Michael adducts of ascorbic acid as inhibitors of protein phosphatase 2A and inducers of apoptosis

Michael adducts of ascorbic acid with alpha,beta-unsaturated carbonyl compounds have been shown to be potent inhibitors of protein phosphatase 1 (PP1) without affecting cell viability at the respective concentrations. Here we were able to show that higher concentrations can partially inhibit PP2A activity and concomitantly induce apoptotic cell death. A nitrostyrene adduct of ascorbic acid proved to be a more potent and effective inhibitor of PP2A as well as a stronger inducer of apoptosis. These adducts only slightly lost their cytotoxic potential in multidrug resistant cells that were 10-fold less sensitive to apoptosis induction by okadaic acid and vinblastine.     

15-Deoxy-Delta(12,14)-prostaglandin J(2) suppresses IL-6-induced STAT3 phosphorylation via electrophilic reactivity in endothelial cells

In this study, the effects of 15d-PGJ(2) were investigated in IL-6-activated endothelial cells (ECs). 15d-PGJ(2) was found to abrogate phosphorylation on tyr705 of STAT3 in IL-6-treated ECs, in a dose- and time-dependent manner, but did not inhibit serine phosphorylation of STAT3 and the upperstream JAK2 phosphorylation. Other PPAR activators, such as WY1643 or ciglitazone, had no effect upon IL-6-induced STAT3 phosphorylation. Additionally, neither orthovanadate nor l-NAME treatment reverses the inhibition of STAT3 phosphorylation by 15d-PGJ(2). Otherwise, the effect of 15d-PGJ(2) requires the alpha,beta-unsaturated carbonyl group in the cyclopentane ring. A 15d-PGJ(2) analog, 9,10-Dihydro-15d-PGJ(2), which lack alpha,beta-unsaturated carbonyl group showed no increase in ROS production and no effect in inhibition of IL-6-induced STAT3 phosphorylation. The electrophilic compound, acrolein, mimics the inhibition effect of 15d-PGJ(2). Among the antioxidants, only NAC and glutathione reversed the effects of 15d-PGJ(2). NAC, glutathione and DTT all reversed the inhibition of STAT3 phosphorylation when preincubated with 15d-PGJ(2). The inhibition of ICAM-1 gene expression by 15d-PGJ(2) was abrogated by NAC and glutathione in IL-6-treated ECs. Taken together, these results suggest that 15d-PGJ(2) inhibits IL-6-stimulated phosphorylation on tyr705 of STAT3 dependent on its own electrophilic reactivity in ECs.     

Induction of apoptosis dependent on caspase activities and growth arrest in HL-60 cells by PGA2

Prostaglandin (PG) A2 has been reported to inhibit the growth or induce apoptosis of various tumor cells. In the present study, PGA2 inhibited the growth of HL-60 cells and concomitantly-induced nuclear condensation and DNA fragmentation, characteristics of apoptosis. Down-regulation of c-myc mRNA, and activation of caspase-3 were observed in the PGA2 -treated cells. PGA2-induced DNA fragmentation was completely abolished in the presence of zVAD-Fmk or zDEVD-Fmk. But, relative cell survival was not improved up to that of untreated cells by pretreatment of caspase inhibitors, and c-myc down-regulation was not recovered by caspase inhibitors, either. Moreover, cytochrome c release and activation of caspase-9 was also observed in apoptotic cells and a specific inhibitor of caspase-9 (zLEHD-Fmk) prevented both DNA fragmentation and activation of caspase-3, but not relative cell survival, implying the upstream mitochondrial event of caspase-3 activation. In addition, antagonistic Fas antibody (ZB4) exerted no effect on the apoptosis. Taken together, these results suggest that PGA2 may induce the apoptosis as well as growth inhibition in HL-60 cells, and cytochrome c release and caspase activation seem to play a critical role in this apoptosis which might be independent or downstream of growth inhibition associated with c-myc down-regulation.     

Aureobasidium pullulans metabolism of prostaglandins of the A-type.

Aureobasidium pullulans, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA2 (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9 alpha or 9 beta-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9 alpha, 15 alpha-dihydroxy-2, 3, 4, 5-tetranor-13-trans-prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9 beta-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9, 15-oxo-2, 3, 4, 5-tetranorprostanoic acid and 9, 15-oxo-2, 3, 4, 5-tetranor-13-trans-prostenoic acid. The water soluble metabolites have not been characterized further. The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that A. pullulans can perform the following transformation: beta-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both delta 10,11 and delta 13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (delta 10, 11 leads to delta 11, 12). A. pullulans metabolizes 15-epimeric PGA2 equally readily with the production of similar products. PGA1 affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. A. pullulans displays some enantioselectivity, PGA2 and 15-epi-PGA2 are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized.     

Regulation of constitutive neutrophil apoptosis by the alpha,beta-unsaturated aldehydes acrolein and 4-hydroxynonenal

Reactive alpha,beta-unsaturated aldehydes are major components of common environmental pollutants and are products of lipid oxidation. Although these aldehydes have been demonstrated to induce apoptotic cell death in various cell types, we recently observed that the alpha,beta-unsaturated aldehyde acrolein (ACR) can inhibit constitutive apoptosis of polymorphonuclear neutrophils and thus potentially contribute to chronic inflammation. The present study was designed to investigate the biochemical mechanisms by which two representative alpha,beta-unsaturated aldehydes, ACR and 4-hydroxynonenal (HNE), regulate neutrophil apoptosis. Whereas low concentrations of either aldehyde (<10 microM) mildly promoted apoptosis in neutrophils (reflected by increased phosphatidylserine exposure, caspase-3 activation, and mitochondrial cytochrome c release), higher concentrations prevented critical features of apoptosis (caspase-3 activation, phosphatidylserine exposure) and caused delayed neutrophil cell death with characteristics of necrosis/oncosis. Inhibition of caspase-3 activation by either aldehyde occurred despite increases in mitochondrial cytochrome c release and occurred in close association with depletion of cellular GSH and with cysteine modifications within caspase-3. However, procaspase-3 processing was also prevented, because of inhibited activation of caspases-9 and -8 under similar conditions, suggesting that ACR (and to a lesser extent HNE) can inhibit both intrinsic (mitochondria dependent) and extrinsic mechanisms of neutrophil apoptosis at initial stages. Collectively, our results indicate that alpha,beta-unsaturated aldehydes can inhibit constitutive neutrophil apoptosis by common mechanisms, involving changes in cellular GSH status resulting in reduced activation of initiator caspases as well as inactivation of caspase-3 by modification of its critical cysteine residue.     

Fatty acid ketodienes and fatty acid ketotrienes: Michael addition acceptors that accumulate in wounded and diseased Arabidopsis leaves

Physical damage and disease are known to lead to changes in the oxylipin signature of plants. We searched for oxylipins produced in response to both wounding and pathogenesis in Arabidopsis leaves. Linoleic acid 9- and 13-ketodienes (KODEs) were found to accumulate in wounded leaves as well as in leaves infected with the pathogen Pseudomonas syringae pv. tomato (Pst). Quantification of the compounds showed that they accumulated to higher levels during the hypersensitive response to Pst avrRpm1 than during infection with a Pst strain lacking an avirulence gene. KODEs are Michael addition acceptors, containing a chemically reactive alpha,beta-unsaturated carbonyl group. When infiltrated into leaves, KODEs were found to induce expression of the GST1 gene, but vital staining indicated that these compounds also damaged plant cells. Several molecules typical of lipid oxidation, including malonaldehyde, also contain the alpha,beta-unsaturated carbonyl reactivity feature, and, when delivered in a volatile form, powerfully induced the expression of GST1. The results draw attention to the potential physiological importance of naturally occurring Michael addition acceptors in plants. In particular, these compounds could act directly, or indirectly via cell damage, as powerful gene activators and might also contribute to host cell death.     

Prostaglandins A and J arrest the cell cycle of cultured vascular smooth muscle cells without suppression of c-myc expression.

The effects of prostaglandins (PGs) A and J, which are anti-tumor eicosanoids, on the proliferation of cultured vascular smooth muscle cells were investigated. Serum-stimulated DNA synthesis was potently inhibited by PGA1, PGA2, PGJ2, and delta 12-PGJ2 in similar dose-dependent fashions. The effects of PGA1 and PGA2 were reversible when they were removed from the culture media, whereas recoveries were only partial in the cells treated with PGJ2 and delta 12-PGJ2. PGs were effective even if they were added immediately before entry into S phase. Inhibition of DNA synthesis was sustained when hydroxyurea, which blocks cell cycle at the G1/S border, was added after the removal of PGA2, and vice versa; PGs blocked DNA synthesis when they were added after the removal of hydroxyurea. Levels of c-myc mRNA formed two peaks during the G1 phase, at 1-2 h and at 8-12 h. The PGs did not affect the first elevation, but enhanced the second and sustained it up to 18-24 h, whereas in controls, c-myc mRNA decreased quickly after entry into S phase. The rate of degradation of c-myc mRNA was much smaller in PG-treated cells than in nontreated cells. We conclude, therefore, that PGA and PGJ inhibit a crucial event(s) in the cell cycle occurring at the G1/S border, but that this inhibition is not accompanied by the reduction in c-myc gene expression in contrast with some types of tumor cells treated with PGs.     

Induction of 68,000-dalton heat shock proteins by cyclopentenone prostaglandins. Its association with prostaglandin-induced G1 block in cell cycle progression

Cyclopentenone prostaglandins (PGs) such as PGA2 and delta 12-PGJ2 act specifically on cells in the G1 phase and induce block of cell cycle progression (Ohno, K., Sakai, T., Fukushima, M., Narumiya, S., and Fujiwara, M. (1988) J. Pharmacol. Exp. Ther. 245, 294-298). In this study, we characterized proteins induced by these PGs in HeLa S3 cells of synchronized growth and examined its association with the cell cycle block. HeLa S3 cells transiently expressed two 68-kDa proteins of isoelectric points of 5.5 and 5.6 in the G1 phase of cell cycle. When G1-enriched cells were incubated with either PGA2 or delta 12-PGJ2, synthesis of these proteins was markedly enhanced. Enhancement by delta 12-PGJ2 was persistent and irreversible, whereas that by PGA2 was reversible. delta 12-PGJ2 also enhanced the synthesis of two additional 68-kDa proteins with isoelectric points of 5.8 and 5.9. On two-dimensional gel electrophoresis, these proteins overlapped exactly with the 68-kDa heat shock proteins induced in cells treated at 43 degrees C for 90 min. They were also indistinguishable from the heat shock proteins in limited proteolysis. When delta 12-PGJ2 was incubated with G2/M phase cells, it induced only a small and transient increase in the 68-kDa proteins. These results suggest that cyclopentenone PGs extensively induce 68-kDa heat shock proteins in the G1 phase HeLa S3 cells and this induction is closely associated with the G1 block of cell cycle progression caused by these PGs.     

Old yellow enzymes protect against acrolein toxicity in the yeast Saccharomyces cerevisiae

Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but particularly affected gene expression, metabolism, and cellular signaling. Surprisingly, the screen did not identify any antioxidants or similar stress-protective molecules, indicating that acrolein toxicity may not be mediated via reactive oxygen species. Most strikingly, a mutant lacking an old yellow enzyme (OYE2) was identified as being acrolein sensitive. Old yellow enzymes are known to reduce alpha,beta-unsaturated carbonyl compounds in vitro, but their physiological roles have remained uncertain. We show that mutants lacking OYE2, but not OYE3, are sensitive to acrolein, and overexpression of both isoenzymes increases acrolein tolerance. Our data indicate that OYE2 is required for basal levels of tolerance, whereas OYE3 expression is particularly induced following acrolein stress. Despite the range of alpha,beta-unsaturated carbonyl compounds that have been identified as substrates of old yellow enzymes in vitro, we show that old yellow enzymes specifically mediate resistance to small alpha,beta-unsaturated carbonyl compounds, such as acrolein, in vivo.     

Urease inhibitory activity of simple alpha,beta-unsaturated ketones

A variety of alpha,beta-unsaturated ketones were evaluated for their effect on the jack bean urease. Of 35 compounds tested, 2-cyclohepten-1-one (1), 2-cyclohexen-1-one (2), 2-cyclopenten-1-one (3), and 5,6-dihydro-2H-pyran-2-one (4) showed potent inhibitory activities against the enzyme. The most potent compound (1) (IC50=0.16 mM) showed similar inhibitory potency to hydroxyurea (IC50=0.095 mM). The inhibitory effects of 1, 2, 3, and 4 were significantly reduced by 2-mercaptoethanol or dithiothreitol. These data suggest that alpha,beta-unsaturated ketones inhibited the urease activity, possibly by a Michael-like addition of a protein SH group to the double bond of the alpha,beta-unsaturated carbonyl group.     

Generation of calcium action potentials in crustacean muscle fibers following exposure to sulfhydryl reagents

The ventroabdominal flexor muscles of the crustacean Atya lanipes, which are normally completely inexcitable, generate trains of overshooting calcium action potentials after exposure to the sulfhydryl reagents known as alpha, beta-unsaturated carbonyl compounds. The chemically induced action potentials are abolished by protein reagents specific for guanidino and amino groups. Attempts to induce excitability by the use of agents that block potassium conductance were without success. It is proposed that calcium channels are made functional by the covalent modification of a calcium protochannel, via the interaction between the introduced carbonyl group and existing arginine residues.     

Specific reaction of alpha,beta-unsaturated carbonyl compounds such as 6-shogaol with sulfhydryl groups in tubulin leading to microtubule damage

6-Shogaol and 6-gingerol are ginger components with similar chemical structures. However, while 6-shogaol damages microtubules, 6-gingerol does not. We have investigated the molecular mechanism of 6-shogaol-induced microtubule damage and found that the action of 6-shogaol results from the structure of alpha,beta-unsaturated carbonyl compounds. alpha,beta-Unsaturated carbonyl compounds such as 6-shogaol react with sulfhydryl groups of cysteine residues in tubulin, and impair tubulin polymerization. The reaction with sulfhydryl groups depends on the chain length of alpha,beta-unsaturated carbonyl compounds. In addition, alpha,beta-unsaturated carbonyl compounds are more reactive with sulfhydryl groups in tubulin than in 2-mercaptoethanol, dithiothreitol, glutathione and papain, a cysteine protease.     

Synergistic effect of 4-hydroxynonenal and PPAR ligands in controlling human leukemic cell growth and differentiation

Peroxisome proliferator-activated receptors play an important role in the differentiation of different cell lines. In this study we demonstrate that PPAR-alpha ligands (clofibrate and ciprofibrate) and PPAR-gamma ligands (troglitazone and 15d-prostaglandin J2) inhibit growth and induce monocytic differentiation in HL-60 cells, whereas only PPAR-gamma ligands inhibit growth of U937 cells. Differentiation was demonstrated by the analysis of surface antigen expression CD11b and CD14, and by the characteristic morphological changes. PPAR-gamma ligands are more effective than PPAR-alpha ligands in the inhibition of cell growth and in the induction of differentiation. The physiological product of lipid peroxidation, 4-hydroxynonenal (HNE), which alone induces granulocytic-like differentiation of HL-60 cells, potentiates the monocytic differentiation induced by ciprofibrate, troglitazone, and 15d-prostaglandin J2. The same HNE treatment significantly inhibits U937 cell growth and potentiates the inhibition of cell growth in PPAR-gamma ligand-treated cells. However, HNE does not induce a significant number of CD14-positive U937 cells. HNE causes a great increase of PPAR-gamma expression in both HL-60 and U937 cells, whereas it does not modify the PPAR-alpha expression. This observation may account for the high synergistic effect displayed by HNE and PPAR-gamma ligands in the inhibition of cell growth and differentiation induction. These results represent the first evidence of the involvement of a product of lipid peroxidation in the modulation of PPAR ligand activity and suggest a relationship between HNE and PPAR ligand pathways in leukemic cell growth and differentiation.     

Modulation of pumpkin glutathione S-transferases by aldehydes and related compounds

Induction of pumpkin (Cucurbita maxima Duch.) glutathione S-transferase (GST, EC 2.5.1.18) by aldehydes and related compounds was examined. All of the tested compounds induced pumpkin GST to different degrees, and it was found that (1) aldehydes induce GST directly and alcohols induce GST indirectly, and (2) alpha,beta-unsaturated aldehydes are the most effective inducers and their potency is related to the Michael acceptors reaction. The results of Western blot analysis showed that the patterns of induction of CmGSTU1, CmGSTU2 and CmGSTU3 were similar to the patterns of activity with the exception of alpha,beta-unsaturated carbonyl compounds. Among the three compounds, crotonaldehyde caused the highest activity induction (9.2-fold), but Western blot expression was the highest only for CmGSTU3. CmGSTF1 was almost non-responsive to all of the tested stresses. Results of induction studies suggested that efficient pumpkin GST inducers have distinctive chemical features. The in vitro activity of the enzyme was inhibited by ethacryanic acid, trans-2-hexenal, crotonaldehyde, and pentanal. Ethacryanic acid was found to be the most potent inhibitor with an apparent I(50) value of 6.90+/-2.06 micro M, while others were weak to moderate inhibitors. The results presented here indicate that plant GSTs might be involved in the detoxification of physiologically and environmentally hazardous aldehydes/alcohols.     

Differences in the biological activity of TNF alpha and TNF beta correlate with their different abilities for binding to the target cells.

TNF alpha and TNF beta were compared regarding their binding to different types of target cells, cytotoxic/cytostatic activity against murine and human tumor cell lines as well as human capillary endothelial cells, their ability to induce differentiation in myeloid leukemia cell lines, and induction of hemorrhagic tumor necrosis and tumor regression as well as lethal toxicity in tumor-bearing mice. The results show considerable quantitative differences in the biological activity between TNF alpha and TNF beta depending on the type of target cell which has been used. TNF beta was 3 fold more cytotoxic than TNF alpha against murine L929 fibroblasts and 3-5 times more active concerning the induction of hemorrhagic tumor necrosis, complete tumor regression and more toxic in tumor-bearing mice. In contrast to this, TNF beta was markedly less cytotoxic against human capillary endothelial cells and the human mammary carcinoma cell line MCF7 and much less cytostatic against the human myeloid leukemia cell lines HL60 and U937. The lesser antiproliferative effect of TNF beta correlated with a lower ability for induction of differentiation in these cell lines. Competitive radioligand binding assays showed that TNF beta was about 4 fold more effective than TNF alpha in competing with 125I-labeled TNF alpha for the binding to murine L929 fibroblasts. But it was 15-20 times less effective in binding to the human MCF7 cells and the human myeloid leukemia cell lines HL60 and U937. This revealed that, at least for these targets, the differences in the biological activity between TNF alpha and TNF beta are due to different abilities for binding to the target cells. Possible mechanisms for these different binding abilities are discussed.     

A 13C NMR approach to categorizing potential limitations of alpha,beta-unsaturated carbonyl systems in drug-like molecules

Compounds that contain an alpha,beta-unsaturated carbonyl moiety are often flagged as potential Michael acceptors. All alpha,beta-unsaturated carbonyl moieties are not equivalent, however, and we sought to better understand this system and its potential implications in drug-like molecules. Measurement of the (13)C NMR shift of the beta-carbon and correlation to in vitro results allowed compounds in our collection to be categorized as potential Michael acceptors, potential substrates for NADPH, or as photoisomerizable.