Integrative proteomic characterization of adenocarcinoma of esophagogastric junction.

The incidence of adenocarcinoma of the esophagogastric junction (AEG) has been rapidly increasing in recent decades, but its molecular alterations and subtypes are still obscure. Here, we conduct proteomics and phosphoproteomics profiling of 103 AEG tumors with paired normal adjacent tissues (NATs), whole exome sequencing of 94 tumor-NAT pairs, and RNA sequencing in 83 tumor-NAT pairs. Our analysis reveals an extensively altered proteome and 252 potential druggable proteins in AEG tumors. We identify three proteomic subtypes with significant clinical and molecular differences. The S-II subtype signature protein, FBXO44, is demonstrated to promote tumor progression and metastasis in vitro and in vivo. Our comparative analyses reveal distinct genomic features in AEG subtypes. We find a specific decrease of fibroblasts in the S-III subtype. Further phosphoproteomic comparisons reveal different kinase-phosphosubstrate regulatory networks among AEG subtypes. Our proteogenomics dataset provides valuable resources for understanding molecular mechanisms and developing precision treatment strategies of AEG.

Downregulation of LINC00515 in high-grade serous ovarian cancer and its relationship with platinum resistance.

Aim: To investigate the expression of long intergenic noncoding RNA 00515 (LINC00515) in high-grade serous ovarian cancer (HGSOC) and its potential correlation with platinum resistance. Patients & methods: Expression of LINC00515 in HGSOC (n = 115) and normal (n = 19) tissues was detected via quantitative real-time PCR (qRT-PCR). We further explored the statistical significance of the relationship between LINC00515 expression and platinum resistance in HGSOC. Results: LINC00515 was gradually downregulated in the order of normal > platinum-sensitive > platinum-resistant tissue (p < 0.05). Results demonstrated that LINC00515 downregulation was correlated with platinum resistance and relapse-free survival (RFS) of HGSOC (p < 0.05). Conclusion: LINC00515 downregulation is correlated with HGSOC development, platinum resistance and RFS, supporting its utility as a potential biomarker to predict platinum resistance and prognosis of RFS.

Simultaneous determination of six tyrosine kinase inhibitors in human plasma using HPLC-Q-Orbitrap mass spectrometry.

AIM: Gefitinib, erlotinib, icotinib, crizotinib, lapatinib and apatinib are targeted cancer therapy agents acting through inhibition of tyrosine kinase. Method for quantifying these six drugs in human plasma of patients was required. MATERIALS & METHODS: An HPLC-Q-Orbitrap method (based on HPLC-MS/MS) was developed and validated for the simultaneous detection and quantitation of six tyrosine kinase inhibitors in human plasma. Sample was extracted by liquid-liquid extraction (ethyl acetate: tert-Butyl methyl ether, 1:1 v/v). The method shows a high level of accuracy and reproducibility. The lower limit of quantification was 0.02 ng/ml for apatinib, 0.1 ng/ml for crizotinib, 2.0 ng/ml for lapatinib and 0.05 ng/ml for erlotinib, gefitinib and icotinib. This method was successfully used for apatinib monitoring in plasma of patients with NSCLC. CONCLUSION: This simple and reproducible method has potential for monitoring of tyrosine kinase inhibitors in patients' plasma.

Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics.

RATIONALE: Mass spectrometry (MS)-based protein identification depends mainly on protein extraction and digestion. Although sodium dodecyl sulfate (SDS) can preclude enzymatic digestion and interfere with MS analysis, it is still the most widely used surfactant in these steps. To overcome these disadvantages, a SDS-compatible proteomic technique for SDS removal prior to MS-based analyses was developed, namely filter-aided sample preparation (FASP). METHODS: Herein, based on the effectiveness of sodium deoxycholate and a detergent removal spin column, we developed a modified FASP (mFASP) method and compared its overall performance, total number of peptides and proteins identified for shotgun proteomic experiments with that of the FASP method. RESULTS: Identification of 4570 ± 392 and 9139 ± 317 peptides and description of 862 ± 46 and 1377 ± 33 protein groups with two or more peptides from the ovarian cancer cell line A2780 was accomplished by FASP and mFASP methods, respectively. The mFASP method (21.2 ± 0.2%) had higher average peptide to protein coverage than FASP method (13.2 ± 0.5%). More hydrophobic peptides were identified by mFASP than by FASP, as indicated by the GRAVY score distribution. CONCLUSIONS: The reported method enables reliable and efficient identification of proteins and peptides in whole-cell extracts containing SDS. The new approach allows for higher throughput (the simultaneous identification of more proteins), a more comprehensive investigation of proteins, and potentially the discovery of new biomarkers. Copyright © 2016 John Wiley & Sons, Ltd.

The Essential Role of H19 Contributing to Cisplatin Resistance by Regulating Glutathione Metabolism in High-Grade Serous Ovarian Cancer.

Primary and acquired drug resistance is one of the main obstacles encountered in high-grade serous ovarian cancer (HGSC) chemotherapy. Cisplatin induces DNA damage through cross-linking and long integrated non-coding RNAs (lincRNAs) play an important role in chemical induced DNA-damage response, which suggests that lincRNAs may be also associated with cisplatin resistance. However, the mechanism of long integrated non-coding RNAs (lincRNAs) acting on cisplatin resistance is not well understood. Here, we showed that expression of lin-RECK-3, H19, LUCAT1, LINC00961, and linc-CARS2-2 was enhanced in cisplatin-resistant A2780-DR cells, while transcriptome sequencing showed decreased Linc-TNFRSF19-1 and LINC00515 expression. Additionally, we verified that different H19 expression levels in HGSC tissues showed strong correlation with cancer recurrence. H19 knockdown in A2780-DR cells resulted in recovery of cisplatin sensitivity in vitro and in vivo. Quantitative proteomics analysis indicated that six NRF2-targeted proteins, including NQO1, GSR, G6PD, GCLC, GCLM and GSTP1 involved in the glutathione metabolism pathway, were reduced in H19-knockdown cells. Furthermore, H19-knockdown cells were markedly more sensitive to hydrogen-peroxide treatment and exhibited lower glutathione levels. Our results reveal a previously unknown link between H19 and glutathione metabolism in the regulation of cancer-drug resistance.

The inhibitory effect of Shaoyao Ruangan formula on mice with transplanted H22 hepatocarcinoma and its mechanism research.

BACKGROUND: The incidence of hepatocellular carcinoma (HCC) is very high in the world. However, a safe and effective strategy is still under research. AIMS: Our aim was to demonstrate the inhibitory effect of Shaoyao Ruangan Formmula (SRF) on the tumor of H22-bearing mice and explore its antitumor mechanisms. SETTINGS AND DESIGN: Corresponding physiological indexes of H22-bearing mice treated with SRF were compared with that of saline treated mice, which could reflect the tumor-suppressing effect of SRF. MATERIALS AND METHODS: After treatment, tumor weight, survival time, related gene expression levels etc., were recorded or detected. STATISTICAL ANALYSIS: Data analyzed using a computer SPSS program. RESULTS AND CONCLUSIONS: Comparing with blank control group, the tumor inhibitor rate (IR) of low, middle and high dose group of SRF was 17.72%, 33.99% and 23.73%, respectively. IR of CTX was 43.95%. The results also showed that each group of SRF could prolong the life span of H22-bearing mice to some extent. In addition, reverse transcription polymerase chain reaction (RT-PCR) results revealed that SRF was able to influence related genes expression in the tumor tissues of H22-bearing mice. The expression of TGF-ß receptor type II (TBRII) gene was significantly upregulated in each SRF group comparing with normal saline group. On the contrary, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was significantly downregulated in each SRF group comparing with normal saline group. In summary, SRF showed tumor-suppressing effect on mice with transplanted H22 hepatocarcinoma. The mechanism of antitumor effect may induced by upregulating TBRII expression and down-regulating NF-κB expression.

Improved conditions for periodate/Schiff's base-based fluorescent staining of glycoproteins with dansylhydrazine in SDS-PAGE.

An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS-PAGE was described. Down to 4-8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16-fold higher than that of original protocol, but similar to that of Pro-Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel-separated glycoproteins.

Fluorescent staining of protein in sodium dodecyl sulfate polyacrylamide gels by salicylaldehyde azine.

As a non-covalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence-based dye for detecting proteins both in 1-D and 2-D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which similars to that of glutaraldehyde (GA)-silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces.

Improved staining of phosphoproteins with high sensitivity in polyacrylamide gels using Stains-All.

An improved Stains-All (ISA) staining method for phosphoproteins in SDS-PAGE was described. Down to 0.5-1 ng phosphoproteins (α-casein, ß-casein, or phosvitin) can be successfully selectively detected by ISA stain, which is approximately 120-fold higher than that of original Stains-All stain, but is similar to that of commonly used Pro-Q Diamond stain. Furthermore, unlike the original Stains-All protocol that was time consuming and light unstable, ISA stain could be completed within 60 min without resorting to protect the gels from light during the whole staining procedure. According to the results, it is concluded that ISA stain is a rapid, sensitive, specific, and economic staining method for a broad application to the research of phosphoproteins.

A method for sensitive staining of DNA in polyacrylamide gels using basic fuchsin.

BACKGROUND: PAGE is a widely used analytical method to resolve components of a DNA mixture based on their size. Various DNA visualization methods including fluorescence, visible dye and silver have been used for the detection of gel-separated DNA, with each having different advantages and disadvantages in terms of sensitivity, safety and simplicity. RESULTS: A fast and sensitive visible dye-based staining method for DNA in polyacrylamide gels using basic fuchsin (BF) is described. As low as 10-20 pg of DNA can be visualized within 10 min; the sensitivity is fourfold more sensitive than that of SYBR® Gold stain, the most sensitive commercial fluorescent probe, but similar to silver staining kit from GE Healthcare. In addition, the mechanism studies suggest that the interaction of BF with DNA is mainly contributed by non-intercalative binding mode. CONCLUSION: By comprehensive studies of this visible dye-based protocol, we concluded that BF stain is a fast and sensitive method currently available for detecting DNA in polyacrylamide gels.

An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels.

A sensitive, brief, and user-friendly silver stain to meet the needs in high-efficiency detection of lipopolysaccharides (LPS) on polyacrylamide gels is described. In this study, the most commonly used formaldehyde-based LPS silver stain, which is potentially hazardous to the operator, is replaced by ascorbic acid (Vc) in alkaline sodium thiosulfate solution. It takes only about 35 min to complete all the protocol, with a detection limit of 4 ng of total LPS. The results indicate that this user-friendly method could be a good choice for LPS visualization on polyacrylamide gels.