Cigarette smoke-induced reduction in binding of the salivary translocator protein is not mediated by free radicals.

Oral cancer is the most common malignancy of the head and neck and its main inducer is exposure to cigarette smoke (CS) in the presence of saliva. It is commonly accepted that CS contributes to the pathogenesis of oral cancer via reactive free radicals and volatile aldehydes. The 18 kDa translocator protein (TSPO) is an intracellular receptor involved in proliferation and apoptosis, and has been linked to various types of cancer. The presence of TSPO in human saliva has been linked to oral cancer, and its binding affinity to its ligand is reduced following exposure to CS. In the present study we wished to further investigate the mechanism behind the CS-induced reduction of TSPO binding by exploring the possible mediatory role of reactive oxygen species (ROS) and volatile aldehydes in this process. We first analyzed TSPO binding in control saliva and in saliva exposed to CS in the presence and absence of various antioxidants. These experiments found that TSPO binding ability was not reversed by any of the antioxidants added, suggesting that CS exerts its effect on TSPO via mechanisms that do not involve volatile aldehydes and free radicals tested. Next, we analyzed TSPO binding in saliva following addition of exogenous ROS in the form of H2O2. These experiments found that TSPO binding was enhanced due to the treatment, once again showing that the CS-induced TSPO binding reduction is not mediated by this common form of ROS. However, the previously reported CS-induced reduction in salivary TSPO binding together with the role of TSPO in cells and its link to cancer strongly suggest that TSPO has a critical role in the pathogenesis of CS-induced oral cancer. The importance of further elucidating the mechanisms behind it should be emphasized.

Gonadotrophin-releasing hormone signalling downstream of calmodulin.

Gonadotrophin-releasing hormone (GnRH) regulates reproduction via binding a G-protein coupled receptor on the surface of the gonadotroph, through which it transmits signals, mostly via the mitogen-activated protein (MAPK) cascade, to increase synthesis of the gonadotrophin hormones: luteinising hormone (LH) and follicle-stimulating hormone (FSH). Activation of the MAPK cascade requires an elevation in cytosolic Ca(2+) levels, which is a result of both calcium influx and mobilisation from intracellular stores. However, Ca(2+) also transmits signals via an MAPK-independent pathway, through binding calmodulin (CaM), which is then able to bind a number of proteins to impart diverse downstream effects. Although the ability of GnRH to activate CaM was recognised over 20 years ago, only recently have some of the downstream effects been elucidated. GnRH was shown to activate the CaM-dependent phosphatase, calcineurin, which targets gonadotrophin gene expression both directly and indirectly via transcription factors such as nuclear factor of activated T-cells and Nur77, the Transducer of Regulated CREB (TORC) co-activators and also the prolyl isomerase, Pin1. Gonadotrophin gene expression is also regulated by GnRH-induced CaM-dependent kinases (CaMKs); CaMKI is able to derepress the histone deacetylase-inhibition of ß-subunit gene expression, whereas CaMKII appears to be essential for the GnRH-activation of all three subunit genes. Asides from activating gonadotrophin gene expression, GnRH also exerts additional effects on gonadotroph function, some of which clearly occur via CaM, including the proliferation of immature gonadotrophs, which is dependent on calcineurin. In this review, we summarise these pathways, and discuss the additional functions that have been proposed for CaM with respect to modifying GnRH-induced signalling pathways via the regulation of the small GTP-binding protein, Gem, and/or the regulator of G-protein signalling protein 2.

Cigarette Smoke Decreases Salivary 18 kDa Translocator Protein Binding Affinity ­ in Association with Oxidative Stress

Reactive oxygen species (ROS) generated by cigarette smoke may contribute to lung and oral cancer. The 18 kDa Translocator protein (TSPO) has been reported to be affected by ROS as well as to participate in ROS generation at mitochondrial levels, and has been implicated in pro-apoptotic and anti-carcinogenic functions. The present study reports the presence of TSPO in the cellular fraction of human saliva. In cells collected from untreated saliva, the specific TSPO ligand [(3)H]PK 11195 showed saturable binding with high affinity, with mean B(max) and K(d) values of 6,471 +/- 501 fmol/mg protein and 6.2 +/- 0.5 nM, respectively. Our study further indicates that the cellular fraction of human saliva possesses TSPO with binding characteristics similar to that of cells from other tissues of human origin. Following exposure of saliva to cigarette smoke a three-fold decrease in the affinity of salivary TSPO to its specific ligand, [(3)H]PK 11195 (p < 0.01) occurred in the cellular fraction of the saliva, in comparison to sham treated control, without significant accompanying changes in TSPO B(max), TSPO protein levels, or general protein levels. The changes in affinity of TSPO from the cellular fraction of saliva exposed to cigarette smoke were accompanied by changes in the mean levels of protein oxidation products (carbonyls) and lipid peroxides, which were three-fold higher (p < 0.01) and two-fold higher (p < 0.01), respectively, compared to those of sham treated controls. Thus, our study shows that TSPO is present in the cellular component of saliva. Interestingly, in vitro this cellular TSPO is affected by exposure of the whole saliva to cigarette smoke, in negative correlation with oxidative stress.

Pleural fluid analysis of lung cancer vs benign inflammatory disease patients.

BACKGROUND: Correct diagnosis of pleural effusion (PE) as either benign or malignant is crucial, although conventional cytological evaluation is of limited diagnostic accuracy, with relatively low sensitivity rates. METHODS: We identified biological markers accurately detected in a simple PE examination. We analysed data from 19 patients diagnosed with lung cancer (nine adeno-Ca, five non-small-cell Ca (not specified), four squamous-cell Ca, one large-cell Ca) and 22 patients with benign inflammatory pathologies: secondary to trauma, pneumonia or TB. RESULTS: Pleural effusion concentrations of seven analysed biological markers were significantly lower in lung cancer patients than in benign inflammatory patients, especially in matrix metalloproteinase (MMP)-9, MMP-3 and CycD1 (lower by 65% (P<0.000003), 40% (P<0.0007) and 34% (P<0.0001), respectively), and in Ki67, ImAnOx, carbonyls and p27. High rates of sensitivity and specificity values were found for MMP-9, MMP-3 and CycD1: 80 and 100%; 87 and 73%; and 87 and 82%, respectively. CONCLUSION: Although our results are of significant merit in both the clinical and pathogenetic aspects of lung cancer, further research aimed at defining the best combination for marker analysis is warranted. The relative simplicity in analysing these markers in any routine hospital laboratory may result in its acceptance as a new diagnostic tool.

Salivary analysis of oral cancer biomarkers.

BACKGROUND: Oral cancer is a common and lethal malignancy. Direct contact between saliva and the oral cancer lesion makes measurement of tumour markers in saliva an attractive alternative to serum testing. METHODS: We tested 19 tongue cancer patients, measuring the levels of 8 salivary markers related to oxidative stress, DNA repair, carcinogenesis, metastasis and cellular proliferation and death. RESULTS: Five markers increased in cancer patients by 39-246%: carbonyls, lactate dehydrogenase, metalloproteinase-9 (MMP-9), Ki67 and Cyclin D1 (CycD1) (P< or =0.01). Three markers decreased by 16-29%: 8-oxoguanine DNA glycosylase, phosphorylated-Src and mammary serine protease inhibitor (Maspin) (P< or =0.01). Increase in salivary carbonyls was profound (by 246%, P=0.012); alterations in CycD1 (87% increase, P=0.000006) and Maspin (29% decrease, P=0.007) were especially significant. Sensitivity values of these eight analysed markers ranged from 58% to 100%; specificity values ranged from 42% to 100%. Both values were especially high for the CycD1 and Maspin markers, 100% for each value of each marker. These were also high for carbonyls, 90% and 80%, respectively, and for MMP-9, 100% and 79%, respectively. CONCLUSION: The significance of each salivary alteration is discussed. As all alterations correlated with each other, they may belong to a single carcinogenetic network. Cancer-related changes in salivary tumour markers may be used as a diagnostic tool for diagnosis, prognosis and post-operative monitoring.