Sodium butyrate-dependent sensitization of human colon adenocarcinoma COLO 205 cells to TNF-alpha-induced apoptosis.

COLO 205 colon adenocarcinoma cells are highly resistant to extrinsic apoptosis induced by immunomodulatory cytokines. One of the antiapoptotic mechanisms is the expression of cFLIP protein, which inhibits TNF-alpha-induced cell death. The use of metabolic inhibitors, such as sodium butyrate (NaBt), the potent repressor of histone deacetylase, sensitizes tumor cells to TNF-alpha-mediated apoptosis. The Western-blot analysis revealed that in COLO 205 cells the susceptibility to apoptogenic stimuli results from time-dependent reduction in cFLIP(L) protein assembled with DISC complex. At the same time, the level of transmembrane TNF-alpha receptor 1 (TNF-R1) was elevated which is consistent with the exaggerated rate of cell death. Since preincubation of COLO 205 cells with N-acetyl-L-cysteine (NAC), or sodium ascorbate (ASC) did not protect cells from combined NaBt- and TNF-alpha-induced apoptosis, we concluded that deletion of cancer cells is not evoked by oxidative stress. Our results suggest that the combination of TNF-alpha with NaBt targets antiapoptotic protein(s) and may provide efficient and non-toxic treatment of colon cancer.

Possible implications of redox-sensitive tumour cell transformation; lessons from cell culture studies.

It is generally accepted that chronic inflammatory disease, either local or generalized, is associated with higher incidence of cancer. Since inflammation is often accompanied by oxidative stress the latter was indicated as the foundation for progressive mutations leading to tumor development (proliferation, invasion, metastasis). Even though, it is very hard to demonstrate by in vitro studies the causal relationship between oxidative stress and cell transformations. From our studies it is clear that cells are more likely to stop divisions and they commit suicide by apoptosis. During last decade, a novel view on the origin of cancer emerged. The so called cancer stem cells (CSC) were found that form the side-population of stem cells (SC) and they are believed to initiate cancer. Are the SC ancestors for CSC? Do SC transform into CSC? These and other questions remain unanswered. We hypothesize that SC might undergo transformation into CSC during prolonged oxidative stress. We claim that several changes in cell biochemistry has to occur to start the molecular modifications leading to neoplasma. These include either hypoxia-promoted apoptosis signal inducing kinase 1 (ASK-1), hypoxia inducing factor 1 alpha (HIF-1alpha) and glycolysis, or normoxia-promoted activating protein-1 (AP-1) or hyperoxia-induced nuclear factor kappa B (NF-kappaB). Next, harsh microenvironment and heterogenous extracellular matrix (ECM) induced by oxidative stress accelerate the selection of clones of cells resistant to apoptogenic signal. HIF-1alpha, protein crucial for transcriptional activation of protooncogene met leads to the overexpression of c-Met receptor that in turn sensitizes cells to hepatocyte growth factor/scatter factor (HGF/SF) mitogen. Finally, both impaired function of mitochondria and hypoxia elevate fibrin protein level and amplify hemostasis as disseminated intracapillary coagulation (DIC). In any case, it is very interesting and remains to be answered whether imbalance in prooxidant-antioxidant homeostasis has causal relationship with transformation of SC to CSC.

Phase II metabolism of benzene.

The hepatic metabolism of benzene is thought to be a prerequisite for its bony marrow toxicity. However, the complete pattern of benzene metabolites formed in the liver and their role in bone marrow toxicity are not fully understood. Therefore, benzene metabolism was studied in isolated rodent hepatocytes. Rat hepatocytes released benzene-1,2-dihydrodiol, hydroquinone (HQ), catechol (CT), phenol (PH), trans-trans-muconic acid, and a number of phase II metabolites such as PH sulfate and PH glucuronide. Pretreatment of animals with 3-methylcholantrene (3-MC) markedly increased PH glucuronide formation while PH sulfate formation was decreased. Likewise, V79 cells transfected with the 3-MC-inducible rat UGT1.6 cDNA showed a considerable rate of PH and HQ glucuronidation. In addition to inducing glucuronidation of phenols, 3-MC treatment (reported to protect rats from the myelotoxicity of benzene) resulted in a decrease of hepatic CYP2E1. In contrast, pretreatment of rats with the CYP2E1-inducer isopropanol strongly enhanced benzene metabolism and the formation of phenolic metabolites. Mouse hepatocytes formed much higher amounts of HQ than rat hepatocytes and considerable amounts of 1,2,4-trihydroxybenzene (THB) sulfate and HQ sulfate. In conclusion, the protective effect of 3-MC in rats is probably due to a shift from the labile PH sulfate to the more stable PH glucuronide, and to a decrease in hepatic CYP2E1. The higher susceptibility of mice toward benzene may be related to the high rate of formation of the myelotoxic metabolite HQ and the semistable phase II metabolites HQ sulfate and THB sulfate.

In vitro metabolism of opioid tetrapeptide agonists in various tissues and subcellular fractions from rats.

The metabolism of three mu-selective opioid tetrapeptide agonists, Tyr-D-Arg-Phe-Nva-NH(2) (TArPN), Tyr-D-Arg-Phe-Phe-NH(2) (TArPP), and Tyr-D-Ala-Phe-Phe-NH(2) (TAPP), was investigated in different rat tissues. High metabolic activity (<20% peptide remaining after 30 min) was found against the three peptides in the kidney homogenate and against TArPN in spleen homogenate. Low metabolic activity (>80% peptide remaining after 30 min) was found for all peptides in brain homogenate and plasma, and for TArPN and TArPP in blood. The other tissue homogenates, prepared from the small and large intestine, liver and lung, all exhibited intermediate metabolic activity (20-80% peptide remaining after 30 min) against the peptides. In all tissues investigated, the tetrapeptides were metabolized at the C-terminal amide by deamidation.A further in depth metabolic investigation was performed in subcellular fractions isolated from three tissues (small intestine, liver and kidney). In the liver, the deamidation was predominantly localized to the mitochondrial/lysosomal fraction, while hydrolysis at the N-terminal Tyr residue was the major metabolic pathway in the microsomal/brush-border membrane fraction from the kidney and small intestine.

Sequencing and tissue distribution of the canine MRP2 gene compared with MRP1 and MDR1.

The effects of xenobiotic drugs and toxic compounds depend largely on their kinetic properties, which can be influenced by transmembrane drug transporters like MDR1/P-glycoprotein and the drug-conjugate transporters multidrug resistance protein (MRP) 1 and 2. As the dog is a preferential species used in the pharmacological and toxicological evaluation of new drugs, we sequenced the canine MRP2 cDNA and investigated its expression in various canine tissues compared with the related transporters MRP1 and P-glycoprotein. The tissue distribution pattern of these ABC-transporters differs partially from the distribution described in humans. So we found relatively high renal and low hepatic canine MRP2 expression levels, relatively high hepatic canine MRP1 expression levels, and quite high levels of MRP1 and P-glycoprotein in the dog brain. The knowledge of the tissue distribution pattern of these transporters will aid to interpret pharmacokinetic and toxicokinetic data gained from dog studies and to extrapolate them to humans.

Justification for antioxidant preconditioning (or how to protect insulin-mediated actions under oxidative stress).

Insulin resistance is characterized by impaired glucose utilization in the peripheral tissues, accelerated muscle protein degradation, impaired antioxidant defences and extensive cell death. Apparently, both insulin and IGF-1 at physiological concentrations support cell survival by phosphatidylinositol 3 kinase-dependent and independent mechanisms. Postprandial hyperglycemia and hyperinsulinemia are found in insulin resistance, which accompanies the so-called noninsulin dependent diabetes mellitus (diabetes type 2). Evidence also indicates that increased susceptibility of muscle cells and cardiomycoytes to oxidative stress is among the harmful complications of insulin resistance and diabetes. Limited knowledge showing benefits of preconditioning with anti- oxidants (vitamin C, E, a-lipoic acid, N-acetylcysteine) in order to protect insulin action under oxidative stress prompted the author to discuss the theoretical background to this approach. It should be stressed that antioxidant preconditioning is relevant to prevention of both diabetes- and insulin resistance-associated side-effects such as low viability and cell deletion. Furthermore, antioxidant conditioning promises to provide higher efficacy for clinical applications in myoblast transfer therapy and cardiomyoplasty.

Investigations of the in-vitro metabolism of three opioid tetrapeptides by pancreatic and intestinal enzymes.

The metabolism of three opioid tetrapeptides, Tyr-D-Arg-Phe-Nva-NH2, Tyr-D-Arg-Phe-Phe-NH2 and Tyr-D-Ala-Phe-Phe-NH2, was investigated in the presence of pure pancreatic enzymes (trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B), as well as in the presence of pure carboxylesterase and aminopeptidase N. The cleavage patterns of the pure pancreatic enzymes were then compared with those found in rat and human jejunal fluid. Metabolism was also studied in homogenates from different intestinal regions (duodenum, jejunum, ileum and colon) and in enterocyte cytosol from rats. The effect of various protease inhibitors was investigated in the jejunal homogenate. The parent peptides were assayed by high-performance liquid chromatography and metabolites were identified by means of liquid chromatography-mass spectrometry. Of the pure enzymes, the quickest hydrolysis of the peptides was observed for the pancreatic enzymes chymotrypsin, trypsin and carboxypeptidase A. In most cases they formed the corresponding deamidated tetrapeptides (chymotrypsin and trypsin) or tripeptides with a missing C-terminal amino acid (carboxypeptidase A). Regional differences in intestinal metabolism rates were found for all three peptides (P < 0.001), with the highest rates observed in jejunal and/or colonic homogenates. The deamidated tetrapeptides were formed both in rat intestinal homogenates and in enterocyte cytosol. Metabolism in the jejunal homogenate was markedly inhibited by some serine and combined serine and cysteine protease inhibitors. In conclusion, the C-terminal amide of these tetrapeptides did not fully stabilise them against intestinal deamidase and carboxypeptidase activities. The significant hydrolysis of the peptides by pure chymotrypsin, trypsin and carboxypeptidase A showed that lumenal pancreatic proteases might be a clear metabolic obstacle in oral delivery even for small peptides such as these tetrapeptides.

Immunohistochemical analysis of bFGF, TGF-beta1 and catalase in rectus abdominis muscle from cattle foetuses at 180 and 260 days post-conception.

The potential for muscle growth depends on myoblast proliferation, which occurs essentially during the first two thirds of the foetal period in cattle. Thereafter, myofibres acquire their contractile and metabolic properties. Proliferation is regulated by molecular growth factors and by the tissue oxidative activity. The aim of this study was the quantification by immunochemistry of basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-beta1) and also of enzyme catalase (CAT) activity in rectus abdominis muscle. Samples were collected from cattle foetuses of different growth potential at 180 and 260 days post-conception (dpc). One major conclusion from this work is that protein contents of the muscle tissue bFGF and, to a lower extent, CAT activity decreased with increasing age during the foetal life. No differences were found between the different genotypes of cattle. However, the CAT to bFGF ratio tended to be lower in fast-growing cattle and increased with foetal age. TGF-beta1 did not change with age and was localised mostly at the vascular bed. CAT was detected in smooth and rough reticulum in striated muscles at 180dpc, and additionally in mitochondria at 260dpc. In conclusion, the balance between intracellular growth factors (bFGF and TGF-beta1) and the activity of antioxidant enzyme CAT may participate in the regulation of the transition from myoblast proliferation to differentiation. Thus, increased ratio of CAT to bFGF might be a good index indicating initiation of muscle maturation in cattle foetus prior to birth.

Expression of apoptosis-related proteins in mammary gland of goat.

The expression of apoptosis-related proteins: TGF-beta1 (auto/paracrine inducer) and its receptor (TGF-betaRIII), Bax (promoter), Bcl-2 (inhibitor) and CPP-32 (executor of apoptosis) as well as the apoptotic cell number in mammary glands of 11 Polish White Improved goats in the course of the lactation cycle (peak of lactation: days 40-70, late lactation: days 208-256, drying off: days 267-340) was investigated. The immunohistochemical study demonstrated a significant increase in TGF-beta1 and TGF-betaRIII expression in the lobuloalveolar tissue from the early lactation to the dry period. Our recent study on HC11 mouse mammary epithelial cells [Cell. Mol. Biol. 46 (2000) 175] has revealed an inhibitory effect of prolactin on TGF-beta1 transcription, which may explain the low TGF-beta1 synthesis during lactogenesis and galactopoiesis and the increase in TGF-beta1 and TGF-betaIIIR expression in late lactation and dry period. Bax expression was the lowest in the peak of lactation, significantly increased in late lactation and remained elevated during drying off. Bcl-2 content was lower than Bax in all examined periods, but it increased significantly at the end of lactation, which suggests the survival of cells with the highest resistance to apoptogenic stimuli. The increase in Bcl-2 level in remnant lobuloalveolar tissue is probably the molecular mechanism that limits the rate of secretory tissue involution. The induction of CPP-32 (caspase 3) from the peak of lactation to dry period was accompanied by a progressive loss of mammary epithelial cells and the increase in apoptotic cell numbers but only in the dry period. The increase in the expression of examined proteins in the late lactation and the dry period indicates their involvement in the induction (TGF-beta1 and TGF-betaRIII), regulation (Bax and Bcl-2) and execution (CPP-32) of programmed cell death in the course of mammary gland involution. The lack of an increase in apoptotic cell number in late lactation, in spite of the evident decrease in total cell number, suggests milk as an alternative route (apart from phagocytosis) of apoptotic cells elimination from the mammary gland. The presented results provide new insights into the molecular mechanism of mammary cell apoptosis in goat and for this reason may have practical implications for control and regulation of mammary gland remodelling, which is a prerequisite for subsequent successful lactation.

Lipid rafts mediate epigallocatechin-3-gallate- and green tea extract-dependent viability of human colon adenocarcinoma COLO 205 cells; clusterin affects lipid rafts-associated signaling pathways.

Epigallocatechin-3-gallate (EGCG) is an important bioactive constituent of green tea extract (GTE) that was widely believed to reduce proliferation of many cancer cell lines. The purpose of this study was to verify the possible pro-apoptotic action of GTE/EGCG in human colon adenocarcinoma COLO 205 cells. The effect of EGCG/GTE treatments on cell viability was studied using methyl thiazolyl tetrazolium (MTT) assay. Cell proliferation was assessed with crystal violet staining, whereas protein expression levels were evaluated by western blotting followed by densitometric analysis. Obtained results were analyzed statistically. Surprisingly, EGCG/GTE dose-dependently up-regulated COLO 205 cells viability and proliferation. Observed effects were mediated by lipid rafts, as cholesterol depletion significantly prevented EGCG/GTE-dependent cell survival. Furthermore, treatment of COLO 205 cells with EGCG/GTE resulted in activation of MEK/ERK1/2, but not Akt1/2/GSK-3ß signaling pathway. The presence of MEK inhibitor - PD98059 but not PI3-K inhibitor - LY294002, both reduced EGCG/GTE-induced ERK1/2 activation and the proliferative effect of catechins. Furthermore, EGCG/GTE stimulated secretory clusterin (sClu) expression level, which underwent complex control through lipid rafts/PKC/Wnt/ß-catenin system. Our studies demonstrated that EGCG and GTE stimulate cell survival and proliferation of COLO 205 cells in a lipid rafts-dependent manner via at least MEK/ERK1/2 signaling pathway. Furthermore, EGCG/GTE mediated positive effects on viability and mitogenicity of COLO 205, while suppression of ß-catenin activity was positively correlated with sClu clusterin expression.

Cholesterol level determines viability and mitogenicity, but it does not affect sodium butyrate-dependent sensitization of Colo 205 cells to TNF-α-induced apoptosis.

Transient treatment of human adenocarcinoma COLO 205 cells with lipit raft (LR) modulators (MßCD, NY, IMP) was followed by the challenge with metabolic inhibitors and selected anti-cancer drugs. To overturn cholesterol chelation, the MßCD, NY treatment was followed by cholesterol conjugates (CHOL-MßCD or CHOL-PEG). The TNF-α- and P(Ser473)-PKB/Akt1/2-mediated effects initiated at LR were evaluated with regard to cell viability and mitogenicity. Cholesterol chelators reversibly reduced cell survival, whereas some of the tested compounds had weak effects (CIS, CLA), stimulated (EGCG) or reduced (NaB) cell survival. Cellular localizations of LR-associated molecules (ceramides, Gαi-2 heterotrimeric protein, and TNF-R1) in different cellular compartments including the plasma membrane were observed in the respective photographs from TEM and SEM. Evidence from SEM also showed that TNF-R1 is clustered on the surface of COLO 205 cells without presence of cognate ligand but clustering is promoted by TNF-α, while it vanished after IMP treatment. COLO 205 cells remained immune to TNF-α-induced apoptosis unless NaB was added, in which case NaB-induced cell death was further potentiated by TNF-α. Combined NaB and TNF-α treatment was associated with marked changes in the expression of pro- and antiapoptotic proteins. In this study, we demonstrated that initial excess of prosurvival signals could be diminished by cholesterol chelators, whereas LR-independent cell survival could be targeted by NaB. Apparently, lipid rafts do not participate in NaB-dependent cell death.

Bisindolylmaleimides in anti-cancer therapy - more than PKC inhibitors.

Bisindolylmaleimide derivatives were originally described as protein kinase C inhibitors. However, several studies have shown that bisindolylmaleimides target several other signaling molecules. The review presents bisindolylmaleimide-mediated PKC-dependent and PKC-independent biological effects, such as reversal of MDR and modulation of Wnt signaling through GSK-3b and b-catenin. Importantly, the potent proapoptotic properties of bisindolylmaleimides are also described. Bis-IX appears as the most efficient activator of intrinsic apoptotic pathway and additionally, facilitates extrinsic apoptosis. Presented molecular mechanisms indicate that bisindolylmaleimides could be useful agents in anticancer therapy. They repress uncontrolled proliferation and restore the sensitivity to chemotherapy which allows eradication of cancer cells.

Crossroads of cytokine signaling--the chase to stop muscle cachexia.

There is no universal approach to stop muscle cachexia in a number of life-threatening diseases. Accordingly, it is uncertain why the body mass is so critical to keep alive patients with cancer, congestive heart failure (CHF), AIDS or sepsis. At present, it is widely believed that excess muscle wasting diminishes lean body mass to the risky level accompanied by anorexia, anemia, lipolysis, acute phase response and insulin resistance. If missed and/or untreated muscle cachexia inevitably leads to death due to cardiac and respiratory failure (almost one-third of all cancer deaths). This complex metabolic disorder is suited by the elevated levels of inflammatory cytokines (TNF-alpha, IFN-gamma, IL-1-beta, IL-6, IL-2) and low levels of anti-inflammatory/ other cytokines (IL-15, leptin). Concurrently, tissue sensitivity to insulin is considerably reduced. Recent findings indicate that entirely few muscle-specific genes (i.e. MyoD and myosin heavy chain, MyHC) and their products must be targeted to initiate muscle wasting. Muscle atrophy occurs at different levels, starting from repressed gene expression and ended with accelerated protein degradation. Muscle growth (myogenesis) is severely compromised and disruption of sarcomere architecture heralds the proteolysis of contractile apparatus. This review aims to synthesize our present knowledge of intracellular mechanisms and molecular regulation of muscle cachexia with respect to cytokine signaling.

Molecular basis of sodium butyrate-dependent proapoptotic activity in cancer cells.

This review outlines the molecular events that accompany the antitumor action of sodium butyrate (NaBt). Butyrate, a low-molecular weight four-carbon chain volatile fatty acid (VFA) has been previously shown to withdraw cells from cell cycle or to promote cell differentiation, and finally to induce programmed cell death. Recent advances in molecular biology indicate, that this product of large bowel microbial fermentation of dietary fiber, might evoke the above-mentioned effects by indirect action on genes. NaBt was shown to inhibit histone deacetylase activity, allowing DNA binding of several transcription factors. Higher genomic activity leads to the higher expression of proapoptotic genes, higher level of their protein products and elevated sensitivity to death ligand-induced apoptosis. Cancer cells might be arrested in G1 phase of cell cycle in a p21-dependent manner. Proapoptotic activity of NaBt includes higher expression of membrane death receptors (DR4/5), higher level and activation of Smad3 protein in TGF-beta-dependent apoptotic pathway, lower level of antiapoptotic proteins (cFLIP, XIAP) and activation ofproapoptotic tBid protein. Thus, both intrinsic and extrinsic apoptotic pathways are stimulated to ampify the apoptotic signals. These effects are specific for tumor but not for regular cells. Unique properties of NaBt make this agent a promising metabolic inhibitor to retard tumorigenesis to suppress tumor growth.

Overview how adenocarcinoma cancer cells avoid immune- and chemotherapy-induced apoptosis.

This review describes some aspects of uncontrolled tumor growth and development. In the past, it has been shown that colon adenocarcinomas use several tactics to avoid cell deletion and to maintain cell viability. In particular, colorectal cancer cells resist death ligands-induced apoptosis by expressing anti-apoptotic proteins. By direct interaction with FADD, the FLIP protein inhibits the signal transmission from death receptors to their cytoplasmic targets in COLO 205 cells. Colorectal cancer cells also stimulate own survival by inhibiting cytotoxic signals induced by interferons. Moreover, IFN-alpha increases immune-resistance of colon cancer cells by activation of NF-kappaB. Additionally, the cytoplasmic retention of proapoptotic protein clusterin also supports viability of cancer cells. Upon suitable stimulation normal cells are featured by clusterin translocation to the nucleus with concomitant cell death. We found that proapoptotic activity of clusterin is dependent on calcium ions, and depletion of intracellular calcium caused extensive death of COLO 205 cells. Other type of strategy to inhibit chemotherapy-dependent cell death is the activity of multidrug resistance proteins (MDR). These cell membrane efflux pumps actively expel the drugs from the cell interior to prevent their action on intracellular targets. The reversal of P-gp efflux pump in chemoresistant COLO 320 cell line was observed upon phenothiazine derivatives. The variety of antiapoptotic mechanisms in colorectal cancer cells makes anticancer therapy a great challenge but detailed knowledge of their complexicity gives promise to sensitize cancer cells to death stimuli.

Cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis in human colorectal cancer cell line COLO 205; role of FLIP and metabolic inhibitors.

Current efforts are focused on revealing the cellular factors that determine the "immune escape" of cancer cells. As an example in our study human colorectal cancer cell line COLO 205 was resistant to TNF-alpha - a death inducing ligand. Nonetheless, co-incubation TNF-alpha (10 ng/ml) with cycloheximide (5 micro g/ml, CHX) caused time-dependent (6, 12, 24 hours) cell death even though, at that concentration cycloheximide did not exert cytotoxic effect unless 24 hour of treatment. After additional pretreatment it is concluded that CHX sensitizes cells to TNF-alpha-induced apoptosis. The aim of this study was to investigate the role and properties of cellular proteins that granted COLO 205 cells survival as well as to assess the effect of several metabolic inhibitors on cell viability at the above-mentioned time-points. PKCs inhibitor staurosporine (5 microM) did not influence, whereas cPKC activator PMA (100 microM) prevented TNF-alpha-induced apoptosis in the presence of CHX. Although EDTA (2 mM) and to a lesser degree EGTA (5 mM) were individually cytotoxic, they exerted protective effect at 6 and partially at 12 hour of combined TNF-alpha and CHX treatment. Ionophore A23187 (1 microM) protected cells against cell death at 12 and 24 but only partially at 6 hour of treatment. On the other hand, AD (10 ng/ml) acted synergistically with TNF-alpha and CHX at 6 and 12 hour. It appears that resistance of COLO 205 cells is determined on genomic level by a few reaction steps in which both Ca(2+)-dependent and antiapoptotic proteins are involved. Further studies revealed that CHX treatment reduces the level of FLIP but not other members of TNF-alpha-dependent death signalosome.

Position of STAT-1 alpha in cycloheximide-dependent apoptosis triggered by TNF-alpha in human colorectal COLO 205 cancer cell line; role of polyphenolic compounds.

"Immune escape" is a crucial instrument used by carcinoma cells to overcome numerous strategies of immune system to delete transformed cells. Cellular factors that make cancer cells immune to defence mechanisms are incompletely understood while some remain ambiguous. Up to date evidence points to some proteins and/or signaling molecules that might be a basis for unusual behavior of cancer cells. In particular STAT kinases are currently in the main focus of attention since they were both shown to accelerate and/or to inhibit apoptosis. In our studies we observed that human colorectal COLO 205 cancer cells were resistant to TNF-alpha- or cycloheximide-induced cytotoxicity. However, when TNF-alpha (10 ng/ml) has been given along with cycloheximide (5 micro g/ml, CHX) COLO 205 cells died extensively from apoptosis. Apparently, cycloheximide sensitized cells to TNF-alpha-induced programmed cell death. To investigate the role of STAT-1 alpha in CHX-mediated TNF-alpha-induced COLO 205 cell death certain polyphenolic compounds were studied if they modulate STAT-1 alpha phosphorylation status and STAT-1 alpha-protein interaction at the level of TNF-alpha signalosome in the 6(th), 12(th), and 24(th) hour of experiment. Neither of phenolic compound, namely PI-3K inhibitor (LY294002, 20 microM) nor MEK inhibitor (PD98059, 50 microM), nor flavonol quercetin or kaempferol (10, 100 microM) in contrast to apigenin (20 microM) influenced COLO 205 cell viability during individual or combined treatment with TNF-alpha and CHX. We conclude, that some antiapoptotic proteins were involved but not STAT-1 alpha kinase to resist TNF-alpha-dependent cell death promoting activity. Summing up, except apigenin, the above-mentioned polyphenolic compounds were unable to modulate survival signal in COLO 205 cells initially believed to be suppressed by STAT-1 alpha.

The relationship between urea and pyrimidine de novo synthesis in ruminant liver.

The influence of ammonia and inhibition of the urea cycle by norvaline on orotic acid synthesis in five Polish Merino ewes was investigated. The sheep were exposed to 60 min of successive infusions of 0.9% NaCl, NH4Cl (20 mumol kg-1 min-1), and NH4Cl with DL-norvaline (10 mumol kg-1 min-1) solutions administered to the mesenteric vein. The average rate of orotic acid output, urea output and ammonia uptake in the liver in control conditions (infusion of 0.9% NaCl) was: 1.09 nmol g-1 min-1, 0.97 mumol g-1 min-1 and 0.94 mumol g-1 min-1, respectively. Ammonia significantly stimulated the orotic acid output, urea output and ammonia uptake. At the same time liver uptake of glutamic acid, alanine and glycine increased. Norvaline as a competitive inhibitor of ornithine transcarbamylase depressed citrulline release and urea output in the liver, and elevated, although not significantly, the orotic acid output. It was concluded that ammonia stimulates the de novo synthesis of pyrimidine in the sheep liver but the participation in this effect of carbamyl phosphate generated in the urea cycle is limited.

[Antioxidative mechanisms in response to reactive forms of oxygen, apoptosis and differentiation of myogenic cells]. / Méchanizmy antyoksydacyjne w odpowiedzi na reaktywne formy tlenu a nekrobiologia i róznicowanie komórek miogennych.

In this paper regard was put on contemporary matters related to apoptosis of myogenic cells of different types (myocardium, visceral, skeletal muscle) and updated theories on the action mechanisms of free radicals and their contribution to this process. Concern was put on the activity of certain growth factors and antioxidants belonging to group of polyphenols on the extent of apoptosis in myoblasts held in culture. Similarities between the pathways depicting the process of muscle differentiation and resistance to programmed cell death are presented. The concept of myogenic cell differentiation and implications to apoptosis induced by particular oxidants is extensively discussed.