Endocan and advanced oxidation protein products in adult population with hypertension.

OBJECTIVE: Hypertension is closely related to oxidative stress and inflammation. Endocan is a new inflammation marker whose role is not completely elucidated in hypertension. The aim of this study was to explore the association between endocan and several oxidative stress markers [i.e., advanced oxidation protein products (AOPP), total protein sulfhydryl (SH-) groups and prooxidant-antioxidant balance (PAB)] in adult population with hypertension. PATIENTS AND METHODS: A total of 90 patients with hypertension were compared with 44 controls. Blood pressure, anthropometric and biochemical parameters were measured. Associations of clinical data with hypertension were tested with univariable and multivariable logistic ordinal regression analysis. RESULTS: Endocan and AOPP were significantly higher in hypertensive patients than in the controls (p=0.006 and p=0.046, respectively). In the multivariable logistic regression analysis, AOPP and endocan kept their independent positive associations with hypertension. As AOPP rose by 1 µmol/L and endocan rose by 1 pg/mL, the probability for hypertension presence rose by 4.2% and 32.2%, respectively and 39.9% of variation in hypertension could be explained with the Model. The area under the Receiver Operating Characteristic curve [(AUC) for AOPP=0.638 (0.550-0.719), p=0.01 and for endocan=0.679 (0.593-0.757), p<0.001] demonstrated sufficient clinical accuracy towards hypertension. On the contrary, the Model showed very good clinical accuracy [AUC= 0.825 (0.749-0.900), p<0.001]. CONCLUSIONS: Endocan and AOPP are independently correlated with hypertension in adult population and these tested markers together could be reliable parameters to discriminate patients with hypertension from normotensive ones.

Total oxidant status and oxidative stress index as indicators of increased Reynolds risk score in postmenopausal women.

OBJECTIVE: Considering the knowledge gap between underlying pathophysiological mechanisms of oxidative stress and increased cardiovascular risk, the present study aimed to examine the potential relationship between total oxidant status (TOS), total antioxidant status (TAS) and oxidative stress index (OSI) and the Reynolds Risk Score (RRS) in the cohort of postmenopausal women. PATIENTS AND METHODS: A total of 126 postmenopausal women participated in this cross-sectional study. Blood pressure, anthropometric and biochemical markers were determined. OSI was calculated as the TOS/TAS ratio. Associations of biochemical parameters with RRS were tested using univariable and multivariable logistic ordinal regression analysis. RESULTS: TOS and OSI were the highest in women in high RRS category compared to moderate and low risk ones (p<0.001, for both). There was no difference in TAS level across RRS categories (p=0.370). Multivariable ordinal regression analysis showed independent association of TOS and OSI with RRS when tested with other clinical variables [OR=2.45; 95% CI (1.08-5.53); p=0.031 and OR=2.84; 95% CI (1.27-6.36); p=0.011, respectively]. CONCLUSIONS: TOS and OSI are associated with the RRS in the cohort of postmenopausal women. Longitudinal studies are needed to confirm whether adding the TOS and OSI to the standard RRS algorithm could improve its potential to predict cardiovascular event.

Inverse association between serum endocan levels and small LDL and HDL particles in patients with type 2 diabetes mellitus.

OBJECTIVE: Determination of lipoprotein size and subclasses distribution can provide more significant information on cardiovascular disease risk than measurement of traditional lipid parameters alone. Accordingly, we aimed to examine their potential relationship with the novel biomarker of endothelial dysfunction, such as endocan in patients with type 2 diabetes mellitus (T2D), since there are no studies concerning this issue. PATIENTS AND METHODS: This case-control study included a total of 42 individuals with T2D and 64 diabetes-free participants. Serum endocan, lipid parameters, and lipoprotein subclasses were measured. RESULTS: Patients with T2D exhibited higher proportion of the smallest high-density lipoprotein (HDL) particles HDL 3c, as compared with diabetes-free participants (p=0.047). Higher serum endocan levels in T2D patients with low small dense low-density lipoprotein (LDL) particles (sdLDL) %, as compared with corresponding group of diabetes-free subjects was shown (p<0.01). Univariate binary logistic analysis revealed significant positive association of endocan and LDL diameter (OR=1.686, p=0.004), and negative associations of endocan with proportions of sdLDL (OR=0.928, p=0.007) and HDL3b (OR=0.789, p=0.009) particles. In a multivariate analysis, LDL diameter and proportions of sdLDL and HDL3b subclasses remained independent predictors of endocan levels in tested population. CONCLUSIONS: The results of our study showed that larger LDL diameters, but lower sdLDL and HDL3b proportions were associated with higher endocan levels in population with T2D. More studies in the future are needed to confirm the observed relationship and to examine its causal nature.

Apoptotic signaling in polyamine analogue-treated SK-MEL-28 human melanoma cells.

N(1),N(11)-Diethylnorspermine (DENSPM) is a polyamine analogue with clinicalrelevance as an experimental anticancer agent and the ability to elicit a profound apoptotic response in certain cell types. Here, we characterize the polyamine effects and apoptotic signaling events initiated by treatment of SK-MEL-28 human melanoma with 10 microM DENSPM. Maximal induction of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine pool depletion were seen by 16 h, whereas early apoptosis was first apparent at 36 h. Intermediate events related to apoptotic signaling were sought between 16 and 36 h. A loss of mitochondrial transmembrane potential (Deltapsi(m)) beginning at 24 h was followed by the release of cytochrome c into the cytosol at 30 h. Loss of mitochondrial integrity was accompanied by caspase-3 activation and poly(ADP-ribose) polymerase digestion from 30 to 36 h. The caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone rendered cells resistant to analogue-induced caspase-3 activation and reduced the apoptotic response in a dose-dependent manner. Because polyamine reduction achieved by inhibitors of polyamine biosynthesis inhibited growth but did not cause apoptosis, we looked for alternative polyamine-related events, focusing on induction of SSAT. Three DENSPM analogues that differentially induced SSAT activity but similarly depleted polyamine pools revealed a close correlation between enzyme induction and cytochrome c release, caspase activation, and apoptosis. Dose-dependent inhibition of polyamine oxidase, an enzyme that oxidizes acetylated polyamines generated by SSAT and releases toxic by-products such as H(2)O(2) and aldehydes, prevented cytochrome c release, caspase activation, and apoptosis. Taken together, the findings indicate that DENSPM-induced apoptosis is at least partially initiated via massive induction of SSAT and related oxidative events and subsequently mediated by the mitochondrial apoptotic signaling pathway as indicated by cytochrome c release and caspase activation.

Polyamine analogue induction of the p53-p21WAF1/CIP1-Rb pathway and G1 arrest in human melanoma cells.

Although polyamines are well recognized for their critical involvement in cell growth, the cell cycle specificity of this requirement has not yet been characterized with respect to the newly delineated regulatory pathways. We recently reported that polyamine analogues having close structural and functional similarities to the natural polyamines produce a distinct G1 and G2-M cell cycle arrest in MALME-3M human melanoma cells. To determine a molecular basis for this observation, we examined the effects of N1,N11-diethylnorspermine on cell cycle regulatory proteins associated with G1 arrest. The analogue is known to deplete polyamine pools by suppressing biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Treatment of MALME-3M cells with 10 microM N1,N11-diethylnorspermine caused an increase in hypophosphorylated Rb, which correlated temporally with the onset of G1 arrest at 16-24 h. Rb hypophosphorylation was preceded by an increase in wild-type p53 (approximately 100-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; approximately 5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D increased slightly, whereas proliferating cell nuclear antigen and p130 remained unchanged. Induction of p21 protein was accompanied by an increase in p21 mRNA, whereas induction of p53 protein was not, suggesting transcriptional activation of the former and posttranscriptional regulation of the latter. SK-MEL-28 human melanoma cells, which contain a mutated p53, failed to induce p53 or p21 and did not arrest in G1. Rather, these cells rapidly underwent programmed cell death within 48 h. Overall, these findings provide the first indication of the cell cycle regulatory pathways by which polyamine antagonists such as analogues might inhibit growth in cells containing wild-type p53 and further suggest a mechanistic basis for differential cellular responses to these agents.

Identification of quantitative trait loci influencing wood specific gravity in an outbred pedigree of loblolly pine.

We report the identification of quantitative trait loci (QTL) influencing wood specific gravity (WSG) in an outbred pedigree of loblolly pine (Pinus taeda L.). QTL mapping in an outcrossing species is complicated by the presence of multiple alleles (> 2) at QTL and marker loci. Multiple alleles at QTL allow the examination of interaction among alleles at QTL (deviation from additive gene action). Restriction fragment length polymorphism (RFLP) marker genotypes and wood specific gravity phenotypes were determined for 177 progeny. Two RFLP linkage maps were constructed, representing maternal and paternal parent gamete segregations as inferred from diploid progeny RFLP genotypes. RFLP loci segregating for multiple alleles were vital for aligning the two maps. Each RFLP locus was assayed for cosegregation with WSG QTL using analysis of variance (ANOVA). Five regions of the genome contained one or more RFLP loci showing differences in mean WSG at or below the P = 0.05 level for progeny as grouped by RFLP genotype. One region contained a marker locus (S6a) whose QTL-associated effects were highly significant (P > 0.0002). Marker S6a segregated for multiple alleles, a prerequisite for determining the number of alleles segregating at the linked QTL and analyzing the interactions among QTL alleles. The QTL associated with marker S6a appeared to be segregating for multiple alleles which interacted with each other and with environments. No evidence for digenic epistasis was found among the five QTL.

Differential post-transcriptional control of ornithine decarboxylase and spermidine-spermine N1-acetyltransferase by polyamines.

Ornithine decarboxylase (ODC) and spermidine/ spermine N1-acetyltransferase (SSAT) are short-lived polyamine enzymes with rate-limiting roles in controlling polyamine biosynthesis and catabolism, respectively. We have found that treatment of MALME-3M human melanoma cells for 6 h with 10 micrograms/ml cycloheximide (CHX) increases ODC and SSAT mRNA 6-9-fold. When cells containing CHX-induced SSAT mRNA were washed and post-incubated for an additional 6 h in drug free media, enzyme activity increased only 2-fold above that in untreated cells despite the > 6-fold increase in accumulated mRNA. Inclusion of 10 microM spermine or spermidine in the post-incubation medium increased SSAT activity approximately 7-fold without further elevating SSAT mRNA levels. This indicates post-transcriptional regulation which, due to the similarity between polyamine-mediated increases in SSAT activity and available mRNA, probably occurs at the level of mRNA translation. In contrast to the SSAT response, polyamines markedly reduced ODC activity (but not mRNA) to one sixth that in cells not exposed to polyamines. The findings illustrate how via post-transcriptional mechanisms, shifts in intracellular polyamine pools can simultaneously and differentially regulate polyamine biosynthesis and catabolism. It is hypothesized that these post-transcriptional responses enable cells to rapidly and sensitively control intracellular spermidine and spermine pools.

Sequence specific antisense oligonucleotide analog interference with spermidine/spermine N1-acetyltransferase gene expression.

Induction of the polyamine acetylating enzyme, spermidine/spermine N1-acetyltransferase (SSAT), is one of several biochemical effects associated with the antiproliferative action of polyamine analogs such as N1, N11 diethylnorspermine (DENSPM). Findings to date indicate that this complex and extremely potent gene response involves increased gene transcription, stabilization of mRNA, enhanced translation and protein stabilization. In this study, SSAT-directed antisense oligonucleotide analogs (AOs) were studied for their ability to prevent enzyme induction by DENSPM. Nine 18-mer fully phosphorothioate modified AOs targeting the start codon, exon 6, stop codon and polyadenylation regions of the human SSAT mRNA were synthesized and evaluated in MALME-3M human melanoma cells prior to and during a 6 hr treatment with 10 microM DENSPM. The most effective AOs were those targeting sequences in the stop codon region. Of these, AO-82 suppressed DENSPM induction of SSAT activity, enzyme protein and mRNA by 70-80%. The quantitative similarity of these effects suggests AO interference with mRNA stabilization, a property apparently mediated by sequences located in the stop codon region. Growth inhibition by DENSPM in the presence of the terminally phophorothioated analogs of AO-82 remained similar to that produced by DENSPM alone. While it is possible that SSAT induction may not be involved in analog-mediated antiproliferative activity, a more likely interpretation is that the approximately 50% suppression of the enzyme response achieved in growth studies is not sufficient to abrogate growth inhibition.

Effects of polyamines, polyamine analogs, and inhibitors of protein synthesis on spermidine-spermine N1-acetyltransferase gene expression.

The key polyamine catabolizing enzyme spermidine-spermine N1-acetyltransferase (SSAT) is among the few genes known to be inducible by the natural polyamines. Certain polyamine analogs markedly exaggerate this response and thus provide useful tools for studying the underlying regulatory mechanisms. As shown here, the analog which most potently induces SSAT activity, N1, N11-diethylnorspermine (DENSPM), increases SSAT mRNA in MALME-3M human melanoma cells to a maximum of > 20-fold and immunodetectable SSAT protein to > 300-fold. By comparison, the natural polyamine spermine is far less effective, increasing SSAT mRNA by approximately 3-fold and protein by approximately 7-fold. In particular, the difference in mRNA accumulation by spermine and the analog was shown to be due to differential effects on both gene transcription and mRNA stabilization. Although the analog DENSPM has been regarded as the most potent inducer of SSAT activity and mRNA, we now report that inhibitors of protein synthesis are capable of increasing SSAT mRNA to nearly comparable levels. Inhibitor-induced accumulation in SSAT mRNA was shown to involve increased gene transcription and mRNA stabilization. This suggests that, under basal conditions, SSAT gene expression is suppressed by a labile protein (or proteins). While induction of SSAT mRNA by inhibitors of protein synthesis only occurred at concentrations which blocked protein synthesis, that by DENSPM took place at concentrations which did not. The combination of either protein inhibitor with DENSPM or spermine produced an additive increase in SSAT mRNA. Taken together, these findings suggest the involvement of two separate but possibly converging pathways in the regulation of SSAT mRNA, one mediated by polyamines and their analogs and the other mediated by a labile repressor of SSAT gene transcription and/or mRNA stabilization. In addition to its apparent regulatory importance, induction of SSAT mRNA by inhibitors of protein synthesis represents a potentially useful system for studying the posttranscriptional regulation of this interesting gene.

Effects of conditional overexpression of spermidine/spermine N1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs.

Acetylation of polyamines by spermidine/spermine N(1)-acetyltransferase (SSAT) has been implicated in their degradation and/or export out of the cell. The relationship of SSAT to polyamine pool dynamics and cell growth is not yet clearly understood. MCF-7 human breast carcinoma cells were transfected with tetracycline-regulated (Tet-off) SSAT human cDNA or murine gene. Doxycycline removal for >2 days caused a approximately 20-fold increase in SSAT RNA and a approximately 10-fold increase in enzyme activity. After 4 days, intracellular putrescine and spermidine pools were markedly lowered, and cell growth was inhibited. Growth inhibition could not be prevented with exogenous polyamines due to a previously unrecognized ability of SSAT to rapidly acetylate influxing polyamines and thereby prevent restoration of the endogenous pools. Instead, cells accumulated high levels of N(1)-acetylspermidine, N(1)-acetylspermine, and N(1), N(12)-diacetylspermine, a metabolite not previously reported in mammalian cells. Doxycycline deprivation before treatment with N(1), N(11)-diethylnorspermine markedly increased analog induction of SSAT mRNA and activity and enhanced growth sensitivity to the analog by approximately 100-fold. Overall, the findings demonstrate that conditional overexpression of SSAT lowers polyamine pools, inhibits cell growth, and markedly enhances growth sensitivity to certain analogs. The enzyme also plays a remarkably efficient role in maintaining polyamine pool homeostasis during challenges with exogenous polyamines.

Structural basis for differential induction of spermidine/spermine N1-acetyltransferase activity by novel spermine analogs.

The spermine analog N1,N11-diethylnorspermine (DE-333, also known as DENSPM or BENSPM) is regarded as the most potent known inducer of the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT), increasing activity by more than 200- to 1000-fold in certain cell types. The relative ability of a series of eight systematically modified DE-333 analogs to affect SSAT expression was examined in Malme-3M human melanoma cells, one of several cell lines known to be especially responsive to induction of this enzyme. In particular, we examined the relative contribution of induction of enzyme mRNA and prolongation of enzyme half-life to analog-mediated increases in enzyme activity. Induction of enzyme mRNA was most influenced by intra-amine carbon distances; relative effectiveness was found to be proportional to the number of three-carbon units. Stabilization of enzyme was most determined by the terminal N-alkyl substituent size; among methyl, ethyl and propyl groups, methyl was least effective. Thus, DE-333, which most potently induces SSAT mRNA and effectively stabilizes SSAT enzyme activity, produces the greatest increase in enzyme activity. Although other contributing mechanisms may be involved, the relative abilities of the various analogs to induce enzyme activity is at least partially attributable to their combined effects on enzyme mRNA and protein half-life. These data reveal the highly sensitive structure-activity relationships that underlie and control spermine analog induction of SSAT activity. Pending further definition of the relationship between SSAT induction and antitumor growth and toxicity in vivo, these relationships may be used to optimize therapeutic efficacy.

Correlation of polyamine and growth responses to N1,N11-diethylnorspermine in primary fetal fibroblasts derived from transgenic mice overexpressing spermidine/spermine N1-acetyltransferase.

A recently generated transgenic mouse line having activated polyamine catabolism due to systemic overexpression of spermidine/spermine N1-acetyltransferase (SSAT) was used to isolate primary fetal fibroblasts as a means to further elucidate the cellular consequences of activated polyamine catabolism. Basal levels of SSAT activity and steady-state mRNA in the transgenic fibroblasts were about approximately 20- and approximately 40-fold higher than in non-transgenic fibroblasts. Consistent with activated polyamine catabolism, there was an overaccumulation of putrescine and N1-acetylspermidine and a decrease in spermidine and spermine pools. Treatment with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) increased SSAT activity in the transgenic fibroblasts approximately 380-fold, whereas mRNA increased only approximately 3-fold, indicating post-mRNA regulation. SSAT activity in the nontransgenic fibroblasts increased approximately 200-fold. By Western blot, enzyme protein was found to increase approximately 46 times higher in the treated transgenic fibroblasts than non-transgenic fibroblasts: a value comparable to 36-fold differential in enzyme activity. With DENSPM treatment, spermidine pools were more rapidly depleted in the transgenic fibroblasts than in nontransgenic fibroblasts. Similarly, transgenic fibroblasts were much more sensitive to DENSPM-induced growth inhibition. This was not diminished by co-treatment with an inhibitor of polyamine oxidase, suggesting that growth inhibition was due to polyamine depletion per se as opposed to oxidative stress. Since the two fibroblasts were genetically identical except for the transgene, the various metabolic and growth response differences are directly attributable to overexpression of SSAT.