Role of Runx2 phosphorylation in prostate cancer and association with metastatic disease.

The osteogenic transcription factor, Runx2, is abnormally expressed in prostate cancer (PCa) and associated with metastatic disease. During bone development, Runx2 is activated by signals known to be hyperactive in PCa including the RAS/MAP kinase pathway, which phosphorylates Runx2 on multiple serine residues including S301 and S319 (equivalent to S294 and S312 in human Runx2). This study examines the role of these phosphorylation sites in PCa. Runx2 was preferentially expressed in more invasive PCa cell lines (PC3>C4-2B>LNCaP). Furthermore, analysis using a P-S319-Runx2-specific antibody revealed that the ratio of P-S319-Runx2/total Runx2 as well as P-ERK/total ERK was highest in PC3 followed by C4-2B and LNCaP cells. These results were confirmed by immunofluorescence confocal microscopy, which showed a higher percentage of PC3 cells staining positive for P-S319-Runx2 relative to C4-2B and LNCaP cells. Phosphorylated Runx2 had an exclusively nuclear localization. When expressed in prostate cell lines, wild-type Runx2 increased metastasis-associated gene expression, in vitro migratory and invasive activity as well as in vivo growth of tumor cell xenografts. In contrast, S301A/S319A phosphorylation site mutations greatly attenuated these Runx2 responses. Analysis of tissue microarrays from 129 patients revealed strong nuclear staining with the P-S319-Runx2 antibody in primary PCas and metastases. P-S319-Runx2 staining was positively correlated with Gleason score and occurrence of lymph node metastases while little or no Runx2 phosphorylation was seen in normal prostate, benign prostate hyperplasia or prostatitis indicating that Runx2 S319 phosphorylation is closely associated with PCa induction and progression towards an aggressive phenotype. These studies establish the importance of Runx2 phosphorylation in prostate tumor growth and highlight its value as a potential diagnostic marker and therapeutic target.

In situ nanomechanical characterization of the early stages of swelling and degradation of a biodegradable polymer.

The interactions of a biodegradable scaffold with cells or living tissues depend on the time-evolution of the nanoscale properties of the scaffold. We present an in situ quantitative study on the early-stage swelling and degradation of poly(lactic-co-glycolic acid) (PLGA). A novel metrology scheme based on force microscopy measurements of the patterns of PLGA nanostructures is developed to characterize the evolution of topography, volume and nanomechanical properties. The volume and nanoscale roughness show an oscillating behaviour during the first eight days of immersion; at a later stage, we observe a continuous decrease of the volume. The effective Young's modulus exhibits a monotonic decrease from an initial value of about 2.4 GPa down to 9 MPa at day 14. The oscillating behaviour of the volume before the onset of full degradation is explained by a coupled diffusion-swelling mechanism. The appearance of a second maximum in the volume evolution results from the competition between swelling and degradation.

Prognostic significance of N-Cadherin expression in oral squamous cell carcinoma.

BACKGROUND: N-Cadherin (CDH2) is a calcium-dependent adhesion protein, whose de novo expression, re-expression, up-regulation and down-regulation in human tumors has been demonstrated. The aim of the present work was to define the prognostic role of N-Cadherin in a large series of oral squamous cell carcinomas (OSCCs). MATERIALS AND METHODS: A total of 94 selected OSCCs were quantitatively and qualitatively analyzed by immunohistochemistry for N-Cadherin. The association between protein expression and clinico-pathological parameters was assessed by statistical analysis. RESULTS: In neoplastic tissue, N-Cadherin levels were more evident than in normal peritumoral epithelium (p<0.05). Protein staining was mainly detected in the neoplastic cells, and only focal nuclear positivity was observed. Expression of cytoplasmic N-Cadherin correlated significantly with poor histological differentiation (p<0.05). Furthermore, we have observed significant a statistical trend for stage and a correlation with worst patient outcome, also confirmed by Kaplan-Meier estimates. CONCLUSION: Our work has underlined the key-role of N-Cadherin in oral carcinogenesis and in the prognostic stratification of patients.

Aurora B expression as a prognostic indicator and possible therapeutic target in oral squamous cell carcinoma.

The aim of this study is to investigate the expression of the chromosomal passenger protein Aurora B and its activated (phosphorylated) form in a large series of human oral squamous cell cancers (OSCC) and to evaluate its clinical and prognostic significance. Western blotting analysis revealed overexpression of both Aurora B and Thr-232 Phopsho-Aurora B in OSCC lines as compared to normal keratinocytes and bladder cancer cells. Furthermore, protein expression was analysed by immunohistochemistry in 101 OSCC of different site, stage and histological grade and in normal peritumoural areas. The intracellular localization of Aurora B in tumour cells was mainly nuclear, especially in proliferative areas, and significant overexpression was found in tumours in comparison to normal peritumoural areas (P=0.012). Staining results were correlated with clinicopathological parameters and long-term follow-up, and a significant association was found between protein expression and tumour stage (stage II, III and IV vs stage I, P=0.030) and size (<2cm vs >2cm, P=0.010). Cox regression analysis confirmed a poorer disease-free survival in cases with high expression of Aurora B protein. Kaplan-Meier curves showed shorter time to progression in patients with high levels of Aurora B expression (p<0.05). Moreover, the tumoral group with nuclear Aurora B immunolocalization had the worst prognosis (P=0.0364 in disease free survival). Our results suggest that assessing Aurora B expression might help in patients’ risk stratification and serve as a novel therapeutic target in advanced OSCCs.

Large oral soft tissue metastasis from anaplastic carcinoma of the lung mimicking a primitive malignancy: case report and brief review of the literature.

Metastatic tumours to the oral region are rare, and those reported in the buccal soft tissues are even less frequent. We describe a case of anaplastic carcinoma of the lung in a 60-year-old man, presenting a huge oral metastasis as the first sign of his primitive lung malignancy. Clinically, the oral lesion mimicked a high-grade primitive carcinoma of the oral cavity. The biopsy established the gingival metastasis from lung cancer which was confirmed by a fine-needle aspiration cytology examination. We report an uncommon case of metastatic lung carcinoma to the gingiva emphasizing the differential diagnosis between primary and metastatic tumours; a short discussion on the pathways of metastatization to oral cavity soft tissues, as well as brief review of the literature are also presented.

Role of calcium in the reaction between pyrroloquinoline quinone and pyridine nucleotides monomers and dimers.

Redox reactions were carried out in aerobiosis and anaerobiosis between NAD(P) dimers or NAD(P)H and pyrroloquinoline quinone (PQQ) in different buffers. The buffer system and pH significantly affected the oxidation rates of nucleotides and the ESR signal intensity of the PQQ(*) radical formed in anaerobiosis by comproportion between the quinone and quinol forms. The relative reactivity of the four nucleotides toward PQQ was affected by pH and buffer nature. PQQ, which behaves as an electron shuttle from nucleotides to oxygen, was first converted to PQQH(2) and then rapidly reoxidized by oxygen, with formation of hydrogen peroxide. Both NAD(P) dimers and NAD(P)H consumed 1 mol of oxygen per mole of reacted molecule of pyridine nucleotide, yielding 1 or 2 mol of NAD(P)(+) from NAD(P)H or from NAD(P) dimers, respectively. Chelating agents such as EDTA and phytate strongly decreased the reaction rate and the PQQ(*) radical signal intensity. Kinetics carried out in the presence of metal ions showed instead an increased reaction rate in the order Ca(2+) >> Mg(2+) > Na(+) >> K(+). Spectrofluorimetric measurements of PQQ with increasing concentrations of Ca(2+) showed a fluorescence quenching and shift of the maximum emission toward lower wavelengths, while other metal ions showed minor effects, if any. Therefore, it is demonstrated that Ca(2+) binds to PQQ, probably forming a complex which is more reactive with both one-electron (NAD(P) dimers) or two-electron donors (NAD(P)H) in nonenzymic reactions. It is important to recall that Ca(2+) was already found to play active role in PQQ-containing enzymes.

Reactions of pyridine coenzyme dimers and monomers with viologens.

DCMV++ (1,1'-dimethyl-2,2'-dicyano-4,4'-bipyridinium, bis-methylsulfate) promotes the aerobic oxidation of the NAD(P) dimers (NADP)2 and (NAD)2 with the formation of 2 mol of NADP+ or NAD+ per mole of dimers. The reaction appears to follow a pseudo-first-order kinetics with respect to the dimer concentration. One mole of oxygen was consumed in the reaction per mole of NAD(P) dimer oxidized and hydrogen peroxide was produced. The monomers NADPH and NADH under the same reaction conditions were not oxidized by DCMV++. In anaerobiosis NAD(P) dimers but not NAD(P)H rapidly reduced DCMV++ to its radical cation DCMV++, which was rapidly back-oxidized by air to its parent dication. Paraquat (MV++) was also able to catalyze the aerobic oxidation of NAD(P) dimers and, at a much lower extent, NADPH and NADH, but only under light irradiation. In anaerobiosis and upon light irradiation all the above nucleotides were able to convert paraquat to its radical cation MV++, reoxidized to MV++ by air admission. This study shows the different ability of NAD(P) dimers and NAD(P)H to undergo one-electron and two-electron oxidation reactions, with different viologens.

1H-NMR study and structure determination of 4,4- and 4,6-dimers from electrochemical reduction of NADP+.

The products arising from one-electron electrochemical reduction of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) have been studied by HPLC chromatography and 1H-NMR spectroscopy. HPLC and NMR analyses have shown seven dimeric species, the most abundant of which (40%) has been isolated and has resulted to be an NADP 4,4-linked dimer. The other two diastereoisomeric 4,4-dimers present for the 25% and 10%, respectively, have been detected in the crude reaction mixture, but have not been isolated. The 4,4-tetrahydrobipyridine structure and the stereochemistry at the ring-ring junction for these three isomers have been determined on the basis of their NMR parameters. Preparative HPLC chromatography also led to two fractions enriched in another four dimers, present in the crude mixture, which turned out to have a 4,6-tetrahydrobipyridine structure. All the chemical shifts and the H,H coupling constants of the 4,4- and 4,6-tetrahydrobipyridine systems have been obtained for the seven compounds. For the most abundant among the 4,4-dimers the NMR analysis also gave the coupling constant values of the ribose-diphosphate chain.

1H-, 13C-, 31P-NMR studies and conformational analysis of NADP+, NADPH coenzymes and of dimers from electrochemical reduction of NADP+.

All H,H, H,P and several C,P coupling constants, including those between C-4' and the vicinal phosphorus atom, have been determined for NADP+, NADPH coenzymes and for a 4,4-dimer obtained from one-electron electrochemical reduction of NADP+. From these data the preferred conformation of the ribose, that of the 1,4-dihydronicotinamide rings, and the conformation about bonds C(4')-C(5') and C(5')-O(5') were deduced. The preferred form of the 1,4- and 1,6-dihydropyridine rings and the conformation about the ring-ring junction were also obtained for all the other 4,4- and 4,6-dimers formed in the same reduction. All the dimers show a puckered structure, i.e., a boat form for the 1,4- and a twist-boat for the 1,6-dihydronicotinamide ring; both protons at the ring-ring junctions are equatorial and have preferred gauche orientation. On the contrary, the reduced coenzyme NADPH displays a planar or highly flexible conformation, rapidly flipping between two limiting boat structures. The conformation of the ribose rings, already suggested for the NADP coenzymes to be an equilibrium mixture of C(2')-endo (S-type) and C(3')-endo (N-type) puckering modes, has been reexamined by using the Altona procedure and the relative proportion of the two modes has been obtained. The S and N families of conformers have almost equal population for the adenine-ribose, whereas for the nicotinamide-ribose rings the S-type reaches the 90%. The rotation about the ester bond C(5')-O(5') and about C(4')-C(5'), defined by torsion angles beta and gamma respectively, displays a constant high preference for the trans conformer beta t (75-80%), whereas the rotamers gamma are spread out in a range of different populations. The values are distributed between the gauche gamma + (48-69%) and the trans gamma t forms (28-73%). The gamma + conformer reaches a 90% value in the case of NADP+ and NMN+. The conformations of the mononucleotides 5'-AMP, NMN+ and NMNH were also calculated from the experimental coupling constant values of the literature.

Adriamycin-catalyzed aerobic photooxidation of NAD dimers to NAD+.

The photooxidation of the dimers of nicotinamide adenine dinucleotide, (NAD)2, is catalyzed by adriamycin under aerobic conditions. (NAD)2 and O2 react in 1:1 molar ratio to yield 2 mol of NAD+. Experiments carried out by irradiating at 340 and 485 nm, corresponding to the absorption maxima of (NAD)2 and adriamycin, respectively, clearly indicate that the process is primed by photoexcitation of adriamycin. The key step of the process is the redox reaction between (NAD)2 and adriamycin with formation of the semiquinone radical anion, identified by the EPR spectrum. The semiquinone is then oxidized back to adriamycin by oxygen with formation of the superoxide radical.

Photocatalyzed anaerobic oxidation of nicotinamide coenzyme dimers to NAD+ by adriamycin.

The nicotinamide adenine dinucleotide dimers (NAD)2 obtained by electrochemical reduction of NAD+ are oxidized by adriamycin in anaerobic photocatalyzed reaction yielding NAD+ and 7-deoxyadriamycinone. Under the same conditions NADH is not oxidized.