Evaluation of capillary leakage after vasopressin resuscitation in a hemorrhagic shock model.

Background: Hemorrhagic shock (HS) is a major threat to patients with trauma and spontaneous bleeding. The aim of the study was to investigate early effects of vasopressin on metabolic and hemodynamic parameters and endothelium permeability by measuring capillary leakage compared to those of other resuscitation strategies in a HS model. Methods: Forty-five Sprague-Dawley rats were randomized into five groups: S group (n = 5), sham-operated rats without shock or resuscitation; HS group (n = 10), HS and no resuscitation; RL group (n = 10), HS and resuscitation with Ringer's lactate (RL); RLB group (n = 10), HS and resuscitation with two-third shed blood plus RL; and vasopressin group (n = 10), HS and resuscitation with RL, followed by continuous infusion of 0.04 U/kg/min vasopressin. The effects of resuscitation on hemodynamic parameters [mean arterial pressure (MAP), superior mesenteric artery blood flow (MBF), and mesenteric vascular resistances (MVR)], arterial blood gases, bicarbonate, base deficit, and lactate levels as well as on capillary leakage in the lung, ileum, and kidney were investigated. Capillary leakage was evaluated with Evans blue dye extravasation. Results: In the vasopressin group, the MAP was higher than in the RL and RLB groups (p < 0.001), while MBF was decreased (p < 0.001). MVR were increased only in the vasopressin group (p < 0.001). Capillary leakage was increased in the lungs of the animals in the vasopressin group compared to that in the lungs of animals in the RLB group (p < 0.05); this increase was associated with the lowest partial pressure of oxygen (p < 0.05). Conversely, decreased capillary leakage was observed with vasopressin in the ileum (p < 0.05). Increased capillary leakage was observed in the kidney in the RLB and vasopressin groups (p < 0.05). Lastly, vasopressin use was associated with higher base deficit and lactate levels when compared to the RL and RLB groups (p < 0.001). Conclusion: Although vasopressin was proposed as a vasoactive drug for provisional hemodynamic optimization in the early phase of HS resuscitation, the overall findings of this experimental study focus on the possible critical side effects of vasopressin on metabolic parameters and endothelium permeability.

The Omega-3 Fatty Acid Docosahexaenoic Acid Modulates Inflammatory Mediator Release in Human Alveolar Cells Exposed to Bronchoalveolar Lavage Fluid of ARDS Patients.

BACKGROUND: This study investigated whether the 1 : 2 ω-3/ω-6 ratio may reduce proinflammatory response in human alveolar cells (A549) exposed to an ex vivo inflammatory stimulus (bronchoalveolar lavage fluid (BALF) of acute respiratory distress syndrome (ARDS) patients). Methods. We exposed A549 cells to the BALF collected from 12 ARDS patients. After 18 hours, fatty acids (FA) were added as docosahexaenoic acid (DHA, ω-3) and arachidonic acid (AA, ω-6) in two ratios (1 : 2 or 1 : 7). 24 hours later, in culture supernatants were evaluated cytokines (TNF-α, IL-6, IL-8, and IL-10) and prostaglandins (PGE2 and PGE3) release. The FA percentage content in A549 membrane phospholipids, content of COX-2, level of PPARγ, and NF-κB binding activity were determined. RESULTS: The 1 : 2 DHA/AA ratio reversed the baseline predominance of ω-6 over ω-3 in the cell membranes (P < 0.001). The proinflammatory cytokine release was reduced by the 1 : 2 ratio (P < 0.01 to <0.001) but was increased by the 1 : 7 ratio (P < 0.01). The 1 : 2 ratio reduced COX-2 and PGE2 (P < 0.001) as well as NF-κB translocation into the nucleus (P < 0.01), while it increased activation of PPARγ and IL-10 release (P < 0.001). Conclusion. This study demonstrated that shifting the FA supply from ω-6 to ω-3 decreased proinflammatory mediator release in human alveolar cells exposed to BALF of ARDS patients.

Unacylated ghrelin induces oxidative stress resistance in a glucose intolerance and peripheral artery disease mouse model by restoring endothelial cell miR-126 expression.

Reactive oxygen species (ROS) are crucial in long-term diabetes complications, including peripheral artery disease (PAD). In this study, we have investigated the potential clinical impact of unacylated ghrelin (UnAG) in a glucose intolerance and PAD mouse model. We demonstrate that UnAG is able to protect skeletal muscle and endothelial cells (ECs) from ROS imbalance in hind limb ischemia-subjected ob/ob mice. This effect translates into reductions in hind limb functional impairment. We show that UnAG rescues sirtuin 1 (SIRT1) activity and superoxide dismutase-2 (SOD-2) expression in ECs. This leads to SIRT1-mediated p53 and histone 3 lysate 56 deacetylation and results in reduced EC senescence in vivo. We demonstrate, using small interfering RNA technology, that SIRT1 is also crucial for SOD-2 expression. UnAG also renews micro-RNA (miR)-126 expression, resulting in the posttranscriptional regulation of vascular cell adhesion molecule 1 expression and a reduced number of infiltrating inflammatory cells in vivo. Loss-of-function experiments that target miR-126 demonstrate that miR-126 also controls SIRT1 and SOD-2 expression, thus confirming its role in driving UnAG-mediated EC protection against ROS imbalance. These results indicate that UnAG protects vessels from ROS imbalance in ob/ob mice by rescuing miR-126 expression, thus emphasizing its potential clinical impact in avoiding limb loss in PAD.

Unacylated ghrelin promotes skeletal muscle regeneration following hindlimb ischemia via SOD-2-mediated miR-221/222 expression.

BACKGROUND: Surgical treatment of peripheral artery disease, even if successful, does not prevent reoccurrence. Under these conditions, increased oxidative stress is a crucial determinant of tissue damage. Given its reported antioxidant effects, we investigated the potential of unacylated-ghrelin (UnAG) to reduce ischemia-induced tissue damage in a mouse model of peripheral artery disease. METHODS AND RESULTS: We show that UnAG but not acylated ghrelin (AG) induces skeletal muscle regeneration in response to ischemia via canonical p38/mitogen-actived protein kinase signaling UnAG protected against reactive oxygen species-induced cell injuries by inducing the expression of superoxide dismutase-2 (SOD-2) in satellite cells. This led to a reduced number of infiltrating CD68(+) cells and was followed by induction of the myogenic process and a reduction in functional impairment. Moreover, we found that miR-221/222, previously linked to muscle regeneration processes, was up-regulated and negatively correlated with p57(Kip2) expression in UnAG-treated mice. UnAG, unlike AG, promoted cell-cycle entry in satellite cells of mice lacking the genes for ghrelin and its receptor (GHSR1a). UnAG-induced p38/mitogen-actived protein kinase phosphorylation, leading to activation of the myogenic process, was prevented in SOD-2-depleted SCs. By siRNA technology, we also demonstrated that SOD-2 is the antioxidant enzyme involved in the control of miR-221/222-driven posttranscriptional p57(Kip2) regulation. Loss-of-function experiments targeting miR-221/222 and local pre-miR-221/222 injection in vivo confirmed a role for miR-221/222 in driving skeletal muscle regeneration after ischemia. CONCLUSIONS: These results indicate that UnAG-induced skeletal muscle regeneration after ischemia depends on SOD-2-induced miR-221/222 expression and highlight its clinical potential for the treatment of reactive oxygen species-mediated skeletal muscle damage.

Increase of palmitic acid concentration impairs endothelial progenitor cell and bone marrow-derived progenitor cell bioavailability: role of the STAT5/PPARγ transcriptional complex.

Metabolic profiling of plasma nonesterified fatty acids discovered that palmitic acid (PA), a natural peroxisome proliferator-activated receptor γ (PPARγ) ligand, is a reliable type 2 diabetes biomarker. We investigated whether and how PA diabetic (d-PA) concentrations affected endothelial progenitor cell (EPC) and bone marrow-derived hematopoietic cell (BM-HC) biology. PA physiologic (n-PA) and d-PA concentrations were used. Proliferating cell nuclear antigen content and signal transducer and activator of transcription 5 (STAT5), PPARγ, cyclin D1, and p21(Waf) expression were evaluated. Small interfering RNA technology, gene reporter luciferase assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and coimmunoprecipitation were exploited. In vivo studies and migration assays were also performed. d-PA, unlike n-PA or physiological and diabetic oleic and stearic acid concentrations, impaired EPC migration and EPC/BM-HC proliferation through a PPARγ-mediated STAT5 transcription inhibition. This event did not prevent the formation of a STAT5/PPARγ transcriptional complex but was crucial for gene targeting, as p21(Waf) gene promoter, unlike cyclin D1, was the STAT5/PPARγ transcriptional target. Similar molecular events could be detected in EPCs isolated from type 2 diabetic patients. By expressing a constitutively activated STAT5 form, we demonstrated that STAT5 content is crucial for gene targeting and EPC fate. Finally, we also provide in vivo data that d-PA-mediated EPC dysfunction could be rescued by PPARγ blockade. These data provide first insights on how mechanistically d-PA drives EPC/BM-HC dysfunction in diabetes.

Des-acyl ghrelin fragments and analogues promote survival of pancreatic ß-cells and human pancreatic islets and prevent diabetes in streptozotocin-treated rats.

Des-acyl ghrelin, although devoid of binding to ghrelin receptor (GRLN), exerts many biological effects, including regulation of glucose and lipid metabolism. Indeed, des-acyl ghrelin promotes pancreatic ß-cell and human islet cell survival and prevents diabetes in streptozotocin (STZ) treated rats. We investigated whether des-acyl ghrelin fragments excluding serine(3), which is essential for binding to GRLN, would display similar actions. Among the different compounds tested, des-acyl ghrelin((6-13)) and des-acyl ghrelin((6-13)) with alanine substitutions or cyclization, but not with d-amino acid substitutions, showed the best survival effect, similar to des-acyl ghrelin. Des-acyl ghrelin((6-13)) even prevented diabetes in STZ-treated rats and protected human circulating angiogenic cells from oxidative stress and senescence, similar to des-acyl ghrelin. These results suggest that not only full-length des-acyl ghrelin but also short des-acyl ghrelin fragments have clear beneficial effects on several tissues in vitro and in vivo.

High glucose via NOX-dependent ROS generation and AKT activity promotes adipose-derived stem cell de-differentiation.

NADPH-oxidase (NOX)-dependent reactive oxygen species (ROS) production is involved in self-renewal of stem and progenitor cells. Herein, we investigated whether high glucose (25 mM/L) (HG)-dependent NOX-mediated ROS generation is involved in self-renewal of visceral adipose tissue-derived stem cells (ASCs) as well. To this end ASCs cultured in HG or normal glucose (5 mM/L) used as control, were evaluated for their stem cell identity. We demonstrated that freshly isolated ASCs are pluripotent as they differentiate into adipocytes in-vitro and form neovessels in-vivo. However, only HG-cultured ASCs expressed octamer-binding transcription factor 4 (Oct4) and Nanog and formed spheroids. The assembly of p47phox and p67phox subunits is crucial for NOX-enzymatic activity. By knock-down of p47phox the role of NOX-generated ROS in driving ASC de-differentiation has been provided. siRNA technology was also applied to demonstrate the role of Akt activity in mediating HG-induced Oct-4 and Nanog expression as well as spheroid formation. Additionally, by knock-down of Oct4 we provided further evidence that Oct4 is essential for HG-mediated stem cell identity. Soluble factors released by ASCs are key elements in their mechanism of action. We found that NOX and Akt activity are required for cytokine production by "spheroids". Finally, as HG-cultured ASCs, diabetic patient-derived ASCs expressed higher levels of Oct-4 and Nanog than ASCs derived from healthy subjects and engaged ROS and Akt activity to turn on their secretion program. Thereby, our data indicate that HG via NOX-dependent Akt activity induces ASC de-differentiation, and suggest that HG pre-conditioning might be exploited for ASC ex-vivo expansion.

Impact of the omega-3 to omega-6 polyunsaturated fatty acid ratio on cytokine release in human alveolar cells.

BACKGROUND: ω-3 polyunsaturated fatty acids (PUFAs) and ω-6 PUFAs have opposing influences on inflammation. The objective was to determine whether lipopolysaccharide (LPS)-induced cytokine release by human alveolar cells was affected by changes in the ω-3/ω-6 ratio of cell membranes induced by different supplies of PUFAs. METHODS: After LPS challenge, PUFAs were added to alveolar cells as docosahexaenoic acid (DHA, ω-3) and arachidonic acid (AA, ω-6) in 4 different DHA/AA ratios (1:1, 1:2, 1:4, and 1:7), and the effects on cytokine release were measured. RESULTS: The supply of 1:1 and 1:2 DHA/AA ratios reversed the baseline predominance of ω-6 over ω-3 in the ω-3/ω-6 PUFA ratio of cell membranes. The release of proinflammatory cytokines (tumor necrosis factor α, interleukin-6, and interleukin-8) was reduced by 1:1 and 1:2 DHA/AA ratios (P < .01 to P < .001) but increased by 1:4 and 1:7 DHA/AA ratios (P < .01 to P < .001) vs control. The 1:1 and 1:2 ratios increased the release of anti-inflammatory interleukin-10 (P < .001). The balance between proinflammatory and anti-inflammatory cytokines showed an anti-inflammatory response with 1:1 and 1:2 ratios and a proinflammatory response with 1:4 and 1:7 ratios (P < .001). CONCLUSIONS: This study showed that proinflammatory cytokine release was dependent on the proportion of ω-3 in the ω-3/ω-6 ratio of alveolar cell membranes, being reduced with the supply of a high proportion of DHA and increased with a high proportion of AA, respectively. These results support the biochemical basis for current recommendations to shift the PUFA supply from ω-6 to ω-3 in nutrition support of patients with acute lung injury.

Unacylated ghrelin rescues endothelial progenitor cell function in individuals with type 2 diabetes.

OBJECTIVE: Acylated ghrelin (AG) is a diabetogenic and orexigenic gastric polypeptide. These properties are not shared by the most abundant circulating form, which is unacylated (UAG). An altered UAG/AG profile together with an impairment of circulating endothelial progenitor cell (EPC) bioavailability were found in diabetes. Based on previous evidence for the beneficial cardiovascular effects of AG and UAG, we investigated their potential to revert diabetes-associated defects. RESEARCH DESIGN AND METHODS: Healthy human subjects, individuals with type 2 diabetes, and ob/ob mice were AG or UAG infused. EPC mobilization in patients and mice was evaluated, and the underlying molecular mechanisms were investigated in bone marrow stromal cells. Recovered EPCs were also evaluated for the activity of senescence regulatory pathways and for NADPH oxidase activation by knocking down p47(phox) and Rac1. Finally, UAG modulation of human EPC vasculogenic potential was investigated in an in vivo mouse model. RESULTS: Neither AG nor UAG had any effect in healthy subjects. However, systemic administration of UAG, but not AG, prevented diabetes-induced EPC damage by modulating the NADPH oxidase regulatory protein Rac1 and improved the vasculogenic potential both in individuals with type 2 diabetes and in ob/ob mice. In addition, unlike AG, UAG facilitated the recovery of bone marrow EPC mobilization. Crucial to EPC mobilization by UAG was the rescue of endothelial NO synthase (eNOS) phosphorylation by Akt, as UAG treatment was ineffective in eNOS knockout mice. Consistently, EPCs expressed specific UAG-binding sites, not recognized by AG. CONCLUSIONS: These data provide the rationale for clinical applications of UAG in pathologic settings where AG fails.

Pyrrolidine dithiocarbamate modulates HSP70, iNOS, and apoptosis during hemorrhagic shock resuscitation in rats.

INTRODUCTION: The aim of this study is to evaluate whether hemorrhage and resuscitation affect liver, intestinal, and renal expressions of heat shock protein 70 (HSP70), inducible nitric oxide synthase (iNOS), and apoptosis indexes (TUNEL, caspase-3 activation) and whether the expression of these proteins can be modulated by pyrrolidine dithiocarbamate (PDTC) after a nonlethal hemorrhagic shock (HS) in rats. METHODS: Forty rats were randomized into four groups: sham-operated, only HS, HS/resuscitation with blood plus normal saline (NS), and HS/resuscitation with blood/NS plus PDTC, 15 mg/kg body weight, intravenously. Rats were subjected to HS by blood removal to a mean arterial pressure of 35-40 mmHg through the femoral artery. After 1 hr of shock, the animals were resuscitated according to the experimental protocol. HSP70, iNOS, cleaved caspase-3 expression, and TUNEL were analyzed in liver, small intestine, and kidney 3 hr after resuscitation. RESULTS: HS upregulated HSP70, iNOS, cleaved caspase-3 expression, and induced apoptosis (TUNEL) (p < .05 to < .001). Resuscitation was not associated with further increase of their expressions. The administration of PDTC during resuscitation decreased liver, intestinal, and renal activation of iNOS and apoptosis indexes (p < .05 to < .001), and was associated with further increase in HSP70 expression (p < .05). CONCLUSIONS: Our results show that HS resuscitation with PDTC modulates several signaling pathways (HSP70, iNOS, TUNEL, and caspase-3) in a rat model. The results suggest that PDTC administration--by reducing apoptosis and iNOS expression--may have a potential role in minimizing organ damage after severe hemorrhage.

Decreased polyunsaturated Fatty Acid content contributes to increased survival in human colon cancer.

Among diet components, some fatty acids are known to affect several stages of colon carcinogenesis, whereas others are probably helpful in preventing tumors. In light of this, our aim was to determine the composition of fatty acids and the possible correlation with apoptosis in human colon carcinoma specimens at different Duke's stages and to evaluate the effect of enriching human colon cancer cell line with the possible reduced fatty acid(s). Specimens of carcinoma were compared with the corresponding non-neoplastic mucosa: a significant decrease of arachidonic acid, PPARalpha, Bad, and Bax and a significant increase of COX-2, Bcl-2, and pBad were found. The importance of arachidonic acid in apoptosis was demonstrated by enriching a Caco-2 cell line with this fatty acid. It induced apoptosis in a dose- and time-dependent manner via induction of PPARalpha that, in turn, decreased COX-2. In conclusion, the reduced content of arachidonic acid is likely related to carcinogenic process decreasing the susceptibility of cancer cells to apoptosis.

Formation of STAT5/PPARgamma transcriptional complex modulates angiogenic cell bioavailability in diabetes.

OBJECTIVE: Circulating angiogenic cells (CACs) expansion is a multistage process requiring sequential activation of transcriptional factors, including STAT5. STAT5, in concert with peroxisome proliferator-activated receptors (PPARs), seems to induce discrete biological responses in different tissues. In the present study we investigated the role of STAT5 and PPARgamma in regulating CAC expansion in normal and diabetic settings. METHODS AND RESULTS: Normal and diabetic CACs were used. siRNA technology, EMSA, and chromatin immunoprecipitation (ChIP) assay as well as site-directed mutagenesis of the STAT5 response element in the PPARgamma promoter enabled us to demonstrate that STAT5 transcriptional activity controls PPARgamma expression. Moreover, FACS analysis, coimmunoprecipitation experiments, and ChIP assay revealed that a STAT5/PPARgamma transcriptional complex controls cyclin D1 expression and CAC progression into the cell-cycle. Conversely, PPARgamma agonists, by preventing the expression of STAT5 and the formation of the STAT5/PPARgamma heterodimeric complex failed to promote CAC expansion. Finally, we demonstrated that diabetic CAC functional capability can be recovered by molecules able to activate the STAT5/PPARgamma transcriptional complex. CONCLUSIONS: Our data identify the STAT5/PPARgamma heterodimers as landmark of CAC expansion and provide evidences for a mechanism that partially rescues CAC bioavailability in diabetic setting.

Interleukin-3 promotes expansion of hemopoietic-derived CD45+ angiogenic cells and their arterial commitment via STAT5 activation.

Interleukin-3 (IL-3) released by infiltrating inflammatory cells in different pathologic settings contributes to organ and tumor angiogenesis. Here we demonstrate that IL-3 expands a subset of CD45+ circulating angiogenic cells clonally derived from the hemopoietic progenitors. Moreover, CD45+ cells exposed to IL-3 acquire arterial specification and contribute to the formation of vessels in vivo. Depletion of signal transducer and activator of transcription 5 (STAT5) provides evidence that IL-3-mediated cell expansion and arterial morphogenesis rely on STAT5 activation. In addition, by means of Tie2-transgenic mice, we demonstrate that STAT5 also regulates IL-3-induced expansion and arterial specification of bone marrow-derived CD45+ cells. Thus, our data provide the first evidence that, in inflammatory microenvironments containing IL-3, angiogenic cells derived from hemopoietic precursors can act as adult vasculogenic cells. Moreover, the characterization of the signaling pathway regulating these events provides the rationale for therapeutically targeting STAT5 in these pathologic settings.

TNF-alpha TGF-beta2 and IL-1beta levels in gingival and peri-implant crevicular fluid before and after de novo plaque accumulation.

AIMS: The aim of this split-mouth study was to investigate levels of tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta2) and interleukin-1 beta (IL-1beta) in gingival crevicular fluid (GCF) and peri-implant crevicular fluid (PICF) after a 21-day-period of de novo plaque accumulation in the same patient. MATERIAL AND METHODS: In 25 patients, samples of GCF and PICF were collected in the sulcus of the tooth and of the implant after professional hygiene. After the no-hygiene phase (21 days), second samples of GCF and PICF were taken. Third samples were collected after 69 days of re-establishment oral hygiene techniques. The crevicular fluids were used to determine the volume and the levels of TNF-alpha, TGF-beta2 and IL-1beta. RESULTS: The volume of the crevicular fluids increased significantly after 21 days of plaque accumulation around teeth and implants and decreased significantly by 69 days. TNF-alpha and TGF-beta2 did not change significantly among the three different samples. A significant increase of IL-1beta was observed after plaque accumulation around the teeth GCF, whereas in the PICF the increase was not statistically significant. CONCLUSIONS: These data suggest that increased volumes of GCF and PICF could be useful markers of early inflammation in gingival and peri-implant tissues. In the presence of de novo plaque, implants showed lower, and nearly significant, levels of IL-1beta compared with teeth.

Effects of dimethyl sulfoxide, pyrrolidine dithiocarbamate, and methylprednisolone on nuclear factor-kappaB and heat shock protein 70 in a rat model of hemorrhagic shock.

BACKGROUND: Nuclear factor kappa B (NF-kappaB) is a transcription factor involved in the inflammatory response. Heat shock protein 70 (HSP70) is involved in the cell protection from various stresses. The aim of this study was to evaluate the effects of dimethyl sulfoxide (DMSO), pyrrolidine dithiocarbamate (PDTC), and methylprednisolone (MP) on liver, renal, and intestinal activation of NF-kappaB and HSP70 in a rat model of hemorrhagic shock (HS). METHODS: Sixty rats were randomized in 6 groups: sham-operated; only HS; HS and resuscitation with blood plus normal saline (NS); HS and resuscitation with blood/NS and 6 mg/kg DMSO; HS and resuscitation with blood/NS and 100 mg/kg PDTC; HS and resuscitation with blood/NS and 30 mg/kg MP. Rats were subjected to HS by blood removal to a mean arterial pressure of 35 to 40 mm Hg through the femoral artery. After 1-hour shock-period, the animals were resuscitated according to the experimental protocol. NF-kappaB and HSP70 expression in liver, kidney, and small intestine was analyzed 1 and 3 hours after resuscitation by immunohistochemistry. RESULTS: HS upregulated NF-kappaB activation and HSP70 expression (p < 0.05). Resuscitation was not associated with a further increase in NF-kappaB and HSP70 activation. DMSO, PDTC, and MP administration resulted in a decreased liver, renal, and intestinal activation of NF-kappaB associated with an increase of HSP70 expression (p < 0.05). CONCLUSIONS: Our results suggest that treatment with DMSO, PDTC, and MP can modulate the expression of NF-kappaB and HSP70 after HS in rats. This modulation may have potential effects in HS through inhibition of the NF-kappaB-dependent production of proinflammatory mediators.

PPARalpha and PP2A are involved in the proapoptotic effect of conjugated linoleic acid on human hepatoma cell line SK-HEP-1.

Conjugated linoleic acid (CLA), found in dairy products, in beef and lamb has been demonstrated to possess anticancer properties protecting several tissues from developing cancer. Moreover, it has been shown to modulate apoptosis in several cancer cell lines. The aim of this study was to investigate which signaling transduction pathways were modulated in CLA-induced apoptosis in human hepatoma SK-HEP-1 cells. The cells exposed to CLA were evaluated for PPARalpha, PP2A, pro-apoptotic proteins Bak, Bad and caspases, and anti-apoptotic proteins Bcl-2 and Bcl-X(L). Cells were also treated with okadaic acid, a PP2A inhibitor, or with Wy-14643, a specific PPARalpha agonist. The CLA-induced apoptosis was concomitant to the increase of percentage of cells in the S phase, PPARalpha, PP2A and pro-apoptotic proteins; simultaneously, antiapoptotic proteins decreased. Inhibition of PP2A prevented apoptosis, and PPARalpha agonist showed similar effect as CLA. The increased PP2A could be responsible for the dephosphorylation of Bcl-2 and Bad, permitting apoptotic activity of Bax and Bad. The increase of caspase 8 and 9 suggested that both the intrinsic and extrinsic apoptotic pathways were induced. PP2A was probably increased by PPARalpha, since putative PPRE sequences were found in genes encoding its subunits. In conclusion, CLA induces apoptosis in human hepatoma SK-HEP-1 cells, by increasing PPARalpha, PP2A and pro-apoptotic proteins.

Arachidonic and docosahexaenoic acids reduce the growth of A549 human lung-tumor cells increasing lipid peroxidation and PPARs.

Polyunsaturated fatty acids (PUFAs) play an important role in both induction and prevention of carcinogenic process. It is well known that several types of neoplastic cells show decreased total PUFA content, contributing to their resistance to chemotherapy and lipid peroxidation. In the light of this, human lung cancer A549 cells, with low PUFA content, were exposed to arachidonic or docosahexaenoic acid to investigate the effect of n-6 and n-3 PUFAs on growth and elucidate underlying mechanisms. The bulk of the results showed that both n-6 PUFAs and n-3 PUFAs decrease human lung-tumor cell growth in a concentration-dependent manner, inducing cell death mainly evident at 100microM concentration. The mechanism underlying the antiproliferative effect of n-6 and n-3 PUFAs appeared to be the same, involving changes in fatty acid composition of biomembranes, production of cytostatic aldehydes derived from lipid peroxidation and able to affect DNA-binding activity of AP-1, and induction of PPAR. From these results it may be hypothesized that n-6 PUFAs, like n-3 PUFAs, are able to inhibit tumor growth.

Arachidonic acid suppresses growth of human lung tumor A549 cells through down-regulation of ALDH3A1 expression.

Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) in certain normal and tumor cells is associated with protection against the growth inhibitory effect of reactive aldehydes generated during membrane lipid peroxidation. We found that human lung tumor (A549) cells, which express high levels of ALDH3A1 protein, were significantly less susceptible to the antiproliferative effects of 4-hydroxynonenal compared to human hepatoma HepG2 or SK-HEP-1 cells that lack ALDH3A1 expression. However, A549 cells became susceptible to lipid peroxidation products when they were treated with arachidonic acid. The growth suppression of A549 cells induced by arachidonic acid was associated with increased levels of lipid peroxidation and with reduced ALDH3A1 enzymatic activity, protein, and mRNA levels. Furthermore, arachidonic acid treatment of the A549 cells resulted in an increased expression of peroxisome proliferator-activated receptor gamma (PPARgamma), whereas NF-kappaB binding activity was inhibited. Blocking PPARgamma using a selective antagonist, GW9662, prevented the arachidonic acid-mediated reduction of ALDH3A1 expression as well as the growth inhibition of A549 cells, suggesting the central role of PPARgamma in these phenomena. The increase in PPARgamma and the reduction in ALDH3A1 were also prevented by exposing cells to vitamin E concomitant with arachidonic acid treatment. In conclusion, our data show that the arachidonic acid-induced suppression of A549 cell growth is associated with increased lipid peroxidation and decreased ALDH3A1 expression, which may be due to activation of PPARgamma.

Effects of di(2-ethylhexyl) phthalate, a widely used peroxisome proliferator and plasticizer, on cell growth in the human keratinocyte cell line NCTC 2544.

Phthalate esters are a widely used class of water-insoluble organic chemicals. The adverse effects of di(2-ethylhexyl) phthalate (DEHP) were chiefly studied in animals, while their potential toxicity to humans has not been properly evaluated. It was hypothesized that the effect of DEHP on human cells depends on the concentration, and this study examined the effects of different concentrations of DEHP on cell growth in cultured human keratinocytes NCTC 2544, together with the possible involvement of peroxisome proliferator-activated receptors (PPARs) in mediating the effects. After exposure to DEHP, the number of NCTC 2544 cells in the monolayer decreased in a concentration-dependent and time-dependent manner, whereas the cells that were detached from the monolayer increased, and died via necrosis. The decrease of cell growth was confirmed by the inhibition of pErk1, pErk2, and changes in the c-myc protein content. With regard to PPARs, the PPARbeta protein content increased, whereas PPARalpha decreased. To demonstrate the involvement of PPARbeta in inhibiting cell growth, the use of an antisense oligonucleotide against this receptor revealed the prevention of DEHP-induced cell growth inhibition. In addition, the treatment of keratinocytes with a specific ligand of PPARbeta (L165041) showed a concentration-dependent inhibition of cell growth, as with DEHP. In conclusion, the effect of DEHP on human keratinocytes is concentration dependent, and this effect is mediated via PPARs.

Conjugated linoleic acid induces apoptosis in MDA-MB-231 breast cancer cells through ERK/MAPK signalling and mitochondrial pathway.

We investigated the molecular mechanisms involved in the anti-proliferative activity exerted by conjugated linoleic acid (CLA) on the estrogen unresponsive MDA-MB-231 human breast cancer cell line. The effects on cell proliferation, cell cycle progression and induction of apoptosis were examined. CLA caused the reduction of cell proliferation along with the accumulation of cells in the S phase of the cycle. The occurrence of apoptosis in these cells was indicated by flow cytometry data and further confirmed by the onset of cells with morphological features typical of apoptosis. ERK1/2 reduction and upregulation of pro-apoptotic protein Bak were induced. These events were associated with: (a) reduced levels of the anti-apoptotic protein Bcl-x(L), (b) the translocation of cytochrome c from the mitochondria to the cytosol, (c) the cleavage of pro-caspase-9 and pro-caspase-3. From the above data, we are induced to think that CLA may trigger apoptosis in the estrogen unresponsive MDA-MB-231 cell line via mechanisms involving above all the mitochondrial pathway.