Mannose receptor-mediated endothelial cell activation contributes to B16 melanoma cell adhesion and metastasis in liver

The role of mannose receptors from hepatic sinusoidal endothelium (HSE) in liver colonization by B16 melanoma (B16M) cells was studied. The expression of high mannose-type oligosaccharides on the surface of B16M cells was enhanced by in vitro treatment with 1-deoximannojirimycin (1-DMM). There was a significant (P < 0.01) enhancement of hepatic metastasis when B16M cells were 1-DMM-treated before being intrasplenically injected into C57BL/6J mice. Intraperitoneal administration of 5 mg/kg recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) inhibited the 1-DMM-induced enhancement of metastasis. Expression of high mannose-type oligosaccharides on the surface of 1-DMM-treated B16M cells and their in vitro adhesion to the HSE was significantly correlated (R = 0.82). The addition of either 100 μg/ml mannan or paraformaldehyde (PFA)-fixed 1-DMM-treated B16M cells to cultured HSE for a period of 12 h significantly (P < 0.01) increased the release of IL-1β from the HSE compared to that liberated by the HSE incubated with either basal medium or PFA-fixed untreated B16M cells. The same HSE treatments also significantly (P < 0.01) increased the degree of adhesion of other B16M cells to HSE, being abrogated by anti-mouse vascular cell adhesion molecule-1 (VCAM-1) antibodies. The conditioned media from HSE cultures, activated by PFA-fixed, 1-DMM-treated B16M cells significantly (P < 0.01) increased B16M cell proliferation when compared to conditioned media from HSE cultures incubated with PFA-fixed, untreated B16M cells. Thus, 1-DMM treatment of B16M cells enhanced the development of hepatic metastasis by IL-1-dependent mechanisms. The mechanism is consistent with in vitro mannose receptor-mediated melanoma cell attachment to the HSE, which subsequently upregulates IL-1β release, VCAM-1-dependent adherence, and melanoma growth factor(s) release by HSE. J. Cell. Physiol. 174:322–330, 1998. © 1998 Wiley-Liss, Inc.     

Neuroprotective effects of aucubin on hydrogen peroxide-induced toxicity in human neuroblastoma SH-SY5Y cells via the Nrf2/HO-1 pathway

BackgroundWhen redox balance is lost in the brain, oxidative stress can cause serious damage that leads to neuronal loss, in congruence with neurodegenerative diseases. Aucubin (AU) is an iridoid glycoside and that is one of the active constituents of Eucommia ulmoides, has many pharmacological effects such as anti-inflammation, anti-liver fibrosis, and anti-atherosclerosis.PurposeThe present study aimed to evaluate the inhibitory effects of AU on cell oxidative stress against hydrogen peroxide (H₂O₂)-induced injury in SH-SY5Y cells in vitro.MethodsSH-SY5Y cells were simultaneously treated with AU and H₂O₂ for 24 h. Cell viability was measured by CCK-8. Additionally, mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, and cell apoptosis were measured by flow cytometry.ResultsThe results showed that AU can significantly increase the H₂O₂-induced cell viability and the mitochondrial membrane potential, decrease the ROS generation, malondialdehyde (MDA), and increase glutathione (GSH) contents and the superoxide dismutase (SOD) activity. We also found that H₂O₂ stimulated the production of nitric oxide (NO), which could be reduced by treatment with AU through inhibiting the inducible nitric oxide synthase (iNOS) protein expression. In H₂O₂-induced SH-SY5Y cells, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) content and cell apoptosis were significantly reduced by AU treatment through nuclear factor E2-related factor 2/hemo oxygenase-1 (Nrf2/HO-1) activation, inhibiting the expression of p-NF-κB/NF-κB and down-regulating MAPK and Bcl-2/Bax pathways.ConclusionThese results indicate that AU can reduce inflammation and oxidative stress through the NF-κB, Nrf2/HO-1, and MAPK pathways.     

Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 mRNA

Studies increasingly indicate that inflammation induced by β-amyloid (Aβ) contributes to the progression of Alzheimer’s disease (AD). How to inhibit the enhanced production of proinflammatory cytokines stimulated by Aβ is an important research subject for the treatment of AD. In this study, we investigated the inhibitory effect and the molecular mechanism of the lipoxin A₄ (LXA₄) on the production of interleukin-1β (IL-1β) and tumor necrosis factorα (TNFα) induced by β-amyloid in the cortex and hippocampus of mice, and in Aβ-stimulated BV2 cells, a mouse microglial cell line. LXA₄ down-regulated the protein expression of IL-1β and TNFα, attenuated the gene expressions of IL-1β and TNFα, inhibited the degradation of IκBα, inhibited translocation of NF-κB p65 subunit into the nucleus induced by β-amyloid in the cortex and hippocampus of mice, and in Aβ-stimulated BV2 cells, and the inhibitory effects were dose dependently elevated. Our findings suggest that LXA₄ inhibits the production of IL-1β and TNFα induced by β-amyloid in the cortex and hippocampus of mice, and in BV2 microglial cells via the NF-κB signal pathway.     

Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H₂O₂ treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H₂O₂-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47^phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.     

Beta-nodavirus B2 protein induces hydrogen peroxide production,leading to Drp1-recruited mitochondrial fragmentation and cell death via mitochondrial targeting

Because the role of the viral B2 protein in the pathogenesis of nervous necrosis virus infection remains unknown, the aim of the present study was to determine the effects of B2 protein on hydrogen peroxide (H₂O₂)-mediated cell death via mitochondrial targeting. Using a B2 deletion mutant, the B2 mitochondrial targeting signal sequence (⁴¹RTFVISAHAA⁵⁰) correlated with mitochondrial free radical production and cell death in fish cells, embryonic zebrafish, and human cancer cells. After treatment of grouper fin cells (GF-1) overexpressing B2 protein with the anti-oxidant drug, N-acetylcysteine (NAC), and overexpression of the antioxidant enzymes, zfCu/Zn superoxide dismutase (SOD) and zfCatalase, decreased H₂O₂ production and cell death were observed. To investigate the correlation between B2 cytotoxicity and H₂O₂ production in vivo, B2 was injected into zebrafish embryos. Cell damage, as assessed by the acridine orange assay, gradually increased over 24 h post-fertilization, and was accompanied by marked increases in H₂O₂ production and embryonic death. Increased oxidative stress, as evidenced by the up-regulation of Mn SOD, catalase, and Nrf2, was also observed during this period. Finally, B2-induced dynamin-related protein 1 (Drp1)-mediated mitochondrial fragmentation and cell death could be reversed by NAC and inhibitors of Drp1 and Mdivi in GF-1 cells. Taken together, betanodavirus B2 induces H₂O₂ production via targeting the mitochondria, where it inhibits complex II function. H₂O₂ activates Drp1, resulting in its association with the mitochondria, mitochondrial fission and cell death in vitro and in vivo.     

Characterization of B16 melanoma-specific cytotoxic T lymphocytes

A B16 melanoma-specific CD8⁺ T cell line (AB1) was established from the spleen cells of C57BL/6 mice cured of B16 melanoma with interleukin (IL)-12 treatment. The AB1 line exclusively used T cell receptor V_β11. The AB1 cells exhibited a cytolytic activity against both syngeneic B16 melanoma and allogeneic P815 mastocytoma, whereas a cold inhibition assay revealed specificity of the AB1 cells against B16 melanoma. Their lostability to kill a class I loss variant of B16 melanoma was restored by the transfection of H-2K^b gene. In addition, their interferon (IFN)-γ production was significantly suppressed by the addition of anti-H-2K^b monoclonal antibody, and RT-PCR analysis showed that the AB1 line expressed the mRNA encoding IFN-γ, but not IL-4 or IL-10. The experiment using synthetic peptides of tyrosinase-related protein-2 (TRP-2) revealed that the AB1 cells could recognize TRP-2_181–188 peptide. Moreover, the AB1 cells showed an in vivo antitumor effect against established pulmonary metastases of B16 melanoma. Overall, these results indicate that the Tc1-type V_β11 ⁺ AB1 cells exert an antitumor activity against syngeneic B16 melanoma through recognition of TRP-2_181–188 peptide in an H-2K^b-restricted manner. Received: 4 June 1998 / Accepted: 21 July 1998     

Eicosanoid pathway expression in bovine endometrial epithelial and stromal cells in response to lipopolysaccharide,interleukin 1 beta,and tumor necrosis factor alpha

During endometrial inflammation, bovine endometrium responds by increasing the production of pro-inflammatory mediators, such as interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNFα), and eicosanoids. The purpose of this study was to establish and characterize an in vitro model of endometrial inflammation using bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines. We evaluated the effects of the infectious agent (bacterial lipopolysaccharide; LPS) and pro-inflammatory mediators (IL-1β and TNFα) on eicosanoid biosynthesis pathway gene expression and production by bEEL and bCSC cells. Based on concentration-response experiments, the optimal concentrations for responses were 1?μg/mL LPS, 10?ng/mL IL-1β and 50?ng/mL TNFα. Real-time PCR results show that there was an upregulation of relative mRNA expression of PTGS2 when bEEL and bCSC were treated with LPS, IL-1β and TNFα. An increase in PTGES3 expression was observed when bEEL cells were treated with LPS and IL-1β and PTGES2 when treated with IL-1β. In bCSC cells, FAAH relative mRNA was decreased upon treatments. Rate of production of PGE₂, PGF_2α, PGE₂-EA and PGF_2α-EA were also determined using liquid chromatography tandem mass spectrometry. Our results show that eicosanoid production was increased in both cell lines in response to LPS, IL-1β, and TNFα. We suggest that the characteristics of bEEL and bCSC cell lines mimic the physiological responses found in mammals with endometrial infection, making them excellent in vitro models for intrauterine environment studies.     

Effect of atrial natriuretic peptide on reactive oxygen species-induced by hydrogen peroxide in THP-1 monocytes: Role in cell growth,migration and cytokine release

Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. The aim of the present study was to investigate in THP-1 monocytes the ANP effect on hydrogen peroxide (H₂O₂)-induced Reactive Oxygen Species (ROS), cell proliferation and migration. A significant increase of H₂O₂-dependent ROS production was induced by physiological concentration of ANP (10⁻¹⁰ M). The ANP action was partially affected by cell pretreatment with PD98059, an inhibitor of mitogen activated-protein kinases (MAPK) as well as by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) and totally suppressed by diphenylene iodonium (DPI), an inhibitor of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The hormone effect was mimicked by cANF and an ANP/NPR-C signaling pathway was studied using pertussis toxin (PTX). A significant increase of H₂O₂-induced cell migration was observed after ANP (10⁻¹⁰ M) treatment, conversely a decrease of THP-1 proliferation, due to cell death, was found. Both ANP actions were partially prevented by DPI. Moreover, H₂O₂-induced release of IL-9, TNF-α, MIP-1α and MIP-1β was not counteracted by DPI, whereas no effect was observed in any experimental condition for both IL-6 and IL-1β. Our results support the view that ANP can play a key role during the inflammatory process.     

Titanium dioxide induces different levels of IL-1β production dependent on its particle characteristics through caspase-1 activation mediated by reactive oxygen species and cathepsin B

Although titanium dioxide (TiO₂) is widely used, its inhalation can induce inflammatory diseases accompanied by interleukin-1β (IL-1β) production. The particle characteristics of TiO₂ are important factors in its biological effects. It is urgently necessary to investigate the relationship between the particle characteristics and biological responses for the development of safe forms of TiO₂. Here, we systematically compared the production of IL-1β in response to various forms of TiO₂ by macrophage-like human THP-1 cells using various sizes (nano to micro), crystal structures (anatase or rutile), and shapes (spherical or spicular) of TiO₂. The production of IL-1β depended dramatically on the characteristics of the TiO₂. Notably, smaller anatase and larger rutile particles provoked higher IL-1β production. In addition, IL-1β production depended on active cathepsin B and reactive oxygen species production independent of the characteristics of TiO₂. Our results provide basic information for the creation of safe and effective novel forms of TiO₂.     

Icariside II,a novel phosphodiesterase 5 inhibitor,protects against H2O2‐induced PC12 cells death by inhibiting mitochondria‐mediated autophagy

Oxidative stress is a major cause of cellular injury in a variety of human diseases including neurodegenerative disorders. Thus, removal of excessive reactive oxygen species (ROS) or suppression of ROS generation may be effective in preventing oxidative stress‐induced cell death. This study was designed to investigate the effect of icariside II (ICS II), a novel phosphodiesterase 5 inhibitor, on hydrogen peroxide (H₂O₂)‐induced death of highly differentiated rat neuronal PC12 cells, and to further examine the underlying mechanisms. We found that ICS II pre‐treatment significantly abrogated H₂O₂‐induced PC12 cell death as demonstrated by the increase of the number of metabolically active cells and decrease of intracellular lactate dehydrogenase (LDH) release. Furthermore, ICS II inhibited H₂O₂‐induced cell death through attenuating intracellular ROS production, mitochondrial impairment, and activating glycogen synthase kinase‐3β (GSK‐3β) as demonstrated by reduced intracellular and mitochondrial ROS levels, restored mitochondrial membrane potential (MMP), decreased p‐tyr216‐GSK‐3β level and increased p‐ser9‐GSK‐3β level respectively. The GSK‐3β inhibitor SB216763 abrogated H₂O₂‐induced cell death. Moreover, ICS II significantly inhibited H₂O₂‐induced autophagy by the reducing autophagosomes number and the LC3‐II/LC3‐I ratio, down‐regulating Beclin‐1 expression, and up‐regulating p62/SQSTM1 and HSP60 expression. The autophagy inhibitor 3‐methyl adenine (3‐MA) blocked H₂O₂‐induced cell death. Altogether, this study demonstrated that ICS II may alleviate oxidative stress‐induced autophagy in PC12 cells, and the underlying mechanisms are related to its antioxidant activity functioning via ROS/GSK‐3β/mitochondrial signalling pathways.     

Immunization of mice with melanoma cells transfected to secrete the superantigen, staphylococcal enterotoxin A

Immunization of mice with a melanoma vaccine coupled with staphylococcal enterotoxin A (SEA) inhibits the growth of primary melanoma tumors in mice. We have now successfully transfected B16 cells with the sea gene and have immunized C57BL/6 mice subcutaneously once per week for 4 weeks prior to tumor challenge with vaccines of irradiated B16 cells or, 4 weeks following tumor challenge of naïve mice with B16 cells, with irradiated B16 cells transfected with the sea gene. Primary tumor growth following both types of treatments was inhibited significantly. To characterize immune responses to these immunogens, we examined the production of antibodies to the B700 melanoma antigen, the stimulation of endogenous IL-2 production, the expression of CD4, CD8, Vβ and CD25 T cell markers, and the induction of NK activity. At 4 weeks following immunization of mice, there was a significant increase (P<0.05) in levels of interleukin-2 production by splenocytes from mice immunized with SEA-secreting B16 cells or with the parental B16 cells, compared to controls. Levels of antibodies to the B700 melanoma antigen were also significantly higher in mice immunized with the SEA-secreting B16 cells, as was expression of CD4, CD8, CD25 and Vβ T cell antigens, particularly CD4. Natural killer cell activity (at various E:T ratios) was tenfold higher in splenocytes of mice immunized with SEA-secreting B16 cells, and fivefold higher in mice immunized with the parental B16 cells, compared to controls.?These data confirm the possibility of using irradiated murine melanoma cells transfected to secrete SEA in vaccines targeted at preventing the development and growth of melanoma.     

IL-1β modulate the Ca2+-activated big-conductance K channels (BK) via reactive oxygen species in cultured rat aorta smooth muscle cells

The large conductance Ca²⁺-activated K⁺ (BK) channel, abundantly expressed in vascular smooth muscle cells, plays a critical role in controlling vascular tone. Activation of BK channels leads to membrane hyperpolarization and promotes vasorelaxation. BK channels are activated either by elevation of the intracellular Ca²⁺ concentration or by membrane depolarization. It is also regulated by a diversity of vasodilators and vasoconstrictors. Interleukin-1β (IL-1β) is one of the cytokines that play important roles in the development and progression of a variety of cardiovascular diseases. The effects of IL-1β on vascular reactivity are controversial, and little is known about the modulation of BK channel function by IL-1β. In this study, we investigated how IL-1β modulates BK channel function in cultured arterial smooth muscle cells (ASMCs), and examined the role of H₂O₂ in the process. We demonstrated that IL-1β had biphasic effects on BK channel function and membrane potential of ASMCs, that is both concentration and time dependent. IL-1β increased BK channel-dependent K⁺ current and hyperpolarized ASMCs when applied for 30 min. While long-term (24–48 h) treatment of IL-1β resulted in decreased expression of α-subunit of BK channel, suppressed BK channel activity, decreased BK channel-dependent K⁺ current and depolarization of the cells. H₂O₂ scavenger catalase completely abolished the early effect of IL-1β, while it only partly diminished the long-term effect of IL-1β. These results may provide important molecular mechanisms for therapeutic strategies targeting BK channel in inflammation-related diseases.     

Cinnamon polyphenols attenuate the hydrogen peroxide-induced down regulation of S100β secretion by regulating sirtuin 1 in C6 rat glioma cells

Aims

It is well established that the brain is particularly susceptible to oxidative damage due to its high consumption of oxygen. The objective of this study was to investigate the protective effects of a water soluble polyphenol-rich extract of cinnamon and the possible mechanisms, under conditions of oxidative stress-induced by hydrogen peroxide, in rat C6 glioma cells.

Main methods

After 24 h of H₂O₂ incubation, the secretion and intracellular expression of S100β were determined by immunoprecitation/immunoblotting and immunofluorescence imaging.

Key findings

Cinnamon polyphenols (CP) counteracted the oxidative effects of H₂O₂ on S100β secretion and expression. CP also enhanced the impaired protein levels of sirtuins 1, 2, and 3, which are deacetylases important in cell survival. H₂O₂ also induced the overexpression of the proinflammatory factors, TNF-α, phospho-NF-κB p65, as well as of Bcl-xl, Bax and Caspase-3, which are all the members of the Bcl-2 family. CP not only suppressed the expression of these proteins but also attenuated the phosphorylation induced by H₂O₂. CP also upregulated the decreased Bcl-2 protein levels in H₂O₂ treated C6 cells. The effects of CP on H₂O₂-induced downregulation of S100β secretion were blocked by SIRT1 siRNA demonstrating that SIRT1 plays a regulatory role in CP-mediated prevention by H₂O₂.

Significance

These data demonstrate that Cinnamon polyphenols may exert neuroprotective effects in glial cells by the regulation of Bcl-2 family members and enhancing SIRT1 expression during oxidative stress.     

The role of oxidative stress in rhinovirus induced elaboration of IL-8 by respiratory epithelial cells

A direct correlation has been reported between the severity of symptoms associated with rhinovirus infection and the concentration of interleukin-8 in nasal secretions. The purpose of these studies was to examine the mechanism of rhinovirus-induced IL-8 elaboration. Rhinovirus infection induced oxidative stress in Beas-2b cells and the concentration of H₂O₂ in supernatant media from rhinovirus challenged cells was 12.5 ± 6.1 μM 1 h after challenge compared to 0.7 ± 0.3 μM in supernatant from control cells. N-acetyl cysteine inhibited RV-induced NF-κB activation and IL-8 elaboration. IL-8 concentrations were 36 ± 2 pg/ml and 10 ± 1 pg/ml 6 h after virus challenge in untreated and NAC-treated (30 mM NAC) cells, respectively. Despite the effects of NAC on IL-8 elaboration and NF-κB activation, RV stimulated increases in supernatant H₂O₂ were not altered by NAC. These data suggest that RV stimulation of IL-8 in respiratory epithelium is mediated through production of oxidative species and the subsequent activation of NF-κB.     

Transforming growth factor β1 inhibits interleukin-1-induced but enhances ionomycin-induced interferon-γ production in a t cell lymphoma: Comparison with the effects of rapamycin

Transforming growth factor β1 (TGF-β1) is a multifunctional cytokine whose potent immunomodulatory activity is well documented. To explore the mechanisms of this activity we examined the effect of TGF-β1 on the production of IFN-γ measured at the mRNA and protein levels in the YAC-1 cell lymphoma. In previous studies, this model proved useful to characterize the mode of action of the immunosuppressant rapamycin (RAP). Here, we found that when induced by IL-1 or IL-1 + PMA, the production of IFN-γ is suppressed by both TGF-β1 (ED₅₀ = 1.9 pM) and RAP (ED₅₀ = 0.2 nM). In contrast, when induced by the calcium ionophore ionomycin, in the absence or in the presence of PMA, this production is enhanced up to 10-fold by TGF-β (ED₅₀ = 1.8 pM) and 1.5—3-fold by RAP. Therefore, in YAC-1 cells, TGF-β1 exerts opposite effects on IFN-γ production depending on the mode of activiation, and these effects parallel those of RAP. To further analyze the mode of action of TGF-β1 in this system, we used okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases. Treatment with OA rendered the expression of IFN-γ mRNA induced by IL-1 insensitive to TGF-β1 or RAP, indicating that activation of a phosphatase may play a role in the suppressive effect of both agents. However, OA did not prevent the augmentation of ionomycin-mediated induction of IFN-β mRNA by either TGF-β1 or RAP. Hence, the up-regulation of IFN-β production by TGF-β1 and RAP may involve a different biochemical mechanism that that mediating their suppressive action. These observations also favor the hypothesis that the two agents act on the same regulatory pathways. This was further supported by the finding that TGF-β1 and RAP modulate IFN-γ production in an additive rather than synergistic fashion. However, their effects could be dissociated in mutants of YAC-1 cells selected for resistance to the inhibition of IL-1-mediated IFN-γ induction by RAP. Moreover, the IFN-γ modulatory action of RAP in YAC-1 cells was accompanied by an antiproliferative effect, whereas TGF-β1 failed to alter the growth of these cells. Therefore, the immunomodulatory action of TGF-β1 may result from the dis ruption of biochemical processes related to, although distinct from, those affected by RAP. © 1994 Wiley-Liss, Inc.