RESUMO
Gedunin is a natural tetranorterpenoid secondary metabolite found in plants of the Meliaceae family, which has been reported for its antiparasitic, antifungal and anticancer activities. Here, we describe the molecular mechanisms underlying the in vitro anti proliferative activity of gedunin (isolated from the mangrove plant Xylocarpus granatum) in human ovarian cancer cells. We observed that gedunin triggered severe ROS generation leading to DNA damage and cell cycle arrest in G2/M phase thus inhibiting cell proliferation. ROS upregulation also led to mitochondrial stress and membrane depolarization, which eventually resulted in mitochondria-mediated apoptosis following cytochrome C release, caspase 9, 3 activation, and PARP cleavage. Transmission electron microscopy of gedunin treated cells revealed sub-cellular features typical of apoptosis. Moreover, an upregulation in stress kinases like phospho-ERK 1/2, phospho-p38 and phospho-JNK was also observed in gedunin treated cells. Free radical scavenger N-Acetyl-L-Cysteine (NAC) reversed all these effects resulting in increased cell survival, abrogation of cell cycle arrest, rescue of mitochondrial membrane potential and suppression of apoptotic markers. Interestingly, gedunin is also an inhibitor of the evolutionarily conserved molecular chaperone Heat Shock Protein 90 (hsp90) responsible for maintaining cellular homeostasis. Targeting this chaperone could be an attractive strategy for developing cancer therapeutics since many oncogenic proteins are also client proteins of hsp90. Collectively, our findings provide insights into the molecular mechanism of action of gedunin, which may aid drug development efforts against ovarian cancer.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Limoninas/farmacologia , Meliaceae/química , Espécies Reativas de Oxigênio/agonistas , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Frutas/química , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Concentração Inibidora 50 , Limoninas/química , Limoninas/isolamento & purificação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Owing to its ability to induce cellular senescence, inhibit PCNA, and arrest cell division cycle by negatively regulating CDKs as well as being a primary target of p53, p21 is traditionally considered a tumor suppressor. Nonetheless, several reports in recent years demonstrated its pro-oncogenic activities such as apoptosis inhibition by cytosolic p21, stimulation of cell motility, and promoting assembly of cyclin D-CDK4/6 complex. These opposing effects of p21 on cell proliferation, supported by the observations of its inconsistent expression in human cancers, led to the emergence of the concept of "antagonistic duality" of p21 in cancer progression. Here we demonstrate that p21 negatively regulates basal autophagy at physiological concentration. Akt activation, upon p21 attenuation, driven ROS accumulation appears to be the major underlying mechanism in p21-mediated modulation of autophagy. We also find p21, as a physiological inhibitor of autophagy, to have oncogenic activity during early events of tumor development while its inhibition favors survival and growth of cancer cells in the established tumor. Our data, thereby, reveal the potential role of autophagy in antagonistic functional duality of p21 in cancer.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Proteína Supressora de Tumor p53 , Humanos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , AutofagiaRESUMO
Naturally occurring cationic antimicrobial peptides (AMPs) mostly adopt α-helical structures in bacterial membrane mimetic environments. To explore the design of novel ß-sheet AMPs, we identified two short cationic amphipathic ß-strand segments from the crystal structure of the innate immune protein, MyD88. Interestingly, of these, the 10-residue arginine-valine-rich synthetic MyD88-segment, KRCRRMVVVV (M3), exhibited ß-sheet structure when bound to the outer membrane Gram-negative bacterial component, LPS. Isothermal titration calorimetric data showed that M3 bound to LPS with high affinity, and the interaction was hydrophobic in nature. Supporting these observations, computational studies indicated strong interactions of multiple and consecutive valine residues of M3 with the acyl chain of LPS. Moreover, M3 adopted nanosheet and nanofibrillar structure in 25% acetonitrile/water and isopropanol, respectively. M3 showed substantial antibacterial activities against both Gram-positive and Gram-negative bacteria which it appreciably retained in the presence of human serum and physiological salts. M3 was non-hemolytic against human red blood cells and non-cytotoxic to 3T3 cells up to 200 µM and to mice in vivo at a dose of 40 mg/kg. Furthermore, M3 neutralized LPS-induced pro-inflammatory responses in THP-1 cells and rat bone marrow-derived macrophages. Consequently, M3 attenuated LPS-mediated lung inflammation in mice and rescued them (80% survival at 10 mg/kg dose) against a lethal dose of LPS. The results demonstrate the identification of a 10-mer LPS-interacting, ß-sheet peptide from MyD88 with the ability to form nanostructures and in vivo activity against LPS challenge in mice. The identified M3-template provides scope for designing novel bioactive peptides with ß-sheet structures and self-assembling properties.
Assuntos
Lipopolissacarídeos , Pneumonia , Camundongos , Humanos , Ratos , Animais , Lipopolissacarídeos/química , Antibacterianos/farmacologia , Conformação Proteica em Folha beta , Endotoxinas , Bactérias Gram-Negativas , Fator 88 de Diferenciação Mieloide , Bactérias Gram-Positivas , Peptídeos Catiônicos Antimicrobianos/farmacologia , Valina , PulmãoRESUMO
High-mobility group box 1 (HMGB1) is a nuclear factor that is released extracellularly as a late mediator of lethality in sepsis as well as after necrotic, but not apoptotic, death. Here we demonstrate that in contrast to the delayed role of HMGB1 in the systemic inflammation of sepsis, HMGB1 acts as an early mediator of inflammation and organ damage in hepatic ischemia reperfusion (I/R) injury. HMGB1 levels were increased during liver I/R as early as 1 h after reperfusion and then increased in a time-dependent manner up to 24 h. Inhibition of HMGB1 activity with neutralizing antibody significantly decreased liver damage after I/R, whereas administration of recombinant HMGB1 worsened I/R injury. Treatment with neutralizing antibody was associated with less phosphorylation of c-Jun NH(2)-terminal kinase and higher nuclear factor-kappaB DNA binding in the liver after I/R. Toll-like receptor 4 (TLR4)-defective (C3H/Hej) mice exhibited less damage in the hepatic I/R model than did wild-type (C3H/HeOuj) mice. Anti-HMGB1 antibody failed to provide protection in C3H/Hej mice, but successfully reduced damage in C3H/Ouj mice. Together, these results demonstrate that HMGB1 is an early mediator of injury and inflammation in liver I/R and implicates TLR4 as one of the receptors that is involved in the process.
Assuntos
Proteína HMGB1/metabolismo , Fígado/metabolismo , Fígado/patologia , Traumatismo por Reperfusão/metabolismo , Animais , Anticorpos/metabolismo , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Hepatócitos/metabolismo , Imuno-Histoquímica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fosforilação , Receptores de Superfície Celular/metabolismo , Traumatismo por Reperfusão/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Receptor 4 Toll-LikeRESUMO
BACKGROUND: Thoracoscopic lobectomy is well established for the treatment of early non-small cell lung cancer (NSCLC). Its safety and efficacy for advanced-stage disease remain uncertain. METHODS: Between January 1, 2002, and July 31, 2007, a total of 125 patients were evaluated for thoracoscopic lobectomy for advanced NSCLC. Thoracoscopic lobectomy was completed in 73 patients. Eleven patients were excluded for extensive chest wall involvement. Open resection was performed in 41 patients, with 19 planned thoracotomies and 22 conversions from an initial thoracoscopic approach. RESULTS: Median operative blood loss, operation time, major complications, and hospital length of stay were all similar for patients undergoing thoracoscopic and open resection. A higher percentage of patients who underwent thoracoscopic lobectomy were able to receive adjuvant therapy compared to the open group (37.2% vs. 5.2%; P = 0.006). The differences between the thoracoscopic and open groups in overall survival (43.7 vs. 22.9 months; P = 0.59) and disease-free survival (34.7 vs. 16.7 months; P = 0.84) were not significant. CONCLUSIONS: Thoracoscopic lobectomy for advanced-stage NSCLC can be performed safely, with results equivalent to open techniques. With continued experience, lower morbidity with resections performed for advanced-stage disease by video-assisted thoracoscopic surgery will be expected, similar to that observed with early-stage disease. This is particularly important given the large number of frail patients with advanced-stage disease who require multimodal therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/cirurgia , Neoplasias Pulmonares/cirurgia , Pneumonectomia , Cirurgia Torácica Vídeoassistida , Toracoscopia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Therapy resistance by evasion of apoptosis is one of the hallmarks of human cancer. Therefore, restoration of cell death by non-apoptotic mechanisms is critical to successfully overcome therapy resistance in cancer. By rational drug design approach, here we try to provide evidence that subtle changes in the chemical structure of spisulosine completely switched its cytotoxic function from apoptosis to autophagy. Our most potent molecule (26b) in a series of 16 synthesized derivatives showed robust autophagic cell death in diverse cancer cells sparing normal counterpart. Compound 26b mediated lethal autophagy induction was confirmed by formation of characteristic autophagic vacuoles, LC3 puncta formation, upregulation of signature autophagy markers like Beclin and Atg family proteins. Altogether, we have detected novel autophagy inducer small molecule which can be tested further for drug discovery research.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Lipídeos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lipídeos/síntese química , Lipídeos/química , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
OBJECTIVES: Given that autophagy inhibition is a feasible way to enhance sensitivity of cancer cells towards chemotherapeutic agents, identifying potent autophagy inhibitor has obvious clinical relevance. Here, we investigated ability of TN-16, a microtubule disrupting agent, on modulation of autophagic flux and its significance in promoting in vitro and in vivo cancer cell death. MATERIALS AND METHODS: The effect of TN-16 on cancer cell proliferation, cell division, autophagic process and apoptotic signalling was assessed by various biochemical (Western blot and SRB assay), morphological (TEM, SEM, confocal microscopy) and flowcytometric assays. In vivo anti-tumour efficacy of TN-16 was investigated in syngeneic mouse model of breast cancer. RESULTS: TN-16 inhibited cancer cell proliferation by impairing late-stage autophagy and induction of apoptosis. Inhibition of autophagic flux was demonstrated by accumulation of autophagy-specific substrate p62 and lack of additional LC3-II turnover in the presence of lysosomotropic agent. The effect was validated by confocal micrographs showing diminished autophagosome-lysosome fusion. Further studies revealed that TN-16-mediated inhibition of autophagic flux promotes apoptotic cell death. Consistent with in vitro data, results of our in vivo study revealed that TN-16-mediated tumour growth suppression is associated with blockade of autophagic flux and enhanced apoptosis. CONCLUSIONS: Our data signify that TN-16 is a potent autophagy flux inhibitor and might be suitable for (pre-) clinical use as standard inhibitor of autophagy with anti-cancer activity.
Assuntos
Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Pirrolidinonas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Pirrolidinonas/farmacologia , Moduladores de Tubulina/farmacologiaRESUMO
Nitric oxide (NO.), an important mediator of inflammation, and beta-catenin, a component of the Wnt-adenomatous polyposis coli signaling pathway, contribute to the development of cancer. We have identified two T-cell factor 4 (Tcf-4)-binding elements (TBE1 and TBE2) in the promoter of human inducible NO synthase 2 (NOS2). We tested the hypothesis that beta-catenin regulates human NOS2 gene. Mutation in either of the two TBE sites decreased the basal and cytokine-induced NOS2 promoter activity in different cell lines. The promoter activity was significantly reduced when both TBE1 and TBE2 sites were mutated (P < 0.01). Nuclear extract from HCT116, HepG2, or DLD1 cells bound to NOS2 TBE1 or TBE2 oligonucleotides in electrophoretic mobility shift assays and the specific protein-DNA complexes were supershifted with anti-beta-catenin or anti-Tcf-4 antibody. Overexpression of beta-catenin and Tcf-4 significantly increased both basal and cytokine-induced NOS2 promoter activity (P < 0.01), and the induction was dependent on intact TBE sites. Overexpression of beta-catenin or Tcf-4 increased NOS2 mRNA and protein expression in HCT116 cells. Lithium chloride (LiCl), an inhibitor of glycogen synthase kinase-3beta, increased cytosolic and nuclear beta-catenin level, NOS2 expression, and NO. production in primary human and rat hepatocytes and cancer cell lines. Treatment with Wnt-3A-conditioned medium increased beta-catenin and NOS2 expression in fetal human hepatocytes. When administered in vivo, LiCl increased hepatic beta-catenin level in a dose-dependent manner with simultaneous increase in NOS2 expression. These data are consistent with the hypothesis that beta-catenin up-regulates NOS2 and suggest a novel mechanism by which the Wnt/beta-catenin signaling pathway may contribute to cancer by increasing NO. production.
Assuntos
Óxido Nítrico Sintase Tipo II/biossíntese , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HCT116 , Humanos , Cloreto de Lítio/farmacologia , Fígado/enzimologia , Masculino , Óxido Nítrico Sintase Tipo II/genética , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais , Fatores de Transcrição TCF/biossíntese , Fatores de Transcrição TCF/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição , Ativação Transcricional , Transfecção , beta Catenina/biossínteseRESUMO
Aß29-40 residues with tryptophan in place of the lone methionine residue and three arginine residues added to its C-terminus exhibited augmented antibacterial activities and protected mice against a lethal dose of LPS. The results show the conversion of a Aß29-40 segment into a cell-selective antimicrobial/anti-endotoxin peptide with nanostructure and cation-π interaction.
Assuntos
Peptídeos beta-Amiloides/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Endotoxinas/antagonistas & inibidores , Nanoestruturas/química , Fragmentos de Peptídeos/química , Animais , Antibacterianos/síntese química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Desenho de Fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Carbon monoxide (CO), a product of heme degradation by heme oxygenase, induces cytoprotection against ischemia/reperfusion (I/R) injury in a variety of organs such as the heart, lung, kidney, and small intestine. We examined whether CO would protect liver grafts against cold I/R injury associated with transplantation. METHODS: Orthotopic liver transplantation (OLT) was performed in syngeneic Lewis rats with 18 hours preservation in cold University of Wisconsin solution. Recipients were exposed to air or CO (100 ppm) for 1 hour before and 24 hours after OLT. Recipients were sacrificed 0.5 to 48 hours post-transplant. RESULTS: CO inhalation significantly decreased serum aspartate aminotransferase and alanine aminotransferase levels and suppressed hepatic necrosis formation and neutrophil accumulation at 6 to 48 hours after OLT, compared with air control. The expressions of tumor necrosis factor alpha, intercellular adhesion molecule 1, and inducible nitric oxide synthase messenger RNA in the liver graft were significantly inhibited in the CO-treated group at 1 hour after reperfusion. Hepatic nuclear factor-kappaB activation did not differ between the groups. CONCLUSIONS: The results demonstrate that exogenous CO treatment suppresses early proinflammatory gene expression and neutrophil infiltration, and efficiently ameliorates hepatic I/R injury. The possible mechanism by which CO protects the liver against cold I/R does not seem to be associated with downregulation of the nuclear factor-kappaB-signaling pathway.
Assuntos
Monóxido de Carbono/farmacologia , Criopreservação , Transplante de Fígado , Traumatismo por Reperfusão/tratamento farmacológico , Administração por Inalação , Animais , Monóxido de Carbono/sangue , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Hemoglobinas/metabolismo , Molécula 1 de Adesão Intercelular/genética , Masculino , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: While thoracoscopic surgical lobectomy is an established operation, the safety of thoracoscopic pneumonectomy (TP) is uncertain. METHODS: From January 1, 2002, to September 30, 2008 at a comprehensive cancer center, 70 patients underwent pneumonectomy. Three patients were excluded for emergent operations. Thoracoscopic pneumonectomy was completed successfully in 24 patients and attempted in 8 others (25% conversion rate). Analysis was done on an intention-to-treat basis. RESULTS: By 2008, 75% of pneumonectomy cases were planned as TP while there were no conversions to thoracotomy. There was no difference in median blood loss between patients undergoing TP versus thoracotomy (325 vs 300 mL, p = 0.52), but operations were longer (286 vs 228 minutes, p < 0.01). Median intensive care unit stay was 2 days in both groups and median hospital stay was 5.0 days in the TP group versus 6.0 days in the thoracotomy group (p = 0.28). Major complications were similar between groups. The TP reoperations were for bleeding (2), bronchopleural fistula (2), empyema (1), and chylothorax (1). The only TP death occurred in an 83-year-old patient from respiratory failure. Neither the use of adjuvant therapy nor the time between surgery and commencement of adjuvant therapy was different between groups. Conversions alone compared with patients undergoing thoracotomy were associated with a moderate increase in blood loss and intensive care unit stay, but not in any major complications. CONCLUSIONS: Thoracoscopic pneumonectomy can be done safely. The availability of this option is important especially in an era of multimodality therapy as more debilitated patients present for surgical therapy.