RESUMEN
Objective: To investigate the protective effects of L-carnitine (LC) on lipopolysaccharide (LPS) - injured mouse pulmonary microvascular endothelial cells (PMVECs) and its effects on autophagy and apoptosis. Methods: Cultured mouse PMVECs were divided into three groups: â Control group, â¡ LPS group (10 µg/ml, 3, 6, 12, 24 h), ⢠LPS (10 µg/ml, 24 h)+LC (2.5, 5.0, 10 µg/ml) (LPS+LC) group. PMVECs apoptosis was examined by Annexin V-FITC/PI double labeling method. Autophagosome was detected by immunofluorescence staining. Levels of autophagy-related protein LC3 and apoptosis-related protein caspase-3 were detected by Western blot. PMVECs viability was measured by CCK-8. Results: â Compared with the control group, LPS treatment inhibited the PMVECs viability significantly, whereas the apoptosis rate and the expression of autophagy protein LC3 II were markedly increased after LPS treatment for 6 h, 12 h and 24 h. â¡ Compared with LPS group (10 µg/ml, 24 h), the PMVECs viability, levels of autophagy protein LC3 II and caspase-3 protein expression as well as apoptosis rate in LPS+LC group were increased significantly. Conclusion: LC can increase the activity of PMVECs injuried by LPS, promote autophagy and inhibit apoptosis of PMVECs.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Carnitina/farmacología , Células Endoteliales , Animales , Células Cultivadas , Lipopolisacáridos , RatonesRESUMEN
Histone H3K27me3 demethylase KDM6B (also known as Jumonji domain-containing protein D3, JMJD3) plays vital roles in the etiology of inflammatory responses; however, little is known about the role of KDM6B in neuroinflammation-induced anxiety-like behavior. The present study aimed to investigate the potential role of KDM6B in lipopolysaccharide (LPS)-induced anxiety-like behavior and to evaluate whether it is associated with the modulation of vestigial-like family member 4 (VGLL4). The elevated plus maze, light-dark box, and open-field test were performed to test the anxiety-like behavior induced by LPS in C57BL/6 J male mice. Levels of relative protein expression in the hippocampus were quantified by western blotting. KDM6B inhibitor GSK-J4 and microglia inhibitor minocycline as well as adeno-associated virus of Vgll4 shRNA were used to explore the underlying mechanisms. We found that KDM6B, VGLL4, interleukin-1ß (IL-1ß), and ionized calcium-binding adaptor molecule-1 (Iba-1, microglia marker) protein levels were increased in LPS-dose dependent manner in the hippocampus but not in prefrontal cortex. GSK-J4 treatment attenuated LPS-induced VGLL4, the signal transducer and activator of transcription 3 (STAT3), IL-1ß and Iba-1 upregulation and anxiety-like behavior. Knockdown VGLL4 with Vgll4 shRNA prevented the increase of anxiety-like behavior and levels of STAT3, IL-1ß, and Iba-1 expression in the hippocampus of LPS-treated mice. Moreover, minocycline, an inhibitor of microglia treatment blunted LPS-induced anxiety-like behavior. Collectively, these results demonstrate that the induction of neuroinflammation by LPS promotes KDM6B activation in the hippocampus, and LPS-induced anxiety-like behavior is associated with upregulation of VGLL4 by KDM6B in the hippocampus.
Asunto(s)
Ansiedad/metabolismo , Conducta Animal/fisiología , Hipocampo/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Enfermedades Neuroinflamatorias/metabolismo , Factores de Transcripción/metabolismo , Animales , Ansiedad/inducido químicamente , Conducta Animal/efectos de los fármacos , Hipocampo/efectos de los fármacos , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades Neuroinflamatorias/inducido químicamente , Regulación hacia ArribaRESUMEN
The MRI technique has been used in diagnosis of manganism in humans and non-human primates. This cross-sectional study was designed to explore whether the pallidal signal intensity in T1-weighted MRI correlated with Mn levels in the blood compartment among Mn-exposed workers and to understand to what extent the MRI signal could reflect Mn exposure. A group of 18 randomly selected male Mn-exposed workers of which 13 were smelting workers with high exposure (mean of airborne Mn in work place: 1.26 mg/m3; range: 0.31-2.93 mg/m3), and 5 power distribution control workers with low exposure (0.66 mg/m3 and 0.23-0.77 mg/m3) from a ferroalloy factory, and another group of 9 male subjects as controls from a non-smelting factory who were office or cafeteria workers (0.01 mg/m3 and 0-0.03 mg/m3) were recruited for neurological tests, MRI examination, and analysis of Mn in whole blood (MnB), plasma (MnP) or red blood cells (MnRBC). No clinical symptoms and signs of manganism were observed among these workers. MRI data showed average increases of 7.4% (p<0.05) and 16.1% (p<0.01) in pallidal index (PI) among low- and high-exposed workers, respectively, as compared to controls. Fourteen out of 18 Mn-exposed workers (78%) had intensified PI values, while this proportion was even higher (85%) among the high Mn-exposed workers. Among exposed workers, the PI values were significantly associated with MnRBC (r=0.55, p=0.02). Our data suggest that the workers exposed to airborne Mn, but without clinical symptoms, display an exposure-related, intensified MRI signal. The MRI, as well as MnRBC, may be useful in early diagnosis of Mn exposure.
Asunto(s)
Encéfalo/patología , Eritrocitos/química , Manganeso/sangre , Metalurgia , Exposición Profesional/efectos adversos , Adulto , Aire/análisis , Biomarcadores , Recuento de Células Sanguíneas , Estudios Transversales , Globo Pálido/efectos de los fármacos , Globo Pálido/metabolismo , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana EdadRESUMEN
This study aims to investigate whether inflammation mediated by NF-κB activation is involved in the induction of anxiety-like behavior in chronic normobaric hypoxia (CNH) exposed rats and to investigate the underlying mechanism. To this end, rats were exposed in a normobaric hypoxic chamber with a fraction of inspired oxygen (FIO2) of â¼ 10%, 23 h/d, continues for 2 weeks. Anxiety-like behavior was tested by elevated plus maze and open field, inflammatory response, nucleus translocation of NF-κB, and signaling pathway in hippocampus were examined. CNH induced a significant increase of anxiety- like behavior and inflammation responses, which were ameliorated by NF-κB inhibitor, PDTC pretreatment, suggesting that the anxiogenic effect induced by inflammation is through NF-κB activation. CNH treatment significantly increased nucleus translocation of p65 and p105 in hippocampus, which was suppressed by PDTC pretreatment. In addition, CNH treatment significantly increased Iba-1, iNOS, COX-2, and p-PKA in hippocampus, which were blocked by PDTC pretreatment, suggesting CNH may activate microglia cells in hippocampus through NF-κB pathway. In conclusion, our results illustrate a mechanism that, activation of NF-κB in hippocampus may trigger the proinflammatory response of microglia cells, and iNOS-PKA pathway may involve in anxiogenic effect in CNH exposed rats.
Asunto(s)
Ansiedad/metabolismo , Hipocampo/metabolismo , Hipoxia/metabolismo , Inflamación/metabolismo , FN-kappa B/metabolismo , Animales , Ciclooxigenasa 2/metabolismo , Hipoxia/psicología , Mediadores de Inflamación/metabolismo , Masculino , Microglía/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
MicroRNAs (miRs) play pivotal roles in carcinogenesis and endoplasmic reticulum (ER) that performs the folding, modification and trafficking of proteins targeted to the secretory pathway. Cancer cells often endure ER stress during tumor progression but use the adaptive ER stress response to gain survival advantage. Here we report: (i) A group of miRs, including miR-30b-5p and miR-30c-5p, are upregulated by proteasome inhibitor PS-341 treatment, in HepG2 and MDA-MB-453 cells. (ii) Two representative PS-341-induced miRs: miR-30b-5p and miR-30c-5p are found to promote cell proliferation and anti-apoptosis in both tumor cells. (iii) eIF2α is confirmed as the congenerous target of miR-30b-5p and miR-30c-5p, essential to the anti-apoptotic function of these miRs. (iv) Upregulation of miR-30b-5p or miR-30c-5p, which occurs latter than the increase of phosphorylated eIF2α (p-eIF2α) in the cell under ER stress, suppresses the p-eIF2α/ATF4/CHOP pro-apoptotic pathway. (v) Inhibition of the miR-30b-5p or miR-30c-5p sensitizes the cancer cells to the cytotoxicity of proteasome inhibition. In conclusion, we unravels a new miRs-based mechanism that helps maintain intracellular proteostasis and promote cell survival during ER stress through upregulation of miR-30b-5p and miR-30c-5p which target eIF2α and thereby inhibit the p-eIF2α/ATF4/CHOP pro-apoptotic pathway, identifying miR-30b-5p and miR-30c-5p as potentially new targets for anti-cancer therapies.
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Carcinogénesis/genética , Factor 2 Eucariótico de Iniciación/biosíntesis , MicroARNs/genética , Neoplasias/genética , Factor de Transcripción Activador 4/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Bortezomib/administración & dosificación , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Factor 2 Eucariótico de Iniciación/genética , Células Hep G2 , Humanos , MicroARNs/biosíntesis , Neoplasias/patología , Inhibidores de Proteasoma/administración & dosificación , Factor de Transcripción CHOP/biosíntesisRESUMEN
Resistance to chemotherapy is a great challenge to improving the survival of patients with diffuse large B-cell lymphoma (DLBCL), especially those with activated B-cell-like DLBCL (ABC-DLBCL). Therefore it is urgent to search for novel agents for the treatment of DLBCL. Gambogic acid (GA), a small molecule derived from Chinese herb gamboges, has been approved for Phase II clinical trial for cancer therapy by Chinese FDA. In the present study, we investigated the effect of GA on cell survival and apoptosis in DLBCL cells including both GCB- and ABC-DLBCL cells. We found that GA induced growth inhibition and apoptosis of both GCB- and ABC-DLBCL cells in vitro and in vivo, which is associated with proteasome malfunction. These findings provide significant pre-clinical evidence for potential usage of GA in DLBCL therapy particularly in ABC-DLBCL treatment.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfoma de Células B Grandes Difuso/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Xantonas/farmacología , Animales , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Masculino , Ratones , Ratones Desnudos , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Verapamil (Ver), an inhibitor of the multidrug resistance gene product, has been proved to be a promising combination partner with other anti-cancer agents including proteasome inhibitor bortezomib. Gambogic acid (GA) has been approved for Phase II clinical trials in cancer therapy in China. We have most recently reported that GA is a potent proteasome inhibitor, with anticancer efficiency comparable to bortezomib but much less toxicity. In the current study we investigated whether Ver can enhance the cytotoxicity of GA. We report that (i) the combination of Ver and GA results in synergistic cytotoxic effect and cell death induction in HepG2 and K562 cancer cell lines; (ii) a combinational treatment with Ver and GA induces caspase activation, endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production; (iii) caspase inhibitor z-VAD blocks GA+Ver-induced apoptosis but not proteasome inhibition; (iv) cysteine-containing compound N-acetylcysteine (NAC) prevents GA+Ver-induced poly(ADP-ribose) polymerase cleavage and proteasome inhibition. These results demonstrate that Ver accelerates GA-induced cytotoxicity via enhancing proteasome inhibition and ROS production. These findings indicate that the natural product GA is a valuable candidate that can be used in combination with Ver, thus representing a compelling anticancer strategy.
Asunto(s)
Inhibidores de Proteasoma/farmacología , Especies Reactivas de Oxígeno/metabolismo , Verapamilo/farmacología , Xantonas/farmacología , Acetilcisteína/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Sinergismo Farmacológico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Hep G2 , Humanos , Células K562 , Inhibidores de Proteasoma/administración & dosificación , Verapamilo/administración & dosificación , Xantonas/administración & dosificaciónRESUMEN
Anacardic acid (6-pentadecylsalicylic acid, AA), a natural compound isolated from the traditional medicine Amphipterygium adstringens, has been reported to possess antitumor activities. However, its molecular targets have not been thoroughly studied. Here, we report that AA is a potent inducer of endoplasmic reticulum (ER) stress, leading to apoptosis in hepatoma HepG2 and myeloma U266 cells. Induction of ER stress by AA was supported by a dose- and time-dependent increase in expression of the ER signaling downstream molecules, such as GRP78/BiP, phosphorylated eIF2α, ATF4 and CHOP in both HepG2 and U266 cell lines. Blockage of ATF4 expression by siRNA partially inhibited, while knockdown of CHOP expression by siRNA slightly increased AA-induced cell death in these cells. In addition, AA suppressed HepG2 xenograft tumor growth, associated with increased ER stress in vivo. These results suggest that AA induces tumor cell apoptosis associated with ATF4-dependent ER stress.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Ácidos Anacárdicos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Factor de Transcripción Activador 4/genética , Animales , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Fosforilación , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Factor de Transcripción CHOP/metabolismo , Transfección , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The proteasome inhibitor-based combinational therapy has been reported to be an efficient cancer treatment. Our recent studies demonstrated that the natural compound gambogic acid (GA) is a tissue-specific proteasome inhibitor, comparable to bortezomib (Bor), and sensitizes malignant cells to the proteasome inhibitor MG132/MG262 both in vitro and in vivo. The aim of this study was to further extend our investigation by combining GA with the clinically used proteasome inhibitor Bor to test their combined efficacy against human hepatoma HepG2 and mouse hepatoma H22 cells. GA and Bor synergistically induced cytotoxicity and cell death in human HepG2 and mouse H22 cells, and accelerated proteasome inhibition, endoplasmic reticulum (ER) stress and caspase activation in HepG2 cancer cells. However, unexpectedly, GA did not enhance or even antagonized Bor-induced tumor growth inhibition in H22 allograft and HepG2 xenograft tumor models. These findings demonstrated that GA increased Bor activity in vitro but limited the efficacy of Bor in vivo. We suggest that the combination of GA and Bor be avoided when administering these drugs to patients.
Asunto(s)
Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Pirazinas/farmacología , Xantonas/farmacología , Animales , Western Blotting , Bortezomib , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy.
Asunto(s)
Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Proteolisis/efectos de los fármacos , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Compuestos Organometálicos/farmacología , Inhibidores de Proteasas/farmacología , Pirazinas/farmacología , Piridinas/farmacología , Ubiquitina/metabolismoRESUMEN
Resistance to Imatinib mesylate (IM) is an emerging problem for patients with chronic myelogenous leukemia (CML). T315I mutation in the Bcr-Abl is the predominant mechanism of the acquired resistance to IM and second generation tyrosine kinase inhibitors (TKI). Therefore it is urgent to search for new measures to overcome TKI-resistance. Auranofin (AF), clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer. In contrast to the reports that AF induces apoptosis by increasing intracellular reactive oxygen species (ROS) levels via inhibiting thioredoxin reductase, our recent study revealed that AF-induced apoptosis depends on inhibition of proteasomal deubiquitinases (UCHL5 and USP14). Here we report that (i) AF induces apoptosis in both Bcr-Abl wild-type cells and Bcr-Abl-T315I mutation cells and inhibits the growth of IM-resistant Bcr-Abl-T315I xenografts in vivo; (ii) AF inhibits Bcr-Abl through both downregulation of Bcr-Abl gene expression and Bcr-Abl cleavage mediated by proteasome inhibition-induced caspase activation; (iii) proteasome inhibition but not ROS is required for AF-induced caspase activation and apoptosis. These findings support that AF overcomes IM resistance through both Bcr/Abl-dependent and -independent mechanisms, providing great clinical significance for cancer treatment.
Asunto(s)
Antirreumáticos/farmacología , Apoptosis/efectos de los fármacos , Auranofina/farmacología , Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Benzamidas/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Humanos , Mesilato de Imatinib , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Trasplante Heterólogo , Ubiquitina Tiolesterasa/antagonistas & inhibidoresRESUMEN
PURPOSE: Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl-T315I is the predominant mutation that causes resistance to imatinib, cytotoxic drugs, and the second-generation tyrosine kinase inhibitors. The emergence of imatinib resistance in patients with CML leads to searching for novel approaches to the treatment of CML. Gambogic acid, a small molecule derived from Chinese herb gamboges, has been approved for phase II clinical trial for cancer therapy by the Chinese Food and Drug Administration (FDA). In this study, we investigated the effect of gambogic acid on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl. EXPERIMENTAL DESIGN: CML cell lines (KBM5, KBM5-T315I, and K562), primary cells from patients with CML with clinical resistance to imatinib, and normal monocytes from healthy volunteers were treated with gambogic acid, imatinib, or their combination, followed by measuring the effects on cell growth, apoptosis, and signal pathways. The in vivo antitumor activity of gambogic acid and its combination with imatinib was also assessed with nude xenografts. RESULTS: Gambogic acid induced apoptosis and cell proliferation inhibition in CML cells and inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Our data suggest that GA-induced proteasome inhibition is required for caspase activation in both imatinib-resistant and -sensitive CML cells, and caspase activation is required for gambogic acid-induced Bcr-Abl downregulation and apoptotic cell death. CONCLUSIONS: These findings suggest an alternative strategy to overcome imatinib resistance by enhancing Bcr-Abl downregulation with the medicinal compound gambogic acid, which may have great clinical significance in imatinib-resistant cancer therapy.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Piperazinas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirimidinas/farmacología , Xantonas/farmacología , Animales , Caspasas/metabolismo , Proliferación Celular , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos , Activación Enzimática , Femenino , Proteínas de Fusión bcr-abl/metabolismo , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Mesilato de Imatinib , Células K562 , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Monocitos/efectos de los fármacos , Monocitos/fisiología , Inhibidores de Proteasoma/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Proteasomes are attractive emerging targets for anti-cancer therapies. Auranofin (Aur), a gold-containing compound clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer but its anti-cancer mechanism is poorly understood. Here we report that (i) Aur shows proteasome-inhibitory effect that is comparable to that of bortezomib/Velcade (Vel); (ii) different from bortezomib, Aur inhibits proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14 rather than the 20S proteasome; (iii) inhibition of the proteasome-associated DUBs is required for Aur-induced cytotoxicity; and (iv) Aur selectively inhibits tumor growth in vivo and induces cytotoxicity in cancer cells from acute myeloid leukemia patients. This study provides important novel insight into understanding the proteasome-inhibiting property of metal-containing compounds. Although several DUB inhibitors were reported, this study uncovers the first drug already used in clinic that can inhibit proteasome-associated DUBs with promising anti-tumor effects.
Asunto(s)
Antirreumáticos/farmacología , Auranofina/farmacología , Neoplasias/tratamiento farmacológico , Inhibidores de Proteasoma/farmacología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Células Hep G2 , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Ubiquitina Tiolesterasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gambogic acid (GA) is the principal active ingredient of gamboges. GA was reported to exert anti-tumor and anti-inflammatory effects both in vitro and in vivo. Previously, we have shown that GA is a more tissue-specific proteasome inhibitor than bortezomib and it is less toxic to peripheral white blood cells compared to bortezomib. Ubiquitous proteasome inhibition was shown by some reports, but not by others, to prevent cardiac remodeling in response to pressure overload by blocking the NF-κB pathway; however, whether GA modulates the development of chronic hypoxia-induced right ventricular hypertrophy has not been investigated yet. Here we report that GA can significantly attenuate right ventricular hypertrophy induced by chronic hypoxia, reduce cardiac fibrosis, and remarkably block the reactivation of bona fide fetal genes in the cardiac tissue. Furthermore, we also investigated the potential molecular targets of GA on right ventricular hypertrophy. The results showed that GA could accumulate the IκB levels associated with decreased proteasomal activity, block the translocation of NF-κB from the cytoplasm to the nucleus, decrease NF-κB DNA-binding activity, and reduce IL-2 levels. In conclusion, GA is capable of preventing the development of chronic hypoxia-induced right ventricular hypertrophy. GA has great potential to be developed into an effective anti-hypertrophy agent.
RESUMEN
Cardiac hypertrophy is a common response of the heart to a variety of cardiovascular stimuli. Pathological cardiac hypertrophy eventually leads to heart failure. Gambogic acid (GA) is a main active ingredient isolated from the gamboge resin of Garcinia hanburyi trees and has potent anti-tumor and anti-inflammatory effects that are associated with inhibition of the NF-κB pathway. We and others recently reported that GA can significantly inhibit the function of the proteasome with much less toxicity than conventional proteasome inhibitors. The increasing lines of evidence indicate that the inhibition of the proteasome can promote the regression of cardiac hypertrophy induced by pressure overload through the blockade of the NF-κB pathway. In the present study, we examined the effect of GA on pressure overload or isoproterenol infusion induced cardiac hypertrophy and fibrosis, and changes in myocardial NF-κB signaling. We observed that the heart weight/body weight ratio, the size of cardiomyocytes, interstitial fibrosis, and the reactivation of fetal genes (α-SK-actin and BNP mRNA) were markedly increased by abdominal aorta constriction (AAC) or isoproterenol infusion (ISO), all of which were effectively inhibited by GA treatment. Furthermore, GA treatment abolished proteasome chymotrypsin-like activity increases induced by AAC or ISO, led to increased myocardial IκB protein, decreased NF-κB p65 subunit levels in the nuclear fraction, decreased NF-κB DNA-binding activity, and reduced IL2 levels in the myocardium of rats subject to AAC or ISO. In conclusion, GA treatment can suppress cardiac hypertrophy and fibrosis induced by pressure overload or isoproterenol possibly through the inhibition of the proteasome and the NF-κB pathway, suggesting that GA treatment may provide a new strategy to treat cardiac hypertrophy.
RESUMEN
Extracellular adenosine (ADE) interacts with cells by two pathways: by activating cell surface receptors at nanomolar/micromolar concentrations; and by interfering with the homeostasis of the intracellular nucleotide pool at millimolar concentrations. Ade shows both cytotoxic and cytoprotective effects; however, the underlying mechanisms remain unclear. In the present study, the effects of adenosine-mediated ATP on cell viability were investigated. Adenosine treatment was found to be cytoprotective in the low intracellular ATP state, but cytotoxic under the normal ATP state. Adenosine-mediated cytotoxicity and cytoprotection rely on adenosine-derived ATP formation, but not via the adenosine receptor pathway. Ade enhanced proteasome inhibition-induced cell death mediated by ATP generation. These data provide a new pathway by which adenosine exerts dual biological effects on cell viability, suggesting an important role for adenosine as an ATP precursor besides the adenosine receptor pathway.
Asunto(s)
Adenosina Trifosfato/metabolismo , Adenosina/metabolismo , Citoprotección , Citotoxinas/metabolismo , Adenosina/farmacología , Adenosina Trifosfato/farmacología , Transporte Biológico , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citosol/metabolismo , Citotoxinas/farmacología , Expresión Génica , Humanos , Células K562 , Receptores Purinérgicos P1/metabolismo , Transducción de SeñalRESUMEN
Gambogic acid (GA) is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues but low in many normal tissues, GA could therefore produce tissue-specific proteasome inhibition and tumor-specific toxicity, with clinical significance for designing novel strategies for cancer treatment.
Asunto(s)
Especificidad de Órganos/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Xantonas/farmacología , Animales , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Células Hep G2 , Humanos , Células K562 , Leucemia/metabolismo , Leucemia/patología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Linfocitos/patología , Ratones , Simulación del Acoplamiento Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , ARN Interferente Pequeño/metabolismo , Análisis de Supervivencia , Tripsina/metabolismo , Ubiquitinación/efectos de los fármacos , Xantonas/químicaRESUMEN
L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21(cip1) gene, mRNA and protein in cancer cells but not p27(kip1); (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21(cip1) gene but not p27(kip1) detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.
Asunto(s)
Carnitina/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias/enzimología , Neoplasias/patología , Acetilación/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Neoplasias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Combinations of proteasome inhibitors and histone deacetylases (HDAC) inhibitors appear to be the most potent to produce synergistic cytotoxicity in preclinical trials. We have recently confirmed that L-carnitine (LC) is an endogenous HDAC inhibitor. In the current study, the anti-tumor effect of LC plus proteasome inhibitor bortezomib (velcade, Vel) was investigated both in cultured hepatoma cancer cells and in Balb/c mice bearing HepG2 tumor. Cell death and cell viability were assayed by flow cytometry and MTS, respectively. Gene, mRNA expression and protein levels were detected by gene microarray, quantitative real-time PCR and Western blot, respectively. The effect of Vel on the acetylation of histone H3 associated with the p21(cip1) gene promoter was examined by using ChIP assay and proteasome peptidase activity was detected by cell-based chymotrypsin-like (CT-like) activity assay. Here we report that (i) the combination of LC and Vel synergistically induces cytotoxicity in vitro; (ii) the combination also synergistically inhibits tumor growth in vivo; (iii) two major pathways are involved in the synergistical effects of the combinational treatment: increased p21(cip1) expression and histone acetylation in vitro and in vivo and enhanced Vel-induced proteasome inhibition by LC. The synergistic effect of LC and Vel in cancer therapy should have great potential in the future clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Carnitina/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Proteasoma/farmacología , Pirazinas/farmacología , Acetilación/efectos de los fármacos , Animales , Bortezomib , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromatina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Sinergismo Farmacológico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células Hep G2 , Histonas/metabolismo , Humanos , Masculino , Ratones , Modelos Biológicos , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Interferente Pequeño/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Proteasome inhibition has emerged as a novel approach to anticancer therapy. Numerous natural compounds, such as gambogic acid, have been tested in vitro and in vivo as anticancer agents for cancer prevention and therapy. However, whether gambogic acid has chemosensitizing properties when combined with proteasome inhibitors in the treatment of malignant cells is still unknown. In an effort to investigate this effect, human leukemia K562 cells, mouse hepatocarcinoma H22 cells and H22 cell allografts were treated with gambogic acid, a proteasome inhibitor (MG132 or MG262) or the combination of both, followed by measurement of cellular viability, apoptosis induction and tumor growth inhibition. We report, for the first time, that: (i) the combination of natural product gambogic acid and the proteasome inhibitor MG132 or MG262 results in a synergistic inhibitory effect on growth of malignant cells and tumors in allograft animal models and (ii) there was no apparent systemic toxicity observed in the animals treated with the combination. Therefore, the findings presented in this study demonstrate that natural product gambogic acid is a valuable candidate to be used in combination with proteasome inhibitors, thus representing a compelling anticancer strategy.