RESUMO
Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD(+) from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD(+) level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.
Assuntos
Apoptose , Cianetos/farmacologia , Guanidinas/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Pentosiltransferases/metabolismo , Espécies Reativas de Oxigênio , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Glutationa/metabolismo , Humanos , NADP/metabolismo , Niacinamida/metabolismo , Oxirredução , Superóxidos/metabolismoRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) have come under scrutiny because of the gastrointestinal, renal, and cardiovascular toxicity associated with prolonged use of these drugs. The purpose of this study was to identify molecular targets for NSAIDs related to cellular toxicity with a view to optimize drug efficacy in the clinic. Coronary artery smooth muscle cells and endothelial cells were treated with low (clinically achievable) and high (typically used in preclinical studies) concentrations of celecoxib, NS398, and ibuprofen for 24 hours. NSAIDs-induced gene expression changes were evaluated by microarray analysis and validated by real-time reverse-transcription polymerase chain reaction and western blotting. The functional significance of differentially expressed genes was evaluated by Ingenuity Pathway Analysis. At high concentrations, NSAIDs altered the expression of genes regulating cell proliferation and cell death. NSAIDs also altered genes associated with cardiovascular functions including inflammation, thrombosis, fibrinolysis, coronary artery disease, and hypertension. The gene expression was most impacted by ibuprofen, celecoxib, and NS398, in that order. This study revealed that NSAIDs altered expression of an array of genes associated with cardiovascular events and emphasizes the potential for fingerprinting drugs in preclinical studies to assess the potential drug toxicity and to optimize the drug efficacy in clinical settings.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Vasos Coronários/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/administração & dosagem , Western Blotting , Celecoxib , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Ibuprofeno/farmacologia , Análise em Microsséries , Terapia de Alvo Molecular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Nitrobenzenos/administração & dosagem , Nitrobenzenos/farmacologia , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologiaRESUMO
Birinapant is a novel SMAC peptidomimetic molecule in clinical development. It suppresses the inhibitor of apoptosis proteins (IAPs) and promotes cytochrome-C/Apaf-1/caspase-9 activation to induce effective apoptosis. Because IAP inhibition has been shown to enhance the sensitivity of cancer cells to radiation, we investigated the role of birinapant in radiosensitization of glioblastoma cells in vitro and in vivo. Two glioblastoma cell lines, U-251 and U-87, were used to analyze radiosensitization in vitro with 7-AAD cell death/apoptosis and clonogenic assays. Subcutaneous flank (U-251 and U-87) and intracranial orthotopic (U-251) xenografts in nude mice were used to evaluate radiosensitization in vivo. TNF-α levels in media and serum were measured using electrochemiluminescence. Radiosensitization in vitro was more prominent for U-251 cells than for U-87 cells. In vivo, in both tumor models, significant tumor growth delay was observed with combination treatment compared to radiation alone. There was a survival benefit with combination treatment in the orthotopic U-251 model. TNF-α levels in media correlated directly with radiation dose in vitro. These findings show that birinapant can enhance the radiosensitivity of glioblastoma cell lines in cell-based assays and tumor models via radiation-induced TNF-α. Further study into the use of birinapant with radiation therapy is warranted.
Assuntos
Dipeptídeos/farmacologia , Glioblastoma/patologia , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Transformação Celular Neoplásica , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Humanos , Camundongos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cyclooxygenase-2 (COX-2) plays a significant role in tumor development and progression. Nonsteroidal anti-inflammatory drugs (NSAID) exhibit potent anticancer effects in vitro and in vivo by COX-2-dependent and COX-2-independent mechanisms. In this study, we used microarray analysis to identify the change of expression profile regulated by a COX-2-specific NSAID NS-398 (0.01 and 0.1 mmol/L), a nonspecific NSAID ibuprofen (0.1 and 1.5 mmol/L) and RNA interference (RNAi)-mediated COX-2 inhibition in PC3 prostate cancer cells. A total of 3,362 differentially expressed genes with 2-fold change and P<0.05 were identified. Low concentrations of NSAIDs and COX-2 RNAi altered very few genes (1-3%) compared with the higher concentration of NS-398 (17%) and ibuprofen (80%). Ingenuity Pathway Analysis was used for distributing the differentially expressed genes into biological networks and for evaluation of functional significance. The top 3 networks for both NSAIDs included functional categories of DNA replication, recombination and repair, and gastrointestinal disease. Immunoresponse function was specific to NS-398, and cell cycle and cellular movement were among the top functions for ibuprofen. Ingenuity Pathway Analysis also identified renal and urologic disease as a function specific for ibuprofen. This comprehensive study identified several COX-2-independent targets of NSAIDs, which may help explain the antitumor and radiosensitizing effects of NSAIDs. However, none of these categories were reflected in the identified networks in PC3 cells treated with clinically relevant low concentrations of NS-398 and ibuprofen or with COX-2 RNAi, suggesting the benefit to fingerprinting preclinical drug concentrations to improve their relevance to the clinical setting.
Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ibuprofeno/farmacologia , Nitrobenzenos/farmacologia , Neoplasias da Próstata/genética , Interferência de RNA , Sulfonamidas/farmacologia , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/genética , Angiopoietinas/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Temperatura Alta , Humanos , Masculino , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.
Assuntos
Antineoplásicos/farmacologia , Camptotecina/farmacologia , Guanidinas/farmacologia , Hidrazonas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Radiação Ionizante , Radiossensibilizantes/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Sítios de Ligação , Western Blotting , Domínio Catalítico , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quinase do Ponto de Checagem 2 , Dano ao DNA , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Citometria de Fluxo , Guanidinas/química , Humanos , Hidrazonas/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Fosforilação , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/biossíntese , Radiossensibilizantes/química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Fosfatases cdc25/metabolismoRESUMO
PURPOSE: Temozolomide, a DNA methylating agent, is currently undergoing clinical evaluation for cancer therapy. Because temozolomide has been shown to increase survival rates of patients with malignant gliomas when given combined with radiation, and there is conflicting preclinical data concerning the radiosensitizing effects of temozolomide, we further investigated the possible temozolomide-induced enhancement of radiosensitivity. EXPERIMENTAL DESIGN: The effects of temozolomide on the in vitro radiosensitivity of U251 (a human glioma) and MDA-MB231BR (a brain-seeking variant of a human breast tumor) cell lines was evaluated using clonogenic assay. DNA damage and repair were evaluated using phosphorylated histone H2AX (gammaH2AX), and mitotic catastrophe was measured using nuclear fragmentation. Growth delay was used to evaluate the effects of temozolomide on in vivo (U251) tumor radiosensitivity. RESULTS: Exposure of each cell line to temozolomide for 1 h before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1 ranging from 1.30 to 1.32. Temozolomide had no effect on radiation-induced apoptosis or on the activation of the G(2) cell cycle checkpoint. As a measure of DNA double strand breaks, gammaH2AX foci were determined as a function of time after the temozolomide + irradiation combination. The number of gammaH2AX foci per cell was significantly greater at 24 h after the combined modality compared with the individual treatments. Mitotic catastrophe, measured at 72 h, was also significantly increased in cells receiving the temozolomide + irradiation combination compared with the single treatments. In vivo studies revealed that temozolomide administration to mice bearing U251 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay with a dose enhancement factor of 2.8. CONCLUSIONS: These results indicate that temozolomide can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair leading to an increase in mitotic catastrophe.
Assuntos
Dacarbazina/análogos & derivados , Radiossensibilizantes/farmacologia , Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Metilação de DNA , Replicação do DNA/efeitos dos fármacos , Dacarbazina/farmacologia , Glioblastoma , Humanos , Mitose/efeitos dos fármacos , Temozolomida , Ensaio Tumoral de Célula-TroncoRESUMO
PURPOSE: Preclinical studies evaluating histone deacetylase (HDAC) inhibitor-induced radiosensitization have largely focused on the preirradiation setting based on the assumption that enhanced radiosensitivity was mediated by changes in gene expression. Our previous investigations identified maximal radiosensitization when cells were exposed to HDAC inhibitors in both the preradiation and postradiation setting. We now expand on these studies to determine whether postirradiation exposure alone affects radiosensitivity. EXPERIMENTAL DESIGN: The effects of the HDAC inhibitor valproic acid (VA) on postirradiation sensitivity in human glioma cell lines were evaluated using a clonogenic assay, exposing cells to VA up to 24 h after irradiation. DNA damage repair was evaluated using gammaH2AX and 53BP1 foci and cell cycle phase distribution was analyzed by flow cytometry. Western blot of acetylated gammaH2AX was done following histone extraction on AUT gels. RESULTS: VA enhanced radiosensitivity when delivered up to 24 h after irradiation. Cells accumulated in G(2)-M following irradiation, although they returned to baseline at 24 h, mitigating the role of cell cycle redistribution in postirradiation sensitization by VA. At 12 h after irradiation, significant gammaH2AX and 53BP1 foci dispersal was shown in the control, although cells exposed to VA after irradiation maintained foci expression. VA alone had no effect on the acetylation or phosphorylation of H2AX, although it did acetylate radiation-induced gammaH2AX. CONCLUSIONS: These results indicate that VA enhances radiosensitivity at times up to 24 h after irradiation, which has direct clinical application.
Assuntos
Inibidores Enzimáticos/farmacologia , Glioma , Inibidores de Histona Desacetilases , Radiossensibilizantes/farmacologia , Ácido Valproico/farmacologia , Neoplasias Encefálicas , Linhagem Celular Tumoral , Humanos , Ácido Valproico/administração & dosagemRESUMO
Overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) in human tumors is associated with poor prognosis and poor outcome to radiation therapy. Inhibition of HIF-1alpha is considered as a promising approach in cancer therapy. The purpose of this study was to test the efficacy of a novel HIF-1alpha inhibitor PX-478 as a radiosensitizer under normoxic and hypoxic conditions in vitro. PC3 and DU 145 prostate carcinoma cells were treated with PX-478 for 20 hr, and HIF-1alpha protein level and clonogenic cell survival were determined under normoxia and hypoxia. Effects of PX-478 on cell cycle distribution and phosphorylation of H2AX histone were evaluated. PX-478 decreased HIF-1alpha protein in PC3 and DU 145 cells. PX-478 produced cytotoxicity in both cell lines with enhanced toxicity under hypoxia for DU-145. PX-478 (20 mumol/L) enhanced the radiosensitivity of PC3 cells irradiated under normoxic and hypoxic condition with enhancement factor (EF) 1.4 and 1.56, respectively. The drug was less effective in inhibiting HIF-1alpha and enhancing radiosensitivity of DU 145 cells compared to PC3 cells with EF 1.13 (normoxia) and 1.25 (hypoxia) at 50 mumol/L concentration. PX-478 induced S/G2M arrest in PC3 but not in DU 145 cells. Treatment of PC3 and DU 145 cells with the drug resulted in phosphorylation of H2AX histone and prolongation of gammaH2AX expression in the irradiated cells. PX-478 is now undergoing Phase I clinical trials as an oral agent. Although the precise mechanism of enhancement of radiosensitivity remains to be identified, this study suggests a potential role for PX-478 as a clinical radiation enhancer.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Compostos de Mostarda/farmacologia , Fenilpropionatos/farmacologia , Neoplasias da Próstata/patologia , Tolerância a Radiação , Western Blotting , Ciclo Celular/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Imunofluorescência , Histonas/metabolismo , Humanos , Masculino , FosforilaçãoRESUMO
PURPOSE: Because ribonucleotide reductase (RR) plays a role in DNA repair, it may serve as a molecular target for radiosensitization. Unlike previously investigated RR inhibitors, Triapine potently inhibits both RR holoenzymes. Therefore, the effects of Triapine on tumor cell radiosensitivity were investigated. EXPERIMENTAL DESIGN: The effects of Triapine on the in vitro radiosensitivity of three human tumor cell lines and one normal cell line were evaluated using a clonogenic assay. Growth delay was used to evaluate the effects of Triapine on in vivo tumor radiosensitivity. The levels of the RR subunits were determined using immunoblot analysis and DNA damage and repair were evaluated using gammaH2AX foci. RESULTS: Exposure of the tumor cell lines to Triapine before or immediately after irradiation resulted in an increase in radiosensitivity. In contrast, Triapine enhanced the radiosensitivity of the normal fibroblast cell line only when the exposure was before irradiation. There were no consistent differences between cell lines with respect to the expression of the RR subunits. Whereas Triapine had no effect on radiation-induced gammaH2AX foci at 1 hour, the number of gammaH2AX foci per cell was significantly greater in the Triapine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Triapine administration to mice bearing tumor xenografts immediately after irradiation resulted in a greater than additive increase in radiation-induced tumor growth delay. CONCLUSIONS: These results indicate that Triapine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair.
Assuntos
Ciclo Celular/efeitos dos fármacos , Piridinas/farmacologia , Radiossensibilizantes/farmacologia , Tiossemicarbazonas/farmacologia , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Inibidores Enzimáticos/farmacologia , Glioma , Humanos , Masculino , Neoplasias Pancreáticas , Neoplasias da Próstata , Ribonucleotídeo Redutases/antagonistas & inibidoresRESUMO
The ability to identify tumors that are susceptible to a given molecularly targeted radiosensitizer would be of clinical benefit. Towards this end, we have investigated the effects of a representative Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG), on the radiosensitivity of a panel of human tumor cell lines. 17DMAG was previously shown to enhance the radiosensitivity of a number of human cell lines, which correlated with the loss of ErbB2. We now report on cell lines in which 17DMAG induced the degradation of ErbB2, yet had no effect on radiosensitivity. In a comparison of ErbB family members, ErbB3 protein was only detectable in cells resistant to 17DMAG-induced radiosensitization. To determine whether ErbB3 plays a casual role in this resistance, short interfering RNA (siRNA) was used to knockdown ErbB3 in the resistant cell line AsPC1. Whereas individual treatments with siRNA to ErbB3 or 17DMAG had no effect on radiosensitivity, the combination, which reduced both ErbB2 and ErbB3, resulted in a significant enhancement in AsPC1 radiosensitivity. In contrast to siRNA to ErbB3 or 17DMAG treatments only, AsPC1 cell exposure to the combination also resulted in a decrease in ErbB1 kinase activity. These results indicate that ErbB3 expression predicts for tumor cell susceptibility to and suggests that the loss of ErbB1 signaling activity is necessary for 17DMAG-induced radiosensitization. However, for cell lines sensitized by 17DMAG, treatment with siRNA to ErbB2, which reduced ErbB1 activity, had no effect on radiosensitivity. These results suggest that, whereas the loss of ErbB1 signaling may be necessary for 17DMAG-induced radiosensitization, it is not sufficient.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias Pancreáticas/radioterapia , Neoplasias da Próstata/radioterapia , Tolerância a Radiação/fisiologia , Receptor ErbB-3/biossíntese , Benzoquinonas , Linhagem Celular Tumoral , Humanos , Lactamas Macrocíclicas , Masculino , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Quinonas/farmacologia , RNA Interferente Pequeno/genética , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Receptor ErbB-2/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/genética , Quinases raf/metabolismoRESUMO
In Saccharomyces cerevisiae, the SIF2 gene product is an integral component of the Set3 complex (SET3C), an assembly of proteins with some homology to the human SMRT and N-CoR corepressor complexes. SET3C has histone deacetylase activity that is responsible for repressing a set of meiotic genes. We have determined the X-ray crystal structure of a 46 kDa C-terminal domain of a SET3C core protein, Sif2p to 1.55 A resolution and a crystallographic R-factor of 19.0%. This domain contains an unusual eight-bladed beta-propeller structure, which differs from other transcriptional corepressor structures such as yeast Tup1p and human groucho (Gro)/TLE1, which have only seven. We have demonstrated intact Sif2p is a tetramer and the N-terminal LisH (Lis-homology)-containing domain mediates tetramerization and interaction with another component of SET3C, Snt1p. Multiple sequence alignments indicate that a surface on the "top" of the protein is conserved among species, suggesting that it may play a common role in binding partner proteins. Since Sif2p appears to be the yeast homolog of human TBL1 and TBLR1, which function in the N-CoR/SMRT complexes, its structural and oligomeric properties are likely to be very similar.
Assuntos
Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , DNA Fúngico/genética , Histona Desacetilases , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Aberrant DNA hypermethylation is a frequent finding in tumor cells, which has suggested that inhibition of DNA methylation may be an effective cancer treatment strategy. Because DNA methylation affects gene expression and chromatin structure, parameters considered to influence radioresponse, we investigated the effects of the DNA methylation inhibitor zebularine on the radiosensitivity of human tumor cells. Three human tumor cell lines were used in this study (MiaPaCa, DU145, and U251) and the methylation status of three genes frequently hypermethylated in tumor cells (RASSF1A, HIC-1, and 14-3-3sigma) was determined as a function of zebularine exposure. Zebularine resulted in DNA demethylation in a time-dependent manner, with the maximum loss of methylation detected by 48 hours. Treatment of cells with zebularine for 48 hours also resulted in an increase in radiosensitivity with dose enhancement factors of >1.5. As a measure of radiation-induced DNA damage, gammaH2AX expression was determined. Whereas zebularine had no effect on radiation-induced gammaH2AX foci at 1 hour, the number of gammaH2AX foci per cell was significantly greater in the zebularine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Zebularine administration to mice reactivated gene expression in U251 xenografts; irradiation of U251 tumors in mice treated with zebularine resulted in an increase in radiation-induced tumor growth delay. These results indicate that zebularine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect may involve an inhibition of DNA repair.
Assuntos
Citidina/análogos & derivados , Citidina/farmacologia , Metilação de DNA/efeitos dos fármacos , Neoplasias Experimentais/terapia , Proteínas 14-3-3 , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta à Radiação , Exonucleases/genética , Exorribonucleases , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fatores de Transcrição Kruppel-Like , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Radiation oncology modalities such as intensity-modulated and image-guided radiation therapy can reduce the high dose to normal tissue and deliver a heterogeneous dose to tumors, focusing on areas deemed at highest risk for tumor persistence. Clinical radiation oncology produces daily doses ranging from 1 to 20 Gy, with tissues being exposed to 30 or more daily fractions. Hypothesizing the cells that survive fractionated radiation therapy have a substantially different phenotype than the untreated cells, which might be exploitable for targeting with molecular therapeutics or immunotherapy, three prostate cancer cell lines (PC3, DU145, and LNCaP) and normal endothelial cells were studied to understand the biology of differential effects of multifraction (MF) radiation of 0.5, 1, and/or 2 Gy fraction to 10 Gy total dose, and a single dose of 5 and 10 Gy. The resulting changes in mRNA, miRNA, and phosphoproteome were analyzed. Significant differences were observed in the MF radiation exposures including those from the 0.5 Gy MF that produces little cell killing. As expected, p53 function played a major role in response. Pathways modified by MF include immune response, DNA damage, cell-cycle arrest, TGF-ß, survival, and apoptotic signal transduction. The radiation-induced stress response will set forth a unique platform for exploiting the effects of radiation therapy as "focused biology" for cancer treatment in conjunction with molecular targeted or immunologically directed therapy. Given that more normal tissue is treated, albeit to lower doses with these newer techniques, the response of the normal tissue may also influence long-term treatment outcome.
Assuntos
Neoplasias/radioterapia , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Humanos , Neoplasias/genética , Tolerância a Radiação , Radioterapia Guiada por Imagem , SobreviventesRESUMO
We assessed changes in cell lines of varying p53 status after various fractionation regimens to determine if p53 influences gene expression and if multifractionated (MF) irradiation can induce molecular pathway changes. LNCaP (p53 wild-type), PC3 (p53 null), and DU145 (p53 mutant) prostate carcinoma cells received 5 and 10 Gy as single-dose (SD) or MF (0.5 Gy x 10, 1 Gy x 10, and 2 Gy x 5) irradiation to simulate hypofractionated and conventionally fractionated prostate radiotherapies, respectively. mRNA analysis revealed 978 LNCaP genes differentially expressed (greater than two-fold change, P < .05) after irradiation. Most were altered with SD (69%) and downregulated (75%). Fewer PC3 (343) and DU145 (116) genes were induced, with most upregulated (87%, 89%) and altered with MF irradiation. Gene ontology revealed immune response and interferon genes most prominently expressed after irradiation in PC3 and DU145. Cell cycle regulatory (P = 9.23 x 10(-73), 14.2% of altered genes, nearly universally downregulated) and DNA replication/repair (P = 6.86 x 10(-30)) genes were most prominent in LNCaP. Stress response and proliferation genes were altered in all cell lines. p53-activated genes were only induced in LNCaP. Differences in gene expression exist between cell lines and after varying irradiation regimens that are p53 dependent. As the duration of changes is ≥24 hours, it may be possible to use radiation-inducible targeted therapy to enhance the efficacy of molecular targeted agents.
RESUMO
We have previously demonstrated that prostate carcinoma cells exposed to fractionated radiation differentially expressed more genes compared to single-dose radiation. To understand the role of miRNA in regulation of radiation-induced gene expression, we analyzed miRNA expression in LNCaP, PC3 and DU145 prostate cancer cells treated with single-dose radiation and fractionated radiation by microarray. Selected miRNAs were studied in RWPE-1 normal prostate epithelial cells by RT-PCR. Fractionated radiation significantly altered more miRNAs as compared to single-dose radiation. Downregulation of oncomiR-17-92 cluster was observed only in the p53 positive LNCaP and RWPE-1 cells treated with single-dose radiation and fractionated radiation. Comparison of miRNA and mRNA data by IPA target filter analysis revealed an inverse correlation between miR-17-92 cluster and several targets including TP53INP1 in p53 signaling pathway. The base level expressions of these miRNAs were significantly different among the cell lines and did not predict the radiation outcome. Tumor suppressor miR-34a and let-7 miRNAs were upregulated by fractionated radiation in radiosensitive LNCaP (p53 positive) and PC3 (p53-null) cells indicating that radiation-induced miRNA expression may not be regulated by p53 alone. Our data support the potential for using fractionated radiation to induce molecular targets and radiation-induced miRNAs may have a significant role in predicting radiosensitivity.
Assuntos
Fracionamento da Dose de Radiação , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Temperatura Alta , Humanos , Imunidade Inata/genética , Imunidade Inata/efeitos da radiação , Masculino , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos da radiação , Reprodutibilidade dos Testes , Transcriptoma/efeitos da radiaçãoRESUMO
To examine the possibility of using fractionated radiation in a unique way with molecular targeted therapy, gene expression profiles of prostate carcinoma cells treated with 10 Gy radiation administered either as a single dose or as fractions of 2 Gy × 5 and 1 Gy × 10 were examined by microarray analysis. Compared to the single dose, the fractionated irradiation resulted in significant increases in differentially expressed genes in both cell lines, with more robust changes in PC3 cells than in DU145 cells. The differentially expressed genes (>twofold change; P < 0.05) were clustered and their ontological annotations evaluated. In PC3 cells genes regulating immune and stress response, cell cycle and apoptosis were significantly up-regulated by multifractionated radiation compared to single-dose radiation. Ingenuity Pathway Analysis (IPA) of the differentially expressed genes revealed that immune response and cardiovascular genes were in the top functional category in PC3 cells and cell-to-cell signaling in DU145 cells. RT-PCR analysis showed that a flexure point for gene expression occurred at the 6th-8th fraction and AKT inhibitor perifosine produced enhanced cell killing after 1 Gy × 8 fractionated radiation in PC3 and DU145 cells compared to single dose. This study suggests that fractionated radiation may be a uniquely exploitable, non-oncogene-addiction stress pathway for molecular therapeutic targeting.
Assuntos
Fracionamento da Dose de Radiação , Neoplasias da Próstata/patologia , Neoplasias da Próstata/radioterapia , Apoptose/efeitos da radiação , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Perfilação da Expressão Gênica , Histonas/metabolismo , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Fosforilação/efeitos da radiação , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Dosagem Radioterapêutica , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Due to an increase in the understanding of molecular radiobiology, strategies for enhancing tumor radiosensitivity have begun to focus on targeting the molecules and processes that regulate cellular radioresponse. Toward this end, histone acetylation has begun to receive considerable attention as a potential target for radiosensitization. Histone acetylation, which is determined by the competing actions of histone acetylases (HATs) and histone deacetylases (HDACs), plays a role in regulating chromatin structure and gene expression--two parameters that have long been considered determinants of radioresponse. As a means of modifying histone acetylation status, considerable effort has been put into the development of inhibitors of HDAC activity, which is often aberrant in tumor cells. This has led to the generation of a relatively large number of structurally diverse compounds that inhibit HDAC activity and result in histone hyperacetylation, and importantly, are applicable to patient treatment. Whereas a number of these HDAC inhibitors have antitumor activity in preclinical cancer models when delivered as single agents, recent studies have indicated that these compounds also significantly enhance tumor cell radiosensitivity. A structurally diverse set of HDAC inhibitors have been shown to enhance the in vitro radiosensitivity of human tumor cell lines generated from a spectrum of solid tumors. Moreover, HDAC inhibitors increased the radiosensitivity of human tumor xenografts. Although the mechanism responsible for this radiosensitization has not been definitely elucidated, data suggest that inhibiting the repair of radiation-induced DNA damage may be involved. Whereas HDAC inhibitors are currently in clinical trials as single modalities and in combination with chemotherapeutic agents, recent results suggest that these compounds may also enhance the antitumor effectiveness of radiotherapy.
Assuntos
Histona Desacetilases , Histonas/metabolismo , Tolerância a Radiação , Acetilação , Animais , Inibidores Enzimáticos/metabolismo , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Neoplasias/radioterapia , Radiossensibilizantes/metabolismo , Radiossensibilizantes/uso terapêuticoRESUMO
Bub3p is a protein that mediates the spindle checkpoint, a signaling pathway that ensures correct chromosome segregation in organisms ranging from yeast to mammals. It is known to function by co-localizing at least two other proteins, Mad3p and the protein kinase Bub1p, to the kinetochore of chromosomes that are not properly attached to mitotic spindles, ultimately resulting in cell cycle arrest. Prior sequence analysis suggested that Bub3p was composed of three or four WD repeats (also known as WD40 and beta-transducin repeats), short sequence motifs appearing in clusters of 4-16 found in many hundreds of eukaryotic proteins that fold into four-stranded blade-like sheets. We have determined the crystal structure of Bub3p from Saccharomyces cerevisiae at 1.1 angstrom and a crystallographic R-factor of 15.3%, revealing seven authentic repeats. In light of this, it appears that many of these repeats therefore remain hidden in sequences of other proteins. Analysis of random and site-directed mutants identifies the surface of Bub3p involved in checkpoint function through binding of Bub1p and Mad3p. Sequence alignments indicate that these surfaces are mostly conserved across Bub3 proteins from diverse species. A structural comparison with other proteins containing WD repeats suggests that these folds may bind partner proteins using similar surface areas on the top and sides of the propeller. The sequences composing these regions are the most divergent within the repeat across all WD repeat proteins and could potentially be modulated to provide specificity in partner protein binding without perturbation of the core structure.
Assuntos
Proteínas de Ciclo Celular/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Proteínas Fúngicas , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares , Ligação Proteica , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fuso Acromático/químicaRESUMO
Valproic acid (VA) is a well-tolerated drug used to treat seizure disorders and has recently been shown to inhibit histone deacetylase (HDAC). Because HDAC modulates chromatin structure and gene expression, parameters considered to influence radioresponse, we investigated the effects of VA on the radiosensitivity of human brain tumor cells grown in vitro and in vivo. The human brain tumor cell lines SF539 and U251 were used in our study. Histone hyperacetylation served as an indicator of HDAC inhibition. The effects of VA on tumor cell radiosensitivity in vitro were assessed using a clonogenic survival assay and gammaH2AX expression was determined as a measure of radiation-induced DNA double strand breaks. The effect of VA on the in vivo radioresponse of brain tumor cells was evaluated according to tumor growth delay analysis carried out on U251 xenografts. Irradiation at the time of maximum VA-induced histone hyperacetylation resulted in significant increases in the radiosensitivity of both SF539 and U251 cells. The radiosensitization was accompanied by a prolonged expression of gammaH2AX. VA administration to mice resulted in a clearly detectable level of histone hyperacetylation in U251 xenografts. Irradiation of U251 tumors in mice treated with VA resulted in an increase in radiation-induced tumor growth delay. Valproic acid enhanced the radiosensitivity of both SF539 and U251 cell lines in vitro and U251 xenografts in vivo, which correlated with the induction of histone hyperacetylation. Moreover, the VA-mediated increase in radiation-induced cell killing seemed to involve the inhibition of DNA DSB repair.