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.

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.

Alterations in cellular proteome and secretome upon differentiation from monocyte to macrophage by treatment with phorbol myristate acetate: insights into biological processes.

Monocyte and macrophage are mainly involved in immune response and inflammatory processes. Monocytes circulate in the bloodstream and migrate to various tissues where they can differentiate to macrophages. However, the molecular basis of biological processes involved in this cellular differentiation remains ambiguous. This study was to investigate alterations in cellular and secreted proteins after this differentiation phase. Macrophage was differentiated from U937 human monocytic cell line by treatment with 100 ng/ml phorbol myristate acetate (PMA) for 48 h. Cellular and secreted proteins extracted from PMA-treated cells (macrophages) were compared with those of untreated cells (monocytes) using 2-DE (n=5 gels/condition; stained with Deep Purple fluorescence dye). Quantitative intensity analysis revealed 81 and 67 protein spots whose levels were significantly altered in cellular proteome and secretome. These proteins were subsequently identified by Q-TOF MS and/or MS/MS analyses. The altered levels of cellular elongation factor-2 (EF-2) and secreted alpha-tubulin were confirmed by Western blot analysis. Global protein network analysis demonstrated that these altered proteins were involved in cell death, lipid metabolism, cell morphology, cellular movement, and protein folding. Our data may provide some insights into molecular mechanisms of biological processes upon differentiation from monocytes to macrophages.

Plasma proteome profiling of von Hippel-Lindau disease after total and subtotal nephrectomy: a preliminary study.

OBJECTIVES: Common treatment of renal cell carcinoma associated with von Hippel-Lindau (VHL) disease is total (bilateral) or subtotal nephrectomy. Whereas total nephrectomy is associated with absolutely no residual renal function, subtotal nephrectomy frequently leads to chronic kidney disease (CKD) with some residual renal functions. However, molecular mechanisms underlying CKD remain unclear and the diagnosis of CKD is frequently accomplished at its late stage. DESIGN AND METHODS: We performed a plasma proteomics study to compare the plasma proteome profile of VHL patient who underwent total nephrectomy to the profiles of VHL patient with subtotal nephrectomy and healthy control. Totally 100 mug proteins from each sample was resolved by two-dimensional electrophoresis (2-DE) in triplicate and visualized with SYPRO Ruby fluorescence stain. RESULTS: The normal plasma proteome profile markedly differed from the profiles of VHL patients. Comparative analysis between total versus subtotal nephrectomized patients revealed significant differences in levels of 20 plasma proteins. Pathway analysis revealed two important networks involving in lipid metabolism, molecular transport, carbohydrate metabolism, cellular growth and proliferation, and small molecule biochemistry, in which these identified and other proteins interplayed. CONCLUSIONS: Our data identified potential biomarkers for CKD. Further characterization of these identified proteins might also lead to better understanding of molecular mechanisms underlying CKD.

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.

Current proteomic analysis and post-translational modifications of biomarkers in human lung cancer materials.

Lung cancer is the leading cause of cancer deaths worldwide and is often diagnosed in the advanced or late stages. The discovery of candidate biomarkers in human lung cancer materials holds clinical potential as well as a significant challenge. Due to a large number of proteins in human materials that form the protein complexity and post-translational modifications (PTMs) of proteins, proteomic analysis of lung cancer biomarkers remains a difficult task. Many proteomic approaches have been used to investigate the biomarkers in human lung cancer materials. In this review, we summarize the current proteomic approaches employed for analysis of proteome profiles in human lung cancer materials and some PTMs causing different proteome profiles and expression levels of biomarkers in lung cancer. In addition, bioinformatic tools for PTMs are useful for prediction of glycosylation and phosphorylation sites in proteins, which can help us further understand the correlation of PTMs and tumor development. However, it is necessary that proteome analysis of lung cancer materials be incorporated with new strategies and technologies to improve the efficiency of the analysis and to investigate novel biomarkers that would be very useful for further diagnostic and therapeutic applications.

Targeting the delivery of glycan-based paclitaxel prodrugs to cancer cells via glucose transporters.

This report describes the synthesis of four novel paclitaxel based prodrugs with glycan conjugation (1-4). Glycans were conjugated using an ester or ether bond as the linker between 2'-paclitaxel and the 2'-glucose or glucuronic acid moiety. These prodrugs showed good water solubility and selective cytotoxicity against cancer cell lines, but showed reduced toxicity toward normal cell lines and cancer cell lines with low expression levels of GLUTs. The ester conjugated prodrug 1 showed the most cytotoxicity among the prodrugs examined and could be transported into cells via GLUTs. Fluorescent and confocal microscopy demonstrated that targeted cells exhibited morphological changes in tubulin and chromosomal alterations that were similar to those observed with paclitaxel treatment. Therefore, these glycan-based prodrugs may be good drug candidates for cancer therapy, and the glycan conjugation approach is an alternative method to enhance the targeted delivery of other drugs to cancer cells that overexpress GLUTs.

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.

Proteomic analysis of host responses in HepG2 cells during dengue virus infection.

Dengue virus infection remains a public health problem worldwide. However, its pathogenic mechanisms and pathophysiology are still poorly understood. We performed proteomic analysis to evaluate early host responses (as indicated by altered proteins) in human target cells during dengue virus infection. HepG2 cells were infected with dengue virus serotype 2 (DEN-2) at multiplicity of infection (MOI) of 0.1, 0.5, and 1.0. Quantitative analyses of DEN-2 infection and cell death at 12, 24, and 48 h postinfection showed that the MOI of 1.0 with 24 h postinfection duration was the optimal condition to evaluate early host responses, as this condition provided the high %Infection ( approximately 80%), while %Cell death ( approximately 20%) was comparable to that of the mock-control cells. Proteins derived from the mock-control and DEN-2-infected cells were resolved by 2-D PAGE ( n = 5 gels for each group) and visualized by SYPRO Ruby stain. Quantitative intensity analysis revealed 17 differentially expressed proteins, which were successfully identified by peptide mass fingerprinting. Most of these altered proteins were the key factors involved in transcription and translation processes. Further functional study on these altered proteins may lead to better understanding of the pathogenic mechanisms and host responses to dengue virus infection, and also to the identification of new therapeutic targets for dengue virus infection.

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.

Gene expression analysis in LLC-PK1 renal tubular cells by atrial natriuretic peptide (ANP): correlation of homologous human genes with renal response.

We used human DNA microarray to explore the differential gene expression profiling of atrial natriuretic peptide (ANP)-stimulated renal tubular epithelial kidney cells (LLC-PK1) in order to understand the biological effect of ANP on renal kidney cell's response. Gene expression profiling revealed 807 differentially expressed genes, consisting of 483 up-regulated and 324 down-regulated genes. The bioinformatics tool was used to gain a better understanding of differentially expressed genes in porcine genome homologous with human genome and to search the gene ontology and category classification, such as cellular component, molecular function and biological process. Four up-regulated genes of ATP1B1, H3F3A, ITGB1 and RHO that were typically validated by real-time quantitative PCR (RT-qPCR) analysis serve important roles in the alleviation of renal hypertrophy as well as other related effects. Therefore, the human array can be used for gene expression analysis in pig kidney cells and we believe that our findings of differentially expressed genes served as genetic markers and biological functions can lead to a better understanding of ANP action on the renal protective system and may be used for further therapeutic application.

Toxicogenomics of A375 human malignant melanoma cells treated with arbutin.

Although arbutin is a natural product and widely used as an ingredient in skin care products, its effect on the gene expression level of human skin with malignant melanoma cells is rarely reported. We aim to investigate the genotoxic effect of arbutin on the differential gene expression profiling in A375 human malignant melanoma cells through its effect on tumorigenesis and related side-effect. The DNA microarray analysis provided the differential gene expression pattern of arbutin-treated A375 cells with the significant changes of 324 differentially expressed genes, containing 88 up-regulated genes and 236 down-regulated genes. The gene ontology of differentially expressed genes was classified as belonging to cellular component, molecular function and biological process. In addition, four down-regulated genes of AKT1, CLECSF7, FGFR3, and LRP6 served as candidate genes and correlated to suppress the biological processes in the cell cycle of cancer progression and in the downstream signaling pathways of malignancy of melanocytic tumorigenesis.

Synthesis of 2'-paclitaxel methyl 2-glucopyranosyl succinate for specific targeted delivery to cancer cells.

A novel glucose-conjugated paclitaxel 5 was synthesized using succinic acid as linker between 2'-paclitaxel and methyl 2'-glucopyranose. 5 has not only improved the pharmaceutical properties of paclitaxel, such as solubility and stability, but also enhanced the specific target delivery to MCF-7 cells without the cytotoxicity against normal cells. Therefore, the glucose conjugation may be potentially used in the targeted delivery of other drugs into cells via glucose transporters (GLUTs) for cancer therapy.

Proteomic identification of altered proteins in skeletal muscle during chronic potassium depletion: Implications for hypokalemic myopathy.

Prolonged potassium depletion is a well-known cause of myopathy. The pathophysiology of hypokalemic myopathy, however, remains unclear. We performed a gel-based, differential proteomics study to define altered proteins in skeletal muscles during chronic potassium depletion. BALB/c mice were fed with normal chow (0.36% K+) or K+-depleted (KD) diet (<0.001% K+) for 8 weeks (n = 5 in each group). Left gastrocnemius muscles were surgically removed from each animal. Histopathological examination showed mild-degree infiltration of polymornuclear and mononuclear cells at the interstitium of the KD muscles. Extracted proteins were resolved with two-dimensional electrophoresis (2-DE), and visualized with Coomassie Brilliant Blue R-250 stain. Quantitative intensity analysis revealed 16 up-regulated protein spots in the KD muscles, as compared to the controls. These differentially expressed proteins were subsequently identified by peptide mass fingerprinting and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). Most of the altered proteins induced by chronic potassium depletion were muscle enzymes that play significant roles in several various metabolic pathways. Other up-regulated proteins included myosin-binding protein H, alpha-B Crystallin, and translationally controlled tumor protein (TCTP). These findings may lead to a new roadmap for research on hypokalemic myopathy, to better understanding of the pathophysiology of this medical disease, and to biomarker discovery.

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.

Different techniques for urinary protein analysis of normal and lung cancer patients.

Many components in urine are useful in clinical diagnosis and urinary proteins are known as important components to define many diseases such as proteinuria, kidney, bladder and urinary tract diseases. In this study, we focused on the comparison of different sample preparation methods for isolating urinary proteins prior to protein analysis of pooled healthy and lung cancer patient samples. Selective method was used for preliminary investigation of some putative urinary protein markers. Urine samples were passed first through a gel filtration column (PD-10 desalting column) to remove high salts and subsequently concentrated. Remaining interferences were removed by ultrafiltration or four precipitation methods. The analysis of urinary proteins by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed many similarities in profiles among preparation methods and a few profiles were different between normal and lung cancer patients. In contrast, the results of two-dimensional gel electrophoresis (2-DE) showed more distinctly different protein patterns. Our finding showed that the sequential preparation of urinary proteins by gel filtration and ultrafiltration could retain most urinary proteins which demonstrated the highest protein spots on 2-D gels and able to identify preliminary urinary protein markers related to cancer. Although sequential preparation of urine samples by gel filtration and protein precipitation resulted in low amounts of proteins on 2-D gels, high Mr proteins were easily detected. Therefore, there are alternative choices for urine sample preparation for studying the urinary proteome and identifying urinary protein markers important for further preclinical diagnostic and therapeutic applications.

Facile synthesis of metal-chelating peptides on chip for protein array.

A unique peptide sequence of HGGHHG screening from a combinatorial synthetic peptide library showed a good chelating ability to bind a transition metal on a chip better than hexa-His peptide. It was directly conjugated with a His-Tagged proteins onto a chip in a mild aqueous solution and can be used this chip as a high throughput technique for protein array in order to detect and purify the His-Tagged proteins.

Proteomic study of cold shock protein in Bacillus stearothermophilus P1: Comparison of temperature downshifts.

The thermophilic bacterium Bacillus stearothermophilus P1 is unique in its ability to thrive in extreme environments such as high temperatures or high pH conditions. The study of cold shock response is very interesting and interpreted as a shock response to express the genes involved in synthesis of specific proteins. This study investigated the study of cold shock protein of B. stearothermophilus P1 when the cell culture temperature shifted from 65 degrees C to 37 degrees C and 25 degrees C. Cell growth at 37 degrees C weakly increased in the previous 3 h and then slowly decreased. In contrast, cell growth at 25 degrees C was slowly decreased. The protein contents after temperature downshifts were analyzed by proteomic techniques using protein chip and two-dimensional (2-D) electrophoresis that are highly effective and useful for protein separation and identification. The different proteins after a temperature decrease from 65 degrees C to 37 degrees C and 25 degrees C were expressed on 2-D gel patterns and the cold shock protein was detected in the acidic area with the isoelectric point and molecular mass approximately 4.5 and 7.3 kDa, respectively. The NH(2)-terminal sequence of a major cold shock protein from B. stearothermophilus P1 was MQRGKVKWFNNEKGFGFIEVEGGSD, similar to other cold shock proteins from Bacillus sp. up to 96% identity, but different from the other bacteria with homology less than 80% identity.