Hydrogen sulfide attenuated tumor necrosis factor-α-induced inflammatory signaling and dysfunction in vascular endothelial cells

Background

Hydrogen sulfide (H₂S), the third physiologically relevant gaseous molecule, is recognized increasingly as an anti-inflammatory mediator in various inflammatory conditions. Herein, we explored the effects and mechanisms of sodium hydrosulfide (NaHS, a H₂S donor) on tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) dysfunction.

Methodology and Principal Findings

Application of NaHS concentration-dependently suppressed TNF-α-induced mRNA and proteins expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), mRNA expression of P-selectin and E-selectin as well as U937 monocytes adhesion to HUVEC. Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of NaHS. Furthermore, TNF-α-induced NF-κB activation assessed by IκBα degradation and p65 phosphorylation and nuclear translocation and ROS production were diminished in cells subjected to treatment with NaHS.

Significance

H₂S can exert an anti-inflammatory effect in endothelial cells through a mechanism that involves the up-regulation of HO-1.     

Multifunction protein staphylococcal nuclease domain containing 1 (SND1) promotes tumor angiogenesis in human hepatocellular carcinoma through novel pathway that involves nuclear factor κB and miR-221

Staphylococcal nuclease domain-containing 1 (SND1) is a multifunctional protein that is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC). Stable overexpression of SND1 in Hep3B cells expressing a low level of SND1 augments, whereas stable knockdown of SND1 in QGY-7703 cells expressing a high level of SND1 inhibits establishment of xenografts in nude mice, indicating that SND1 promotes an aggressive tumorigenic phenotype. In this study we analyzed the role of SND1 in regulating tumor angiogenesis, a hallmark of cancer. Conditioned medium from Hep3B-SND1 cells stably overexpressing SND1 augmented, whereas that from QGY-SND1si cells stably overexpressing SND1 siRNA significantly inhibited angiogenesis, as analyzed by a chicken chorioallantoic membrane assay and a human umbilical vein endothelial cell differentiation assay. We unraveled a linear pathway in which SND1-induced activation of NF-κB resulted in induction of miR-221 and subsequent induction of angiogenic factors Angiogenin and CXCL16. Inhibition of either of these components resulted in significant inhibition of SND1-induced angiogenesis, thus highlighting the importance of this molecular cascade in regulating SND1 function. Because SND1 regulates NF-κB and miR-221, two important determinants of HCC controlling the aggressive phenotype, SND1 inhibition might be an effective strategy to counteract this fatal malady.     

Glial tumor necrosis factor alpha (TNFα) generates metaplastic inhibition of spinal learning

Injury-induced overexpression of tumor necrosis factor alpha (TNFα) in the spinal cord can induce chronic neuroinflammation and excitotoxicity that ultimately undermines functional recovery. Here we investigate how TNFα might also act to upset spinal function by modulating spinal plasticity. Using a model of instrumental learning in the injured spinal cord, we have previously shown that peripheral intermittent stimulation can produce a plastic change in spinal plasticity (metaplasticity), resulting in the prolonged inhibition of spinal learning. We hypothesized that spinal metaplasticity may be mediated by TNFα. We found that intermittent stimulation increased protein levels in the spinal cord. Using intrathecal pharmacological manipulations, we showed TNFα to be both necessary and sufficient for the long-term inhibition of a spinal instrumental learning task. These effects were found to be dependent on glial production of TNFα and involved downstream alterations in calcium-permeable AMPA receptors. These findings suggest a crucial role for glial TNFα in undermining spinal learning, and demonstrate the therapeutic potential of inhibiting TNFα activity to rescue and restore adaptive spinal plasticity to the injured spinal cord. TNFα modulation represents a novel therapeutic target for improving rehabilitation after spinal cord injury.     

Androgens inhibit tumor necrosis factor-α-induced cell adhesion and promote tube formation of human coronary artery endothelial cells

Endothelial cells contribute to the function and integrity of the vascular wall, and a functional aberration may lead to atherogenesis. There is increasing evidence on the atheroprotective role of androgens. Therefore, we studied the effect of the androgens-testosterone and dihydrotestosterone-and estradiol on human coronary artery endothelial cell (HCAEC) function. We found by MTT assay that testosterone is not cytotoxic and enhances HCAEC proliferation. The effect of testosterone (10-50 nM), dihydrotestosterone (5-50 nM), and estradiol (0.1-0.4 nM) on the adhesion of tumor necrosis factor-α (TNF-α)-stimulated HCAECs was determined at different time points (12-96 h) by assessing their binding with human monocytic THP-1 cells. In addition, the expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), was determined by ELISA and Western blot analysis. Both testosterone and dihydrotestosterone attenuated cell adhesion and the expression of VCAM-1 and ICAM-1 in a dose- and time-dependent manner. Furthermore, androgen treatment for a longer duration inhibited cell migration, as demonstrated by wound-healing assay, and promoted tube formation on a Matrigel. Western blot analysis demonstrated that the expression of phosphorylated endothelial nitric oxide synthase (eNOS) increased, whereas that of inducible nitric oxide synthase (iNOS) decreased following the 96-h steroid treatment of TNF-α-stimulated HCAECs. Our findings suggest that androgens modulate endothelial cell functions by suppressing the inflammatory process and enhancing wound-healing and regenerative angiogenesis, possibly through an androgen receptor (AR)-dependent mechanism.     

Preparation and evaluation of a new releasable PEGylated tumor necrosis factor-α(TNF-α) conjugate for therapeutic application

To design a releasable PEGylated TNF-α(rPEG-TNF-α ), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF- (PEG-TNF- ), facilitating its clinical use for anti-tumor therapy. Comparative pharmaco- kinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were ~60-fold greater than that of unmodified TNF-α . In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9- fold more potent, respectively, than PEG-TNF-α . Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α .     

Oxidatively truncated phospholipids are required agents of tumor necrosis factor α (TNFα)-induced apoptosis

TNFα generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the (hydro)peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNFα receptor signaling to mitochondrial damage and apoptotic death.     

Synergic action between tumor necrosis factor and endotoxins or poly(A·U) on cultured bovine endothelial cells

Summary In order to investigate whether direct effects on tumor vasculature may contribute to induction of necrosis of solid tumors in vivo, agents and combinations with an established different capacity to induce tumor necrosis were studied for their effects on endothelial cells in vitro. Tumor necrosis serum caused a marked inhibition of [³H]thymidine incorporation by bovine umbilical cord endothelial cells after 4h coincubation. Endotoxin was less inhibitory, whereas detoxified endotoxin and recombinant human tumor necrosis factor (rTNF) were hardly active in concentrations that can be achieved in vivo. Combinations of rTNF and (detoxified) endotoxin caused synergic inhibition. By 24h effects of the separate agents and synergic effects of the combinations were much stronger. The nontoxic dsRNA, poly(A·U), also had inhibitory activity, and acted synergistically with rTNF. Morphologically, a combination of endotoxin and rTNF but not the separate constituents induced marked cell detachment by 24 h, an indication of cell death. Whereas both endotoxin and rTNF inhibited DNA synthesis of human endothelial cells, the agents did not act synergistically on these cells. The ability of the agents and the combinations to affect endothelial cells in culture appeared to be well in line with their capacity to induce tumor necrosis. Data suggest that direct (synergic) effects on endothelium may contribute to the induction of vascular damage in tumors by (combinations of) the agents. The fact that endothelial cell death is only induced by the combinations and not by the separate agents in vivo, may be a cause of the greater therapeutic activity of the combinations in vivo. The synergy between rTNF and the other agents indicates that the agents act by different mechanisms.Supported by a grant of the Stichting Koningin Wilhelmina Fonds, Netherlands Cancer Foundation     

The mycotoxin fumonisin B1 transiently activates nuclear factor-κB, tumor necrosis factor α and caspase 3 via protein kinase Cα-dependent pathway in porcine renal epithelial cells

Fumonisin B1 (FB1) is a toxic mycotoxin produced by Fusarium verticillioides, predominantly present in corn. The principal biochemical responses of FB1 involve disruption of sphingolipid metabolism from the inhibition of ceramide synthesis leading to accumulation of free sphingoid bases, particularly sphinganine. The ability of FB1 to modulate signal transduction pathways plays a role in its toxicity. We recently reported that FB1 selectively and transiently activates protein kinase Calpha (PKCalpha) in porcine renal epithelial cells (LLC-PK1). The aim of current study was to investigate the effect of PKCalpha activation by FB1 on NF-kappaB activation and subsequently on TNFalpha gene expression and caspase 3 induction in LLC-PK1 cells. FB1 (1 micromol/L for 5 min) transiently activated PKCalpha and increased nuclear translocation of NF-kappaB, followed by their down-regulation at later time points. Preincubating the cells with the PKC inhibitor, calphostin C, prevented the activation of NF-kappaB by FB1. TNFalpha mRNA expression was increased after 15 min exposure to FB1 or the PKC activator, phorbol 12-myristate 13-acetate. In addition, an increase in caspase 3 activity was observed after addition of FB1 for 1 h. Calphostin C prevented both the FB1-induced increase in TNFalpha expression and caspase 3 activation. In summary, we hereby demonstrate that the FB1 activation of NF-kappaB and sequential induction of TNFalpha expression resulting in the subsequent increase in caspase 3 activity are all dependent on PKCalpha stimulation in LLC-PK1 cells.     

Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor-α-induced shock

Tumor necrosis factor receptor (TNFR) signaling may result in survival, apoptosis or programmed necrosis. The latter is called necroptosis if the receptor-interacting protein 1 (RIP1) inhibitor necrostatin-1 (Nec-1) or genetic knockout of RIP3 prevents it. In the lethal mouse model of TNFα-mediated shock, addition of the pan-caspase inhibitor zVAD-fmk (zVAD) accelerates time to death. Here, we demonstrate that RIP3-deficient mice are protected markedly from TNFα-mediated shock in the presence and absence of caspase inhibition. We further show that the fusion protein TAT-crmA, previously demonstrated to inhibit apoptosis, also prevents necroptosis in L929, HT29 and FADD-deficient Jurkat cells. In contrast to RIP3-deficient mice, blocking necroptosis by Nec-1 or TAT-crmA did not protect from TNFα/zVAD-mediated shock, but further accelerated time to death. Even in the absence of caspase inhibition, Nec-1 application led to similar kinetics. Depletion of macrophages, natural killer (NK) cells, granulocytes or genetic deficiency for T lymphocytes did not influence this model. Because RIP3-deficient mice are known to be protected from cerulein-induced pancreatitis (CIP), we applied Nec-1 and TAT-crmA in this model and demonstrated the deterioration of pancreatic damage upon addition of these substances. These data highlight the importance of separating genetic RIP3 deficiency from RIP1 inhibition by Nec-1 application in vivo and challenge the current definition of necroptosis.     

Isoflavone genistein protects human vascular endothelial cells against tumor necrosis factor-α-induced apoptosis through the p38β mitogen-activated protein kinase

Isoflavone genistein may have beneficial effects on vascular function, but the mechanism is unclear. Here, we investigated whether genistein protects vascular endothelial cells against apoptosis induced by tumor necrosis factor-α. We show that genistein significantly inhibited TNF-α-induced apoptosis in human aortic endothelial cells as determined by caspase-3 activation, 7-amino actinomycin D staining, in situ apoptotic cell detection and DNA laddering. The anti-apoptotic effect of genistein was associated with an enhanced expression of Bcl-2 protein and its promoter activity. Inhibition of extracellular signal-regulated kinase 1/2, protein kinase A, or estrogen receptors had no effect on the cytoprotective effect of genistein. However, inhibition of p38 mitogen-activated protein kinase (p38) completely abolished this genistein effect. Accordingly, stimulation of HAECs with genistein resulted in rapid activation of p38β, but not p38α. These findings provide the evidence that genistein acts as a survival factor for vascular ECs to protect cells against apoptosis via activation of p38β. Preservation of the functional integrity of the endothelial monolayer may represent an important mechanism by which genistein exerts its vasculoprotective effect.     

Preparation and evaluation of a new releasable PEGylated tumor necrosis factor-α (TNF-α) conjugate for therapeutic application

To design a releasable PEGylated TNF-α (rPEG-TNF-α), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF-α (PEG-TNF-α), facilitating its clinical use for anti-tumor therapy. Comparative pharmacokinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were ～60-fold greater than that of unmodified TNF-α. In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9-fold more potent, respectively, than PEG-TNF-α. Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α.     

Establishment of a cell-based assay for examining the expression of tumor necrosis factor alpha (TNF-α) gene

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine produced by activated macrophages and lymphocytes and involved in many inflammatory diseases. Preventing the production or action of TNF-alpha is a potent therapeutic strategy for these inflammatory diseases. Since there is a lack of rapid and effective assay for examining the expression TNF-alpha in macrophages, we attempt to establish a reporter system to assess TNF-alpha gene expression through measuring luciferase activity. In this study, mouse macrophage cell line RAW 264.7 was stably transfected with a luciferase reporter pGL3-TNFPro-UTR, which contains TNF-alpha promoter and 3'-untranslated region (3'-UTR). The TNF-alpha-luciferase reporter cell line is used for assessing the expression of TNF-alpha gene induced by LPS in the presence or absence of chemicals that inhibit the biosynthesis of TNF-alpha such as dexamethasone and emodin, and also for measuring change of expression of TNF-alpha gene under downregulation of the expression of steroid receptor coactivator-3, a modulator for TNF-alpha. The luciferase activity correlated well with the ELISA results for TNF-alpha production, therefore, the TNF-alpha-luciferase reporter cell line is a sensitive, effective tool for studying the expression of TNF-alpha gene.     

Importin β1 protein-mediated nuclear localization of death receptor 5 (DR5) limits DR5/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cell death of human tumor cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor 5 (DR5)-mediated cell death plays an important role in the elimination of tumor cells and transformed cells. Recently, recombinant TRAIL and agonistic anti-DR5 monoclonal antibodies have been developed and applied to cancer therapy. However, depending on the type of cancer, the sensitivity to TRAIL has been reportedly different, and some tumor cells are resistant to TRAIL-mediated apoptosis. Using confocal microscopy, we found that large amounts of DR5 were localized in the nucleus in HeLa and HepG2 cells. Moreover, these tumor cells were resistant to TRAIL, whereas DU145 cells, which do not have nuclear DR5, were highly sensitive to TRAIL. By means of immunoprecipitation and Western blot analysis, we found that DR5 and importin β1 were physically associated, suggesting that the nuclear DR5 was transported through the nuclear import pathway mediated by importin β1. Two functional nuclear localization signals were identified in DR5, the mutation of which abrogated the nuclear localization of DR5 in HeLa cells. Moreover, the nuclear transport of DR5 was also prevented by the knockdown of importin β1 using siRNA, resulting in the up-regulation of DR5 expression on the cell surface and an increased sensitivity of HeLa and HepG2 cells to TRAIL. Taken together, our findings suggest that the importin β1-mediated nuclear localization of DR5 limits the DR5/TRAIL-induced cell death of human tumor cells and thus can be a novel target to improve cancer therapy with recombinant TRAIL and anti-DR5 antibodies.     

Role of nitric oxide and nuclear factor-κB in the CYP2E1 potentiation of tumor necrosis factor α hepatotoxicity in mice

Induction of CYP2E1 by pyrazole (PY) potentiated the hepatotoxicity induced by TNFα in mice. We evaluated the role of nitrosative and oxidative stress and the NF-κB activation pathway in this liver injury. The iNOS inhibitor N-(3-aminomethyl)benzylacetamindine (1400W) or the antioxidant N-acetyl-l-cysteine (NAC) prevented this liver injury. TNFα plus PY treatment triggered radical stress in the liver with increased lipid peroxidation and decreased glutathione and caused mitochondrial damage as reflected by elevated membrane swelling and cytochrome c release. The radical stress and mitochondrial damage were prevented by 1400W and NAC. TNFα plus PY treatment elevated 3-nitrotyrosine adduct formation and induced NOS2 in the liver; 1400W and NAC blocked these changes. A lower extent of liver injury and oxidative stress was found in NOS2^?/? mice treated with TNFα plus PY compared with wild-type controls. Neither 1400W nor NAC modified CYP2E1 activity or protein. Activation of JNK and p38MAPK was weaker in TNFα plus PY-treated NOS2^?/? mice and 1400W and NAC blocked the activation of JNK and p38MAPK in wild-type mice. IKKα/β protein levels were decreased by TNFα plus PY treatment, whereas IκBα and IκBβ protein levels were elevated compared with saline, PY, or TNFα alone. NF-κB DNA binding activity was increased by TNFα alone but lowered by TNFα plus PY. All these changes were blocked by 1400W and NAC. NF-κB activation products such as Bcl-2, Bcl-X_L, cFLIP_S, cFLIP_L, and Mn-SOD were reduced by TNFα plus PY and restored by 1400W or NAC. We conclude that TNFα plus CYP2E1 induces oxidative/nitrosative stress, which plays a role in the activation of JNK or p38MAPK and mitochondrial damage. These effects combine with the blunting of the NF-κB activation pathways and the synthesis of protective factors to cause liver injury.