Systematic discovery of drug action mechanisms by an integrated chemical genomics approach: identification of functional disparities between azacytidine and decitabine.

Polypharmacology (the ability of a drug to affect more than one molecular target) is considered a basic property of many therapeutic small molecules. Herein, we used a chemical genomics approach to systematically analyze polypharmacology by integrating several analytical tools, including the LINCS (Library of Integrated Cellular Signatures), STITCH (Search Tool for Interactions of Chemicals), and WebGestalt (WEB-based GEne SeT AnaLysis Toolkit). We applied this approach to identify functional disparities between two cytidine nucleoside analogs: azacytidine (AZA) and decitabine (DAC). AZA and DAC are structurally and mechanistically similar DNA-hypomethylating agents. However, their metabolism and destinations in cells are distinct. Due to their differential incorporation into RNA or DNA, functional disparities between AZA and DAC are expected. Indeed, different cytotoxicities of AZA and DAC toward human colorectal cancer cell lines were observed, in which cells were more sensitive to AZA. Based on a polypharmacological analysis, we found that AZA transiently blocked protein synthesis and induced an acute apoptotic response that was antagonized by concurrently induced cytoprotective autophagy. In contrast, DAC caused cell cycle arrest at the G2/M phase associated with p53 induction. Therefore, our study discriminated functional disparities between AZA and DAC, and also demonstrated the value of this chemical genomics approach that can be applied to discover novel drug action mechanisms.

Differential proteomics to explore the inhibitory effects of acidic, slightly acidic electrolysed water and sodium hypochlorite solution on Vibrio parahaemolyticus.

Slightly acidic electrolysed water (SlAEW) and acidic electrolysed water (AEW) have been demonstrated to effectively inactivate food-borne pathogens. However, the underlying mechanism of inactivation remains unknown. Therefore, in this study, a differential proteomic platform was used to investigate the bactericidal mechanism of SlAEW, AEW, and sodium hypochlorite (NaOCl) solutions against Vibrio parahaemolyticus. The upregulated proteins after SlAEW, AEW, and NaOCl treatments were identified as outer membrane proteins K and U. The downregulated proteins after the SlAEW, AEW, and NaOCl treatments were identified as adenylate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and enolase, all of which are responsible for energy metabolism. Protein synthesis-associated proteins were downregulated and identified as elongation factor Tu and GAPDH. The inhibitory effects of SlAEW and AEW solutions against V. parahaemolyticus may be attributed to the changes in cell membrane permeability, protein synthesis activity, and adenosine triphosphate (ATP) biosynthesis pathways such as glycolysis and ATP replenishment.

A low toxicity synthetic cinnamaldehyde derivative ameliorates renal inflammation in mice by inhibiting NLRP3 inflammasome and its related signaling pathways.

Uncontrolled inflammation is a leading cause of various chronic diseases. Cinnamaldehyde (CA) is a major bioactive compound isolated from the essential oil of the leaves of Cinnamomum osmophloeum kaneh that exhibits anti-inflammatory activity; however, the use of CA is limited by its cytotoxicity. Here, we synthesized three CA derivatives and identified 4-hydroxycinnamaldehyde-galactosamine (HCAG) as a low toxicity anti-inflammatory compound in vitro (HCAG IC50 ≫ 1600 µM; CA IC50=40 µM) and in vivo. HCAG reduced pro-inflammatory mediator expression in LPS-activated macrophages by inhibiting MAPK and PKC-α/δ phosphorylation, decreasing ROS generation and reducing NF-κB activation. HCAG also reduced NLRP3 inflammasome-derived IL-1ß secretion by inhibiting the ATP-mediated phosphorylation of AKT and PKC-α/δ. In a mouse model of LPS-induced renal inflammation, we observed reduced albuminuria and a mild degree of glomerular proliferation, glomerular sclerosis and periglomerular inflammation in the HCAG-treated mice compared with the vehicle-treated mice. The underlying mechanisms for these renoprotective effects involved: (1) inhibited NLRP3 inflammasome activation; (2) decreased superoxide anion levels and apoptosis; and (3) suppressed activation of NF-κB and related downstream inflammatory mediators.

Glycosylation-dependent interaction between CD69 and S100A8/S100A9 complex is required for regulatory T-cell differentiation.

Cluster of differentiation (CD)69 is a leukocyte activation receptor involved in the maintenance of immune homeostasis and is positively selected in activated regulatory T (Treg) cells, implicating its role during Treg-cell differentiation. By RNA interference, we show that CD69 is not sufficient to support the conversion of CD4(+) naive T cells into Treg cells, whereas it does that of human peripheral blood mononuclear cells (hPBMCs) (P < 0.01), suggesting that a ligand-receptor interaction is required for CD69 function. Using immunoprecipitation and mass spectrometry, we identified the S100A8/S100A9 complex as the natural ligand of CD69 in hPBMCs. CD69 specifically associates with S100A8/S100A9 complex as confirmed by in vitro binding and competition assay, and the treatment of CD69 with peptide-N-glycosidase significantly abolishes such association. In agreement, the glycomics analysis determines the glycosylation site and the N-glycan composition of CD69, and terminal removal of sialic acid from that N-linked glycans reverses the generation of forkhead box P3-positive Treg cells (23.21%; P < 0.05). More specifically, we showed that CD69-S100A8/S100A9 association is required for the up-regulation of suppressor of cytokine signaling 3 resulting in inhibited signaling of signal transducer and activator of transcription 3 (36.54% increase upon CD69 silencing; P < 0.01). This might in turn support the secretion of key regulator TGF-ß (∼ 3.28-fold decrease upon CD69 silencing; P < 0.05), leading to reduced production of IL-4 in hPBMCs. Our results demonstrate the functional and mechanistic interplays between CD69 and S100A8/S100A9 in supporting Treg-cell differentiation.

GM2-activator protein: a new biomarker for lung cancer.

INTRODUCTION: Effective biomarkers for early diagnosis of lung cancer are needed. A recent study demonstrated that urinary GM2-activator protein (GM2AP) level was increased in lung cancer patients. This study aims to validate the potential application of GM2AP as a biomarker for diagnosis of lung cancer. METHODS: Serum and urine samples were obtained from 189 participants (133 patients for treatment naive lung cancer, 26 healthy volunteers for urine, and 30 healthy volunteers for serum). GM2AP level was detected by Western blotting and quantified using enzyme-linked immunosorbent assay (ELISA). The GM2AP expression in tumors and nontumor parts of lung tissues from 143 nonsmall cell lung cancers was detected by immunohistochemical stains. RESULTS: There was an 8.11 ± 1.36 folds increase in urine and a 5.41 ± 0.73 folds increase in serum level of GM2AP in lung cancer patients compared with healthy volunteers (p < 0.0001), achieving a 0.89 AUC value in urine and 0.90 AUC value in serum for the receiver-operating characteristic curves. Both serum and urine levels of GM2AP correlated significantly with pathology stages (urine, p = 0.009; serum, p < 0.0001). Using immunohistochemical, positive expression of GM2AP was found at 83.9% of nonsmall cell lung cancers patients and none in normal tissue. The GM2AP expression was significantly correlated with pathology stage (p = 0.0001). Patients with higher GM2AP expression had shorter overall survival (p = 0.045) and disease-free survival (p = 0.049) than lower GM2AP expression. Moreover, the multivariate analysis suggested GM2AP as an independent predictors of disease-free survival and overall survival. CONCLUSIONS: Our study demonstrates that GM2AP might serve as potential diagnostic and prognostic biomarkers in patients with lung cancer.

MicroRNA-21-3p, a berberine-induced miRNA, directly down-regulates human methionine adenosyltransferases 2A and 2B and inhibits hepatoma cell growth.

Methionine adenosyltransferase (MAT) is the cellular enzyme that catalyzes the synthesis of S-adenosylmethionine (SAM), the principal biological methyl donor and a key regulator of hepatocyte proliferation, death and differentiation. Two genes, MAT1A and MAT2A, encode 2 distinct catalytic MAT isoforms. A third gene, MAT2B, encodes a MAT2A regulatory subunit. In hepatocellular carcinoma (HCC), MAT1A downregulation and MAT2A upregulation occur, known as the MAT1A:MAT2A switch. The switch is accompanied with an increasing expression of MAT2B, which results in decreased SAM levels and facilitates cancer cell growth. Berberine, an isoquinoline alkaloid isolated from many medicinal herbs such as Coptis chinensis, has a wide range of pharmacological effects including anti-cancer effects. Because drug-induced microRNAs have recently emerged as key regulators in guiding their pharmacological effects, we examined whether microRNA expression is differentially altered by berberine treatment in HCC. In this study, we used microRNA microarrays to find that the expression level of miR-21-3p (previously named miR-21*) increased after berberine treatment in the HepG2 human hepatoma cell line. To predict the putative targets of miR-21-3p, we integrated the gene expression profiles of HepG2 cells after berberine treatment by comparing with a gene list generated from sequence-based microRNA target prediction software. We then confirmed these predictions through transfection of microRNA mimics and a 3' UTR reporter assay. Our findings provide the first evidence that miR-21-3p directly reduces the expression of MAT2A and MAT2B by targeting their 3' UTRs. In addition, an overexpression of miR-21-3p increased intracellular SAM contents, which have been proven to be a growth disadvantage for hepatoma cells. The overexpression of miR-21-3p suppresses growth and induces apoptosis in HepG2 cells. Overall, our results demonstrate that miR-21-3p functions as a tumor suppressor by directly targeting both MAT2A and MAT2B, indicating its therapeutic potential in HCC.

Benzoxazinoids from Scoparia dulcis (sweet broomweed) with antiproliferative activity against the DU-145 human prostate cancer cell line.

Sweet broomweed (Scoparia dulcis) is an edible perennial medicinal herb widely distributed in tropical and subtropical regions of Asia, Africa, and the Americas. Four compounds, (2R)-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-O-ß-galactopyranoside [(2R)-HMBOA-2-O-Gal], 3,6-dimethoxy-benzoxazolin-2(3H)-one (3,6-M2BOA), 3-hydroxy-6-methoxy-2-benzoxazolinone (3-OH-MBOA), and scutellarein 7-O-ß-glucuronamide, along with eight known compounds, including two 7-methoxy-1,4-benzoxazin-3(2H)-one 3-O-hexopyranosides [(2R)-HMBOA-2-O-Glc and (2R)-HDMBOA-2-O-Glc], 6-methoxy-benzoxazolin-2(3H)-one (MBOA), acteoside, sodium scutellarin, p-coumaric acid, and two monosaccharides (fructose and glucose), were isolated from the aqueous extract of S. dulcis. Antiproliferative activities of the six benzoxazinoid compounds against the DU-145 human prostate cancer cell line were assayed, and one of these displayed an IC50 of 65.8 µg/mL.

Chemical and biological evaluation of nephrocizin in protecting nerve growth factor-differentiated PC12 cells by 6-hydroxydopamine-induced neurotoxicity.

The neurotoxin 6-hydroxydopamine (6-OHDA) has been widely used to generate an experimental model of Parkinson's disease. This model is crucial in the search for compounds that diminish 6-OHDA-induced nerve growth factor (NGF)-differentiated PC12 cell death. Nephrocizin (luteolin-7-O-ß-D-glucopyranoside), a flavone glycoside, was isolated from widely distributed plants. The protective effects of pre-treatment with nephrocizin on the induced neurotoxicity in PC12 cells by 6-OHDA and its oxidative products, H2O2-, and p-quinone, were evaluated herein. Nephrocizin promoted cell viability, scavenged ROS-related products, increased cellular glutathione (GSH) levels, and reduced caspase-3 and -8 activities in 6-OHDA-, H2O2-, or p-quinone-treated PC12 cells. Furthermore, nephrocizin-conjugated metabolites in PC12 cells were identified with the boronate-affinity method and LC-MS technology, and preferential regioselectivity at the C2' and C5' positions by the nephrocizin-GSH (or NAC) adduct method was observed. These lines of evidence established that nephrocizin could form a dimer to diminish the intracellular ROS. These results demonstrate the first neuroprotective mechanism of nephrocizin against 6-OHDA-, H2O2- or p-quinone-induced cytotoxicity in PC12 cells via chemical and biological studies. These dietary antioxidants are potential candidates for use in intervention in neurodegenerative diseases.

Improved purity and immunostimulatory activity of ß-(1→3)(1→6)-glucan from Pleurotus sajor-caju using cell wall-degrading enzymes.

The objective of this work was to improve the purity of ß-(1→3)(1→6)-glucan in the native triple helical structure from the fruiting bodies of Pleurotus sajor-caju for effective biological function using cell wall-degrading enzymes. A crude carbohydrate was extracted with hot water, then treated with crude xylanase and cellulase from Paenibacillus curdlanolyticus B-6. ß-Glucan in the extract was purified to homogeneity with a single and symmetrical peak using 650M DEAE Toyopearl and Sepharose CL-6B column chromatography. The purity of ß-glucan was confirmed by high-performance size-exclusion chromatography. Purified ß-glucan was obtained at a purity of up to 90.2%. The Congo red reaction and atomic force microscopy indicated that the purified ß-glucan exhibited a triple helix conformation. Purified ß-glucan was able to effectively up-regulate the functions of macrophages such as nitric oxide (NO) and tumor necrosis factor (TNF-α) production.

Anti-inflammatory and free radial scavenging activities of the constituents isolated from Machilus zuihoensis.

A new biflavonol glycoside, quercetin-3-O-ß-D-glucopyranoside-(3'→O-3''')-quercetin-3-O-ß-D-galactopyranoside (9), together with eight known compounds was isolated for the first time from the leaves of Machilus zuihoensis Hayata (Lauraceae). The structure of compound 9 was elucidated by various types of spectroscopic data analysis. Analysis of the biological activity assay found that compound 9 showed significant superoxide anion scavenging activity (IC50 is 30.4 µM) and markedly suppressed LPS-induced high mobility group box 1 (HMGB-1) protein secretion in RAW264.7 cells. In addition, the HMGB-1 protein secretion was also inhibited by quercitrin (3), ethyl caffeate (6), and ethyl 3-O-caffeoylquinate (7) treatment. In the LPS-stimulated inducible nitric oxide synthase (iNOS) activation analysis, two known compounds, quercetin (1) and ethyl caffeate (6), were found to markedly suppress nitric oxide (NO) production (IC50 value, 27.6 and 42.9 µM, respectively) in RAW264.7 cells. Additionally, it was determined that ethyl caffeate (6) down-regulated mRNA expressions of iNOS, IL-1ß, and IL-10 in the LPS-treatment of RAW264.7 cells via a suppressed NF-kB pathway. These results suggested for the first time that the new compound 9 and other constituents isolated from M. zuihoensis have potential anti-inflammatory and superoxide anion scavenging effects. These constituents may be useful for treating various inflammatory diseases.

Glycoproteomics analysis to identify a glycoform on haptoglobin associated with lung cancer.

Glycosylation is a common protein modification that is of interest in current cancer research because altered carbohydrate moieties are often found during cancer progress. A search for biomarkers in human lung cancer serum samples using glycoproteomic approaches identified fucosylated haptoglobin (Hp) significantly increased in serum of each subtype of lung cancer compared to normal donors. In addition, MS provided evidence of an increase of Hp fucosylation; the glycan structure was determined to be an α 2,6-linked tri-sialylated triantennary glycan containing α1,3-linked fucose attached to the four-linked position of the three-arm mannose of N-linked core pentasaccharide. These preliminary findings suggest that the specific glycoform of Hp may be useful as a marker to monitor lung cancer progression.

Calcium oxalate dihydrate crystal induced changes in glycoproteome of distal renal tubular epithelial cells.

Calcium oxalate dihydrate (COD) crystals can adhere onto the apical surface of renal tubular epithelial cells. This process is associated with crystal growth and aggregation, resulting in kidney stone formation. Glycoproteins have been thought to play roles in response to crystal adhesion. However, components of the glycoproteome that are involved in this cellular response remain largely unknown. Our present study therefore aimed to identify altered glycoproteins upon COD crystal adhesion onto tubular epithelial cells representing distal nephron, the initiating site of kidney stone formation. Madin-Darby Canine Kidney (MDCK) cells were maintained in culture medium with or without COD crystals for 48 h (n = 5 flasks per group). Cellular proteins were extracted, resolved by 2-DE and visualized by SYPRO Ruby total protein stain, whereas glycoproteins were detected by Pro-Q Emerald glycoprotein dye. Spot matching and quantitative intensity analysis revealed 16 differentially expressed glycoprotein spots, whose corresponding total protein levels were not changed by COD crystal adhesion. These altered glycoproteins were successfully identified by Q-TOF MS and/or MS/MS analyses, and potential glycosylation sites were identified by the GlycoMod tool. For example, glycoforms of three proteasome subunits (which have a major role in regulating cell-cell dissociation) were up-regulated, whereas a glycoform of actin-related protein 3 (ARP3) (which plays an important role in cellular integrity) was down-regulated. These coordinated changes implicate that COD crystal adhesion induced cell dissociation and declined cellular integrity in the distal nephron. Our findings provide some novel insights into the pathogenic mechanisms of kidney stone disease at the molecular level, particularly cell-crystal interactions.

Down-regulation of granulocyte-macrophage colony-stimulating factor by 3C-like proteinase in transfected A549 human lung carcinoma cells.

BACKGROUND: Severe Acute Respiratory Syndrome (SARS) is a severe respiratory illness caused by a novel virus, the SARS coronavirus (SARS-CoV). 3C-like protease (3CLpro) of SARS-CoV plays a role in processing viral polypeptide precursors and is responsible of viral maturation. However, the function of 3CLpro in host cells remains unknown. This study investigated how the 3CLpro affected the secretion of cytokines in the gene-transfected cells. RESULTS: From immunofluorescence microscopy, the localization of c-myc tagged 3CLpro was detected both in the cytoplasm and nucleus of transfected A549 cells. Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly decreased in 3CLpro-transfected cells by both RT-PCR and ELISA, but without changes in other cytokines, i.e., IL-1ß, IL-6, IL-8, IL12p40, TNF-α, and TGF-ß. Furthermore, the protein levels of NF-kB decreased in 3CLpro-transfected A549 cells when compared to EGFP transfected cells. CONCLUSIONS: Our results suggest that the 3CLpro may suppress expression of GM-CSF in transfected A549 cells through down-regulation of NF-kB production.

Coregulation of transcription factors and microRNAs in human transcriptional regulatory network.

BACKGROUND: MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression at the post-transcriptional level. Recent studies have suggested that miRNAs and transcription factors are primary metazoan gene regulators; however, the crosstalk between them still remains unclear. METHODS: We proposed a novel model utilizing functional annotation information to identify significant coregulation between transcriptional and post-transcriptional layers. Based on this model, function-enriched coregulation relationships were discovered and combined into different kinds of functional coregulation networks. RESULTS: We found that miRNAs may engage in a wider diversity of biological processes by coordinating with transcription factors, and this kind of cross-layer coregulation may have higher specificity than intra-layer coregulation. In addition, the coregulation networks reveal several types of network motifs, including feed-forward loops and massive upstream crosstalk. Finally, the expression patterns of these coregulation pairs in normal and tumour tissues were analyzed. Different coregulation types show unique expression correlation trends. More importantly, the disruption of coregulation may be associated with cancers. CONCLUSION: Our findings elucidate the combinatorial and cooperative properties of transcription factors and miRNAs regulation, and we proposes that the coordinated regulation may play an important role in many biological processes.

One-pot tandem copper-catalyzed library synthesis of 1-thiazolyl-1,2,3-triazoles as anticancer agents.

One-pot multicomponent synthesis to assemble compounds has been an efficient method for constructing a compound library. We have developed one-pot tandem copper-catalyzed azidation and CuAAC reactions that afford 1-thiazolyl-1,2,3-triazoles with anticancer activity. By utilizing this one-pot synthetic strategy, we constructed a library of 1-thiazolyl-1,2,3-triazoles in search of the potent lead compound. Furthermore, 1-thiazolyl-1,2,3-triazoles were evaluated for anticancer activity against the multidrug-resistant cancer cells MES-SA/Dx5. Most of the 1-thiazolyl-1,2,3-triazoles revealed cytotoxic effect against cancer cells at micromolar to low micromolar range. Testing some of the most potent compounds (5{4,2-4} and 5{5,1-3}) against the normal cell line Vero showed no significant toxicity (except 5{4,2}) to normal cells. This result indicates that compounds 5{4,3-4} and 5{5,1-3} possessed good potency and selectivity to cancer cells over normal cells.

Ganoderma lucidum Polysaccharides Induce Macrophage-Like Differentiation in Human Leukemia THP-1 Cells via Caspase and p53 Activation.

Differentiation therapy by induction of tumor cells is an important method in the treatment of hematological cancers such as leukemia. Tumor cell differentiation ends cancer cells' immortality, thus stopping cell growth and proliferation. In our previous study, we found that fucose-containing polysaccharide fraction F3 extracted from Ganoderma lucidum can bring about cytokine secretion and cell death in human leukemia THP-1 cells. This prompted us to further investigate on how F3 induces the differentiation in human leukemia cells. We integrated time-course microarray analysis and network modeling to study the F3-induced effects on THP-1 cells. In addition, we determined the differentiation effect using Liu's staining, nitroblue tetrazolium (NBT) reduction assay, flow cytometer, western blotting and Q-PCR. We also examined the modulation and regulation by F3 during the differentiation process. Dynamic gene expression profiles showed that cell differentiation was induced in F3-treated THP-1 cells. Furthermore, F3-treated THP-1 cells exhibited enhanced macrophage differentiation, as demonstrated by changes in cell adherence, cell cycle arrest, NBT reduction and expression of differentiation markers including CD11b, CD14, CD68, matrix metalloproteinase-9 and myeloperoxidase. In addition, caspase cleavage and p53 activation were found to be significantly enhanced in F3-treated THP-1 cells. We unraveled the role of caspases and p53 in F3-induced THP-1 cells differentiation into macrophages. Our results provide a molecular explanation for the differentiation effect of F3 on human leukemia THP-1 cells and offer a prospect for a potential leukemia differentiation therapy.

Neurocytoprotective effects of the bioactive constituents of Pueraria thomsonii in 6-hydroxydopamine (6-OHDA)-treated nerve growth factor (NGF)-differentiated PC12 cells.

Chronic neurodegenerative disorders are having an increasing impact on public health as human longevity increases. Parkinson's disease (PD) is a degenerative disorder of the central nervous system and is characterized by motor system disorders resulting in loss of dopamine-producing brain cells. Pueraria thomsonii Benth. (Fabaceae) is an herbal medicine that has traditionally been used as an antipyretic agent. In the present study, the active constituents, daidzein and genistein, were isolated from P. thomsonii. Both compounds exhibited neurocytoprotective effects against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in nerve growth factor (NGF)-differentiated PC12 cells. Neither daidzein nor genistein affected 6-OHDA-induced cellular reactive oxygen species (ROS) generation according to flow cytometric analysis. Rather, they inhibited caspase-8 and partially inhibited caspase-3 activation, providing a protective mechanism against 6-OHDA-induced cytotoxicity in NGF-differentiated PC12 cells. The present results imply that daidzein and genistein may be useful in the development of future strategies for the treatment of PD.

Structure and functions of gamma-dodecalactone isolated from Antrodia camphorata for NK cell activation.

The preserved fungal species Antrodia camphorata has diverse health-promoting effects and has been popularly used in East Asia as a traditional herb. We isolated a volatile compound from the culture medium of A. camphorata and identified it as gamma-dodecalactone (gamma-DDL). Cytomic screening for immune-modulating activity revealed that gamma-DDL can activate human NK cells to express the early activation marker CD69. Further experiments showed that gamma-DDL not only can induce NK cells to express CD69 but also stimulate NK cells to secrete cytotoxic molecules (FasL and granzyme B) and Th1 cytokines (TNF-alpha and INF-gamma). Measuring the distribution of gamma-DDL in the subcellular compartments of NK cells revealed that gamma-DDL has been converted to 4-hydroxydodecanoic acid (an acyclic isomer of gamma-DDL) in a time-dependent manner in the cytoplasm. Synthetic (R,S)-4-hydroxydodecanoic acid activated NK cells to express CD69 mRNA within 10min, in contrast to gamma-DDL, which activated NK cells to express CD69 within 50min. This faster activation suggests that gamma-DDL has converted to 4-hydroxydodecanoic acid and to stimulate the NK cells to express CD69. Optically pure (R)-(+)-4-hydroxydodecanoic acid and (S)-(-)-4-hydroxydodecanoic acid were obtained via: (1) synthesis of its diastereomeric esters of (R,S)-4-hydroxydodecanoic (R)-(-)-2-phenylpropionate; (2) separation of diastereomers via preparative HPLC, and (3) subsequent hydrolysis of the obtained optical pure ester of (R)-(+)-4-hydroxydodecanoic acid (R)-(-)-2-phenylpropionate and (R)-(-)-4-hydroxydodecanoic acid (R)-(-)-2-phenylpropionate, respectively. Further assays of NK cells activation using each enantiomer showed that only the (R)-(+)-4-hydroxydodecanoic acid can activate NK cells.

Different sample preparation and detection methods for normal and lung cancer urinary proteome analysis.

The urinary proteome is known to be a valuable field of study related to human physiological functions because many components in urine provide an alternative to blood plasma as a potential source of disease biomarkers useful in clinical diagnosis and therapeutic application. Due to the variability and complexity of urine, sample preparation is very important for decreasing the dynamic range of components and isolating specific urinary proteins prior to analysis. We discuss many useful sample preparation methods in this chapter, including those of lung cancer urine samples. In addition, protein detection methods are also crucial in visualizing protein profiles and for quantification of protein content in urine samples from both normal donor and lung cancer patients. This chapter also provides alternative choices of urine sample preparation and detection methods for selective use in urinary proteome analysis and for identifying urinary protein markers in lung cancer and other diseases.

Protective effect of mangosteen extract against beta-amyloid-induced cytotoxicity, oxidative stress and altered proteome in SK-N-SH cells.

Beta-amyloid (A beta) plays a key role in the pathogenesis of Alzheimer's disease (AD) by inducing neurotoxicity and cell death mainly through production of reactive oxygen species (ROS). Garcinia mangostana L. (mangosteen) has been recognized as a major source of natural antioxidants that could decrease ROS. However, its role in protection of A beta-induced cytotoxicity and apoptosis in neuronal cells remains unclear. We therefore examined such a protective effect of mangosteen extract (ME) by evaluating cell viability using MTT test, ROS level, caspase-3 activity, and cellular proteome. Treating SK-N-SH cells with 5-20 microM A beta((1-42)) for 24 h caused morphologically cytotoxic changes, decreased cell viability and increased ROS level, whereas preincubation with 50-400 microg/mL ME 30 min before the induction by A beta((1-42)) successfully prevented such cytotoxic effects in a dose-dependent manner (completely at 400 microg/mL). The A beta-induced increase in caspase-3 activity was also preventable by 400 microg/mL ME. Proteomic analysis using 2-D gel electrophoresis (n = 5 gels/group) followed by mass spectrometry revealed 63 proteins whose levels were significantly altered by A beta((1-42)) induction. Interestingly, changes in 10 proteins were successfully prevented by the ME pretreatment. In summary, we report herein the significant protective effects of ME against A beta-induced cytotoxicity, increased ROS, and increased caspase activity in SK-N-SH cells. Moreover, proteomic analysis revealed some proteins that might be responsible for these protective effects by ME. Further characterizations of these proteins may lead to identification of novel therapeutic targets for successful prevention and/or decreasing the severity of AD.