Amprenavir inhibits the migration in human hepatocarcinoma cell and the growth of xenografts.

The introduction of HAART (highly-active-antiretroviral-therapy) has resulted in extended survival of HIV positive patients. Conversely, due to the prolonged expectancy of life and the ageing of the HIV positive population, tumors are now one of the major cause of death, and among them hepatocellular carcinoma (HCC) has become a growing concern in these patients. Considering the potential anti-tumoral effects of HIV protease inhibitors, we decided to evaluate the anti-tumoral activity of Amprenavir on liver carcinoma and to evaluate its potential synergistic effects in combination with standard chemoterapic drugs, such as Doxorubicin. Our results indicate that Amprenavir had direct inhibitory effects on invasion of Huh-7 hepatocarcinoma cell lines, inhibiting MMP proteolytic activation. Amprenavir was able to delay the growth of hepatocarcinoma xenografts in nude mice and had a synergistic effect with Doxorubicin. Furthermore, Amprenavir was able to promote regression of hepatocarcinoma growth in vivo by anti-angiogenetic and overall anti-tumor activities, independently by PI3K/AKT related pathways that at today is one of the more suggestive hypothesis to explain the anti-tumor effects of the different protease inhibitors. In summary these findings suggest novel anti-neoplastic action of Amprenavir on liver cancer showing the possibility of novel combination therapies.

Role of FAP48 in HIV-associated lipodystrophy.

The highly active antiretroviral therapy (HAART) can cause a metabolic syndrome consisting of lipodystropy/lipoatrophy, dyslipidemia, and type 2 diabetes mellitus with an increased cardiovascular risk. The pathogenetic bases of HAART-associated lipodystrophy are poorly known. A genetic screen was used to evaluate proteins that are modulated in HIV-1-infected patients with or without lipodystrophy syndrome, that are routinely treated with HAART regimens. The most significant modulation was represented by FAP48 expression. Stable over-expression of FAP48 was able to alter, in vitro, adipogenesis, acting both on calcineurin and glucocorticoid pathways. Finally, we demonstrated that FAP48 over-expression was able to influence the capacity of some HIV drugs, Saquinavir and Efavirenz, but not Stavudine, Amprenavir, and Indinavir to inhibit adipocyte formation. In conclusion, this molecule could be a potential target for novel therapeutic approaches to the HAART related lipodystrophy in HIV patients.

The Helicobacter pylori protein HspB interferes with Nrf2/Keap1 pathway altering the antioxidant response of Ags cells.

BACKGROUND: Helicobacter pylori infection causes chronic oxidative stress on gastric mucosa, thereby causing mucosal damage and increasing the risk of gastric adenocarcinoma. Nrf2 is an important transcription factor, regulating the antioxidant response in the cells. Nrf2 signaling is repressed by Keap1 at basal condition and induced by oxidative stress. The aim of our study was to analyze whether the H. pylori proteins interfered in the Nrf2/Keap1 pathway. MATERIAL AND METHODS: Gene expression in AGS cells transiently and stably transfected was analyzed by real-time PCR. Immunoprecipitation and immunofluorescence assays were performed to investigate the ability of H. pylori proteins to interfere with the Nrf2 pathway. RESULTS: We demonstrated that the H. pylori HspB protein interferes with Nrf2/Keap1 pathway. When HspB was transiently transfected in AGS cells, a significant increase in Keap1 gene expression was induced. The same result was observed when AGS cells were HspB stably transfected. In this case, the increase in Keap1 was associated with reduced gene expression of Nrf2, and of the antioxidant enzymes superoxide dismutase, hemeoxygenase-1, and phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase-1. Immunoprecipitation and immunofluorescence assays confirmed the ability of HspB protein to interfere with the Nrf2 pathway. Lastly, in HspB-transfected AGS cells, sustained activation of IL-8, COX2, MMP3, and MMP7 was demonstrated. CONCLUSION: The results here reported suggest that inhibited nuclear translocation of Nrf2, associated with induced inflammation and increased production of MMPs, might represent a condition enhancing the risk of gastric adenocarcinoma.

Propionibacterium acnes in urine and semen samples from men with urinary infection.

OBJECTIVE: Propionibacterium acnes has been implicated in the pathogenesis of prostate disease as acute and chronic prostatic inflammation, benign prostatic hyperplasia and prostate cancer although it should still be clarified if Propionibacterium acnes (P. acnes) is a commensal or accidental prostate pathogen. Aiming to evaluate the pathogenic potential for genitourinary tract of Propionibacterium acnes, we investigated the frequency of P. acnes genome in urine or semen samples from men with recurrent symptoms of urinary infection and negative testing for the most common urinary tract pathogens and sexually transmitted infections (STI) agents as Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum. MATERIALS AND METHODS: The DNA extracted from urine and semen samples was analyzed for evaluating the P. acnes genome presence by real-time polymerase chain reaction (PCR). Infections were treated with vancomycin and cephalosporins antibiotics and then the search for the P.acnes genome by realtime PCR was repeated. RESULTS: The P. acnes qualitative real-time PCR revealed the genome in 73 out of 159 samples examined (108 urine and 51 semen). After antibiotic therapy, P. acnes was never detected. CONCLUSIONS: These results suggested that P. acnes genome determination should be performed in cases of chronic inflammation in the urinary tract to identify an unknown potential pathogen of genitourinary tract.

Selective class II HDAC inhibitors impair myogenesis by modulating the stability and activity of HDAC-MEF2 complexes.

Histone deacetylase (HDAC) inhibitors are promising new epi-drugs, but the presence of both class I and class II enzymes in HDAC complexes precludes a detailed elucidation of the individual HDAC functions. By using the class II-specific HDAC inhibitor MC1568, we separated class I- and class II-dependent effects and defined the roles of class II enzymes in muscle differentiation in cultured cells and in vivo. MC1568 arrests myogenesis by (i) decreasing myocyte enhancer factor 2D (MEF2D) expression, (ii) by stabilizing the HDAC4-HDAC3-MEF2D complex, and (iii) paradoxically, by inhibiting differentiation-induced MEF2D acetylation. In vivo MC1568 shows an apparent tissue-selective HDAC inhibition. In skeletal muscle and heart, MC1568 inhibits the activity of HDAC4 and HDAC5 without affecting HDAC3 activity, thereby leaving MEF2-HDAC complexes in a repressed state. Our results suggest that HDAC class II-selective inhibitors might have a therapeutic potential for the treatment of muscle and heart diseases.

Role of NEDD8 in HIV-associated lipodystrophy.

The pathogenetic bases of HAART-associated lipodystrophy are still poorly known, even if it is clear that adipose tissue and its metabolism are sensitive to antiretroviral therapy alone and/or in combination with HIV infection. The NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression, morphogenesis and tumorigenesis. We investigated the possible involvement of NEED8 in adipogenesis and, consequently, in HIV-related lipodystrophy. One hundred HIV-1-infected patients were included in the study. Using an in vitro model of adipogenesis we evaluated the effects on adipogenesis of the forced expression of NEDD8 together with efavirenz, stavudine, saquinavir, amprenavir and indinavir, belonging to the three main classes of anti-HIV medications. We showed that NEDD8 expression level is higher in the peripheral blood of HIV patients developing lipodystrophy. Coherently, forced expression of NEDD8 in an in vitro model of adipogenesis was able to perturb expression of some key proteins involved in adipogenesis, such as C/EBPalpha and PPARgamma, possibly acting throughout the NEDD8/p27/beta-catenin pathway. Moreover, three out of five evaluated drugs were able to affect adipocyte differentiation: efavirenz, stavudine and saquinavir. Finally, we have shown that NEDD8 was expressed in the fat tissue of lipodystrophic patients, being significantly higher in the lipodystrophic patients with respect to the controls, thus further confirming the altered NEDD8 expression in the fat tissue of HIV-infected patients affected by lipodystrophy. Taken together, our data support the hypothesis of an implication of NEDD8 through p27 and beta-catenin pathways in the disruption of adipogenesis and consequent lipodystrophy in patients affected by HIV infection under HAART therapy with qualitative and quantitative differences according to diverse antiretroviral treatments. These evidences indicate the NEDD8/beta-catenin/p27 pathway as a possible molecular target for prevention of lipodystrophy development in patients under HAART therapy.

OPLA scaffold, collagen I, and horse serum induce an higher degree of myogenic differentiation of adult rat cardiac stem cells.

In the last few years, a major goal of cardiac research has been to drive stem cell differentiation to replace damaged myocardium. Several research groups have attempted to differentiate potential cardiac stem cells (CSCs) using bi- or three-dimensional systems supplemented with growth factors or molecules acting as differentiating substances. We hypothesize that these systems failed to induce a complete differentiation because they lacked an architectural space. In the present study, we isolated a pool of small proliferating and fibroblast-like cells from adult rat myocardium. The phenotype of these cells was assessed and the characterized cells were cultured in a collagen I/OPLA scaffold with horse serum to obtain fine myocardial differentiation. C-Kit(POS)/Sca-1(POS) CSCs fully differentiated in vitro when an environment more similar to the CSC niche was created. These experiments demonstrated an important model for the study of the biology of CSCs and the biochemical pathways that lead to myocardial differentiation. The results pave the way for a new surgical approach.

Helicobacter pylori heat shock protein B (HspB) localizes in vivo in the gastric mucosa and MALT lymphoma.

Heat shock protein B (HspB) is one of the dominant proteins recognized by most Helicobacter pylori-infected persons and is being considered as potential candidates for subunit vaccines. In the present study we describe the generation of an antibody against HspB and its use in immunohistochemical assays on gastric biopsies. We have demonstrated that our rabbit polyclonal antibody against HspB did not recognize any protein in lysates from a lung human epithelial cell (H1299) line and did not cross-react with the other members of human heat shock proteins. Secondly, we have observed that in gastric biopsies, HspB immunostaining was present inside the cytoplasm of human epithelial cells with a particular localization in the apical portion of gastric epithelial cells other than in the extracellular spaces among gastric cells of human stomach. Finally, we have demonstrated a cytoplasmic HspB immunostaining in groups of neoplastic cells of MALT lymphoma. In conclusion, our observations suggest a possible involvement of HspB in the pathogenesis of H. pylori-related pathologies such as gastritis, ulcer and gastric cancer.

The pattern of expression of Notch protein members in normal and pathological endometrium.

The objective of this study was to investigate the pattern of expression and the localization of Notch-1, Notch-4 and Jagged-1 in physiological and pathological human endometrium and to evaluate the expression levels of two major regulators of the G1 checkpoint, namely cyclin D1 and p21. Sixty samples of physiological endometrium and 60 samples of pathological endometrium were used for the study. Evaluation of the expression level and the distribution of Notch pathway members and cell-cycle proteins was performed by immunohistochemistry. In the physiological endometrium we observed an increase of Notch-1 and Jagged-1 from proliferative to secretory phase and an opposite trend for Notch-4. In menopause, the level of expression of all three members of the Notch pathway decreased. We also observed a cyclin D1 increase from proliferative to secretory phase. By contrast, p21 showed a slight increase from proliferative to secretory phase. In the pathological endometrium, we observed an increase of Notch-1 expression from polyps to carcinoma and decrease for Notch-4 and Jagged-1. Moreover, we observed a higher expression of cyclin D1 in all the endometrial pathologies. By contrast, the expression level of p21 slightly increased from polyps to carcinoma. We concluded that in human endometrium Notch-4 seems to be more involved in controlling proliferation, whereas Notch-1 seems to be more involved in differentiation programming. Deregulation of these functions may induce the onset of several endometrial pathologies from polyps to cancer.

Modulation of Bax expression in physiological and pathological human placentas throughout pregnancy.

Apoptosis is intimately involved in placental homeostasis, growth and remodelling, and apoptotic rates increase progressively during normal pregnancy as part of normal placental development. Moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction and diabetes. In the present study, we describe differences in the expression of proapoptotic protein Bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. We first observed a strong increase of Bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate Bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. Secondly, we showed a more intense immunopositivity for Bax in the third trimester spontaneous birth with respect to the third trimester caesarean birth. Thirdly, we observed an increase of Bax expression in preeclamptic placentas compared to the normal full-term placentas. In contrast, we observed a moderate Bax expression in diabetic placentas only slightly lower than the normal full-term placentas. Our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies.

Molecular evidence of apoptotic pathway activation in semen samples with high DNA fragmentation.

BACKGROUND/AIM: Male infertility is diagnosed by semen parameters, such as concentration, motility and morphology; however, these are not sufficient for the prediction of male fertility capacity. In the clinical routine, several other sperm functions have been introduced, including the sperm DNA fragmentation test. The objective of the present study was to evaluate sperm chromatin integrity in semen samples. MATERIALS AND METHODS: Sperm chromatin dispersion test (SCD) was used in ejaculates from men divided into five groups: normozoospermic, oligozoospermic, asthenozoospermic, oligoasthenozoospermic and cryptozoospermic. RESULTS: The data obtained showed that the SCD percentage appeared to be significantly associated with oligozoospermia diagnosis. We also evaluated total testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and inhibin B serum hormonal levels in all samples examined, in order to assess whether DNA fragmentation increase could correlate with abnormal hormonal values. Finally we selected certain samples with an increasing DNA fragmentation and analyzed the molecular activated apoptotic pathways. CONCLUSION: A significant relationship was found between caspase-3 activation and increased DNA fragmentation.

Suppression of pre adipocyte differentiation and promotion of adipocyte death by anti-HIV drugs.

In the present study, we investigated the ability of anti-HIV drugs to interfere with normal cell cycle progression and to induce oxidative stress by perturbing the redox environment. Our results provide evidence that anti-HIV drugs have a differential effect on adipocyte cell cycle and differentiation, being able to modify the response to oxidative stress through an increase of reactive oxygen species (ROS) that compromises the induction of phase-2 and antioxidant enzymes. In detail, saquinavir, efavirenz, and stavudine exert antiadipogenic influences on the model 3T3-L1 cell line, perturbing the oxidative response and inducing of apoptosis. When considered together, the effects of anti-HIV drugs on 3T3-L1 pre adipocytes are distinct but commonly antiadipogenic, thus suggesting another additional possible mechanism by which antiretroviral therapies could contribute to lipoatrophy.

Localization and distribution of wolframin in human tissues.

Wolframin is a transmembrane glycoprotein of 890 aminoacids, encoded by WFS1 gene. WFS1 mutations are responsible for Wolfram syndrome, an autosomal recessive disorder. In the present paper, we first characterized the polyclonal wolframin antibody by dot blot. Secondly, we verified antibody specificity by western blotting using different human cell lines. Thirdly, we studied wolframin localization in human foetal (14-35 weeks) and adult tissues by immunohistochemistry. Wolframin expression was distributed in many organs, with different tissue and cell localization and expression levels. In foetal systems, wolframin expression was faint at 14-16 weeks and increased when development proceeded. In adult human tissues a variable positive staining was observed in both simple and stratified epithelia. A moderate wolframin expression was observed in liver and in the endocrine portion of the pancreas. In conclusion, our data suggest that this protein may have important roles in a number of different tissues, including many that are not known to be affected by WFS1-linked diseases. The immunopositivity in adult human tissues suggests that it may function maintaining physiological cellular homeostasis.

Comparative transcriptional profiling in HIV-infected patients using human stress arrays: clues to metabolic syndrome.

Highly active antiretroviral therapy (HAART therapy) for HIV-1 infection has significantly increased the survival and quality of life of patients with this disease. However, in several epidemiological studies the onset of metabolic syndrome is a phenomenon reported to be extremely frequent. In the present study, genes involved in the molecular cascade responsible for the alteration of fat tissue and of lipid and glucose metabolism in patients with HIV-1 infection treated with antiretroviral therapy were identified. Towards this goal, hybridization using Atlas cDNA Expression Arrays allowed simultaneous monitoring of the expression levels of approximately 250 genes and identification of a panel of changes in relation to different therapeutic groups and in the presence of metabolic syndrome, with some genes being up-regulated, while others are down-regulated in the different subgroups of patients. The results of this analysis have shown a panel of transcriptional changes associated with oxidative stress mechanisms that provide a basis for further studies on understanding of mechanisms that, in vivo, are the foundation the metabolic disorders in patients with HIV infection.

Identification of protein-protein interactions of human HtrA1.

The human heat shock protein HtrA1, a member of the HtrA family of serine proteases, is a evolutionarily highly conserved factor which displays a widespread pattern of expression. The yeast two-hybrid technique was employed to identify new cellular proteins physically interacting with HtrA1, and thus potential targets of this serine protease. An enzymatically inactive HtrA1 point mutant, HtrA1-S328A, was generated and used as bait in a yeast two-hybrid system. Fifty-two plasmids were isolated from primary positive yeast clones. Subsequent sequencing and BLAST analysis revealed cDNAs encoding for 13 different proteins. These putative binding partners of HtrA1 appeared to be a) components of extracellular matrix; b) factors related to signal pathways, and c) unknown proteins. Among the 13 positive clones identified and reported here, it is worth of note that the interaction of HtrA1 with tubulin and collagen (extracellular matrix proteins) and with tuberin (cytoplasmic protein) is confirmed by other studies, and this further supports previous findings in which HtrA1 can be found active as an intracytoplasmic protein or as secreted protein as well.

The serine protease HtrA1 specifically interacts and degrades the tuberous sclerosis complex 2 protein.

Hamartin and tuberin are products of the tumor suppressor genes TSC1 and TSC2, respectively. Mutations affecting either gene result in the tuberous sclerosis syndrome, a neurologic genetic disorder characterized by the formation of multiple benign tumors or hamartomas. In this study, we report the identification of TSC2, but not TSC1, as a substrate of HtrA1, a member of the human HtrA family proteins of serine proteases. We show the direct interaction and colocalization in the cytoplasm of HtrA1 and TSC2 and that HtrA1 cleaves TSC2 both in vitro and in vivo. Finally, we show that alterations in HtrA1 expression cause modifications in phosphorylation status of two downstream targets of TSC2: 4E-BP1 and S6K. Our data suggest that, under particular physiologic or pathologic conditions, HtrA1 degrades TSC2 and activates the downstream targets. Considering that HtrA1 levels are significantly increased during embryogenesis, we speculate that one of the targets of HtrA1 activity during fetal development is the TSC2-TSC1 pathway.

Human papillomavirus-16 E7 interacts with Siva-1 and modulates apoptosis in HaCaT human immortalized keratinocytes.

The viral factor E7 plays a key role in the well-established association between "high-risk" Human Papillomavirus (HPV) infection and the development of epithelial malignant tumors, as uterine cervix and ano-genital cancer. To delve into the molecular mechanisms of HPV-mediated cell transformation, we searched for novel potential cellular targets of the HPV-16 E7 oncoprotein, by means of the yeast two-hybrid technique, identifying a protein-protein interaction between HPV-16 E7 and the pro-apoptotic cellular factor Siva-1. Using co-precipitation assays and the "PepSets" technique, we confirmed this physical interaction and mapped accurately, for both proteins, the amino acid residues involved. Additionally, we found that HPV-16 E7 competed in vitro with the binding of the Bcl-X(L) anti-apoptotic factor to Siva-1, an interaction that has a major inference in UV radiation-induced apoptosis. In HaCaT immortalized human keratinocytes, forced HPV-16 E7 expression by retroviral infection caused Siva-1 transcript up-regulation, detected by cDNA macroarray hybridization and real-time quantitative PCR, paralleled by an increased amount of protein. Confirming the anti-apoptotic role of HPV-16 E7 in the HaCaT cellular model, evaluated by nuclear morphology, we also found that Siva-1 expression produced a significant increase of the apoptotic rate in UV radiation-exposed HaCaT cells, and that this effect resulted explicitly counteracted by HPV-16 E7. Being apoptosis a key physiological process for the elimination of irreversibly injured cells, the anti-apoptotic role of HPV-16 E7, performed at least by its interference with Siva-1, can be considered an additional mechanism for the survival of damaged, potentially transforming, cell clones.

Distribution of Notch protein members in normal and preeclampsia-complicated placentas.

Notch proteins are a transmembrane receptor family that is structurally and functionally conserved from worms to humans. The mammalian family of Notch proteins consists of several genes encoding Notch receptors and related Notch ligands. Notch signaling is involved in different aspects of the cell-fate decision tree: differentiation, proliferation, and apoptosis. These three processes are finely regulated in human placenta in order to allow a successful pregnancy and correct fetal growth. Notch and its ligands also participate in vascular remodeling and stabilization. Vasculogenesis and blood regulation are of importance in the human placenta for normal fetal development and growth; any disorder of these systems leads to preeclampsia. Drawing on this background, we have investigated the expression of Notch-1, Notch-4, and Jagged-1, together with two members related to the Notch pathway in angiogenesis: VEGF and p21. Normal and preeclamptic human placentas have been evaluated by immunohistochemistry. In preeclamptic samples, a down-regulation of Notch pathway members occurs with a weak/moderate expression of the Notch protein members in all components of placenta compared with physiological placentas that, at term, exhibit the strong expression of Jagged-1 and a moderate expression of both Notch-1 and Notch-4 in all compartments of the placental villi. Moreover, preeclamptic samples also reveal a down-regulation of VEGF expression, together with a moderate nuclear expression of p21(Cip1) in the syncytiotrophoblast, cytotrophoblast, and endothelial cells. This down-regulation of VEGF in preeclamptic placentas, in turn, probably decreases Notch protein expression in placental compartments and in endothelial cells and could offer an ethiopathogenetic explanation for the onset of this pathology.

Pkn is a novel partner of cyclin T2a in muscle differentiation.

With the aim to find novel partners of human Cyclin T2a, we performed a two-hybrid screening in yeast using the full-length cDNA of this cyclin as bait, and a human heart cDNA library as preys source. Upon several interesting genes selected, our attention has been focused on the cDNA coding for PKNalpha, a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. Co-immunoprecipitation and in vitro pull-down assays independently confirmed the interaction between the two proteins. Luciferase assays, performed on NIH3T3 cell extracts after transfection with a MyoD-responsive promoter, pointed out that PKNalpha was able to enhance MyoD-dependent transcription, and that this effect was further increased when cyclin T2a was co-overexpressed. Finally, overexpression of both Cyclin T2a and PKNalpha in C2C12 cells strongly enhanced the expression of myogenic differentiation markers, such as Myogenin and Myosin Heavy Chain, during starvation-induced differentiation. Taken together, our data strengthen the hypothesis that Cyclin T2a plays a role in muscle differentiation, and propose PKNalpha as a novel partner of Cyclin T2a in this process.