000 Llewellyn CD, Johnson NW, Warnakulasuriya KAAS: Risk factors for squamous cell carcinoma of the oral cavity in young people – a comprehensive literature review. Oral Oncol 37: 401–418, 2001 Miller BS, Shankavaram UT, Horney MJ, Gore ACS, Kurtz DT, Rosenzweig SA: Activation of cJun NH2-terminal kinase/stressactivated protein kinase by insulin. Biochemistry 35: 8769–8775, 1996 Moxham CM, Tabrizchi A, Davis RJ, Malbon CC: Jun N-terminal kinase mediates activation of skeletal muscle glycogen synthase by insulin in vivo. J Biol Chem 271: 30765–30773, 1996 Purves T, Middlemas A, Agthong S, Jude EB, Boulton AJ, Fernyhough P, Tomlinson DR: A role for mitogen-activated protein kinases in the etiology of diabetic neuropathy. FASEB J 15: 2508–2514, 2001 Pyo SW, Hashimoto M, Kim YS, Kim CH, Lee SH, Johnson KR, Wheelock MJ, Park JU: Expression of E-cadherin, P-cadherin and N-cadherin in oral squamous cell carcinoma: correlation with the clinicopathologic features and patient outcome. J Craniomaxillofac Surg 35: 1–9, 2007 Schimming R, Reusch P, Kuschnierz J, Schmelzeisen R: Angiogenic factors in squamous cell carcinoma of the oral cavity: do they have prognostic relevance? J Craniomaxillofac Surg 32: 176–181, 2004 Scully C, Field JK, Tanzawa H: Genetic aberrations in oral and head and neck squamous cell carcinoma (SCCHN) 1: carcinogen metabolism, DNA repair and cell cycle control. Oral Oncol 36: 256–263, 2000
Szabo E, Riffe ME, Steinberg SM, Birrer MJ, Linnoila RI: Altered c-jun expression:an early event in human lung carcinogenesis. Cancer Res 56: 305–315, 1996 Ujpal M, Matos O, Bibok G, Somogyi A, Szabo G, Suba Z: Diabetes and oral tumors in Hungary. Epidemiological correlations. Diabetes Care 27: 770–774, 2004 Vairaktaris E, Goutzanis L, Kalokerinos G, Yannopoulos A, Yapijakis C, Derka S, Vassiliou S, Spyridonidou S, Vylliotis A, Nkenke E, Papageorgiou G, Lazaris AC, Tesseromatis C, Tsigris C, Patsouris E: Diabetes increases both N-ras and ets-1 expression during oral oncogenesis resulting in enhanced cell proliferation and metastatic potential. In Vivo 21: 615–621, 2007 Vasilevskaya I, O’Dwyer PJ: Role of Jun and Jun kinase in resistance of cancer cells to therapy. Drug Resist Updat 6: 147–156, 2003 Virkamaki A, Ueki K, Kahn CR: Protein–protein interaction in insulin signalling and the molecular mechanisms of insulin resistance. J Clin Invest 103: 931–943, 1999 Vogt PK: Jun, the oncoprotein. Oncogene 20: 2365–2377, 2001 Weston CR, Davis RJ: The JNK signal transduction pathway. Curr Opin Genet Dev 12: 14–21, 2002 White MF: IRS proteins and the common path to diabetes. Am J Physiol Endocrinol Metab 283: 413–422, 2002 Wolff D, Hassfeld S, Hofele C: Influence of marginal and segmental mandibular resection on the survival rate in patients with squamous cell carcinoma of the inferior parts of the oral cavity. J Craniomaxillofac Surg 32: 318–323, 2004 Zayzafoon M, Stell C, Irwin R, McCabe LR: Extracellular glucose influences osteoblast differentiation and c-jun expression. J Cell Biochem 79: 301–310, 2000
Prof. Dr. Eleftherios VAIRAKTARIS, MD, DDS, PhD, PhD. Department of Oral and Maxillofacial Surgery University of Athens Medical School ‘‘Attikon’’ Hospital Rimini 1 Athens 12462 Greece Tel.: +30 210 6443035 Fax: +30 210 6443803 E-mail: [email protected] Paper received 7 September 2006 Accepted 18 May 2007