RESUMO
While IgE is considered the primary mediator of mast cell activation, IL-33 contributes substantially in asthma, allergic rhinitis, and atopic dermatitis. To develop effective treatments for allergic disease, it is important to understand the role of therapeutic agents on IL-33 activation. We examined the effect of Didox (3,4-dihydroxybenzohydroxamic acid), an antioxidant and ribonucleotide reductase (RNR) inhibitor, on IL-33-mediated mast cell activation. Didox suppressed IL-6, IL-13, TNF, and MIP-1α (CCL3) production in bone marrow derived mast cells following IL-33 activation. This suppression was observed in different genetic backgrounds and extended to peritoneal mast cells. The antioxidant N-acetylcysteine mimicked the suppression of Didox, albeit at a much higher dose, while the RNR inhibitor hydroxyurea had no effect. Didox substantially suppressed IL-33-mediated NFκB and AP-1 transcriptional activities. These results suggest that Didox attenuates IL-33-induced mast cell activation and should be further studied as a potential therapeutic agent for inflammatory diseases involving IL-33.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Imunossupressores/farmacologia , Interleucina-33/farmacologia , Mastócitos/efeitos dos fármacos , Acetilcisteína/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Quimiocina CCL3/antagonistas & inibidores , Quimiocina CCL3/genética , Quimiocina CCL3/imunologia , Feminino , Regulação da Expressão Gênica/imunologia , Genes Reporter , Hidroxiureia/farmacologia , Interleucina-13/antagonistas & inibidores , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-33/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Luciferases/genética , Luciferases/imunologia , Masculino , Mastócitos/citologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/imunologia , Cultura Primária de Células , Transdução de Sinais , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Mast cell activation via the high-affinity IgE receptor (FcεRI) elicits production of inflammatory mediators central to allergic disease. As a synthetic antioxidant and a potent ribonucleotide reductase (RNR) inhibitor, Didox (3,4-dihyroxybenzohydroxamic acid) has been tested in clinical trials for cancer and is an attractive therapeutic for inflammatory disease. We found that Didox treatment of mouse bone marrow-derived mast cells (BMMC) reduced IgE-stimulated degranulation and cytokine production, including IL-6, IL-13, TNF and MIP-1a (CCL3). These effects were consistent using BMMC of different genetic backgrounds and peritoneal mast cells. While the RNR inhibitor hydroxyurea had little or no effect on IgE-mediated function, high concentrations of the antioxidant N-acetylcysteine mimicked Didox-mediated suppression. Furthermore, Didox increased expression of the antioxidant genes superoxide dismutase and catalase, and suppressed DCFH-DA fluorescence, indicating reduced reactive oxygen species production. Didox effects were not due to changes in FcεRI expression or cell viability, suggesting it inhibits signaling required for inflammatory cytokine production. In support of this, we found that Didox reduced FcεRI-mediated AP-1 and NFκB transcriptional activity. Finally, Didox suppressed mast cell-dependent, IgE-mediated passive systemic anaphylaxis in vivo. These data demonstrate the potential use for Didox asa means of antagonizing mast cell responses in allergic disease.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Ácidos Hidroxâmicos/farmacologia , Hipersensibilidade/tratamento farmacológico , Imunoglobulina E/imunologia , Mastócitos/imunologia , NF-kappa B/genética , Fator de Transcrição AP-1/genética , Acetilcisteína/farmacologia , Animais , Células da Medula Óssea/imunologia , Catalase/biossíntese , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL3/biossíntese , Hipersensibilidade/imunologia , Interleucina-13/biossíntese , Interleucina-6/biossíntese , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/biossíntese , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Axonal pathology is a key contributor to long-term disability in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS), but the mechanisms that underlie axonal pathology in MS remain elusive. Evidence suggests that axonal pathology is a direct consequence of demyelination, as we and others have shown that the node of Ranvier disassembles following loss of myelin. In contrast to the node of Ranvier, we now show that the axon initial segment (AIS), the axonal domain responsible for action potential initiation, remains intact following cuprizone-induced cortical demyelination. Instead, we find that the AIS is disrupted in the neocortex of mice that develop experimental autoimmune encephalomyelitis (EAE) independent of local demyelination. EAE-induced mice demonstrate profound compromise of AIS integrity with a progressive disruption that corresponds to EAE clinical disease severity and duration, in addition to cortical microglial reactivity. Furthermore, treatment with the drug didox results in attenuation of AIS pathology concomitantly with microglial reversion to a less reactive state. Together, our findings suggest that inflammation, but not demyelination, disrupts AIS integrity and that therapeutic intervention may protect and reverse this pathology. GLIA 2016;64:1190-1209.
Assuntos
Segmento Inicial do Axônio/fisiologia , Axônios/patologia , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica/fisiologia , Microglia/metabolismo , Animais , Animais Geneticamente Modificados , Doenças Autoimunes do Sistema Nervoso/induzido quimicamente , Doenças Autoimunes do Sistema Nervoso/tratamento farmacológico , Doenças Autoimunes do Sistema Nervoso/patologia , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Morte Celular/fisiologia , Células Cultivadas , Cuprizona/toxicidade , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ácidos Hidroxâmicos/uso terapêutico , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Inibidores da Monoaminoxidase/toxicidade , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A hallmark of cancer is the dysregulation of the cell cycle. The CDK4/6 inhibitor palbociclib is approved for treating advanced estrogen-receptor-positive breast cancer, but its success is limited by the development of acquired resistance owing to long-term therapy despite promising clinical outcomes. This situation necessitates the development of potential combination strategies. Here, we report that didox, an inhibitor of ribonucleotide reductase in combination with palbociclib, can overcome palbociclib resistance in ER-positive and ER-negative breast cancers. This study shows didox downregulates an element of the cell cycle checkpoint, cyclin D1, accompanied by a reduction in NF-κB activity in vitro and tumor growth inhibition of palbociclib-resistant ER positive breast cancer tumor growth in vivo. Furthermore, didox induces cell cycle arrest at G1 as well as reduces ROS generated by on-target effects of palbociclib on the cell cycle. Our current study also reports that the CCND1 and RRM2 upregulation associated with palbociclib-resistant breast cancers decreases upon ribonucleotide reductase inhibition. Our data present a novel and promising biomarker-driven combination therapeutic approach for the treatment of ER-positive and ER-negative breast cancers that involves the inhibition of the CDK4/6-cyclinD1/pRb cell cycle axis that merits further clinical investigation in human models.
RESUMO
Doxorubicin and other anthracycline derivatives are frequently used as part of the adjuvant chemotherapy regimen for triple-negative breast cancer (TNBC). Although effective, doxorubicin is known for its off-target and toxic side effect profile, particularly with respect to the myocardium, often resulting in left ventricular (LV) dysfunction and congestive heart failure when used at cumulative doses exceeding 400 mg/m2 Previously, we have observed that the ribonucleotide reductase subunit M2 (RRM2) is significantly overexpressed in estrogen receptor (ER)-negative cells as compared with ER-positive breast cancer cells. Here, we inhibited RRM2 in ER-negative breast cancer cells as a target for therapy in this difficult-to-treat population. We observed that through the use of didox, a ribonucleotide reductase inhibitor, the reduction in RRM2 was accompanied by reduced NF-κB activity in vitro When didox was used in combination with doxorubicin, we observed significant downregulation of NF-κB proteins accompanied by reduced TNBC cell proliferation. As well, we observed that protein levels of mutant p53 were significantly reduced by didox or combination therapy in vitro Xenograft studies showed that combination therapy was found to be synergistic in vivo, resulting in a significantly reduced tumor volume as compared with doxorubicin monotherapy. In addition, the use of didox was also found to ameliorate the toxic myocardial effects of doxorubicin in vivo as measured by heart mass, LV diameter, and serum troponin T levels. The data present a novel and promising approach for the treatment of TNBC that merits further clinical evaluation in humans.
Assuntos
Terapia de Alvo Molecular/métodos , NF-kappa B/metabolismo , Ribonucleosídeo Difosfato Redutase/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Nus , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Gallic acid (GA) is a naturally occurring polyhydroxyphenolic compound and an excellent free radical scavenger. In this study, we examined its cytotoxic and biochemical effects on the human HL-60 promyelocytic leukemia cell line. GA caused a significant imbalance of deoxynucleosidetriphosphate (dNTP) pool sizes, indicating ribonucleotide reductase inhibition. Moreover, GA induced dose-dependent apoptosis in HL-60 cells (80microM GA led to the induction of apoptosis in 39% of cells) and attenuated progression from G0/G1 to the S phase of the cell cycle (60microM GA doubled the number of cells in G0/G1 phase from 22 to 44% when compared to untreated controls). We further determined IC(50) values of 3.5 and 4.4nM for the inhibition of cyclooxygenases I and II, respectively. When cells were simultaneously treated with GA and trimidox, another inhibitor of RR, highly synergistic growth inhibitory effects could be observed. Taken together, we identified novel biochemical effects of GA which could be the basis for further preclinical and in vivo studies.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Ácido Gálico/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Ribonucleotídeo Redutases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Benzamidinas/química , Benzamidinas/farmacologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citidina Trifosfato/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ácido Gálico/química , Guanosina Trifosfato/metabolismo , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/enzimologia , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Estrutura Molecular , Ribonucleotídeo Redutases/metabolismo , Nucleotídeos de Timina/metabolismoRESUMO
We describe the use of the new ribonucleotide reductase inhibitor, trimidox (TDX), in combination chemotherapy under in vitro and in vivo conditions with cisplatin and cyclophosphamide. In vitro, the combination of TDX and cisplatin was tested in L1210 cells. The combination caused concentration dependent antagonistic or additive effects. However, the combination of TDX-cisplatin-cyclophosphamide in vivo is highly synergistic in both, the L1210 and P388D1 leukemia mouse models. Both combinations, TDX with cisplatin or TDX with cyclophosphamide were also synergistic in the L1210 and P388D1 leukemia animal models.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzamidinas/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Leucemia L1210/tratamento farmacológico , Leucemia P388/tratamento farmacológico , Ribonucleotídeo Redutases/antagonistas & inibidores , Animais , Cisplatino/administração & dosagem , Ciclofosfamida/administração & dosagem , Sinergismo Farmacológico , Masculino , Camundongos , Camundongos Endogâmicos DBARESUMO
The most common and widely transplanted tissue world wide is blood, which in 2000 resulted in the transfusion of 12.5 million units of blood in the US alone [Goodnough LT, Shander A, Brecher ME. Transfusion medicine: looking to the future. Lancet 2003;361:161-9]. The current use of donated blood products is relatively safe; however, there are inherent problems with allogeneic blood transfusions. The wide spread use of blood in procedures results in problems involving inadequate supply exacerbated in times of war and disasters and by the limited storage life of blood donations (30-42 days). Blood contamination due to patient pre-disposition, poor collection, sterilization, or storage is the second most common cause of death from transfusion in the US [Hillyer CD, Josephson CD, Blajchman MA, Vostal JG, Epstein JS, Goodman JL. Bacterial contamination of blood components: risks, strategies, and regulation: joint ASH and AABB educational session in transfusion medicine. Hematology (Am Soc Hematol Educ Program) 2003:575-89]. Blood is a complex tissue involved in a plethora of homeostatic roles, including immunity, wound healing and the transport of nourishment, electrolytes, hormones, vitamins, heat, oxygen and the removal of metabolic waste products. However, by far the principle role of blood transfusions is the replacement of red cell volume and the maintenance of oxygen levels within the circulation. Creation of investigational new drugs (INDs) which would function as oxygen carriers and prolong shelf life is now a very active arena of scientific research. Several such IND products are now in clinical trials. This article gives an easy to follow concise evaluation of major areas of focus and current testing for each type of blood substitution molecule.
Assuntos
Substitutos Sanguíneos/química , Transfusão de Sangue/métodos , Oxigênio/metabolismo , Infecções Bacterianas , Sangue/microbiologia , Bancos de Sangue , Transfusão de Componentes Sanguíneos , Preservação de Sangue/métodos , Coleta de Amostras Sanguíneas , Substitutos Sanguíneos/farmacologia , Desenho de Fármacos , Hemoglobinas , Humanos , Modelos QuímicosRESUMO
The ribonucleotide reductase inhibitor hydroxyurea (HU) has demonstrated some benefit as a component of drug cocktails for the treatment of HIV-1 infection. However, HU is notoriously myelosuppressive and often administered only as salvage therapy to patients with late-stage disease, potentially exacerbating the bone marrow toxicity of HU. In this report we have compared the antiviral effects of HU and two novel RR inhibitors trimidox (3,4,5-trihydroxybenzamidoxime) and didox (3,4-dihydroxybenzohydroxamic acid) in combination with didanosine (2,3-didoxyinosine; ddI) in the LPBM5 MuLV retrovirus model (murine AIDS). We also evaluated the effects of these drug combinations on the hematopoietic tissues of LPBM5 MuLV-infected animals. The combination of RR inhibitors and ddI was extremely effective (DX>TX>HU) in inhibiting development of retrovirus-induced disease (splenomegaly, hypergammaglobulinemia, activated B-splenocytes and loss of splenic architecture). In addition, relative levels of proviral DNA were significantly lower in combination drug-treated animals compared to infected controls. Evaluation of femur cellularity, numbers of marrow-derived myeloid progenitor cells (CFU-GM and BFU-E) and peripheral blood indices revealed that TX and DX in combination with ddI were well-tolerated. However, treatment with HU and ddI induced moderate myelosuppression. These data demonstrate that RR inhibitors in combination with ddI provide significant protection against retroviral disease in murine AIDS. Moreover, the novel RR inhibitors TX and DX appear to be more effective and less myelosuppressive than HU when administered with ddI in this model.
Assuntos
Antivirais/uso terapêutico , Benzamidinas/uso terapêutico , Didanosina/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Hidroxiureia/uso terapêutico , Síndrome de Imunodeficiência Adquirida Murina/tratamento farmacológico , Inibidores da Transcriptase Reversa/uso terapêutico , Animais , Antivirais/administração & dosagem , Linfócitos B/imunologia , Benzamidinas/administração & dosagem , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/uso terapêutico , Feminino , Ácidos Hidroxâmicos/administração & dosagem , Hidroxiureia/administração & dosagem , Vírus da Leucemia Murina/efeitos dos fármacos , Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/virologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Síndrome de Imunodeficiência Adquirida Murina/virologia , Infecções por Retroviridae/tratamento farmacológico , Infecções por Retroviridae/virologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Resultado do Tratamento , Infecções Tumorais por Vírus/tratamento farmacológico , Infecções Tumorais por Vírus/virologiaRESUMO
Tamoxifen is widely used as an adjuvant therapy for patients with estrogen receptor (ERα)-positive tumors. However, the clinical benefit is often limited because of the emergence of drug resistance. In this study, overexpression of ribonucleotide reductase M2 (RRM2) in MCF-7 breast cancer cells resulted in a reduction in the effectiveness of tamoxifen, through downregulation of ERα66 and upregulation of the 36-kDa variant of ER (ERα36). We identified that NF-κB, HIF1α, and MAPK/JNK are the major pathways that are affected by RRM2 overexpression and result in increased NF-κB activity and increased protein levels of EGFR, HER2, IKKs, Bcl-2, RelB, and p50. RRM2-overexpressing cells also exhibited higher migratory and invasive properties. Through time-lapse microscopy and protein profiling studies of tamoxifen-treated MCF-7 and T-47D cells, we have identified that RRM2, along with other key proteins, is altered during the emergence of acquired tamoxifen resistance. Inhibition of RRM2 using siRRM2 or the ribonucleotide reductase (RR) inhibitor didox not only eradicated and effectively prevented the emergence of tamoxifen-resistant populations but also led to the reversal of many of the proteins altered during the process of acquired tamoxifen resistance. Because didox also appears to be a potent inhibitor of NF-κB activation, combining didox with tamoxifen treatment cooperatively reverses ER-α alterations and inhibits NF-κB activation. Finally, inhibition of RRM2 by didox reversed tamoxifen-resistant in vivo tumor growth and decreased in vitro migratory and invasive properties, revealing a beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , NF-kappa B/antagonistas & inibidores , Ribonucleosídeo Difosfato Redutase/antagonistas & inibidores , Tamoxifeno/farmacologia , Animais , Antineoplásicos/farmacologia , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Humanos , Estimativa de Kaplan-Meier , Células MCF-7 , Camundongos Nus , NF-kappa B/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Didox (3,4-dihydroxy-benzohydroxamic acid), is a synthetic ribonucleotide reductase (RR) inhibitor derived from polyhydroxy-substituted benzohydroxamic acid, and originally developed as an anti-cancer agent. Some studies indicate that didox may have anti-oxidative stress-like properties, while other studies hint that didox may have anti-inflammatory properties. Using nitric oxide production in response to LPS treatment as a sensitive screening assay for anti-inflammatory compounds, we show that didox is very potent at levels as low as 6.25 µM, with maximal inhibition at 100 µM. A qRT-PCR array was then employed to screen didox for other potential anti-inflammatory and anti-oxidative stress-related properties. Didox was very potent in suppressing the expression of these arrayed mRNA in response to LPS, and in some cases didox alone suppressed expression. Using qRT-PCR as a follow up to the array, we demonstrated that didox suppresses LPS-induced mRNA levels of iNOS, IL-6, IL-1, TNF-α, NF-κß (p65), and p38-α, after 24h of treatment. Treatment with didox also suppresses the secretion of nitric oxide, IL-6, and IL-10. Furthermore, oxidative stress, as quantified by intracellular ROS levels in response to macrophage activators LPS and phorbol ester (PMA), and the glutathione depleting agent BSO, is reduced by treatment with didox. Moreover, we demonstrate that nuclear translocation of NF-κß (p65) in response to LPS is inhibited by didox. These findings were supported by qRT-PCR for oxidative stress genes SOD1 and catalase. Overall, this study supports the conclusion that didox may have a future role in managing acute and chronic inflammatory diseases and oxidative stress due to high production of ROS.
Assuntos
Anti-Inflamatórios/farmacologia , Sequestradores de Radicais Livres/farmacologia , Ácidos Hidroxâmicos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Estresse Oxidativo/efeitos dos fármacos , Receptor 4 Toll-Like/imunologia , Animais , Catalase/genética , Linhagem Celular , Ciclo-Oxigenase 2/genética , Regulação para Baixo/efeitos dos fármacos , Interleucina-6/genética , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/genética , Espécies Reativas de Oxigênio/imunologia , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Fator de Necrose Tumoral alfa/genética , Regulação para Cima/efeitos dos fármacosRESUMO
In this study, we investigated the influence of Bcl-2 overexpression on the radiosensitizing potential of Didox (DX; 3,4-Dihydroxybenzohydroxamic acid), a novel ribonucleotide reductase inhibitor, in p53-null prostate cancer cell line PC-3. The PC-3 cells were transfected with vector alone or ectopically overexpressed with CMV-Bcl-2 construct. The effect of radiation (IR) or DX alone and in combination (pre and post IR exposure of DX) on cell survival was determined by colony-forming assay. The impact of these two treatments on the cell cycle was determined by flow cytometry. To further understand the molecular mechanism of DX-mediated radiosensitization, induction of pro-survival and pro-apoptotic factors were determined by Western blot and gel-shift assays respectively. When compared to PC-3/Bcl-2 cells (SF(2)=0.84; D(0)=437cGy), the PC-3/vector cells (SF(2)=0.4; D(0)=235cGy) were significantly sensitive to ionizing radiation (p<0.001). Exposure of DX at 5 microM concentration prior or post to radiation in both PC-3/vector and PC-3/Bcl-2 transfectants caused an increase in radiation enhancement ratios. A significant reduction in G(2)M phase was observed in cells exposed to DX post IR when compared to cells exposed to IR alone. Exposure to DX after radiation in PC-3/vector significantly abrogated radiation-induced Bcl-2 upregulation, with a concomitant induction of bax protein. In PC-3/Bcl-2 transfectants, DX exposure after IR caused an induction of bax protein. Gel shift assays indicated that in PC-3/vector cells when exposed to IR caused an induction of NFkappa-B activity however, DX down regulated the NFkappa-B activity. Radiation-induced NFkappa-B activity was abrogated in pre and post DX exposure in combination with IR. These findings indicate that DX mediates a potent radiosensitizing effect in p53 null prostate cancer cells by overcoming radiation induced NFkappa-B activity and Bcl-2 expression.
Assuntos
Antineoplásicos/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Neoplasias da Próstata/radioterapia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Tolerância a Radiação , Apoptose/efeitos da radiação , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Vetores Genéticos , Humanos , Masculino , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , NF-kappa B/efeitos dos fármacos , NF-kappa B/efeitos da radiação , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/efeitos da radiação , Proteínas Proto-Oncogênicas c-bcl-2/efeitos da radiação , Radiação Ionizante , Transfecção , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Mechanisms of acquired resistance to three purine analogues, 2-chloro-2'-deoxyadenosine (cladribine, CdA), 9-beta-D-arabinofuranosyl-2-fluoroadenine (fludarabine, Fara-A), and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (clofarabine, CAFdA) were investigated in a human T-lymphoblastic leukemia cell line (CCRF-CEM). These analogues are pro-drugs and must be activated by deoxycytidine kinase (dCK). The CdA and CAFdA resistant cell lines exhibited increased resistance to the other nucleoside analogues activated by dCK. This was also the case for the Fara-A resistant cells, except that they were sensitive to CAFdA and guanosine analogues. The CdA and CAFdA resistant cells displayed a deficiency in dCK activity (to <5%) while the Fara-A resistant cells showed only a minor reduction of dCK activity (20% reduction). The activity of high K(m) 5'-nucleotidase (5'-NT) (cN-II) using IMP as substrate, was 2-fold elevated in the resistant cell lines. The amount of the small subunit R2 of ribonucleotide reductase (RR) was higher in the Fara-A resistant cells, which translated into a higher RR activity, while CdA and CAFdA cells had decreased activity compared to the parental cells. Expression of the recently identified RR subunit, p53R2 full-size protein, in CAFdA cells was low compared to parental cells, but a protein of lower molecular weight was detected in CdA and CAFdA cells. Co-incubation of Fara-A with the RR inhibitor 3,4-dihydroxybenzohydroxamic acid (didox) enhanced cytotoxicity in the Fara-A resistant cells by a factors of 20. Exposure of the cells to the nucleoside analogues studied here also caused structural and numerical instability of the chromosomes; the most profound changes were recorded for CAFdA cells, as demonstrated by SKY and CGH analysis. We conclude that down-regulation of dCK in cells resistant to CdA and CAFdA and increased activity of RR in cells resistant to Fara-A contribute to resistance.
Assuntos
Antineoplásicos/farmacologia , Arabinonucleosídeos/farmacologia , Cladribina/farmacologia , Desoxicitidina Quinase/metabolismo , Ribonucleotídeo Redutases/metabolismo , Vidarabina/análogos & derivados , Vidarabina/farmacologia , Nucleotídeos de Adenina , Linhagem Celular , Clofarabina , Citogenética , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Humanos , Leucemia/patologia , Fenótipo , Fosforilação , Ribonucleotídeo Redutases/antagonistas & inibidoresRESUMO
Ribonucleotide reductase is the rate-limiting enzyme for the de novo synthesis of deoxynucleoside triphosphates and therefore represents a good target for cancer chemotherapy. Trimidox (3,4,5-trihydroxybenzamidoxime) was identified as a potent inhibitor of this enzyme and was shown to significantly decrease deoxycytidine triphosphate (dCTP) pools in HL-60 leukemia cells. We now investigated the ability of trimidox to increase the antitumor effect of 1-beta-D-arabinofuranosyl cytosine (Ara-C). Ara-C is phosphorylated by deoxycytidine kinase, which is subject to negative allosteric regulation by dCTP. Therefore, a decrease of dCTP may cause increased Ara-C phosphorylation and enhanced incorporation of Ara-C into DNA. Ara-C incorporation indeed increased 1.51- and 1.89-fold after preincubation with 75 and 100 microM trimidox, respectively. This was due to the significantly increased 1-beta-D-arabinofuranosyl cytosine triphosphate pools (1.9- and 2.5-fold) after preincubation with trimidox. We also investigated the effects of a combination of trimidox and Ara-C on the colony formation of HL-60 cells. A synergistic potentiation of the effect of Ara-C could be observed, when trimidox was added. Trimidox, which decreases intracellular deoxynucleoside triphosphate concentrations thus leading to apoptosis, enhanced the induction of apoptosis caused by Ara-C. We conclude, that trimidox is capable of synergistically enhancing the effects of Ara-C and therefore this drug combination might be further tested in animals.
Assuntos
Benzamidinas/farmacologia , Citarabina/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Apoptose , Arabinofuranosilcitosina Trifosfato/metabolismo , Divisão Celular/efeitos dos fármacos , Citarabina/metabolismo , DNA/efeitos dos fármacos , DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Células HL-60 , Humanos , Leucemia Promielocítica Aguda , Inibidores da Síntese de Ácido Nucleico/metabolismoRESUMO
Inhibition of ribonucleotide reductase (RR) has gained attention as a potential strategy for HIV-1 therapy through the success of hydroxyurea (HU) to potentiate the activity of the nucleoside reverse transcriptase inhibitor (NRTI) didanosine (ddI) in clinical trials. However, the use of HU has been limited by its development of hematopoietic toxicity. In this study, the novel RR inhibitors didox (DX; 3,4-dihydroxybenzohydroxamic acid), and trimidox (TX; 3,4,5-trihydroxybenzamidoxime) were evaluated along with HU for anti-retroviral efficacy in LPBM5-induced retro-viral disease (MAIDS) both as monotherapeutic regimens and in combination with the guanine containing NRTI abacavir (ABC). Anti-retroviral drug efficacy was determined by measuring inhibition of splenomegaly, hypergammaglobulinemia, and splenic levels of proviral DNA. In this study, all RRIs tested showed the ability to improve the efficacy of ABC in the MAIDS model by reducing splenomegaly, hypergammaglobulinemia, and splenic proviral DNA levels.
Assuntos
Antivirais/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Hidroxiureia/uso terapêutico , Síndrome de Imunodeficiência Adquirida Murina/tratamento farmacológico , Ribonucleotídeo Redutases/antagonistas & inibidores , Animais , Antivirais/farmacologia , Benzamidinas/uso terapêutico , Didesoxinucleosídeos/uso terapêutico , Modelos Animais de Doenças , Quimioterapia Combinada , Células-Tronco Hematopoéticas/efeitos dos fármacos , Ácidos Hidroxâmicos/uso terapêutico , Hidroxiureia/efeitos adversos , Hidroxiureia/química , Camundongos , Camundongos Endogâmicos C57BL , Síndrome de Imunodeficiência Adquirida Murina/sangue , Síndrome de Imunodeficiência Adquirida Murina/imunologia , Síndrome de Imunodeficiência Adquirida Murina/patologia , Baço/patologia , EsplenomegaliaRESUMO
Recently, the use of the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU) in combination with nucleoside analogs has gained attention as a potential strategy for anti-HIV-1 therapy. However, appeal for the long-term use of HU in HIV-1 infection may be limited by its propensity to induce hematopoietic toxicity. We report a comparison of the efficacy and bone marrow toxicity of HU (400 and 200 mg/kg/day) with the novel RR inhibitors and free radical-scavenging compounds didox (DX; 3,4-dihydroxybenzohydroxamic acid; 350 mg/kg/day) and trimidox (TX; 3,4,5-trihydroxybenzamidoxime; 175 mg/kg/day) in the murine AIDS (LPBM5 MuLV) model of retrovirus infection. Infected mice received daily drug treatment for 8 weeks. Efficacy was determined by measuring drug effects on retroviral-induced disease progression (i.e. development of splenomegaly and hypergammaglobulinemia) and by evaluating splenic levels of proviral DNA. Bone marrow toxicity was evaluated by measuring peripheral blood indices (WBC, hematocrit and reticulocyte counts), femoral cellularity and by determining the numbers of hematopoietic progenitor cells (CFU-GM, BFU-E) per femur and spleen. Compared to infected controls receiving no drug treatment, disease progression was significantly suppressed by TX, DX and HU. However, HU was associated with mortality and induced significant hematopoietic toxicity in a time- and dose-dependent manner. Conversely, TX and DX effectively inhibited retrovirus-induced disease but did not induce hematopoietic toxicity. These results suggest that due to their reduced hematopoietic toxicity and ability to inhibit disease progression in murine AIDS, TX and DX may offer effective alternatives to HU therapy in HIV-1 infection.
Assuntos
Benzamidinas/efeitos adversos , Células da Medula Óssea/efeitos dos fármacos , Sequestradores de Radicais Livres/efeitos adversos , Ácidos Hidroxâmicos/efeitos adversos , Hidroxiureia/efeitos adversos , Vírus da Leucemia Murina/efeitos dos fármacos , Leucemia Experimental/patologia , Síndrome de Imunodeficiência Adquirida Murina/patologia , Infecções por Retroviridae/patologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Infecções Tumorais por Vírus/patologia , Animais , Benzamidinas/química , Benzamidinas/uso terapêutico , DNA Viral , Feminino , Fêmur/citologia , Fêmur/efeitos dos fármacos , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/uso terapêutico , Células-Tronco Hematopoéticas/efeitos dos fármacos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/uso terapêutico , Hidroxiureia/química , Hidroxiureia/uso terapêutico , Hipergamaglobulinemia/tratamento farmacológico , Vírus da Leucemia Murina/genética , Leucemia Experimental/sangue , Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Síndrome de Imunodeficiência Adquirida Murina/sangue , Síndrome de Imunodeficiência Adquirida Murina/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida Murina/imunologia , Provírus/genética , Infecções por Retroviridae/sangue , Infecções por Retroviridae/tratamento farmacológico , Infecções por Retroviridae/imunologia , Baço/patologia , Esplenomegalia , Infecções Tumorais por Vírus/sangue , Infecções Tumorais por Vírus/tratamento farmacológico , Infecções Tumorais por Vírus/imunologiaRESUMO
PURPOSE: Ribonucleotide reductase (RR) is the rate-limiting enzyme of de novo DNA synthesis and has been shown to be upregulated linked with proliferation and malignant transformation. It was therefore identified as an excellent target for antitumor therapy. In the present study we investigated the biochemical and cytotoxic effects of didox, an inhibitor of RR, as a single agent and in combination with BCNU, an alkylating anticancer drug, in 9L rat gliosarcoma cells and DAOY human medulloblastoma cells. METHODS: The effect of didox on the intracellular concentrations of deoxynucleosidetriphosphates (dNTPs) was studied in 9L cells. Pool sizes were determined by HPLC. In addition, the cytotoxic effects of didox and BCNU as single drugs and in equimolar combination were tested in 9L and in DAOY cells. Combination effects were determined according to the equation of Chou and Talalay. The expression of DNA repair-related genes was determined after exposure of 9L cells to BCNU, didox and a combination of the two compounds, using a cDNA array. RESULTS: Incubation of 9L cells with 30 microM didox for 24 h significantly decreased the intracellular concentrations of the DNA precursors dCTP (61% of control) and dGTP (17% of control), and significantly increased the concentration of dATP (155% of control). This dNTP imbalance compromised DNA synthesis and repair and might therefore have been, at least in part, responsible for the highly synergistic cytotoxic effects seen when BCNU was used simultaneously with didox in 9L and in DAOY cells. With almost all combinations tested, highly synergistic effects were seen, as indicated by combination indices of <1 according to the equation of Chou and Talalay. In 9L cells, BCNU upregulated the expression of DNA repair-associated genes, whereas coincubation of the cells with didox reduced overexpression of some of these repair-related genes. CONCLUSION: A combination of BCNU and didox was proven to act in a synergistic manner in two cell lines, 9L rat gliosarcoma and DAOY human medulloblastoma cells. Further in vivo tests using these two compounds systemically and/or locally at the tumor site might be warranted.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/toxicidade , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Neoplasias Encefálicas/patologia , Carmustina/farmacologia , Carmustina/toxicidade , Neoplasias Cerebelares/patologia , Gliossarcoma/patologia , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/toxicidade , Meduloblastoma/patologia , Animais , Interações Medicamentosas , Humanos , Ratos , Células Tumorais CultivadasRESUMO
Amidox, a new polyhydroxy-substituted benzoic acid derivative, is a potent inhibitor of the enzyme ribonucleotide reductase (RR), which catalyses the de novo synthesis of DNA. RR is considered to be an excellent target for anti cancer chemotherapy. We investigated the biochemical and antineoplastic effects of amidox as a single agent and in combination with Ara-C in human HL-60 promyelocytic leukemia cells. Amidox inhibited the growth of HL-60 cells in a growth inhibition assay with an IC50 of 25 microM. In a soft agar colony forming assay, amidox yielded a 50% inhibition of colony formation at 13 microM. We also investigated the effects of amidox treatment on the formation of deoxynucleosidetriphosphates. Amidox (50 and 75 microM for 24 hours) could significantly decrease intracellular concentrations of dCTP, dATP and dGTP pools, whereas dTTP levels increased. We then tested the combination effects of amidox with Ara-C; this combination yielded additive cytotoxic effects both in growth inhibition and in soft agar colony formation assays. This effect was due to the increased formation of Ara-CTP, the active metabolite of Ara-C, after preincubation with amidox. Preincubation of HL-60 cells with 75 and 100 microM amidox for 24 hours caused an increase in the intracellular Ara-CTP concentrations by 576% and 1143%, respectively. Therefore amidox might offer an additional option for the treatment of leukemia and thus be further investigated in in vivo studies as a single agent and in combination with Ara-C.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Oximas/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Arabinofuranosilcitosina Trifosfato/metabolismo , Divisão Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Desoxirribonucleotídeos/metabolismo , Inibidores do Crescimento/metabolismo , Células HL-60 , HumanosRESUMO
Acute Myeloid Leukemia (AML) is an aggressive malignancy which leads to marrow failure, and ultimately death. There is a desperate need for new therapeutics for these patients. Ribonucleotide reductase (RR) is the rate limiting enzyme in DNA synthesis. Didox (3,4-Dihydroxybenzohydroxamic acid) is a novel RR inhibitor noted to be more potent than hydroxyurea. In this report we detail the activity and toxicity of Didox in preclinical models of AML. RR was present in all AML cell lines and primary patient samples tested. Didox was active against all human and murine AML lines tested with IC50 values in the low micromolar range (mean IC50 37 µM [range 25.89-52.70 µM]). It was active against primary patient samples at concentrations that did not affect normal hematopoietic stem cells (HSCs). Didox exposure resulted in DNA damage and p53 induction culminating in apoptosis. In syngeneic, therapy-resistant AML models, single agent Didox treatment resulted in a significant reduction in leukemia burden and a survival benefit. Didox was well tolerated, as marrow from treated animals was morphologically indistinguishable from controls. Didox exposure at levels that impaired leukemia growth did not inhibit normal HSC engraftment. In summary, Didox was well tolerated and effective against preclinical models of AML.