Pharmacological Properties of White Mulberry (Morus alba L.) Leaves: Suppressing Migratory and Invasive Activities Against A549 Lung Cancer Cells.

Morus alba known as a white mulberry is a medicinal plant that has been used in food ingredients and traditional medicine. M. alba leaves contain various bioactive phenolic compounds, in particular chlorogenic acid (CGA), which is a major bioactive ingredient. Their anticancer potency of M. alba leaf extracts derived from Soxhlet extraction was evaluated based on cytotoxicity and antimigratory and antiinvasive properties. The dichloromethane extract exhibited the highest nitric oxide radical scavenging activity with a half-maximal inhibitory concentration (IC50) value of 780 µg/mL, promising cytotoxicity against HuCCA-1, MCF-7, and A-549 cells with IC50 values of 59.18, 62.20, and 103.25 µg/mL, respectively. CGA selectively inhibited the growth of MCF-7 cells with an IC50 value of 26.75 µg/mL and showed potent radical scavenging activity against DPPH radicals (IC50 = 18.85 µg/mL). An ethanolic extract derived from the gradient Soxhlet extraction suppressed A549 lung cancer cell migration and invasion more effectively than CGA with no migratory inhibition effect on noncancerous HaCaT cells. Furthermore, the ethanolic extract and CGA accelerated HaCaT wound closure at 20 µg/mL, which was the same as allantoin. Bioactive ingredients including triterpenes, steroids, phenolics, and flavonoids were mainly detected in all extracts. The highest content of CGA (52.23 g/100 g dry weight) was found in the ethanolic extract derived from the gradient Soxhlet extraction. These findings show the potency of the dichloromethane extract as a cytotoxic agent against various cancer types and the ethanolic extract as an antimetastatic agent by their antimigratory and antiinvasive activities.

Proteomic profiling of circulating ß-thalassaemia/haemoglobin E extra-cellular vesicles reveals that association with immunoglobulin induces membrane vesiculation.

Splenectomised ß-thalassaemia/haemoglobin E (HbE) patients have increased levels of circulating microparticles or medium extra-cellular vesicles (mEVs). The splenectomised mEVs play important roles in thromboembolic complications in patients since they can induce platelet activation and endothelial cell dysfunction. However, a comprehensive understanding of the mechanism of mEV generation in thalassaemia disease has still not been reached. Thalassaemic mEVs are hypothesised to be generated from cellular oxidative stress in red blood cells (RBCs) and platelets. Therefore, a proteomic analysis of mEVs from splenectomised and non-splenectomised ß-thalassaemia/HbE patients was performed by liquid chromatography with tandem mass spectrometry. A total of 171 proteins were identified among mEVs. Interestingly, 72 proteins were uniquely found in splenectomised mEVs including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)-bearing mEVs in splenectomised patients were significantly increased. Furthermore, complement C1q was detected in both mEVs with IgG binding and mEVs without IgG binding. Interestingly, the percentage of mEVs generated from RBCs with IgG binding was approximately 15-20 times higher than the percentage of RBCs binding with IgG. This suggested that the vesiculation of thalassaemia mEVs could be a mechanism of RBCs to eliminate membrane patches harbouring immune complex and may consequently prevent cells from phagocytosis and lysis.

The redox-sensing mechanism of Agrobacterium tumefaciens NieR as a thiol-based oxidation sensor for hypochlorite stress.

NieR is a TetR family transcriptional repressor previously shown to regulate the NaOCl-inducible efflux pump NieAB in Agrobacterium tumefaciens. NieR is an ortholog of Escherichia coli NemR that specifically senses hypochlorite through the redox switch of a reversible sulfenamide bond between C106 and K175. The amino acid sequence of NieR contains only one cysteine. NieR has C104 and R166, which correspond to C106 and K175 of NemR, respectively. The aim of this study was to investigate the redox-sensing mechanism of NieR under NaOCl stress. C104 and R166 were subjected to mutagenesis to determine their roles. Although the substitution of R166 by alanine slightly reduced its DNA-binding activity, NieR retained its repressor function. By contrast, the DNA-binding and repression activities of NieR were completely lost when C104 was replaced by alanine. C104 substitution with serine only partially impaired the repressor function. Mass spectrometry analysis revealed an intermolecular disulfide bond between the C104 residues of NieR monomers. This study demonstrates the engagement of C104 in the mechanism of NaOCl sensing. C104 oxidation induced the formation of a disulfide-linked dimer that was likely to alter conformation, thus abolishing the DNA-binding ability of NieR and derepressing the target genes.

Arsenic induces the global hypophosphorylation of insulin receptor substrate proteins in differentiated human neuroblastoma SH-SY5Y cells.

We recently reported that arsenic disrupted neuronal insulin signaling. Here, we further investigated the effect of arsenic on insulin receptor substrate (IRS) proteins, which are crucial downstream signaling molecules of insulin in differentiated human neuroblastoma SH-SY5Y cells. We also found that prolonged arsenic treatment accelerated the migration of IRS1 and IRS2 on SDS-PAGE. Treatment with phosphatases abolished the arsenic-induced increased mobility of IRS, suggesting that the electrophoretic mobility shift of IRS on SDS-PAGE by arsenic was phosphorylation-dependent. By using label-free mass spectrometry, the phosphorylation sites of IRS1 were found to be S24, S345, S636, T774, S1057, S1058, and S1070, while those of IRS2 were at S645, Y653, T657, S665, S667, S669, S672, S915, and S1203, which were at least 2-fold lower than found in the control. These findings indicated a global hypophosphorylation of IRS proteins after prolonged arsenic treatment. In addition, four novel phosphorylation sites were identified on IRS1 (T774, S1057, S1058, and S1070), with another two on IRS2 (S665 and S667). As basal IRS phosphorylation plays an important role in insulin signaling, the reduction of IRS phosphorylation on multiple residues may underlie arsenic-impaired insulin signaling in neurons.

Translational Proteomic Approach for Cholangiocarcinoma Biomarker Discovery, Validation, and Multiplex Assay Development: A Pilot Study.

Cholangiocarcinoma (CCA) is a highly lethal disease because most patients are asymptomatic until they progress to advanced stages. Current CCA diagnosis relies on clinical imaging tests and tissue biopsy, while specific CCA biomarkers are still lacking. This study employed a translational proteomic approach for the discovery, validation, and development of a multiplex CCA biomarker assay. In the discovery phase, label-free proteomic quantitation was performed on nine pooled plasma specimens derived from nine CCA patients, nine disease controls (DC), and nine normal individuals. Seven proteins (S100A9, AACT, AFM, and TAOK3 from proteomic analysis, and NGAL, PSMA3, and AMBP from previous literature) were selected as the biomarker candidates. In the validation phase, enzyme-linked immunosorbent assays (ELISAs) were applied to measure the plasma levels of the seven candidate proteins from 63 participants: 26 CCA patients, 17 DC, and 20 normal individuals. Four proteins, S100A9, AACT, NGAL, and PSMA3, were significantly increased in the CCA group. To generate the multiplex biomarker assays, nine machine learning models were trained on the plasma dynamics of all seven candidates (All-7 panel) or the four significant markers (Sig-4 panel) from 45 of the 63 participants (70%). The best-performing models were tested on the unseen values from the remaining 18 (30%) of the 63 participants. Very strong predictive performances for CCA diagnosis were obtained from the All-7 panel using a support vector machine with linear classification (AUC = 0.96; 95% CI 0.88-1.00) and the Sig-4 panel using partial least square analysis (AUC = 0.94; 95% CI 0.82-1.00). This study supports the use of the composite plasma biomarkers measured by clinically compatible ELISAs coupled with machine learning models to identify individuals at risk of CCA. The All-7 and Sig-4 assays for CCA diagnosis should be further validated in an independent prospective blinded clinical study.

Global Analysis of Protein Expression in A549 Cells After Prolonged Nicotine Exposure by Using Label-free Quantification.

BACKGROUND/AIM: Lung cancer is the leading cause of cancer death worldwide. Cigarette smoke is the most important risk factor for cancer development. Growing evidence indicates that prolonged nicotine exposure is a potential factor associated with tumorigenesis. Here, the effect of prolonged nicotine exposure on A549 cells was investigated, using label-free quantitative proteomics. MATERIALS AND METHODS: Selection of an invasive subpopulation from the A549 cell line was performed to reveal the differential expression of proteins in relation to prolonged nicotine exposure, using Boyden chamber assays in combination with a proteomics approach. RESULTS: One hundred proteins from the NicoA549-L5 subline showed significant change in expression compared to those from the A549-L5 subline and their A549 parental cell line. Heat shock protein, protein disulfide isomerase A3, profilin-1 and legumain were expressed at higher levels in A549 cells after prolonged nicotine exposure. CONCLUSION: These aberrant proteins might serve as novel cancer biomarkers for cigarette smokers.

Serum Proteomic Profiling Reveals Differentially Expressed IGHG3 and A1AG1 as Potential Predictors of Chemotherapeutic Response in Advanced Non-small Cell Lung Cancer.

BACKGROUND: This study aimed to identify differentially expressed proteins in the serum of advanced non-small cell lung cancer (NSCLC) patients responding to carboplatin (CAR) plus paclitaxel (PTX) chemotherapy compared to non-responders. MATERIALS AND METHODS: Serum from 8 responders and 6 non-responders was subjected to proteomic analysis by label-free liquid chromatography tandem mass spectrometry and validated by western blotting. CAR/PTX-resistant human H1792 and A549 cells were used for evaluating gene expression. RESULTS: Fifty-two proteins were differentially expressed between responders and non-responders. Alpha 1 antitrypsin antibody, alpha 1 acid glycoprotein (A1AG1), afamin, protein S100-A9 and immunoglobulin heavy constant gamma 3 (IGHG3) were validated. IGHG3 was elevated (p=0.037) while A1AG1 was reduced (p=0.003) in responders as compared to non-responders. Gene expression of IGHG3 and ORM1 in resistant cells showed consistent results with the proteomics profiles. CONCLUSION: Serum expression levels of IGHG3 and A1AG1 proteins may be useful to recruit an NSCLC subpopulation that can benefit from CAR plus PTX standard therapy.

Proteomic analysis reveals plasma haptoglobin, interferon-γ, and interleukin-1ß as potential biomarkers of pediatric refractory epilepsy.

BACKGROUND: Children with refractory epilepsy (RE) are associated with increased mortality rate, nonfatal injuries, disability, and diminished quality of life. Biomarkers for the early prediction of RE is still an unmet need. METHODS: Eighteen children with RE and six age-matched unrelated controls were included in this study. Plasma samples were prefractionated by the optimized thermal treatment before proteomic analysis using 2DE-LC-MS/MS. Bioinformatic analysis was carried out using STRING protein network. Immunoassay of unprocessed plasma was applied to confirm changes of proteins of interest. P-value < 0.05 was considered statistically significant. RESULTS: Proteomic analysis (n = 6 each group) revealed nine differentially expressed proteins, i.e., haptoglobin, S100A9, serpin B1, apolipoprotein A-I, apolipoprotein A-IV, apolipoprotein C-II, alpha-1-acid glycoprotein 1 and 2, and transthyretin. Western immunoblotting confirmed haptoglobin upregulation in the RE group. STRING protein network predicted the inflammatory cytokines, i.e., interferon gamma (IFN-γ), interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), play roles in pathophysiology in RE patients. Cytokine immunoassay (n = 24, 18 RE vs. 6 controls) exhibited plasma IFN-γ was upregulated in RE patients as compared to the healthy individuals (median [IQR]; 2.9 [2.9, 4.9] vs. 1.32 [0.8, 1.5] pg/mL, p = 0.0013), and plasma IL-1ß was significantly downregulated in patients (1.0 [0.2, 1.9] vs. 4.5 [1.9, 11.0] pg/mL, p = 0.01). TNF-α had no difference between groups. The results suggest that haptoglobin may be associated with oxidative brain damage, while IFN-γ and IL-1ß may be involved with neuroinflammation. CONCLUSIONS: Alterations in plasma haptoglobin, IFN-γ, and IL-1ß were associated with RE patients. Future studies using a combination of these candidate biomarkers may help predict the intractability of epilepsy in pediatric populations.

Detection of superoxide dismutase (Cu-Zn) isoenzymes in leaves and pseudobulbs of Bulbophyllum morphologlorum Kraenzl orchid by comparative proteomic analysis.

Typically, biological systems are protected from the toxic effect of free radicals by antioxidant defense. Extracts from orchids have been reported to show high levels of exogenous antioxidant activity including Bulbophyllum orchids but so far, there have been no reports on antioxidant enzymes. Therefore, differences in protein expression from leaves and pseudobulbs of Bulbophyllum morphologlorum Kraenzl and Dendrobium Sonia Earsakul were studied using two-dimensional gel electrophoresis and mass spectrometry (LC/MS/MS). Interestingly, the largest group of these stress response proteins were associated with antioxidant defense and temperature stress, including superoxide dismutase (Cu-Zn) and heat shock protein 70. The high expression of this antioxidant enzyme from Bulbophyllum morphologlorum Kraenzl was confirmed by activity staining on native-PAGE, and the two Cu/Zn-SODs isoenzymes were identified as Cu/Zn-SOD 1 and Cu/Zn-SOD 2 by LC/MS/MS. The results suggested that Bulbophyllum orchid can be a potential plant source for medicines and natural antioxidant supplements.

Identification of potential cervical cancer serum biomarkers in Thai patients.

Cervical cancer is one of the most common causes of cancer-associated mortality in females worldwide. Serum biomarkers are important tools for diagnosis, disease staging, monitoring treatment and detecting recurrence in different types of cancer. However, only a small number of established biomarkers have been used for clinical diagnosis of cervical cancer. Therefore, the identification of minimally invasive, sensitive and highly specific biomarkers for detection of cervical cancer may improve outcomes. In the present pilot study, changes in disease-relevant proteins in 31 patients with cervical cancer were compared with 16 healthy controls. The Human 14 Multiple Affinity Removal system was used to deplete the 14 most abundant serum proteins to decrease sample complexity and to enrich proteins that exhibited decreased levels of abundance in the serum samples. Immunoaffinity-depleted serum samples were analyzed by in-gel digestion, followed by liquid chromatography mass spectrometry analysis and data processing. Automated quantitative western blot assays and receiver operating characteristic (ROC) curves were used to evaluate the differential protein expression levels between the two groups. Capillary electrophoresis-based western blot analysis was performed to quantitatively determine serum levels of the candidate biomarkers. Significantly increased levels of α-1-antitrypsin (A1AT) and pyrroline-5-carboxylate reductase 2 (PYCR2) were detected, whereas the levels of transthyretin (TTR), apolipoprotein A-I (ApoA-I), vitamin D binding protein (VDBP) and multimerin-1 (MMRN1) were significantly decreased in patients with cervical cancer compared with the healthy controls. ROC curve analysis indicated that the sensitivity and specificity was improved through the combination of the 6 candidate biomarkers. In summary, the results demonstrated that 6 candidate biomarkers (A1AT, PYCR2, TTR, ApoA-I, VDBP and MMRN1) exhibited significantly different expression between serum samples from healthy controls and patients with cervical cancer. These proteins may represent potential biomarkers for distinguishing patients with cervical cancer from healthy controls and for differentiation of patient subgroups.

Optimization and Standardization of Thermal Treatment as a Plasma Prefractionation Method for Proteomic Analysis.

Prefractionation is a prerequisite step for deep plasma proteomics. Highly abundant proteins, particularly human serum albumin (HSA) and immunoglobulin G (IgG), typically interfere with investigation of proteins with lower abundance. A relatively simple preparation method based on high temperature can precipitate thermolabile proteins, providing a strategic window to access the thermostable plasma subproteome. This study aimed to optimize thermal treatment as a reliable prefractionation method and to compare it with two commercial kits, including HSA and IgG immunodepletion (IMDP) and combinatorial peptide ligand libraries (CPLL), using untreated plasma as a control condition. By varying the temperature and the incubation period, the optimal condition was found as treatment at 95°C for 20 min, which maintained about 1% recovery yield of soluble proteins. Consistency and reproducibility of thermal treatment-derived plasma subproteome were checked by two-dimensional electrophoresis. The coefficient of variation regarding protein spot numbers was less than 10% among three independent specimens. Highly abundant protein depletion of the thermal treatment was evaluated by immunoblotting against HSA and IgG as compared to the untreated plasma, IMDP, and CPLL. Multidimensional comparison based on 489 unique peptides derived from the label-free quantitative mass spectrometry revealed that the thermal treatment, IMDP, and CPLL provided distinct sets of plasma subproteome compared to untreated plasma, and these appeared to be complementary to each other. Comparing the characteristics of the three procedures suggested that thermal treatment was more cost-effective and less time-consuming than IMDP and CPLL. This study proposes the use of thermal treatment as a reliable and cost-effective method for plasma prefractionation which provides benefits to large-scale proteomic projects and biomarker studies.

Urinary biomarkers for the diagnosis of cervical cancer by quantitative label-free mass spectrometry analysis.

Due to the invasive procedure associated with Pap smears for diagnosing cervical cancer and the conservative culture of developing countries, identifying less invasive biomarkers is of great interest. Quantitative label-free mass spectrometry was performed to identify potential biomarkers in the urine samples of patients with cervical cancer. This technique was used to study the differential expression of urinary proteomes between normal individuals and cancer patients. The alterations in the levels of urinary proteomes in normal and cancer patients were analyzed by Progenesis label-free software and the results revealed that 60 proteins were upregulated while 73 proteins were downregulated in patients with cervical cancer. This method could enrich high molecular weight proteins from 100 kDa. The protein-protein interactions were obtained by Search Tool for the Retrieval of Interacting Genes/Proteins analysis and predicted the biological pathways involving various functions including cell-cell adhesion, blood coagulation, metabolic processes, stress response and the regulation of morphogenesis. Two notable upregulated urinary proteins were leucine-rich α-2-glycoprotein (LRG1) and isoform-1 of multimerin-1 (MMRN1), while the 3 notable downregulated proteins were S100 calcium-binding protein A8 (S100A8), serpin B3 (SERPINB3) and cluster of differentiation-44 antigen (CD44). The validation of these 5 proteins was performed by western blot analysis and the biomarker sensitivity of these proteins was analyzed individually and in combination with receiver operator characteristic curve (ROC) analysis. Quantitative mass spectrometry analysis may allow for the identification of urinary proteins of high molecular weight. The proteins MMRN1 and LRG1 were presented, for the first time, to be highly expressed urinary proteins in cervical cancer. ROC analysis revealed that LRG1 and SERPINB3 could be individually used, and these 5 proteins could also be combined, to detect the occurrence of cervical cancer.

Phosphoproteome Profiling of Isogenic Cancer Cell-Derived Exosome Reveals HSP90 as a Potential Marker for Human Cholangiocarcinoma.

The northeastern region of Thailand is well known to have a high incidence and mortality of cholangiocarcinoma (CCA). Protein phosphorylation status has been reported to reflect a key determinant of cellular physiology, but identification of phosphoproteins can be a problem due to the presence of phosphatase. Exosomes are stable toward circulating proteases and other enzymes in human blood and can be recognized before the onset of cancer progression. Here an in vitro metastatic model of isogenic CCA cells is used to provide insight into the phosphorylation levels of exosomal proteins derived from highly invasive cells. Gel-based and gel-free proteomics approaches are used to reveal the proteins differentially phosphorylated in relation to tumor cell phenotypes. Forty-three phosphoproteins are identified with a significant change in phosphorylation level. Phos-tag western blotting and immunohistochemistry staining are then employed to validate the candidate phosphoproteins. Heat shock protein 90 is successfully confirmed as being differentially phosphorylated in relation to tumor malignancy. Importantly, the aberrant phosphorylation of exosomal proteins might serve as a promising tool for the development of a biomarker for metastatic CCA.

Differentially expressed plasma proteins of ß-thalassemia/hemoglobin E patients in response to curcuminoids/vitamin E antioxidant cocktails.

OBJECTIVE: Iron overload and oxidative stress are the major causes of serious complications and mortality in thalassemic patients. Our previous work supports the synergistic effects of antioxidant cocktails (curcuminoids or vitamin E, N-acetylcysteine, and deferiprone) in treatment of ß-thalassemia/Hb E patients. This further 2-DE-based proteomic study aimed to identify the plasma proteins that expressed differentially in response to antioxidant cocktails. METHODS: Frozen plasma samples of ten normal subjects and ten ß-thalassemia/Hb E patients at three-time points (baseline, month 6, and month 12) were reduced the dynamic range of proteome using ProteoMiner kit and separated proteins by two-dimensional gel electrophoresis. Differentially expressed proteins were identified using tandem mass spectrometry. Several plasma proteins were validated by ELISA and Western blot analysis. RESULTS: Thirteen and 11 proteins were identified with altered expression levels in the curcuminoids- and vitamin E cocktail groups, respectively. The associations between vitronectin (VTN) expression and total bilirubin levels, as well as between serum paraoxonase/arylesterase 1 (PON1) expression and blood reactive oxygen species were observed. Validation results were consistent with proteomics results. DISCUSSION AND CONCLUSIONS: These plasma proteins may provide better understanding of the mechanisms underlying the therapeutic effects of antioxidant cocktails in thalassemic patients.

Effect of Derris scandens extract on a human hepatocellular carcinoma cell line.

The incidence rate of hepatocellular carcinoma (HCC) remains high in numerous countries, including Thailand. There are numerous different lines of HCC treatment; however, various side effects and the resistance of cancer cells during treatment remain issues. At present, traditionally used herb plants have been widely used as alternatives to cancer therapy. Derris scandens is a Thai traditional herb which is commonly found in Thailand and widely used as a traditional medicine for numerous different diseases. The cytotoxicity of D. scandens ethanolic extract on a HCC cell line (HCC-S102) was determined using an MTT assay. Following treatment with D. scandens ethanolic extract, the induction of apoptosis was determined by Annexin V and dead cell assays, and then confirmed by the upregulation of cleaved poly(ADP-ribose) polymerase. Furthermore, a proteomic approach was used in order to study protein alteration upon treatment with D. scandens ethanolic extract coupled with liquid chromatography-tandem mass spectrometry analysis for protein identification. The results suggested that D. scandens ethanolic extract resulted in cytotoxicity against HCC-S102 cells, as the half-maximal inhibitory concentration values were 36.0±1.0, 29.6±0.6, and 22.6±1.5 µg/ml at 24, 48 and 72 h, respectively. Apoptotic cells were induced following treatment with D. scandens. The comparative proteomic profiles of D. scandens ethanolic extract-treated and untreated cells revealed various protein targets for anticancer activity including heterogeneous nuclear ribonucleoprotein (hnRNP) K, hnRNP A2/B1, stomatin-like 2 and GAPDH. In the present study, the anticancer activity of D. scandens ethanolic extract was demonstrated to affect the cell proliferation of HCC-S102 via an apoptotic pathway. The alteration in these proteins provides a better understanding of the mechanism of action of D. scandens, which may be a promising anticancer agent for the treatment of patients with HCC in the future.

Apigenin inhibits growth and induces apoptosis in human cholangiocarcinoma cells.

A promising nutraceutical, apigenin, was recently revealed to exhibit biological activity in inhibiting several types of cancer. The effects of apigenin on the growth inhibition and apoptosis of the cholangiocarcinoma HuCCA-1 cell line were investigated. Protein alterations subsequent to apigenin treatment were studied using a proteomic approach. The values of 20, 50 and 90% inhibition of cell growth (IC20, IC50 and IC90) were determined by MTT cell viability assay. Apoptotic cell death was detected using two different methods, a flow cytometric analysis (Muse Cell Analyzer) and DNA fragmentation assay. A number of conditions including attached and detached cells were selected to perform two-dimensional gel electrophoresis (2-DE) to study the alterations in the expression levels of treated and untreated proteins and identified by liquid chromatography (LC)/tandem mass spectrometry (MS/MS). The IC20, IC50 and IC90 values of apigenin after 48 h treatment in HuCCA-1 cells were 25, 75 and 200 µM, respectively, indicating the cytotoxicity of this compound. Apigenin induced cell death in HuCCA-1 cells via apoptosis as detected by flow cytometric analysis and exhibited, as confirmed with DNA fragmentation, characteristics of apoptotic cells. A total of 67 proteins with altered expression were identified from the 2-DE analysis and LC/MS/MS. The cleavage of proteins involved in cytoskeletal, cytokeratin 8, 18 and 19, and high expression of S100-A6 and S100-A11 suggested that apoptosis was induced by apigenin via the caspase-dependent pathway. Notably, two proteins, heterogeneous nuclear ribonucleoprotein H and A2/B1, disappeared completely subsequent to treatment, suggesting the role of apigenin in inducing cell death. The present study indicated that apigenin demonstrates an induction of growth inhibition and apoptosis in cholangiocarcinoma cells and the apoptosis pathway was confirmed by proteomic analysis.

Secretomic profiling of cells from hollow fiber bioreactor reveals PSMA3 as a potential cholangiocarcinoma biomarker.

Cholangiocarcinoma (CCA), derived from the bile duct, occurs with a relatively high incidence in Northeast Thailand. Early diagnosis is still hampered by the lack of sufficient biomarkers. In recent years, biomarker discovery using secretomes has provided interesting results, including our studies on CCA secretomes, especially with three-dimensional cell cultures. Thus, cells cultured using the hollow fiber bioreactor (HFB) with 20 kDa molecular weight cut-off (MWCO) yielded higher quality and quantity of secretomes than those from conditioned media of the monolayer culture (MNC) system. In this study, we employed the HFB culture system with 5 kDa MWCO and compared conditioned media from the HFB and MNC systems using two-dimensional gel electrophoresis, followed by identifying proteins of interest by liquid chromatography and mass spectrometry (LC/MS/MS). Two out of 4 spots of NGAL or lipocalin-2 were found to show highest increase in expression of 19.93-fold and 18.79-fold in HFB compared to MNC. Interestingly, all 14 proteasome subunits including proteasome subunit α type-1 to type-7 and ß type-1 to type-7 showed 2.92-fold to 12.13-fold increased expression in HFB. The protein-protein interactions of upregulated proteins were predicted, and one of the main interaction clusters involved 20S proteasome subunits. Proteasome activity in the HFB conditioned media was also found to be higher than that in MNC conditioned media. Three types of proteasome subunit were also validated by immunoblotting and showed higher expression in the HFB system compared to MNC system. Proteasome subunit α type-3 (PSMA3) showed the highest level in plasma of cholangiocarcinoma patients compared to normal and hepatocellular carcinoma patients by immunodetection, and is of interest as a potential biomarker for cholangiocarcinoma.

Elevated O-GlcNAcylation of Extracellular Vesicle Proteins Derived from Metastatic Colorectal Cancer Cells.

BACKGROUND: O-GlcNAcylation is a single sugar attachment of serine and/or threonine residues on intracellular proteins. Recent reports reveal that it can modify several secretory proteins; however, the underlying mechanisms are largely unexplored. MATERIALS AND METHODS: To investigate whether extracellular vesicles (EVs) carry secretory O-GlcNAc-modified proteins that were isolated from colorectal cancer (CRC) cells, two-dimensional gel electrophoresis followed with O-GlcNAc immunoblotting and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied. RESULTS: It was revealed that the O-GlcNAc modification of many EV proteins was increased in metastatic cells. Among these, transitional endoplasmic reticulum ATPase (TER ATPase) and RuVB-like1 were successfully confirmed for the O-GlcNAc modification in which the levels were significantly higher in EVs of metastatic CRC cell line. CONCLUSION: These data, demonstrate that proteins carried by EVs are O-GlcNAc-modified. Importantly, elevated aberrant O-GlcNAcylation of EV proteins might serve as a potential biomarker of metastatic CRC.

Syringe-push membrane absorption as a simple rapid method of urine preparation for clinical proteomics.

BACKGROUND: The analysis of urinary proteome might reveal biomarkers of clinical value. However, current methods of urine preparation for down-stream proteomic analysis are complicated, time-consuming, and/or expensive. This study aims to develop a robust, simple, inexpensive and readily accessible urine preparation method to facilitate clinical proteomic workflow. RESULT: Syringe-push membrane absorption (SPMA) was successfully developed by a combination of 5-ml medical syringe and protein-absorbable membrane. Comparing three membranes i.e., nitrocellulose, polyvinylidene difluoride (PVDF) and Whatman no.1, nitrocellulose combined with SPMA (nitrocellulose-SPMA) provided the greatest quality of proteome profile as demonstrated by 2-DE. The quality of the proteome profile and the performance of nitrocellulose-SPMA were systematically compared with three current methods of urine preparation (i.e., ultrafiltration, dialysis/lyophilization and precipitation). While different methods of urine preparation provided comparable proteome quality, nitrocellulose-SPMA had better working performance due to acceptable recovery yield, less workload, short working time, high accessibility and low unit cost. In addition, protein absorbed on nitrocellulose harvested from the SPMA procedure could be stored as a dried membrane at room temperature for at least 1-month without protein degradation or modification. CONCLUSIONS: SPMA is a simple rapid method of preparing urine for downstream proteomic analysis. Because of it is highly accessible and has long storage duration, this technique holds potential benefit for large-scale multi-center research and future development of clinical investigation based upon urinary proteomic analysis.

Role of curcuminoids in ameliorating oxidative modification in ß-thalassemia/Hb E plasma proteome.

Thalassemic patients often exhibit high levels of oxidative stress and iron overload, which can lead to hazardous complications. Curcuminoids, extracted from the spice turmeric, are known to have antioxidant and iron-chelating properties and have been proposed as a potential upstream therapy of thalassemia. Here we have applied proteomic techniques to study the protein profile and oxidative damage in the plasma of ß-thalassemia/Hb E patients before and after treatment with curcuminoids. In this study, 10 ß-thalassemia/Hb E patients were treated with 500 mg curcuminoids daily for 12 months. The plasma protein profile and protein carbonyl content were determined at baseline, 6 and 12 months using two-dimensional fluorescence difference gel electrophoresis and carbonyl immunoblotting, respectively. Other hematological, clinical, and biochemical parameters were also analyzed. Twenty-six spots, identified as coagulation factors and proteins involved in iron homeostasis, showed significantly decreased intensity in thalassemic plasma, compared to those of normal subjects. Treatment with curcuminoids up-regulated the plasma levels of these proteins and reduced their oxidative damage. Serum non-transferrin bound iron, platelet factor-3 like activity, oxidative stress parameters and antioxidant enzymes were also improved after curcuminoids treatment. This study is the first proteomic study of plasma in the thalassemic state and also shows the ameliorating role of curcuminoids towards oxidative stress and iron overload in the plasma proteome.