Role of AGAP2 in the profibrogenic effects induced by TGFß in LX-2 hepatic stellate cells.

Liver damage induces hepatic stellate cells (HSC) activation, characterised by a fibrogenic, proliferative and migratory phenotype. Activated HSC are mainly regulated by transforming growth factor ß 1 (TGFß1), which increases the production of extracellular matrix proteins (e.g. collagen-I) promoting the progression of hepatic fibrosis. AGAP2 (ArfGAP with GTPase domain, ankyrin repeat and PH domain 2) is a GTPase/GTP-activating protein involved in the actin remodelling system and receptor recycling. In the present work the role of AGAP2 in human HSC in response to TGFß1 was investigated. LX-2 HSC were transfected with AGAP2 siRNA and treated with TGFß1. AGAP2 knockdown prevented to some extent the proliferative and migratory TGFß1-induced capacities of LX-2 cells. An array focused on human fibrosis revealed that AGAP2 knockdown partially prevented TGFß1-mediated gene expression of the fibrogenic genes ACTA2, COL1A2, EDN1, INHBE, LOX, PDGFB, TGFΒ12, while favored the expression of CXCR4, IL1A, MMP1, MMP3 and MMP9 genes. Furthermore, TGFß1 induced AGAP2 promoter activation and its protein expression in LX-2. Moreover, AGAP2 protein levels were significantly increased in liver samples from rats with thioacetamide-induced fibrosis. In addition, AGAP2 silencing affected TGFß1-receptor 2 (TGFR2) trafficking in U2OS cells, blocking its effective recycling to the membrane. AGAP2 silencing in LX-2 cells prevented the TGFß1-induced increase of collagen-I protein levels, while its overexpression enhanced collagen-I protein expression in the presence or absence of the cytokine. AGAP2 overexpression also increased focal adhesion kinase (FAK) phosphorylated levels in LX-2 cells. FAK and MEK1 inhibitors prevented the increase of collagen-I expression caused by TGFß1 in LX-2 overexpressing AGAP2. In summary, the present work shows for the first time, that AGAP2 is a potential new target involved in TGFß1 signalling, contributing to the progression of hepatic fibrosis.

NADPH oxidase 5 promotes proliferation and fibrosis in human hepatic stellate cells.

NADPH oxidase (Nox) variants Nox1, Nox2 and Nox4 are implicated in the progression of liver fibrosis. However, the role of Nox5 is not yet known, mainly due to the lack of this enzyme in rat and mouse genomes. Here we describe the expression and functional relevance of Nox5 in the human cell line of hepatic stellate cells (HSC) LX-2. Under basal conditions, three long (Nox5-L: Nox5α, -ß, and -δ) and a short (Nox5-S or Nox5ε) splice variants were detected, which were silenced with specific siRNAs for Nox5. The most abundant isoform was Nox5-S, accounting for more than 90% of Nox5 protein. Overexpression of Nox5ß generated reactive oxygen species (ROS) in the presence of calcium, as judged by the production of hydrogen peroxide, L-012 luminescence and cytochrome c reduction. Nox5ε did not generated ROS under these conditions, and a reduced ROS production was observed when co-expressed with Nox5ß. In contrast, dihydroethidium oxidation was increased by Nox5ß or Nox5ε, suggesting that Nox5ε induced intracellular oxidative stress by an unknown mechanism. Functional studies showed that both Nox5ß and Nox5ε stimulated the proliferation of LX-2 cells and the collagen type I levels, while Nox5 siRNAs inhibited these effects. Interestingly, TGF-ß and angiotensin II upregulated Nox5 expression, which was reduced in cells pre-incubated with catalase. Further studies silencing Nox5 in TGF-ß-treated cells resulted in a reduction of collagen levels via p38 MAPK. Collectively, these results show for the first time that Nox5 can play a relevant role in the proliferation and fibrosis on human HSC.

Unfolded protein response induced by Brefeldin A increases collagen type I levels in hepatic stellate cells through an IRE1α, p38 MAPK and Smad-dependent pathway.

Unfolded protein response (UPR) triggered as a consequence of ER stress has been shown to be involved in the development of different pathologies, including fibrotic disorders. In the present paper we explore the role played by UPR on a key fibrogenic parameter in the liver: collagen type I levels in activated hepatic stellate cells (HSC). Using Brefeldin A (BFA) as an ER stress inducer we found that UPR correlated with enhanced mRNA and protein levels of collagen type I in a cell line of immortalized non-tumoral rat HSC. Analysis of the three branches of UPR revealed the activation of IRE1α, PERK and ATF6 in response to BFA, although PERK activation was shown not to be involved in the fibrogenic action of BFA. BFA also activated p38 MAPK in an IRE1α-dependent way and the p38 MAPK inhibitor SB203580 prevented the increase in collagen type I mRNA and protein levels caused by BFA, suggesting the involvement of this kinase on this effect. Analysis of Smad activation showed that phosphorylated nuclear levels of Smad2 and 3 were increased in response to BFA treatment. Inhibition of Smad3 phosphorylation by SIS3 prevented the enhancement of collagen type I levels caused by BFA. Pretreatment with IRE1α and p38 MAPK inhibitors also prevented the increased p-Smad3 accumulation in the nucleus, suggesting an IRE1α-p38 MAPK-Smad pathway to be responsible for the fibrogenic action of BFA on HSC.

Oxidation pathways underlying the pro-oxidant effects of apigenin.

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (H(2)DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H(2)DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H(2)DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H(2)DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.

Fibronectin peptides as potential regulators of hepatic fibrosis through apoptosis of hepatic stellate cells.

The turnover of extracellular matrix (ECM) components can generate signals that regulate several cellular functions such as proliferation, differentiation, and apoptosis. During liver injury, matrix metalloproteases (MMPs) production is enhanced and increased levels of peptides derived from extracellular matrix proteins can be generated. Synthetic peptides with sequences present in extracellular matrix proteins were previously found to induce both stimulating and apoptotic effects on several cell types including the inflammatory cells monocytes/macrophages. Therefore, in inflammatory liver diseases, locally accumulated peptides could be also important in regulating hepatic fibrosis by inducing apoptosis of hepatic stellate cells (HSC), the primary cellular source of extracellular matrix components. Here, we describe the apoptotic effect of fibronectin peptides on the cell line of human hepatic stellate cells LX-2 based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion, and accumulation of Bax protein. We also found that these peptides trigger the activation of Src kinase, which in turn mediated the increase of JNK and p38 activities. By the use of specific inhibitors we demonstrated the involvement of Src, JNK, and p38 in apoptosis induced by fibronectin peptides on HSC. Moreover, fibronectin peptides increased iNOS expression in human HSC, and specific inhibition of iNOS significantly reduced the sustained activity of JNK and the programmed cell death caused by these peptides. Finally, the possible regulatory effect of fibronectin peptides in liver fibrosis was further supported by the ability of these peptides to induce metalloprotease-9 (MMP-9) expression in human monocytes.

Cytotoxic and proapototic activities of imidoselenocarbamate derivatives are dependent on the release of methylselenol.

In the search for new molecules with potential antiangiogenic activity, we found that several imidoselenocarbamate derivatives effectively suppressed the expression of vascular endothelial growth factor (VEGF) induced by hypoxia in NCI-H157 tumor cells. Mechanistic studies indicated that these compounds inhibited STAT3 phosphorylation triggered by hypoxia, suggesting that inhibition of STAT3 function may play a role in VEGF inhibition. Moreover, these molecules showed interesting proapoptotic and antiproliferative effects. Both the presence of selenium, but not sulfur, and the nature of the radical substituents were important for activity. Interestingly, under hypoxic conditions, several methyl imidoselenocarbamate derivatives released methylselenol, a highly reactive and cytotoxic gas, which was responsible for their biological activities. The kinetics of the release of methylselenol by these molecules was highly dependent on the nature of the substituent radicals and correlated with their early proapoptotic activity. Our results support the notion that pharmacological activities reported for methyl imidoselenocarbamate derivatives are dependent on the release of methylselenol. Given the well-known antitumor activities of this compound, imidoselenocarbamate derivatives represent a promising approach to develop new drugs that release methylselenol in a controlled way.

Flavonoids inhibit hypoxia-induced vascular endothelial growth factor expression by a HIF-1 independent mechanism.

Flavonoids are a group of polyphenolic dietary compounds that have been proposed to possess chemopreventive properties against lung cancer. In this work we analyzed the effect of a group of 20 structurally related flavonoids, including flavones, flavonols and isoflavones, on the production of vascular endothelial growth factor (VEGF) induced by hypoxia in NCI-H157 cells. VEGF is the main regulator of physiological and pathological angiogenesis and is highly stimulated by hypoxia-inducible factor 1 (HIF-1). We found that apigenin, luteolin, fisetin and quercetin inhibited hypoxia-induced VEGF expression in the low micromolar range. Structure-activity relationships demonstrated that flavone derivatives were the most active compounds and that hydroxylation of the A ring at the positions 5 and 7 and of the B ring at the 4' position were important for this activity. Interestingly, only a group of VEGF inhibitors, including apigenin, flavone and 4',7-dihydroxiflavone, reduced the expression of HIF-1alpha under these conditions, whereas others, such as fisetin, luteolin, galangin or quercetin, induced HIF-1alpha expression while reducing those of VEGF. When cells were exposed to hypoxia in the presence of these flavonoids, HIF-1alpha translocated to the nucleus and interacted with p300/CBP, but this complex was transcriptionally inactive. Taken together these findings indicate that flavonoids impair VEGF transcription by an alternative mechanism that did not depend on nuclear HIF levels. We also found that flavonoids suppressed hypoxia-induced STAT3 tyrosine phosphorylation and that this activity correlated with their potency as VEGF inhibitors, suggesting that inhibition of STAT3 function may play a role in this process.

A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template-primer.

Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PP(i)-dependent phosphorolysis catalysed by wild-type and AZT (3'-azido-3'-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template-primer (K(d)=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template-primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues.

Selective excision of chain-terminating nucleotides by HIV-1 reverse transcriptase with phosphonoformate as substrate.

A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3'-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP(i)) analogs on PP(i)- and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP(i)- and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT.template/primer complex was hindered by the presence of a chain terminator at the 3'-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZT-triphosphate in the presence of PP(i) or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.

Inhibition of phosphorolysis catalyzed by HIV-1 reverse transcriptase is responsible for the synergy found in combinations of 3'-azido-3'-deoxythymidine with nonnucleoside inhibitors.

In spite of the growing attention to the combined chemotherapy in the treatment of AIDS, the molecular mechanisms underlying the antiviral synergy of combinations of reverse transcriptase (RT) inhibitors are in most cases unknown. Most combinations of nonnucleoside inhibitors (NNRTI) with nucleoside analogues synergistically inhibit HIV-1 replication in cell culture, though they fail to show synergy in enzymatic assays. In this work we have examined the mechanisms mediating the synergy in combinations of AZTTP with NNRTIs on HIV-1 RT and their possible relevance in antiretroviral therapy. We found that if two inhibitors bind either to different sites on the RT or to the same site but to different mechanistic forms, it is always possible to find conditions in which their combination results in synergistic inhibition of DNA polymerase activity. Though these analyses are interesting from a biochemical point of view, this kind of synergy is unlikely to play any role in vivo, since this positive interaction is lost under the conditions present in viral replication. Here we describe that the synergy found for combinations of NNRTI with AZT is due not to the inhibition of the DNA polymerase activity but to the inhibition of the RT-catalyzed phosphorolysis by the NNRTI. While phosphorolytical removal of the AZT-terminated primer has been related to the mechanism of resistance toward AZT, our data suggest that a basal phosphorolysis occurs even with the wild-type enzyme, and that the inhibition of this activity could explain the synergy found in antiviral assays.

Flavonoids induce apoptosis in human leukemia U937 cells through caspase- and caspase-calpain-dependent pathways.

Flavonoids are polyphenolic phytochemicals that are ubiquitous in plants and present in the common human diet. They may exert diverse beneficial effects, including antioxidant and anticarcinogenic activities. In this study we tested the apoptotic activity of 22 flavonoids and related compounds in leukemic U937 cells. Several flavones but none of the isoflavones or flavanones tested induced apoptotic cell death under these conditions, as determined by reduction in cell viability, flow cytometry, and oligonucleosomal DNA fragmentation. Structure-activity relationship showed that at least two hydroxylations in positions 3, 5, and 7 of the A ring were needed to induce apoptosis, whereas hydroxylation in 3' and/or 4' of the B ring enhanced proapoptotic activity. At lower concentrations, these compounds were also able to sensitize these cells to apoptosis induced by tumor necrosis factor-alpha. Regarding the mechanisms, galangin, luteolin, chrysin, and quercetin induced apoptosis in a way that required the activation of caspases 3 and 8, but not caspase 9. In contrast, an active role of calpains in addition to caspases was demonstrated in apoptosis induced by fisetin, apigenin, and 3,7-dihydroxyflavone. Our data show evidence of the proapoptotic properties of some flavonoids that could support their rational use as chemopreventive and therapeutic agents against carcinogenic disease.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase inhibit phosphorolysis and resensitize the 3'-azido-3'-deoxythymidine (AZT)-resistant polymerase to AZT-5'-triphosphate.

Removal of 3'-azido-3'deoxythymidine (AZT) 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP) from the terminated primer mediated by the human HIV-1 reverse transcriptase (RT) has been proposed as a relevant mechanism for the resistance of HIV to AZT. Here we compared wild type and AZT-resistant (D67N/K70R/T215Y/K219Q) RTs for their ability to unblock the AZTMP-terminated primer by phosphorolysis in the presence of physiological concentrations of pyrophosphate or ATP. The AZT-resistant enzyme, as it has been previously described, showed an increased ability to unblock the AZTMP-terminated primer by an ATP-dependent mechanism. We found that only mutations in the p66 subunit were responsible for this ability. We also found that three structurally divergent non-nucleoside reverse transcriptase inhibitor (NNRTI), nevirapine, TIBO, and a 4-arylmethylpyridinone derivative, were able to inhibit the phosphorolytic activity of the enzyme, rendering the AZT-resistant RT sensitive to AZTTP. The 4-arylmethylpyridinone derivative proved to be about 1000-fold more potent in inhibiting phosphorolysis than nevirapine or TIBO. Moreover, combinations of AZTTP with NNRTIs exhibited an exceptionally high degree of synergy in the inhibition of AZT-resistant enzyme only when ATP or PPi were present, indicating that inhibition of phosphorolysis was responsible for the synergy found in the combination. Our results not only demonstrate the importance of phosphorolysis concerning HIV-1 RT resistance to AZT but also point to the implication of this activity in the strong synergy found in some combinations of NNRTIs with AZT.