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Giardino Torchia M.L.,U.S. National Institutes of Health | Conze D.B.,U.S. National Institutes of Health | Conze D.B.,Spherix | Ashwell J.D.,U.S. National Institutes of Health
PLoS ONE | Year: 2013

Cellular Inhibitors of Apoptosis 1 and 2 (c-IAP1 and c-IAP2) are ubiquitin protein ligases (E3s) that constitutively ubiquitinate and induce proteasomal-mediated degradation of NF-κB Inducing Kinase (NIK) and repress non-canonical NF-κB activation. Mice expressing an E3-inactive c-IAP2 mutant (c-IAP2H570A) have constitutive activation of non-canonical NF-κB, resulting in B cell hyperplasia and T cell costimulation-independence. If, and if so to what extent, c-IAP1 and c-IAP2 are redundant in NF-κB regulation in these mice is not known. Here we have generated mice expressing a mutant c-IAP1 that lacks E3 activity (c-IAP1H582A). These mice were phenotypically normal and did not have constitutive NF-κB activation in B cells or MEFs. siRNA-mediated knockdown of c-IAP2 showed that accumulated c-IAP2, resulting from lack of c-IAP1-dependent degradation, compensated for absent c-IAP1 E3 activity. Surprisingly, c-IAP1H582A T cells had a lower p100/p52 ratio than wild type T cells, and in the absence of costimulation proliferated to a degree intermediate between wild type and c-IAP2H570A T cells. Therefore, although c-IAP1 and c-IAP2 both can repress constitutive NF-κB activation, the relative importance of each varies according to cell type.

Sahpazidou D.,Symeonidion Research Center | Geromichalos G.D.,Symeonidion Research Center | Stagos D.,University of Thessaly | Apostolou A.,Agricultural University of Athens | And 6 more authors.
Toxicology Letters | Year: 2014

A major part of the wineries' wastes is composed of grape stems which are discarded mainly in open fields and cause environmental problems due mainly to their high polyphenolic content. The grape stem extracts' use as a source of high added value polyphenols presents great interest because this combines a profitable venture with environmental protection close to wine-producing zones. In the present study, at first, the Total Polyphenolic Content (TPC) and the polyphenolic composition of grape stem extracts from four different Greek Vitis vinifera varieties were determined by HPLC methods. Afterwards, the grape stem extracts were examined for their ability to inhibit growth of colon (HT29), breast (MCF-7 and MDA-MB-23), renal (786-0 and Caki-1) and thyroid (K1) cancer cells. The cancer cells were exposed to the extracts for 72h and the effects on cell growth were evaluated using the SRB assay. The results indicated that all extracts inhibited cell proliferation, with IC50 values of 121-230μg/ml (MCF-7), 121-184μg/ml (MDA-MD-23), 175-309μg/ml (HT29), 159-314μg/ml (K1), 180-225μg/ml (786-0) and 134->400μg/ml (Caki-1). This is the first study presenting the inhibitory activity of grape stem extracts against growth of colon, breast, renal and thyroid cancer cells. © 2014 Elsevier Ireland Ltd.

Henrich C.J.,NCI Inc | Henrich C.J.,SAIC | Thomas C.L.,NCI Inc | Brooks A.D.,SAIC | And 8 more authors.
Apoptosis | Year: 2012

Cucurbitacins B and D were among the compounds identified as sensitizers of cancer cells to TRAILmediated apoptosis in a high-throughput screen. Therefore a series of cucurbitacins was further investigated for TRAIL sensitization and possible mechanisms of action. A total of six cucurbitacins promoted TRAIL-induced apoptosis(B, I, E, C, D, and K) and one (P) was inactive. Sensitization of renal adenocarcinoma cells to TRAIL was apparent after as little as 1-4 h pretreatment and did not require continued presence of cucurbitacin. Active cucurbitacins induced caspase-8 activation only after subsequent TRAIL addition and caspase activation was required for apoptosis suggesting amplified proximal signaling from TRAIL death receptors. Cucurbitacin-sensitized TRAILinduced cytotoxicity was inhibited by N-acetyl cysteine. Structure-activity relationship analysis in comparison to published studies suggests that TRAIL-sensitizing and general cytotoxic activities of cucurbitacins may be decoupled. Cucurbitacins are reported to be inhibitors of STAT3 activation. However, their TRAIL-sensitizing activity is STAT3-independent. Treatment of renal carcinoma cells with active cucurbitacins produced rapid and dramatic changes in cell morphology and cytoskeletal organization (also prevented by NAC). Therefore, cucurbitacins may be useful as tools for investigating the molecular mechanism(s) of action of TRAIL sensitizers, particularly with regard to temporal aspects of sensitization and modulation of TRAIL signaling by cell morphology, and could form the basis for future therapeutic development in combination with TRAIL death receptor agonists. © 2011 Springer Science+Business Media, LLC.

Kerasioti E.,University of Thessaly | Stagos D.,University of Thessaly | Priftis A.,University of Thessaly | Aivazidis S.,University of Thessaly | And 4 more authors.
Food Chemistry | Year: 2014

In the present study, the in vitro scavenging activity of sheep whey protein against free radicals, as well as its reducing power were determined and compared with that of beef protein, soy protein and cow whey protein. Moreover, the possible protective effects of sheep whey protein from tert-butyl hydroperoxide (tBHP)-induced oxidative stress in muscle C2C 12 cells were determined by assessing oxidative stress markers by flow cytometry and spectrophotometry. The results showed that sheep whey protein scavenged DPPH, ABTS+ and OH radicals with IC50 values of 3.1, 4.1 and 1.8 mg of protein/ml. Moreover, the reducing power activity assessed with potassium ferricyanide of sheep whey protein was 1.3 mg/ml. As regards to the antioxidant effects in muscle cell line, sheep whey protein at 0.78, 1.56, 3.12 and 6.24 mg of protein/ml increased GSH levels up to 138%, lowered TBARS levels up to 25% and decreased ROS levels up to 41.4%. © 2014 Elsevier Ltd. All rights reserved.

Apostolou A.,Agricultural University of Athens | Stagos D.,University of Thessaly | Galitsiou E.,University of Thessaly | Spyrou A.,University of Thessaly | And 7 more authors.
Food and Chemical Toxicology | Year: 2013

Grape extracts and wine have been studied widely due to their beneficial effects on human health. However, there are only few studies from grape stems extracts. Therefore, the main objective of the present study was the assessment in stem extracts from Greek Vitis vinifera varieties of the total polyphenolic content (TPC), the identification of the polyphenols present in them, and the evaluation of their antioxidant activity, protection against ROS-induced DNA damage and inhibition of liver (HepG2) and cervical (HeLa) cancer cell growth. The range of the TPC in grape stem extracts was from 345 to 584mg GAE/g dry weight. Moreover, stem extracts contained different classes of polyphenols as flavonols, flavanols, procyanidins, phenolic acids and stilbenes. In DPPH and ABTS assays, the IC50 values of the stem extracts had an average of 7.8±2.8 and 5.4±2.6μg/mL respectively. Also, all stem extracts inhibited OH- and ROO-induced DNA damage dose dependent with average IC50 values of 478±217 and 1.15±0.85μg/mL respectively. Furthermore, stem extracts inhibited at low concentrations the growth of HepG2 and HeLa cancer cells with average IC50 values of 50±12 and 32±16μg/mL respectively. The above activities of grape stem extracts were comparable to those of seed extracts. © 2013 Elsevier Ltd.

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