Genetic Variations Related to Angiotensin II Production and Risk for Basal Cell Carcinoma.

Basal cell carcinoma (BCC) is the most prevalent human neoplasm, with constantly increasing annual incidence. Despite its slow growth, BCC is locally invasive and, if left untreated, can cause severe complications, including metastasis and death. The renin-angiotensin system (RAS) plays a key role in electrolyte balance, atrial pressure, tissue development, homeostasis, and inflammation, but also in cancer development. After binding to its type 1 receptor (AT1R), angiotensin II (ANGII), the system's principal hormonal effector, regulates cancer pathways spanning from the formation of the initial cancer cell to the construction and nutrition of the tumor microenvironment, angiogenesis, proliferation, and metastasis. Although the role of RAS in the development of skin pathologies has not been widely researched, RAS-targeting antihypertensive medications have been shown to have a chemoprotective effect against BCC. Based on those findings, our group conducted a series of genetic association studies to investigate the association between common functional variations in key genes related to ANGII production (AGT, ACE, ACE2, AT1R, AT2R, and CMA1) and the risk of BCC occurrence. This review provides a summary of the current understanding of the ANGII involvement in BCC development. The reliable and easily assessed pool of genetic biomarkers may be used for predictive testing and prevention purposes in high-risk individuals.

EGFR and HER-2 oncogenes expression in an experimental model of two-stage chemically induced carcinogenesis in mouse skin.

The aim of the study was to investigate the expression of EGFR and HER-2 oncogenes using an experimental two stage chemically induced carcinogenesis protocol on the dorsal skin in FVB/N mice. Forty female FVB/N mice 4 weeks old, were grouped into one control (n = 8) and two experimental groups (Group A: n = 16, Group B: n = 16) following a randomization process. Two-stage carcinogenesis protocol, was implicated, including an initial treatment with 97.4 nmol DMBA on their shaved dorsal skin and subsequent treatments of 32.4 nmol TPA applications after 13 weeks for Group A and after 20 weeks for Group B. The control group C, received no treatment. Skin was examined weekly for tumor development. Post-experiment, animals were euthanized for tissue analysis. The histological status of the skin lesions in the experimental groups corresponded well with tumour advancement (from dysplasia to poorly-differentiated carcinoma). Tumour sections were evaluated histologically and immunohistochemically. EGFR expression was found significantly higher in precancerous and malignant tumours (p = 042 and p = 008 respectively), while tended to be higher in benign tumours (p = 079), compared to normal histology. Moreover, mean percentage of EGFR positive expression in malignant tumours was significantly higher than in benign tumours (p < 001). HER-2 expression was found significantly higher in precancerous and malignant tumours (p = 042 and p = 015 respectively), while tended to be higher in benign tumours (p = 085), compared to normal histology. Furthermore, mean percentage of HER-2 positive expression in malignant tumours was significantly higher than in benign tumours (p = 005). The study demonstrated that in FVB/N mice subjected to a two-stage chemically induced carcinogenesis protocol, there was a significant increase in the expression of EGFR and HER-2 oncogenes in precancerous and malignant skin lesions compared to normal tissue. This suggests a potentially early role of these oncogenes in the progression of skin tumours in this model.

Neurobiology of bruxism: The impact of stress (Review).

Bruxism is a non-functional involuntary muscle activity that affects more than one-third of the population at some point in their lives. A number of factors have been found to be related to the etiopathogenesis of bruxism; therefore, the condition is considered multifactorial. The most commonly accepted factor is stress. Stress has long been considered to increase muscle tone and to reduce the pain threshold. Current evidence indicates that exposure to chronic stress, distress and allostatic load ignite neurological degeneration and the attenuation of critical neuronal pathways that are highly implicated in the orofacial involuntary muscle activity. The present review discusses the negative effects that chronic stress exerts on certain parts of the central nervous system and the mechanisms through which these changes are involved in the etiopathogenesis of bruxism. The extent of these morphological and functional changes on nerves and neuronal tracts provides valuable insight into the obstacles that need to be overcome in order to achieve successful treatment. Additionally, particular emphasis is given on the effects of bruxism on the central nervous system, particularly the activation of the hypothalamic-pituitary-adrenal axis, as this subsequently induces an increase in circulating corticosterone levels, also evidenced by increased levels of salivary cortisol, thereby transforming bruxism into a self-reinforcing loop.

Angiotensinogen, Angiotensin-Converting Enzyme, and Chymase Gene Polymorphisms as Biomarkers for Basal Cell Carcinoma Susceptibility.

INTRODUCTION: The intake of angiotensin-converting enzyme (ACE) inhibitors and specific antagonists of angiotensin II receptors, widely used as antihypertensive drugs, significantly reduces the risk of developing basal cell carcinoma (BCC), highlighting the possible tumorigenic role of angiotensin II (AngII). We present here the investigated genetic association between the development of BCC and functional DNA polymorphisms M235T, I/D, and A1903G in the genes of angiotensinogen (AGT), angiotensin-converting enzyme (ACE), and chymase (CMA1), which mediate AngII production levels. METHODS: DNA samples of 203 unrelated Greeks were studied, including 100 patients with BCC and 103 matched healthy controls. RESULTS: The MT genotype of the AGT-M235T polymorphism was significantly more prevalent in the patient group (78.0%) versus the healthy control group (28.3%; p < 0.001). The DD genotype of the ACE-I/D polymorphism was also increased in BCC patients (72.8%) compared to controls (46.2%; p = 0.001). The heterozygous AG genotype of CMA1-A1903G was significantly more frequent in the BCC group (86%) than in the healthy controls (50.5%; p < 0.001). CONCLUSIONS: The MT, DD, and AG genotypes of the AGT- M235T, ACE-I/D, and CMA1-A1903G polymorphisms, respectively, were significantly increased in frequency within the group of cancer patients compared to the healthy controls. All three genotypes correspond to increased enzyme levels or activity and result in increased levels of AngII; therefore, they may be potentially utilized as reliable biomarkers associated with an individual's increased risk for BCC development.

Association Between a High Gene Expression Variant of Chymase and Increased Risk for Basal Cell Carcinoma.

BACKGROUND/AIM: Previous studies have associated certain variations in genes encoding factors of renin-angiotensin system (RAS), indirectly leading to higher angiotensin II (AngII) levels, with greater risk for basal cell carcinoma (BCC) development. Chymase (CMA1) is the main regulator of the RAS-independent AngII generation pathway and numerous studies have shown its oncogenic potential in several cancer types including BCC. In this study, we investigated the possible association between BCC pathogenesis and the functional DNA polymorphism A1903G (rs1800875) that affects expression of the CMA1 gene. PATIENTS AND METHODS: We genotyped 199 DNA samples, isolated from 100 BCC patients and 99 age, sex, and ethnicity-matched healthy controls for the CMA1 A1903G polymorphism. Genotyping was performed with PCR amplification, followed by MboI enzyme digestion and agarose gel electrophoresis of the resulted DNA fragments. RESULTS: The variant G allele that possibly increases CMA1 gene expression was not detected at a significantly different frequency between the groups of BCC patients and healthy controls. However, the AG heterozygous genotype was significantly increased in BCC patients compared with controls (p<0.001). CONCLUSION: The high expression CMA1 G allele carriers have an increased risk for BCC and elevated levels of chymase in the skin may have a carcinogenic effect.

Lack of Association Between the ACE2 G8790A Gene Variation and Risk for Basal Cell Carcinoma.

BACKGROUND/AIM: The G8790A (rs2285666) functional polymorphism of the angiotensin-converting enzyme 2 (ACE2) gene influences alternative mRNA splicing and quantitatively affects the enzyme's production. Specifically, the presence of the A allele has been associated with higher ACE2 plasma levels. In this study, we investigated the possible association of the functional polymorphism ACE2-G8790A with the pathogenesis of basal cell carcinoma (BCC). PATIENTS AND METHODS: A total of 190 DNA samples were studied, including 91 BCC patients and 99 controls of Greek origin. Molecular genotyping for the ACE2 G8790A polymorphism was carried out by PCR amplification, followed by AluI enzyme digestion and agarose gel electrophoresis of the DNA fragments. RESULTS: The allelic and genotypic frequencies presented no statistical difference between the patient and the control group. CONCLUSION: There is no association between the ACE2 G8790A polymorphism and pathogenesis of BCC.

Association study indicates combined effect of interleukin-10 and angiotensin-converting enzyme in basal cell carcinoma development.

Cytokines involved in inflammatory and immune response have been associated with risk for development of basal cell carcinoma (BCC). In this study, three functional DNA polymorphisms affecting gene expression were investigated in 54 BCC patients and 111 healthy controls: interleukin-1b (IL-1b) +3953C/T, interleukin-10 (IL-10) - 1082G/A and angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphisms. Significant increase of the variant alleles was observed in IL-10 - 1082G (P = 0.019) and in ACE D (P = 0.003) in BCC patients in comparison to controls. Multivariate logistic regression models evaluated the contribution of homozygous and heterozygous variant polymorphisms to the risk for BCC development. The studied polymorphisms influencing the expression of IL-10 and ACE genes were recognized as potential predictive factors for BCC. These findings suggest a possible molecular mechanism leading to BCC development that is likely to involve the activation of angiotensin receptors in combination with increased plasma levels of IL-10 in patients.

The Hamster Model of Sequential Oral Carcinogenesis: An Update.

Animal models are valuable tools for studying human cancer as well as for preclinical trials. The hamster model of chemically induced sequential oral carcinogenesis was developed by our group a decade ago in order to study the multistep process of alterations in gene expression during carcinogenesis. The purpose of this review was to discuss the utility of the hamster model of sequential oral carcinogenesis regarding the deciphering of the main pathways altered. An extended search for articles that cited that specific animal models was performed. Many studies have used the hamster model of sequential oral carcinogenesis either for evaluation of the expression of biomarkers alone, or for applying chemopreventive compounds and other therapeutic methods, or combining the use of biomarkers with the anticancer effect of some compounds. It seems that this animal model is indeed a useful tool that enables the study of cell biology, pathology and therapeutics of oral cancer.

ACE Gene Variant Causing High Blood Pressure May Be Associated With Medication-related Jaw Osteonecrosis.

The association of the high blood pressure D variant of the angiotensin-converting enzyme (ACE) gene with medication-related jaw osteonecrosis (MRONJ) is described in two Greek patients. The first patient, a 73-year-old man, took zolendronate, 4 mg/100 ml IV once per month for two years for prostate cancer and bone metastases. Three months after drug discontinuation, extraction of the first premolar was performed. After the intervention, he suffered from osteonecrosis of the mandible. He presented with hypertension and genetic testing revealed that he was homozygous for the high blood pressure D variant of the ACE gene. The second patient, a 65 years old woman, took denosumab, 120 mg subcutaneously once per month for 6 months for possible bone metastases from breast cancer. Three months after extraction of the first molar, she suffered from MRONJ. He also presented with hypertension and genetic testing revealed that she had the high blood pressure D variant of the ACE gene in a heterozygous state, which moderately predisposes to hypertension. To our knowledge, this is the first report indicating that genetic predisposition to hypertension may increase risk for MRONJ.

Effect of Olmesartan on the Level of Oral Cancer Risk Factor PAI1.

AIM: To study if the angiotensin receptor blocker olmesartan reduces levels of plasminogen activator inhibitor 1 (PAI1), a risk factor for oral cancer, in a mouse model and therefore whether it could be used in the treatment of this malignancy. MATERIALS AND METHODS: Twelve transgenic PAI1 mice aged 16-20 weeks were divided in two groups each containing six animals. One group was given olmesartan every day for 30 days in drinking water in an amount corresponding to their weight, 0.005 mg/g, while the second group did not receive any medication (control group). Blood samples were obtained from animals of both groups, before and after one month of olmesartan administration and plasma PAI1 levels were measured using enzyme-linked immunosorbent assay. RESULTS: In the olmesartan-treated group, a significant decrease of PAI1 level was found after 1 month of treatment (11.9±8.6 vs. 21.7±7.2 ng/ml, respectively; p=0.028). However, no statistically significant difference was observed in PAI1 levels between the olmesartan-treated and control groups after one month, (p=0.177). CONCLUSION: Olmesartan did not significantly affect PAI1 levels in this mouse model.

A serious complication in the temporomandibular region due to insufficient follow-up.

The aim of this case report is to point out follow-up insufficiency as a contributing factor of ankylotic development after condylar fractures as well as the significance of clinicians' familiarity with this complication and its prevention. Condylar fractures require close follow-up due to the potential emergence of delayed and distressing complications, such as ankylosis, regardless of their proper initial treatment. Regular follow-up for a minimum of 18 months is of crucial importance for the prevention of ankylosis. The clinician's contribution in alerting his patients could be considerable, given he or she is aware of the development of this complication. The case of a 17-year-old patient with bilateral condylar fractures and a mental fracture is presented. He was successfully treated with mental osteosynthesis and intermaxillary fixation. Strict instructions for kinesiotherapy were given and constant re-examinations were made, but the patient's compliance was poor. This resulted in his readmission 2 years later with a great limitation of mouth opening (0.5 cm), demanding more serious surgical procedures. Follow-up insufficiency could be identified as a contributing factor to traumatic temporomandibular joint ankylosis. Intense surveillance and harmonious collaboration is dictated from both the clinician and patient to prevent any untoward development.

H-ras and c-fos exhibit similar expression patterns during most stages of oral oncogenesis.

BACKGROUND: H-ras and c-fos oncogenes interact in signalling pathways but their level and time course of expression during oral cancer development are unclear. The present study used an animal model for the simultaneous investigation of H-Ras and c-Fos expression in sequential stages of oral oncogenesis. MATERIALS AND METHODS: Three experimental groups of Syrian golden hamsters (A, B and C; 10 animals each) and one control group (7 animals) were used. The buccal pouches of hamsters in groups A, B and C were treated with 0.5% of the carcinogen 9,10-dimethyl-1,2-benzanthracene and were excised at 10, 14 and 19 weeks, respectively. The biopsies, which included tissue stages ranging from normal oral mucosa to moderately differentiated carcinoma, were studied immunohistochemically. RESULTS: A reduction in both H-Ras and c-Fos expression was observed from group A to B and from hyperplasias to early tumour stages, while a simultaneous increase was noted from group B to C and from well-differentiated to moderately-differentiated carcinomas. The H-ras/c-fos expression ratio had a value of approximately (1.09 +/- 0.21) in five out of seven studied tissue stages. CONCLUSION: H-Ras and c-Fos exhibit a similar expression pattern throughout most stages of oral carcinogenesis, an observation supported by the known molecular pathway connecting H-ras signalling with subsequent c-fos gene transcription.

Metastases following biopsy of oral carcinoma in hamsters and the role of local prebiopsy bleomycin.

BACKGROUND: This animal study researches the effect of biopsy on metastasis of oral carcinoma. MATERIALS AND METHODS: Sixty hamsters developed oral tumors after treatment with 9,10-dimethyl-1,2-benzanthracene and were then divided into six groups. Animals of groups 1 and 2 did not receive any treatment, while groups 3-6 were biopsied at the end of the 14th week and groups 5 and 6 also received a prebiopsy intratumoral injection of bleomycin. Animals of groups 1, 3, 5 and 2, 4, 6 were sacrificed at the 17th and 19th week respectively. Specimens of tumors, ipsilateral cervical lymph nodes, lungs and livers were obtained from all animals and histologically examined. RESULTS: All animals developed oral squamous cell carcinomas. In group 4, four out of ten animals showed metastases to the cervical lymph nodes, and one out of four showed a distant metastasis to the lung. CONCLUSION: Delay of treatment following biopsy can increase the risk of cervical lymph node metastases which can be reduced by an intratumoral administration of bleomycin.

Maxillofacial injuries sustained during soccer: incidence, severity and risk factors.

A very popular sport worldwide, soccer generates a great number of maxillofacial injuries, mainly fractures, resulting in esthetic or functional problems. The aim of this retrospective study was to contribute to the knowledge of soccer-related maxillofacial injuries, and call attention to the risk factors that favor these injuries. A total of 108 patients, who attended hospital because of maxillofacial injuries during soccer within a period of 8 years, were included in this study. The relationship of the patients with soccer, the type, the site, the severity, the mechanism of the injuries and the applied treatment were analyzed. The injured were all males, and were principally amateurs. Around 89.8% of the patients suffered maxillofacial fractures while 10.2% presented only soft tissue injuries; 13.9% had multiple fractures; 50% of the maxillofacial fractures concerned the zygomatic complex and 38.2% the mandible where the majority occurred at the angle. The prevailing mechanism was the direct impact of players. Head to head impact outnumbered. Elbow to head impact caused contusions of the temporomandibular joint. Kick to head impact was the main cause of multiple fractures. The treatment of fractures was mainly surgical (68.2%). These findings support the fact that maxillofacial injuries sustained during soccer tend to be severe, demanding surgical treatment. The mandibular angle is in danger due to the usual existence of impacted and semi-impacted third molars. There should be a preventive intervention on the above contributors, and mainly coaches and sports physicians should be properly informed about the specificity of the maxillofacial injuries.

EGFR and c-Jun exhibit the same pattern of expression and increase gradually during the progress of oral oncogenesis.

BACKGROUND: Epidermal growth factor receptor (EGFR) and c-Jun oncogenes are implicated in the same pathway of signal transduction affecting cell differentiation. In order to investigate their possible correlation with sequential histological stages of OSCC formation, we established an experimental model of induced oral carcinogenesis in Syrian golden hamsters. MATERIALS AND METHODS: Thirty-seven animals were divided into one control group (n=7) and three experimental groups (n = 10 each), which were treated with a carcinogen and sacrificed at 10, 14 and 19 weeks after treatment. Tumour sections were studied using monoclonal antibodies against EGFR and c-Jun proteins. RESULTS: The same pattern of expression was observed for both oncogenes, with a significant gradual increase of positively stained cells throughout oral carcinogenesis. CONCLUSION: Since EGFR and c-Jun are implicated in the same molecular pathway of signal transduction, it may be assumed that an increase in EGFR levels leads to increased activation of phospholipase Cy signal transduction cascade, which in turn activates c-Jun protein. Therefore, c-Jun expression in oral cancer seems to be increased through the EGFR-PLCy-Raf-MEK-ERK pathway and not the H-ras-Raf-MEK-ERK/JNK pathway.

Evaluation of apoptosis in nasal and buccal cells of septic patients.

BACKGROUND: Inhibition of lung cell apoptosis in the bronchoalveolar lavage (BAL) of septic patients may have a prognostic value for the severity of sepsis. The present study evaluated apoptosis in the nasal and buccal mucosa of septic patients as an alternative and less invasive approach for studying the cells involved in bronchial inflammation. PATIENTS AND METHODS: A prospective study was designed. Nasal and buccal mucosa brushings were obtained from 20 consecutive septic patients who were admitted to two intensive care units. Twenty-four patients scheduled to undergo surgery for colorectal cancer or laparascopic cholocystectomy were the control group. Apoptosis was evaluated using a TUNEL assay, while BCL-2 and BAX expression were evaluated by immunohistochemistry. RESULTS: Significantly reduced apoptosis in the nasal mucosa of septic patients compared to the control group (p=0.043) was detected only by the TUNEL assay. CONCLUSION: Reduced apoptosis was found during sepsis in the nasal mucosa in accordance with the reduced apoptosis in the lungs of septic patients. In contrast to septic lungs the underlying mechanism leading to apoptosis in the nasal mucosa was unrelated to the expression of two apoptosis-related genes BCL-2 and BAX.

Solvent-dehydrated fascia lata allograft for covering intraoral defects: our experience.

Mucosal defects in the oral cavity as a result of tumors, preprosthetic surgical procedure, or trauma are always a concern for surgeons. The aim of this study is to present our experience and discuss the advantages and problems arising with the use of solvent-dried human fascia lata allografts in oral mucosal defects, thus evaluating its clinical efficacy. Sixteen intraoral lesions were removed from 15 patients. The rehabilitation of the mucosal defects was achieved using solvent-dehydrated human fascia lata allografts. No graft rejection or infections were detected. The material was effective for enhancing the hemostasis, relieving the pain, and inducing rapid epithelization. The final result was excellent, even though in 2 cases complications were experienced. Hence, the use of the material proved to be reliable, practical, and safe.

Expression of ets-1 is not affected by N-ras or H-ras during oral oncogenesis.

PURPOSE: To determine whether ras-activated cascades lead to activation of ets-1 expression in sequential histological stages of oral oncogenesis in an experimental animal model. METHODS: Thirty-seven Syrian golden hamsters were divided into three experimental groups (A, B, C) and one control group. The hamsters' buccal pouches in experimental groups were treated with 0.5% 9, 10-dimethyl-1, 2-benzanthracene (DMBA) for 14 weeks and were excised at 10, 14, 19 weeks, respectively. The biopsies were classified pathologically (normal mucosa, hyperkeratosis, hyperplasia, dysplasia, early invasion, well and moderately differentiated carcinoma) and studied immunohistochemically. The two-tailed Student's t test was performed for each animal group and for each histological category. RESULTS: The ets-1 expression increased in early stages of oral tumor formation and invasion. The expression of N-ras gradually decreased during oral oncogenesis, as previously observed with H-ras. CONCLUSIONS: Neither N-ras nor H-ras affects ets-1 expression in contrast to other types of cancer in which N-ras and ets-1 are implicated in the same signalling pathway. Therefore, the existing pathway implicating these proteins might be somehow altered in oral cancer. It seems that ets-1 is a good prognostic marker for invasiveness and progression of oral cancer.

The co-expression of c-myc and p53 increases and reaches a plateau early in oral oncogenesis.

BACKGROUND: The balance between cell proliferation and apoptosis plays a significant role in cancer development. The expressions of the p53 and c-myc genes, both strongly related to cell proliferation and apoptosis, were studied in sequential histological grades of oral carcinogenesis in an animal model. MATERIALS AND METHODS: Thirty-seven hamsters were divided into three groups (A,B,C), which were treated with 9,10-dimethyl-1,2-benzanthracene and sacrificed at 10,14 or 19 weeks, respectively, after treatment. The histological status of the oral lesions in the experimental groups corresponded well with tumour advancement (from dysplasia to moderately-differentiated carcinoma). Tumour sections were studied immunohistochemically. RESULTS: The expressions of both p53 and c-myc increased significantly in precancer stages and then reached a plateau. The same pattern was observed in the animal groups with the culmination of expression of both genes in group A. CONCLUSION: The coexpression of p53 and c-myc proteins in the earlier stages of oral oncogenesis may be used for the early detection of premalignant lesions.

FGFR-2 and -3 play an important role in initial stages of oral oncogenesis.

BACKGROUND: FGFR-2 and FGFR-3 (fibroblast growth factor receptors) have been shown to play an important role in several processes including carcinogenesis. This study was designed to determine gradual FGFR-2 and FGFR-3 expression in sequential stages of oral carcinogenesis in an experimental animal system of Syrian golden hamsters. MATERIALS AND METHODS: Tissue sections ranging from normal mucosa to squamous cell carcinoma were studied using monoclonal antibodies against FGFR-2 and FGFR-3 proteins. RESULTS: A significant elevation was revealed in both FGFR-2 and FGFR-3 expression during the stages of dysplasia and early invasion, while in the later stages of oral carcinogenesis the expression of both FGFR-2 and FGFR-3 decreased although not significantly. CONCLUSION: Our findings indicate that FGFR-2 and FGFR-3 seem to play an important role in the initial stages of oral cancer progression.