Unfolded protein response, a link between endometrioid ovarian carcinoma and endometriosis: A pilot study.

The aim of the present study was to analyze the expression profile of unfolded protein response (UPR) genes in endometrioid ovarian carcinoma and to evaluate its possible involvement in the neoplastic progression of endometriosis. An experimental retrospective pilot study was conducted on women with a diagnosis of endometrioid ovarian carcinoma at FIGO stage IA, ovarian endometriotic cysts or healthy subjects without a previous diagnosis of endometriosis. The expression profiles of UPR genes (ATF6, GRP78, CHOP and XBP1) were compared among ovaries with endometrioid ovarian cancer, endometriotic ovarian cysts, healthy contralateral ovaries and eutopic and healthy endometrial tissues. A significantly higher expression of ATF6 and GRP78 was detected in the affected ovaries in comparison with the healthy contralateral ovaries, while CHOP and XBP1 exhibited a significantly lower expression. XBP1 was overexpressed in endometrial tissues and its expression gradually decreased in endometriosis cysts and endometrioid ovarian carcinoma. These results support the hypothesis that alterations in the UPR genes CHOP and XBP1 are involved in the neoplastic progression of endometrioid ovarian cancer and are acquired following ovarian localization of ectopic endometrial cells.

Expression of extracellular matrix and adhesion proteins in pelvic organ prolapse.

Pelvic organ prolapse (POP) is a common disorder in women. It is characterized by the descent of the vaginal wall with consequent drop of pelvic organs. Pregnancy, labour and childbirth seem to be important events leading to the development of POP, since they are associated with prolonged stretch and mechanical stress of muscles, ligaments and connective tissue supporting pelvic organs. In pubocervical fascia, we explored the expression level of extracellular matrix and adhesion molecules. Tissue samples were obtained from twenty patients with POP who underwent cystocele repair, and from twenty control subjects during hysterectomy surgery. The PCR array analysis was performed and data were confirmed by Real-Time PCR and Western Blot.  Real-Time PCR results showed a significant upregulation for extracellular matrix protein 1 (ECM1) and integrin beta 3 (ITGB3) and a significant downregulation for FBLN5 in POP group. The decreased mRNA expression of FBLN5 in pathological samples was paralleled by a quantitative decrease in the corresponding protein, as Western Blot test highlighted. Our data provide an understanding of molecular mechanisms involved in POP-related pathophysiological processes and might represent an important tool to develop novel therapeutic agents for the treatment of this condition.

Exosomal miR-126 as a circulating biomarker in non-small-cell lung cancer regulating cancer progression.

Lung cancer is one of the leading causes of cancer-related deaths. It is diagnosed mostly at the locally advanced or metastatic stage. Recently, micro RNAs (miRs) and their distribution in circulation have been implicated in physiological and pathological processes. In this study, miR-126 was evaluated in serum, exosome and exosome-free serum fractions in non-small cell lung cancer (NSCLC) patients at early and advanced stages, and compared with healthy controls. Down-regulation of miR-126 was found in serum of advanced stage NSCLC patients. In healthy controls, circulating miR-126 was equally distributed between exosomes and exosome-free serum fractions. Conversely, in both early and advanced stage NSCLC patients, miR-126 was mainly present in exosomes. Different fractions of miR-126 in circulation may reflect different conditions during tumour formation. Incubation of exosomes from early and advanced NSCLC patients induced blood vessel formation and malignant transformation in human bronchial epithelial cells. On the other hand, exosome-enriched miR-126 from normal endothelial cells inhibited cell growth and induces loss of malignancy of NSCLC cells. These findings suggest a role of exo-miRs in the modulation of the NSCLC microenvironmental niche. Exosome-delivered miRs thus hold a substantial promise as a diagnostics biomarker as well as a personalized therapeutic modality.

Potential Role of Placental Klotho in the Pathogenesis of Preeclampsia.

The aim of this study was to analyze the placental expression and allele status of promoter region of Klotho in association with preeclampsia, which represents the most common hypertensive disease of pregnancy. Klotho mRNA and protein levels were determined using real-time PCR and Western blot, respectively, in placental tissue samples obtained from 34 patients affected with preeclampsia and 34 controls. A PCR-based genotyping analysis was carried out in the promoter region of Klotho gene. Moreover, expression levels of pluripotency markers, Nanog and Oct4, and telomere length were assessed using real-time PCR. Klotho mRNA and protein levels were reduced in preeclamptic placentas compared with controls. -744delA single-nucleotide polymorphism was significantly associated with preeclampsia. In pathological placentas, there was a downregulation of pluripotency markers and a reduced telomere length. This study is the first to evaluate the placental expression level of Klotho in association with preeclampsia. Further analyses will clarify its role in the pathogenesis of this pregnancy hypertensive disorder.

Placental expression of endothelial and inducible nitric oxide synthase and NO metabolism in gestational hypertension: a case-control study.

OBJECTIVE: Hypertension is one of the most common medical disorders in pregnancy and a role of nitric oxide (NO) metabolism has been described. Thus, the present work aimed at determining placental gene expression of eNOS and iNOS, to measure NO and ONOO(-) levels in patients with gestational hypertension (GH). METHODS: Fifteen patients with GH and 15 healthy pregnant controls were enrolled in the study. Placental tissue was taken immediately after delivery and was stored at -80 °C until analysis. A piece of frozen tissue was homogenized in the appropriate buffer. Total RNA was extracted and was reverse transcribed to obtain complementary DNA that was used for real-time PCR for iNOS and eNOS expression, whereas NO and ONOO(-) production were measured by commercially available kits. RESULTS: Placental eNOS and iNOS mRNA levels were significantly reduced in GH when compared to controls. NO and ONOO(-) production were both significantly higher in GH than controls. CONCLUSIONS: The reduced eNOS and iNOS gene expression in women with GH reinforces the hypothesis that the mechanisms involving NO pathways, may promote oxidative damage, by contributing to the reduced blood flow and increased resistance in the feto-maternal circulation and suggests the use of NO modulators as useful tools in GH management.

Identification and characterization of cancer stem cells from head and neck squamous cell carcinoma cell lines.

BACKGROUND/AIMS: Head and neck squamous cell carcinoma (HNSCC) ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs), has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. METHODS: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed in several malignancies. RESULTS: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. CONCLUSION: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy.

Clues to apoptosis pathway involvement in hemolysis, elevated liver enzyme, and low platelet (HELLP) syndrome and intrauterine growth restriction (IUGR).

OBJECTIVE: The neurotrophin family comprises molecules involved in growth, differentiation, survival, regeneration, normal functions of the neuronal system, and in angiogenesis. We have investigated the expression pattern of neurotrophic signaling molecules in pregnancies complicated by elevated liver enzyme, and low platelet (HELLP) syndrome and intrauterine growth restriction (IUGR). METHODS: Placentas from normal and pathological pregnancies were collected. Macroarray analysis was performed and the data were confirmed by real-time PCR. RESULTS: Real-time PCR analyses (pathological vs. normal pregnancies) confirmed a significant down-regulation for IL-6, STAT3α, STAT3ß, and Bcl-2. The expression of Mcl-1 isoform 1 (long) was significantly increased. CONCLUSIONS: We suggest that decreased expression of IL-6 could mean that abnormalities in the immunological system function involve inflammatory cytokines other than IL-6 in examined pathological pregnancies. The STAT3α and STAT3ß down-regulation lead to a marked reduction of cellular transcriptional activity. Decreased expression of IL-6 is associated with a down-regulation of Bcl-2 but not of Mcl-1 isoform 1, suggesting that these two antiapoptotic proteins may function independently and that Mcl-1 may have a distinct role in controlling apoptotic pathway.

PP008. Placental klotho gene in preeclampsia.

INTRODUCTION: An aging-suppressor gene, klotho, is a candidate factor for vascular disease because its deficiency leads to impaired endothelium-dependent vasodilation and impaired angiogenesis. This protein is involved in several metabolic pathways such as the insulin-like growth factor 1 (IGF-1), apoptosis, angiotensin-II-induced events in the kidney and oxidative stress. OBJECTIVES: The aim of this study was to determine the difference of klotho genotiping and expression in the placentas of women with normal and preeclamptic pregnancies. METHOD/DESIGN: Placental tissue was collected from normal pregnancies (n=12) and pregnancies complicated by Preeclampsia (n=12), matched for gestational age. Klotho genotyping and expression was determined using real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. RESULTS: A polymorphism for -744 G/A mutation was significantly more common in the pathological group, with an odds ratio (OR) of 3.00 (1.02-8.81; 95% CI). The expression levels of both klotho isoforms and of the short klotho isoform were lower (80%) in the Preeclampsia group as compared to matched controls. Results of Western Blot agreed with those from Real-Time PCR. CONCLUSION: In preeclamptic pregnancies there are a genotyping polymorphism and a reduced expression of klotho gene. Given its role in cardiovascular disease in aging, it may link preeclamptic mothers and their offsprings to long term cardiovascular outcomes.

The involvement of inflammatory cytokines in the pathogenesis of recurrent miscarriage.

OBJECTIVE: To investigate the inflammatory cytokine expression pattern in trophoblastic tissue from women with unexplained recurrent miscarriage (RM). STUDY DESIGN: Trophoblasts were obtained during uterine evacuation from 11 women with RM and from 20 healthy pregnant women undergoing elective termination of pregnancy, who served as controls. The array was performed using GEArray Q Series Human Inflammatory Cytokines & Receptors Gene Array HS-015 membranes. Data were confirmed by quantitative real-time PCR. The Mann-Whitney U test was performed for statistical analysis. RESULTS: Microarray analysis identified three genes that were differentially expressed between RM patients and controls. We observed significant downregulation of Transforming Growth Factor beta 3 (TGF-ß3) and Interleukin 25 (IL-25) (5-fold reduction and 2.5-fold reduction, respectively) and significant upregulation of CD-25, also known as Interleukin 2 receptor alpha (IL-2RA) (7-fold increase) in women with RM compared with controls. The median ΔC(t) of TGF-ß3 was 8.2 (interquartile range, 7.67-8.9) in RM patients vs. 5.85 (interquartile range, 5.3-6.09) in controls; the median ΔC(t) of IL-25 was 5.18 (interquartile range, 4.46-5.76) in RM patients vs. 3.85 (interquartile range, 3.6-4.51) in controls, and the median ΔC(t) of CD-25 was 9.62 (interquartile range, 7.81-12.42) in RM patients vs. 12.44 (interquartile range, 11.02-13.86) in controls. DISCUSSION: Our results suggest that the immunological and inflammatory regulation mechanisms of the placental environment play a key role in recurrent miscarriage. The observed trophoblast cytokine expression pattern at the maternal-fetal interface confirms the immunotrophic theory, as demonstrated by a switch from a T-helper-1 (Th1) profile to a T-helper-2 (Th2) profile in women who experience recurrent miscarriages.

Bioinformatic analyses to select phenotype affecting polymorphisms in HTR2C gene.

OBJECTIVE: Single nucleotide polymorphisms (SNPs) in serotonin related genes influence mental disorders, responses to pharmacological and psychotherapeutic treatments. In planning association studies, researchers that want to investigate new SNPs have to select some among a large number of candidates. Our aim is to guide researchers in the selection of the most likely phenotype affecting polymorphisms. Here, we studied serotonin receptor 2C (HTR2C) SNPs because, till now, only relatively few of about 2000 are investigated. METHODS: We used the most updated and assessed bioinformatic tools to predict which variations can give rise to biological effects among 2450 HTR2C SNPs. RESULTS: We suggest 48 SNPs that are worth considering in future association studies in the field of psychiatry, psychology and pharmacogenomics. Moreover, our analyses point out the biological level probably affected, such as transcription, splicing, miRNA regulation and protein structure, thus allowing to suggest future molecular investigations. CONCLUSIONS: Although few association studies are available in literature, their results are in agreement with our predictions, showing that our selection methods can help to guide future association studies.

Placental expression of endothelial and inducible nitric oxide synthase and nitric oxide levels in patients with HELLP syndrome.

OBJECTIVE: To determine placental gene expression of endothelial and inducible nitric oxide synthases and measure nitric oxide levels in patients with hemolysis, elevated liver enzyme levels, and low platelet count syndrome. STUDY DESIGN: Preterm placentas were obtained from 15 patients with hemolysis, elevated liver enzyme levels, and low platelet count syndrome and 30 controls matched for age, parity, and gestational age. mRNA levels were evaluated by real-time polymerase chain reaction, whereas nitric oxide and peroxynitrite production was measured by a commercially available kit. RESULTS: Placental gene expression of inducible nitric oxide and endothelial nitric oxide synthases were significantly lower in the hemolysis, elevated liver enzyme levels, and low platelet count syndrome group than in controls, whereas nitric oxide and peroxynitrite production were significantly higher in hemolysis, elevated liver enzyme levels, and low platelet count syndrome compared with controls. CONCLUSION: The reduced endothelial nitric oxide and inducible nitric oxide synthases gene expression in women with hemolysis, elevated liver enzyme levels, and low platelet count syndrome may indicate extreme placental dysfunction that is unable to compensate the endothelial derangement and the related hypertension. The higher nitric oxide formation found in hemolysis, elevated liver enzyme levels, and low platelet count syndrome placentas could be explained as a counteraction to the impaired fetoplacental perfusion, typical of the syndrome.

Asbestos exposure affects poly(ADP-ribose) polymerase-1 activity: role in asbestos-induced carcinogenesis.

Asbestos is known to induce malignant mesothelioma (MM) and other asbestos-related diseases. It is directly genotoxic by inducing DNA strand breaks and cytotoxic by promoting apoptosis in lung target cells. Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear zinc-finger protein with a function as a DNA damage sensor. To determine whether PARP1 is involved in asbestos-induced carcinogenesis, PARP1 expression and activity as well as DNA damage and repair were evaluated in circulating cells of asbestos-exposed subjects, MM patients and age-matched controls. PARP1 expression and activity were also evaluated in pleural biopsies of MM patients and compared with normal tissue. Accumulation of the pre-mutagenic 8-hydroxy-2'-deoxyguanosine and elevated PARP1 expression were found both in asbestos-exposed subjects and MM patients. Although PARP1 was highly expressed, its activity was relatively low. Low DNA repair efficiency was observed in lymphocytes from MM patients. High expression of PARP1 associated with low PARP activity was also found in MM biopsies. To mimic PARP1 dysfunction, PARP1 expression and activity were induced in immortalised mesothelial cells by their exposure to asbestos in the presence of a PARP1 inhibitor, which resulted in transformation of the cells. We propose that exposure to asbestos inhibits the PARP1 activity possibly resulting in higher DNA instability, thus causing malignant transformation.

Thrombomodulin is silenced in malignant mesothelioma by a poly(ADP-ribose) polymerase-1-mediated epigenetic mechanism.

Malignant mesothelioma (MM) is often complicated by thromboembolic episodes, with thrombomodulin (TM) playing a critical role in the anticoagulant process. Heterogeneous expression of TM has been observed in cancer, and low or no TM expression in cancer cells is associated with poor prognosis. In this study, we analyzed TM expression in biopsies of MM patients and compared them with normal mesothelial tissue. The role of DNA methylation-associated gene silencing in TM expression was investigated. To evaluate poly(ADP-ribose) polymerase-1 (PARP1) as responsible for gene promoter epigenetic modifications, nonmalignant mesothelial cells (Met-5A) and MM cells (H28) were silenced for PARP1 and the DNA methylation/acetylation-associated TM expression evaluated. A correlation between low TM expression and high level of TM promoter methylation was found in MM biopsies. Low expression of TM was restored in MM cells by their treatment with 5-aza-2'-deoxycytidine and, to a lesser extent, with trichostatin, whereas the epigenetic agents did not affect TM expression in Met-5A cells. Silencing of PARP1 resulted in a strong down-regulation of TM expression in Met-5A cells, while restoring TM expression in H28 cells. PARP1 silencing induced TM promoter methylation in Met-5A cells and demethylation in MM cells, and this was paralleled by corresponding changes in the DNA methyltransferase activity. We propose that methylation of the TM promoter is responsible for silencing of TM expression in MM tissue, a process that is regulated by PARP1.

HLA-G and pregnancy adverse outcomes.

There is growing evidence that pregnancy complications such as preeclampsia, recurrent pregnancy loss (RPL), IUGR, and premature birth could be associated with abnormal immunologic interactions at the fetal-maternal interface. The restricted expression of HLA-G to the subpopulation of trophoblast cells which invade the uterus has generated much interest. The alternative splicing of HLA-G primary transcript, gives origin to seven isoforms, including both membrane-bound forms (HLA-G1, G2, G3, G4) and soluble forms (sHLA-G: sHLA-G5, G6, G7). sHLA-G consists predominantly of sHLA-G1 after its shedding by metalloproteinases, and secreted sHLA-G5 representing the quantitatively dominating and full-length isoforms. HLA-G expression and HLA-G genetic variations in both the mother and the embryo/fetus may be important for pregnancy outcome. It is also intuitively apparent that a gene with putative immunosuppressive and immunotolerant potential might be functional in both the mother and the embryo/fetus/placenta. Reduced or aberrant HLA-G expression seems to be associated with certain complications of pregnancy, among which preeclampsia and possibly the risk of miscarriage, and that this may be further linked to HLA-G polymorphisms. Most of the studies aimed at assessing the role of HLA-G in pregnant diseases have considered only the maternal genotype and ignored the contribution of the fetus. In this regard, the mother, placenta and the fetus form a synthesis. Therefore, studies on placental diseases should address HLA-G expression and genetic variations also to the fetus/placenta.

Unexplained fetal loss: the fetal side of thrombophilia.

Carrier status of the fetus for factor V polymorphism or double homozygosity for mutant alleles of the PAI-1 4 G/4 G and MTHFR T677 T polymorphisms must be considered risk factors for intrauterine fetal death. The clinical implications of these data need to be addressed in a prospective study to confirm our preliminary data and to answer the question of whether or not double homozygous individuals should be treated with low molecular-weight heparin and/or low-dose aspirin.

The role of p38alpha mitogen-activated protein kinase gene in the HELLP syndrome.

Mitogen-activated protein kinase (MAPK) p38alpha was shown to be implicated in the organogenesis of the placenta, and such placental alteration is crucial for the development of hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. We aimed to analyze for the first time human placental expression of MAPK p38alpha in pregnancies complicated by HELLP. The placental expression of MAPK p38alpha was investigated by semiquantitative polymerase chain reaction using cDNA extracted from placental tissue of 15 pregnancies with HELLP syndrome and 15 gestational age-matched controls. Seven patients with HELLP also had intrauterine fetal growth restriction (IUGR). In placenta from pregnancy complicated by HELLP, the expression of MAPK p38alpha is significantly decreased compared to the group with normal pregnancy (p < 0.001), while no difference was found between the HELLP and HELLP with IUGR subpopulations. Our study shows for the first time that MAPK p38alpha is expressed in the human placenta. Pregnancies with placental dysfunction and hypertensive complications are characterized by a significantly decreased expression of MAPK p38alpha. Our observations suggest that p38 MAPK signaling may be essential in placental angiogenesis and functioning.

Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque.

BACKGROUND: The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. RESULTS: A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of approximately 42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. CONCLUSION: This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.

Effect of acidic phospholipids on the structural properties of recombinant cytosolic human glyoxalase II.

A peculiar characteristic of highly concentrated cytosolic recombinant human glyoxalase II (GII) solutions is to undergo partial precipitation. Previous work indicated that anionic phospholipids (PLs) exert a noncompetitive inhibition on the enzymatic activity of the soluble enzyme. In this study, FTIR spectroscopy was used to analyze the structural properties and the thermal stability of the soluble protein in the absence and in the presence of liposomes made of different phospholipids (PLs). The structural analysis was performed on the precipitate as well. The interaction of acidic PLs with GII lowered the thermal stability of the enzyme and inhibited protein intermolecular interactions (aggregation) brought about by thermal denaturation. Infrared data indicated that ionic and hydrophobic interactions occur between GII and acidic PLs causing small changes in the secondary structure of the enzyme. No interactions of the protein with egg phosphatidylcholine liposomes were detected. The results are consistent with the destabilization of the protein tertiary structure, and indicate that GII possesses hydrophobic part(s) that interact with the acyl chains of PLs. Data on precipitated GII did not show remarkable modification of secondary structure, suggesting that hydrophobic stretches of the enzyme may also be involved in the protein-protein association (precipitation) at high GII concentration. The alterations in the GII structure and the noncompetitive inhibition exerted by acidic PLs are strictly related.