RESUMO
Plastics have become ubiquitous in both their adoption as materials and as environmental contaminants. Widespread pollution of these versatile, man-made and largely petroleum-derived polymers has resulted from their long-term mass production, inappropriate disposal and inadequate end of life management. Polyethylene (PE) is at the forefront of this problem, accounting for one-third of plastic demand in Europe in part due to its extensive use in packaging. Current recycling and incineration processes do not represent sustainable solutions to tackle plastic waste, especially once it becomes littered, and the development of new waste-management and remediation technologies are needed. Mycoremediation (fungal-based biodegradation) of PE has been the topic of several studies over the last two decades. The utility of these studies is limited by an inconclusive definition of biodegradation and a lack of knowledge regarding the biological systems responsible. This review highlights relevant features of fungi as potential bioremediation agents, before discussing the evidence for fungal biodegradation of both high- and low-density PE. An up-to-date perspective on mycoremediation as a future solution to PE waste is provided.
Assuntos
Plásticos , Polietileno , Biodegradação Ambiental , Fungos , Humanos , ReciclagemRESUMO
Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli.
Assuntos
Glicosiltransferases/metabolismo , Fenotiazinas/farmacologia , Acetatos/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/química , Sítios de Ligação/genética , Domínio Catalítico/genética , Parede Celular/metabolismo , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Glicosiltransferases/antagonistas & inibidores , Glicosiltransferases/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Muramidase/antagonistas & inibidores , Muramidase/metabolismo , Peptidoglicano/metabolismo , Fenotiazinas/metabolismo , Conformação Proteica/efeitos dos fármacosRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed when metastatic events have occurred. Cancer stem cells (CSCs) play an important role in tumor initiation, metastasis, chemoresistance and relapse. A growing number of studies have suggested that CSCs exist in a dynamic equilibrium with more differentiated cancer cells via a bidirectional regeneration that is dependent on the environmental stimuli. In this investigation, we obtain, by using a selective medium, PDAC CSCs from five out of nine PDAC cell lines, endowed with different tumorsphereforming ability. PDAC CSCs were generally more resistant to the action of five anticancer drugs than parental cell lines and were characterized by an increased expression of EpCAM and CD44v6, typical stem cell surface markers, and a decreased expression of Ecadherin, the main marker of the epithelial state. PDAC CSCs were able to redifferentiate into parental cells once cultured in parental growth condition, as demonstrated by reacquisition of the epithelial morphology, the decreased expression levels of EpCAM and CD44v6 and the increased sensitivity to anticancer drugs. Finally, PDAC CSCs injected into nude mice developed a larger subcutaneous tumor mass and showed a higher metastatic activity compared to parental cells. The present study demonstrates the ability to obtain CSCs from several PDAC cell lines and that these cells are differentially resistant to various anticancer agents. This variability renders them a model of great importance to deeply understand pancreatic adenocarcinoma biology, to discover new biomarkers and to screen new therapeutic compounds.
Assuntos
Carcinoma Ductal Pancreático/patologia , Desdiferenciação Celular , Diferenciação Celular , Células-Tronco Neoplásicas/fisiologia , Neoplasias Pancreáticas/patologia , Animais , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/fisiopatologia , Desdiferenciação Celular/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/fisiopatologiaRESUMO
The aim of this work was the preparation, characterization, and preliminary evaluation of the targeting ability toward pancreatic adenocarcinoma cells of liposomes containing the gemcitabine lipophilic prodrug [4-(N)-lauroyl-gemcitabine, C12GEM]. Hyaluronic acid (HA) was selected as targeting agent since it is biodegradable, biocompatible, and can be chemically modified and its cell surface receptor CD44 is overexpressed on various tumors. For this purpose, conjugates between a phospholipid, the 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and HA of two different low molecular weights 4800 Da (12 disaccharidic units) and 12,000 Da (32 disaccharidic units), were prepared, characterized, and introduced in the liposomes during the preparation. Different liposomal formulations were prepared and their characteristics were analyzed: size, Z potential, and TEM analyses underline a difference in the HA-liposomes from the non-HA ones. In order to better understand the HA-liposome cellular localization and to evaluate their interaction with CD44 receptor, confocal microscopy studies were performed. The results demonstrate that HA facilitates the recognition of liposomes by MiaPaCa2 cells (CD44(+)) and that the uptake increases with increase in the polymer molecular weight. Finally, the cytotoxicity of the different preparations was evaluated and data show that incorporation of C12GEM increases their cytotoxic activity and that HA-liposomes inhibit cell growth more than plain liposomes. Altogether, the results demonstrate the specificity of C12GEM targeting toward CD44-overexpressing pancreatic adenocarcinoma cell line using HA as a ligand.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antimetabólitos Antineoplásicos/administração & dosagem , Desoxicitidina/análogos & derivados , Ácido Hialurônico/química , Neoplasias Pancreáticas/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Desoxicitidina/química , Desoxicitidina/farmacologia , Sistemas de Liberação de Medicamentos , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Lipossomos , Peso Molecular , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosfatidiletanolaminas/química , Pró-FármacosRESUMO
Pancreatic adenocarcinoma is often diagnosed when metastatic events have occurred. The early spread of circulating cancer cells expressing the CD44 receptor may play a crucial role in this process. In this study, we have investigated the cellular delivery ability and both in vitro and in vivo anti-tumoral activity of liposomes conjugated with two different low molecular weight hyaluronic acids (HA 4.8kDa and HA 12kDa), the primary ligand of CD44, and containing a lipophilic gemcitabine (GEM) pro-drug. By confocal microscopy and flow cytometry analyses, we demonstrate that the cellular uptake into a highly CD44-expressing pancreatic adenocarcinoma cell line is higher with HA-conjugated (12kDa>4.8kDa) than non-conjugated liposomes. Consistently, in vitro cytotoxic assays display an increased sensitivity towards GEM containing HA-liposomes, compared to non-conjugated liposomes. Conversely, CD44 non-expressing normal cells show a similar uptake and in vitro cytotoxicity with both HA-conjugated and non-conjugated liposomes. Furthermore, we demonstrate that the HA-liposomes are taken up into the cells via lipid raft-mediated endocytosis. All the liposome formulations containing GEM show a higher antitumoral activity than free GEM in a mouse xenograft tumor model of human pancreatic adenocarcinoma. The 12kDa HA-liposomes have the strongest efficiency, while non-conjugated liposomes and the 4.8kDa HA-liposomes are similarly active. Taken together, our results provide a strong rationale for further development of HA-conjugated liposomes to treat pancreatic adenocarcinoma.
Assuntos
Adenocarcinoma/tratamento farmacológico , Desoxicitidina/análogos & derivados , Lipossomos/química , Neoplasias Pancreáticas/tratamento farmacológico , 1,2-Dipalmitoilfosfatidilcolina/química , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colesterol/química , Desoxicitidina/química , Desoxicitidina/farmacologia , Citometria de Fluxo , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Lipossomos/metabolismo , Camundongos , Camundongos Nus , Microscopia Confocal , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosfatidiletanolaminas/química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
TP53 mutations compromising p53 transcriptional function occur in more than 50 % of human cancers, including pancreatic adenocarcinoma, and render cancer cells more resistant to conventional therapy. In the last few years, many efforts have been addressed to identify p53-reactivating molecules able to restore the wild-type transcriptionally competent conformation of the mutated proteins. Here, we show that two of these compounds, CP-31398 and RITA, induce cell growth inhibition, apoptosis, and autophagy by activating p53/DNA binding and p53 phosphorylation (Ser15), without affecting the total p53 amount. These effects occur in both wild-type and mutant p53 pancreatic adenocarcinoma cell lines, whereas they are much less pronounced in normal human primary fibroblasts. Furthermore, CP-31398 and RITA regulate the axis SESN1-2/AMPK/mTOR by inducing AMPK phosphorylation on Thr172, which has a crucial role in the autophagic response. The protective role of autophagy in cell growth inhibition by CP-31398 and RITA is supported by the finding that the AMPK inhibitor compound C or the autophagy inhibitors chloroquine or 3-methyladenine sensitize both pancreatic adenocarcinoma cell lines to the apoptotic response induced by p53-reactivating molecules. Our results demonstrate for the first time a survival role for autophagy induced by p53-reactivating molecules, supporting the development of an anti-cancer therapy based on autophagy inhibition associated to p53 activation.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Pirimidinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática , Furanos/farmacologia , Humanos , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Mitochondrial uncoupling protein 2 (UCP2) can moderate oxidative stress by favoring the influx of protons into the mitochondrial matrix, thus reducing electron leakage from respiratory chain and mitochondrial superoxide production. Here, we demonstrate that UCP2 inhibition by genipin or UCP2 siRNA strongly increases reactive oxygen species (ROS) production inhibiting pancreatic adenocarcinoma cell growth. We also show that UCP2 inhibition triggers ROS-dependent nuclear translocation of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), formation of autophagosomes, and the expression of the autophagy marker LC3-II. Consistently, UCP2 over-expression significantly reduces basal autophagy confirming the anti-autophagic role of UCP2. Furthermore, we demonstrate that autophagy induced by UCP2 inhibition determines a ROS-dependent cell death, as indicated by the apoptosis decrease in the presence of the autophagy inhibitors chloroquine (CQ) or 3-methyladenine (3-MA), or the radical scavenger NAC. Intriguingly, the autophagy induced by genipin is able to potentiate the autophagic cell death triggered by gemcitabine, the standard chemotherapeutic drug for pancreatic adenocarcinoma, supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to standard chemotherapy. Our results demonstrate for the first time that UCP2 plays a role in autophagy regulation bringing new insights into mitochondrial uncoupling protein field.
Assuntos
Adenocarcinoma/patologia , Autofagia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Canais Iônicos/antagonistas & inibidores , Iridoides/farmacologia , Proteínas Mitocondriais/antagonistas & inibidores , Neoplasias Pancreáticas/patologia , Espécies Reativas de Oxigênio/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colagogos e Coleréticos/farmacologia , Imunofluorescência , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Desacopladora 2RESUMO
Cancer cells exhibit an endogenous constitutive oxidative stress higher than that of normal cells, which renders tumours vulnerable to further reactive oxygen species (ROS) production. Mitochondrial uncoupling protein 2 (UCP2) can mitigate oxidative stress by increasing the influx of protons into the mitochondrial matrix and reducing electron leakage and mitochondrial superoxide generation. Here, we demonstrate that chemical uncouplers or UCP2 over-expression strongly decrease mitochondrial superoxide induction by the anticancer drug gemcitabine (GEM) and protect cancer cells from GEM-induced apoptosis. Moreover, we show that GEM IC(50) values well correlate with the endogenous level of UCP2 mRNA, suggesting a critical role for mitochondrial uncoupling in GEM resistance. Interestingly, GEM treatment stimulates UCP2 mRNA expression suggesting that mitochondrial uncoupling could have a role also in the acquired resistance to GEM. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing strongly enhances GEM-induced mitochondrial superoxide generation and apoptosis, synergistically inhibiting cancer cell proliferation. These events are significantly reduced by the addition of the radical scavenger N-acetyl-l-cysteine or MnSOD over-expression, demonstrating a critical role of the oxidative stress. Normal primary fibroblasts are much less sensitive to GEM/genipin combination. Our results demonstrate for the first time that UCP2 has a role in cancer cell resistance to GEM supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to GEM treatment.
Assuntos
Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/genética , Iridoides/farmacologia , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Neoplasias/enzimologia , Neoplasias/genética , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Desacopladores , Proteína Desacopladora 2 , GencitabinaRESUMO
UNLABELLED: PRESENTATION AND LESION LOCALISATION: Seven adult domestic shorthair cats were presented with a 1- to 6-day history of progressive neurological signs. A focal skin puncture and subcutaneous swelling over the dorsal part of the head were detected on physical examination. Neurological examination indicated lesion(s) in the right forebrain in four cats, multifocal forebrain in one cat, left forebrain in one cat, and multifocal forebrain and brainstem in the remaining cat. In all cats, magnetic resonance imaging revealed a space-occupying forebrain lesion causing a severe mass effect on adjacent brain parenchyma. CLINICAL APPROACH AND OUTCOME: All cats were managed with a combination of medical and surgical treatment. At surgery a small penetrating calvarial fracture was detected in all cats, and a tooth fragment was found within the content of the abscess in two cats. The combination of surgical intervention, intensive care and intravenous antimicrobials led to a return to normal neurological function in five cats. PRACTICAL RELEVANCE: As this series of cases indicates, successful resolution of a brain abscess due to a bite injury depends on early recognition and combined used of antimicrobials and surgical intervention. A particular aim of surgery is to remove any skull and foreign body (tooth) fragments that may represent a continuing focus of infection.
Assuntos
Mordeduras e Picadas/veterinária , Abscesso Encefálico/veterinária , Doenças do Gato/diagnóstico , Doenças do Gato/terapia , Gatos/lesões , Traumatismos Cranianos Penetrantes/veterinária , Animais , Mordeduras e Picadas/complicações , Abscesso Encefálico/diagnóstico , Abscesso Encefálico/etiologia , Abscesso Encefálico/terapia , Doenças do Gato/patologia , Feminino , Traumatismos Cranianos Penetrantes/complicações , Imageamento por Ressonância Magnética/veterinária , Masculino , Exame Neurológico/veterinária , Prosencéfalo , Resultado do TratamentoRESUMO
Pancreatic adenocarcinoma is a common malignancy that remains refractory to all available therapies, including the gold standard drug gemcitabine (GEM). We investigated the effect of the combination of GEM and each of the ionophore compounds pyrrolidine dithiocarbamate (PDTC) and disulfiram [DSF; 1-(diethylthiocarbamoyldisulfanyl)-N,N-diethylmethanethioamide] on p53(-/-) pancreatic adenocarcinoma cell growth. PDTC or DSF synergistically inhibited cell proliferation when used in combination with GEM by inducing apoptotic cell death. This effect was associated with an increased mitochondrial O(2)(â¢-) production and was further enhanced by zinc ions. Basal levels of mitochondrial O(2)(â¢-) or manganese superoxide dismutase (MnSOD) strictly correlated with the IC(50) for GEM or the percentage of synergism. Thus, the most relevant values of the antiproliferative synergism were obtained in GEM-resistant pancreatic adenocarcinoma cell lines. Interestingly, the GEM-sensitive T3M4 cells transfected with MnSOD expression vector showed mitochondrial O(2)(â¢-) and IC(50) for GEM similar to those of resistant cell lines. In vivo experiments performed on nude mice xenotransplanted with the GEM-resistant PaCa44 cell line showed that only the combined treatment with GEM and DSF/Zn completely inhibited the growth of the tumoral masses. These results and the consideration that DSF is already used in clinics strongly support the GEM and DSF/Zn combination as a new approach to overcoming pancreatic cancer resistance to standard chemotherapy.
Assuntos
Adenocarcinoma/patologia , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/patologia , Tiocarbamatos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Humanos , GencitabinaRESUMO
"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B. L., et al. (2007) Proteins 66, 183-195], were further characterized and tested for their possible in vitro cytotoxic activity. Results obtained are the following. Besides dimers, also trimers and higher oligomers could be identified among the products of the covalently linking reaction, and the "zero-length" dimers prepared by us appear not to be a unique species. The product was indeed heterogeneous, and results obtained with two RNase A mutants, E9A and K66A, indicated that amino and carboxyl groups others than those belonging to Lys66 and Glu9 are involved in the amide bond. As for their functional properties, the "zero-length" dimers degrade poly(A).poly(U) (dsRNA) with an activity that increases with the increase of the oligomer's basicity and yeast RNA (ssRNA) with an activity that instead decreases with the increase of oligomer's basicity, which is in agreement with previous data. No cytotoxicity of the RNase A "zero-length" dimers could be evidenced in assays performed with various tumor cells lines; the dimers, instead, become cytotoxic if cationized by conjugation with polyethylenimine (PEI) [Futami et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, PEI derivatives of RNase A "zero-length" dimers and PEI derivatives of native RNase A resulted to be equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, the acquired cytotoxicity does not seem to be specific for tumor cells: PEI-cationized native RNase A was also cytotoxic toward human monocytes.
Assuntos
Ribonuclease Pancreático/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dimerização , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Monócitos/citologia , Mutação , Polietilenoimina/química , Polietilenoimina/farmacologia , Ribonuclease Pancreático/metabolismo , Relação Estrutura-AtividadeRESUMO
The aim of the present study was to analyse the molecular mechanisms involved in the Interleukin-6 (IL-6) silencing in pancreatic adenocarcinoma cell lines. Our results demonstrate that TNF-alpha, a major IL-6 inducer, is able to induce IL-6 only in three out of six cell lines examined. 5-aza-2'-deoxycytidine (DAC), but not trichostatin A (TSA), activates the expression of IL-6 in all cell lines, indicating that DNA methylation, but not histone deacetylation, plays an essential role in IL-6 silencing. Indeed, the IL-6 upstream region shows a methylation status that correlates with IL-6 expression and binds MeCP2 and H3meK9 only in the non-expressing cell lines. Our results suggest that critical methylations located from positions -666 to -426 relative to the transcription start site of IL-6 may act as binding sites for MeCP2.
Assuntos
Adenocarcinoma/genética , Azacitidina/análogos & derivados , Inativação Gênica , Interleucina-6/antagonistas & inibidores , Proteína 2 de Ligação a Metil-CpG/metabolismo , Neoplasias Pancreáticas/genética , Azacitidina/farmacologia , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , DNA/metabolismo , Metilação de DNA , Decitabina , Humanos , Ácidos Hidroxâmicos/farmacologia , Interleucina-6/genética , Interleucina-6/metabolismo , NF-kappa B/metabolismoRESUMO
BACKGROUND: Despite the importance of the BCL2L11 (BIM) protein in various apoptotic processes in development and disease, little is known of the promoter structure of the human BCL2L11 locus and of the cis-acting elements regulating expression of the human gene. RESULTS: In the search for novel promoter sequences in the human BCL2L11 locus, we have identified previously unrecognized genomic sequences displaying promoter activity and E2F responsiveness, and driving the expression of BCL2L11 coding transcripts. In man, transcripts originating from this novel putative promoter contribute significantly to total BCL2L11 mRNA expression in testis, heart and liver. In HEK293 cells, this novel candidate promoter originates BCL2L11 transcripts whose expression can be modulated by a known modulator of BCL2L11 expression (Trichostatin A) and by E2F, a characterized transcriptional regulator of BCL2L11 expression. CONCLUSION: The identification of a novel putative human BCL2L11 promoter provides new insights into the structure and regulation of the BCL2L11 locus.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Perfilação da Expressão Gênica , Proteínas de Membrana/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Sequência de Bases , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Biologia Computacional , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Éxons/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Fígado/metabolismo , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Miocárdio/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testículo/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Polyspermine-ribonuclease A (PS-RNase A) and polyspermine-dimeric ribonuclease A (PS-dimeric RNase A) were prepared by cross-linking ribonuclease A or its covalently linked dimer to polyspermine (PS) using dimethyl suberimidate. The two RNase A derivatives were tested for a possible antitumor action. The in vitro and in vivo cytotoxic activity of PS-RNase A, although strong, is not higher than that known for free polyspermine. PS-dimeric RNase A, which was characterized by mass spectroscopy, titration of free amine groups, and enzymatic assays, proved instead to be a definitely more efficient antitumor agent, both in vitro and in vivo. This result could tentatively be explained in view of the importance of positive charges for ribonuclease activity, considering the higher basicity of PS-dimeric RNase A compared to that of PS-(monomeric)RNase A. It must be also taken into account that the dimeric RNase A moiety of PS-dimeric RNase A could evade the cytoplasmic ribonuclease inhibitor, which instead could trap the monomeric RNase A moiety of the other derivative. The two RNase A derivatives degrade poly(A).poly(U) under conditions where native RNase A is inactive. The results of this work demonstrate once again the importance of positive charges for the functions of mammalian pancreatic type ribonucleases in general, in particular for RNase A derivatives, and the potential therapeutic use of the ribonuclease A derivatives.
Assuntos
Ribonuclease Pancreático/química , Ribonuclease Pancreático/toxicidade , Espermina/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cromatografia em Gel , Reagentes de Ligações Cruzadas/química , Dimerização , Humanos , Estrutura Molecular , RNA/metabolismo , Ribonuclease Pancreático/isolamento & purificação , Ribonuclease Pancreático/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We investigated the ability of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) to interact with gemcitabine (GEM) in inducing pancreatic cancer cell death. The combined treatment with TSA and GEM synergistically inhibited growth of four pancreatic adenocarcinoma cell lines and induced apoptosis. This effect was associated with the induction of reactive oxygen species (ROS) by GEM, increased expression of the pro-apoptotic BIM gene by both TSA and GEM and downregulation of the 5'-nucleotidase UMPH type II gene by TSA. The expression of other genes critical for GEM resistance (nucleoside transporters, deoxycytidine kinase, cytidine deaminase, and ribonucleotide reductase genes) was not affected by TSA. The functional role of ROS in cell growth inhibition by GEM was supported by (i) a significantly reduced GEM-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine, and (ii) a positive correlation between the basal level of ROS and sensitivity to GEM in 10 pancreatic cancer cell lines. The functional role of both Bim and 5'-nucleotidase UMPH type II in cell growth inhibition by TSA and GEM was assessed by RNA interference assays. In vivo studies on xenografts of pancreatic adenocarcinoma cells in nude mice showed that the association of TSA and GEM reduced to 50% the tumour mass and did not cause any apparent form of toxicity, while treatments with TSA or GEM alone were ineffective. In conclusion, the present study demonstrates a potent anti-tumour activity of TSA/GEM combination against pancreatic cancer cells in vitro and in vivo, strongly supporting the use of GEM in combination with an HDAC inhibitor for pancreatic cancer therapy.
Assuntos
Adenocarcinoma/metabolismo , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Inibidores Enzimáticos , Ácidos Hidroxâmicos , Neoplasias Pancreáticas/metabolismo , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Desoxicitidina/metabolismo , Desoxicitidina/farmacologia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/metabolismo , Ácidos Hidroxâmicos/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , GencitabinaRESUMO
We present evidence that pyrrolidine dithiocarbamate (PDTC) inhibits growth of p53-negative pancreatic adenocarcinoma cell lines via cell cycle arrest in the S-phase, while it has no effect on primary fibroblast proliferation. Growth inhibition of cancer cells is dependent on ROS and ERK1/2 induction as indicated by a significantly reduced PDTC-associated growth inhibition by the free radical scavenger N-acetyl-L-cysteine (NAC) or the MEK/ERK1/2 inhibitor (PD98059). Moreover, ERK1/2 induction is dependent on ROS production as demonstrated by a complete removal of PDTC-mediated ERK1/2 phosphorylation by NAC. p21(WAF1/CIP1) activation has a central role in growth inhibition by PDTC, as revealed by P21(WAF1/CIP1) silencing experiments with antisense oligonucleotide, and occurs via increased mRNA stability largely mediated by ROS/ERK induction. Conversely, PDTC does not affect P21(WAF1/CIP1) gene expression in primary fibroblasts, although it is able to activate p53 and the p53-regulated antioxidant SESN2. These results suggest that the resistance of fibroblasts to the cytotoxic action of PDTC may be related to the up-regulation of p53-dependent antioxidant genes. Finally, in vivo studies on PaCa44 cells subcutaneously xenografted in nude mice show that treatment with 100 or 200 mg/kg PDTC reduces of 30% or 60% the tumour volume, respectively, and does not cause any apparent form of toxicity.
Assuntos
Adenocarcinoma/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Pirrolidinas/farmacologia , Estabilidade de RNA , Tiocarbamatos/farmacologia , Acetilcisteína/farmacologia , Animais , Linhagem Celular Tumoral/citologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Primers do DNA , Fibroblastos , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Immunoblotting , Camundongos , Oligonucleotídeos Antissenso , Pirrolidinas/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiocarbamatos/uso terapêuticoRESUMO
Pancreatic cancer is an aggressive neoplasia, and standard chemotherapies are by and large ineffective. The purpose of this work was to get a comprehensive preclinical study on the ability of anticancer drug combinations that best inhibit growth of pancreatic adenocarcinoma cells. We evaluated the in vitro growth inhibition of ten pancreatic cancer cell lines to gemcitabine and 5-fluorouracil, newer generation cytotoxic agents (oxaliplatin, irinotecan), targeted therapy (gefitinib) and a histone deacetylase (HDAC) inhibitor (trichostatin A). Cells were treated with the single drug alone and all pairwise drug association. Our results demonstrate that TSA can effectively increase the drug sensitivity of all the cell lines studied. The association of TSA and irinotecan determines an increase in growth inhibition on the highest percentage of cell lines (80%). Our findings may represent an experimental basis for potential clinical application of HDAC inhibitors, in particular in association with drugs used in cancer clinical treatment, supporting the idea that HDAC inhibitors could act as sensitizers for chemotherapy.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores da Síntese de Proteínas/farmacologia , Adenocarcinoma/patologia , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Irinotecano , Neoplasias Pancreáticas/patologiaRESUMO
The histone deacetylase inhibitor trichostatin A (TSA) has been previously shown to block cellular growth in G2 and induce apoptosis in human pancreatic cancer cell lines. In order to better understand this phenomenon, we have analyzed the gene expression profiles in PaCa44 cells after treatment with TSA using microarrays containing 22,283 probesets. TSA was found to cause both the induction and repression of a large number of genes, although the number whose expression was up-regulated was greater than the number of genes that were down-regulated. When a threshold value of 3 was used as a cutoff level, a total of 306 (3.4%) of the detectable genes had altered expression. When categorized according to cellular function, the differentially expressed genes were found to be involved in a wide variety of cellular processes, including cell proliferation, signaling, regulation of transcription, and apoptosis. Moreover, Sp1/Sp3 transcription factor binding sites were significantly more abundant among TSA-induced genes. One prominent feature was the increased ratio between the levels of expression of pro-apoptotic (BIM) and anti-apoptotic (Bcl-XL and Bcl-W) genes. This result was confirmed in eight additional pancreatic cancer cell lines after treatment with TSA, suggesting that this event may be a strong determinant for the induction of apoptosis by TSA.
Assuntos
Adenocarcinoma/genética , Apoptose/genética , Perfilação da Expressão Gênica , Histona Desacetilases/fisiologia , Ácidos Hidroxâmicos/farmacologia , Neoplasias Pancreáticas/genética , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The human IL-6 promoter contains multiple regulatory elements such as those binding transcription factors belonging to the NF-kappaB (-75/-63), C/EBP (-158/-145 and -87/-76) and AP-1 (-283/-277) families. Herein, we report that ectopic expression of c-Jun, C/EBPdelta, and the p65 subunit of NF-kappaB synergistically activates an IL-6 promoter construct containing only a TATA box and a kappaB binding site. These results suggest that interactions among NF-kappaB, C/EBP, and AP-1, which are all activated by the most powerful physiological inducers of the IL-6 gene, namely TNF-alpha and IL-1, may be crucial for maximal activation of the IL-6 promoter in response to the two cytokines. Furthermore, we show that a mutated form of c-Jun lacking the transactivation domain (TAM-67) was a much stronger activator of the IL-6 promoter than c-Jun. In combination with p65 and/or C/EBPdelta, TAM-67 also synergistically activated the IL-6 promoter, while it inhibited TNF-alpha induced AP-1 activity directing an AP-1-responsive reporter plasmid. Lastly, electrophoretic mobility shift assay (EMSA) results strongly suggest the formation of complexes between p65, C/EBPdelta, and/or c-Jun or TAM-67 on the kappaB site, supporting the idea that the functional synergism is determined by a physical interaction. These data provide new insight into the molecular mechanisms regulating the formation of the transcription complex responsible for IL-6 promoter activation.
Assuntos
Proteínas de Ligação ao Cálcio , Interleucina-6/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/genética , Fatores de Transcrição , Sítios de Ligação , Proteína delta de Ligação ao Facilitador CCAAT , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Genes Reporter , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Mutação , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sinaptotagmina I , Sinaptotagminas , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
In cells with an altered p53 gene, the expression of p21(WAF1/CIP1), a potent inhibitor of cyclin-dependent kinases, can be induced by histone deacetylase (HDAC) inhibitors via a p53-independent pathway, which may play a critical role in arrest of cell growth. Accordingly, HDAC inhibitors such as trichostatin A (TSA) have potential utility in pancreatic cancer, as most of these tumors possess mutations in p53, which in fact is the main cause of chemoresistance to 5-fluorouracil. We have analyzed the effect of TSA on the proliferation of nine pancreatic adenocarcinoma cell lines, all containing a mutated p53 gene. TSA strongly inhibited the cellular growth of all these cell lines at submicromolar concentrations. The cellular mechanisms underlying this effect consisted of cell cycle arrest at the G2 phase and apoptotic cell death. The expression of p21(WAF1/CIP1) normally induced at the transcriptional level by p53 was also strongly activated by TSA. These findings suggest that inhibitors of HDAC may represent a novel therapeutic strategy for treatment of pancreatic cancer.