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.

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.

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.

mirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets.

Techniques to isolate the small RNA fraction (<200nt) by column-based methods are commercially available. However, their use is limited because of the relatively high cost. We found that large RNA molecules, including mRNAs and rRNAs, are aggregated together in the presence of salts when RNA pellets are over-dried. Moreover, once RNA pellets are over-dried, large RNA molecules are barely soluble again during the elution process, whereas small RNA molecules (<100nt) can be eluted. We therefore modified the acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA extraction protocol by skipping the 70% ethanol washing step and over-drying the RNA pellet for 1 hour at room temperature. We named this novel small RNA isolation method "mirRICH." The quality of the small RNA sequences was validated by electrophoresis, next-generation sequencing, and quantitative PCR, and the findings support that our newly developed column-free method can successfully and efficiently isolate small RNAs from over-dried RNA pellets.

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.

Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma.

Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma.

Effective enrichment of cholangiocarcinoma secretomes using the hollow fiber bioreactor culture system.

The Northeastern region of Thailand is well known to have high incidence of bile duct cancer known as cholangiocarcinoma. So there is a continued need to improve diagnosis and treatment, and discovery of biomarkers for early detection of bile duct cancer should greatly improve treatment outcome for these patients. The secretome, a collection of proteins secreted from cells, is a useful source for identifying circulating biomarkers in blood secreted from cancer cells. Here a Hollow Fiber Bioreactor culture system was used for enrichment of cholangiocarcinoma secretomes, since this culture system mimics the dense three-dimensional microenvironment of the tumor found in vivo. Two-dimensional fluorescence difference gel electrophoresis using a sensitive Fluor saturation dye staining, followed by LC/MS/MS, was used to compare protein expression in the secretomes of cells cultured in the Hollow Fiber system and cells cultured in the monolayer culture system. For the first time, the 2D-patterns of cholangiocarcinoma secretomes from the two culture systems could be compared. The Hollow Fiber system improved the quality and quantity of cholangiocarcinoma secreted proteins compared to conventional monolayer system, showing less interference by cytoplasmic proteins and yielding more secreted proteins. Overall, 75 spots were analyzed by LC/MS/MS and 106 secreted proteins were identified. Two novel secreted proteins (C19orf10 and cystatin B) were found only in the Hollow Fiber system and were absent from the traditional monolayer culture system. Among the highly expressed proteins, 22 secreted soluble proteins were enriched by 5 fold in Hollow Fiber system compared to monolayer culture system. The Hollow Fiber system is therefore useful for preparing a wide range of proteins from low-abundance cell secretomes.