Altered expression of integrins and basement membrane proteins in malignant and pre-malignant lesions of oral mucosa

Integrins are transmembrane receptors that regulate cell-cell and cell-extracellular matrix (ECM) contact. In epithelial tissues, they interact with ECM components of the basement membrane (BM) to maintain the homeostasis and the architecture of the tissue. This interaction controls several cell functions such as adhesion, migration, proliferation, differentiation, and therefore has a key role in cancer development and metastasis. We studied the expression of integrins and ECM components of the BM by immunohistochemistry in frozen specimens of malignant squamous cell carcinoma (SCC), pre-malignant lesions of the oral mucosa (leucoplakia) and oral lichen planus. In invasive SCC, we observed altered polarity and distribution of alpha2beta1, alpha6beta4 and alpha3beta1 integrins, whereas in the in situ carcinoma alpha6beta4 and alpha3beta1 patterns only were altered. Immunostaining for ECM components such as Laminin-1 (Ln-1), Ln-5, and Collagen IV (Coll IV) was discontinuous and interrupted in invasive SCC, whereas it was normal in the in situ carcinoma. In both pre-malignant lesions and lichen planus specimens, integrins were expressed in a polarized manner in the presence of a normal BM, whereas were abnormally distributed in those tissues with altered staining patterns of the ECM components. In conclusion, we suggest that abnormal re-distribution of alpha3beta1 and alpha6beta4 integrins and expression of ECM components such as Ln-5 could play an important role in SCC invasion and metastasis.     

The corticosteroid hormone induced factor: a new modulator of KCNQ1 channels?

The corticosteroid hormone induced factor (CHIF) is a member of the one-transmembrane segment protein family named FXYD, which also counts phospholemman and the Na,K-pump gamma-subunit. Originally it was suggested that CHIF could induce the expression of the I(Ks) current when expressed in Xenopus laevis oocytes, but recently CHIF has attracted attention as a modulatory subunit of the Na,K-pump. In renal and intestinal epithelia, the expression of CHIF is dramatically up-regulated in response to aldosterone stimulation, and regulation of epithelial ion channels by CHIF is an attractive hypothesis. To study a potential regulatory effect of the CHIF subunit on KCNQ1 channels, co-expression experiments were performed in Xenopus laevis oocytes and mammalian CHO-K1 cells. Electrophysiological characterization was obtained by two-electrode voltage-clamp and patch-clamp, respectively. In both expression systems, we find that CHIF drastically modulates the KCNQ1 current; in the presence of CHIF, the KCNQ1 channels open at all membrane potentials. Thereby, CHIF is the first accessory subunit shown to be capable of modulating both the Na,K-pump and an ion channel. To find a possible physiological function of the constitutively open KCNQ1/CHIF complex, the precise localization of KCNQ1 and CHIF in distal colon and kidney from control and salt-depleted rats was determined by confocal microscopy. However, in these tissues, we did not detect an obvious overlap in expression between KCNQ1 and CHIF. In conclusion, the hormone-regulated subunit CHIF modulates the voltage sensitivity of the KCNQ channels, but so far evidence for an actual co-localization of CHIF and KCNQ1 channels in native tissue is lacking.     

The complexity of targeting EGFR signalling in cancer: from expression to turnover

The epidermal growth factor receptor (ErbB1 or EGFR) has been found to be altered in a variety of human cancers. A number of agents targeting these receptors, including specific antibodies directed against the ligand-binding domain of the receptor and small molecules that inhibit kinase activity are either in clinical trials or are already approved for clinical treatment. However, identifying patients that are likely to respond to such treatments has been challenging. As a consequence, it still remains important to identify additional alterations of the tumor cell that contribute to the response to EGFR-targeted agents. While EGFR-mediated signalling pathways have been well established, there is still a rather limited understanding of how intracellular protein-protein interactions, ubiquitination, endocytosis and subsequent degradation of EGFR contribute to the determination of sensitivity to EGFR targeting agents and are emerging areas of investigation. This review primarily focuses on the basic signal transduction pathways mediated through activated membrane bound and/or endosomal EGFR and emphasizes the need to co-target additional proteins that function either upstream or downstream of EGFR to improve cancer therapy.     

Substrate specificity of human kallikrein 6: salt and glycosaminoglycan activation effects

Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km 38,667 s(-1) mm(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Abeta amyloid peptide, reactive center loop of alpha1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1-4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.     

Modeling H3 histone N-terminal tail and linker DNA interactions

Molecular dynamics computer simulations were performed for the 25-residue N-terminal tail of the H3 histone protein in the proximity of a DNA segment of 10 base pairs (bp), representing a model for the linker DNA in chromatin. Several least biased configurations were used as initial configurations. The secondary structure content of the protein was increased by the presence of DNA close to it, but the locations of the secondary motifs were different for different initial orientations of the DNA grooves with respect to the protein. As a common feature to all simulations, the electrostatic attraction between negatively charged DNA and positively charged protein was screened by the water solvent and counterbalanced by the intrinsic compaction of the protein due to hydrophobic effects. The protein secondary structure limited the covering of DNA by the protein to 4-5 bp. The degree of compaction and charge density of the bound protein suggests a possible role of H3 tail in a nonspecific bending and plasticity of the linker DNA when the protein is located in the crowded dense chromatin.     

Effects of water garlic extracts on cell cycle and viability of HepG2 hepatoma cells

Garlic extracts, either aqueous or oily, are commonly employed to prepare garlic derivative supplements used as nutraceuticals for the treatment of different pathologies. In this study, we investigated the effects of water garlic extracts from two different areas of Italy well known for garlic production, Latina (GEL) and Sulmona (GES), on cell cycle and death of HepG2 hepatoma cells. The effects of the treatments with GEL and GES were also compared with the oil-soluble sulfur compound of garlic, diallyl disulfide (DADS). GEL and GES induced a p53/p21-dependent cell cycle arrest in G2/M phase and apoptosis, although to a different extent, whereas DADS, under the experimental conditions used, was not detrimental to HepG2 cells. GEL and GES committed HepG2 cells to apoptosis by the activation of c-Jun-NH(2) terminal kinase (JNK)/c-Jun phosphorylative cascade without a detectable increase in the flux of reactive oxygen species. Moreover, differentiation of HepG2 cells induced by retinoic acid determined resistance to GEL and GES treatments without the activation of JNK signaling pathway. Overall, the results obtained indicate that water-soluble garlic extracts are more inhibitory of the growth of transformed hepatoma cells than the oil-soluble isolated compound DADS, and that their antiproliferative properties are different depending on the area of origin of the starting material.     

Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.     

Neurohormones and inflammatory mediators in patients with heart failure undergoing cardiac resynchronization therapy: time courses and prediction of response

Despite interest in neurohormonal activation as a determinant of prognosis in chronic heart failure (CHF) and as a target for pharmacological treatments, data are lacking on the time-related effects of electrical cardiac resynchronization therapy (CRT) on a broad spectrum of neurohormones and cytokines. The aim of this study was to assess time-courses and extents of changes within the neurohormonal profile of CHF patients treated with CRT. We performed a prospective follow-up study in 32 patients with NYHA class III-IV CHF to investigate the effects of CRT on a broad panel of neurohormones proposed for characterization of CHF patients. Levels of atrial and brain natriuretic peptides (ANP, BNP), epinephrine, norepinephrine, aldosterone, plasma renin activity, IL-6, TNF, soluble receptors sTNFR1 and 2, and chromogranin A were assessed before implantation and after 3 months of CRT; when feasible, measurements were also performed at 1 week, 1 month and 12 months (clinical evaluation, echocardiography and ECG were also performed at each time-point). The results showed that at 3 months improvement in NYHA class and echographically assessed left ventricular (LV) reverse structural remodeling were accompanied by significant reductions versus baseline in ANP and BNP, but not in other neurohormones. Moreover a baseline ANP concentration < or = 150 pg/ml was a good predictor of response to CRT in terms of NYHA class reduction and reverse LV remodeling. In conclusion 3 months of CRT significantly reduce natriuretic peptides concentrations, while values of other neurohormones and inflammatory cytokines are relatively unvaried. A baseline ANP concentration < or = 150 pg/ml might be a clinically useful predictor of medium-term response to CRT.