Putative probiotics decrease cell viability and enhance chemotherapy effectiveness in human cancer cells: role of butyrate and secreted proteins.

Recent advances have highlighted probiotic role in preventing colorectal cancer, by promoting differentiation, inhibiting proliferation, and inducing apoptosis in colonocytes. Here, three ascertained probiotics (L. rhamnosus GG ATCC 53103, L. reuterii DSM 17938 and L. johnsonii LC1) and four food-isolated putative probiotics (L. plantarum S2, L. plantarum O2, L. pentosus S3, L. rhamnosus 14E4) were investigated for their ability to adhere to HT29 cancer cells and to inhibit their and the chemoresistant counterpart (HT29-dx cells) proliferation. Three putative probiotics (S2, S3 and 14E4) were able to decrease viability of both sensitive and chemo-resistant HT-29 cells. Supposing this effect related to secreted metabolites (namely short chain fatty acids (SCFA), exopolysaccharides (EPS) and extracellular proteins) we tested the efficacy of extracellular extracts and butyrate with or without the chemotherapeutic agent doxorubicin (DOXO) (10 µM, 4 h). Increased production of mitochondrial reactive oxygen species (ROS) in HT29 and HT29-dx cells was observed. Moreover, cell exposure to DOXO (10 µM, 24 h) and extracellular extracts (48 h) reduced cell viability. Comparative phenotypic and secretome analyses on the effective/non effective strains, revealed quantitative/qualitative differences in EPS content and protein profiles, suggesting that P40, phage-tail-like and capsid-like proteins may be also involved. These results suggest that food-isolated bacteria releasing bioactive compounds (butyrate, EPS and peculiar proteins) may control cancer cell proliferation and improve their response to chemotherapy.

Cryopreservation effects on Wharton's Jelly Stem Cells proteome.

Cryopreservation is the only method for long-term storage of viable cells and tissues used for cellular therapy, stem cell transplantation and/or tissue engineering. However, the freeze-thaw process strongly contributes to cell and tissue damage through several mechanisms, including oxidative stress, cell injury from intracellular ice formation and altered physical cellular properties. Our previous proteomics investigation was carried out on Wharton's Jelly Stem Cells (WJSCs) having similar properties to adult mesenchymal stem cells and thus representing a rich source of primitive cells to be potentially used in regenerative medicine. The aim of the present work was to investigate molecular changes that occur in WJSCs proteome in different experimental conditions: fresh primary cell culture and frozen cell. To analyze changes in protein expression of WJSCs undergoing different culturing procedures, we performed a comparative proteomic analysis (2DE followed by MALDI-TOF MS/MS nanoESI-Q-TOF MS coupled with nanoLC) between WJSCs from fresh and frozen cell culturing, respectively. Frozen WJSCs showed qualitative and quantitative changes compared to cells from fresh preparation, expressing proteins involved in replication, cellular defence mechanism and metabolism, that could ensure freeze-thaw survival. The results of this study could play a key role in elucidating possible mechanisms related to maintaining active proliferation and maximal cellular plasticity and thus making the use of WJSCs in cell therapy safe following bio-banking.

Proteomic analysis of the carotid body: a preliminary study.

We present a proteomic analysis of the rat carotid body (CB) preparation by comparison between normoxia and hypoxia. Proteomic investigation would be helpful to identify the stress-induced protein during hypoxia and to know what O(2) species are being sensed by CB cells. Adult Wistar rats were used, one group was kept in room air (21% O(2)) as control, and the other was kept in a Plexiglas chamber for 12 days in chronic hypoxia (10-11% inspired oxygen). A total protein extract for each lysated tissue was separated using a broad pH range no-linear IPG strip (3-10) and the second dimension was performed on a 9-16% polyacrylamide gel. Exposure to hypoxia for 12 days produced significant changes in protein expression, providing an initial insight into the mechanism underlying differences in susceptibility to hypoxia. Further investigation is needed to have an overview of the specific set of proteins present in the CB and the functions of such proteins in signal transduction and adaptation during hypoxia.

Effects of beta-naphthoflavone, phenobarbital and dichlobenil on the drug-metabolizing system of liver and nasal mucosa of Italian water frogs.

In this study, we have examined the presence and inducibility of phase I and II drug-metabolizing enzymes in the liver and nasal mucosa of Italian water frogs of control and pretreated with beta-naphthoflavone, phenobarbital and dichlobenil by using typical substrates for these enzymes along with polyclonal antibodies mainly raised against mammalian enzymes. The CYP content and various monooxygenase and phase II enzyme activities in the liver of this frog were found similar, when reported, to those of largely aquatic and semiaquatic frogs. The treatment with beta-naphthoflavone resulted in an induction in the liver of a CYP1A and the induction was manifested by (a) immunoblot analysis using anti-rat CYP1A1, (b) an increase of CYP1A-mediated methoxyresorufin-O-demethylase and ethoxyresorufin-O-deethylase activities. The treatments with both phenobarbital and dichlobenil did not produce in the liver any effect on the assayed enzymes. When the nasal mucosa of water frogs was analyzed, various monooxygenase and phase II enzymatic activities, generally comparable to those of liver, were determined. However, by using antibodies anti-three GST different classes, we found a different reactivity into the cytosol of the two tissues indicating a differential tissue susceptibility to toxic effects of xenobiotics. In the nasal mucosa, a protein immunorelated to CYP2A and monooxygenase activities (i.e. ethoxycoumarin-O-deethylase and coumarin-7-hydroxylase) linked in mammals to this isoform have also been found. The treatment of water frogs with the herbicide dichlobenil decreased both the above-mentioned activities and the immunoreactive CYP2A apoprotein. The pretreatment with metyrapone, a CYP inhibitor, protected the CYP2A apoprotein and its linked activities from toxic effect of dichlobenil indicating a key role of this enzyme in the bioactivation of this herbicide. The findings of the present work suggest that the hepatic CYP1A induction and the nasal CYP2A-like inhibition profiles might provide two potential biomarkers of the Italian water frogs exposure to environmental and aquatic pollutants.

Purification and characterization of glutathione transferases from the sea bass (Dicentrarchus labrax) liver.

Two forms of glutathione transferase were purified from liver cytosol of the sea bass (Dicentrarchus labrax) by GSH-Sepharose affinity chromatography followed by chromatofocusing. The major enzyme (DL-GST-6.7; 75% of total activity bound to the column) has a pI value of 6.7 and is composed of two subunits of apparent molecular mass 26.5 kDa. The minor enzyme (DL-GST-8.2; 25% of total activity bound to the column) has a pI value of 8.2 and is composed of two subunits of molecular mass 23.5 kDa. Both isoenzymes appear to have blocked N-terminal. The purified proteins were characterized with respect to substrate specificity, CD spectra, TNS binding properties (with 2-toluidinylnaphthalene 6-sulfonate), and immunological reactivity. Partial internal amino acid sequence was also determined for each isoenzyme. The results obtained suggest that DL-GST-6.7 and DL-GST8.2 are novel GSTs belonging, respectively, to theta and alpha classes.

Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi.

The gene coding for a novel glutathione S-transferase (GST) has been isolated from the bacterium Ochrobactrum anthropi. A PCR fragment of 230 bp was obtained using oligonucleotide primers deduced from N-terminal and 'internal' sequences of the purified enzyme. The gene was obtained by screening of a genomic DNA partial library from O. anthropi constructed in pBluescript with a PCR fragment probe. The gene encodes a protein (OaGST) of 201 amino acids with a calculated molecular mass of 21738 Da. The product of the gene was expressed and characterized; it showed GST activity with substrates 1-chloro-2, 4-dinitrobenzene (CDNB), p-nitrobenzyl chloride and 4-nitroquinoline 1-oxide, and glutathione-dependent peroxidase activity towards cumene hydroperoxide. The overexpressed product of the gene was also confirmed to have in vivo GST activity towards CDNB. The interaction of the recombinant GST with several antibiotics indicated that the enzyme is involved in the binding of rifamycin and tetracycline. The OaGST amino acid sequence showed the greatest identity (45%) with a GST from Pseudomonas sp. strain LB400. A serine residue in the N-terminal region is conserved in almost all known bacterial GSTs, and it appears to be the counterpart of the catalytic serine residue present in Theta-class GSTs. Substitution of the Ser-11 residue resulted in a mutant OaGST protein lacking CDNB-conjugating activity; moreover the mutant enzyme was not able to bind Sepharose-GSH affinity matrices.

Glyoxalases activity during Bufo bufo embryo development.

In this work we have investigated the expression of glyoxalase I (GLO I) and glyoxalase II (GLO II) activities during Bufo bufo embryo development and in some tissues of both male and female adult animals, in order to study how they correlate with cell proliferation and differentiation. The results show that both the activities are expressed at significant levels from the earliest developmental stages, reaching the highest values at the end of embryonic development (stage 25). The GLO I/GLO II ratio is very high at the beginning of the development and then gradually decreases as the development goes on. These data emphasize the importance of GLO I activity in the phases in which elevated cell division is taking place. In the differentiated tissues, a peculiar sexual dimorphism in both GLO I and GLO II activities, with higher values in female than in male, was found. GLO I embryonic activity levels are comparable to those found in female differentiated tissues, but significantly higher than those detected in male differentiated tissues. On the contrary, the GLO II activities found in the adult tissues were always higher than those found in embryos. These results further support the idea that high GLO I/GLO II ratios are a characteristic of the proliferative status, which assures a good scavenging action against the potentially cytotoxic and cytostatic effect of methylglyoxal.

Spatial distribution of glutathione, glutathione-related and antioxidant enzymes in cultured mouse embryos.

The present study was undertaken to evaluate the detoxifying capacity of organogenesis-stage murine concepti cultured in vitro. Investigative attention was particularly focused on the embryonic tissue distribution of cytoprotective pathways. Glutathione (GSH) status, GSH-related and antioxidant enzymes were assayed in the embryo proper (EP), visceral yolk sac (VYS) and ectoplacental cone (EC) of 29.44 +/- 1.56 (mean +/- SD) somite pairs concepti. All the tissues displayed significant and comparable concentrations of GSH, further supporting this tripeptide as critical in protection against embryotoxicants. The totality of enzymatic activities was detectable in the selected embryonic compartments. In terms of spatial distribution analysis, maximal activities were found in EC (glutathione peroxidase, glutathione reductase, superoxide dismutase and glyoxalase I and II), and VYS (glutathione transferase and catalase). These results indicate: (1) the organogenesis-stage conceptus, in addition to significant amounts of GSH, expresses constitutive activities of GSH-related and antioxidant enzymes; (2) maximal activity levels are detectable in the embryonic sites which, at the developmental stage selected for assay, serve (VYS) or are evolving to serve (EC) embryo/maternal exchange, and thus represent the primary sites of interaction with foreign compounds.

Purification and characterization of three Pi class glutathione transferase from monkey (Macaca fascicularis) placenta.

Three forms of glutathione transferase (GST) with an apparent isoelectric point of pH 4.65 (GST I), 4.75 (GST II) and 4.9 (GST III) were resolved from the monkey (Macaca fascicularis) placenta after GSH-affinity chromatography followed by chromatofocusing. Substrate specificity, immunological reactivity, as well as N-terminal aminoacid sequences indicate that the three enzymes belongs to the pi class of GST. Reverse phase HPLC analysis indicates that the three GST arise from the combination of two different subunits eluting respectively at 29.60 +/- 0.10 min and 32.43 +/- 0.13 min. GST I is an homodimer of the 29.60 +/- 0.10 min subunit, GST III is an homodimer of the 32.43 +/- 0.13 min subunit, whereas the GST II is an heterodimer of the 29.60 +/- 0.10 min and 32.43 +/- 0.13 min subunits. Our results strongly suggest that unlike human, multiple forms of pi class GST exist in monkey placenta.

Interaction of glutathione transferase P1-1 with captan and captafol.

Glutathione transferase (GST, EC 2.5.1.18) P1-1 was strongly inhibited by captan and captafol in a time- and concentration-dependent manner. The IC50 values for captan and captafol were 5.8 microM and 1.5 microM, respectively. Time-course inactivation of GSTP1-1 by two pesticides was prevented by 3 microM of hexyl-glutathione, but not by methylglutathione. The fact that the inactivated enzyme recovered all the 5,5'-dithiobis(2-nitrobenzoic acid) titrable thiol groups, with concomitant recovery of all its original activity after treatment with 100 microM dithiothreitol, suggested that captan and captafol were able to induce the formation of disulfide bonds. That the inactivation of GSTP1-1 by captan and captafol involves the formation of disulfide bonds between the four cysteinil groups of the enzymes was confirmed by the SDS-PAGE experiments on nondenaturant conditions. In fact, on SDS-PAGE, GSTP1-1 as well as the cys47ala, cys101ala, and cys47ala/cys101ala GSTP1-1 mutants treated with captan and captafol showed several extra bands, with apparent molecular masses higher and lower than the molecular mass of native GSTP1-1 (23.5 kDa), indicating that both intra- and inter-subunit disulfide bonds were formed. These extra bands returned to the native 23.5 kDa band with concomitant restoration of activity when treated with dithiothreitol.

Alteration of glutathione transferase subunits composition in the liver of young and aged rats submitted to hypoxic and hyperoxic conditions.

In the present work, we have studied glutathione transferase (GST) activity and GST subunits distribution in the liver of young and aged rats kept under hypoxic or hyperoxic normobaric conditions as model of oxidative stress. A significant decrease of GST activity was detected in young hypoxic rat liver, whereas a significant increase occurred in aged hypoxic liver. No significant alteration of activity was obtained in both young and aged rat livers subjected to hyperoxic treatment. Substrate specificity measurements, SDS/PAGE analysis and reverse-phase HPLC, of GSH-affinity purified fractions were used to study the changes in the GST subunits pattern occurring in the liver of rat as a consequence of hypoxic and hyperoxic treatment. The results demonstrate that young and aged rat liver has a different constitutive GST subunit pattern which are markedly and differentially altered in hypoxia or hyperoxia. The hyperoxic treatment caused an increase of GST subunit 3 in aged, but not in young liver. In aged liver, both the hypoxic and hyperoxic treatment produced a decrease of GST subunit 4. After hypoxic treatment GST subunit 3 significantly increased in both young and aged liver. GST subunit 1a increased in both young and adult liver after hyperoxia. Following hypoxia a decrease of subunit 1a was seen in both young and aged liver. After hypoxic treatment, subunit 6 doubled in young, but not in aged, livers. It was concluded that the alterations in GST subunit expression occurring in the liver as a consequence of hypoxic or hyperoxic treatment respond to the necessity of a better protection of liver against the products of oxidative metabolism.

Analysis by limited proteolysis of domain organization and GSH-site arrangement of bacterial glutathione transferase B1-1.

Limited proteolysis method has been used to study the structure-function relationship of bacterial glutathione transferase (GSTB1-1). In absence of three-dimensional structural data of prokaryote GST, the results represent the first information concerning the G-site and domains organization of GSTB1-1. The tryptic cleavages occur mainly at the peptide bonds Lys35-Lys36 and Phe43-Leu44, generating two major molecular species of 20-kDa, 3-kDa and traces of 10-kDa. 1-chloro-2,4-dinitrobenzene favoured the proteolysis of the 20-kDa fragment markedly enhancing the production of the 10-kDa peptide by cleaving the chemical bonds Lys87-Ala88 and Arg91-Tyr92. The tryptic cleavage sites of GSTB1-1 was found to be located close to those previously found for the mammalian GSTP1-1 isozyme. It was concluded that despite their low sequence homology (18%), GSTB1-1 and GSTP1-1 displayed similar structural features in their G-site regions and probably a common organization in structural domains.

Glyoxalase activities in tumor and non-tumor human urogenital tissues.

Glyoxalase I and glyoxalase II activities have been measured in human tumor and non-tumor samples of 15 kidneys, 15 bladders, 4 testes, 2 adrenals as well as in 4 samples of prostatic adenomas. In all tissues examined glyoxalase I and glyoxalase II activity values varied widely from one patient to another. No significant difference in glyoxalase I activity between the tumor and non-tumor samples was found. When comparison was made between normal and neoplastic tissues of the same patients, glyoxalase I activity was found to be lower in tumor tissues of 10 out of 15 kidneys, and 2 out of 8 bladders and 1 out of 3 testes. A significant (P < 0.004) decrease of glyoxalase II activity was found only in tumor kidney. The possibility of using the present data to predict the relative sensitivity of human tumor tissues to glyoxalase-related chemotherapy is discussed.

Glutathione peroxidase and glutathione reductase activities in cancerous and non-cancerous human kidney tissues.

Selenium-dependent (Se-GSH-Px), selenium-independent (non-Se-GSH-Px) glutathione peroxidase and glutathione reductase (GSSG-Rx) activities have been determined in cancerous and non-cancerous human adult kidney. Large inter-individual variation in the activities of all enzymes tested were found in both tumour and non-tumour specimens. In general a significant decrease in the activities of the three enzymes was found in tumours. When a comparison was made between cancerous and non-cancerous tissues of the same individual, Se-GSH-Px activity was found to be lower in tumour in 17 cases out of 29, and the non-Se-GSH-Px activity in 20. In 20 cases out of 29 GSSG-Rx was found to be lower in tumour. It was concluded that changes in the factors involved in the anti-oxidative protection actually occur in human kidney tumour.

Structural and functional properties of the 34-kDa fragment produced by the N-terminal chymotryptic cleavage of glutathione transferase P1-1.

Limited proteolysis of glutathione transferase P1-1 (GSTP1-1) by chymotrypsin generates a 34-kDa GSTP1-1 fragment (a dimer of the 17-kDa subunit composed by residues 48-207) containing the whole C-terminal domain and a part (about 15%) of the N-terminal domain (residues 48-76, i.e., the structural elements beta 3, beta 4, and alpha C). The structural and functional properties of this large fragment have been investigated by analyzing its binding properties to 2-p-toluidinylnaphthalene-6-sulfonate (TNS) extrinsic probe, the TNS displacement technique, and the molecular modeling approach. The results obtained indicated that the 34-kDa GSTP1-1 fragment maintains an hydrophobic pocket with the same structural properties of the corresponding GSTP1-1 hydrophobic binding site. In addition, the 34-kDa GSTP1-1 binds a number of hydrophobic compounds such as 1-chloro-2,4-dinitrobenzene, hemin, and bilirubin with the same affinity of the native enzyme. Being structurally and functionally autonomous, this fragment, mostly constituted by domain II, appears as an independent folding unit in the protein. Nevertheless, in the entire native protein, interdomain interactions occur and are responsible for the major solvent exposure of the H-site in the presence of glutathione.

Time-dependent and tissue-specific variations of glutathione transferase activity during gestation in the mouse.

Glutathione transferases (GSTs; EC. 2.1.5.18) activity was measured in maternal liver and conceptal tissues during gestation. In maternal liver, maximum activity was found at gestational day (GD) 9 after which it slowly decreased up to the end of gestation. The placental GSTs activity at GD18 was three times lower than that found at GD14. Conversely, fetal liver GSTs at GD14 was about 75% that at GD18. It was also observed that GSTs activity at GD9 and GD10 was higher in visceral yolk sac than in embryo proper. Substrate specificity measurements, SDS PAGE analysis and HPLC runs, carried out on GSH-affinity purified fractions, revealed that with the progress of gestation in maternal liver an increase in pi class GSTs subunit occurs, with a concomitant decrease in alpha class GSTs. With respect to the time of gestation, a significant change in alpha, mu and pi class GSTs expression also occurred in fetal liver and in chorioallantoic placenta. It was concluded that during gestation the GSTs system is subjected to a time-dependent and tissue-specific modulation which may play a protective role against developmental toxicants.

Immunogold localization of glutathione transferase B1-1 in Proteus mirabilis.

By using the immunolabelling technique, the cellular localization of glutathione transferase in Proteus mirabilis was investigated. Evidence was obtained indicating a significant higher content of glutathione transferase in the periplasmic than cytoplasmic space. This result further support the idea that bacterial glutathione transferase is involved in xenobiotic detoxication.

Glutathione transferase isoenzymes in olfactory and respiratory epithelium of cattle.

Glutathione transferase (GST) was investigated in the olfactory and respiratory epithelium of cattle. A significantly more abundant GST in terms of either protein amount or activity was found in the olfactory rather than in the respiratory epithelium. No apparent qualitative differences in the isoelectric focusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC profiles were noted in the reduced glutathione (GSH) affinity purified GST pool of olfactory and respiratory epithelium. Both tissues have at least six GST isoenzymes with isoelectric point values of 4.9 (peak I), 5.3 (peak II), 5.95 (peak III), 6.5 (peak IV), 7.1 (peak V) and 9.3 (peak VI). From both tissues at least seven different GST subunits can be resolved by HPLC analysis. The GST isoenzymes having pI at 5.3 and 9.3 were predominantly expressed in the olfactory than in the respiratory epithelium. These latter forms conjugate GSH efficiently with alkenals and hydroperoxides, respectively. Kinetic, immunological and structural properties, including HPLC analysis and N-terminal region amino acid sequence seem to indicate that the bovine nasal mucosa tissue in addition to a GST subunit which is orthologue to rat subunit 8 (alpha class) express tissues specific subunits.

Developmental aspects of Bufo bufo embryo glutathione transferases.

The expression of glutathione transferase isoenzymes has been studied during the development of Bufo bufo embryo. By analysing the GSH-affinity purified materials in terms of substrate specificities, SDS-PAGE pattern, HPLC elution profile, we conclude that, up to stage 22, no significant changes in the expression of glutathione transferases isoenzymes occurred during Bufo bufo embryo development. At stage 25 the distribution of glutathione transferases was found to be slightly different from those of all other foregoing stages. A marked decrease of embryonic glutathione transferases subunits with a parallel appearance of new structurally and immunologically different subunits was noted in toad liver and kidney. Toad ovary continued to express embryonic glutathione transferase subunits.

Purification and characterization of the major glutathione transferase from adult toad (Bufo bufo) liver.

Five forms of glutathione transferase (GST) were resolved from the cytosol of adult common toad (Bufo bufo) liver by GSH-affinity chromatography followed by isoelectric focusing. The major enzyme (GST-7.64; 55% of total activity bound to the column) has a pI value of 7.64, is composed of two subunits each with a molecular mass of 23 kDa, and has the N-terminal amino acid residue blocked. GST-7.64 has also been characterized with respect to amino acid composition, substrate specificity, inhibition characteristics, c.d. spectra and immunological reactivity. The N-terminal sequence of some peptides obtained after tryptic digestion has also been determined. All together the results obtained suggest that the major toad liver GST is distinct from any known GST, including microbial, plant and mammalian GSTs.