Patra Vezyraki

Hsp70 translocates to the nuclei and nucleoli, binds to XRCC1 and PARP-1, and protects HeLa cells from single-strand DNA breaks

For many years, there has been uncertainty concerning the reason for Hsp70 translocation to the nucleus and nucleolus. Herein, we propose that Hsp70 translocates to the nucleus and nucleoli in order to participate in pathways related to the protection of the nucleoplasmic DNA or ribosomal DNA from single-strand breaks. The absence of Hsp70 in HeLa cells, via Hsp70 gene silencing (knockdown), indicated the essential role of Hsp70 in DNA integrity. Therefore, HeLa Hsp70 depleted cells were very sensitive in heat treatment and their DNA breaks were multiple compared to that of control HeLa cells. The molecular mechanism with which Hsp70 performs its role at the level of nucleus and nucleolus during stress was examined. Hsp70 co-localizes with PARP1 in the nucleus/nucleoli as was observed in confocal studies and binds to the BCRT domain of PARP1 as was revealed with protein–protein interaction assays. It was also found that Hsp70 binds simultaneously to XRCC1 and PARP-1, indicating that Hsp70 function takes place at the level of DNA repair and possibly at the base excision repair system. Making a hypothetical model, we have suggested that Hsp70 is the molecule that binds and interrelates with PARP1 creating the repair proteins simultaneously, such as XRCC1, at the single-strand DNA breaks. Our data partially clarify a previously unrecognized cellular response to heat stress. Finally, we can speculate that Hsp70 plays a role in the quality and integrity of DNA.

Selenium in serum and neoplastic tissue in breast cancer: correlation with CEA.

Trace element selenium (Se) is regarded to be a breast cancer preventive factor involved in multiple protective pathways. In all, 80 women with breast cancer who underwent a radical mastectomy were enrolled in the study. Serum Se and carcinoembryonic antigen levels were measured using a fluorometric and IRMA assay, respectively. Se tissue concentration was determined by a tissue extracting fluorometric assay. For statistical analysis purposes t-test was used and P-values <0.001 were regarded as statistically significant. Serum Se was 42.5±7.5 μg l−1 in breast cancer patients and 67.6±5.36 μg l−1 in the age-matched control group of healthy individuals. Serum carcinoembryonic antigen in patients was 10±1.7 U ml−1 (normal <2.5 U ml−1 in nonsmokers/<3.5 U ml−1 in smokers). A statistically significant difference was found for both serum Se and CEA between two groups studied (P<0.001). Neoplastic tissue Se concentration was 2660±210 mg g−1 tissue; its concentration in the adjacent non-neoplastic tissue was 680±110 mg g−1 tissue (P<0.001). An inverse relationship between Se and CEA serum levels was found in the two groups studied (r=−0.794). There was no correlation between serum/tissue Se concentration and stage of the disease. The decrease in serum Se concentration as well as its increased concentration in the neoplastic breast tissue is of great significance. These alterations may reflect part of the defence mechanisms against the carcinogenetic process.

Effect of Plant Polyphenols on Adipokine Secretion from Human SGBS Adipocytes

Introduction. Adipose tissue contributes to atherosclerosis with mechanisms related to adipokine secretion. Polyphenols may exhibit antiatherogenic properties. The aim of the study was to investigate the effects of three polyphenols, namely, quercetin, epigallocatechin gallate (EGCG), and resveratrol on adipokine secretion from cultured human adipocytes. Methods. Human SGBS adipocytes were treated with quercetin, EGCG, and resveratrol for 24 and 48 hours. Visfatin, leptin, and adiponectin were measured in the supernatant. Results. Visfatin secretion was inhibited by quercetin 10 μM by 16% and 24% at 24 and 48 hours respectively. The corresponding changes for quercetin 25 μM were 47% and 48%. Resveratrol 25 μM reduced visfatin by 28% and 38% at 24 and 48 hours. EGCG did not have an effect on visfatin. None of tested polyphenols influenced leptin and adiponectin secretion. Conclusion. Quercetin and resveratrol significantly decreased visfatin secretion from SGBS adipocytes. This effect may contribute to their overall antiatherogenic properties.

Rosiglitazone improves endothelial function in patients with type 2 diabetes treated with insulin

An increased incidence of myocardial infarction with rosiglitazone in patients with type 2 diabetes mellitus (T2DM) has been reported. This study aimed to assess the effect of rosiglitazone on endothelial function, assessed by flow-mediated dilation (FMD), in 34 patients with advanced T2DM treated with insulin without known cardiovascular disease. Patients were randomised into two groups: no additional treatment was given in 17 patients, while 17 patients were given rosiglitazone for 6 months. Addition of rosiglitazone significantly reduced glycosylated haemoglobin (HbA1c) (p < 0.0005) and fasting glucose (p < 0.05) and improved FMD (p < 0.005). No significant changes were observed in the insulin-only group. The single independent predictor of FMD improvement was rosiglitazone treatment (p = 0.048). These results show that, in patients with advanced T2DM treated with insulin, addition of rosiglitazone may have a beneficial effect on endothelial function. Further research is needed to investigate why this beneficial effect does not translate into improved cardiovascular prognosis in these patients.

Increased Vascular Inflammation in Early Menopausal Women Is Associated with Hot Flush Severity

CONTEXT Menopause has been related to an increased atherosclerotic risk. Presence and severity of hot flushes in menopausal women have been associated with impaired endothelial function and advanced subclinical atherosclerosis. OBJECTIVE The objective of the study was to evaluate the effect of menopausal transition on vascular inflammation indices and investigate the association of hot flush severity with these indices in early menopausal women. DESIGN, SETTING, AND PARTICIPANTS This was a cross-sectional study that included 120 early menopausal women (age range 42-55 yr, <3 yr in menopause) recruited from the menopause outpatient clinic of an academic hospital and 24 age-matched premenopausal women (controls). MAIN OUTCOMES Serum high-sensitivity C-reactive protein, P-selectin, and soluble CD40 ligand (sCD40L) levels were measured. RESULTS P-selectin and sCD40L were increased in early menopausal compared with control women (P = 0.006 and P = 0.02 respectively), whereas high-sensitivity C-reactive protein levels did not differ (P = 0.4) between the groups. Hot flush severity was the most important independent predictor of P-selectin levels (P = 0.011) in early menopausal women. Women with moderate/severe/very severe hot flushes had increased P-selectin compared with women with no/mild hot flushes or controls (P < 0.05 for both). The sCD40L levels were also higher in menopausal women with moderate/severe/very severe hot flushes compared with controls (P = 0.03) but did not differ significantly compared with women with no/mild hot flushes (P = 0.2). CONCLUSIONS Increased indices of vascular inflammation in early menopausal compared with age-matched premenopausal women may indicate a higher atherosclerotic risk. Increased severity of hot flushes was associated with adverse changes in vascular inflammation, further supporting the emerging role of hot flushes in cardiovascular prognosis in these women.

Effects of pioglitazone and metformin on vascular endothelial function in patients with type 2 diabetes treated with sulfonylureas

Pioglitazone and metformin are insulin sensitisers used for the treatment of T2DM. The effects of pioglitazone and metformin on endothelial function, assessed by FMD, in T2DM patients treated with sulfonylureas were compared. Patients were randomised to receive pioglitazone (n = 15) 30 mg once daily or metformin (n = 16) 850 mg twice daily for six months. Pioglitazone significantly decreased fasting insulin, HbA1C and HOMA-IR (p < 0.05 for all) and increased FMD (p = 0.002). Metformin induced a significant decrease in HbA1C (p = 0.02) and only a trend for increase in FMD (p = 0.08). The greater improvement in FMD with pioglitazone, compared with metformin, did not reach significance (p = 0.11). Treatment-induced changes in FMD were not associated with the effects of the two insulin sensitisers on glycaemic control or insulin resistance. The beneficial effects of pioglitazone and metformin on endothelial function in T2DM patients did not differ greatly. Larger studies are needed to explore whether a potentially greater benefit with pioglitazone may exist.

Vitamin C transiently arrests cancer cell cycle progression in S phase and G2/M boundary by modulating the kinetics of activation and the subcellular localization of Cdc25C phosphatase

Regulation of cell cycle progression involves redox (oxidation–reduction)‐dependent modification of proteins including the mitosis‐inducing phosphatase Cdc25C. The role of vitamin C (ascorbic acid, ASC), a known modulator of the cellular redox status, in regulating mitotic entry was investigated in this study. We demonstrated that vitamin C inhibits DNA synthesis in HeLa cells and, mainly the form of dehydroascorbic acid (DHA), delays the entry of p53‐deficient synchronized HeLa and T98G cancer cells into mitosis. High concentrations of Vitamin C caused transient S and G2 arrest in both cell lines by delaying the activation of the M‐phase promoting factor (MPF), Cdc2/cyclin‐B complex. Although vitamin C did not inhibit the accumulation of cyclin‐B1, it may have increased the level of Cdc2 inhibitory phosphorylation. This was achieved by transiently maintaining Cdc25C, the activator of Cdc2, both in low levels and in a phosphorylated on Ser216 inactive form that binds to 14‐3‐3 proteins contributing thus to the nuclear exclusion of Cdc25C. As expected, vitamin C prevented the nuclear accumulation of Cdc25C in both cell lines. In conclusion, it seems that vitamin C induces transient cell cycle arrest, at least in part, by delaying the accumulation and the activation of Cdc25C.

Emotional and learning behaviour in mice overexpressing heat shock protein 70.

The effects of inducible heat shock protein 70 (HSP70) on emotional and learning behaviour as well as hippocampal long-term potentiation was investigated in transgenic HSP70 overexpressing mice. In active two-way avoidance learning (shuttle box) as well as spatial 8-arm radial maze learning, the HSP70 overexpressing mice showed diminished learning performance. In several tests there was no indication of differences in anxiety behaviour between transgenic mice and wild-type mice. This suggests that impairment in learning behaviour is unrelated to the learning task and motivational aspects of behaviour. To investigate the neurophysiological correlate of learning, long-term potentiation experiments were performed. In transversal hippocampal slices, an enhanced amplitude of the population spike was found in HSP70 overexpressing mice. It was hypothesised that enhanced potentiation in conjunction with potentiation effects due to learning led to learning impairment.

Cytotoxic and anticancer effects of the triorganotin compound [(C6H5)3Sn(cmbzt)]: An in vitro, ex vivo and in vivo study

Since the initial success of cisplatin, metal complexes and organometallic compounds have been gaining growing interest in cancer therapy. It is well known that organotin(IV) compounds display strong biological activity. The triorganotin compound [(C(6)H(5))(3)Sn(cmbzt)] (cmbzt=5-chloro-2-mercaptobenzothiazole) (SnCMB), was tested for its antiproliferative and antitumour activities. Two sets of experimental procedures were followed: (1) In vitro and ex vivo procedures included the study of the cytotoxic activity of the complex against leiomyosarcoma cells (LMS) and on a normal human fibroblast line (MRC5) by the MTT assay (cell proliferation), colony formation efficiency and flow cytometric analysis with Annexin V-FITC. The anticoagulation properties of the complex were also studied. (2) In vivo procedures included acute toxicity studies and finally administration of the complex to tumour bearing Wistar rats. The results showed that the complex exhibited potent cytotoxic activity (LMS IC(50)=155 nM) and induced significant apoptosis against LMS cells. Acute toxicity studies on Wistar rats presented kidney and liver toxicity at a single dose of 40 mg/kg body wt. Furthermore, antitumour activity studies on sarcoma bearing Wistar rats revealed that SnCMB complex, administrated in two different therapeutic schemes (treated with 4 × 2 mg/kg body wt every 5 days and 3 × 2.67 mg/kg body wt every 10 days of SnCMB complex), prolonged mean survival time (by 50% and 70% respectively), but failed to decrease the mean tumour growth rate (MTGR) compared to the control group (p<0.01). In conclusion, the organic complex SnCMB possess potent cytotoxic and antimetastatic effects, and low toxicity introducing it as possible successor of organometallic compounds used nowadays in chemotherapy.

FSH, TSH, T3 and T4 changes in relation to plasma copper and zinc alterations during D-penicillamine administration in female Wistar rats

Abstract Zinc and copper are considered to antagonize each other in several organs, while they play a significant role in the regulation and function of many systems. D-penicillamine was administered to 19 female Wistar rats for 68 days, 1 g/d/kg B.W. Plasma copper and zinc levels, as well as serum T3, T4, TSH and FSH were measured at days 50, 53, 54, 58, 62 and 68. Copper concentration presented a significant decrease. Zinc showed an initial increase by day 54, followed by a subsequent decrease. FSH levels decreased, presenting a pattern identical with that of copper. T3 and —especially— T4 alterations were similar to that of zinc and copper, whereas TSH alterations were inverse to them. It is concluded that copper and zinc alterations, induced by d-penicillamine administration, can significantly affect pituitary sex hormones (TSH) and hypothalamic-pituitary-thyroid axis in rats.