Fucoxanthin decreases lipopolysaccharide-induced acute lung injury through the inhibition of RhoA activation and the NF-κB pathway.

Fucoxanthin is a natural pigment widely distributed in macroalgae and microalgae. An orange-colored xanthophyll, it has several bioactive effects, including anticancer, anti-obesity, oxidative stress reduction, and anti-inflammation. Acute lung injury (ALI) caused by acute infections or injurious stimuli to the lung tissues is a severe pulmonary inflammatory disease. To date, no evidence has shown ALI to be reduced by fucoxanthin through activation of Ras homolog family member A (RhoA) and the nuclear factor (NF)-κB pathway in lipopolysaccharide (LPS)-treated mice. Pretreatment with fucoxanthin inhibited histopathological changes in lung tissues and neutrophil infiltration into bronchoalveolar lavage fluid induced by LPS in ALI mice. Moreover, LPS-induced proinflammatory cytokine expression and neutrophil infiltration were inhibited by fucoxanthin in a concentration-dependent manner. Pretreatment of mice with fucoxanthin inhibited NF-κB phosphorylation and IκB degradation in the lungs of mice with LPS-induced ALI. We further found that phosphorylation of Akt and p38 mitogen-activated protein KINASE (MAPK) was inhibited by fucoxanthin. By contrast, the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase was not inhibited by fucoxanthin. Furthermore, we found that the activation of RhoA was inhibited by fucoxanthin in LPS-induced ALI. On the basis of these results, we propose that fucoxanthin disrupts the RhoA activation-mediated phosphorylation of Akt and p38 MAPK, leading to NF-κB activation in mice with LPS-induced ALI.

Proinflammatory Responses of 1-Nitropyrene against RAW264.7 Macrophages through Akt Phosphorylation and NF-κB Pathways.

Air pollution is a major environmental and public health problem worldwide. A nitro-polycyclic aromatic hydrocarbon and the most abundant air pollutant in diesel engine exhaust, 1-nitropyrene (1-NP), is caused by the incomplete combustion of carbonaceous organic substances. Macrophages are effector cells of the innate immune cells that provide resistance in the peripheral tissue. The overactivation of macrophages results in inflammation. The generation of proinflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumour necrosis factor alpha, is induced by 1-NP in a concentration-dependent manner in macrophages. In this study, the production of proinflammatory mediators, such as nitrogen oxide and prostaglandin E2, was induced by 1-NP in a concentration-dependent manner through the expression of iNOS and COX2. The generation of proinflammatory cytokines, iNOS, and COX2 was induced by 1-NP through nuclear factor (NF)-κB p65 phosphorylation and the degradation of its upstream factor, IκB. Finally, Akt phosphorylation was induced by 1-NP in a concentration-dependent manner. These findings suggest that 1-NP exhibits a proinflammatory response through the NF-κB pathway activation due to Akt phosphorylation.

Genotoxic effects of 1-nitropyrene in macrophages are mediated through a p53-dependent pathway involving cytochrome c release, caspase activation, and PARP-1 cleavage.

Genotoxic stress from environmental pollutants plays a critical role in cytotoxicity. The most abundant nitro-polycyclic aromatic hydrocarbon in environmental pollutants, 1-nitropyrene (1-NP), is generated during fossil fuel, diesel, and biomass combustion under sunlight. Macrophages, the key regulators of the innate immune system, provide the first line of defense against pathogens. The toxic effects of 1-NP on macrophages remain unclear. Through a lactate dehydrogenase assay, we measured the cytotoxicity induced by 1-NP. Our results revealed that 1-NP induced genotoxicity also named DNA damage, including micronucleus formation and DNA strand breaks, in a concentration-dependent manner. Furthermore, 1-NP induced p53 phosphorylation and nuclear accumulation; mitochondrial cytochrome c release; caspase-3 and -9 activation and cleavage; and poly (ADP-ribose) polymerase-1 (PARP-1) cleavage in a concentration-dependent manner. Pretreatment with the PARP inhibitor, 3-aminobenzamide, significantly reduced cytotoxicity, genotoxicity, and PARP-1 cleavage induced by 1-NP. Pretreatment with the caspase-3 inhibitor, z-DEVD-fmk, significantly reduced cytotoxicity, genotoxicity, PARP-1 cleavage, and caspase 3 activation induced by 1-NP. Pretreatment with the p53 inhibitor, pifithrin-α, significantly reduced cytotoxicity, genotoxicity, PARP-1 cleavage, caspase 3 activation, and p53 phosphorylation induced by 1-NP. We propose that cytotoxicity and genotoxicity induced by 1-NP by PARP-1 cleavage via caspase-3 and -9 activation through cytochrome c release from mitochondria and its upstream p53-dependent pathway in macrophages.

Nerolidol Suppresses the Inflammatory Response during Lipopolysaccharide-Induced Acute Lung Injury via the Modulation of Antioxidant Enzymes and the AMPK/Nrf-2/HO-1 Pathway.

Acute lung injury (ALI) is a life-threatening disease that is characterised by the rapid onset of inflammatory responses. Lipopolysaccharide (LPS) is an endotoxin that plays an important role in triggering ALI via pneumonia and sepsis. However, no effective therapeutic strategies are currently available to treat ALI. Nerolidol is an aliphatic sesquiterpene alcohol that is found in the essential oils of many flowers as well as floral plants. It has been shown to exhibit anti-inflammatory, antioxidant, and anticancer properties. Herein, we show that nerolidol pretreatment counteracted the histopathological hallmarks in LPS-induced ALI mice. Indeed, nerolidol pretreatment inhibited LPS-induced alveolar-capillary barrier disruption, lung edema, and lipid peroxidation. Moreover, nerolidol pretreatment prevented the LPS from decreasing the enzymatic activities of superoxide dismutase, catalase, and glutathione peroxidase. Importantly, nerolidol treatment enhanced phosphorylation of AMP-activated protein kinase (AMPK) and expression of nuclear factor erythroid-derived 2-related factor 2 (Nrf-2) and heme oxygenase-1 (HO-1). Taken together, our study reveals the novel protective effects of nerolidol in LPS-induced ALI via the induction of antioxidant responses and activation of the AMPK/Nrf-2/HO-1 signalling pathway.

Cadmium nitrate-induced neuronal apoptosis is protected by N-acetyl-l-cysteine via reducing reactive oxygen species generation and mitochondria dysfunction.

Cigarette smoking is a well-established risk factor for various diseases, such as cardiovascular diseases, neurodegeneration, and cancer. Cadmium nitrate (Cd(NO3)2) is one of the major products from the cigarette smoke. Up to now, no supporting evidence on Cd(NO3)2-induced apoptosis and its related working mechanism in neurons has been found. In present study, the mode of cell death, caspase activities, reactive oxygen species (ROS) generation, and mitochondrial dysfunction in N2a cells, which are neuron-like cells, were assessed by Annexin V-FITC and PI assays, caspase fluorometric assay, DCFH-DA fluorescence assay, and JC-1 fluorescence assay respectively. The results showed that not only Cd(NO3)2 induced apoptosis and necrosis but also the activities of caspase-3 and -9 expressed in a concentration-dependent manner. In addition, Cd(NO3)2 also induced both mitochondrial dysfunction and ROS generation in a concentration-dependent manner. All these indicated that in N2a cells parallel trends could be observed in apoptosis, caspase-3 and -9 activities, mitochondrial dysfunction, and ROS generation when induced by Cd(NO3)2. Furthermore, Cd(NO3)2-induced apoptosis, caspases activities, mitochondrial dysfunction, and ROS generation were reduced by N-acetyl-l-cysteine (NAC). These results indicated that Cd(NO3)2-induced neuronal apoptosis was reduced by NAC via intrinsic apoptotic caspase cascade activities and their up-stream factors, including mitochondrial dysfunction and ROS generation.

Zerumbone from Zingiber zerumbet Ameliorates Lipopolysaccharide-Induced ICAM-1 and Cytokines Expression via p38 MAPK/JNK-IκB/NF-κB Pathway in Mouse Model of Acute Lung Injury.

Acute lung injury (ALI) is a clinical syndrome with high morbidity and mortality rates mainly caused by Gram-negative bacteria. Nevertheless, an effective treatment strategy for ALI is yet to be developed. Zerumbone, a sesquiterpene isolated from Zingiber zerumbet Smith, possesses several advantageous bioeffects such as antioxidation, anti-inflammation, and antiulcer. Pretreatment of zerumbone inhibited lipopolysaccharide (LPS)-induced arterial blood gas exchange, neutrophils infiltration, and increased pulmonary vascular permeability. LPS-induced expression of intercellular adhesion molecule-1 (ICAM-1) was inhibited by zerumbone at a lower concentration than that of vascular cell adhesion molecule-1 (VCAM-1). In addition, proinflammatory cytokines, such as interleukin (IL)-1ß and macrophage inflammatory protein (MIP)-2 were suppressed by zerumbone. The phosphorylation of nuclear factor (NF)-κB, a proinflammatory transcription factor, and degradation of inhibitor of κB (IκB), an inhibitor of NF-κB, were also reduced by zerumbone. Furthermore, we found the inhibitory concentration of zerumbone on phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was lower than that of extracellular signal-regulated kinase (ERK). In conclusion, zerumbone could be a potential protective agent for ALI, possibly via expression of ICAM-1, IL-1ß, and MIP-2. The protective mechanism of zerumbone was by reversing the activation of p38 MAPK/JNK-IκB/NF-κB pathway.

Bioactive Phenolic Components from the Twigs of Atalantia buxifolia.

Five new compounds named buxifoximes A-C (1-3), buxifobenzoate (4), and 7- O-(7'-peroxygeranyl) coumarin (5), together with 25 known compounds, were identified from the twigs of Atalantia buxifolia. Compounds 1-3 are unique secondary metabolites with the aldoxime functionality. The structures of the isolates were determined on the basis of spectroscopic data analyses, and the structure of 1 was confirmed by an X-ray single-crystallographic analysis. With respect to bioactivity, antidengue virus, anti-inflammatory, and cytotoxic activities of all purified compounds were tested and evaluated. Compound 1 showed a significant anti-inflammatory effect by inhibiting superoxide anion generation with an IC50 value of 4.8 ± 0.7 µM. Among the acridone alkaloids, 5-hydroxy- N-methylseverifoline (23) exhibited antidengue activity (IC50 = 5.3 ± 0.4 µM), and atalaphyllinine (20) demonstrated cytotoxicity (IC50 = 6.5 ± 0.0 µM) against the human liver cancer cell line, HepG2.

Zerumbone reduced the inflammatory response of acute lung injury in endotoxin-treated mice via Akt-NFκB pathway.

Zerumbone, a cyclic eleven-membered sesquiterpene, is the major component of the essential oil isolated from the wild ginger, Zingiber zerumbet. There are several beneficial pharmacological activities of zerumbone including anti-inflammatory, antioxidant, and anticancer activities. Acute lung injury (ALI) is an acute pulmonary inflammatory disorder with high morbidity and mortality rate. In present study, we aimed to investigate the protective effects and mechanisms of zerumbone on endotoxin, lipopolysaccharide (LPS)-induced ALI. Mice were pretreated with zerumbone at various concentrations for 30 min followed by intratracheal administration of LPS for 6 h. Pretreatment with zerumbone not only reduced leukocytes infiltration into the alveolar space but also inhibited lung edema in LPS-induced ALI. Decreased secretion of proinflammatory cytokines such as TNFα and IL-6 caused by LPS were reversed by zerumbone. LPS-induced expressions of proinflammatory mediators, iNOS and COX-2, were inhibited by zerumbone. In addition, NFκB activation and Akt phosphorylation were inhibited by zerumbone in LPS-induced ALI. All these results suggested that the protective mechanisms of zerumbone on endotoxin-induced ALI were via inhibition of Akt-NFκB activation.

Protective effect of zerumbone reduces lipopolysaccharide-induced acute lung injury via antioxidative enzymes and Nrf2/HO-1 pathway.

Acute lung injury (ALI) is a serious disease with high morbidity and mortality rate. Although there are effective strategies for treatment of ALI; a widely accepted specific pharmacotherapy has not yet established. Zerumbone, the major active phytochemical compound from Zingiber zerumbet Smith, exhibits various beneficial biological and pharmacological activities, such as antioxidation, anti-inflammation, immunomodulation, and anti-cancer. We aimed to study the potential protective effects and mechanisms of zerumbone in mouse model of lipopolysaccharide (LPS)-induced ALI. Pretreatment with zerumbone inhibited the histopatholgical changes such as neutrophils infiltration, increased in alveolar barrier thickness, hemorrhage, and hyaline membrane formation occurred in lungs in LPS-induced ALI. In addition, not only LPS-induced activation of myeloperoxidase (MPO) and metallopeptidase-9 (MMP-9) was suppressed by zerumbone, but also lipid peroxidation in lungs was inhibited as well. Moreover, pretreatment with zerumbone reversed the antioxidative enzymes activities, including superoxide dismutase, catalase, and glutathione peroxidase, decreased by LPS and enhanced the expression of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase (HO-1) induced by LPS. These results from present study suggested that the protective mechanisms of zerumbone on LPS-induced ALI were via up-regulation of antioxidative enzymes and Nrf2/HO-1 pathway.

Complementary usage of Rhodiola crenulata (L.) in chronic obstructive pulmonary disease patients: the effects on cytokines and T cells.

Although chronic obstructive pulmonary disease (COPD) is an inflammatory disease predominantly involving T cells, no study of Rhodiola as an immunomodulator in COPD patients has been reported. In this study, COPD patients took Rhodiola crenulata 500 mg (n = 38) or placebo (starch/phosphate buffered saline) (n = 19) daily for 12 weeks and were compared with untreated, age-matched, and sex-matched non-COPD control subjects. Our results showed that serum levels of IL-2, IL-10, and IFN-γ in COPD patients before treatment are significantly higher than levels in non-COPD controls (p < 0.05). A significant decrease in IFN-γ was seen in the Rhodiola treatment group (p < 0.05) but not in the placebo group (p > 0.05). The results suggested that Rhodiola treatment had beneficial antiinflammation effects, lower COPD assessment test score and decreased high-sensitivity C-reactive protein, on COPD patients (p < 0.05). The effects of Rhodiola treatment on COPD patients were shown to decrease the IFN-γ concentration and CD8(+) count but increase the expressions of CD4(+) CD25(+) FOXP3(+) and CD4(+) CD25(+) CD45(+) FOXP3(+) in the blood significantly (p < 0.05). This is the first trial using Rhodiola as a complementary therapy for COPD patients. T cells play an important role in the pathogenesis of COPD through the increased expression of CD8(+) T cells and IFN-γ and may be a viable target for potential therapy.

Selective apoptotic cell death effects of oral cancer cells treated with destruxin B.

BACKGROUND: Recent studies have revealed that destruxins (Dtx) have potent cytotoxic activities on individual cancer cells, however, data on oral cancer cells especial human are absent. METHODS: Destruxin B (DB) was isolated and used to evaluate the selective cytotoxicity with human oral cancer cell lines, GNM (Neck metastasis of gingival carcinoma) and TSCCa (Tongue squamous cell carcinoma) cells, and normal gingival fibroblasts (GF) were also included as controls. Cells were tested with different concentrations of DB for 24, 48, and 72 h by MTT assay. Moreover, the mechanism of cytotoxicity was investigated using caspase-3 Immunofluorescence, annexin V/PI staining, and the expression of caspase-3, Bax, and Bcl-2 by western blotting after treated with different concentrations of DB for 72 h as parameters for apoptosis analyses. RESULTS: The results show that DB exhibited significant (p < 0.01) and selective time- and dose-dependent inhibitory effects on GNM and TSCCa cells viability but not on GF cells. The data suggested that DB is capable to induce tumor specific growth inhibition in oral GNM and TSCCa cancer cells via Bax/Bcl-2-mediated intrinsic mitochondrial apoptotic pathway in time- and dose-dependent manners. CONCLUSIONS: This is the first report on the anti-proliferation effect of DB in oral cancer cells. The results reported here may offer further evidences to the development of DB as a potential complementary chemotherapeutic target for oral cancer complications.

Reduction of TLR4 mRNA stability and protein expressions through inhibiting cytoplasmic translocation of HuR transcription factor by E2 and/or ERα in LPS-treated H9c2 cardiomyoblast cells.

Our previous results have indicated that Akt mediates 17ß-estradiol (E2) and/or estrogen receptor α (ERα) to inhibit lipopolysaccharide (LPS)-induced JNK activity, tumor necrosis factor α (TNFα) protein expression, and exhibits cardioprotective effects. Toll-like receptor 4 (TLR4) mRNAs often contain AU-rich elements (AREs) in their 3'-untranslated regions (3'UTR) which have a high affinity for RNA-binding proteins. It is not known whether E2 and ERα affect TLR4 mRNA stability and TLR4 protein expression through regulating the RNA-binding proteins, human antigen R (HuR), tristetraprolin (TTP) and AU-binding factor 1 (AUF-1) in myocardial cells. Therefore, we investigated if the LPS in- duces these RNA-binding proteins to regulate TLR4 mRNAs of cardiomyocytes, and whether the E2/ERα reduces the TLR4 mRNA stability induced by LPS through the inhibition of RNA-binding protein expression. Using a doxycycline (Dox)-induced Tet-On ERα H9c2 myocardic cell model, we also aimed to identify whether E2 and/or ERα regulate LPS-induced TLR4 mRNA stability. The results of Western blotting and reverse transcription-PCR assays demonstrated that LPS significantly in- creased the level of cytoplasmic HuR protein and the stability of TLR4 mRNA, and farther induced TLR4 protein expression in H9c2 cells, an effect mediated through the JNK pathway. Interestingly, E2 and/or ERα decreased the cytoplasmic HuR protein level and TLR4 mRNA stability, and farther decreased the level of TLR4 protein induced by LPS in H9c2 cardiomyoblast cells. Therefore, LPS triggered HuR expression which led to enhanced TLR4 mRNA and upregulated TLR4 expression through JNK1/2 in myocardial cells.

Glycoproteomic analysis of WGA-bound glycoprotein biomarkers in sera from patients with lung adenocarcinoma.

Differential protein expression profiles in the serum samples from patients with lung adenocarcinoma may be associated with glycosylation during cancer development. In this study, we used various glycoproteomic approaches to investigate the different glycoproteomic profiles of human normal and lung adenocarcinoma serum samples and to investigate putative altered glycoprotein biomarkers. In our preliminary screening, FITC-labeled lectin staining was used for the detection of specific glycoprotein profiles. wheat germ agglutinin (WGA) lectin had the highest level of specific binding to glycoproteins in both samples. We enriched for glycoproteins in the serum samples using WGA lectin affinity and then performed co-immunoprecipitation with anti-haptoglobin and 2-DE, 2-D difference in-gel electrophoresis and MS analyses. From these analyses, we identified 39 differentially expressed proteins, including 27 up-regulated proteins and 12 down-regulated proteins. Bioinformatics tools were used to search for protein ontology, category classifications and prediction of glycosylation sites. In addition, three up-regulated glycoproteins (adiponectin, cerulolasmin and glycosylphosphatidyl-inositol-80) and two down-regulated glycoproteins (cyclin H and Fyn) that were found to be correlated with lung cancer development were validated by Western blot analysis. We suggest that these altered glycoproteins may be useful as biomarkers for lung cancer development and progression.

Proteomics analysis of A375 human malignant melanoma cells in response to arbutin treatment.

Although the toxicogenomics of A375 human malignant melanoma cells treated with arbutin have been elucidated using DNA microarray, the proteomics of the cellular response to this compound are still poorly understood. In this study, we performed proteomic analyses to investigate the anticancer effect of arbutin on the protein expression profile in A375 cells. After treatment with arbutin (8 microg/ml) for 24, 48 and 72 h, the proteomic profiles of control and arbutin-treated A375 cells were compared, and 26 differentially expressed proteins (7 upregulated and 19 downregulated proteins) were identified by MALDI-Q-TOF MS and MS/MS. Among these proteins, 13 isoforms of six identical proteins were observed. Bioinformatic tools were used to search for protein function and to predict protein interactions. The interaction network of 14 differentially expressed proteins was found to be correlated with the downstream regulation of p53 tumor suppressor and cell apoptosis. In addition, three upregulated proteins (14-3-3G, VDAC-1 and p53) and five downregulated proteins (ENPL, ENOA, IMDH2, PRDX1 and VIME) in arbutin-treated A375 cells were validated by RT-PCR analysis. These proteins were found to play important roles in the suppression of cancer development.

Proteomics analysis of kojic acid treated A375 human malignant melanoma cells.

Although the toxicogenomics of kojic acid treated A375 human malignant melanoma cells has been elucidated, the proteomics of cellular response is still poorly understood. We performed proteomic analysis to investigate the anticancer effect of kojic acid on protein expression profile in A375 cells. A375 cells were treated with kojic acid at 8 microg/mL for 24, 48, and 72 h. With the use of 2-D PAGE and MALDI-Q-TOF MS and MS/MS analyses, proteomic profiles of A375 cells between control and kojic acid treatment were compared, and 30 differentially expressed proteins, containing 2 up-regulated proteins and 28 down-regulated proteins, were identified. Among these proteins, 17 isoforms of 5 identical proteins were observed and 11 chaperone proteins showed the high proportion of protein spots with 36.7% of total proteins. Bioinformatic tools were used to search for protein function and prediction of protein interaction. Sixteen differentially expressed proteins exhibited interaction network linked to the downstream regulations of p53 tumor suppressor and cell apoptosis, which may lead to suppress the melanogenesis and tumorigenesis of kojic acid treated A375 cells. In addition, GRP75, VIME and 2AAA were validated by Western blot analysis, whereas GRP75, 2AAA, HS90B, ENPL and KPYM were validated by RT-PCR. Therefore, these proteins play the important roles in cancer progression and may be potential biomarkers that are useful for diagnostic and therapeutic applications of malignant melanoma cancer.

Toxicogenomics of A375 human malignant melanoma cells.

Toxicogenomics applications are increasingly applied to the evaluation of preclinical drug safety, and to explain toxicities associated with compounds at the mechanism level. In this review, we aim to describe the application of toxicogenomics tools for studying the genotoxic effect of active compounds on the gene-expression profile of A375 human malignant melanoma cells, through the other molecular functions of target genes, regulatory pathways and mechanisms of malignant melanomas. It also includes the current systems biology approaches, which are very useful for analyzing the biological system and understanding the entire mechanisms of malignant melanomas. We believe that this review would be very potent and useful for studying the toxicogenomics of A375 melanoma cells, and for further diagnostic and therapeutic applications.

Toxicogenomics of kojic acid on gene expression profiling of a375 human malignant melanoma cells.

Kojic acid is a natural product and normally used as a food additive and preservative, a skin-whitening agent in cosmetics, a plant growth regulator and a chemical intermediate. Using DNA microarray technology, the overall biological effects of kojic acid on the gene expression profiling of a human skin A375 malignant melanoma cells were examined. After treatment with kojic acid, a total of 361 differentially expressed genes were distinctively changed with 136 up-regulated genes and 225 down-regulated genes. We used the bioinformatics tool to search the gene ontology and category classification of differentially expressed genes that provided the useful information of expressed genes belonging to cellular component, molecular function and biological process in regulation of melanogenesis. Seven down-regulated genes of APOBEC1, ARHGEF16, CD22, FGFR3, GALNT1, UNC5C and ZNF146 that were typically validated by the real-time quantitative PCR (RT-qPCR) analysis technology showed to be the tumor suppressor genes in melanoma cancer cells. Thus, microarray technology coupled with RT-qPCR offered a high throughput method to explore the number of differentially expressed genes responding to kojic acid and their biological functions, and led to more understanding of kojic acid effects on skin cancer therapy and related side effects. Moreover, the differentially expressed genes may become useful markers of skin malignant melanoma for further diagnostic and therapeutic applications.