Parthenolide and DMAPT exert cytotoxic effects on breast cancer stem-like cells by inducing oxidative stress, mitochondrial dysfunction and necrosis.

Triple-negative breast cancers (TNBCs) are aggressive forms of breast carcinoma associated with a high rate of recidivism. In this paper, we report the production of mammospheres from three lines of TNBC cells and demonstrate that both parthenolide (PN) and its soluble analog dimethylaminoparthenolide (DMAPT) suppressed this production and induced cytotoxic effects in breast cancer stem-like cells, derived from dissociation of mammospheres. In particular, the drugs exerted a remarkable inhibitory effect on viability of stem-like cells. Such an effect was suppressed by N-acetylcysteine, suggesting a role of reactive oxygen species (ROS) generation in the cytotoxic effect. Instead z-VAD, a general inhibitor of caspase activity, was ineffective. Analysis of ROS generation, performed using fluorescent probes, showed that both the drugs stimulated in the first hours of treatment a very high production of hydrogen peroxide. This event was, at least in part, a consequence of activation of NADPH oxidases (NOXs), as it was reduced by apocynin and diphenylene iodinium, two inhibitors of NOXs. Moreover, both the drugs caused downregulation of Nrf2 (nuclear factor erythroid 2-related factor 2), which is a critical regulator of the intracellular antioxidant response. Prolonging the treatment with PN or DMAPT we observed between 12 and 24 h that the levels of both superoxide anion and hROS increased in concomitance with the downregulation of manganese superoxide dismutase and catalase. In addition, during this phase dissipation of mitochondrial membrane potential occurred together with necrosis of stem-like cells. Finally, our results suggested that the effect on ROS generation found in the first hours of treatment was, in part, responsible for the cytotoxic events observed in the successive phase. In conclusion, PN and DMAPT markedly inhibited viability of stem-like cells derived from three lines of TNBCs by inducing ROS generation, mitochondrial dysfunction and cell necrosis.

Parthenolide generates reactive oxygen species and autophagy in MDA-MB231 cells. A soluble parthenolide analogue inhibits tumour growth and metastasis in a xenograft model of breast cancer.

Triple-negative breast cancers (TNBCs) are clinically aggressive forms associated with a poor prognosis. We evaluated the cytotoxic effect exerted on triple-negative MDA-MB231 breast cancer cells both by parthenolide and its soluble analogue dimethylamino parthenolide (DMAPT) and explored the underlying molecular mechanism. The drugs induced a dose- and time-dependent decrement in cell viability, which was not prevented by the caspase inhibitor z-VAD-fmk. In particular in the first hours of treatment (1-3 h), parthenolide and DMAPT strongly stimulated reactive oxygen species (ROS) generation. The drugs induced production of superoxide anion by activating NADPH oxidase. ROS generation caused depletion of thiol groups and glutathione, activation of c-Jun N-terminal kinase (JNK) and downregulation of nuclear factor kB (NF-kB). During this first phase, parthenolide and DMAPT also stimulated autophagic process, as suggested by the enhanced expression of beclin-1, the conversion of microtubule-associated protein light chain 3-I (LC3-I) to LC3-II and the increase in the number of cells positive to monodansylcadaverine. Finally, the drugs increased RIP-1 expression. This effect was accompanied by a decrement of pro-caspase 8, while its cleaved form was not detected and the expression of c-FLIPS markedly increased. Prolonging the treatment (5-20 h) ROS generation favoured dissipation of mitochondrial membrane potential and the appearance of necrotic events, as suggested by the increased number of cells positive to propidium iodide staining. The administration of DMAPT in nude mice bearing xenografts of MDA-MB231 cells resulted in a significant inhibition of tumour growth, an increment of animal survival and a marked reduction of the lung area invaded by metastasis. Immunohistochemistry data revealed that treatment with DMAPT reduced the levels of NF-kB, metalloproteinase-2 and -9 and vascular endothelial growth factor, while induced upregulation of phosphorylated JNK. Taken together, our data suggest a possible use of parthenolide for the treatment of TNBCs.

Parthenolide induces superoxide anion production by stimulating EGF receptor in MDA-MB-231 breast cancer cells.

The sesquiterpene lactone parthenolide (PN) has recently attracted considerable attention because of its anti-microbial, anti-inflammatory and anticancer effects. However, the mechanism of its cytotoxic action on tumor cells remains scarcely defined. We recently provided evidence that the effect exerted by PN in MDA-MB-231 breast cancer cells was mediated by the production of reactive oxygen species (ROS). The present study shows that PN promoted the phosphorylation of EGF receptor (phospho-EGFR) at Tyr1173, an event which was observed already at 1 h of incubation with 25 µM PN and reached a peak at 8-16 h. This effect seemed to be a consequence of ROS production, because N-acetylcysteine (NAC), a powerful ROS scavenger, prevented the increment of phospho-EGFR levels. In addition fluorescence analyses performed using dihydroethidium demonstrated that PN stimulated the production of superoxide anion already at 2-3 h of incubation and the effect further increased prolonging the time of treatment, reaching a peak at 8-16 h. Superoxide anion production was markedly hampered by apocynin, a well known NADPH oxidase (NOX) inhibitor, suggesting that the effect was dependent on NOX activity. The finding that AG1478, an EGFR kinase inhibitor, substantially blocked both EGFR phosphorylation and superoxide anion production strongly suggested that phosphorylation of EGFR can be responsible for the activation of NOX with the consequent production of superoxide anion. Therefore, EGFR phosphorylation can exert a key role in the production of superoxide anion and ROS induced by PN in MDA-MB-231 cells.

Phase transitions in strongly coupled three-dimensional Z(N) lattice gauge theories at finite temperature.

We perform an analytical and numerical study of the phase transitions in three-dimensional Z(N) lattice gauge theories at finite temperature for N>4, exploiting equivalence of these models with a generalized version of the two-dimensional vector Potts models in the limit of vanishing spatial coupling. In this limit the Polyakov loops play the role of Z(N) spins. The effective couplings of these two-dimensional spin models are calculated explicitly. It is argued that the effective spin models have two phase transitions of BKT type. This is confirmed by large-scale Monte Carlo simulations. Using a cluster algorithm we locate the position of the critical points and study the critical behavior across both phase transitions in details. In particular, we determine various critical indices and compute the helicity modulus, the average action, and the specific heat. A scaling formula for the critical points with N is proposed.

Phase transitions in two-dimensional Z(N) vector models for N>4.

We investigate both analytically and numerically the renormalization group equations in two-dimensional (2D) Z(N) vector models. The position of the critical points of the two phase transitions for N>4 is established and the critical index ν is computed. For N=7 and 17 the critical points are located by Monte Carlo simulations, and some of the corresponding critical indices are determined. The behavior of the helicity modulus is studied for N=5, 7, and 17. Using these and other available Monte Carlo data we discuss the scaling of the critical points with N and some other open theoretical problems.

Numerical study of the phase transitions in the two-dimensional Z(5) vector model.

We investigate the critical properties of the two-dimensional Z(5) vector model. For this purpose, we propose a cluster algorithm, valid for Z(N) models with odd values of N. The two-dimensional Z(5) vector model is conjectured to exhibit two phase transitions with a massless intermediate phase. We locate the position of the critical points and study the critical behavior across both phase transitions in details. In particular, we determine various critical indices and compare the results with analytical predictions.

Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications.

Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP(C). In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP(C) at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

Early pregnancy loss in celiac women: The role of genetic markers of thrombophilia.

BACKGROUND: Adverse pregnancy outcomes are more frequent in celiac than in non-celiac women. AIMS: To investigate a possible role of genetic prothrombotic variants in early pregnancy loss of celiac women. METHODS: Thirty-nine celiac women who had experienced early pregnancy losses (at least two losses within the first 3 months of pregnancy), and 72 celiac women with a history of one or more normal pregnancies and no pregnancy loss (controls) entered the study, at the moment of diagnosis for celiac disease. A clinical history was obtained from each woman. DNA from leukocytes was tested for: factor V Leiden (mutation G1691A), factor V R2 (H1299R), factor II (G20210A), methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), beta-fibrinogen (-455 G>A), PAI-1 alleles 4G/5G, factor XIII (V34L), and HPA-1 (L33P). RESULTS: Age at diagnosis was significantly higher (p=0.002) in the celiac women with pregnancy losses than in controls. Of the gene variants studied, the allelic frequency of 4G variant of PAI-1, and the frequency of mutant genotypes were significantly more frequent in the group of celiac women with early pregnancy loss (p=0.00003 and 0.028, respectively). Surprisingly, the beta-fibrinogen -455 G>A genotype distribution (but not the allelic frequency of the variant allele) significantly differed between the two groups, since variant genotypes were more frequent in the control group (p=0.009). CONCLUSION: The 4G variant of the PAI-I gene may predispose to miscarriage a subset of celiac women; these data should be verified on larger populations.

RAS and MTHFR gene polymorphisms in a healthy exercise-trained population: association with the MTHFR (TT) genotype and a lower hemoglobin level.

The aim of this study was to determine the frequencies of ACE (I/D), AGT (M235T), AT1R (A1166C) and MTHFR (C677T) polymorphisms in a well-defined (in regards to health and nutritional status and lifestyle) population of young, healthy, exercise-trained subjects (no. 100) from the Campania region of Southern Italy. We also investigated whether there was any correlation between these polymorphisms and biochemical, hematological and hemostatic parameters in this "low-risk" population. Gene polymorphisms were analyzed with the polymerase chain reaction and restriction enzyme analysis. Allele frequencies of the genotypes examined were in Hardy-Weinberg equilibrium and agree with those reported in the Italian population. No associations were found between ACE, AGT, AT1R gene polymorphisms and anthropometric, clinical and laboratory parameters. However, the MTHFR (C677T) polymorphism was significantly associated with lower hemoglobin plasma levels in TT vs. CC + CT females (p < 0.016). This report is the first to describe the frequencies of RAS and MTHFR gene polymorphisms in young, exercise-trained volunteers from Campania and to identify an association between the MTHFR gene polymorphisms and lower hemoglobin plasma levels in young healthy females.

Polymorphism p.402Y>H in the complement factor H protein is a risk factor for age related macular degeneration in an Italian population.

AIMS: To evaluate the complement factor H (CFH) p.402Y>H polymorphism as a risk factor in age related macular degeneration (AMD) in an Italian population. METHODS: 104 unrelated Italian AMD patients and 131 unrelated controls were screened for the CFH polymorphism p.402Y>H (c.1277 T>C), which has been associated with AMD. Retinography was obtained for patients and controls; the AMD diagnosis was confirmed by fluorescein angiograms. The c.1277 T>C polymorphism was genotyped with the TaqMan real time polymerase chain reaction single nucleotide polymorphism assay. RESULTS: The frequency of c.1277C allele was higher in AMD patients than in controls (57.2% v 39.3%; p<0.001). The odds ratio (OR; logistic regression analysis) for AMD was 3.9 (95% confidence interval (CI): 1.9 to 8.2) for CC homozygotes. The CC genotype conferred a higher risk for sporadic (OR 4.6; CI: 2.0 to 10.5) than for familial AMD (OR 2.9; CI: 1.0 to 8.4). Genotypes were not related to either age at AMD diagnosis or to AMD phenotype. However, geographic atrophy and choroidal neovascularisation were more frequent in sporadic than in familial AMD (p = 0.027). Overall, the percentage of population attributable risk for the CC genotype was 28% (95% CI:18% to 33%). CONCLUSION: The association between the p.402Y>H (c.1277T>C) polymorphism and AMD applies to the Italian population and the CC genotype is more frequent in sporadic than in familial AMD cases.

[Epidemiologic analysis of patients afferent to the ICU]. / Studio epidemiologica sull'attività del centro di rianimazione.

BACKGROUND: To understand in- and out-patients flow to and from an ICU during a year (1998). The setting of the study was an 8-beds Intensive Care Unit of a 480-beds General Hospital with an Emergency Department. METHODS: Retrospective analysis by a specific designed software of all patient data extrapolated from the hospital database, in order to: 1) Divide all ICU patients in four groups, according to the first admission Department; 2) Classify all ICU patients into 3 subgroups: a) medical; b) surgical; c) trauma; 3) Evaluate the different needs of ICU resources in these different patient populations. RESULTS: Two hundred and fifty-four patients were admitted to our ICU during the study period (1.2% of all admissions). The mean duration of ICU stay was 10.4 days. Thirty-five per cent of ICU admissions came from the Emergency Department, 61% of ICU patients were discharged to another hospital ward, while the remaining 7% had to be transferred to a different hospital; 2.8% of our patients had ICU re-admissions. The overall mortality rate was 32%. CONCLUSIONS: Compared with previously reported data, a lower re-admission rate (3%), a longer mean stay in the ICU (>10 days) and a higher occupancy rate (91.4%) were observed. These data suggest that a large part of the available resources for the intensive care in our hospital are devoted to the in-hospital patient care. The hypothesis is suggested that this could be mainly due to the lack of sub-critical care areas.

Isolation and characterization of two cDNA clones coding for isoforms of the Parietaria judaica major allergen Par j 1.0101.

Two cDNA clones named P9* and P1* of 794 and 631 bp, respectively, were isolated from a lambda ZAP cDNA expression library using Parietaria judaica (Pj) pollen-specific IgE antibodies from a pool of sera (n = 23) of patients allergic to Pj. Sequence analysis showed open reading frames of 176 and 138 amino acids. Both clones contain a putative signal peptide giving two mature processed proteins named Par j 1.0102 of 14,726 D and Par j 1.0201 of 10,677 D. These proteins represent isoallergenic forms of the major Pj allergen Par j 1.0101 (clone P5) previously reported. The Par j 1.0102 shared 98% amino acid sequence homology with the P5, while the Par j 1.0201 shared 89% homology. Since P1, P5 and P9 clones were expressed in Escherichia coli, and since the three allergenic proteins shared a very high degree of sequence identity and comparable binding to the Pj-specific IgE, we decided to analyze in more detail the immunological properties of only one allergen, the recombinant Par j 1.0101. The allergenic activity determined by the histamine release assay ranged between 9 and 56%, depending on the allergic patient analyzed, while it blocked approximately 40% of all the Pj-specific IgE antibodies, as detected after ELISA and cross-absorption analysis.

cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen.

A clone (P2) coding for an allergen of Parietaria judaica (Pj) pollen has been isolated and sequenced from a cDNA library in lambda ZAP using a pool of 23 sera from Pj-allergic patients. The clone contained an insert of 622 nucleotides with an open reading frame of 133 amino acids (aa) and a putative signal peptide of 31 aa giving a deduced mature processed protein of 102 aa with a molecular mass of 11344 Da. The expressed recombinant protein, named rPar j 2.0101, was a major allergen since it reacted with IgE of 82% (23/28) of the sera of Pj-allergic subjects analyzed. It was shown to be a new allergen since (i) the amino acid sequence homology with the already reported recombinant allergen Par j 1.0101 was 45% and (ii) there was no cross-inhibition between rPar j 2.0101 and rPar j 1.0101. In addition, rPar j 2.0101 inhibited 35% of the specific IgE for 10-14 kDa native allergens and preincubation of sera from Pj-allergic patients with both rPar j 2.0101 and rPar j 1.0101 fully abolished the IgE recognition of the 10-14 kDa native allergen region, suggesting that these two allergens contributed to the region.

Detection of HCV RNA in serum and seminal fluid from HIV-1 co-infected intravenous drug addicts.

The presence of hepatitis C virus (HCV) RNA in serum and seminal fluid was investigated in eleven drug addicts coinfected with HIV-1 and HCV. Serum and seminal fluid were taken from each patient at the same time point. HCV RNA was found in ten of the eleven serum samples tested, but only in one of the semen samples. No relationship was observed between CD4 cell counts, the stage of HIV infection, extent of liver damage and the presence of HCV RNA in serum and semen. The results indicate that HCV is not usually present in the semen and provide further evidence against sexual transmission as an important mode of transmission of HCV infection.