Locoregional management of in-transit metastasis in melanoma: an Ontario Health (Cancer Care Ontario) clinical practice guideline.

Objective: The purpose of this guideline is to provide guidance on appropriate management of satellite and in-transit metastasis (itm) from melanoma. Methods: The guideline was developed by the Program in Evidence-Based Care (pebc) of Ontario Health (Cancer Care Ontario) and the Melanoma Disease Site Group. Recommendations were drafted by a Working Group based on a systematic review of publications in the medline and embase databases. The document underwent patient- and caregiver-specific consultation and was circulated to the Melanoma Disease Site Group and the pebc Report Approval Panel for internal review; the revised document underwent external review. Recommendations: "Minimal itm" is defined as lesions in a location with limited spread (generally 1-4 lesions); the lesions are generally superficial, often clustered together, and surgically resectable. "Moderate itm" is defined as more than 5 lesions covering a wider area, or the rapid development (within weeks) of new in-transit lesions. "Maximal itm" is defined as large-volume disease with multiple (>15-20) 2-3 cm nodules or subcutaneous or deeper lesions over a wide area.■ In patients presenting with minimal itm, complete surgical excision with negative pathologic margins is recommended. In addition to complete surgical resection, adjuvant treatment may be considered.■ In patients presenting with moderate unresectable itm, consider using this approach for localized treatment: intralesional interleukin 2 or talimogene laherparepvec as 1st choice, topical diphenylcyclopropenone as 2nd choice, or radiation therapy as 3rd choice. Evidence is insufficient to recommend intralesional bacille Calmette- Guérin or CO2 laser ablation outside of a research setting.■ In patients presenting with maximal itm confined to an extremity, isolated limb perfusion, isolated limb infusion, or systemic therapy may be considered. In extremely select cases, amputation could be considered as a final option in patients without systemic disease after discussion at a multidisciplinary case conference.■ In cases in which local, regional, or surgical treatments for itm might be ineffective or unable to be performed, or if a patient has systemic metastases at the same time, systemic therapy may be considered.

Spatial learning and memory impairment and increased locomotion in a transgenic amyloid precursor protein mouse model of Alzheimer's disease.

This study provides an examination of spatial learning and a behavioral assessment of irritability and locomotion in TgCRND8 mice, an amyloid precursor protein transgenic model of Alzheimer's disease. Performance was assessed using the Barnes maze, the touch escape test, and an open-field test. While past research focused primarily on 2-5-month-old TgCRND8 mice, the present study used an older age cohort (9-month-old female mice), in addition to a 4-month-old cohort of both transgenic (Tg) and wildtype female mice. Both younger and older Tg mice displayed poor spatial learning in the Barnes maze task compared to their wildtype littermates, as demonstrated by significantly longer latencies and more errors both during acquisition and at a 2-week retest. No differences in irritability were found between Tg and control mice in the younger cohort; however, older Tg mice displayed significantly higher irritability compared with wildtype littermates, as measured by the touch escape test. Additionally, Tg mice of both age cohorts showed increased locomotion and slowed habituation during a 60-min open-field test over 3 days of testing. These results demonstrate that TgCRND8 mice show significant deficits in spatial and nonspatial behavioral tasks at advanced stages of amyloid pathology.

Low energy laser light (632.8 nm) suppresses amyloid-ß peptide-induced oxidative and inflammatory responses in astrocytes.

Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid ß-peptides (Aß) in mediating these processes. In astrocytes, oligomeric Aß induces the assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complexes resulting in its activation to produce anionic superoxide. Aß also promotes production of pro-inflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by Aß. Primary rat astrocytes were exposed to Helium-Neon laser (λ=632.8 nm), followed by the treatment with oligomeric Aß. Primary rat astrocytes were used to measure Aß-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47(phox) subunit and the membrane gp91(phox) subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A(2) cPLA(2) and expressions of pro-inflammatory factors including interleukin-1ß (IL-1ß) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed Aß-induced superoxide production, colocalization between NADPH oxidase gp91(phox) and p47(phox) subunits, phosphorylation of cPLA(2,) and the expressions of IL-1ß and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD.

Influence of amino acids, organic solvents and surfactants for phenylalanine ammonia lyase activity in recombinant Escherichia coli.

AIM: To improve phenylalanine ammonia lyase (E.C.4.3.1.5-PAL) activity in recombinant Escherichia coli. Some methods for enrichment of PAL activity in recombinant E. coli JM109 were described. In an effort to create a rich enzyme source these methods would lead to improvements in the production of L-phenylalanine. METHODS AND RESULTS: The possibilities of enriching PAL activity in recombinant E. coli was investigated by using individual and combinations of amino acids, organic solvents and surfactants. PAL activity was induced by adding combination of L-phenylalanine and L-tyrosine, activities as high as 64.3 U g(-1)of cells were obtained and enzyme activity was enriched by over 3.5-fold in comparison with the control. Permeabilization with cetyl trimethyl ammonium bromide or the acetone significantly enriched cellular PAL activity, which improved over 8.2- and 9.0-fold compared with the control, as high as 148.5 and 164.5 U g(-1)of cells respectively. CONCLUSION: These efforts may provide some effective methods for enhancing L-phenylalanine ammonia lyase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: These approaches for manipulating recombinant E. coli in an effort to create a rich enzyme source would serve as a biotechnologically important protocol for production of L-phenylalanine.

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents.

Upregulation and activation of phospholipases A2 (PLA2) and cyclooxygenases (COX) leading to prostaglandin E2(PGE2) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE2 production in primary rat astrocytes in response to agents that activate PLA2 including pro-inflammatory cytokines (IL-1beta, TNFalpha and IFNgamma), the P2 nucleotide receptor agonist ATP, and oxidants (H2O2 and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE2 production that was marked by increased expression of secretory sPLA2 and COX-2, but not COX-1 and cytosolic cPLA2. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA2 phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE2. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H2O2 or menadione, markedly increased PGE2 production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE2 production and may contribute to chronic inflammation seen in Alzheimer's disease.

Ethanol and oxidative stress.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was Albert Y. Sun. The presentations were (1) Ethanol-inducible cytochrome P-4502E1 in alcoholic liver disease, by Magnus Ingelman-Sundberg and Etienne Neve; (2) Regulation of NF-kappaB by ethanol, by H. Matsumoto, Y. Nishitani, Y. Minowa, and Y. Fukui; (3) Chronic ethanol consumption increases concentration of oxidized proteins in rat liver, by Shannon M. Bailey, Vinood B. Patel, and Carol C. Cunningham; (4) Antiphospholipids antibodies and oxidized modified low-density lipoprotein in chronic alcoholic patients, by Tomas Zima, Lenka Fialova, Ludmila Mikulikova, Ptr Popov, Ivan Malbohan, Marta Janebova, and Karel Nespor; and (5) Amelioration of ethanol-induced damage by polyphenols, by Albert Y. Sun and Grace Y. Sun.

The C. elegans gon-2 gene encodes a putative TRP cation channel protein required for mitotic cell cycle progression.

The C. elegans gon-2 gene is required for the post-embryonic mitotic cell divisions of the gonadal precursor cells. A single major transcript of approximately 6.7 kb is derived from the gon-2 locus. This mRNA encodes a protein related to the TRP family of cation channels and has a high degree of similarity to several vertebrate genes, including melastatin. Mutant alleles of gon-2 affect evolutionarily conserved amino acid residues. Northern analyses suggest that gon-2 expression is not limited to gonadal tissues.

Ethanol and oxidative mechanisms in the brain.

There is strong evidence showing that chronic and excessive ethanol consumption may enhance oxidative damage to neurons and result in cell death. Although not yet well understood, ethanol may enhance ROS production in brain through a number of pathways including increased generation of hydroxyethyl radicals, induction of CYP2E1, alteration of the cytokine signaling pathways for induction of iNOS and sPLA(2), and production of prostanoids through the PLA(2)/COX pathways. Since many neurodegenerative diseases are also associated with oxidative and inflammatory mechanisms in the brain, it would be important to find out whether chronic and excessive ethanol consumption may exacerbate the progression of these diseases. There is evidence that the polyphenolic antioxidants, especially those extracted from grape skin and seed, may protect the brain from neuronal damage due to chronic ethanol administration. Among the polyphenols from grapes, resveratrol seems to have unique antioxidant properties. The possible use of this compound as a therapeutic agent to ameliorate neurodegenerative processes should be further explored.

Oxidized lipoproteins, beta amyloid peptides and Alzheimer's disease.

Recent studies have provided strong evidence for the involvement of oxidative stress in the pathogenesis of Alzheimer's disease (AD) and beta-amyloid peptides (ABeta) have been implicated to play an important role in mediating these oxidative events. Lipoproteins (LP) in the brain are likely targets of oxidative insult and together enhance ABeta -mediated toxicity to neurons. We hypothesize that uptake of oxidized LP by neuron leads to an acceleration of the intracellular oxidative pathways and exacerbation of neuron cell death. In our previous studies, we demonstrated the ability of oxidized low-density LP from plasma to induce cell death in PC12 cells. In this study, a synthetic LP fraction was prepared using lipids extracted from rat brain and incubated with albumin and apoE. This brain lipid-derived LP (BLP) was subjected to oxidation by incubation with Fe(3+)and subsequently tested with primary cortical neurons in culture. To study uptake of the BLP, native and oxidized BLP containing apoE3 or apoE4 were labeled with [(14)C]cholesterol or the fluorescent probe 3,3-dioctadecylindo-carbocyanine (Di-I) prior to exposing to cultured neurons. Results showed that regardless of the labeling method, oxidized BLP were more effectively taken up by the neurons than the native BLP. Cell viability was assessed by assaying the release of lactate dehydrogenase (LDH) into the medium and by determining the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), an agent depicting mitochondrial activity. While exposure of neurons to oxidized BLP and aggregated ABeta (1-42) alone could result in MTT reduction (24%), greater reduction (40%) could be observed when oxidized LP was added together with ABeta. Neuronal cell death due to oxidized BLP could be ameliorated by resveratrol, a polyphenolic compound known for its antioxidant properties. Taken together, these results are in agreement with the notion that ABeta and oxidized BLP can synergistically enhance oxidative damage in neurons and antioxidants such as resveratrol can ameliorate these damages.

Dietary supplementation of grape polyphenols to rats ameliorates chronic ethanol-induced changes in hepatic morphology without altering changes in hepatic lipids.

Increase in oxidative stress after chronic ethanol consumption can result in hepatic injury. Because polyphenolic compounds can offer antioxidant protection to the cardiovascular system, this study was designed to investigate whether dietary supplementation of polyphenols from grapes may ameliorate hepatic injury resulting from chronic ethanol consumption. Male Sprague-Dawley rats were administered the following diets for 2 mo: 1) Lieber-DeCarli (L-D) diet with isocaloric amount of maltose instead of ethanol (Basal), 2) the L-D diet with 50g/L ethanol (EtOH); 3) L-D diet with 50 mg/L of grape polyphenols (GP) and 4) ethanol diet with GP (EtOH + GP). Rats given EtOH or EtOH + GP diets had significantly more hepatic triacylglycerols (P < 0.0001) and lipid peroxidation products (P < 0.01) compared with those given the Basal and GP diets. In addition, ethanol ingestion also decreased significantly (P < 0.01) the proportion of 16:0 and increased 18:0 and 18:1 in hepatic phospholipids, suggesting a perturbation of the de novo fatty acid biosynthesis pathways. However, GP supplementation alone and GP added to the ethanol diet did not alter the lipid changes mediated by ethanol except for the levels of 22:6(n-3) which were significantly (P < 0.05) higher in the EtOH + GP group than in the EtOH group. Despite a lack of gross lipid changes, histologic assessment showed significantly (P < 0.05) less hepatic damage in the GP + EtOH group compared with the EtOH group. These results clearly distinguished ethanol-mediated changes in hepatic morphology from the changes in hepatic lipids and further demonstrated the ability of GP to ameliorate hepatic damage resulting from chronic ethanol consumption.

Paraquat induced activation of transcription factor AP-1 and apoptosis in PC12 cells.

Drugs and certain environmental toxins may be responsible for the pathogenesis of Parkinson's disease. We have used paraquat as a model toxin for this study since paraquat has been shown to make its way to the nerve terminals and cause cell death of dopamine neurons by oxidative injury. We have shown by the electrophoretic mobility shift assay that paraquat, together with low concentrations of chelated iron (Fe++/DETAPAC), induced the activation of transcription factor AP-1 binding activity to DNA. Under similar conditions we also found by both a DNA laddering assay procedure and by terminal deoxynucleotidyl transferase assay (TUNEL assay) that paraquat also induces apoptotic cell death. Interestingly, both apoptotic cell death and AP-1/DNA binding activity induced by paraquat were blocked by cyclohexamide and genistein, indicating that both the AP-1/DNA binding activation and apoptosis induced by paraquat are closely related. Moreover, cells were also protected from paraquat toxicity in the presence of antioxidant defense enzymes SOD and catalase. The results support the hypothesis that oxidative stress may be contributing to the apoptotic cell death of dopaminergic neurons, leading to the manifestation of Parkinson's disease. Since paraquat was an important herbicide in the mid 20th Century, our results have the important implication that exposure to environmental toxins such as paraquat may induce Parkinson's disease.

Grape polyphenols protect neurodegenerative changes induced by chronic ethanol administration.

Increased oxidative stress in the brain due to chronic ethanol consumption is known to result in a number of neurodegenerative changes. This study was designed to test whether dietary supplementation of grape polyphenols (GP) can offer protection to the neurodegenerative changes resulting from chronic ethanol consumption. Sprague-Dawley rats were fed a Leiber-DeCarli liquid diet with ethanol or isocaloric amount of maltose, and with or without GP for 2 months. Chronic ethanol caused significant decreases in synaptosomal Na,K-ATPase (20.5%) and dopamine uptake (22.8%) activities compared with pair-fed controls. Although GP alone did not alter activities of these membrane-bound proteins, GP supplementation was able to completely protect the decrease in synaptic protein function elicited by chronic ethanol consumption.

Oxidative stress and neurodegenerative disorders.

Oxidative insults, whether over-excitation, excessive release of glutamate or ATP caused by stroke, ischemia or inflammation, exposure to ionizing radiation, heavy-metal ions or oxidized lipoproteins may initiate various signaling cascades leading to apoptotic cell death and neurodegenerative disorders. Among the various reactive oxygen species (ROS) generated in the living organism, hydroxyl and peroxynitrite are the most potent and can damage proteins, lipids and nucleic acids. It appears that some natural antioxidants (tocopherol, ascorbic acid and glutathione) and defense enzyme systems (superoxide dismutase, catalase and glutathione peroxidase) may provide some protection against oxidative damage. Recent findings indicate several polyphenols and antioxidant drugs (probucol, seligilline) are effective in protecting the cells from ROS attack. Further development of these antioxidant molecules may be of value in preventing the development of neurodegenerative diseases.

Paraquat-induced cell death in PC12 cells.

Paraquat was taken up by PC12 cells in a carrier-mediated, saturable manner. When PC12 cells were permeabilized with digitonin (50 microg/ml) lipid peroxidation was observed after paraquat treatment in the presence of NADPH and chelated iron. The fact that lipid peroxidation preceded the appearance of LDH release provides positive evidence that lipid peroxidation may be one of the important factors leading to cytotoxicity of cells. Furthermore, the fact that addition of superoxide dismutase, catalase and promethazine efficiently blocked the malondialdehyde formation and attenuated the cell death indicated the involvement of reactive oxygen radicals in mediating the cytotoxicity induced by paraquat. Taken together the results present in vitro evidence that neurotoxicity of paraquat may be a consequence of cellular lipid peroxidation, which leads to cell death and may have great implications in assessing the risk of exposure to paraquat in Parkinson's disease.

Protection of PC12 cells glutathione peroxidase in L-DOPA induced cytotoxicity.

L-DOPA may cause side-effects during the treatment of Parkinson's disease. We investigated the role of glutathione peroxidase (GSHPx) in cellular defense against L-DOPA cytotoxicity. A line of PC12 cells overexpressing GSHPx with plasmid pRc/CMV-GSHPx was established and stable transfectants overexpressing GSHPx were used for this study. GSHPx activity was found to be 1.5-fold higher in GSHPx-transfectants than in mock-controlled transfectants. Transfectants over expressing GSHPx were also significantly more resistant to exposure to either L-DOPA or t-butyl hydroperoxide than mock-transfected cells. Results suggested that L-DOPA may cause neuronal cell death by an oxidative pathway and GSHPx may play an important role in cellular defense against oxidative stress.

Oxidized lipoproteins may play a role in neuronal cell death in Alzheimer disease.

Oxidative stress in the central nervous system (CNS) may cause oxidation of lipoprotein particles. The oxidized lipoproteins may damage cellular and subcellular membranes, leading to tissue injury and cell death. Human low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) are oxidized by transition metal ions, such as Cu2+. Using PC 12 cells, we tested the cytotoxicity of oxidized LDL and VLDL. Cell death was increased in a dose-dependent manner. Antioxidants added to the incubation medium, such as vitamins E or C, or resveratrol showed some protection. Results indicated that oxidized lipoproteins may serve as an oxidative stressor, which may initiate the neuronal cell death leading to the manifestation of Alzheimer disease (AD).

Activation of transcription factor AP-1 by extracellular ATP in PC12 cells.

We have previously shown that extracellular ATP caused cell death in PC12 cells through activation of its receptors. Oxidative stress has been implicated as a mechanism of cell death caused by extracellular ATP. In the present study we examined the possible signal transduction cascades leading to cell death by extracellular ATP. We found, using the electrophoretic mobility shift assay, that transcription factor AP-1 DNA binding activity was stimulated by extracellular ATP. Northern blot analysis showed that mRNA levels of c-fos, c-jun were elevated after treatment with ATP. The stimulation was receptor mediated, since it was blocked by the ATP receptor antagonist, suramin. The stimulated AP-1 binding was also blocked by the antioxidant N-acetyl-L-cysteine, indicating that reactive oxygen species generated following ATP stimulation were involved in the induction of AP-1 activity. It appears that both translational and posttranslational events contributed to the increased AP-1 DNA binding since cyclohexamide (a protein synthesis inhibitor), genistein (tyrosine kinase inhibitor) and staurosporine (PKC inhibitor) each partially blocked the AP-1 activation. Changes in AP-1 DNA binding activity may modulate expression of target genes involved in cell death pathways.

Paraquat-induced free radical reaction in mouse brain microsomes.

Paraquat has been implicated as an environmental toxin which may induce the syndrome of Parkinson's disease after exposure to this agent. However, the biochemical mechanism by which paraquat causes cell death and neurodegeneration has not been extensively studied. Paraquat was rapidly taken up by nerve terminals isolated from mouse cerebral cortices. It induced lipid peroxidation in a concentration dependent manner in the presence of NADPH and ferrous ion. The maximal stimulation effect was obtained at a paraquat concentration around 100 microM and the Km value for paraquat was 46.7 microM. The lipid peroxidation required microsomal enzymes. Antioxidants, such as superoxide dismutase, catalase and promethazine significantly inhibited paraquat-induced lipid peroxidation. Due to its structural similarity to the pyridinium compound MPP+ (N-methyl-4-phenyl pyridium ion), it may be taken up by dopamine neurons and cause lipid peroxidation and cell death resulting in the manifestation of Parkinsonian syndrome.

Oxidized lipoproteins activate NF-kappaB binding activity and apoptosis in PC12 cells.

Oxidative stress in the central nervous system may cause oxidation of lipoproteins. The oxidized lipoproteins may in turn damage cellular and subcellular membranes and other biomolecules, leading to tissue injury and cell death. Recently, we have demonstrated that oxidized LDL and VLDL induced cell death in a dose-dependent manner. The present study examined the possible signal transduction cascade leading to cell death by oxLDL and oxVLDL in PC12 cells. Using the electrophoretic mobility shift assay, we found that both oxLDL and oxVLDL activated the binding of NF-kappaB to the consensus sequence in the promoter region of the target genes, followed by apopototic cell death. Resveratrol protects the cells from both the activation of NF-kappa-B/ DNA binding activity and apoptotic cell death. Results indicated that oxidized lipoproteins may serve as an oxidative mediator and may activate apoptosis through a nuclear signalling pathway contributing to the pathology in Alzheimer's disease.

Extracellular ATP-induced apoptosis in PC12 cells.

Studies in our laboratory indicate that extracellular ATP (ATP)o may induce cell death by reactive oxygen insults. We have also shown that the Ca(2+)-induced oxidative stress as elicited by ATP may lead to an activation of a specific AP-1 activity. Since early impairment of mitochondria constitutes a critical event of the apoptotic cell death, we have examined whether (ATP)o will affect mitochondrial damage and cell injury by using mitochondrial specific probes, dihydrorhodamine and 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). We have found that (ATP)o induced cell death in a concentration dependent manner by MTT assay. The (ATP)o induced cell death correlated well with the reactive oxygen species (ROS) generation in mitochondria, since (ATP)o enhanced both cell death and ROS production and antioxidant blocked both of these processes. We found (ATP)o treatment led to apoptotic cell death by examining DNA laddering and the TUNEL assay. Interestingly, vitamin C and vitamin E combined treatment appeared to attenuate the (ATP)o-induced apoptosis. Results indicated that (ATP)o may cause oxidative damage of mitochondria leading to apoptotic cell death. Antioxidants may be useful in preventing apoptosis by preventing ROS formation in mitochondria.