Svetlana Dinić

Methanol extract from the stem of Cotinus coggygria Scop., and its major bioactive phytochemical constituent myricetin modulate pyrogallol-induced DNA damage and liver injury

The present study was undertaken to investigate the hepatoprotective effect of the methanol extract of Cotinus coggygria Scop. in rats exposed to the hepatotoxic compound pyrogallol. Assessed with the alkaline version of the comet assay, 1000 and 2000mg/kg body weight (bw) of the extract showed a low level of genotoxicity, while 500mg/kg bw of the extract showed no genotoxic potential. Quantitative HPLC analysis of phenolic acids and flavonoids in the methanol extract of C. coggygria showed that myricetin was a major component. To test the hepatoprotective effect, a non-genotoxic dose of the C. coggygria extract and an equivalent amount of synthetic myricetin, as present in the extract, were applied either 2 or 12h prior to administration of 100mg/kg bw of pyrogallol. The extract and myricetin promoted restoration of hepatic function by significantly reducing pyrogallol-induced elevation in the serum enzymes AST, ALT, ALP and in total bilirubin. As measured by the decrease in total score and tail moment, the DNA damage in liver was also reduced by the extract and by myricetin. Our results suggest that pro-surviving Akt activity and STAT3 protein expression play important roles in decreasing DNA damage and in mediating hepatic protection by the extract. These results suggest that myricetin, as a major component in the extract, is responsible for the antigenotoxic and hepatoprotective properties of the methanol extract of C. coggygria against pyrogallol-induced toxicity.

Decreased O -GlcNAcylation of the key proteins in kinase and redox signalling pathways is a novel mechanism of the beneficial effect of α-lipoic acid in diabetic liver

The present study aimed to investigate the effects of the treatment with a-lipoic acid (LA), a naturally occurring compound possessing antioxidant activity, on liver oxidant stress in a rat model of streptozotocin (STZ)-induced diabetes by examining potential mechanistic points that influence changes in the expression of antioxidant enzymes such as catalase (CAT) and CuZn/Mn superoxide dismutase(s) (SOD). LA was administered for 4 weeks by daily intraperitoneal injections (10 mg/kg) to STZ-induced diabetic rats, starting from the last STZ treatment. LA administration practically normalised the activities of the indicators of hepatocellular injury, alanine and aspartate aminotransferases, and lowered oxidative stress, as observed by the thiobarbituric acid-reactive substance assay, restored the reduced glutathione:glutathione disulphide ratio and increased the protein sulfhydryl group content. The lower level of DNA damage detected by the comet assay revealed that LA reduced cytotoxic signalling, exerting a hepatoprotective effect. The LA-treated diabetic rats displayed restored specific enzymatic activities of CAT, CuZnSOD and MnSOD. Quantitative real-time PCR analysis showed that LA restored CAT gene expression to its physiological level and increased CuZnSOD gene expression, but the gene expression of MnSOD remained at the diabetic level. Although the amounts of CAT and CuZnSOD protein expression returned to the control levels, the protein expression of MnSOD was elevated. These results suggested that LA administration affected CAT and CuZnSOD expression mainly at the transcriptional level, and MnSOD expression at the post-transcriptional level. The observed LA-promoted decrease in the O-GlcNAcylation of extracellular signal-regulated kinase, protein 38 kinase, NF-kB, CCAAT/enhancer-binding protein and the antioxidative enzymes themselves in diabetic rats suggests that the regulatory mechanisms that supported the changes in antioxidative enzyme expression were also influenced by post-translational mechanisms.

STAT3/NFκB Interplay in the Regulation of α2‐Macroglobulin Gene Expression During Rat Liver Development and the Acute Phase Response

The synthesis of alpha‐2‐macroglobulin (α2M) is low in adult rat liver and elevated in fetal liver. During the acute‐phase (AP) response it becomes significantly increased in both adult and fetal liver. In this work, the cross talk of STAT3 and NF‐κB transcription factors during α2M gene expression was analysed. Using immunoblotting, their cellular compartmentalization was examined by comparing the cytoplasmic levels of STAT3 and NF‐κB with their active equivalents, the 86 and 91 kDa isoforms and p65‐subunit, respectively, in the nuclear extract and nuclear matrix. Different partitioning dynamics of the transcription factors were observed. At the level of protein‐DNA interactions, studied by α2M promoter affinity chromatography, it was established that different ratios of promoter‐binding STAT3 isoforms participated in elevated hepatic transcription in the basal state fetus and the AP‐adult, but only the 91 kDa isoform in the AP‐fetus. Unchanged levels of DNA‐bound p65 in the control and AP‐fetus suggest that it participated in constitutive transcription. The promoter‐binding of p65 observed in the AP‐adult suggests that it was involved in transcriptional stimulation of α2M expression. The selective enrichment of the AP‐adult nuclear matrix with promoter‐binding STAT3 disclosed the importance of this association in the induction of transcription. Protein‐protein interactions were examined by co‐immunoprecipitation. Interactions between the 86 kDa STAT3 isoform and p65 that were observed in the control and AP‐fetus and of both the 86 and 91 kDa STAT3 isoforms with p65 in the AP‐adult, suggest that protein‐protein interactions were functionally connected to increased transcription. We concluded that α2M gene expression is driven by developmental‐ and AP‐related mechanisms that rely on STAT3/NF‐κB interplay. IUBMB Life, 59: 170‐178, 2007

The protein composition of the hepatocyte nuclear matrix is differentiation-stage specific.

The protein composition of hepatocyte nuclear matrices was examined in rats from the 16th day of gestation to 75 days after birth (adult). An overall increase in size of the nuclear matrix was accompanied by quantitative and qualitative changes in its protein content. Quantitative changes of the major proteins of the peripheral lamina surrounding the isolated nuclear matrix were detected. By Western analysis we established that in pre- and postnatal nuclear matrices the relative concentrations of lamin C were greater than lamin A. After birth, the relative concentrations of both lamins progressively increased. In the adult nuclear matrix, the concentration of lamin A was greater than lamin C. In contrast, the relative concentrations of lamin B remained unchanged throughout development and growth. The relative concentrations of two nuclear matrix-associated regulatory proteins studied changed with development and growth: transcription factor C/EBPalpha isoforms, which were detected during the gestation period, increased notably after the first postnatal day, attaining a maximum at the adult stage; the high concentrations of the proliferating cell nuclear antigen (PCNA) perceptibly decreased after the 21st prenatal day. Changes in the composition of the nuclear matrix protein suggest that this structure coordinates nuclear functioning during cell differentiation.

Saquinavir-NO-targeted S6 protein mediates sensitivity of androgen-dependent prostate cancer cells to TRAIL

We previously reported that the NO-modified form of HIV protease inhibitor Saquinavir (Saq) is a potent antitumoral agent efficient against numerous tumor cell lines in vitro and in vivo. In acute toxicity studies, doses of Saq-NO equivalent to DL100 of the parental drug were completely nontoxic. Beside direct effect on malignant cell growth, Saq-NO sensitizes certain type of cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cell death. In this study, we evaluated the effects of Saq-NO on androgen-dependent prostate cancer LNCaP. Saq-NO inhibited both the growth of LNCaP cells in vitro and in xenograft models. Suppression of tumor growth was accompanied with cell cycle arrest in G0/G1 phase and established a persistent inhibition of proliferation. Furthermore, Saq-NO reverted sensitivity of LNCaP cells to TRAIL but not to TNF. Treatment of cells with Saq-NO induced transient upregulation of Akt and ERK1/2. This, however, did not represent the primary mode of action of Saq-NO, as elimination with specific inhibitors did not compromise the chemotherapic efficacy of the drug. However, permanent abrogation of phosphorylation of the S6 protein, which is the downstream target of both signaling pathways, was observed. Diminished S6 phosphorylation was associated with re-established sensitivity to TRAIL and reduction of X-linked inhibitor of apoptosis protein (XIAP). In summary, NO modification of Saq led to a new chemical entity with stronger and more pleiotropic antitumor activity than the parental drug.

Proteolytic events in cryonecrotic cell death: Proteolytic activation of endonuclease P23 ☆

Although cryosurgery is attaining increasing clinical acceptance, our understanding of the mechanisms of cryogenic cell destruction remains incomplete. While it is generally accepted that cryoinjured cells die by necrosis, the involvement of apoptosis was recently shown. Our studies of liver cell death by cryogenic temperature revealed the activation of endonuclease p23 and its de novo association with the nuclear matrix. This finding is strongly suggestive of a programmed-type of cell death process. The presumed order underlying cryonecrotic cell death is addressed here by examining the mechanism of p23 activation. To that end, nuclear proteins that were prepared from fresh liver, which is devoid of p23 activity, were incubated with protein fractions isolated from liver exposed to freezing/thawing that possessed a presumed p23 activation factor. We observed that the activation of p23 was the result of a proteolytic event in which cathepsin D played a major role. Different patterns of proteolytic cleavage of nuclear proteins after in vitro incubation of nuclei and in samples isolated from frozen/thawed liver were observed. Although both processes induced p23 activation, the incubation experiments generated proteolytic hallmarks of apoptosis, while freezing/thawing of whole liver resulted in typical necrotic PARP-1 cleavage products and intact lamin B. As an explanation we offer a hypothesis that after freezing, cells possess the potential to die through necrotic as well as apoptotic mechanisms, based on our finding that the cytosol of cells exposed to cryogenic temperatures contains both necrotic and apoptotic executors of cell death.

C/EBPα AND C/EBPβ ARE PERSISTENTLY ASSOCIATED WITH THE RAT LIVER NUCLEAR MATRIX THROUGHOUT DEVELOPMENT AND THE ACUTE PHASE RESPONSE

The partitioning of C/EBPα and C/EBPβ on the nuclear matrix structure was examined during the different transcriptional activities accompanying liver development and the acute phase (AP) response. The presence of C/EBPα and C/EBPβ was established on the nuclear matrix. Their relative concentrations on the matrix always reflected the developmental stage‐ and AP‐related fluctuations observed in the nuclear extract. Thus, they progressively increased as development proceeded, whereas during the AP response, C/EBPα decreased and C/EBPβ increased. In addition, the levels of both transcription factors were always notably higher in the nuclear matrix than in the extracts. We conclude that the observed changes and overall enrichment of the nuclear matrix with regulatory proteins is a reflection of the importance of such interactions for the in vivo functioning of C/EBP proteins.

The Importance of the CXCL12/CXCR4 Axis in Therapeutic Approaches to Diabetes Mellitus Attenuation

The pleiotropic chemokine (C–X–C motif) ligand 12 (CXCL12) has emerged as a crucial player in several diseases. The role of CXCL12 in diabetes promotion and progression remains elusive due to its multiple functions and the overwhelming complexity of diabetes. Diabetes is a metabolic disorder resulting from a failure in glucose regulation due to β-cell loss and/or dysfunction. In view of its ability to stimulate the regeneration, proliferation, and survival of β-cells, as well as its capacity to sustain local immune-isolation, CXCL12 has been considered in approaches aimed at attenuating type 1 diabetes. However, a note of caution emerges from examinations of the involvement of CXCL12 in the development of diabetes and its complications, as research data indicate that CXCL12 displays effects that range from protective to detrimental. Therefore, as a beneficial effect of CXCL12 in one process could have deleterious consequences in another, a more complete understanding of CXCL12 effects, in particular its functioning in the cellular microenvironment, is essential before CXCL12 can be considered in therapies for diabetes treatment.

C/EBPα and C/EBPβ Regulate Haptoglobin Gene Expression during Rat Liver Development and the Acute-phase Response

The participation of C/EBPα and C/EBPβ in the transcriptional regulation of the haptoglobin (Hp) gene throughout liver development and the acute-phase (AP) response was examined. Western immunoblot analysis revealed that the relative concentrations of C/EBPα and C/EBPβ increased during differentiation in two nuclear protein fractions – the nuclear extract and nuclear matrix. The AP reaction was accompanied by a decrease of the relative concentration of C/EBPα and an increase of C/EBPβ during development in both protein fractions. Using Western analysis after DNA-affinity chromatography it was observed that a 45 kDa C/EBPα isoform displayed a binding affinity towards the Hp gene hormone responsive element (HRE) in both pre- and postnatal livers. In the course of the AP response DNA binding of the 45 kDa isoform was detected only in the adult, when its binding affinity decreased. The 35 kDa C/EBPβ isoform exhibited a binding affinity towards the Hp HRE after the second week from birth, whereas the AP response promoted an enhanced binding of 35 kDa isoform after the first postnatal week. These results indicate that Hp gene transcription is regulated by C/EBPα during normal liver development, whereas C/EBPβ is involved in the AP regulation during the later phase of differentiation and in the adult.

CXCL12 protects pancreatic β-cells from oxidative stress by a Nrf2-induced increase in catalase expression and activity

Due to intrinsically low levels of antioxidant enzyme expression and activity, insulin producing pancreatic β-cells are particularly susceptible to free radical attack. In diabetes mellitus, which is accompanied by high levels of oxidative stress, this feature of β-cells significantly contributes to their damage and dysfunction. In light of the documented pro-survival effect of chemokine C-X-C Ligand 12 (CXCL12) on pancreatic β-cells, we examined its potential role in antioxidant protection. We report that CXCL12 overexpression enhanced the resistance of rat insulinoma (Rin-5F) and primary pancreatic islet cells to hydrogen peroxide (H2O2). CXCL12 lowered the levels of DNA damage and lipid peroxidation and preserved insulin expression. This effect was mediated through an increase in catalase (CAT) activity. By activating downstream p38, Akt and ERK kinases, CXCL12 facilitated Nrf2 nuclear translocation and enhanced its binding to the CAT gene promoter, inducing constitutive CAT expression and activity that was essential for protecting β-cells from H2O2.