Recombinant heat shock protein 27 (HSP27/HSPB1) protects against cadmium-induced oxidative stress and toxicity in human cervical cancer cells.

Cadmium (Cd) is a carcinogen with several well-described toxicological effects in humans, but its molecular mechanisms are still not fully understood. Overexpression of heat shock protein 27 (HSP27/HSPB1)-a multifunctional protein chaperone-has been shown to protect cells from oxidative damage and apoptosis triggered by Cd exposure. The aims of this work were to investigate the potential use of extracellular recombinant HSP27 to prevent/counteract Cd-induced cellular toxicity and to evaluate if peroxynitrite was involved in the development of Cd-induced toxicity. Here, we report that the harmful effects of Cd correlated with changes in oxidative stress markers: upregulation of reactive oxygen species, reduction in nitric oxide (NO) bioavailability, increment in lipid peroxidation, peroxynitrite (PN), and protein nitration; intracellular HSP27 was reduced. Treatments with Cd (100 µM) for 24 h or with the peroxynitrite donor, SIN-1, decreased HSP27 levels (~50%), suggesting that PN formation is responsible for the reduction of HSP27. Pre-treatments of the cells either with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a pharmacological inhibitor of NO synthase) or with recombinant HSP27 (rHSP27) attenuated the disruption of the cellular metabolism induced by Cd, increasing in a 55 and 52%, respectively, the cell viability measured by CCK-8. Cd induced necrotic cell death pathways, although apoptosis was also activated; pre-treatment with L-NAME or rHSP27 mitigated cell death. Our findings show for the first time a direct relationship between Cd-induced toxicity and PN production and a role for rHSP27 as a potential therapeutic agent that may counteract Cd toxicity.

HER2 and ß-catenin protein location: importance in the prognosis of breast cancer patients and their correlation when breast cancer cells suffer stressful situations.

In human breast cancer, ß-catenin localization has been related with disease prognosis. Since HER2-positive patients are an important subgroup, and that in breast cancer cells a direct interaction of ß-catenin/HER2 has been reported, in the present study we have explored whether ß-catenin location is related with the disease survival. The study was performed in a tumor bank from patients (n = 140) that did not receive specific anti-HER2 therapy. The proteins were detected by immunohistochemistry in serial sections, 47 (33.5%) patients were HER2-positive with a long follow-up. HER2-positive patients that displayed ß-catenin at the plasma membrane (completely surrounding the tumour cells) showed a significant better disease-free survival and overall survival than the patients showing the protein on other locations. Then we explored the dynamics of the co-expression of ß-catenin and HER2 in human MCF-7 and SKBR3 cells exposed to different stressful situations. In untreated conditions MCF-7 and SKBR3 cells showed very different ß-catenin localization. In MCF-7 cells, cadmium administration caused a striking change in ß-catenin localization driving it from plasma membrane to cytoplasmic and perinuclear areas and HER2 showed a similar localization patterns. The changes induced by cadmium were compared with heat shock, H2O2 and tamoxifen treatments. In conclusion, this study shows the dynamical associations of HER2 and ß-catenin and their changes in subcellular localizations driven by stressful situations. In addition, we report for the first time the correlation between plasma membrane associated ß-catenin in HER2-positive breast cancer and survival outcome, and the importance of the protein localization in breast cancer samples.

Cytoprotective mechanisms in rats lung parenchyma with zinc deprivation.

Suboptimal intake of Zinc (Zn) is one of the most common worldwide nutritional problems. The aim of this study is to provide new evidence on the relation between moderate Zn restriction, and cytoprotective functions in airway epithelium. We analyzed the effect of moderate Zn deficiency (ZD) on the expression of several pro and anti-apoptotic proteins and cytoprotective factors (Hsp27 and Hsp 70i), as well as the effect of restoring Zn during the refeeding period. Adult male rats were divided into three groups: Zn-adequate control group, Zn-deficient group and Zn-refed group. Our previous findings showed an important oxidative and nitrosative stress during ZD, this situation is accompanied by inflammation and alterations in the expression of matrix extracellular proteins. We observed a strong immunopositive area of anti and pro-apoptotics proteins in ZD groups. The mRNA levels of Nrf-2, Bax and Bad were increased in ZD, while in ZD refed group its levels were similar to the control values. The increased expression of Nrf-2 is likely to be critical for protection of lung under inflammatory process triggered during ZD. Hsp27 and Hsp 70i showed an increase of immunostaining area but they were not significant. During the supplementation period, heat-shock proteins increased significantly. In conclusion, our results provide further evidence of the pathways involved in cytoprotection and apoptosis caused by ZD. Additional studies are required in order to investigate whether Hsp27 and Hsp70 are consistently associated with cellular stress and inflammation in lung. There may be a beneficial role for improved Zn nutrition or Zn supplements early in lung pathology.

Alterations of the extracellular matrix of lung during zinc deficiency.

Suboptimal intake of Zn is one of the most common nutritional problems worldwide. Previously, we have shown that Zn deficiency (ZD) produces oxidative and nitrosative stress in the lung of rats. We analyse the effect of moderate ZD on the expression of several intermediate filaments of the cytoskeleton, as well as the effect of restoring Zn during the refeeding period. Adult male rats were divided into three groups: Zn-adequate control (CO) group; ZD group; Zn-refeeding group. CerbB-2 and proliferating cell nuclear antigen (PCNA) expression was increased in the ZD group while the other parameters did not change. During the refeeding time, CerbB-2, cytokeratins, vimentin and PCNA immunostaining was higher than that in the CO group. The present findings indicate that the overexpression of some markers could lead to the fibrotic process in the lung. Perhaps ZD implications must be taken into account in health interventions because an inflammation environment is associated with ZD in the lung.

Alteration in the expression of inflammatory parameters as a result of oxidative stress produced by moderate zinc deficiency in rat lung.

Suboptimal intake of dietary zinc (Zn) is one of the most common nutritional problems worldwide. Previously, the authors have shown that zinc deficiency (ZD) produces oxidative and nitrosative stress in lung of male rats. The goal of this study is to test the effect of moderate ZD on insulin-like growth factor (IGF)-1, IGF-binding protein (IGFBP)-5, NADH oxidase (NOX)-2, tumor necrosis factor alpha (TNFalpha), as well as the effect of restoring zinc during the refeeding period. Adult male rats were divided into 3 groups: Zn-adequate control group, Zn-deficient group, and Zn-refeeding group. eNOS, metallothionein (MT) II, and NOX-2 was increased in ZD group. The authors observed an increased gene transcription of superoxide dismutase (SOD)-2 and gluthathione peroxidase (GPx)-1 in ZD group, as well as in ZD-refeeding group, but catalase (CAT) transcription did not change in the treated groups. Proinflammatory factors, such as TNFalpha and vascular cell adhesion molecular (VCAM)-1 increased in ZD, whereas it decreased in ZD refeeding. However, peroxisome proliferator-activated receptor gamma (PPARgamma) and IGF-1 gene transcription decreased in ZD, whereas IGFBP-5 decreased in the ZD group. These parameters are associated to alterations in the lung histoarchitecture. The zinc supplementation period is brief (only 10 days), but it is enough to inhibit some proinflammatory factors. Perhaps, zinc deficiency implications must be taken into account in health interventions because inflammation and prooxidant environment are associated with ZD in lung.

Cadmium exposure modifies lactotrophs activity associated to genomic and morphological changes in rat pituitary anterior lobe.

Cadmium (Cd) is widely used in industrial applications and is an important contaminant of agricultural products. As an endocrine disruptor, Cd modifies the hormone release of pituitary anterior lobe (PAL). This work was undertaken to evaluate a possible association between phospholipase D (PLD) and prolactin mRNA expressions and the activity of lactotrophs and folliculostellate cells (FSC) in PAL of Cd exposed adult male Wistar rats (Cd, 0.133 mM per liter for 2 months). The PALs were submitted to immunohistochemical and morphometric analysis to determine the percentage of lactotrophs (PRL-ir) and FSC (S-100-ir). Cultured PAL cells were stained with Hoechst 33258 to determine the presence of alterations in nuclear morphology consistent with apoptosis. The expressions of PLD and prolactin mRNA were assessed by RT-PCR. Cd treated rats showed a decrease of PLD mRNA levels that can be associated to both high number of apoptotic cells and increase of S-100 protein expression in FSC. Cd decreased prolactin mRNA expression, number of lactotrophs and percentage of PRL-ir suggesting a low availability of prolactin to be secreted from PAL. Cd modifies the lactotrophs activity of pituitary gland through biochemical, genomic and morphological changes and contributes directly or indirectly to the levels of serum prolactin.

Zn-limited diet modifies the expression of the rate-regulatory enzymes involved in phosphatidylcholine and cholesterol synthesis.

Suboptimal intake of Zn is one of the most common nutritional worldwide problems. Previously, we showed that Zn deficiency produces alterations in lung lipid metabolism in rats. We studied the effect of a Zn-limited (ZL) diet on the expression of the enzymes involved in phosphatidylcholine and cholesterol synthesis. After 2 months of treatment with a ZL diet we found important variations in the lipid content of Wistar male rats: triacylglycerol (TG) decreased 60% (P<0.001) while esterified cholesterol (EC), free cholesterol and phospholipids (PL) increased 66%, 24 % and 25% respectively. We also observed a decrease of 40 % in the amount of (3)H incorporated into TG and an increase of 47% and 28% in the (3)H incorporated to PL and EC respectively. Fatty acid synthase and glucose-6-phosphate dehydrogenase activity was increased (P<0.01 and P<0.05 respectively). Glycerol-3-phosphate acyltransferase, lipoprotein lipase, diacyl glycerol acyl transferase and 3-hydroxy-3-methylglutaryl CoA reductase expression decreased (P<0.01 in all cases), while acetyl CoA carboxylase and cholinephosphate cytidylyltransferase increased (P<0.01 and P<0.005 respectively). These results suggest that ZL alters the expression of enzymes involved in phosphatidylcholine and cholesterol synthesis, which could lead to increased PL and cholesterol and decreased TG. This study suggests that major changes in the lipid composition of lung are induced by a ZL condition. Therefore, Zn deficiency must be taken into account in order to design therapies and public health interventions, such as Zn supplementation for high-risk subjects or certain diseases, such as asthma.

Overexpression of inducible nitric oxide synthase and cyclooxygenase-2 in rat zinc-deficient lung: Involvement of a NF-kappaB dependent pathway.

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of pulmonary diseases. The goal of this study was to measure the response of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 enzymes (COX-2) in lung with moderate zinc deficiency. Adult male Wistar rats were divided into two groups receiving (1) a zinc-deficient diet (ZD) or (2) a zinc-adequate control diet. After 2 months of treatment, the zinc-deficient group showed a significant pulmonary edema. This was associated to a reduction of protein thiols and to a significant increase of metallothionein and glutathione disulfide levels. In addition, a higher serum and lung NO production in ZD group was positively related to the higher activity and expression of iNOS and COX-2 found in lungs. Western blot analysis revealed increased IkappaBalpha degradation, an indicator of NF-kappaB activation in ZD lungs. Anatomopathologic analysis of ZD lungs showed an increase of connective tissue fibers with an influx of polymorphonuclear cells. These cells and type II cells from the alveoli showed specific immunohistochemical signals for iNOS. The conclusion is that, during the development of zinc-deficiency, iNOS activity increases in lung and contributes to lung injury. Zinc deficiency implications must be taken into account to design therapies and public health interventions involving targeted zinc supplementation for high-risk subjects or certain diseases, such as asthma.