乳腺癌组织中蛋白质二级结构的Fourier变换红外光谱研究

根据乳腺大汗腺癌、小管癌、黏液癌、浸润导管癌、单纯癌和髓样癌组织Ｆｏｕｒｉｅｒ变换红外光谱酰胺Ⅰ带的去卷积和拟合分析,获得组织中蛋白质二级结构的数目及其组成。结果表明：这些组织中蛋白质二级结构的数目及其组成存在着显著的差异,并与组织的类型、分化、坏死等密切相关其中,髓样癌的差异性最大;高分化癌中螺旋结构的含量及非典型螺旋和α－螺旋含量的比值均比低分化癌的相应值高;肿瘤边缘坏死的乳腺癌组织也表现出明     

Mass Spectrometric Characterization of Protein Structure Details Refines the Proteome Signature for Invasive Ductal Breast Carcinoma

Early diagnosis as well as individualized therapies are necessary to reduce the mortality of breast cancer, and personalized patient care strategies rely on novel prognostic or predictive factors. In this study, with six breast cancer patients, 2D gel analysis was applied for studying protein expression differences in order to distinguish invasive ductal breast carcinoma, the most frequent breast tumor subtype, from control samples. In total, 1203 protein spots were assembled in a 2D reference gel. Differentially abundant spots were subjected to peptide mass fingerprinting for protein identification. Twenty proteins with their corresponding 38 differentially expressed 2D gel spots were contained in our previously reported proteome signature, suggesting that distinct protein forms were contributing. In-depth MS/MS measurements enabled analyses of protein structure details of selected proteins. In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified. Principle component analysis and hierarchical clustering with normalized quantitative data from the 38 spots resulted in accurate separation of tumor from control samples. Thus, separation of tissue samples as in our initial proteome signature could be confirmed even with a different proteome analysis platform. In addition, detailed protein structure investigations enabled refining our proteome signature for invasive ductal breast carcinoma, opening the way to structure-/function studies with respect to disease processes and/or therapeutic intervention.     

Spatially Resolved Genetic Analysis of Tissue Sections Enabled by Microscale Flow Confinement Retrieval and Isotachophoretic Purification

We have developed a method for spatially resolved genetic analysis of formalin‐fixed paraffin‐embedded (FFPE) cell block and tissue sections. This method involves local sampling using hydrodynamic flow confinement of a lysis buffer, followed by electrokinetic purification of nucleic acids from the sampled lysate. We characterized the method by locally sampling an array of points with a circa 200 μm diameter footprint, enabling the detection of single KRAS and BRAF point mutations in small populations of RKO and MCF‐7 FFPE cell blocks. To illustrate the utility of this approach for genetic analysis, we demonstrate spatially resolved genotyping of FFPE sections of human breast invasive ductal carcinoma.     

Towards a proteome signature for invasive ductal breast carcinoma derived from label-free nanoscale LC-MS protein expression profiling of tumorous and glandular tissue

As more and more alternative treatments become available for breast carcinoma, there is a need to stratify patients and individual molecular information seems to be suitable for this purpose. In this study, we applied label-free protein quantitation by nanoscale LC-MS and investigated whether this approach could be used for defining a proteome signature for invasive ductal breast carcinoma. Tissue samples from healthy breast and tumor were collected from three patients. Protein identifications were based on LC-MS peptide fragmentation data which were obtained simultaneously to the quantitative information. Hereby, an invasive ductal breast carcinoma proteome signature was generated which contains 60 protein entries. The on-column concentrations for osteoinductive factor, vimentin, GAP-DH, and NDKA are provided as examples. These proteins represent distinctive gene ontology groups of differentially expressed proteins and are discussed as risk markers for primary tumor pathogenesis. The developed methodology has been found well applicable in a clinical environment in which standard operating procedures can be kept; a prerequisite for the definition of molecular parameter sets that shall be capable for stratification of patients.     

Use of Legendre moments for the fast comparison of two-dimensional polyacrylamide gel electrophoresis maps images

In this paper, Legendre moments are calculated to extract the global information from a set of two-dimensional polyacrylamide gel electrophoresis map images. The dataset contains 18 samples belonging to two different cell lines (PACA44 and T3M4) of control (untreated) and drug-treated pancreatic ductal carcinoma cells. The aim of this work was to obtain the correct classification of the 18 samples, using the Legendre moments as discriminant variables. For each image the Legendre moments up to a maximum order of 100 were computed. The stepwise linear discriminant analysis (LDA) was performed in order to select the moments with the highest discriminating power. The results demonstrate that the Legendre moments can be successfully applied for fast classification purposes and similarity analysis.     

Application of organometallic chemistry in the formulation of a modern therapeutic drug by Ag nanoparticles green-mediated by Allium to treat the breast cancer

In the recent study, we decided to survey the capacities of metallic nanoparticles formulated by Allium monanthum (AgNPs) as a novel chemotherapeutic drug in the treatment of several types of breast cancers. Characterization of AgNPs was done by UV–Visible Spectroscopy (UV–Vis), Fourier Transformed Infrared Spectroscopy (FT‐IR), Transmission Electron Microscopy (TEM), and Field Emission Scanning Electron Microscopy (FE‐SEM). For investigating the antioxidant properties of AgNO₃, Allium monanthum, and AgNPs, the DPPH test was used in the presence of butylated hydroxytoluene as the positive control. To survey the cytotoxicity and anti-breast cancer effects of AgNO₃, Allium monanthum, and AgNPs, MTT assay was used on the breast adenocarcinoma (MCF7), breast carcinoma (Hs 578Bst), infiltrating ductal cell carcinoma (Hs 319.T), infiltrating lobular carcinoma of breast (UACC-3133), inflammatory carcinoma of the breast (UACC-732), and metastatic carcinoma (MDA-MB-453) cell lines. DPPH test revealed similar antioxidant potentials for Allium monanthum, AgNPs, and butylated hydroxytoluene. Silver nanoparticles had very low cell viability and anti-breast cancer properties dose-dependently against MCF7, Hs 578Bst, Hs 319.T, UACC-3133, UACC-732, and MDA-MB-453 cell lines without any cytotoxicity on the normal cell line. The best result of anti-breast cancer properties of AgNPs against the above cell lines was seen in the case of the UACC-3133 cell line. According to the above findings, the silver nanoparticles containing Allium monanthum aqueous extract can be administrated in humans for the treatment of several types of breast cancer especially breast adenocarcinoma, breast carcinoma, infiltrating ductal cell carcinoma, infiltrating lobular carcinoma of breast, inflammatory carcinoma of the breast, and metastatic carcinoma.     

Identification of the regulatory proteins in human pancreatic cancers treated with Trichostatin A by 2D-PAGE maps and multivariate statistical analysis

In this paper, principal component analysis (PCA) is applied to a spot quantity dataset comprising 435 spots detected in 18 samples belonging to two different cell lines (Paca44 and T3M4) of control (untreated) and drug-treated pancreatic ductal carcinoma cells. The aim of the study was the identification of the differences occurring between the proteomic patterns of the two investigated cell lines and the evaluation of the effect of the drug Trichostatin A on the protein content of the cells. PCA turned out to be a successful tool for the identification of the classes of samples present in the dataset. Moreover, the loadings analysis allowed the identification of the differentially expressed spots, which characterise each group of samples. The treatment of both the cell lines with Trichostatin A therefore showed an appreciable effect on the proteomic pattern of the treated samples. Identification of some of the most relevant spots was also performed by mass spectrometry.     

The use of Au@SiO2 shell-isolated nanoparticle-enhanced Raman spectroscopy for human breast cancer detection

This study uses the powerful fingerprint features of Raman spectroscopy to distinguish different types of breast tissues including normal breast tissues (NB), fibroadenoma (FD), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC). Thin frozen tissue sections of fresh breast tissues were measured by Raman spectroscopy. Due to the inherent low sensitivity of Raman spectra, Au@SiO₂ shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) technique was utilized to provide supplementary and more informative spectral features. A total of 619 Raman spectra were acquired and compared to 654 SHINERS spectra. The maximum enhancement effect of distinct and specific bands was characterized for different tissue types. When applying the new criteria, excellent separation of FD, DCIS, and IDC was obtained for all tissue types. Most importantly, we were able to distinguish ADH from DCIS. Although only a preliminary distinction was characterized between ADH and NB, the results provided a good foundation of criteria to further discriminate ADH from NB and shed more light toward a better understanding of the mechanism of ADH formation. This is the first report to detect the premalignant (ADH and DCIS) breast tissue frozen sections and also the first report exploiting SHINERS to detect and distinguish breast tissues. The results presented in this study show that SHINERS can be applied to accurately and efficiently identify breast lesions. Further, the spectra can be acquired in a minimally invasive procedure and analyzed rapidly facilitating early and accurate diagnosis in vivo/in situ. Figure

Human breast cancer detection with Au@SiO₂ SHINERS     

Bioinformatics analysis and verification of gene targets for renal clear cell carcinoma

BackgroundIt is estimated that there are 338,000 new renal-cell carcinoma releases every year in the world. Renal cell carcinoma (RCC) is a heterogeneous tumor, of which more than 70% is clear cell renal cell carcinoma (ccRCC). It is estimated that about 30% of new renal-cell carcinoma patients have metastases at the time of diagnosis. However, the pathogenesis of renal clear cell carcinoma has not been elucidated. Therefore, it is necessary to further study the pathogenesis of ccRCC.MethodsTwo expression profiling datasets (GSE68417, GSE71963) were downloaded from the GEO database. Differentially expressed genes (DEGs) between ccRCC and normal tissue samples were identified by GEO2R. Functional enrichment analysis was made by the DAVID tool. Protein-protein interaction (PPI) network was constructed. The hub genes were excavated. The clustering analysis of expression level of hub genes was performed by UCSC (University of California Santa Cruz) Xena database. The hub gene on overall survival rate (OS) in patients with ccRCC was performed by Kaplan-Meier Plotter. Finally, we used the ccRCC renal tissue samples to verify the hub genes.Results1182 common DEGs between the two datasets were identified. The results of GO and KEGG analysis revealed that variations in were predominantly enriched in intracellular signaling cascade, oxidation reduction, intrinsic to membrane, integral to membrane, nucleoside binding, purine nucleoside binding, pathways in cancer, focal adhesion, cell adhesion molecules. 10 hub genes ITGAX, CD86, LY86, TLR2, TYROBP, FCGR2A, FCGR2B, PTPRC, ITGB2, ITGAM were identified. FCGR2B and TYROBP were negatively correlated with the overall survival rate in patients with ccRCC (P < 0.05). RT-qPCR analysis showed that the relative expression levels of CD86, FCGR2A, FCGR2B, TYROBP, LY86, and TLR2 were significantly higher in ccRCC samples, compared with the adjacent renal tissue groups.ConclusionsIn summary, bioinformatics technology could be a useful tool to predict the progression of ccRCC. In addition, there are DEGs between ccRCC tumor tissue and normal renal tissue, and these DEGs might be considered as biomarkers for ccRCC.     

Lipidomic study and diagnosis of hepatocellular carcinoma tumor with rapid evaporative ionization mass spectrometry

Liver cancer is generally considered the leading cause of cancer deaths worldwide, and hepatocellular carcinoma (HCC) contributes to more than 90% of liver cancers. The altered lipid metabolism for rapid cancer cell growth and tumor formation has been frequently proven. In this study, an ambient ionization mass spectrometry technique, rapid evaporative ionization mass spectrometry (REIMS) using a monopolar electric knife, called iKnife, was systematically optimized and employed for ex vivo analysis of 12 human HCC tumor tissue specimens together with the paired paracancerous tissue (PT) and noncancerous liver tissue (NCT) specimens. Nine free fatty acids and 34 phospholipids were tentatively identified according to their extract masses and/or tandem mass spectra. With the help of statistical methods, 7 free fatty acids and 10 phospholipids were distributed differently in 3 types of liver tissue specimens (95% confidence interval). The box plots showed these characterized lipid metabolites varied in PT, HCC, and NCT. Compared with PT and NCT, the upregulations of four common fatty acids FA 18:0, FA 20:4, FA 16:0, and FA 18:1, together with phospholipids PC 36:1, PE 38:3, PE (18:0/20:4), PA (O-36:1), PC (32:1), PC 32:0, PE 34:0, and PC (16:0/18:1), were found in HCC specimens. The sensitivity and specificity of the established statistic model for real-time HCC tumor diagnosis were 100% and 90.5%, respectively. This study demonstrated that the described REIMS technique is a potential method for rapid lipidomic analysis and characterization of HCC tumor tissue.     

Proteome study of colorectal carcinogenesis

Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post-translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco-2 and DLD-1 originating from well-differentiated and poorly differentiated colon carcinoma, respectively.     

PHOTODYNAMIC THERAPY IN THE TREATMENT OF PANCREATIC CARCINOMA: DIHEMATOPORPHYRIN ETHER UPTAKE and PHOTOBLEACHING KINETICS

Abstract Results of dihematoporphryin ether (DHE) uptake and fluorescence kinetics show that the concentration in the pancreas is on the order of 40-60 μg DHE g⁻¹ of tissue at an injected dose of 40 mg kg⁻¹. Previously concentrations on this order have primarily been found in organs of the reticuloendothelial system. Two intra-pancreatic carcinoma models, one of acinar origin (rat) and one of ductal origin (hamster), were studied. Both showed equal or higher concentrations of DHE as compared with normal pancreas when fluorescence measurements and chemical extraction procedures were performed. Photodynamic therapy (PDT) treatment of the normal pancreas and pancreatic tumors yielded atypical results. When the normal pancreas with DHE present is exposed to 630-nm light from a dye laser (75 mW cm⁻², 30 min), the normal photobleaching measurable by fluorescence decay does not occur. Yet the pancreatic tumor responds with a relatively normal fluorescence decay pattern, with hemorrhaging and a resultant loss of measurable DHE concentration.     

Expression Levels of RFP in Normal and Cancer Human Tissues via Real-time RT-PCR Detection

IntroductionRet finger protein(RFP) was first identified as apart of the human recombined transforming gene, ret,and was a member of the tripartite motif(TRIM) pro-tein family. Itwas postulated thatmultimeric complexesmade of TRIM proteins defined differe…     

Green formulation of Ag nanoparticles by Hibiscus rosa-sinensis: Introducing a navel chemotherapeutic drug for the treatment of liver cancer

An eco-friendly biosynthesized Ag NPs immobilized Hibiscus rosa-sinensis extract has been introduced. The as-prepared nanoparticles were characterized using UV–Vis, SEM, and FT-IR analysis. In the FT-IR test, the presence of many antioxidant compounds with related bonds caused the excellent condition for reducing of silver in the silver nanoparticles. In UV–Vis, the clear peak in the wavelength of 428 nm indicated the formation of silver nanoparticles. The synthesized nanoparticles had very low cell viability and high anti-liver cancer activities dose-dependently against pleomorphic hepatocellular carcinoma (SNU-387), hepatic ductal carcinoma (LMH/2A), morris hepatoma (McA-RH7777), and novikoff hepatoma (N1-S1 Fudr) cell lines without any cytotoxicity on the normal cell line (HUVEC). The synthesized nanoparticles inhibited half of the DPPH molecules in the concentration of 78 µg/mL. Perhaps notable anti-liver cancer activities of the synthesized nanoparticles against common liver cancer cell lines are linked to their antioxidant activities.     

Using MALDI-TOF MS coupled with a high-mass detector to directly analyze intact proteins in thyroid tissues

Protein analysis is vital for biological and clinical research, but the measurement of unseparated, intact and high-mass proteins is also a challenging task by mass spectrometry-based methods. Here, we present a protocol for rapid and high-throughput analysis of intact proteins in tissue samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDITOF MS) combined with a high-mass detector platform. The method involves tissue specimens that undergo a simple protein extraction before MALDI-MS analysis. Using this method, the high abundance proteins in human thyroid carcinoma and paracarcinoma tissues were successfully investigated, and the mass spectra of the tissues of the 30 illustrated thyroid cancers showed remarkable differences. The peak intensity revealed a significant increase in human albumin in thyroid carcinoma tissues(p0.05). To validate the feasibility and credibility of this method, label-free proteomics quantitative analysis and Western blotting were used to relatively quantify the proteins in these tissues. Those results demonstrated a nearly 3-fold difference in human albumin levels between thyroid carcinoma and para-carcinoma tissues, which were consistent with the results of our method. The advantages of our method are easy sample handling, remarkable reproducibility and the ability to analyze high-mass proteins without digestion, which make them have the potential to be used in biological research and in clinical practice.     

Expression of high-abundance proteins in sera of patients with endometrial and cervical cancers: analysis using 2-DE with silver staining and lectin detection methods

The expression of high-abundance serum proteins in newly diagnosed patients with endometrial adenocarcinoma (EACa), squamous cell cervical carcinoma (SCCa) and cervical adenocarcinoma (ACCa), relative to control female subjects, was analyzed by subjecting serum samples to 2-DE followed by image analysis of the silver-stained protein profiles. The three cohorts of cancer patients demonstrated different altered expression of serum high-abundance proteins compared to negative control women. The expression of alpha1-antitrypsin, alpha1-B glycoprotein, cleaved high-molecular-weight kininogen (light chain) and antithrombin III were consistently altered in all the patients. However, clusterin was upregulated only in the patients with EACa, while those with SCCa and ACCa were typically characterized by the upregulated expression of zinc alpha-2-glycoprotein. The aberrant expression of selective serum proteins in the various cohorts of cancer patients was validated by competitive ELISA as well as by lectin detection. Analysis by using the champedak galactose binding lectin further highlighted an unidentified protein that may be differently glycosylated in the sera of the EACa patients that were studied.     

Multivariate statistical differentiation of renal cell carcinomas based on lipidomic analysis by ambient ionization imaging mass spectrometry

Desorption electrospray ionization (DESI) mass spectrometry (MS) was used in an imaging mode to interrogate the lipid profiles of thin tissue sections of 11 sample pairs of human papillary renal cell carcinoma (RCC) and adjacent normal tissue and nine sample pairs of clear cell RCC and adjacent normal tissue. DESI-MS images showing the spatial distributions of particular glycerophospholipids (GPs) and free fatty acids in the negative ion mode were compared to serial tissue sections stained with hematoxylin and eosin (H&E). Increased absolute intensities as well as changes in relative abundance were seen for particular compounds in the tumor regions of the samples. Multivariate statistical analysis using orthogonal projection to latent structures treated partial least square discriminate analysis (PLS-DA) was used for visualization and classification of the tissue pairs using the full mass spectra as predictors. PLS-DA successfully distinguished tumor from normal tissue for both papillary and clear cell RCC with misclassification rates obtained from the validation set of 14.3% and 7.8%, respectively. It was also used to distinguish papillary and clear cell RCC from each other and from the combined normal tissues with a reasonable misclassification rate of 23%, as determined from the validation set. Overall DESI-MS imaging combined with multivariate statistical analysis shows promise as a molecular pathology technique for diagnosing cancerous and normal tissue on the basis of GP profiles.     

Alterations of HLA class I and II antigen expression in preinvasive, invasive and metastatic cervical cancers

HLA expression is altered in a large variety of human cancers. We performed immunohistochemical staining on tissues from normal, preinvasive, invasive and metastatic cervical cancer tissues using anti-HLA class I or class II antibody. In tissues from normal squamous epithelium, carcinoma in situ (CIS) and microinvasive carcinoma (MIC), the expressions of HLA-B, C heavy chains and class II heavy chain were significantly decreased as disease progressed. When the expression patterns were compared between primary and metastatic squamous cell carcinoma (SCC) lesions, statistically significant down-regulation of HLA class I and class II antigen in metastatic lesions was observed. The rates of HLA-B, C heavy chains and class II heavy chain expressions were all significantly down-regulated compared to the down-regulation rate of class I beta2-microglobulin (beta2m) in invasive squamous lesions, and the expressions of class II heavy chain in metastatic lesions was decreased further than that in primary lesions. Unlike SCC, the degree of HLA class I and class II loss was not evident as disease progressed in early stage of adenocarcinoma. In invasive adenocarcinoma lesions, only the expression of HLA-B, C heavy chains was decreased and no differences were seen in HLA-B, C heavy chain expression patterns between primary and metastatic lesions. These results suggest that alterations of HLA class I and II expressions seem to occur at a particular step in cervical cancer development and depend on tissue types: when the tumor becomes invasive and starts to metastasize.     

Volatile organic compounds in head and neck squamous cell carcinoma—An in vitro pilot study

Owing to the lack of specific symptoms, diagnosis of head and neck squamous cell carcinoma (HNSCC) may be delayed. We evaluated volatile organic compounds in tumor samples from patients suffering from HNSCC and tested the hypothesis that there is a characteristic altered composition in the headspace of HNSCC compared with control samples from the same patient with normal squamous epithelium. These results provide the basis for future noninvasive breath analysis in HNSCC. Headspace air of suspected tumor and contralateral control samples in 20 patients were analyzed using ion-mobility spectrometry. Squamous cell carcinoma was diagnosed in 16 patients. In total, we observed 93 different signals in headspace measurements. Squamous cell carcinomas revealed significantly higher levels of volatile cyclohexanol (0.54 ppb_v, 25th to 75th percentiles 0.35–0.86) compared with healthy squamous epithelium (0.24 ppb_v, 25th to 75th percentiles 0.12–0.3; p < 0.001). In conclusion, head and neck squamous cell carcinoma emitted significantly higher levels of volatile cyclohexanol in headspace compared with normal squamous epithelium. These findings form the basis for future breath analysis for diagnosis, therapy control and the follow-up of HNSSC to improve therapy and aftercare.