RESUMEN
It is known that p53 is an important transcription factor and plays a central role in ionizing radiation (IR)-induced DNA damage responses such as cell cycle arrest, DNA repair and apoptosis. We previously reported that regulating p53 protein is an effective strategy for modulating cell fate by reducing the acute side effects of radiation therapy. Herein, we report on the discovery of STK160830 as a new radioprotector from a chemical library at The University of Tokyo and the design, synthesis and biological evaluation of its derivatives. The radioprotective activity of STK160830 itself and its derivatives that were synthesized in this work was evaluated using a leukemia cell line, MOLT-4 cells as a model of normal cells that express the p53 protein in a structure-activity relationships (SAR) study. The experimental results suggest that a direct relationship exists between the inhibitory effect of these STK160830 derivatives on the expression level of p53 and their radioprotective activity and that the suppression of p53 by STK160830 derivatives contribute to protecting MOLT-4 cells from apoptosis that is induced by exposure to radiation.
Asunto(s)
Apoptosis , Proteína p53 Supresora de Tumor , Daño del ADN , Reparación del ADN , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Epo-C12 is a synthetic derivative of epolactaene, isolated from Penicillium sp. BM 1689-P. Epo-C12 induces apoptosis in human acute lymphoblastoid leukemia BALL-1 cells. In our previous studies, seven proteins that bind to Epo-C12 were identified by a combination of pull-down experiments using biotinylated Epo-C12 (Bio-Epo-C12) and mass spectrometry. In the present study, the effect of Epo-C12 on peroxiredoxin 1 (Prx 1), one of the proteins that binds to Epo-C12, was investigated. Epo-C12 inhibited Prx 1 peroxidase activity. However, it did not suppress its chaperone activity. Binding experiments between Bio-Epo-C12 and point-mutated Prx 1s suggest that Epo-C12 binds to Cys52 and Cys83 in Prx 1. The present study revealed that Prx 1 is one of the target proteins through which Epo-C12 exerts an apoptotic effect in BALL-1 cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Peroxirredoxinas/antagonistas & inhibidores , Animales , Antineoplásicos/química , Línea Celular Tumoral , Inhibidores Enzimáticos , Compuestos Epoxi/química , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Estructura Molecular , Mutación , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Polienos/químicaRESUMEN
Hyperthermia induced by heat stress (HS) is known to inhibit proliferation and induce cell death in cancer. We previously demonstrated that checkpoint kinase 1 (Chk1) contributes to G2/M arrest and cell survival under HS; however, the role of Chk2, a functional analog of Chk1, in regulation of the cell cycle and cell death under HS is still unknown. Here, we addressed the role of Chk2 using Molt-4 cells with p53-targeted shRNA (Molt-4/shp53) and parental control cells (Molt-4/V). Chk2 inhibition suppressed C-terminal acetylation of p53 and delayed the induction of p53-target genes in Molt-4/V cells under HS; however, Chk2 inhibition failed to inhibit apoptosis induced by HS, indicating that Chk2 was dispensable for p53-dependent apoptosis under HS. In contrast, Chk2 inhibition abrogated G2/M arrest and promoted cell death induced by HS in HeLa cells and Molt-4/shp53 cells. Thus, we demonstrated for the first time that Chk2 was required for cell cycle arrest and cell survival, particularly in cells with p53 defects under HS. These findings indicated that Chk2 may be a selective target for p53-mutated or -deficient cancer treated with hyperthermia.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Quinasa de Punto de Control 2/fisiología , Calor , Estrés Fisiológico/genética , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/fisiología , Línea Celular Tumoral , Supervivencia Celular/genética , Quinasa de Punto de Control 2/antagonistas & inhibidores , Quinasa de Punto de Control 2/genética , Daño del ADN , Citometría de Flujo , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Mutación , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria and that this occurs in a DDR-independent manner.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Hepatoblastoma/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocondrias Hepáticas/metabolismo , Daño del ADN , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Activación Enzimática/efectos de los fármacos , Células Hep G2 , Hepatoblastoma/genética , Humanos , Peróxido de Hidrógeno/farmacología , Neoplasias Hepáticas/genética , Estrés Oxidativo , Peroxisomas/metabolismoRESUMEN
In a previous study, we reported that some tetradentate zinc(II) chelators inhibit p53 through the denaturation of its zinc-requiring structure but a chelator, Bispicen, a potent inhibitor of in vitro apoptosis, failed to show any efficient radioprotective effect against irradiated mice because the toxicity of the chelator to mice. The unsuitability of using tetradentate chelators as radioprotectors prompted us to undertake a more extensive search for p53-inhibiting agents that are weaker zinc(II) chelators and therefore less toxic. Here, we show that an 8-hydroxyquinoline (8HQ) derivative, AS-2, suppresses p53-dependent apoptosis through a transcription-independent mechanism. A mechanistic study using cells with different p53 characteristics revealed that the suppressive effect of AS-2 on apoptosis is specifically mediated through p53. In addition, AS-2 was less effective in preventing p53-mediated transcription-dependent events than pifithrin-µ (PFTµ), an inhibitor of transcription-independent apoptosis by p53. Fluorescence visualization of the extranuclear distribution of AS-2 also supports that it is ineffective on the transcription-dependent pathway. Further investigations revealed that AS-2 suppressed mitochondrial apoptotic events, such as the mitochondrial release of intermembrane proteins and the loss of mitochondrial membrane potential, although AS-2 resulted in an increase in the mitochondrial translocation of p53 as opposed to the decrease of cytosolic p53, and did not affect the apoptotic interaction of p53 with Bcl-2. AS-2 also protected mice that had been exposed to a lethal dose of ionizing radiation. Our findings indicate that some types of bidentate 8HQ chelators could serve as radioprotectors with no substantial toxicity in vivo.
Asunto(s)
Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Radiación Ionizante , Protectores contra Radiación/farmacología , Ésteres del Ácido Sulfúrico/farmacología , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/fisiología , Línea Celular Transformada , Ratones , Microscopía Fluorescente , Mitocondrias/fisiologíaRESUMEN
In radiation therapy, adverse side effects are often induced due to the excessive cell death that occurs in radiosensitive normal cells. The radiation-induced cell death of normal cells is caused, at least in part, by apoptosis, which undergoes via activation of p53 and increase in the p53 protein, a zinc-containing transcriptional factor, in response to cellular damage. Therefore, radioprotective drugs that can protect normal cells from radiation and thus suppress adverse side effects would be highly desirable. We report herein on the radioprotective activity of 8-hydroxyquinoline (8HQ) derivatives that were initially designed so as to interact with the Zn(2+) in p53. Indeed, the 5,7-bis(methylaminosulfonyl)-8HQ and 8-methoxyquinoline derivatives considerably protected MOLT-4 cells against γ-ray radiation (10 Gy), accompanied by a low cytotoxicity. However, mechanistic studies revealed that the interaction of these drugs with p53 is weak and the mechanism for inhibiting apoptosis appears to be different from that of previously reported radioprotectors such as bispicen, which inhibits apoptosis via the denaturation of p53 as well as by blocking both transcription-dependent and -independent apoptotic pathways.
Asunto(s)
Diseño de Fármacos , Oxiquinolina/química , Protectores contra Radiación/síntesis química , Zinc/química , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Rayos gamma , Humanos , Oxiquinolina/síntesis química , Oxiquinolina/toxicidad , Protectores contra Radiación/química , Protectores contra Radiación/toxicidad , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized type I membrane glycoprotein, and promotes dendritic filopodia formation. Although we have determined the N-glycan structures of ICAM-5 in a previous report, their function is unknown. Here, we produced fifteen ICAM-5 gene constructs, in which each potential N-glycosylation site was mutated, to elucidate the function of the N-glycans of ICAM-5, and observed the effects of transfection of them on a neuronal cell line, Neuro-2a (N2a). Only the N54Q mutant, which is the mutant for the most N-terminal glycosylation site, failed to induce filopodia-like protrusions in N2a cells. Immunofluorescence staining and cell surface biotinylation revealed that N54Q ICAM-5 was confined to the ER and also could not be expressed on the cell surface. This is further supported by the biochemical evidence that almost all N-glycans of N54Q ICAM-5 were digested by Endo glycosidase H and peptide:N-glycanase, indicating that almost all of them retain high-mannose-type structures in ER. In additon, it also failed to form disulfide bonds or functional protein complexes. The stable transformants of N54Q ICAM-5 showed retarded cell growth, but it was interesting that there was no apparent ER stress, because the mutant was sequentially degraded via ER associated degradation pathway by comparing the susceptibilities of the responses to various inhibitors of this pathway in wild-type and N54Q ICAM-5 transfectants. Taken together, the Asn(54)-linked glycan is necessary for normal trafficking and function of ICAM-5, but is unassociated with ER-associated degradation of it.
Asunto(s)
Asparagina/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Polisacáridos/metabolismo , Pliegue de Proteína , Animales , Asparagina/química , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Glicósido Hidrolasas/metabolismo , Glicosilación , Humanos , Inmunohistoquímica , Manosa/metabolismo , Ratones , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida/métodos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Polisacáridos/química , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Seudópodos/química , Ratas , Transfección/métodosRESUMEN
3-(3-Phenoxybenzyl)amino-ß-carboline 2h showed extremely-high activity; the IC(50) value was 0.074 µM. To verify 2h-induced cell death types, we observed the chromatin condensation, the DNA fragmentation and activated caspase-3 using Hoechst 33342, agarose electrophoresis and western blot, and suggesting 2h-induced cell death type was apoptosis. Flow cytometry showed that 2h-treated cell was induced SubG1 cell population after G2/M cell cycle arrest. In addition, using affinity chromatography and peptide mass fingerprinting, we found that interacting protein with this compound was α-tubulin protein.
Asunto(s)
Antineoplásicos/farmacología , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Estructura Molecular , Estereoisomerismo , Relación Estructura-Actividad , Tubulina (Proteína)/químicaRESUMEN
RNA synthesis inhibitors and protein synthesis inhibitors are useful for investigating whether biological events with unknown mechanisms require transcription or translation; however, the dependence of RNA synthesis has been difficult to verify because many RNA synthesis inhibitors cause adverse events that trigger a p53 response. In this study, we screened a library containing 9600 core compounds and obtained STK160830 that shows anti-apoptotic effects in irradiated wild-type-p53-bearing human T-cell leukemia MOLT-4 cells and murine thymocytes. In many of the p53-impaired cells and p53-knockdown cells tested, STK160830 did not show a remarkable anti-apoptotic effect, suggesting that the anti-apoptotic activity is p53-dependent. In the expression analysis of p53, p53-target gene products, and reference proteins by immunoblotting, STK160830 down-regulated the expression of many of the proteins examined, and the downregulation correlated strongly with its inhibitory effect on cell death. mRNA expression analyses by qPCR and nascent RNA capture kit revealed that STK160830 showed a decreased mRNA expression, which was similar to that induced by the RNA synthesis inhibitor actinomycin D but differed to some extent. Furthermore, unlike other RNA synthesis inhibitors such as actinomycin D, p53 accumulation by STK160830 alone was negligible, and a DNA melting-curve analysis showed very weak DNA-intercalating activity, indicating that STK160830 is a useful inhibitor for RNA synthesis without triggering p53-mediated damage responses.
RESUMEN
PURPOSE: Our previous study indicated that sodium orthovanadate (vanadate), a strong inhibitor of p53, effectively suppressed the lethality from the hematopoietic (HP) and gastrointestinal (GI) syndromes after 12 Gy total-body irradiation (TBI) in mice. This conclusion, however, was inconsistent with the fact that p53 plays a radioprotective role in the intestinal epithelium. The death after TBI of around 12 Gy was attributed to a combined effect of HP and GI syndromes. To verify the effect from prophylactic administration of p53 inhibitor on protection of HP and GI syndromes, in this study, the radioprotective effects from vanadate were investigated in TBI and lower half-body irradiation (partial-body irradiation: PBI) mouse models. METHODS: Female ICR mice were given a single injection of vanadate or vehicle, followed by a lethal dose of TBI or PBI. Radioprotective effects of vanadate against the irradiations were evaluated by analyzing survival rate, body weight, hematopoietic parameters, and histological changes in the bone marrow and intestinal epithelium. RESULTS: TBI-induced HP syndrome was effectively suppressed by vanadate treatment. After TBI, the vanadate-treated mice retained better bone marrow cellularity and showed markedly higher survival rate compared to the vehicle-treated animals. In contrast, vanadate did not relieve loss of intestinal crypts and failed to rescue mice from GI death after PBI. CONCLUSION: Vanadate is a p53 inhibitor that has been shown to be beneficial as a radiation protective agent against HP but was not effective in protecting against acute GI radiation injury.
Asunto(s)
Protectores contra Radiación/química , Protectores contra Radiación/farmacología , Sodio/química , Vanadatos/química , Vanadatos/farmacología , Irradiación Corporal Total/efectos adversos , Animales , Médula Ósea/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Tracto Gastrointestinal/efectos de la radiación , Ratones , Ratones Endogámicos ICR , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Flavonoids are a subclass of polyphenols which are attractive, due to possessing various physiological activities, including a radioprotective effect. Tumor suppressor p53 is a primary regulator in the radiation response and is involved in the pathogenesis of radiation injuries. In this study, we revealed that isorhamnetin inhibited radiation cell death, and investigated its action mechanism focusing on DNA damage response. Although isorhamnetin moderated p53 activity, it promoted phosphorylation of ataxia telangiectasia mutated (ATM) and enhanced 53BP1 recruitment in irradiated cells. The radioprotective effect of isorhamnetin was not observed in the presence of ATM inhibitor, indicating that its protective effect was dependent on ATM. Furthermore, isorhamnetin-treated mice survived gastrointestinal death caused by a lethal dose of abdominal irradiation. These findings suggested that isorhamnetin enhances the ATM-dependent DNA repair process, which is presumably associated with the suppressive effect against GI syndrome.
Asunto(s)
Síndrome de Radiación Aguda/tratamiento farmacológico , Mucosa Intestinal/efectos de los fármacos , Quercetina/análogos & derivados , Protectores contra Radiación/uso terapéutico , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Femenino , Células Hep G2 , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos ICR , Fosforilación , Quercetina/farmacología , Quercetina/uso terapéutico , Protectores contra Radiación/farmacologíaRESUMEN
Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized membrane glycoprotein expressed in tissues distinct from those expressing other ICAMs. Here, we determined the N-glycan structure of ICAM-5 purified from adult rat brain and compared it with that of other ICAMs. N-glycans were released by N-glycosidase F digestion and labeled with p-amino benzoic octylester (ABOE). ABOE-labeled glycans were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. The N-glycans obtained from rat brain ICAM-5 consisted of approximately 85% neutral, 10.2% sialylated-only, 2.8% sulfated-only, and 1.2% sialylated and sulfated glycans. Compared with the N-glycan structures of human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells, rat brain ICAM-5 had less highly branched glycans, sialylated glycans, and N-acetyllactosamine structures. In contrast, high-mannose-type N-glycans and Lewis X were more commonly found in rat brain ICAM-5 than in human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells. In addition, sulfated glycans contained GlcNAc 6-O-sulfate on the non-reducing terminal side. Our data will be important for the elucidation of the roles of the N-glycans expressed in neural cells, including those present on ICAM-5.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Polisacáridos/química , Ácido 4-Aminobenzoico/química , Animales , Células CHO , Secuencia de Carbohidratos , Moléculas de Adhesión Celular/química , Cromatografía Líquida de Alta Presión/métodos , Cricetinae , Cricetulus , Humanos , Conformación Molecular , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Moléculas de Adhesión de Célula Nerviosa/química , Neuronas/metabolismo , Polisacáridos/metabolismo , Conejos , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , para-AminobenzoatosRESUMEN
Activin A is a multifunctional homo-dimeric protein that belongs to the transforming growth factor (TGF)-beta superfamily. In neurons, activin has neuroprotective effects both in vitro and in vivo, but it inhibits neuronal differentiation in some cell lines. Here we report that activin A can promote neuronal differentiation in particular cases. We examined activin A-induced neuronal differentiation and survival in a selected subpopulation of a human neuroblastoma cell line, SK-N-SH, grown in low-serum (differentiation-inducing) conditions. Activin A caused dramatic neurite outgrowth, and increased the expression of neuronal markers and the transactivation of dopamine beta-hydroxylase. We demonstrated that the activin A signal is transduced through the activin A type 1 receptor, ALK4, and transactivates several TGF-beta target genes in a SMAD-independent manner. That is, activin A did not induce the phosphorylation of SMAD2/3, the interaction of SMAD2/3 with SMAD4, the binding of SMAD2/3 to the promoter of TGF-beta target genes, or the accumulation of SMAD2/3 in the nucleus. These results suggest that, in particular cases, activin A can induce neuronal differentiation and support neuronal survival in vitro. These findings may reflect previously unknown functions of activin A in neuronal cells in vivo.
Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Activinas/metabolismo , Neurogénesis , Neuronas/citología , Proteínas Smad/metabolismo , Activinas/farmacología , Adulto , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular , Humanos , Masculino , Neuronas/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteína Smad4/metabolismo , Activación Transcripcional , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
We previously reported on a 8-quinolinol-pendant cyclen (L(5)) as a Zn(2+) fluorophore (cyclen = 1,4,7,10-tetraazacyclododecane) and its caged derivative, 8-(benzenesulfonyloxy)-5-(N,N-dimethylaminosulfonyl)quinolin-2-ylmethyl-pendant cyclen (BS-caged-L(5)), which can be reactivated by hydrolysis of benzenesulfonyl group upon complexation with Zn(2+) at neutral pH to give a 1:1 Zn(2+)-L(5) complex (Zn(H(-1)L(5))). We report herein on the synthesis of 5,7-bis(N,N-dimethylaminosulfonyl)-8-hydroxyquinolin-2-ylmethyl-pendant cyclen (L(6)) and its caged derivative (BS-caged-L(6)) for more sensitive and more efficient cell-membrane permeability than those of L(5) and BS-caged-L(5). By potentiometric pH, (1)H NMR, and UV-vis spectroscopic titrations, the deprotonation constants pK(a1)-pK(a6) of H(5)L(6) were determined to be <2, <2, <2, 2.5 +/- 0.1 (for the 8-OH group of the quinoline moiety), 9.7 +/- 0.1, and 10.8 +/- 0.1 at 25 degrees C with I = 0.1 (NaNO(3)). The results of (1)H NMR, potentiometric pH, UV-vis, and fluorescent titrations showed that L(6) rapidly forms a 1:1 complex with Zn(2+) (Zn(H(-1)L(6))), the dissociation constant of which is 50 fM at pH 7.4. The fluorescent emission of Zn(H(-1)L(6)) at 478 nm is 32 times as large as that of L(6) (excitation at 370 nm), and the fluorescent quantum yield of Zn(H(-1)L(6)) (Phi(F) = 0.41) is much greater than that of Zn(H(-1)L(5)) (Phi(F) = 0.044). The BS-caged-L(6) was reactivated by hydrolysis of the benzenesulfonyl moiety more rapidly (completes in 30 min at pH 7.4 at 37 degrees C) than BS-caged-L(5), presumably enabling the practical detection of Zn(2+) in sample solutions and living cells. The photochemical deprotection of BS-caged-L(6) and the cell membrane permeability of L(6) and BS-caged-L(6) are also described.
Asunto(s)
Colorantes Fluorescentes/síntesis química , Compuestos Organometálicos/síntesis química , Sulfonas/química , Zinc/química , Colorantes Fluorescentes/química , Hidrólisis , Ligandos , Estructura Molecular , Compuestos Organometálicos/química , FotoquímicaRESUMEN
Radiation damage to normal tissues is one of the most serious concerns in radiation therapy, and the tolerance dose of the normal tissues limits the therapeutic dose to the patients. p53 is well known as a transcription factor closely associated with radiation-induced cell death. We recently demonstrated the protective effects of several p53 regulatory agents against low-LET X- or γ-ray-induced damage. Although it was reported that high-LET heavy ion radiation (>85 keV/µm) could cause p53-independent cell death in some cancer cell lines, whether there is any radioprotective effect of the p53 regulatory agents against the high-LET radiation injury in vivo is still unclear. In the present study, we verified the efficacy of these agents on bone marrow and intestinal damages induced by high-LET heavy-ion irradiation in mice. We used a carbon-beam (14 keV/µm) that was shown to induce a p53-dependent effect and an iron-beam (189 keV/µm) that was shown to induce a p53-independent effect in a previous study. Vanadate significantly improved 60-day survival rate in mice treated with total-body carbon-ion (p < 0.0001) or iron-ion (p < 0.05) irradiation, indicating its effective protection of the hematopoietic system from radiation injury after high-LET irradiation over 85 keV/µm. 5CHQ also significantly increased the survival rate after abdominal carbon-ion (p < 0.02), but not iron-ion irradiation, suggesting the moderate relief of the intestinal damage. These results demonstrated the effectiveness of p53 regulators on acute radiation syndrome induced by high-LET radiation.
Asunto(s)
Iones Pesados/efectos adversos , Traumatismos por Radiación/prevención & control , Radiación Ionizante , Proteína p53 Supresora de Tumor/efectos de los fármacos , Animales , Humanos , Transferencia Lineal de Energía , RatonesRESUMEN
BACKGROUND: The prognosis for patients with unresectable advanced hepatocellular carcinoma (HCC) is poor. Miriplatin is a hydrophobic platinum compound that has a long retention time in lesions after transarterial chemoembolization (TACE). We investigated anti-tumor activity of miriplatin combined with irradiation on HCC cells, and its underlying mechanism of apoptosis. We also analyzed the effectiveness of miriplatin-TACE and radiotherapy for locally advanced HCC. METHODS: Human HCC cell lines HepG2 and HuH-7 were treated with DPC (active form of miriplatin) and radiation, and synergy was evaluated using a combination index (CI). Apoptosis-related proteins and cell cycles were analyzed by western blotting and flowcytometry. We retrospectively analyzed treatment outcomes in 10 unresectable HCC patients with vascular/bile duct invasion treated with miriplatin-TACE and radiotherapy. RESULTS: DPC or X-ray irradiation decreased cell viability dose-dependently. DPC plus irradiation decreased cell viability synergistically in both cell lines (CI < 1, respectively). Cleaved PARP expression was induced much more strongly by DPC plus irradiation than by each treatment alone. Expression of p53 up-regulated modulator of apoptosis (PUMA) was significantly induced by the combination, and knockdown of PUMA with siRNA significantly decreased apoptosis in both cell lines. DPC plus irradiation caused sub-G1, G2/M, and S phase cell arrest in those cells. The combination of miriplatin-TACE and radiotherapy showed a high response rate for patients with locally advanced HCC despite small number of patients. CONCLUSIONS: Miriplatin plus irradiation had synergistic anti-tumor activity on HCC cells through PUMA-mediated apoptosis and cell cycle arrest. This combination may possibly be effective in treating locally advanced HCC.
Asunto(s)
Antineoplásicos/administración & dosificación , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Compuestos Organoplatinos/administración & dosificación , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proteínas Reguladoras de la Apoptosis/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Terapia Combinada , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Compuestos Organoplatinos/farmacología , Proteínas Proto-Oncogénicas/genética , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Radiation damage to normal tissues is a serious concern in radiation therapy. Advances in radiotherapeutic technology have improved the dose distribution of the target volumes and risk organs, but damage to risk organs that are located within the irradiation field still limits the allowable prescription dose. To overcome this dose-limiting toxicity, and to further improve the efficacy of radiotherapy, the development of drugs that protect normal tissues but not cancer tissues from the effects of radiation are expected to be developed based on molecular target-based drugs. p53 is a well-known transcription factor that is closely associated with radiation-induced cell death. In radiation-injured tissues, p53 induces apoptosis in hematopoietic lineages, whereas it plays a radioprotective role in the gastrointestinal epithelium. These facts suggest that p53 inhibitor would be effective for radioprotection of the hematopoietic system, and that a drug that upregulates the radioprotective functions of p53 would enhance the radioresistance of gastrointestinal tissues. In this review, we summarize recent progress regarding the prevention of radiation injury by regulating p53 and provide new strategic insights into the development of radioprotectors in radiotherapy. J. Med. Invest. 66 : 219-223, August, 2019.
Asunto(s)
Desarrollo de Medicamentos , Tolerancia a Radiación/efectos de los fármacos , Protectores contra Radiación/farmacología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Apoptosis/efectos de la radiación , Quelantes/farmacología , Cloroquinolinoles/farmacología , Humanos , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Epolactaene, isolated from cultured Penicillium sp. BM 1689-P mycelium, induces neurite outgrowth and arrests the cell cycle of the human neuroblastoma cell line, SH-SY5Y, at the G1 phase. We have found that epolactaene and its derivatives induce apoptosis in the human leukemia B-cell line, BALL-1. In this study, we prepared fluorescent and biotinylated epolactaene derivatives. We characterized the cellular location and the identification of BALL-1 proteins that reacted with these compounds. The results obtained from the reaction of epolactaene or its derivative with N-acetylcysteine methyl ester indicate that these compounds induce the disulfide formation and the alpha-position of the epoxylactam core is the reactive site.
Asunto(s)
Biotina/química , Disulfuros/química , Acetilcisteína/análogos & derivados , Acetilcisteína/química , Biotinilación , Muerte Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Compuestos Epoxi/síntesis química , Compuestos Epoxi/química , Compuestos Epoxi/farmacología , Fluorescencia , Humanos , Estructura Molecular , Polienos/síntesis química , Polienos/química , Polienos/farmacología , Estereoisomerismo , Células Tumorales CultivadasRESUMEN
Inhibiting p53-dependent apoptosis by inhibitors of p53 is an effective strategy for preventing radiation-induced damage in hematopoietic lineages, while p53 and p21 also play radioprotective roles in the gastrointestinal epithelium. We previously identified some zinc(II) chelators, including 8-quinolinol derivatives, that suppress apoptosis in attempts to discover compounds that target the zinc-binding site in p53. We found that 5-chloro-8-quinolinol (5CHQ) has a unique p53-modulating activity that shifts its transactivation from proapoptotic to protective responses, including enhancing p21 induction and suppressing PUMA induction. This p53-modulating activity also influenced p53 and p53-target gene expression in unirradiated cells without inducing DNA damage. The specificity of 5CHQ for p53 and p21 was demonstrated by silencing the expression of each protein. These effects seem to be attributable to the sequence-specific alteration of p53 DNA-binding, as evaluated by chromatin immunoprecipitation and electrophoretic mobility shift assays. In addition, 5-chloro-8-methoxyquinoline itself had no antiapoptotic activity, indicating that the hydroxyl group at the 8-position is required for its antiapoptotic activity. We applied this remarkable agonistic activity to protecting the hematopoietic and gastrointestinal system in mouse irradiation models. The dose reduction factors of 5CHQ in total-body and abdominally irradiated mice were about 1.2 and 1.3, respectively. 5CHQ effectively protected mouse epithelial stem cells from a lethal dose of abdominal irradiation. Furthermore, the specificity of 5CHQ for p53 in reducing the lethality induced by abdominal irradiation was revealed in Trp53-KO mice. These results indicate that the pharmacologic upregulation of radioprotective p53 target genes is an effective strategy for addressing the gastrointestinal syndrome. Mol Cancer Ther; 17(2); 432-42. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."
Asunto(s)
Protectores contra Radiación/uso terapéutico , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Modelos Animales de Enfermedad , Humanos , Ratones , Protectores contra Radiación/farmacología , Activación TranscripcionalRESUMEN
Bisdemethoxycurcumin (BDMC), which is isolated from the rhizomes of Curcuma longa, has anti-inflammatory and anti-carcinogenic activities. Here we found that BDMC enhanced X-ray-induced apoptosis in human T-cell leukemia MOLT-4 cells. Knockdown of p53 significantly attenuated the radiosensitizing effect of BDMC. However, BDMC did not enhance X-ray-mediated activation of the p53 signaling pathway via p53's transactivation or mitochondrial translocation. On the other hand, BDMC promoted the X-ray-induced dephosphorylation at Ser 70 in Bcl-2's flexible loop regulatory domain and Bcl-2 binding to p53. Overexpressing Bcl-2 completely blocked the BDMC's radiosensitization effect. Our results indicate that BDMC stimulates the dephosphorylation and p53-binding activity of Bcl-2 and suggest that BDMC may induce a neutralization of Bcl-2's anti-apoptotic function, thereby enhancing X-ray-induced apoptosis.