Changes in electric charge and phospholipids composition in human colorectal cancer cells.
ABSTRACT Cancer cells perform their malicious activities through own cell membranes that screen and transmit inhibitory and stimulatory signals out of the cells and into them. This work is focused on changes of phospholipids content (PI-phosphatidylinositol, PS-phosphatidylserine, PE-phosphatidylethanolamine, PC-phosphatidylcholine) and electric charge that occur in cell membranes of colorectal cancer of pT 3 stage, various grades (G2, G3) and without/with metastasis. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC (high-performance liquid chromatography). The surface charge density of colorectal cancer cell membranes was measured using electrophoresis. The measurements were carried out at various pH of solution. It was shown that the process of cancer transformation was accompanied by an increase in total amount of phospholipids as well as an increase in total positive charge at low pH and total negative charge at high pH. A malignant neoplasm cells with metastases are characterized by a higher PC/PE ratio than malignant neoplasm cells without metastases.
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ABSTRACT: Negative ion desorption electrospray ionization (DESI) was used for the analysis of an ex vivo tissue sample set comprising primary colorectal adenocarcinoma samples and colorectal adenocarcinoma liver metastasis samples. Frozen sections (12 μm thick) were analyzed by means of DESI imaging mass spectrometry (IMS) with spatial resolution of 100 μm using a computer-controlled DESI imaging stage mounted on a high resolution Orbitrap mass spectrometer. DESI-IMS data were found to predominantly feature complex lipids, including phosphatidyl-inositols, phophatidyl-ethanolamines, phosphatidyl-serines, phosphatidyl-ethanolamine plasmalogens, phosphatidic acids, phosphatidyl-glycerols, ceramides, sphingolipids, and sulfatides among others. Molecular constituents were identified based on their exact mass and MS/MS fragmentation spectra. An identified set of molecules was found to be in good agreement with previously reported DESI imaging data. Different histological tissue types were found to yield characteristic mass spectrometric data in each individual section. Histological features were identified by comparison to hematoxylin-eosin stained neighboring sections. Ions specific to certain histological tissue types (connective tissue, smooth muscle, healthy mucosa, healthy liver parenchyma, and adenocarcinoma) were identified by semi-automated screening of data. While each section featured a number of tissue-specific species, no potential global biomarker was found in the full sample set for any of the tissue types. As an alternative approach, data were analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA) which resulted in efficient separation of data points based on their histological types. A pixel-by-pixel tissue identification method was developed, featuring the PCA/LDA analysis of authentic data set, and localization of unknowns in the resulting 60D, histologically assigned LDA space. Novel approach was found to yield results which are in 95% agreement with the results of classical histology. KRAS mutation status was determined for each sample by standard molecular biology methods and a similar PCA/LDA approach was developed to assess the feasibility of the determination of this important parameter using solely DESI imaging data. Results showed that the mutant and wild-type samples fully separated. DESI-MS and molecular biology results were in agreement in 90% of the cases.Analytical and Bioanalytical Chemistry 03/2012; 403(8):2315-25. · 3.66 Impact Factor
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ABSTRACT: Alyteserin-2a (ILGKLLSTAAGLLSNL.NH(2)) is a cationic, amphipathic α-helical cell-penetrating peptide, first isolated from skin secretions of the midwife toad Alytes obstetricans. Structure-activity relationships were investigated by synthesizing analogs of alyteserin-2a in which amino acids on the hydrophobic face of the helix were replaced by L-tryptophan and amino acids on the hydrophilic face were replaced by one or more L-lysine or D-lysine residues. The Trp-containing peptides display increased cytotoxic activity against non-small cell lung adenocarcinoma A549 cells (up to 11-fold), but hemolytic activity against human erythrocytes increases in parallel. The potency of the N15K analog against A549 cells (LC(50) = 13 μM) increases sixfold relative to alyteserin-2a and the therapeutic index (ratio of LC(50) for erythrocytes and tumor cells) increases twofold. Incorporation of a D-Lys(11) residue into the N15K analog generates a peptide that retains potency against A549 cells (LC(50) = 15 μM) but whose therapeutic index is 13-fold elevated relative to the native peptide. [G11k, N15K] alyteserin-2a is also active against human hepatocarcinoma HepG2 cells (LC(50) = 26 μM), breast adenocarcinoma MDA-MB-231 cells (LC(50) = 20 μM), and colorectal adenocarcinoma HT-29 cells (LC(50) = 28 μM). [G11k, N15K] alyteserin-2a, in concentrations as low as 1 μg/mL, significantly (P < 0.05) inhibits the release of the immune-suppressive cytokines IL-10 and TGF-β from unstimulated and concanavalin A-stimulated peripheral blood mononuclear cells. The data suggest a strategy of increasing the cationicity while reducing the helicity of naturally occurring amphipathic α-helical peptides to generate analogs with improved cytotoxicity against tumor cells but decreased activity against non-neoplastic cells.Amino Acids 09/2012; · 3.91 Impact Factor
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ABSTRACT: Breast cancer is the leading cause of cancer-related deaths in women. Altered cellular functions of cancer cells lead to uncontrolled cellular growth and morphological changes. Cellular biomembranes are intimately involved in the regulation of cell signaling; however, they remain largely understudied. Phospholipids (PLs) are the main constituents of biological membranes and play important functional, structural and metabolic roles. The aim of this study was to establish if patterns in the PL profiles of mammary epithelial cells and breast cancer cells differ in relation to degree of differentiation and metastatic potential. For this purpose, PLs were analyzed using a lipidomic approach. In brief, PLs were extracted using Bligh and Dyer method, followed by a separation of PL classes by thin layer chromatography, and subsequent analysis by mass spectrometry (MS). Differences and similarities were found in the relative levels of PL content between mammary epithelial and breast cancer cells and between breast cancer cells with different levels of aggressiveness. When compared to the total PL content, phosphatidylcholine levels were reduced and lysophosphatydilcholines increased in the more aggressive cancer cells; while phosphatidylserine levels remained unchanged. MS analysis showed alterations in the classes of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, and phosphatidylinositides. In particular, the phosphatidylinositides, which are signaling molecules that affect proliferation, survival, and migration, showed dramatic alterations in their profile, where an increase of phosphatdylinositides saturated fatty acids chains and a decrease in C20 fatty acids in cancer cells compared with mammary epithelial cells was observed. At present, information about PL changes in cancer progression is lacking. Therefore, these data will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for therapy.Breast Cancer Research and Treatment 10/2011; 133(2):635-48. · 4.47 Impact Factor