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.

Protein Modification via Nitrile Oxide-Dehydroalanine Cycloaddition: Formation of Isoxazoline Ring on the Protein Backbone.

Here we describe a novel catalyst-free 1,3-dipolar cycloaddition bioconjugation approach for chemical modification of proteins. The dehydroalanine (Dha)-containing protein reacts with nitrile oxides generated in situ through 1,3-dipolar cycloaddition in fully aqueous-buffered systems. This leads to the formation of a new isoxazoline ring at a pre-defined site (Dha) of the protein. Furthermore, the 1-pyrene isoxazoline-installed annexin V acts as a fluorescent probe, which successfully labels the outer cellular membranes of human cholangiocarcinoma (HuCCA-1) cells for detection of apoptosis.

Label-free quantitative proteomics reveals aberrant expression levels of LRG, C9, FN, A1AT and AGP1 in the plasma of patients with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the major causes of cancer-related death worldwide. Although commercial biomarkers of CRC are currently available, they are still lacking in terms of sensitivity and specificity; thus, searching for reliable blood-based biomarkers are important for the primary screening of CRC. METHODS: Plasma samples of patients with non-metastatic (NM) and metastatic (M) CRC and healthy controls were fractionated using MARS-14 immunoaffinity chromatography. The flow-through and elute fractions representing low- and high-abundant proteins, respectively, were analyzed by label-free quantitative proteomics mass spectrometry. The functional analysis of the proteins with greater than 1.5-fold differential expression level between the CRC and the healthy control groups were analyzed for their biological processes and molecular functions. In addition, the levels of plasma proteins showing large alterations in CRC patients were confirmed by immunoblotting using two independent cohorts. Moreover, receiver operating characteristic (ROC) curve analysis was performed for individual and combinations of biomarker candidates so as to evaluate the diagnostic performance of biomarker candidates. RESULTS: From 163 refined identifications, five proteins were up-regulated and two proteins were down-regulated in NM-CRC while eight proteins were up-regulated and three proteins were down-regulated in M-CRC, respectively. Altered plasma proteins in NM-CRC were mainly involved in complement activation, while those in M-CRC were clustered in acute-phase response, complement activation, and inflammatory response. Results from the study- and validation-cohorts indicate that the levels of leucine-rich alpha-2-glycoprotein-1(LRG), complement component C9 (C9), alpha-1-acid glycoprotein 1 (AGP1), and alpha-1-antitrypsin (A1AT) were statistically increased, while fibronectin (FN) level was statistically decreased in CRC patients compared to healthy controls, with most alterations found in a metastatic stage-dependent manner. ROC analysis revealed that FN exhibited the best diagnostic performance to discriminate CRC patients and healthy controls while AGP1 showed the best discrimination between the disease stages in both cohorts. The combined biomarker candidates, FN + A1AT + AGP1, exhibited perfect discriminatory power to discriminate between the CRC population and healthy controls whereas LRG + A1AT + AGP1 was likely to be the best panel to discriminate the metastatic stages in both cohorts. CONCLUSIONS: This study identified and quantified distinct plasma proteome profiles of CRC patients. Selected CRC biomarker candidates including FN, LRG, C9, A1AT, and AGP1 may be further applied for screening larger cohorts including disease groups from other types of cancer or other diseases.

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.

Toxicity of DDT to the hooded oyster Saccostrea cucullata: Mortality, histopathology and molecular mechanisms as revealed by a proteomic approach.

Dichlorodiphenyltrichloroethane (DDT), a persistent organochlorine pesticide, has been linked to adverse biological effects in organisms. However, there is limited knowledge about its toxic effects on marine organisms and the underlying molecular mechanisms. This study investigated the toxic effects of DDT in the hooded oyster Saccostrea cucullata. The oysters were exposed to DDT at concentrations of 0, 10, 50, 100, 500, 1000 and 2000 µg/L for 96 h and the LC50 (96 h) was 891.25 µg/L. Two sublethal concentrations (10 and 100 µg/L) were used to investigate the histopathological effects and the proteome response. Histopathological results showed that DDT caused the alteration of mantle tissue. This included the induction of mucocytes in the epithelium and the inflammatory effect in the connective tissue indicated by the enlargement of blood sinus and hemocyte aggregation within the sinus. Proteomic results showed that, amongst approximately 500 protein spots that were detected across 2DE gels, 51 protein spots were differentially expressed (P < 0.01; fold change > 1.2). Of these, 29 protein spots were identified by LC-MS/MS. These included 23 up-regulated, 5 down-regulated and 1 fluctuating spots. Thus, we observed that stress response and cytoskeletal proteins are the central targets of DDT action. Furthermore, DDT alters the expression of proteins involved in energy metabolism, calcium homeostasis and other proteins of unknown function. Additionally, proteomic results clearly elucidated the molecular response of the histopathological changes which were driven by the alteration of cytoskeletal proteins. Our results improve the current knowledge of toxicity of the DDT to histology and molecular response of oyster proteome to DDT exposure. In addition, histopathological changes will be beneficial for the development of an appropriate guideline for health assessment of this species in ecotoxicological context.

HMP-S7 Is a Novel Anti-Leukemic Peptide Discovered from Human Milk.

Chemotherapy in childhood leukemia is associated with late morbidity in leukemic survivors, while certain patient subsets are relatively resistant to standard chemotherapy. It is therefore important to identify new agents with sensitivity and selectivity towards leukemic cells, while having less systemic toxicity. Peptide-based therapeutics has gained a great deal of attention during the last few years. Here, we used an integrative workflow combining mass spectrometric peptide library construction, in silico anticancer peptide screening, and in vitro leukemic cell studies to discover a novel anti-leukemic peptide having 3+ charges and an alpha helical structure, namely HMP-S7, from human breast milk. HMP-S7 showed cytotoxic activity against four distinct leukemic cell lines in a dose-dependent manner but had no effect on solid malignancies or representative normal cells. HMP-S7 induced leukemic cell death by penetrating the plasma membrane to enter the cytoplasm and cause the leakage of lactate dehydrogenase, thus acting in a membranolytic manner. Importantly, HMP-S7 exhibited anti-leukemic effects against patient-derived leukemic cells ex vivo. In conclusion, HMP-S7 is a selective anti-leukemic peptide with promise, which requires further validation in preclinical and clinical studies.

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.

Molecular characterization of Thai patients with phenylalanine hydroxylase deficiency and in vitro functional study of two novel PAH variants.

Phenylketonuria (PKU) is an autosomal recessive amino acid metabolism disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH; EC1.14.16.1). This study aimed to assess the specific heterogeneity of PAH variants found in Thai population as well as evaluate enzyme activity and expression of novel variants. PAH gene from 13 patients was analyzed by PCR amplification and direct Sanger-sequencing of 13 exons of the coding region. The novel variants were transiently transfected in COS-7 cells for functional verification. Eleven different PAH variants were identified: all pathogenic variants were missense variants, of which the most frequent variant was p.R169L, accounting for 24% (6/25) of all identified alleles. Two novel variants p.R169L and p.Y317N and previously reported variants with mutated residues at the same positions (p.R169H and p.Y317H) were expressed in COS-7 cells. These showed mildly impaired residual activity levels (42.3-63.1% of wild type), while the protein levels were well expressed (82.8-110%), except for p.R169L, which showed decreased protein expression of 55.7% compared to the wild type enzyme. All subjects with p.R169L identified in at least one of pathogenic alleles (one case is homozygous) had a metabolic phenotype of mild hyperphenylalaninemia (HPA). Our data has expanded the information on the genetic heterogeneity of Thai patients with PAH deficiency. This finding emphasizes the importance of genotyping in patients with HPA, and in vitro studies can provide additional information for prediction of phenotype.

Aberrant proteins expressed in skin fibroblasts of Parkinson's disease patients carrying heterozygous variants of glucocerebrosidase and parkin genes.

Parkinson's disease (PD) is a neurodegenerative disorder that affects movement, and its development is associated with environmental and genetic factors. Genetic variants in GBA and PARK2 are important risk factors implicated in the development of PD; however, their precise roles have yet to be elucidated. The present study aimed to identify and analyse proteins from the skin fibroblasts of patients with PD carrying heterozygous GBA and PARK2 variants, and from healthy controls. Liquid chromatography coupled with tandem mass spectrometry and label-free quantitative proteomics were performed to identify and compare differential protein expression levels. Moreover, protein-protein interaction networks were assessed using Search Tool for Retrieval of Interacting Genes analysis. Using these proteomic approaches, 122 and 119 differentially expressed proteins from skin fibroblasts of patients with PD carrying heterozygous GBA and PARK2 variants, respectively, were identified and compared. According to the results of protein-protein interaction and Gene Ontology analyses, 14 proteins involved in the negative regulation of macromolecules and mRNA metabolic processes, and protein targeting to the membrane exhibited the largest degree of differential expression in the fibroblasts of patients with PD with a GBA variant, whereas 20 proteins involved in the regulation of biological quality, NAD metabolic process and cytoskeletal organization exhibited the largest degree of differential expression in the fibroblasts of patients with PD with a PARK2 variant. Among these, the expression levels of annexin A2 and tubulin ß chain, were most strongly upregulated in the fibroblasts of patients with GBA-PD and PARK2-PD, respectively. Other predominantly expressed proteins were confirmed by western blotting, and the results were consistent with those of the quantitative proteomic analysis. Collectively, the results of the present study demonstrated that the proteomic patterns of fibroblasts of patients with PD carrying heterozygous GBA and PARK2 variants are different and unique. Aberrant expression of the proteins affected by these variants may reflect physiological changes that also occur in neurons, resulting in PD development and progression.

Aberrant RL2 O-GlcNAc antibody reactivity against serum-IgA1 of patients with colorectal cancer.

O-GlcNAcylation, a single attachment of N-acetylglucosamine (GlcNAc) on serine and threonine residues, plays important roles in normal and pathobiological states of many diseases. Aberrant expression of O-GlcNAc modification was found in many types of cancer including colorectal cancer (CRC). This modification mainly occurs in nuclear-cytoplasmic proteins; however, it can exist in some extracellular and secretory proteins. In this study, we investigated whether O-GlcNAc-modified proteins are present in serum of patients with CRC. Serum glycoproteins of CRC patients and healthy controls were enriched by wheat germ agglutinin, a glycan binding protein specifically binds to terminal GlcNAc and sialic acid. Two-dimensional gel electrophoresis, RL2 O-GlcNAc immunoblotting, affinity purification, and mass spectrometry were performed. The results showed that RL2 O-GlcNAc antibody predominantly reacted against serum immunoglobulin A1 (IgA1). The levels of RL2-reacted IgA were significantly increased while total IgA were not different in patients with CRC compared to those of healthy controls. Analyses by ion trap mass spectrometry using collision-induced dissociation and electron-transfer dissociation modes revealed one O-linked N-acetylhexosamine modification site at Ser268 located in the heavy constant region of IgA1; unfortunately, it cannot be discriminated whether it was N-acetylglucosamine or N-acetylgalactosamine because of their identical molecular mass. Although failed to demonstrate unequivocally it was O-GlcNAc, these data indicated that serum-IgA had an aberrantly increased reactivity against RL2 O-GlcNAc antibody in CRC patients. This specific glycosylated form of serum-IgA1 will expand the spectrum of aberrant glycosylation which provides valuable information to cancer glycobiology.

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.

Quantitative proteomic analysis of the association between decreasing O­GlcNAcylation and metastasis in MCF­7 breast cancer cells.

Breast cancer is the most common type of cancer and leading cause of cancer­associated mortality in women worldwide. O­linked N­acetyl glucosaminylation (O­GlcNAcylation) is a dynamic post­translational modification of nuclear, cytoplasmic and mitochondrial proteins. Mounting evidence suggests that abnormal O­GlcNAcylation status is associated with cancer malignancy. In our previous study, it was reported that O­GlcNAc and O­GlcNAc transferase (OGT; an enzyme responsible for the addition of O­GlcNAc) were upregulated in breast cancer tissues and cells. Moreover, O­GlcNAcylation was required for resistance to anoikis and the anchorage­independent growth of breast cancer cells. However, the precise roles of this modification on the development of malignancy are yet to be elucidated. Therefore, in the present study, the effects of inhibiting O­GlcNAc on the malignant transformation of MCF­7 breast cancer cells under different culture conditions were determined, using monolayer (primary growth), anoikis resistance (spheroid growth) and reseeding (secondary growth) to mimic the metastatic process. Decreasing O­GlcNAc levels using small interfering (si)RNA targeting OGT resulted in a reduction in cell viability and invasiveness in anoikis resistant and reseeding conditions. Furthermore, gel­free quantitative proteomics was performed to identify the proteins affected by a reduction of O­GlcNAc. A total of 317 proteins were identified and compared, and the expression of 162 proteins was altered >1.5 fold in the siOGT treated cells compared with the siScamble (siSC) treated cells. Notably, 100 proteins involved in cellular metabolism, cellular localization, stress responses and gene expression were significantly altered in the reseeding condition. Among these differentially expressed proteins, the levels of small nuclear ribonucleoprotein Sm D1 exhibited the largest decrease in expression following knockdown of OGT, and this reduction in expression was associated with a significant decrease in the levels of mTOR expression, a protein which promotes tumor growth and progression. Taken together, the results of the present study demonstrate that decreasing O­GlcNAcylation altered protein expression, and ultimately influenced the metastatic processes, particulary regarding the invasion and reattached growth of MCF­7 breast cancer cells.

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.