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.

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.

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.

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.

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.

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.

Protein profiling of osteosarcoma tissue and soft callus unveils activation of the unfolded protein response pathway.

Oncogenic drivers of osteosarcoma remain controversial due to the complexity of the genomic background of the disease. There are limited novel therapeutic options, and the survival rate of patients with osteosarcoma has not improved in decades. Genomic instability leads to complexity in various pathways, which is potentially revealed at the protein level. Therefore, the present study aimed to identify the mechanisms involved in the oncogenesis of osteosarcoma using proteomics and bioinformatics tools. As clinical specimens from patients are the most relevant disease­related source, expression patterns of proteins in osteosarcoma tissues were compared with soft tissue callus from donors containing high numbers of osteoblastic cells. Two­dimensional electrophoresis and liquid chromatography­tandem mass spectrometry (LC­MS/MS) successfully identified 33 differentially expressed proteins in the osteosarcoma tissues compared with the soft tissue callus. Among these proteins, 29 proteins were significantly upregulated in osteosarcoma. A functionally grouped network of the overexpressed proteins, that was created using the ClueGo and CluePedia applications, demonstrated that the unfolded protein response (UPR) pathway was activated mainly through the activating transcription factor 6 arm in osteosarcoma. The results of proteomics analysis were confirmed by elevated expression of UPR­related chaperone proteins, including 78 kDa glucose­related protein (GRP78), endoplasmin, calreticulin and prelamin­A/C, in the patient­derived primary cells and osteosarcoma cell lines. Furthermore, the expression of GRP78, a master regulator of the UPR, was enhanced in the osteosarcoma tissues of patients that were resistant to double regimen of doxorubicin and a platinum­based drug. The findings of the present study suggest that targeting the UPR pathway may be promising for the treatment of osteosarcoma.

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.

Oncogenic roles of serine-threonine kinase receptor-associated protein (STRAP) in osteosarcoma.

PURPOSE: To validate the presence of serine-threonine kinase receptor-associated Protein (STRAP) in osteosarcoma tissue and to investigate the oncological role of STRAP in osteosarcoma. METHODS: Expression of STRAP protein in osteosarcoma tissue compared to soft callus (hyperactive bone healing tissue) and in multiple cell lines was examined using western blot analysis. Effects of STRAP silencing on cell proliferation, invasion, migration and re-implantability in chick chorioallantoic membrane (CAM) were observed in osteosarcoma cell lines (MNNG-HOS, 143B, and U2OS). RESULTS: The result demonstrated that STRAP was highly up-regulated in osteosarcoma tissues compared with the normal physiological bone healing tissue (soft callus). Expression level of STRAP was markedly high in osteosarcoma cell lines with aggressive phenotype. Upon STRAP silencing, invasion and migration, but not proliferative activity, were selectively modulated in high-expression-STRAP cell lines. In addition, STRAP silencing reduced the success rate of tumor implantation and growth of MNNG-HOS cells in CAM model. CONCLUSIONS: Serine-threonine kinase receptor-associated protein is up-regulated during osteosarcoma progression. The presence of STRAP enhances osteosarcoma cell invasion, migration and re-implantation ability, factors which play a critical role in metastasis. Serine-threonine kinase receptor-associated protein and its related pathway are worthy for further exploration as a novel target for anti-metastasis agents.

Tumor Susceptibility Gene 101 Mediates Anoikis Resistance of Metastatic Thyroid Cancer Cells.

BACKGROUND/AIM: Resistance to anoikis is a pre-requisite step in metastasis, a major cause of death in patients with cancer, including thyroid cancer. Impairing anoikis resistance is a possible strategy for therapy of metastatic cancer. We, therefore, we aimed to investigate the key players of anoikis resistance. MATERIALS AND METHODS: Papillary-type (BCPAP), follicular-type (FTC133), and anaplastic-type (ARO) thyroid carcinoma cells, cultured in poly(2-hydroxyethyl methacrylate)-coated plates to mimic circulating cells, were used as model systems in this study. Flow cytometry and soft-agar assays were used to determine cells exhibiting anoikis resistance. Proteomics was used to identify candidate proteins and validated using western blot and siRNA knockdown. RESULTS: Only ARO cells showed both anoikis resistance potential and anchorage-independent growth ability. Tumor susceptibility gene 101 protein (TSG101) was identified to be potentially important in anoikis resistance, which was confirmed by an increase in anoikis and expression of a pro-apoptotic protein (BCL-2 like protein 4) and an apoptotic marker (cleaved poly-ADP ribose polymerase) in floating siTSG101-knockdown cells. CONCLUSION: To our knowledge, this is the first study that implicates the importance of TSG101 in anoikis resistance of thyroid cancer.