Hydroxytyrosol produced by engineered Escherichia coli strains activates Nrf2/HO-1 pathway: An in vitro and in vivo study.

This study explores the biological effects of hydroxytyrosol (HT), produced by the metabolic engineering of Escherichia coli, in a series of in vitro and in vivo experiments. In particular, a metabolically engineered Escherichia coli strain capable of producing HT was constructed and utilized. HEK293 and HeLa cells were exposed to purified HT to determine non-toxic doses that can offer protection against oxidative stress (activation of Nrf2/HO-1 signaling pathway). Male CD-1 mice were orally supplemented with HT to evaluate (1) renal and hepatic toxicity, (2) endogenous system antioxidant response, and (3) activation of Nrf2/HO-1 system in the liver. HT protected cells from oxidative stress through the activation of Nrf2 regulatory network. Activation of Nrf2 signaling pathway was also observed in the hepatic tissue of the mice. HT supplementation was safe and produced differential effects on mice's endogenous antioxidant defense system. HT biosynthesized from genetically modified Escherichia coli strains is an alternative method to produce high-quality HT that exerts favorable effects in the regulation of the organism's response to oxidative stress. Nonetheless, further investigation of the multifactorial action of HT on the antioxidant network regulation is needed.

Heat shock protein 70 is a key molecule to rescue imbalance caused by low-frequency noise.

A previous study showed that people living in urban areas are generally exposed to low-frequency noise (LFN) with frequencies below 100 Hz and sound levels of 60-110 dB in daily and occupational environments. Exposure to LFN has been shown to affect balance in humans and mice. However, there is no information about prevention of LFN-mediated imbalance because of a lack of information about the target region based on health risk assessment of LFN exposure. Here, we show that acute exposure to LFN at 100 Hz, 95 dB, but not at 85 dB or 90 dB, for only 1 h caused imbalance in mice. The exposed mice also had decreased cervical vestibular-evoked myogenic potential (cVEMP) with impaired activity of vestibular hair cells. Since imbalance in the exposed mice was irreversible, morphological damage in the vestibules of the exposed mice was further examined. The exposed mice had breakage of the otoconial membrane in the vestibule. LFN-mediated imbalance and breakage of the otoconial membrane in mice were rescued by overexpression of a stress-reactive molecular chaperone, heat shock protein 70 (Hsp70), which has been shown to be induced by exposure of mice to 12 h per day of LFN at 95 dB for 5 days. Taken together, the results of this study demonstrate that acute exposure to LFN at 100 Hz, 95 dB for only 1 h caused irreversible imbalance in mice with structural damage of the otoconial membrane as the target region for LFN-mediated imbalance, which can be rescued by Hsp70.

The increased expression of the inducible Hsp70 (HSP70A1A) in serum of patients with heart failure and its protective effect against the cardiotoxic agent doxorubicin.

The aim of this study was to examine the potential association between the expression of Hsp70 protein and heart failure and to investigate the possible protective effect of Hsp70 against the doxorubicin-induced toxicity. Initially, at clinical level, the expression levels of the inducible Hsp70 were quantified in serum from patients with heart failure. Our results showed that in heart failure, Hsp70 concentration appeared to be increased in blood sera of patients compared to that of healthy individuals. The enhanced expression of Hsp70 in serum of patients with heart failure seemed to be associated with various features, such as gender, age and the type of heart failure, but not with its etiology. Next, in our study at cellular level, we used primary cell cultures isolated from embryos of Hsp70-transgenic mice (Tg/Tg) overexpressing human HSP70 and wild-type mice (F1/F1). After exposure to a wide range of doxorubicin concentrations and incubation times, the dose- and time-dependent toxicity of the drug, which appeared to be reduced in Tg/Tg cells, was demonstrated. In addition, doxorubicin administration appeared to result in a dose- and time-dependent decrease in the activity of two of the major endogenous antioxidant enzymes (SOD and GPx). The increased activity of these enzymes in Tg/Tg cells compared to the control F1/F1 cells was obvious, suggesting that the presence of Hsp70 confers enhanced tolerance against DOX-induced oxidative stress. Overall, it has been indicated that Hsp70 protein exerts a very important protective action and renders cells more resistant to the harmful effects of doxorubicin.

Hsp70 (HSP70A1A) downregulation enhances the metastatic ability of cancer cells.

Heat shock protein 70 (Hsp70; also known as HSP70A1A) is one of the most induced proteins in cancer cells; however, its role in cancer has not yet been fully elucidated. In the present study, we proposed a hypothetical model in which the silencing of Hsp70 enhanced the metastatic properties of the HeLa, A549 and MCF7 cancer cell lines. We consider that the inability of cells to form cadherin­catenin complexes in the absence of Hsp70 stimulates their detachment from neighboring cells, which is the first step of anoikis and metastasis. Under these conditions, an epithelial­to­mesenchymal transition (EMT) pathway is activated that causes cancer cells to acquire a mesenchymal phenotype, which is known to possess a higher ability for migration. Therefore, we herein provide evidence of the dual role of Hsp70 which, according to international literature, first establishes a cancerous environment and then, as suggested by our team, regulates the steps of the metastatic process, including EMT and migration. Finally, the trigger for the anti­metastatic properties that are acquired by cancer cells in the absence of Hsp70 appears to be the destruction of the Hsp70­dependent heterocomplexes of E­cadherin/catenins, which function like an anchor between neighboring cells.

Lipidome of plasma lipoproteins and liver is zinc- modulated in High fat diet treated mice.

Zinc (Zn) and Zn-transcription Factors regulate the metabolic pathways of lipids and glucose, consequently nutritional zinc deficiency or excess, activates stress pathways and deranges the hepatic metabolism of lipids. High fat diet (HFD) also leads to lipids' profile disorders as well as to intracellular free radicals (FR) accumulation and finally to metabolic stress-syndrome. Study of nutritional Zn effects on the lipidome of plasma lipoproteins and liver, in HFD-mice, was the aim of the present research. Three Zn enriched HF-Diets as follows, 3 mg/kg feed (Zn deficient diet), 30 mg/kg feed (Zn sufficient diet), 300mgZn /kg feed (Zn excess diet) (Mucedola s.r.l Italy-55% cal) were applied respectively to three groups of male wild type (wt) mice (Hybrid F1/F1),C57Bl/6xCBA, one month old, for 10 weeks. Accordingly, mice body weight rate and 1H-NMR spectrum analysis of liver extracts and plasma HDL and non-HDL lipoproteins were evaluated at the end of the experimental period. It is concluded that Zn sufficient diet (30 mg/Kg Feed) creates a highly protective lipidomic profile on plasma and liver lipoproteins of HFD-mice, related to significantly increased antiatherogenic indicators in lipids' composition, compared to mice in nutritional Zn deficiency or excess.

Increased expression level of Hsp70 in the inner ears of mice by exposure to low frequency noise.

Previous studies showed that people in urban areas are possibly exposed to 60-110 dB of low frequency noise (LFN) defined as noise of ≤100 Hz in their daily life. Previous studies also showed increased health risks by exposure to high levels (130-140 dB) of LFN in animals. However, little is known about the health effects of exposure to an ordinary level of LFN. We biochemically and immunohistochemically assessed the effects of exposure to inaudible LFN for mice (12 h/day of 100 Hz LFN at 95 dB for 5 days), at a level to which people are possibly exposed in daily life, on a murine inner ear by targeting 9 stress-reactive molecules. There was more than a 5-fold increased transcript level of heat shock protein 70 (Hsp70) in the whole inner ear exposed to LFN. However, the transcript levels of the other 8 stress-reactive molecules including Hsp27 and Hsp90 were comparable in LFN-exposed and unexposed murine inner ears. Only the transcript level of Cebpß among the previously reported 4 transcriptional activators for Hsp70 expression was more than 3-fold increased by LFN exposure. Hsp70 transcript expression levels in the inner ears 3 days after LFN exposure were comparable to those in unexposed inner ears. The protein level of Hsp70, but not the levels of Hsp27 and Hsp90, was also increased in the vestibule by LFN exposure. However, hearing levels as well as expression levels of Hsp70 protein in the cochleae were comparable in LFN-exposed mice and unexposed mice. Our results demonstrated that the inner ear might be one of the organs that is negatively affected by stress from inaudible LFN exposure. Moreover, LFN exposure might increase Hsp70 expression level via Cebpß in the inner ear. Thus, Hsp70 and Cebpß levels could be candidates of biomarkers for response to LFN exposure.

Hsp70 regulates the doxorubicin-mediated heart failure in Hsp70-transgenic mice.

The aim of this study was to investigate the potential protective effect of the Hsp70 protein in the cardiac dysfunction induced by doxorubicin (DOX) and the mechanisms of its action. For this purpose, we used both wild-type mice (F1/F1) and Hsp70-transgenic mice (Tg/Tg) overexpressing human HSP70. Both types were subjected to chronic DOX administration (3 mg/kg intraperitoneally every week for 10 weeks, with an interval from weeks 4 to 6). Primary cell cultures isolated from embryos of these mice were also studied. During DOX administration, the mortality rate as well as weight reduction were lower in Tg/Tg compared to F1/F1 mice (P < 0.05). In vivo cardiac function assessment by transthoracic echocardiography showed that the reduction in left ventricular systolic function observed after DOX administration was lower in Tg/Tg mice (P < 0.05). The study in primary embryonic cell lines showed that the apoptosis after incubation with DOX was reduced in cells overexpressing Hsp70 (Tg/Tg), while the apoptotic pathway that was activated by DOX administration involved activated protein factors such as p53, Bax, caspase-9, caspase-3, and PARP-1. In myocardial protein extracts from identical mice with DOX-induced heart failure, the particular activated apoptotic pathway was confirmed, while the presence of Hsp70 appeared to inhibit the apoptotic pathway upstream of the p53 activation. Our results, in this DOX-induced heart failure model, indicate that Hsp70 overexpression in Tg/Tg transgenic mice provides protection from myocardial damage via an Hsp70-block in p53 activation, thus reducing the subsequent apoptotic mechanism.

Arsenic induces VL30 retrotransposition: the involvement of oxidative stress and heat-shock protein 70.

Arsenic is an environmental contaminant with known cytotoxic and carcinogenic properties, but the cellular mechanisms of its action are not fully known. As retrotransposition consists a potent mutagenic factor affecting genome stability, we investigated the effect of arsenic on retrotransposition of an enhanced green fluorescent protein (EGFP)-tagged nonautonomous long terminal repeat (LTR)-retrotransposon viral-like 30 (VL30) in a mouse NIH3T3 cell culture-retrotransposition assay. Flow cytometry analysis of assay cells treated with 2.5-20µM sodium arsenite revealed induction of retrotransposition events in a dose- and time-dependent manner, which was further confirmed as genomic integrations by PCR analysis and appearance of EGFP-positive cells by UV microscopy. Specifically, 20µM sodium arsenite strongly induced the VL30 retrotransposition frequency, which was ~90,000-fold higher than the natural one and also VL30 RNA expression was ~6.6-fold. Inhibition of the activity of endogenous reverse transcriptases by efavirenz at 15µM or nevirapine at 375µM suppressed the arsenite-induced VL30 retrotransposition by 71.16 or 79.88%, respectively. In addition, the antioxidant N-acetyl-cysteine reduced the level of arsenite-induced retrotransposition, which correlated with the rescue of arsenite-induced G2/M cell cycle arrest and cell toxicity. Treatment of assay cells ectopically overexpressing the human heat-shock protein 70 (Hsp70) with 15µM sodium arsenite resulted in an additional ~4.5-fold induction of retrotransposition compared with normal assay cells, whereas treatment with 20µM produced a massive cell death. Our results show for the first time that arsenic both as an oxidative and heat-shock mimicking agent is a potent inducer of VL30 retrotransposition in mouse cells. The impact of arsenic-induced retrotransposition, as a cellular response, on contribution to or explanation of the arsenic-associated toxicity and carcinogenicity is discussed.

Spinal cord early ischemic preconditioning activates the stabilized fraction of ß-catenin after thoracoabdominal aortic occlusion in pigs.

BACKGROUND: Paraplegia after thoracoabdominal aortic surgery is a devastating complication attributed to motor neurons loss and dysfunction, due to spinal cord ischemia. ß-Catenin is a protein that has been associated with cell survival and healing and many studies have correlated this protein with late ischemic preconditioning (IPC). Herein we investigate the potential contribution of ß-catenin in an early IPC animal model, and its relationship with heat shock protein 70 (Hsp70), suggesting a possible role of this protein as a first window of protection. METHODS: A total of 42 pigs were used in an experimental thoracoabdominal aortic occlusion model. Twelve animals were used for neurologic evaluation and were randomly assigned to 2 groups (A and B). The remaining 30 animals were used in experiments for biologic measurements and innunohistochemical studies, and were randomly assigned to 5 groups (1-5). Western blotting analysis and immunoprecipitations were performed to study the levels of ß-catenin and its binding relationship with Hsp70. The cellular distribution of ß-catenin at various time-points was investigated by immunohistochemical studies. RESULTS: According to neurologic evaluation, the animals in the IPC+ischemia group had significantly better neurologic scores compared with those in the ischemia group, indicating a protective role for IPC. The biologic measurements demonstrated a significant (P=0.03) increase in ß-catenin levels and translocation of the protein in the nucleus at the end of ischemic preconditioning. CONCLUSIONS: Our results suggest a significant role of ß-catenin in early IPC protection of spinal cord after thoracoabdominal occlusion, as IPC seems to trigger the activation of the ß-catenin stabilized fraction and, thus, its survival pathway.

Loss of p14(ARF) confers resistance to heat shock- and oxidative stress-mediated cell death by upregulating ß-catenin.

The p14(ARF) is a key tumor suppressor induced mainly by oncogenic stimuli. Although p14(ARF) does not seem to respond to DNA damage, there are very few data regarding its role in other forms of stress, such as heat shock (HS) and oxidative stress (OS). Here, we report that suppression of p14(ARF) increased resistance to cell death when cells were treated with H(2) O(2) or subjected to HS. In this setting, protection from cell death was mediated by elevated levels and activity of ß-catenin, as downregulation of ß-catenin alleviated the protective role of p14(ARF) silencing. Moreover, Hsp70 was shown to regulate ß-catenin protein levels by interacting with p14(ARF) , suggesting that Hsp70, p14(ARF) and ß-catenin form a regulatory network. This novel pathway triggers cell death signals when cells are exposed to HS and OS.

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.

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.

Antirestenotic effects of a novel polymer-coated d-24851 eluting stent. Experimental data in a rabbit iliac artery model.

Experimental and clinical data suggest that stents eluting antiproliferative agents can be used for the prevention of in-stent restenosis. Here we investigate in vitro the antiproliferative and apoptotic effect of D-24851 and evaluate the safety and efficacy of D-24851-eluting polymer-coated stents in a rabbit restenosis model (n = 53). Uncoated stents (n = 6), poly (DL: -lactide-co-glycolide) (PLGA)-coated stents (n = 7), and PLGA-coated stents loaded with 0.08 +/- 0.0025 microM (31 +/- 1 mug; low dose; n = 7), 0.55 +/- 0.02 microM (216 +/- 8 mug; high dose; n = 6), and 4.55 +/- 0.1 microM (1774 +/- 39 mug; extreme dose; n = 5) of D-24851 were randomly implanted in New Zealand rabbit right iliac arteries and the animals were sacrificed after 28 days for histomorphometric analysis. For the assessment of endothelial regrowth in 90 days, 12 rabbits were subjected to PLGA-coated (n = 3), low-dose (n = 3), high-dose (n = 3), and extreme-dose (n = 3) stent implantation. In vitro studies revealed that D-24851 exerts its growth inhibitory effects via inhibition of proliferation and induction of apoptosis without increasing the expression of heat shock protein-70, a cytoprotective and antiapoptotic protein. Treatment with low-dose D-24851 stents was associated with a significant reduction in neointimal area and percentage stenosis only compared with bare metal stents (38% [P = 0.029] and 35% [P = 0.003] reduction, respectively). Suboptimal healing, however, was observed in all groups of D-24851-loaded stents in 90 days in comparison with PLGA-coated stents. We conclude that low-dose D-24851-eluting polymer-coated stents significantly inhibit neointimal hyperplasia at 28 days through inhibition of proliferation and enhancement of apoptosis. In view of the suboptimal re-endothelialization, longer-term studies are needed in order to establish whether the inhibition of intimal growth is maintained.

Pentylenetetrazol-kindling in mice overexpressing heat shock protein 70.

Kindling induced by the convulsant pentylenetetrazol (PTZ) is an accepted model of primary generalized epilepsy. Because seizures represent a strong distressing stimulus, stress-induced proteins such as heat shock proteins might counteract the pathology of increased neuronal excitation. Therefore, the aim of the present study was to determine whether PTZ kindling outcome parameters are influenced by heat shock protein 70 (Hsp70) overexpression in Hsp70 transgenic mice as compared to the respective wild-type mice. Kindling was performed by nine intraperitoneal injections of PTZ (ED(16) for induction of clonic-tonic seizures, every 48 h); control animals received saline instead of PTZ. Seven days after the final injection, all mice received a PTZ challenge dose. Outcome parameters included evaluation of seizure stages and overall survival rates. In addition, histopathological findings such as cell number in hippocampal subfields CA1 and CA3 were determined. The onset of the highest convulsion stage was delayed in Hsp70 transgenic mice as compared to wild-type mice, and overall survival during kindling was improved in Hsp70 transgenic mice as compared to wild-type mice. In addition, a challenge dose after termination of kindling produced less severe seizures in Hsp70 transgenic mice than in wild-type mice. PTZ kindling did not result in significant subsequent neuronal cell loss in CA1 or CA3 neither in wild-type mice nor in the Hsp70 transgenic mice. The results of the present experiments clearly demonstrate that overexpression of Hsp70 exerts protective effects regarding seizure severity and overall survival during PTZ kindling. In addition, the decreased seizure severity in Hsp70 transgenic mice after a challenge dose suggests an interference of Hsp70 with the developmental component of kindling.

Involvement of heat shock protein-70 in the mechanism of hydrogen peroxide-induced DNA damage: the role of lysosomes and iron.

Heat shock protein-70 (Hsp70) is the main heat-inducible member of the 70-kDa family of chaperones that assist cells in maintaining proteins functional under stressful conditions. In the present investigation, the role of Hsp70 in the molecular mechanism of hydrogen peroxide-induced DNA damage to HeLa cells in culture was examined. Stably transfected HeLa cell lines, overexpressing or lacking Hsp70, were created by utilizing constitutive expression of plasmids containing the functional hsp70 gene or hsp70-siRNA, respectively. Compared to control cells, the Hsp70-overexpressing ones were significantly resistant to hydrogen peroxide-induced DNA damage, while Hsp70-depleted cells showed an enhanced sensitivity. In addition, the "intracellular calcein-chelatable iron pool" was determined in the presence or absence of Hsp70 and found to be related to the sensitivity of nuclear DNA to H(2)O(2). It seems likely that the main action of Hsp70, at least in this system, is exerted at the lysosomal level, by protecting the membranes of these organelles against oxidative stress-induced destabilization. Apart from shedding additional light on the mechanistic details behind the action of Hsp70 during oxidative stress, our results indicate that modulation of cellular Hsp70 may represent a way to make cancer cells more sensitive to normal host defense mechanisms or chemotherapeutic drug treatment.

Heat shock protein 70 chaperone overexpression ameliorates phenotypes of the spinal and bulbar muscular atrophy transgenic mouse model by reducing nuclear-localized mutant androgen receptor protein.

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR). The nuclear inclusions consisting of the mutant AR protein are characteristic and combine with many components of ubiquitin-proteasome and molecular chaperone pathways, raising the possibility that misfolding and altered degradation of mutant AR may be involved in the pathogenesis. We have reported that the overexpression of heat shock protein (HSP) chaperones reduces mutant AR aggregation and cell death in a neuronal cell model (Kobayashi et al., 2000). To determine whether increasing the expression level of chaperone improves the phenotype in a mouse model, we cross-bred SBMA transgenic mice with mice overexpressing the inducible form of human HSP70. We demonstrated that high expression of HSP70 markedly ameliorated the motor function of the SBMA model mice. In double-transgenic mice, the nuclear-localized mutant AR protein, particularly that of the large complex form, was significantly reduced. Monomeric mutant AR was also reduced in amount by HSP70 overexpression, suggesting the enhanced degradation of mutant AR. These findings suggest that HSP70 overexpression ameliorates SBMA phenotypes in mice by reducing nuclear-localized mutant AR, probably caused by enhanced mutant AR degradation. Our study may provide the basis for the development of an HSP70-related therapy for SBMA and other polyQ diseases.

Mydj2 as a potent partner of hsc70 in mammalian cells.

Dj2 is a member of the DnaJ family of proteins, which regulate the chaperoning function of the hsp70s. We isolated a monkey cDNA dj2 clone corresponding to the large mRNA species encoded by the gene. This mRNA differs from the small mRNA produced by the same gene in that it contains a long 3' untranslated region. Both messages were found to be equally stable and to produce the same protein, which is susceptible to farnesylation. Studies in mouse tissues and various cell lines revealed that these messages and their products are differentially expressed. Surprisingly, we found that only the nonfarnesylated form of dj2 is capable of translocating to the cell nucleus, especially after heat shock. Finally, based on protein interaction studies, our results indicate that dj2 is a specific partner for hsc70 and not for hsp70.