Interaction of vaginal Lactobacillus strains with HeLa cells plasma membrane.

Vaginal lactobacilli offer protection against recurrent urinary and vaginal infections. The precise mechanisms underlying the interaction between lactobacilli and the host epithelium remain poorly understood at the molecular level. Deciphering such events can provide valuable information on the mode of action of commensal and probiotic bacteria in the vaginal environment. We investigated the effects exerted by five Lactobacillus strains of vaginal origin (Lactobacillus crispatus BC1 and BC2, Lactobacillus gasseri BC9 and BC11 and Lactobacillus vaginalis BC15) on the physical properties of the plasma membrane in a cervical cell line (HeLa). The interaction of the vaginal lactobacilli with the cervical cells determined two kinds of effects on plasma membrane: (1) modification of the membrane polar lipid organisation and the physical properties (L. crispatus BC1 and L. gasseri BC9); (2) modification of α5ß1 integrin organisation (L. crispatus BC2, L. gasseri BC11 and L. vaginalis BC15). These two mechanisms can be at the basis of the protective role of lactobacilli against Candida albicans adhesion. Upon stimulation with all Lactobacillus strains, we observed a reduction of the basal oxidative stress in HeLa cells that could be related to modifications in physical properties and organisation of the plasma membrane. These results confirm the strictly strain-specific peculiarities of Lactobacillus and deepen the understanding of the mechanisms underlying the health-promoting role of this genus within the vaginal ecosystem.

Modulation of apoptotic signalling by 9-hydroxystearic acid in osteosarcoma cells.

9-hydroxystearic acid (9-HSA) belongs to the class of endogenous lipid peroxidation by-products that greatly diminish in tumors, causing as a consequence the loss of one of the control mechanisms on cell division. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity. In this paper our attention has not only been focused on HDAC1 inhibition but also on the hyperacetylation of other substrates such as p53, that is involved in inducing cell cycle arrest and/or apoptosis, and whose activity and stability are known to be regulated by posttranslational modifications, particularly by acetylation at the C-terminus region. 9-HSA administration to U2OS, an osteosarcoma cell line p53 wt, induces a growth arrest of the cells in G2/M and apoptosis via a mitochondrial pathway. In particular hyperacetylation of p53 induced by the HDAC1 inhibitory activity of 9-HSA has been demonstrated to increase Bax synthesis both at the transcriptional and the translational level. The subsequent translocation of Bax to the mitochondria is associated to a significant increase in caspase 9 activity. Our data demonstrate that the effects of 9-HSA on U2OS correlate with posttranslational modifications of p53.

A new EGFR inhibitor induces apoptosis in colon cancer cells.

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, mAbs and EGFR tyrosine kinase inhibitors seemed to be the most promising. However they have demonstrated scarce utility in therapy, the former being effective only at toxic doses, the latter resulting inefficient in colon cancer. This paper presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphthoquinone core as shikonin, an agent with great anti-tumor potential. In HT29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 microM, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer.

Histone proteins determined in a human colon cancer by high-performance liquid chromatography and mass spectrometry.

The application of reversed-phase high-pressure liquid chromatography under gradient conditions and electrospray ion trap mass spectrometry (LC-ESI-MS) to the analysis of global modification levels of core histones is described. The optimised LC-ESI-MS method was applied for the first time to the characterisation of histones extracted from HT29, a human colon cancer cell line. Eight histones (H1-1, H1-2, H2A-1, H2A-2, H2B, H3-1, H3-2, H4) were separated on a C4 stationary phase with complete resolution, never reached in previous HPLC-MS methods, by using a gradient elution with the combined presence of heptafluorobutyric acid and formic acid as acidic modifiers in the mobile phase. Heptafluorobutyric acid was found to improve selectivity, whereas the presence of formic acid decreased ion suppression. Histones eluted from the column were detected with an ion trap mass spectrometer with an electrospray source. The peak averaged mass spectra were reconstructed by Mag Tran 1.0 software and the mass of the various isoforms of histones were derived. Method validation was conducted by performing the same sample analysis by coupling LC-ESI to a quadrupole-time-of-flight mass spectrometer (Q-TOF). The number of histone forms and their mass were found to differ not significantly from those obtained by ion trap mass spectrometer. Also the relative modifications abundance within the same histone type was found following the same trend as the two mass analysers. This method was then applied to the characterisation of changes in histone modification in HT29, never analysed by LC-MS before, treated with histone deacetylase inhibitors such as valproate and sodium butyrate, also used in preclinical trials as anticancer drugs. In particular, both the inhibitors produced a significant increase in H4 histone acetylated forms: 89% increase of the diacetyl dimethyl H4 form was observed with 1mM valproate supplementation, whereas 5 mM butyrate led to a 68% increase of the same form. Triacetyl monomethyl H4 (11,377 Da) and triacetyl dimethyl H4 (11,390 Da) were found only in cells treated with butyrate. Selective changes of H3 histone were detected with butyrate, in agreement with recently reported western blotting studies. Modifications in the H2A histone degree of acetylation were revealed by treatment of the cells with butyrate (H2A-1, H2A-2) and valproate (H2A-2). The results of the proposed methodology confirmed that inhibition of histone deacetylases caused histone hyperacetylation, responsible for decondensation and reorganization of interphase dynamic chromatin. This method resulted in selective and sensitive method to monitor variations in the acetylation and methylation state of histones after treatment of HT29 with inhibitors, and is therefore suitable for further application in new drug discovery for tumour therapy.

9-Hydroxystearic acid upregulates p21(WAF1) in HT29 cancer cells.

Growing evidence supports the critical role of lipid peroxidation products in the control of cell proliferation. In previous studies we demonstrated the efficient restriction of the proliferation rate in several cell lines resulting from the in vitro treatment with endogenous lipid polar components of cell membranes. Among these, 9-hydroxystearic acid (9-HSA), a primary intermediate of lipid peroxidation, induced a significant arrest in G0/G1 in HT29 colon cancer cells. In response to 9-HSA treatment of HT29 we observed cell growth arrest and increase in p21(WAF1) expression both at the transcriptional and the translational levels. Growth of p21(WAF1)-deleted HCT116 human colon carcinoma cells was not inhibited by 9-HSA. We present evidence that p21(WAF1) is required for 9-HSA mediated growth arrest in human colon carcinoma cells.

Cytotoxic and cytostatic effects induced by 4-hydroxynonenal in human osteosarcoma cells.

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Furthermore, numerous results demonstrate that lipid peroxidation products affect central biochemical pathways and intracellular signalling at physiological concentrations. 4-Hydroxynonenal (HNE) is one of the most active products of lipid peroxidation. This work has focused on the evaluation of HNE nuclear content, so far never directly measured, by electrospray-ionization-mass-spectrometry (ESI/MS) and on the correlation between its concentration and the induced effects after exogenous administration. In a human osteosarcoma cell line (SaOS2), HNE exhibited an early cytotoxic effect characterized by apoptosis, cytostatic and differentiating effects characterized by slow growth, increase in alkaline phosphatase (ALP), and alpha5 integrin subunit content with decrease in tumorigenicity.

Gas chromatography/mass spectrometric assay of endogenous cellular lipid peroxidation products: quantitative analysis of 9- and 10-hydroxystearic acids.

A sensitive, specific, accurate and reproducible gas chromatography/mass spectrometry method was developed for the assay of 9- and 10-hydroxystearic acids in samples obtained as cell extracts. The preparation of the samples required specific procedures to allow the analysis of both the free and the conjugated hydroxy acids as the corresponding methyl esters. The quantification used propyl-paraben as the internal standard and monitoring of a specific fragment of each isomeric hydroxy acid methyl ester, and allowed quantification of the conjugate and the free fractions of both 9- and 10-hydroxystearic acids. This method is suitable for identification and quantification (LOQ 1.8 and 4.4 ng, respectively) of these important metabolites of lipid peroxidation. In particular the development of an assay for the free 9-hydroxystearic acid methyl ester makes the method a reliable analytical tool for investigations of the role of this metabolite in the mechanisms of tumour cell proliferation.

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).

Chromosome aberrations, micronuclei, and activity of superoxide dismutases in human lymphocytes after irradiation in vitro.

The goal of this study was to provide data on the dose-dependent production of dicentrics and micronuclei in human lymphocytes irradiated with 22.6 MeV protons and to estimate the possible contribution of intracellular superoxide dismutases (SOD) to the relative biological effectiveness (RBE) of protons. For the dose-response study, heparinized whole blood of a healthy volunteer was irradiated with protons and X-rays employing radiation doses of 0.5-4 Gy. Three biological endpoints were analyzed: chromosomal aberrations, micronuclei, and specific activity of cytosolic (CuZnSOD) and mitochondrial (MnSOD) superoxide dismutases in harvested human blood cells. Dicentric dose-response curves fit a linear-quadratic form (alpha = 0.094 +/- 0.006, beta = 0.032 +/- 0.001) induced with X-rays and (alpha = 0.119 +/- 0.057, beta = 0.029 +/- 0.014) for 22.6 MeV protons. Protons were more effective than X-rays in producing exchanges, particularly at 0.5 and 1 Gy. In contrast to X-ray irradiated samples where a significant increase in the specific activity of MnSOD was recorded (up to a radiation dose of 1 Gy), irradiation with protons markedly reduced its activity. As a consequence of the reduced activity of MnSOD, the chromosomal dose-response curve became quadratic. The RBE for dicentrics varies with dose (from 2.2 to 1.01) and reduced activity of MnSOD is an important contributor to the RBE of protons. SODs, particularly MnSOD, play an important role in defending DNA from reactive oxygen species. A reduced activity of SOD, particularly MnSOD, is an important contributor to the RBE of protons.

Determination of catecholamines in human plasma by high-performance liquid chromatography with electrochemical detection.

In the present study, assays were improved for the determination of catecholamines in human plasma. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase, and the detection potential, was investigated. An accurate solid-phase extraction procedure, after catecholamine complexation with diphenylborate, was developed. The efficiency yield for all catecholamines was in the range 92-98%. Relative standard deviation values for repeatability and for intermediate precision were less than 2% and 3%, respectively, for all three analytes.

Determination of 4-hydroxy-2-nonenal at cellular levels by means of electrospray mass spectrometry.

trans-4-Hydroxy-2-nonenal (HNE) is an end-product of lipid peroxidation in biological systems which produces a variety of powerful biological effects. A method based on electrospray mass spectrometry was developed for the determination of 4-HNE at cellular levels. Quantification was carried out by using HNE-d(11) as internal standard; the mass chromatograms were acquired in the single ion monitoring mode (SIM) on the [M + H](+) monoisotopic species for HNE and HNE-d(11). With this approach a higher precision and lower detection limit and biological sample size than those typical of the methods so far employed are achieved. Furthermore the determination of the analyte from the cell extract is directly performed without the need of any HNE derivatization. As a first application the method was used to identify and quantify HNE in human T cell leukemia extracts.

Effect of hydrostatic pressure and cholesterol depletion on the expression of a tumor-associated antigen.

The molecular events related to the expression of three tumor-associated epitopes, Ca-MOv17, Ca-MOv18 and Ca-MOv19 have been addressed. The epitopes are carried by a 38-kDa glycoprotein (gp38), recently cloned and identified as a human folate-binding protein. They were found to be coexpressed on the surface of the ovarian carcinoma cell line OVCA432, while they are not coordinately expressed on other adenocarcinoma cell lines (IGROV1, HT-29). This lack of coexpression was investigated from a molecular point of view. We studied three carcinoma cell lines, characterized by a different reactivity with the three relevant monoclonal antibodies MOv17, MOv18 and MOv19. The epitope expression was examined after modifying the membrane properties by using hydrostatic pressure and/or the variation of cholesterol content. Measurement of the expression after cell labelling by mAbs was performed by indirect immunofluorescence, using both fluorescence microscopy and flow cytometry. At variance with HT-29 cells, treatment of ovarian carcinoma IGROV1 cells with hydrostatic pressure failed to exert any effect. On IGROV1, instead, cholesterol depletion affected the expression Ca-MOv17, increasing, in the indirect immunofluorescence tests, the proportion of positive cells from 0 to 66 +/- 9%. Moreover, restoring the cholesterol content of the plasma membrane did not reverse the induced epitope expression. In parallel, immunoprecipitation experiments confirmed that, on IGROV1 surface, gp38 was recognized by all three mAbs. The data presented suggest that in IGROV1 cells the selective lacking of the epitope expression is related to the physical state of the plasma membrane. An explanation is provided by the model of membrane microdomains in which epitope expression may be influenced by the cholesterol level of different plasma membrane regions.

Preparation of lucifer yellow fluorescent liposomes: A method for cells' membrane labeling.

This report describes a method to conjugate lucifer yellow to the external surface of liposomes. The heterobifunctional cross-linking reagentN-succinimidyl 3-(2-pyridyldithio)propionate has been used to activate DMPE molecules. The DMPE-dithiopyridine product has been mixed with DMPC to prepare liposome vesicles. These have been reduced by DTT and finally reacted with lucifer yellow-iodoacetamide to produce the fluorescence-labeled vesicles. The quenching of their fluorescence intensity by Kl is consistent with fully exposed fluorophores. The decay of the fluorescence intensity of the lipid-bound lucifer yellow is biexponential (τ1=7.9 ns; τ2=1.1 ns), with a relative yield of 0.16. When the fluorescent liposomes are mixed with cells, the lucifer yellow-DMPE derivative is transferred. Boar spermatozoa and peripheral human blood lymphocytes have been used as cellular models. The extent of incorporation is dependent on the incubation time and temperature. At 36°C, lucifer yellow fluorescence appears in the spermatozoa cells after 10 min of incubation and reaches its maximum at about 60 min. The fluorescent phospholipid derivative seems to incorporate specifically into membrane structures. The highest labeling ratio is observed with integer, scarcely motile, spermatozoa. A poorer labeling yield (≈15%) is found with lymphocytes. Interestingly, photobleaching due to epiillumination of the labeled cells is apparently negligible and cells are clearly visible after irradiation times ranging from several minutes to few hours.