Human metapneumovirus establishes persistent infection in lung microvascular endothelial cells and primes a th2-skewed immune response

Human metapneumovirus (HMPV) is a major cause of lower respiratory tract infections. HMPV infection has been hypothesized to alter dendritic cell (DC) immune response; however, many questions regarding HMPV pathogenesis within the infected lung remain unanswered. Here, we show that HMPV productively infects human lung microvascular endothelial cells (L-HMVECs). The release of infectious virus occurs for up to more than 30 days of culture without producing overt cytopathic effects and medium derived from persistently HMPV-infected L-HMVECs (secretome) induced monocyte-derived DCs to prime naïve CD4 T-cells toward a Th2 phenotype. Moreover, we demonstrated that infected secretomes trigger DCs to up-regulate OX40L expression and OX40L neutralization abolished the pro-Th2 effect that is induced by HMPV-secretome. We clarified secretome from HMPV by size exclusion and ultracentrifugation with the aim to characterize the role of viral particles in the observed pro-Th2 effect. In both cases, the percentage of IL-4-producing cells and expression of OX40L returned at basal levels. Finally, we showed that HMPV, per se, could reproduce the ability of secretome to prime pro-Th2 DCs. These results suggest that HMPV, persistently released by L-HMVECs, might take part in the development of a skewed, pro-Th2 lung microenvironment

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, a specific glutathione S-transferase inhibitor, overcomes the multidrug resistance (MDR)-associated protein 1-mediated MDR in small cell lung cancer

In the present work, we have investigated the antitumor activity of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on aggressive small cell lung cancer. NBDHEX not only is cytotoxic toward the parental small cell lung cancer H69 cell line (LC50 of 2.3 +/- 0.6 mu mol/L) but also overcomes the multidrug resistance of its variant, H69AR, which overexpresses the ATP-binding cassette transporter multidrug resistance-associated protein 1 (MRP1; LC50 of 4.5 +/- 0.9 mu mol/L). Drug efflux experiments, done in the presence of a specific inhibitor of MRP1, confirmed that NBDHEX is not a substrate for this export pump. Interestingly, NBDHEX triggers two different types of cell death: a caspase-dependent apoptosis in the H69AR cells and a necrotic phenotype in the parental H69 cells. The apoptotic pathway triggered by NBDHEX in H69AR cells is associated with c-Jun NH2-terminal kinase and c-Jun activation, whereas glutathione oxidation and activation of p38(MAPK) is observed in the NBDHEX-treated H69 cells. In contrast to the parental cells, the higher propensity to die through apoptosis of the H69AR cell line may be related to the lower expression of the antiapoptotic protein Bcl-2. Therefore, down-regulation of a factor crucial for cell survival makes H69AR cells more sensitive to the cytotoxic action of NBDHEX, which is not a MRP1 substrate. We have previously shown that NBDHEX is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines. Therefore, NBDHEX seems a very promising compound in the search for new molecules able to overcome the ATP-binding cassette family of proteins, one of the major mechanisms of multidrug resistance in cancer cells

Targeting GSTP1-1 induces JNK activation and leads to apoptosis in cisplatin-sensitive and -resistant human osteosarcoma cell lines

The effect of the glutathione transferase P1-1 (GSTP1-1) targeting has been investigated in both sensitive (U-2OS) and cisplatin-resistant (U-2OS/CDDP4μg) human osteosarcoma cell lines. Despite the different enzyme’s content, inhibition of GSTP1-1 by 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) causes the activation of c-Jun N-terminal kinase (JNK) and apoptosis in both cell lines. However, different time courses of JNK activation and cell responses are observed. Whereas in the U-2OS/CDDP4μg cell line drug treatment results in an early increase of caspase activity and secondary necrosis, in the U-2OS cells it mainly causes cell cycle arrest followed by apoptosis. Thereafter, we detailed the action mechanism of NBDHEX in the U-2OS cell line. We report evidence of the interaction between GSTP1-1 and the TNF receptor associated factor 2 (TRAF2) and we demonstrate that NBDHEX is able to dissociate the GSTP1-1:TRAF2 complex. This restores the TRAF2:ASK1 signaling, thereby leading to the simultaneous and prolonged activation of JNK and p38. These mitogen-activated protein kinases (MAPKs) mediate different effects: JNK is crucial for apoptosis, whereas p38 causes an increase in the p21 level and a concomitant cell cycle arrest. Our study shows that GSTP1-1 plays an important regulatory role in TRAF signaling of osteosarcoma and discloses new features of the action mechanism of NBDHEX that suggest potentially practical consequences of these finding

In vitro and in vivo efficacy of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on human melanoma

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is a powerful inhibitor of the glutathione transferase P1-1 (GSTP1-1) and causes the disruption of the complex between GSTP1-1 and c-Jun N-terminal Kinase (JNK). This induces JNK activation and apoptosis in tumour cells. in the present work we assess the in vitro and in vivo effectiveness of NBDHEX on two human melanoma cell lines, Me501 and A375. NBDHEX shows IC50 values in the low micromolar range (IC50 of 1.2 +/- 0.1 mu M and 2.0 +/- 0.2 mu M for Me501 and A375, respectively) and is over 100 times more cytotoxic to these cell lines than temozolomide. Apoptosis is observed in Me501 cells within 3 h of the addition of NBDHEX, while in A375 cells the apoptotic event is rather late, and is preceded by a G2/M phase arrest. In both melanoma cell lines, INK activity is required for the ability of NBDHEX to trigger apoptosis, confirming that the JNK pathway is an important therapeutic target for this tumour. NBDHEX is also both effective and well tolerated in in vivo tumour models. A tumour inhibition of 70% is observed in vivo against Me501 human melanoma and a similar result is obtained on A375 model, with 63% of turnout inhibition. These findings indicate that the activation of the JNK pathway, through a selective GSTP1-1 targeting, could prove to be a promising new strategy for treating melanoma, which responds poorly to conventional therapies. (C) 2009 Elsevier Ltd. All rights reserved

Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin: IDENTIFICATION OF THE HEPARIN-BINDING MOTIF OF p17 AS A TARGET FOR THE DEVELOPMENT OF MULTITARGET ANTAGONISTS

Once released by HIV cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (Kd 190 nM) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg3Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highlyN,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists

Characterization of a new trabectedin-resistant myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and β, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin

Inhibition of DNA Repair Mechanisms and Induction of Apoptosis in Triple Negative Breast Cancer Cells Expressing the Human Herpesvirus 6 U94

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancers. In spite of initial good response to chemotherapy, the prognosis of TNBC remains poor and no effective specific targeted therapy is readily available. Recently, we demonstrated the ability of U94, the latency gene of human herpes virus 6 (HHV-6), to interfere with proliferation and with crucial steps of the metastatic cascade by using MDA-MB 231 TNBC breast cancer cell line. U94 expression was also associated with a partial mesenchymal-to-epithelial transition (MET) of cells, which displayed a less aggressive phenotype. In this study, we show the ability of U94 to exert its anticancer activity on three different TNBC cell lines by inhibiting DNA damage repair genes, cell cycle and eventually leading to cell death following activation of the intrinsic apoptotic pathway. Interestingly, we found that U94 acted synergistically with DNA-damaging drugs. Overall, we provide evidence that U94 is able to combat tumor cells with different mechanisms, thus attesting for the great potential of this molecule as a multi-target drug in cancer therapy

p17 from HIV induces brain endothelial cell angiogenesis through EGFR-1-mediated cell signalling activation

© 2018, United States & Canadian Academy of Pathology. HIV-associated neurocognitive disorder in HIV patients substantially reduces their quality of life. We previously showed that the HIV matrix protein, p17 could stimulate lymph-angiogenesis in vitro potentially contributing to lymphoma tumour growth and in addition is associated with vascular activation in neuro-degenerating brain tissue; here, therefore, we have investigated the detailed molecular mechanisms of this action. We performed in vitro cell culture, angiogenesis experiments, phospho-protein microarrays and Western blotting to identify cellular signalling induced by p17 within human brain endothelial cells (HbMEC), and inhibitor studies to block p17-induced vascular growth. We also characterised the effects of hippocampal CA1 injection of p17 on epidermal growth factor receptor-1 (EGFR1) expression linked to our murine model of dementia. p17 strongly induced angiogenesis of HbMEC (migration, tube formation and spheroid growth). p17 concomitantly increased phosphorylation of EGFR1 as well as down-stream intermediates ERK1/2, FAK, PLC-γ and PKC-β whilst an inhibitor peptide of EGFR, blocked cell signalling and angiogenesis. Finally, Mice that showed reduced cognitive function and behavioural deficiencies after p17 injection, demonstrated that p17 localised in cortical microvessels and also neurones many of which stained positive for p-EGFR1 by histology/IHC. This work provides strong support that p17 may be involved in initiating and/or perpetuating vascular tissue pathophysiology associated with comorbidity in HIV patients

Opposite Effects of HIV-1 p17 Variants on PTEN Activation and Cell Growth in B Cells

The HIV-1 matrix protein p17 is a structural protein that can act in the extracellular environment to deregulate several functions of immune cells, through the interaction of its NH2-terminal region with a cellular surface receptor (p17R). The intracellular events triggered by p17/p17R interaction have been not completely characterized yet. In this study we analyze the signal transduction pathways induced by p17/p17R interaction and show that in Raji cells, a human B cell line stably expressing p17R on its surface, p17 induces a transient activation of the transcriptional factor AP-1. Moreover, it was found to upregulate pERK1/2 and downregulate pAkt, which are the major intracellular signalling components involved in AP-1 activation. These effects are mediated by the COOH-terminal region of p17, which displays the capability of keeping PTEN, a phosphatase that regulates the PI3K/Akt pathway, in an active state through the serin/threonin (Ser/Thr) kinase ROCK. Indeed, the COOH-terminal truncated form of p17 (p17Δ36) induced activation of the PI3K/Akt pathway by maintaining PTEN in an inactive phosphorylated form. Interestingly, we show that among different p17s, a variant derived from a Ugandan HIV-1 strain, named S75X, triggers an activation of PI3K/Akt signalling pathway, and leads to an increased B cell proliferation and malignant transformation. In summary, this study shows the role of the COOH-terminal region in modulating the p17 signalling pathways so highlighting the complexity of p17 binding to and signalling through its receptor(s). Moreover, it provides the first evidence on the presence of a p17 natural variant mimicking the p17Δ36-induced signalling in B cells and displaying the capacity of promoting B cell growth and tumorigenesis