HSP70 Inhibition Leads to the Activation of Proteasomal System under Mild Hyperthermia Conditions in Young and Senescent Fibroblasts.

Aging has been characterized with the accumulation of oxidized proteins, as a consequence of progressive decline in proteostasis capacity. Among others, proteasomal system is an efficient protein turnover complex to avoid aggregation of oxidized proteins. Heat shock protein 70 (HSP70) is another critical player that is involved in some key processes including the correct folding of misfolded proteins and targeting aggregated proteins to the proteasome for rapid degradation. The aim of this study was to determine the role of proteasomal system and heat shock proteins to maintain proteome balance during replicative senescence in mild hyperthermia conditions. Our results demonstrated that HSP40/70 machinery is induced by mild hyperthermia conditions independent from senescence conditions. Since HSP70 is largely responsible for the rapidly inducible cell protection following hyperthermia, the activation of "heat shock response" resulted in the elevation of HSP40/70 expressions as well as the proteasome activity. Interestingly, when HSP70 expression was inhibited, increased proteasomal activation was shown to be responsive to mild hyperthermia. Since HSP70 is involved in various stress-related pathways such as oxidative and endoplasmic reticulum stress, depletion of HSP70 expression may induce proteasomal degradation to maintain proteome balance of the cell. Thus, our data suggests that in mild heat stress conditions, molecular chaperone HSP70 plays an important role to avoid protein oxidation and aggregation; however, activities of proteasomal system are induced when HSP70 expression is depleted.

The effect of temporary aneurysm clip on the common carotid artery of atherosclerotic rabbits.

BACKGROUND: We compared the effect of temporary aneurysm clips on atherosclerotic and nonatherosclerotic CCA of rabbits by morphometric and ultrastructural methods. METHODS: The rabbits (N = 12) were divided into 2 groups: the first group was fed a 2% cholesterol diet, and the second group, a normal diet for 4 weeks. Atherosclerotic lesions developed after 4 weeks. Temporary aneurysm clips were placed on the left CCA of both groups; the right CCA of both groups served as control. Thus, a total of 4 groups were used: atherosclerotic (A), atherosclerotic/clip (AC), nonatherosclerotic (NA), and nonatherosclerotic/clip (NAC). Temporary aneurysm clips were applied for 1, 5, and 10 minutes in the AC and NAC groups. No temporary clip was placed on the right CCA (A and NA groups). The affected parts of the CCA via clips were examined under light microscope and SEM. RESULTS: Comparison of atherosclerotic and nonatherosclerotic CCA of rabbits under light microscope indicated that the wall of atherosclerotic CCA was thicker than that of nonatherosclerotic CCA. The difference between the thickness of atherosclerotic and nonatherosclerotic CCAs was significant. SEM analyses showed that in nonatherosclerotic CCAs, the effect of temporary aneurysm clips was seen after 10 minutes, but in atherosclerotic CCAs, the effect was seen within the 1st minute of clipping and continued in the 5th and 10th minutes. CONCLUSION: The duration of temporary clipping should be decreased for the neurovascular surgery of atherosclerotic patients.

Oxidative stress related changes in the brain of hypercholesterolemic rabbits.

In recent years, there has been increasing evidence that cholesterol plays a role in the pathology of Alzheimer disease. Since hypercholesterolemia was reported to increase the levels of reactive oxygen species and Alzheimer disease has clearly involved an oxidative component, it is possible that hypercholesterolemia is via increased oxidant production facilitating the disease development of the neurodegenerative disease. Therefore, we tested in an established model of enhanced cholesterol feed in rabbits the effects of serum cholesterol increase on oxidative stress parameters as well in serum as in the brain. In addition to that we tested the effects of vitamin E on the cholesterol-induced oxidative stress. Since Alzheimer disease is largely connected with increased protein oxidation whereas cholesterol is rather connected with lipid peroxidation processes, we tested both protein carbonyl levels and the formation of malondialdehyde, a marker of lipid peroxidation. We could clearly demonstrate an increase in serum malondialdehyde due to high cholesterol feeding, which is accompanied by an increase in protein oxidation parameters in the brain, especially in the hippocampus. Therefore, we suggest that specific neuropathological changes occur during the feeding of hypercholesterolemic diet.

Antitumor and antimetastatic effects of walnut oil in esophageal adenocarcinoma cells.

BACKGROUND: Walnuts contain many components including specific fatty acids, which could be active against cancer. Even though the anticarcinogenic effect of some of the individual fatty acids in walnut oil has been described, the effect of walnut oil itself on esophageal cancer cells hasn't yet been investigated. OBJECTIVE: We aimed to investigate whether walnut oil affects tumor growth and metastatic potential in esophageal cancer cells. METHODS: The human esophageal adenocarcinoma cell line, OE19, was treated with different doses of walnut oil and cell viability, apoptosis/necrosis and cell cycle analyses were performed using WST-1 assay and flow cytometry respectively. Adhesion, colony formation and wound healing assays were performed to assess the antimetastatic effects of walnut oil. NFkB expression was evaluated with western blot analysis. RESULTS: Walnut oil decreased the cell viability of esophageal cancer cells in a dose-dependent manner. 20 mg/mL walnut oil reduced cell viability by ∼50% when compared with control. The analysis revealed that necrosis and accumulation of cells in G0/G1 phase was induced in the cells treated with high doses of walnut oil. It also down-regulated the protein levels of NFkB. Walnut oil suppressed the adhesion, migration and colony formation of the cells. CONCLUSIONS: High-dose short-term administration of walnut oil reduces the cell viability and metastatic ability of esophageal cancer cells, while exhibiting anticarcinogenic effect by inducing necrosis and cell cycle arrest at the G0/G1 phase, probably through suppression of the NFkB pathway. These data indicate that walnut oil, and by extension walnut consumption, may have beneficial effects in esophageal cancer in humans. This should be tested by clinical trials in the future.

What evidence is there for the existence of individual genes with antagonistic pleiotropic effects?

Classical evolutionary theory predicts the existence of genes with antagonistic effects on longevity and various components of early-life fitness. Quantitative genetic studies have provided convincing evidence that such genes exist. However, antagonistic pleiotropic effects have rarely been attributed to individual loci. We examine several classes of longevity-assurance genes: those involved in regulation of the gonad; the insulin-like growth factor pathway; free-radical scavenging; heat shock proteins and apoptosis. We find initial evidence that antagonistic pleiotropic effects are pervasive in each of these classes of genes and in various model systems--although most studies lack explicit studies of fitness components. This is particularly true of human studies. Very little is known about the early-life fitness effects of longevity loci. Given the possible medical importance of such effects we urge their future study.

Homocysteine induces DNA synthesis and proliferation of vascular smooth muscle cells by interfering with MAPK kinase pathway.

Hyperhomocysteinemia has been identified as an important and independent risk factor for cerebral, coronary and peripheral atherosclerosis. However the mechanisms by which homocysteine promote atherosclerotic plaque formation are not clearly defined. Earlier reports have suggested that homocysteine exert its effect via the H2O2 produced during its metabolism. To evaluate which signalling molecules are involved in homocysteine induced atherosclerotic changes during the pathogenesis of vascular diseases, we examined homocysteine induced smooth muscle cell proliferation in the presence of different signal transduction inhibitors. We show that MAPK kinase pathway is involved in homocysteine induced DNA synthesis and proliferation of vascular smooth muscle cells in the presence of the peroxide scavenging enzyme, catalase. Our data suggest that homocysteine induces smooth muscle cell growth through a pathway that is independent of H2O2, that involves MAPK kinase activation, and that results in accelerated atherosclerosis.

Molecular mechanisms of cholesterol or homocysteine effect in the development of atherosclerosis: Role of vitamin E.

The development of atherosclerosis is a multifactorial process in which both elevated plasma cholesterol levels and proliferation of smooth muscle cells play a central role. Numerous studies have suggested the involvement of oxidative processes in the pathogenesis of atherosclerosis and especially of oxidized low density lipoprotein. Some epidemiological studies have shown an association between high dietary intake and high serum concentrations of vitamin E and lower rates of ischemic heart disease. Cell culture studies have shown that alpha-tocopherol brings about inhibition of smooth muscle cell proliferation. This takes place via inhibition of protein kinase C activity. alpha-Tocopherol also inhibits low density lipoprotein induced smooth muscle cell proliferation and protein kinase C activity. The following animal studies showed that vitamin E protects development of cholesterol induced atherosclerosis by inhibiting protein kinase C activity in smooth muscle cells in vivo. Elevated plasma levels of homocysteine have been identified as an important and independent risk factor for cerebral, coronary and peripheral atherosclerosis. However the mechanisms by which homocysteine promotes atherosclerotic plaque formation are not clearly defined. Earlier reports have been suggested that homocysteine exert its effect via H2O2 produced during its metabolism. To evaluate the contribution of homocysteine in the pathogenesis of vascular diseases, we examined whether the homocysteine effect on vascular smooth muscle cell growth is mediated by H2O2. We show that homocysteine induces DNA synthesis and proliferation of vascular smooth muscle cells in the presence of peroxide scavenging enzyme, catalase. Our data suggest that homocysteine induces smooth muscle cell growth through the activation of an H2O2 independent pathway and accelerate the progression of atherosclerosis. The results indicate a cellular mechanism for the atherogenicity of cholesterol or homocysteine and protective role of vitamin E in the development of atherosclerosis.

CD36 as a biomarker of atherosclerosis.

Atherosclerosis is a chronic inflammatory disorder occurs as a result of mononuclear lymphocyte infiltration to the arterial wall accompanied by smooth muscle cell proliferation and damage in the arterial wall caused by extracellular matrix accumulation. Besides several genetic and environmental factors, increased serum cholesterol and oxidized low density lipoproteins are considered to be major risk factors of the disease. During atherosclerosis, lipoproteins such as LDL become trapped at the site of lesion and are converted to oxLDL. Smooth muscle cells become activated by oxLDL, start to proliferate, and migrate into the intima of the arterial wall. OxLDL provokes a cascade of cellular responses at the atherosclerotic lesion, ultimately leading to formation of atherosclerotic plaques. In this process, scavenger receptors could play a critical role because of their ability to bind oxLDL and their function in transporting lipids and cholesterol into and out of the cells. Scavenger receptors are expressed on macrophages and foam cells in atherosclerotic lesions. CD36 is the most important one among them playing role in atherosclerotic process. CD36 is a raft associated glycosylated protein with an 88kDa molecular weight and various ligands such as oxLDL, apoptotic cells, advanced glycation end products bind to this receptor. It has been shown that mice knockouts for the apolipoprotein E exhibited a marked decrease in atherosclerotic lesions if CD36 gene was made inactive. Previously we have shown that the aortas of cholesterol-fed rabbits showed typical atherosclerotic lesions, detected by macroscopic and microscopic examination, and exhibited an increase in CD36 mRNA expression. In this study, we planned to compare CD36 mRNA expressions in the aortic tissue and peripheral blood mononuclear cells in cholesterol induced atherosclerosis. Our results suggests that CD36 mRNA levels in peripheral blood mononuclear cells reflect the levels in aorta and this might be used as a marker for diagnosis of atherosclerosis.

The role of hypercholesterolemic diet and vitamin E on Nrf2 pathway, endoplasmic reticulum stress and proteasome activity.

Hypercholesterolemia is the major risk factor for the development of atherosclerosis and vitamin E is suggested to have a preventive role in this process (1), although the mechanism of action still remains unclear.The ubiquitin-proteasome system (UPS) may in?uence atherosclerosis by affecting disease-relevant cellular processes such as apoptosis, proliferation, and differentiation, or by affecting cellular stress responses and/or adaptive phenomena, such as ER stress, in?ammation, and redox homeostasis (2). NF-E2-related factor 2 (Nrf2) is a transcription factor that controls the expression of phase II detoxi?cation and antioxidant genes. Nrf2 signaling has additionally been shown to upregulate the expression of the proteasome catalytic subunits (3). In the present study, we investigated the role of Nrf2 pathway on oxidative and ER stress conditions induced by cholesterol diet and the effects of vitamin E on related signaling pathways in in vivo model of atherosclerosis. All experimental procedures were approved by the Marmara University Ethics Committee. Twenty-one male albino rabbits (23 months old) were assigned randomly to four groups fed for 8 weeks: (i) vitamin E deficient diet, (ii) vitamin E deficient diet containing 2% cholesterol, and (iii) vitamin E deficient diet containing 2% cholesterol with daily intramuscular injections of vitamin E (50mg/kg), (iv) vitamin E deficient diet with daily intramuscular injections of vitamin E (50mg/kg). In order to elucidate in vivo role of oxidative stress and ER stress in cardiovascular system of hypercholesterolemic rabbits, we investigated serum levels of cholesterol, MDA and vitamin E and Nrf2, GST-1, GRP78, GRP94, PERK, IRE1 protein levels and the proteasomal activity in aortic tissues will be discussed.

Heat shock proteins and proteasomal degradation in normal and tumor cells.

Proteasomal degradation of oxidized proteins is a crucial mechanism to prevent the accumulation of cellular damage. The removal of the damage is generally a required process for healthy organisms to keep the integrity while in cancer cells the situation may be different. In normal conditions, cancer cells have higher proteasome activity compared to normal cells. During cancer treatment, cellular damage by chemotherapy is an expected process to be able to kill the tumor cells. And the accumulation of this damage accompanied by the decrease in protein repair and removal systems may increase the efficacy of the cancer therapy. Heat shock proteins (Hsp) as molecular chaperones are involved in the folding, activation and assembly of a variety of proteins. Among these Hsp40, Hsp70 and Hsp90 are believed to act as a chaperone system to regulate the proteasomal degradation. In this study, we tested the role of heat stress response on the proteasomal degradation of oxidized proteins. We used two different cell lines to observe the difference in normal and tumor cells. First the effect of heat stress (42°C, 1h) were tested in terms of protein oxidation tested by protein carbonyl formation and proteasomal degradation. The results were extremely different in normal fibroblast cells and hippocampal tumor cells. In the same direction, the expressions of Hsp40, Hsp70 and Hsp90 were affected in a different manner in two cell lines, will be discussed in detail. Supported by TUBITAK COST-CM1001-110S281.

The effect of Irisin on antioxidant system in liver.

Nonalcoholic fatty liver disease (NAFLD) is a global health problem and lead to subacute liver failure, cirrhosis and/or hepatocellular carcinoma. An increased generation of reactive oxygen species (ROS) and antioxidant depletion is found in the liver of obese patients with NAFLD. Irisin is a recently identified exercise-induced myokine. It increases total energy consumption, reduces body weight, and insulin resistance. It was shown that irisin levels were significantly lower in patients with NAFLD. The aim of the present study was to investigate the effect of irisin on prooxidant-antioxidant balance in liver. In the first phase; AML12 liver cells were divided into 4 groups: control, hydrogen peroxide (H2O2)-treated, 10nM irisin-treated and 50nM irisin-treated groups. ROS accumulation in these groups was analyzed by FACS. In the second phase; to see if there is any protective role of irisin on ROS production in the liver, AML12 liver cells were divided into 4 groups: control, H2O2 -treated, H2O2+10nM irisin-treated and H2O2+50nM-irisin treated groups. After measuring ROS accumulation again in these groups, the levels of enzymes related with prooxidant-antioxidant balance via oxidative stress in liver were measured by western blotting. In H2O2 treatment groups, ROS production was increased in AML12 liver cells, on the other hand in irisin treatment groups ROS production was slightly changed. Irisin might be a potential target for metabolic diseases like NAFLD.

The effect of resveratrol on signal transduction pathways and the role of pro-apoptotic Bax protein on apoptosis in HCT- 116 colon carcinoma cell lines.

Colon carcinoma is the third among the cancer related deaths. The role of pro-apoptotic Bax protein on the resveratrol related apoptosis, mitochondrial membrane potential and signal pathways has not been identified in colon carcinoma cells. In this direction, HCT-116 bax positive and HCT-116 negative cell lines were utilized to detect the apoptotic effect and I?B, MEK1 and STAT 3 signal transduction pathways of resveratrol. The impact on the cell viability and IC50 value of resveratrol has been determined via WST-1 viability assay. The ratio of apoptosis has been evaluated via flow cytometry following Annexin V/Propidium iodide (PI) double staining. Changes in the mitochondrial membrane potential have been analyzed by flow cytometry and JC-1 fluorometric staining. IkB, MEK1 and STAT3 molecules were measured by Enspire device. Data showed the IC50 value for resveratrol as 50M. According to the flow cytometry, apoptosis ratio has been determined as 29.65% in the experimental group of bax positive cells, as 13.98% in the experimental group of bax negative cells. Changes in the membrane potential has been established as 8.62% in the experimental group of bax positive cells, as 97.98% in the experimental group of bax negative cells. When the obtained data from Enspire device was reviewed; bax positive cells I?B phosphorylations were found as as 5.22 for experimental groups; MEK1 phosphorylations were found as and as 1.15 for experimental groups; STAT 3 phosphorylations were found as and as 2.52 for experimental groups. In HCT-116 bax negative cells, I?B phosphorylation were and 2.71 in experimental groups; MEK1 phosphorylation were 1.18 in experimental groups; STAT 3 phosphorylation were 1.54 in experimental groups. Our data show that Bax protein plays role in the apoptotic effect of resveratrol by altering mitochondrial membrane potential and mitochondrial membrane permeability, signal transduction and the absence of Bax increase the sensitivity of HCT-116 colon carcinoma cells to apoptosis.

Lipid rafts and redox regulation of cellular signaling in cholesterol induced atherosclerosis.

Redox mediated signaling mechanisms play crucial roles in the pathogenesis of several cardiovascular diseases. Atherosclerosis is one of the most important disorders induced mainly by hypercholesterolemia. Oxidation products and related signaling mechanisms are found within the characteristic biomarkers of atherosclerosis. Several studies have shown that redox signaling via lipid rafts play a significant role in the regulation of pathogenesis of many diseases including atherosclerosis. This review attempts to summarize redox signaling and lipid rafts in hypercholesterolemia induced atherosclerosis.