Search

Background According to GLOBOSCAN, hepatocellular carcinoma (HCC) claimed 782,000 lives in 2018. The tyrosine kinase inhibitor sofafenib is used to treat HCC, but new anticancer agents targeting different pathways are urgently needed to improve outcomes for patients with advanced disease. The aberrant metabolism and aggressive growth of cancer cells can render them particularly susceptible to proteasome inhibition, as demonstrated by bortezomib treatment of multiple myeloma. However, resistance does emerge, and this 20S proteasome inhibitor has not proven active against HCC. The bis-benzylidine piperidone RA190 represents a novel class of proteasome inhibitor that covalently binds to cysteine 88 of RPN13, an ubiquitin receptor subunit of the proteasome’s 19S regulatory particle. RA190 treatment inhibits proteasome function, causing rapid accumulation of polyubiquitinated proteins. Considerable evidence suggests that nuclear factor κB (NF-κB) signaling, which is dependent upon the proteasome, is a major driver of inflammation-associated cancers, including HCC. Methods Human HCC cell lines were treated with titrations of RA190. The time course of endoplasmic reticulum stress and NF-κB-related mechanisms by which RA190 may trigger apoptosis were assessed. The therapeutic activity of RA190 was also determined in an orthotopic HCC xenograft mouse model. Results RA190 is toxic to HCC cells and synergizes with sofafenib. RA190 triggers rapid accumulation of polyubiquitinated proteins, unresolved endoplasmic reticulum stress, and cell death via apoptosis. RA190 blocks proteasomal degradation of IκBα and consequent release of NF-κB into the nuclei of HCC cells. Treatment of mice bearing an orthotopic HCC model with RA190 significantly reduced tumor growth. Conclusions RA190 has therapeutic activity in a xenograft model, and with sorafenib exhibited synergetic killing of HCC cells in vitro, suggesting further exploration of such a combination treatment of HCC is warranted.

Background and purpose Haem oxygenase-1 (HO-1) is induced by thiazolidinediones including rosiglitazone and exerts anti-inflammatory effects in various models. However, the molecular mechanisms underlying rosiglitazone-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). Experimental approach HO-1 expression was determined by real time-PCR, Western blotting and promoter reporter analyses. Signalling pathways were investigated using pharmacological inhibitors or specific siRNAs. Interactions between nuclear factor erythroid-2-related factor (Nrf2) and antioxidant response elements (ARE) binding site of the HO-1 promoter were investigated with chromatin immunoprecipitation assays. Key results Up-regulation of HO-1 in HPAEpiCs or in mice by rosiglitazone blunted ICAM-1 expression and monocyte adhesion to HPAEpiCs challenged with LPS. Rosiglitazone-induced HO-1 expression was significantly attenuated by NADPH oxidase (NOX) inhibitors (apocynin and diphenyleneiodonium) or ROS scavenger (N-acetyl cysteine). The involvement of NOX activity and ROS generation in rosiglitazone-induced HO-1 expression was confirmed by transfection with p47phox or NOX2 siRNA. Moreover, pretreatment with the inhibitors of c-Src (c-Srci II), proline-rich tyrosine kinase 2 (Pyk2) (PF431396), Akt (Akti VIII) or PPARγ (GW9662) and transfection with siRNA of c-Src, Pyk2, Akt or PPARγ abolished the rosiglitazone-induced HO-1 expression in HPAEpiCs. Subsequently, Nrf2 was activated by phosphorylation of c-Src, Pyk2 and Akt, which turned on transcription of HO-1 gene by binding to AREs binding site and enhancing ARE promoter activity. Conclusions and implications Rosiglitazone induces HO-1 expression via either NOX/ROS/c-Src/Pyk2/Akt-dependent Nrf2 activation or PPARγ in HPAEpiCs and suppresses LPS-mediated inflammatory responses, suggesting that PPARγ agonists may be useful for protection against pulmonary inflammation.

Background: Mevastatin (MVS), a 3-hydroxy-3-methylglutaryl coenzyme, a reductase (HMG-CoA) inhibitor, has anti-inflammatory effects potentially via up-regulation of heme oxygenase-1 (HO-1). However, the mechanisms underlying MVS-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs). Methods: HO-1 and intercellular adhesion molecule (ICAM)-1 expression were determined using real-time PCR, Western blotting, and promoter reporter analyses. The signaling components were investigated using pharmacological inhibitors or specific small interfering RNA (siRNA)s. Interaction between Nrf2 and the antioxidant response element (ARE) binding site for the HO-1 promoter was determined by chromatin immunoprecipitation (ChIP) assay. Results: Upregulation of HO-1 by MVS attenuated the tumor necrosis factor (TNF)-&alpha, stimulated ICAM-1 expression associated with THP-1 adhesion to HPAEpiCs. These inhibitory effects of HO-1 were reversed by tin protoporphyrin (SnPP)IX or by transfection with HO-1 siRNA. MVS-induced HO-1 expression was mediated via NADPH oxidase (Nox)-derived reactive oxygen species (ROS) generation. Activation of Nox2/ROS further stimulated the phosphorylation of p47phox, proto-oncogene tyrosine-protein kinase (c-Src), platelet-derived growth factor receptor (PDFGR)&alpha, protein kinase B (Akt), and Nrf2, which were inhibited by siRNAs. Pretreatment with pharmacological inhibitors, including diphenyleneiodonium (DPI), apocynin (APO), N-acetyl-L-cysteine (NAC), PP1, AG1296, or LY294002, reduced the MVS-activated Nrf2 nuclear-translocation binding to the ARE on the HO-1 promoter. Conclusions: MVS-induced HO-1 is, at least in part, mediated through a p47phox/Nox2/ROS-dependent activation of c-Src/PDGFR&alpha, /PI3K/Akt-regulated Nrf2/ARE axis and suppresses the TNF-&alpha, mediated inflammatory responses in HPAEpiCs.

The upregulation of heme oxygenase-1 (HO-1) by the carbon monoxide-releasing molecule (CORM)-2 may be mediated through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [Nox] and reactive oxygen species (ROS) generation, which could provide cytoprotection against various cellular injuries. However, the detailed mechanisms of CORM-2-induced HO-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain largely unknown. Therefore, we dissected the mechanisms underlying CORM-2-induced HO-1 expression in HPAEpiCs. We found that the administration of mice with CORM-2 attenuated the tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule-1 (ICAM-1) expression and leukocyte count as revealed by immunohistochemical staining, western blot, real-time polymerase chain reaction (PCR), and cell count. Furthermore, TNF-α-induced ICAM-1 expression associated with monocyte adhesion to HPAEpiCs was attenuated by infection with adenovirus (adv)-HO-1 or incubation with CORM-2. These inhibitory effects of HO-1 were reversed by pretreatment with hemoglobin (Hb). Moreover, CORM-2-induced HO-1 expression was mediated via the phosphorylation of p47phox, c-Src, epidermal growth factor receptor (EGFR), Akt, and NF-E2-related factor 2 (Nrf2), which were inhibited by their pharmacological inhibitors, including diphenyleneiodonium (DPI) or apocynin (APO), ROS [N-acetyl-L-cysteine (NAC)], PP1, AG1478, PI3K (LY294002), or Akt (SH-5), and small interfering RNAs (siRNAs). CORM-2-enhanced Nrf2 expression, and anti-oxidant response element (ARE) promoter activity was also inhibited by these pharmacological inhibitors. The interaction between Nrf2 and AREs was confirmed with a chromatin immunoprecipitation (ChIP) assay. These findings suggest that CORM-2 increases the formation of the Nrf2 and AREs complex and binds with ARE-binding sites via Src, EGFR, and PI3K/Akt, which further induces HO-1 expression in HPAEpiCs. Thus, the HO-1/CO system might suppress TNF-α-mediated inflammatory responses and exert a potential therapeutic strategy in pulmonary diseases.

Tumor necrosis factor-alpha (TNF-alpha) triggers activation of cytosolic phospholipase A2 (cPLA2) and then enhancing the synthesis of prostaglandin (PG) in inflammatory diseases. However, the detailed mechanisms of TNF-alpha induced cPLA2 expression were not fully defined in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that TNF-alpha-stimulated increases in cPLA2 mRNA (5.2 folds) and protein (3.9 folds) expression, promoter activity (4.3 folds), and PGE2 secretion (4.7 folds) in HPAEpiCs, determined by Western blot, real-time PCR, promoter activity assay and PGE2 ELISA kit. These TNF-alpha-mediated responses were abrogated by the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], ROS [N-acetyl cysteine, (NAC)], NF-kappaB (Bay11-7082) and transfection with siRNA of ASK1, p47phox, TRAF2, NIK, IKKalpha, IKKbeta, or p65. TNF-alpha markedly stimulated NADPH oxidase activation and ROS including superoxide and hydrogen peroxide production which were inhibited by pretreatment with a TNFR1 neutralizing antibody, APO, DPI or transfection with siRNA of TRAF2, ASK1, or p47phox. In addition, TNF-alpha also stimulated p47phox phosphorylation and translocation in a time-dependent manner. On the other hand, TNF-alpha induced TNFR1, TRAF2, ASK1, and p47phox complex formation in HPAEpiCs, which were attenuated by a TNF-alpha neutralizing antibody. We found that pretreatment with NAC, DPI, or APO also attenuated the TNF-alpha-stimulated IKKalpha/beta and NF-kappaB p65 phosphorylation, NF-kappaB (p65) translocation, and NF-kappaB promoter activity in HPAEpiCs. Finally, we observed that TNF-alpha-stimulated NADPH oxidase activation and ROS generation activates NF-kappaB through the NIK/IKKalpha/beta pathway. Taken together, our results demonstrated that in HPAEpiCs, up-regulation of cPLA2 by TNF-alpha is, at least in part, mediated through the cooperation of TNFR1, TRAF2, ASK1, and NADPH oxidase leading to ROS generation and ultimately activates NF-kappaB pathway.

Upregulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Lipopolysaccharide (LPS) has been shown to play a key role in inflammation via adhesion molecule induction and then causes lung injury. However, the mechanisms underlying LPS-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. We showed that LPS induced ICAM-1 expression in HPAEpiCs, revealed by Western blotting, RT-PCR, real-time PCR, and promoter assay. Pretreatment with the inhibitor of c-Src (protein phosphatase-1, PP1), reactive oxygen species (ROS) (Edaravone), NADPH oxidase (apocynin and diphenyleneiodonium chloride), EGFR (AG1478), PDGFR (AG1296), phosphatidylinositol-3-kinase (PI3K) (LY294002), MEK1/2 (U0126), or NF-κB (Bay11-7082) and transfection with siRNAs of c-Src, EGFR, PDGFR, Akt, p47 phox, Nox2, Nox4, p42, and p65 markedly reduced LPS-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with LPS. In addition, we established that LPS stimulated phosphorylation of c-Src, EGFR, PDGFR, Akt, or p65, which was inhibited by pretreatment with their respective inhibitors. LPS induced Toll-like receptor 4 (TLR4), MyD88, TNF receptor-associated factor 6 (TRAF6), c-Src, p47 phox, and Rac1 complex formation 2, which was attenuated by transfection with c-Src or TRAF6 siRNA. Furthermore, LPS markedly enhanced NADPH oxidase activation and intracellular ROS generation, which were inhibited by PP1. We established that LPS induced p42/p44 MAPK activation via a c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt-dependent pathway in these cells. Finally, we observed that LPS significantly enhanced NF-κB and IκBα phosphorylation, NF-κB translocation, and NF-κB promoter activity, which were inhibited by PP1, Edaravone, apocynin, diphenyleneiodonium chloride, AG1478, AG1296, LY294002 , or U0126. These results demonstrated that LPS induces p42/p44 MAPK activation mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt pathway, which in turn initiates the activation of NF-κB and ultimately induces ICAM-1 expression in HPAEpiCs.