Histone acetyltransferase p300 is induced by p38MAPK after photodynamic therapy: the therapeutic response is increased by the p300HAT inhibitor anacardic acid

Oxidative stress mediated by photodynamic therapy (PDT) mediates the tumoricidal effect, but has also been shown to induce the expression of prosurvival molecules, such as cyclooxygenase-2 (COX-2), which is involved in tumor recurrences after PDT. However, the molecular mechanism is still not fully understood. In this study, we found that activated p38MAPK could significantly up-regulate the activity and expression of histone acetyltransferase p300 (p300HAT) in A375 and C26 cells treated with ALA-and chlorin e6 (Ce6)-mediated photodynamic treatment. A colony-formation assay showed that PDT-induced cytotoxicity was dramatically elevated in the presence of the p300HAT inhibitor anacardic acid (AA). Further studies showed that increased p300HAT acetylates histone H3 and NF-κB p65 subunit to up-regulate the COX-2 expression, which was reduced by AA or p300HAT shRNA. Using chromatin immunoprecipitation analysis, we found that the augmented acetylation of histone H3 and NF-κB increases their binding to the COX-2 promoter region. These in vitro findings were further verified in mice bearing murine C26 and human A375 tumors treated with liposomal Ce6 mediated PDT. Meanwhile, the combination of PDT and AA resulted in greater tumor regression in BALB/c mice bearing C26 tumors, compared with PDT only or combined with COX-2 inhibitor. Finally, we demonstrated that suppression of the PDT-induced p300HAT activity also resulted in the decreased expression of survivin, restoring caspase-3 activity and sensitizing PDT-treated cells from autophagy to apoptosis due to the Becline-1 cleavage. This study demonstrates for the first time the molecular mechanisms involved in histone modification induced by PDT-mediated oxidative stress, suggesting that HAT inhibitors may provide a novel therapeutic approach for improving PDT response.     

Effects of physiological quercetin metabolites on interleukin-1β-induced inducible NOS expression

Cytokines released by inflammatory cells around the pancreatic islets are implicated in the pathogenesis of diabetes mellitus. Specifically, interleukin-1β (IL-1β) is known to be involved in islet β-cell damage by activation of nuclear factor-κB (NF-κB)-mediated inducible nitric oxide synthase (iNOS) gene expression. Though most flavonoids are shown to have various beneficial effects, little is known about the anti-inflammatory effects of their metabolites. Therefore, we investigated the effects of quercetin and its metabolites quercetin 3'-sulfate, quercetin 3-glucuronide and isorhamnetin 3-glucuronide on IL-1β-stimulated iNOS gene expression in RINm5F β-cells. The nitrite level, iNOS protein and its mRNA expression levels and iNOS promoter activity were measured. In addition, IκBα protein phosphorylation, nuclear translocation of nuclear factor-κB (NF-κB) and NF-κB DNA binding activity were determined. Adenosine 5'-triphosphate disodium salt-induced insulin release was also measured. Quercetin significantly reduced IL-1β-induced nitrite production, iNOS protein and its mRNA expression levels, and it also inhibited IL-1β-induced IκBα phosphorylation, NF-κB activation and iNOS promoter activity. Additionally, quercetin significantly restored the inhibition of insulin secretion by IL-1β. Meanwhile, quercetin metabolites did not show any effect on IL-1β-induced iNOS gene expression and also on insulin secretion. Therefore, in terms of iNOS expression mechanism, dietary ingestion of quercetin is unlikely to show anti-inflammatory effects in rat islet β-cells exposed to IL-1β.     

Molecular mechanisms of gastrointestinal protection by quercetin against indomethacin-induced damage: role of NF-κB and Nrf2

The aim of this study was to determine the gastrointestinal protection by quercetin against indomethacin-induced oxidative stress and inflammation, with specific interest in studying the underlying molecular mechanisms. We hypothesized that the quercetin-protective effect relies on its antioxidant and antiinflammatory properties. Rats were pretreated with quercetin (50- or 100-mg/kg, ig single dose), 30 min before INDO administration (40-mg/kg ig single dose). Caco-2 cells were treated with INDO (250 and 500 μM) in the absence or presence of quercetin (10 μg/ml). Quercetin prevented the decrease in nuclear translocation of Nrf2, a key regulator of the antioxidant response, and the increase in reactive oxygen species levels induced by INDO by inhibiting the enhancement of NADPH oxidase and xanthine oxidase activities as well as the reduction in superoxide dismutase and glutathione peroxidase activities in gastric and ileal tissues. Quercetin also prevented INDO-induced ICAM-1 and P-selectin expressions and the increase of myeloperoxidase activity in gastric and ileal tissues and NF-κB activation and IL-8 production in Caco-2 cells. Quercetin did not affect the inhibition of TNFα-mediated production of prostaglandin E₂ induced by INDO in Caco-2 cells. The protective effects of quercetin observed in the gastric and ileal mucosa of rats as well as in Caco-2 cells relied on the ability of this flavonol to prevent NF-κB activation and increase Nrf2 translocation. This study supports the concept that quercetin may be useful in the prevention and/or treatment of nonsteroidal antiinflammatory drug-associated side effects, without interfering with their therapeutic efficacy.     

Discovery of lesser known flavones as inhibitors of NF-κB signaling in MDA-MB-231 breast cancer cells—A SAR study

Seventeen flavonoids with different substitutions were evaluated for inhibition of nuclear factor-κB (NF-κB) signaling in the invasive breast cancer cell line MDA-MB-231. They were screened using an engineered MDA-MB-231 cell line reporting NF-κB activation. The modulation of expression of two NF-κB regulated genes involved in tumorigenesis, matrix metalloproteinase-9 (MMP-9), and cyclooxygenase-2 (COX-2) were also analyzed in these cells. Among the compounds tested, all except gossypetin and quercetagetin inhibited the activation of NF-κB, and the expression of MMP-9 and COX-2 to different degree. Methylated flavone, chrysoeriol (luteolin-3′-methylether), was found to be the most potent inhibitor of MMP-9 and COX-2 expressions. The effect of chrysoeriol on cell proliferation, cell cycle, apoptosis and metastasis was analyzed by established methods. Chrysoeriol caused cell cycle arrest at G2/M and inhibited migration and invasion of MDA-MB-231 cells. The structure–activity relations amongst the flavonoids as NF-κB signaling inhibitors was studied. The study indicates differences between the actions of various flavonoids on NF-κB activation and on the biological activities of breast cancer cells. Flavones in general, were more active than the corresponding flavonols.     

Up-regulation of breast cancer resistance protein plays a role in HER2-mediated chemoresistance through PI3K/Akt and nuclear factor-kappa B signaling pathways in MCF7 breast cancer cells

Human epidermal growth factor receptor 2 (HER2/neu, also known as ErbB2) overexpression is correlated with the poor prognosis and chemoresistance in cancer. Breast cancer resistance protein (BCRP and ABCG2) is a drug efflux pump responsible for multidrug resistance (MDR) in a variety of cancer cells. HER2 and BCRP are associated with poor treatment response in breast cancer patients, although the relationship between HER2 and BCRP expression is not clear. Here, we showed that transfection of HER2 into MCF7 breast cancer cells (MCF7/HER2) resulted in an up-regulation of BCRP via the phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappa B (NF-κB) signaling. Treatment of MCF/HER2 cells with the PI3K inhibitor LY294002, the IκB phosphorylation inhibitor Bay11-7082, and the dominant negative mutant of IκBα inhibited HER2-induced BCRP promoter activity. Furthermore, we found that HER2 overexpression led to an increased resistance of MCF7 cells to multiple antitumor drugs such as paclitaxel (Taxol), cisplatin (DDP), etoposide (VP-16), adriamycin (ADM), mitoxantrone (MX), and 5-fluorouracil (5-FU). Moreover, silencing the expression of BCRP or selectively inhibiting the activity of Akt or NF-κB sensitized the MCF7/HER2 cells to these chemotherapy agents at least in part. Taken together, up-regulation of BCRP through PI3K/AKT/NF-κB signaling pathway played an important role in HER2-mediated chemoresistance of MCF7 cells, and AKT, NF-κB, and BCRP pathways might serve as potential targets for therapeutic intervention.     

Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-κB acetylation in fibroblast-like synoviocyte MH7A cells

Histone acetyltransferase (HAT) inhibitors (HATi) isolated from dietary compounds have been shown to suppress inflammatory signaling, which contributes to rheumatoid arthritis. Here, we identified a novel HATi in Punica granatum L. known as delphinidin (DP). DP did not affect the activity of other epigenetic enzymes (histone deacetylase, histone methyltransferase, or sirtuin1). DP specifically inhibited the HAT activities of p300/CBP. It also inhibited p65 acetylation in MH7A cells, a human rheumatoid arthritis synovial cell line. DP-induced hypoacetylation was accompanied by cytosolic accumulation of p65 and nuclear localization of IKBα. Accordingly, DP treatment inhibited TNFα-stimulated increases in NF-κB function and expression of NF-κB target genes in these cells. Importantly, DP suppressed lipopolysaccharide-induced pro-inflammatory cytokine expression in Jurkat T lymphocytes, demonstrating that HATi efficiently suppresses cytokine-mediated immune responses. Together, these results show that the HATi activity of DP counters anti-inflammatory signaling by blocking p65 acetylation and that this compound may be useful in preventing inflammatory arthritis.     

Adenosine triphosphate regulates NADPH oxidase activity leading to hydrogen peroxide production and COX-2/PGE2 expression in A549 cells

Non-small cell lung carcinoma (NSCLC) accounts for most of all lung cancers, which is the leading cause of mortality in human beings. High level of cyclooxygenase-2 (COX-2) is one of the features of NSCLC and related to the low survival rate of NSCLC. However, whether extracellular nucleotides releasing from stressed resident tissues contributes to the expression of COX-2 remains unclear. Here, we showed that stimulation of A549 cells by adenosine 5'-O-(3-thiotriphosphate) (ATPγS) led to an increase in COX-2 gene expression and prostaglandin E(2) (PGE(2)) synthesis, revealed by Western blotting, RT-PCR, promoter assay, and enzyme-linked immunosorbent assay. In addition, ATPγS induced intracellular reactive oxygen species (ROS) generation through the activation of NADPH oxidase. The increase of ROS level resulted in activation of the c-Src/epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor (NF)-κB cascade. We also found that activated Akt was translocated into the nucleus and recruited with NF-κB and p300 to form a complex. Thus, activation of p300 modulated the acetylation of histone H4 via the NADPH oxidase/c-Src/EGFR/PI3K/Akt/NF-κB cascade stimulated by ATPγS. Our results are the first to show a novel role of NADPH oxidase-dependent Akt/p65/p300 complex formation that plays a key role in regulating COX-2/PGE(2) expression in ATPγS-treated A549 cells. Taken together, we demonstrated that ATPγS stimulated activation of NADPH oxidase, resulting in generation of ROS, which then activated the downstream c-Src/EGFR/PI3K/Akt/NF-κB/p300 cascade to regulate the expression of COX-2 and synthesis of PGE(2) in A549 cells. Understanding the regulation of COX-2 expression and PGE(2) release by ATPγS on A549 cells may provide potential therapeutic targets of NSCLC.     

Resveratrol inhibits urban particulate matter-induced COX-2/PGE2 release in human fibroblast-like synoviocytes via the inhibition of activation of NADPH oxidase/ROS/NF-κB

Human fibroblast-like synoviocytes (FLSs) play a role in joint synovial inflammation in rheumatoid arthritis (RA). Some evidence indicates that particulate matter (PM) in air pollution could contribute to the progression of RA. However, more research is needed to clarify this relationship. Up-regulation of cyclooxygenase (COX)-2 and its metabolite prostaglandin E₂ (PGE₂) are implicated in various inflammatory diseases. Resveratrol, a polyphenol found mainly in grapes and red wine, has antioxidant and anti-inflammatory activities. In the present study, we demonstrated that resveratrol reduced PM-induced COX-2/PGE₂ expression in human FLSs, and attenuated PM-enhanced NADPH oxidase activity and ROS generation. In addition, PM induced Akt, ERK1/2, or p38 MAPK activation, which was inhibited by resveratrol. Finally, we demonstrated that PM enhanced NF-κB p65 phosphorylation and the NF-κB promoter activity, which were reduced by pretreatment with a ROS inhibitor or resveratrol. Thus, we concluded that resveratrol functions as a suppressor of PM-induced inflammatory signaling pathways by inhibiting COX-2/PGE₂ expression.