RESUMO
Conventional cancer treatments often have little impact on the course of advanced pancreatic cancer. Although cancer gene therapy with adenoviruses is a promising developmental approach, the primary receptor is poorly expressed in pancreatic cancers which might compromise efficacy and thus targeting to other receptors could be beneficial. Extended stealth delivery, combination with standard chemotherapy or circumvention of host antiadenoviral immune response might improve efficacy further. In this work, capsid-modified adenoviruses were studied for transduction of cell lines and clinical normal and tumor tissue samples. The respective oncolytic viruses were tested for oncolytic activity in vitro and in vivo. Survival was studied in a peritoneally disseminated pancreas cancer model, with or without concurrent gemcitabine while silica implants were utilized for extended intraperitoneal virus delivery. Immunocompetent mice and Syrian hamsters were used to study the effect of silica mediated delivery on antiviral immune responses and subsequent in vivo gene delivery. Capsid modifications selectively enhanced gene transfer to malignant pancreatic cancer cell lines and clinical samples. The respective oncolytic viruses resulted in increased cell killing in vitro, which translated into a survival benefit in mice. Early proinfammatory cytokine responses and formation of antiviral neutralizing antibodies was partially avoided with silica implants. The implant also shielded the virus from pre-existing neutralizing antibodies, while increasing the pancreas/liver gene delivery ratio six-fold. In conclusion, capsid modified adenoviruses would be useful for testing in pancreatic cancer trials. Silica implants might increase the safety and efficacy of the approach.
Assuntos
Adenovírus Humanos , Desoxicitidina/análogos & derivados , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos , Neoplasias Pancreáticas/terapia , Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Animais , Anticorpos Neutralizantes , Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Terapia Combinada , Cricetinae , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Feminino , Humanos , Camundongos , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/imunologia , Sílica Gel , Transfecção , GencitabinaRESUMO
We have previously shown that ethanol (EtOH) induces protective NF-kappaB activation in gastric surface epithelial cells. This study investigates the defense systems in rat gastric mucosal cells (RGM-1) exposed simultaneously to EtOH and taurocholate (TC) or acetylsalicylic acid (ASA). Simultaneous exposure to ASA and EtOH increased EtOH-induced caspase-3 activity and decreased cell viability, indicating synergetic damaging action. Simultaneous exposure to TC (5 mM) with EtOH (5%) increased EtOH-induced NF-kappaB activation, opposing EtOH-induced decrease in cell membrane integrity and in cell viability as shown by decreasing RelA expression with siRNA technique. Low doses of TC decreased the EtOH (5%) induced caspase-3 activity independently from NF-kappaB pathway and inhibited EtOH-induced decrease in caspase-3 precursor protein levels, also indicating the inhibition of caspase-3 pathway. The TC (5 mM)-induced protection in EtOH exposed tissues seems to have two distinct pathways, inhibition of apoptosis and enhancement of NF-kappaB signaling.
Assuntos
Caspase 3/metabolismo , Células Epiteliais/efeitos dos fármacos , Etanol/toxicidade , Mucosa Gástrica/efeitos dos fármacos , NF-kappa B/metabolismo , Ácido Taurocólico/metabolismo , Animais , Apoptose/efeitos dos fármacos , Aspirina/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Mucosa Gástrica/enzimologia , Mucosa Gástrica/patologia , NF-kappa B/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismoRESUMO
CONTEXT: Since benign and malignant mucin-producing tumors of the pancreas may be difficult to distinguish from each other; preoperative methods for differential diagnosis would reduce unnecessary surgery. OBJECTIVE: To compare syndecan-1 and tenascin immunoexpression in benign and malignant cystic pancreatic tumors. DESIGN: We used immunohistochemical staining for syndecan-1 and tenascin antibodies in tumor tissue samples. SETTING: Helsinki University Central Hospital. PATIENTS: Tissue material came from 33 patients undergoing surgery from 1979 to 2005 for cystic pancreatic tumors. RESULTS: A statistically significant difference appeared in syndecan-1 expression between benign (mucinous cystic neoplasms and intraductal papillary mucinous neoplasms) and mucinous carcinomas, but there was no significant difference in tenascin immunoexpression between these tumor groups. CONCLUSION: Our findings suggest that low syndecan-1 expression might serve as a predictive factor for malignancy in cystic tumors of the pancreas.
Assuntos
Cisto Pancreático/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Sindecana-1/biossíntese , Tenascina/biossíntese , Adulto , Idoso , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Cisto Pancreático/classificação , Cisto Pancreático/metabolismo , Neoplasias Pancreáticas/classificação , Neoplasias Pancreáticas/metabolismo , Valor Preditivo dos TestesRESUMO
We performed an integrated array comparative genomic hybridization (aCGH) and expression microarray analysis of 8 normal gastric tissues and 38 primary tumors, including 25 intestinal and 13 diffuse gastric adenocarcinomas to identify genes whose expression is deregulated in association with copy number alteration. Our aim was also to identify molecular genetic alterations that are specific to particular clinicopathological characteristics of gastric cancer. Distinct molecular genetic profiles were identified for intestinal and diffuse gastric cancers and for tumors obtained from 2 different locations of the stomach. Interestingly, the ERBB2 amplification and gains at 20q13.12-q13.33 almost exclusively discriminated intestinal cancers from the diffuse type. In addition, the 17q12-q25 gain was characteristic to cancers located in corpus and the 20q13.12-q13.13 gain was more common in the antrum. Statistical analysis was performed using integrated copy number and expression data to identify genes showing differential expression associated with a copy number alteration. Genes with the highest statistical significance included ERBB2, MUC1, GRB7, PPP1R1B and PPARBP with concomitant changes in copy number and expression. Immunohistochemical analysis of ERBB2 and MUC1 on a tissue microarray containing 78 independent gastric tissues showed statistically significant differences (p < 0.05 and <0.001) in immunopositivity in the intestinal (31 and 70%) and diffuse subtypes (14 and 41%), respectively. In conclusion, our results demonstrate that intestinal and diffuse type gastric cancers as well as cancers located in different sites of the stomach have distinct molecular profiles which may have clinical value.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Dosagem de Genes , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Intestinais/metabolismo , Mucina-1/biossíntese , Mucina-1/genética , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Neoplasias Gástricas/metabolismoRESUMO
PURPOSE: Metastatic gastric cancer remains a common and devastating disease without curative treatment. Recent proof-of-concept clinical trials have validated gene therapy with adenoviruses as an effective and safe modality for the treatment of cancer. However, expression of the primary coxsackie-adenovirus receptor is variable in advanced cancers, and therefore, the use of heterologous receptors could be advantageous. EXPERIMENTAL DESIGN: Here, we used capsid-modified adenoviruses for increasing the transduction and subsequent antitumor efficacy. 5/3 chimeric viruses have a serotype 3 knob which allows binding to a receptor distinct from coxsackie-adenovirus receptor. The fiber of Ad5lucRGD is modified with an integrin-targeted motif. Polylysine motifs, pK7 and pK21, bind to heparan sulfates. Oncolytic adenoviruses replicate in and kill tumor cells selectively. Gastric cancer cell lines and fresh clinical samples from patients were infected with transductionally targeted viruses. Capsid-modified oncolytic adenoviruses were used in cell killing experiments. To test viral transduction and therapeutic efficacy in vivo, we developed orthotopic mouse models featuring i.p. disseminated human gastric cancer, which allowed the evaluation of biodistribution and antitumor efficacy in a system similar to humans. RESULTS: Capsid modifications benefited gene transfer efficiency and cell killing in gastric cancer cell lines and clinical samples in vitro and in vivo. Modified oncolytic adenoviruses significantly increased the survival of mice with orthotopic gastric cancer. CONCLUSIONS: These preclinical data set the stage for the clinical evaluation of safety and efficacy in patients with disease refractory to current modalities.
Assuntos
Adenoviridae/patogenicidade , Capsídeo/metabolismo , Técnicas de Transferência de Genes , Terapia Genética/métodos , Terapia Viral Oncolítica , Neoplasias Gástricas/terapia , Animais , Morte Celular , Sobrevivência Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Feminino , Camundongos , Neoplasias Experimentais , Receptores Virais , Neoplasias Gástricas/patologia , Transdução Genética , Células Tumorais CultivadasRESUMO
AIM: To explore whether preoperative chemoradiation therapy improves survival of patients with pancreatic cancer undergoing resectional surgery. METHODS: Forty-seven patients with a malignant pancreatic tumor localized in the head or uncinate process of the pancreas underwent radical pancreatico-duodenectomy. Twenty-two received chemoradiation therapy (gemcitabine and radiation dose 50.4 Gy) before surgery (CRR) and 25 patients underwent surgery only (RO). The study was non-randomised. Patients were identified from a prospective database. RESULTS: The median survival time was 30.2 mo in the CRR group and 35.9 mo in the RO group. No statistically significant differences were found in subclasses according to lymph node involvement, TNM stages, tumor size, or perineural invasion. The one, three and five year survival rates were 81%, 33% and 33%, respectively, in the CRR group and 72%, 47% and 23%, respectively, in the RO group. In ductal adenocarcinoma, the median survival time was 27 mo in the CRR group and 20 mo in the RO group. No statistically significant differences were found in the above subclasses. The one, three and five year survival rates were 79%, 21% and 21%, respectively, in the CRR group and 64%, 50% and 14%, respectively, in the RO group. The overall hospital mortality rate was 2%. The morbidity rate was 45% in the CRR group and 32% (NS) in the RO group. CONCLUSION: Major multicenter randomized studies are needed to conclusively assess the impact of neoadjuvant treatment in the management of pancreatic cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/radioterapia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/radioterapia , Adenocarcinoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/uso terapêutico , Quimioterapia Adjuvante , Terapia Combinada , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Relação Dose-Resposta à Radiação , Feminino , Mortalidade Hospitalar , Humanos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Neoplasias Pancreáticas/cirurgia , Pancreaticoduodenectomia , Radioterapia Adjuvante , Taxa de Sobrevida , GencitabinaRESUMO
AIM: To assess the effects of ulcerogenic agents on actin cytoskeleton and cell motility and the contribution of oxidative stress. METHODS: Rat gastric mucosal cell monolayers were cultured on coverslips. The cells were exposed, with or without allopurinol (2 mmol/L), for 15 min to ethanol (10-150 mL/L), ASA (1-20 mmol/L) or taurocholate (1-20 mmol/L), then the cells were processed for actin and vinculin staining. Cell migration after wounding was also measured. RESULTS: Exposure to 10 mL/L ethanol caused divergence of zonula adherens-associated actin bundles of adjacent cells and decreased rate of migration. These actions were opposed by xanthine oxidase inhibitor allopurinol. Exposure to 50 mL/L ethanol induced degradation and divergence of zonula adherens-associated vinculin from adjacent cells, which was, again, partially reverted by allopurinol. With 1 mmol/L ASA actin filaments became shorter and thicker. However, higher concentrations (10, 20 mmol/L) of ASA returned microfilaments thinner and longer, and decreased rate of migration. Zonula adherens-associated actin bundles were moderately distorted with 10 mmol/L ASA and with 10 mmol/L taurocholate. Exposure to taurocholate provoked changes resembling those of ASA. Taurocholate 5-20 mmol/L decreased the rate of migration dose dependently. The effects of ASA and taurocholate were not prevented by allopurinol. CONCLUSION: All ulcerogenic agents decreased the rate of migration dose dependently and induced divergence of zonula adherens-associated actin bundles of adjacent cells. In addition, ethanol and ASA caused degradation of actin cytoskeleton. Oxidative stress seems to underlie ethanol, but not ASA or taurocholate, induced cytoskeletal damage.
Assuntos
Actinas/metabolismo , Aspirina/toxicidade , Citoesqueleto/efeitos dos fármacos , Etanol/toxicidade , Mucosa Gástrica/patologia , Úlcera Gástrica/induzido quimicamente , Ácido Taurocólico/toxicidade , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , RatosRESUMO
Ethanol is a well-established "barrier breaker" in gastric mucosa, but its detailed effects at the cellular level remain unclear. We have previously shown that the intracellular free calcium concentration is increased, gap junctions are closed, and cell volume is decreased after exposure to 5% (v/v) ethanol in primarily cultured rabbit gastric epithelial cells. Rat gastric mucosal (RGM) cells were grown to confluence on a coverslip or on a filter membrane. Gap junctional diffusion was measured in 5-carboxyfluorescein-loaded cells by bleaching a small area with a laser and measuring the recovery with confocal microscope. Intracellular calcium was measured spectrofluorometrically in fura-2-loaded cells. For cell volume measurements the cell monolayer was loaded with calcein and imaged along the Z-axis with a confocal microscope. The changes in fluorescence intensity were intercepted as a measure of cell volume change. TMB-8 was used to inhibit intracellular calcium release and lanthanum to block plasma membrane calcium selective ion channels, while BABTA served as an intracellular calcium chelating agent. Results showed that ethanol (7.5%, v/v) exposure increased intracellular calcium from 69± 7 to 142± 11 nM (N = 5; P < 0.05), decreased cell volume by -23± 5% (N = 8; P < 0.05), and induced gap junction closure (fluorescence recovery from 37± 9 to 15± 3%; N = 6; P < 0.05). A serosal potassium channel blocker, quinine, almost completely prevented the ethanol-induced cell volume decrease (from -23± 5 to -3± 3%), suggesting that opening of basolateral potassium channels underlies cell shrinkage. BABTA inhibited completely (from 35± 3 to 39± 4 nM; N = 6; P < 0.05), and TMB-8 + lanthanum partially (from 60± 6 to 92± 12 nM; N = 6; P < 0.05), the ethanol-induced intracellular calcium increase. BABTA also abolished the ethanol-induced volume decrease (from -23± 5 to 1± 4%; N = 6; P < 0.05), while TMB-8 + lanthanum had a lesser effect on it (from -23± 5 to -11± 3%; N = 9; P < 0.05). They also abolished the closure of gap junctions induced by ethanol (fluorescence recovery, 38± 5% for BABTA and 30± 4% for TMB-8 + lanthanum). We conclude that luminal ethanol opens basolateral calcium-dependent potassium selective channels with resultant shrinkage of the cells and blocks the intercellular gap junctions. These actions are mediated by intracellular calcium signaling.
RESUMO
Gastric cancer is the fourth most common cancer worldwide and the second leading cause of cancer mortality. Three hereditary gastric cancer syndromes have been described; hereditary diffuse gastric cancer (HDGC), familial intestinal gastric cancer (FIGC) and gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS). Thirty per cent of HDGC families have heterozygous germline mutations in CDH1, which encodes E-cadherin. A germline truncating mutation in the gene encoding α-E-catenin (CTNNA1) was also recently discovered in a family with HDGC, but no other genes specifically predisposing to gastric cancer have been identified, leaving the majority of cases showing familial aggregation without a known genetic cause. The aim of this study was to find the putative gastric cancer predisposing gene defect in a family with HDGC that had previously been tested negative for mutations in CDH1. In this family, there were six cases of diffuse gastric cancer in two generations. Exome sequencing was applied to two affected family members. The shared variants which were predicted deleterious in silico and could not be found in databases or in a control set of over 4,000 individuals were Sanger sequenced in a third family member. Three candidate variants were identified: p.Glu1313Lys in Insulin receptor (INSR), p.Arg81Pro in F-box protein 24 (FBXO24) and p.Pro1146Leu in DOT1-like histone H3K79 methyltransferase (DOT1L). These variants and adjacent regions were screened for in an additional 26 gastric cancer patients with a confirmed (n = 13) or suspected (n = 13) family history of disease, but no other non-synonymous mutations were identified. This study identifies INSR, FBXO24 and DOT1L as new candidate diffuse gastric cancer susceptibility genes, which should be validated in other populations. Of these genes, INSR is of special interest as insulin signaling was recently shown to affect tumor cell invasion capability by modulating E-cadherin glycosylation.
Assuntos
Exoma , Proteínas F-Box/genética , Predisposição Genética para Doença , Metiltransferases/genética , Receptor de Insulina/genética , Neoplasias Gástricas/genética , Antígenos CD , Caderinas/genética , Feminino , Histona-Lisina N-Metiltransferase , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , alfa Catenina/genéticaRESUMO
BACKGROUND: Distinguishing between pancreatic cancer and chronic pancreatitis (CP) is often difficult. Certain (5-6%) CP cases are autoimmune in nature, and these patients respond to corticosteroid treatment, making surgery avoidable. Our aim was to evaluate the incidence of autoimmune pancreatitis (AIP) among patients operated on for a pancreatic mass with a final histology of CP. PATIENTS AND METHODS: A total of 33 patients were operated on at the Tampere or Helsinki University Hospital for suspicion of cancer, but with final histopathological diagnosis of CP. The median age was 58 (31-81) years; 26 patients (79%) were male. There were 28 pancreaticoduodenectomes and five left pancreatic resections. Surgical specimens were re-evaluated by experienced pathologists, with representative samples chosen for immunohistochemistry Each sample was scored as positive or negative for immunoglobulin G4 (IgG4) independently by two pathologists. Honolulu consensus criteria served for AIP sub-typing. RESULTS: Out of the 33 specimens, 10 (30%) were positive for IgG4. Histopathological re-evaluation of these revealed all to be type 1 AIP. CONCLUSION: The proportion of AIP, according to IgG4-positive immunohistochemistry and histological re-evaluation, was much higher than expected. This suggests that by focusing on diagnosis of AIP preoperatively, certain patients might be treated with corticosteroids and possibly avoid unnecessary surgery.
Assuntos
Doenças Autoimunes/diagnóstico , Biomarcadores/sangue , Imunoglobulina G/sangue , Neoplasias Pancreáticas/diagnóstico , Pancreatite Crônica/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Autoimunes/sangue , Doenças Autoimunes/cirurgia , Diagnóstico Diferencial , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Pancreatectomia , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/cirurgia , Pancreatite Crônica/sangue , Pancreatite Crônica/cirurgia , Prognóstico , Estudos RetrospectivosRESUMO
The International Gastric Cancer Linkage Consortium (IGCLC) predicted that up to 25% of families fulfilling the criteria for hereditary diffuse gastric cancer (HDGC) would harbor CDH1 germline mutations. This was based on observations from the low number of diffuse gastric cancer families described at the time, and its validation would require analysis of larger numbers. Here we report the results of germline CDH1 mutation screening in 39 kindred with familial aggregation of gastric cancer, a subset of which fulfills the criteria defined by the IGCLC for HDGC. CDH1 germline mutations were detected in four of 11 (36.4%) HDGC families. No mutations were identified in 63.6% of HDGC families or in kindred with familial aggregation of gastric cancer not fulfilling criteria for HDGC. These results add support to the evidence that only HDGC families harbor germline mutations in CDH1 and that genes other than CDH1 remain to be identified.
Assuntos
Caderinas/genética , Testes Genéticos/métodos , Mutação em Linhagem Germinativa/genética , Neoplasias Gástricas/genética , Adulto , Idoso , Neoplasias da Mama/genética , Neoplasias da Mama Masculina/genética , Neoplasias do Colo/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , LinhagemRESUMO
PURPOSE: Define the maximum tolerated dose (MTD), tolerability, and efficacy of gemcitabine given concomitantly with radiotherapy in patients with locally advanced pancreatic cancer. METHODS AND MATERIALS: Patients were required to have locally advanced T1-T3 resectable pancreatic cancer. Gemcitabine, given twice weekly before irradiation as a 30-min infusion, was tested at 3 dose levels: 20, 50, and 100 mg/m(2). The radiation dose was 50.4 Gy (ICRU) in 28 fractions. The targeted irradiation volume included the tumor, edema, and a 1-cm margin. RESULTS: Twenty-eight of 34 patients was eligible for analysis of the treatment. The median age was 67 years (range 38-82). Six patients had T1, 9 had T2, and 19 had T3 diseases (AJCC). Dose-limiting toxicities were Grade 4, fatigue and nausea; Grade 3, thrombocytopenia, diarrhea, and infection. The MTD established was at the 50-mg/m(2) gemcitabine dose. A total of 21 of 28 patients underwent surgery: 18 had pancreaticoduodenectomy, 2 had total pancreatectomy, and 1 for palliative surgery. At the time of analysis, 13 of 28 (46%) were disease-free. The estimated median survival was 25 months and overall survival rate at 2 years (Kaplan-Meier) was 55%. CONCLUSION: Gemcitabine 50 mg/m(2) given twice weekly with concomitant irradiation induces acceptable and manageable toxicity and might prolong survival.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/radioterapia , Desoxicitidina/análogos & derivados , Desoxicitidina/administração & dosagem , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/radioterapia , Radiossensibilizantes/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/efeitos adversos , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/cirurgia , Terapia Combinada , Desoxicitidina/efeitos adversos , Feminino , Humanos , Excisão de Linfonodo , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Terapia Neoadjuvante , Pancreatectomia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Pancreaticoduodenectomia , Radiossensibilizantes/efeitos adversos , Dosagem Radioterapêutica , GencitabinaRESUMO
Tylosis with esophageal cancer (TOC) is a rare familial cancer syndrome inherited in an autosomal-dominant manner and characterized by esophageal cancer susceptibility and hyperkeratotic skin lesions. Two heterozygous missense mutations in the RHBDF2 gene were recently reported to be associated with TOC in three families: a p.Ile186Thr mutation was found in families from the UK and the US and a p.Pro189Leu mutation was detected in a German TOC family. We aimed to validate these novel results in an independent material by screening RHBDF2 in a previously unreported Finnish TOC family. We identified a new missense mutation, p.Asp188Asn, segregating with TOC in the Finnish family, and interestingly the detected mutation alters a codon located between the two previously reported mutation sites. Thus, we confirmed RHBDF2 mutations as the underlying cause of the TOC syndrome and our results suggest that the TOC associated mutations might be specific for this particular site in the RHBDF2 gene. These results enable the genetic counseling and diagnostic mutation screening of the members of TOC families.
Assuntos
Proteínas de Transporte/genética , Neoplasias Esofágicas/genética , Ceratodermia Palmar e Plantar Difusa/genética , Mutação de Sentido Incorreto , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Finlândia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
Ceramides may mediate saturated fat-induced insulin resistance, but there are no data comparing ceramide concentrations between human tissues. We therefore performed lipidomic analysis of human subcutaneous (SCfat) and intra-abdominal (IAfat) adipose tissue, the liver, and serum in eight subjects. The liver contained (nmol/mg tissue) significantly more ceramides (1.5-3-fold), sphingomyelins (7-8-fold), phosphatidylethanolamines (10-11-fold), lysophosphatidylcholines (7-12-fold), less ether-linked phosphatidylcholines (2-2.5-fold) but similar amounts of diacylglycerols as compared to SCfat and IAfat. The amounts of ceramides and their synthetic precursors, such as palmitic (16:0) free fatty acids and sphingomyelins, differed considerably between the tissues. The liver contained proportionally more palmitic, stearic (18:0), and long polyunsaturated fatty acids than adipose tissues. Stearoyl-CoA desaturase 1 (SCD1) activity reflected by serum, estimated from the 16:1/16:0-ratio, was closely related to that in the liver (r = 0.86, P = 0.024) but not adipose tissues. This was also true for estimated elongase (18:1/16:1, r = 0.89, P = 0.01), and Delta5 (20:4/20:3, r = 0.89, P = 0.012) and Delta6 (18:3[n-6]/18:2, r = 1.0, P < 0.001) desaturase activities. We conclude that the human liver contains higher concentrations of ceramides and saturated free fatty acids than either SCfat or IAfat.
Assuntos
Gordura Intra-Abdominal/química , Lipídeos/análise , Lipídeos/sangue , Fígado/química , Gordura Subcutânea/química , Adulto , Análise de Variância , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Seleção de PacientesRESUMO
OBJECTIVE: To determine whether 1) hepatic ceramide and diacylglycerol concentrations, 2) SCD1 activity, and 3) hepatic lipogenic index are increased in the human nonalcoholic fatty liver. RESEARCH DESIGN AND METHODS: We studied 16 subjects with (n = 8) and without (n = 8) histologically determined nonalcoholic fatty liver (NAFL(+) and NAFL(-)) matched for age, sex, and BMI. Hepatic concentrations of lipids and fatty acids were quantitated using ultra-performance liquid chromatography coupled to mass spectrometry and gas chromatography. RESULTS: The absolute (nmol/mg) hepatic concentrations of diacylglycerols but not ceramides were increased in the NAFL(+) group compared with the NAFL(-) group. The livers of the NAFL(+) group contained proportionally less long-chain polyunsaturated fatty acids as compared with the NAFL(-) group. Liver fat percent was positively related to hepatic stearoyl-CoA desaturase 1 (SCD1) activity index (r = 0.70, P = 0.003) and the hepatic lipogenic index (r = 0.54, P = 0.030). Hepatic SCD1 activity index was positively related to the concentrations of diacylglycerols (r = 0.71, P = 0.002) but not ceramides (r = 0.07, NS). CONCLUSIONS: We conclude that diacylglycerols but not ceramides are increased in NAFL. The human fatty liver is also characterized by depletion of long polyunsaturated fatty acids in the liver and increases in hepatic SCD1 and lipogenic activities.
Assuntos
Ceramidas/metabolismo , Diglicerídeos/metabolismo , Fígado Gorduroso/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Adolescente , Adulto , Idoso , Cromatografia Líquida , Fígado Gorduroso/enzimologia , Fígado Gorduroso/patologia , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Adulto JovemRESUMO
We have here elucidated whether ulcerogenic agents affect the production of NO and reactive oxygen species (ROS). The ulcerogenic agents dose dependently induced NO and ROS production in mouse gastric epithelial cells. Taurocholate (TC, 5 mM) exposure did not affect cell viability, but it increased inducible nitric oxide synthase (iNOS) expression, NO production, ROS production, and epithelial permeability. Epithelial permeability was inhibited with NOS inhibitors or antioxidants. Oxidative stress induced by acetylsalicylic acid (ASA) and ethanol was not inhibited with NOS inhibitors. ASA induced ROS production even at low concentrations (1 mM), which did not affect cell viability. Ethanol-induced ROS production was linked to cell viability, suggesting direct oxidative stress caused by ethanol. Taurocholate-induced NO signaling and the ensuing production of ROS might contribute to initiation of defensive or adaptive cellular mechanisms. ASA-induced ROS signaling might have similar effects, whereas ethanol induced direct oxidative stress, having an influence on cell viability.
Assuntos
Colagogos e Coleréticos/farmacologia , Mucosa Gástrica/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ácido Taurocólico/farmacologia , Animais , Antioxidantes/farmacologia , Aspirina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Etanol/farmacologia , Mucosa Gástrica/citologia , Camundongos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Solventes/farmacologiaRESUMO
Despite the high prevalence of nonalcoholic fatty liver disease (NAFLD), little is known of its pathogenesis based on study of human liver samples. By the use of Affymetrix GeneChips (17,601 genes), we investigated gene expression in the human liver of subjects with extreme steatosis due to NAFLD without histological signs of inflammation (liver fat 66.0 +/- 6.8%) and in subjects with low liver fat content (6.4 +/- 2.7%). The data were analyzed by using sequence-based reannotation of Affymetrix probes and a robust model-based normalization method. We identified genes involved in hepatic glucose and lipid metabolism, insulin signaling, inflammation, coagulation, and cell adhesion to be significantly associated with liver fat content. In addition, genes involved in ceramide signaling (MAP2K4) and metabolism (UGCG) were found to be positively associated with liver fat content. Genes involved in lipid metabolism (PLIN, ACADM), fatty acid transport (FABP4, CD36), amino acid catabolism (BCAT1), and inflammation (CCL2) were validated by real-time PCR and were found to be upregulated in subjects with high liver fat content. The data show that multiple changes in gene expression characterize simple steatosis.
Assuntos
Fígado Gorduroso/genética , Perfilação da Expressão Gênica , Acil-CoA Desidrogenase/genética , Adulto , Antígenos CD36/genética , Metabolismo dos Carboidratos/genética , Proteínas de Transporte , Quimiocina CCL2/genética , Regulação para Baixo/genética , Proteínas da Matriz Extracelular/genética , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Humanos , Inflamação/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metabolismo dos Lipídeos/genética , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Perilipina-1 , Fosfoproteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transaminases/genética , Regulação para Cima/genéticaRESUMO
Pancreatic adenocarcinoma is known for early aggressive local invasion, high metastatic potential, and a low 5-year survival rate. Matrix metalloproteinases (MMPs) play important roles in tumor growth and invasion. Earlier studies on pancreatic cancer have found increased expression of certain MMPs to correlate with poorer prognosis, short survival time or presence of metastases. We studied the expression of MMP-21, -26, and tissue inhibitor of matrix metalloproteinases (TIMP)-4 in 50 tissue samples, including 25 adenocarcinomas, seven other malignant pancreatic tumors, and 18 control samples of non-neoplastic pancreatic tissue with immunohistochemistry. The regulation of MMP-21, -26, and TIMP-4 mRNAs by cytokines was studied with RT-PCR in pancreatic cancer cell lines PANC-1, BxPC-3, and AsPC-1. MMP-21, -26, and TIMP-4 were detected in cancer cells in 64, 40, and 60% of tumors, respectively. MMP-21 expressed in well-differentiated cancer cells and occasional fibroblasts, like TIMP-4, tended to diminish in intensity from grade I to grade III tumors. Patients with metastatic lymph nodes had increased expression of MMP-26 in actual tumor samples. All cultured cancer cell lines expressed MMP-21 basally at low levels, and presence of the protein was confirmed immunohistochemically in cultured cells. MMP-21 expression was induced by epidermal growth factor (EGF) in PANC-1 cells. MMP-26 was neither expressed basally nor induced by tumor necrosis factor alpha, transforming growth factor beta-1 (TGFbeta1), EGF, or interferon gamma. Basal TIMP-4 expression was lowest in the poorly differentiated cancer cell line PANC-1 compared to better-differentiated BxPC-3 and AsPC-1 cells. TIMP-4 expression was induced by TGFbeta1 in PANC-1 cells and by EGF in BxPC-3 cells. Our findings suggest that MMP-21 is not a marker of invasiveness, but rather of differentiation, in pancreatic cancer and it may be upregulated by EGF. The putative role of MMP-26 as a marker of metastases warrants further studies. Unlike other TIMPs, TIMP-4 was not upregulated in relation to aggressiveness of pancreatic cancer.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/análise , Metaloproteinases da Matriz Secretadas/biossíntese , Metaloproteinases da Matriz/biossíntese , Neoplasias Pancreáticas/metabolismo , Inibidores Teciduais de Metaloproteinases/biossíntese , Adenocarcinoma/patologia , Adulto , Idoso , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/fisiopatologia , Neoplasias Pancreáticas/patologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
Ethanol is a well-established irritant inducing inflammation in gastric mucosa, but the effects at the cellular level remain unclear. This study investigates NF-kappaB activation in gastric mucosal cells by ethanol and assesses the effects of heat shock pretreatment in this ulcerogenic situation. Rat gastric mucosal epithelia were exposed to ethanol for different time periods. Heat shock was induced by incubating the cells at 42 degrees C for 1 h prior to the experiments. For evaluation of NF-kappaB activation, the nuclear fraction of the cell lysates was analyzed with an EMSA or an ELISA-based assay. Caspase-3 (a promoter of apoptosis) activity was measured with a time-resolved fluorescence based assay, cell viability with a tetrazolium assay, and cell membrane integrity with a LDH assay. Ethanol (1-5%) induced NF-kappaB activation, reaching a maximum after 3 h, and also led to moderately increased COX-2 expression. Heat shock pretreatment and the intracellular calcium chelator BAPTA were able to inhibit ethanol-induced NF-kappaB activation. Heat shock pretreatment decreased ethanol-induced caspase-3 activation, decreased cell membrane damage, and retained cellular viability. Inhibition of NF-kappaB activation by NEMO-binding peptide, by decreasing RelA expression, or by inhibiting COX-2 activity by CAY-14040 promoted the effects of ethanol, such as increased caspase-3 activity and decreased cell viability. In conclusion, ethanol induces NF-kappaB activation via a calcium-dependent pathway and induces COX-2 expression. Inhibition of the NF-kappaB activation or COX-2 activity potentiates apoptosis and cell damage induced by ethanol, suggesting a protective role for NF-kappaB activation and COX-2 expression.