RESUMO
The incidences of chronic inflammatory disorders have increased considerably over the past three decades. Recent shifts in dietary consumption may have contributed importantly to this surge, but how dietary consumption modulates inflammatory disease is poorly defined. Pstpip2(cmo) mice, which express a homozygous Leu98Pro missense mutation in the Pombe Cdc15 homology family protein PSTPIP2 (proline-serine-threonine phosphatase interacting protein 2), spontaneously develop osteomyelitis that resembles chronic recurrent multifocal osteomyelitis in humans. Recent reports demonstrated a crucial role for interleukin-1ß (IL-1ß) in osteomyelitis, but deletion of the inflammasome components caspase-1 and NLRP3 failed to rescue Pstpip2(cmo) mice from inflammatory bone disease. Thus, the upstream mechanisms controlling IL-1ß production in Pstpip2(cmo) mice remain to be identified. In addition, the environmental factors driving IL-1ß-dependent inflammatory bone erosion are unknown. Here we show that the intestinal microbiota of diseased Pstpip2(cmo) mice was characterized by an outgrowth of Prevotella. Notably, Pstpip2(cmo) mice that were fed a diet rich in fat and cholesterol maintained a normal body weight, but were markedly protected against inflammatory bone disease and bone erosion. Diet-induced protection against osteomyelitis was accompanied by marked reductions in intestinal Prevotella levels and significantly reduced pro-IL-1ß expression in distant neutrophils. Furthermore, pro-IL-1ß expression was also decreased in Pstpip2(cmo) mice treated with antibiotics, and in wild-type mice that were kept under germ-free conditions. We further demonstrate that combined deletion of caspases 1 and 8 was required for protection against IL-1ß-dependent inflammatory bone disease, whereas the deletion of either caspase alone or of elastase or neutrophil proteinase 3 failed to prevent inflammatory disease. Collectively, this work reveals diet-associated changes in the intestinal microbiome as a crucial factor regulating inflammasome- and caspase-8-mediated maturation of IL-1ß and osteomyelitis in Pstpip2(cmo) mice.
Assuntos
Dieta Hiperlipídica , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Microbiota/efeitos dos fármacos , Osteomielite/dietoterapia , Osteomielite/patologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Peso Corporal/efeitos dos fármacos , Caspase 1/deficiência , Caspase 1/genética , Caspase 8/genética , Caspase 8/metabolismo , Colesterol/farmacologia , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Feminino , Inflamassomos/metabolismo , Inflamação/dietoterapia , Inflamação/patologia , Interleucina-1beta/sangue , Interleucina-1beta/metabolismo , Intestinos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mieloblastina/deficiência , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Elastase Pancreática/deficiência , Prevotella/crescimento & desenvolvimento , Prevotella/isolamento & purificaçãoRESUMO
The introduction of cultured p185(BCR-ABL)-expressing (p185+) Arf (-/-) pre-B cells into healthy syngeneic mice induces aggressive acute lymphoblastic leukemia (ALL) that genetically and phenotypically mimics the human disease. We adapted this high-throughput Philadelphia chromosome-positive (Ph(+)) ALL animal model for in vivo luminescent imaging to investigate disease progression, targeted therapeutic response, and ALL relapse in living mice. Mice bearing high leukemic burdens (simulating human Ph(+) ALL at diagnosis) entered remission on maximally intensive, twice-daily dasatinib therapy, but invariably relapsed with disseminated and/or central nervous system disease. Although relapse was frequently accompanied by the eventual appearance of leukemic clones harboring BCR-ABL kinase domain (KD) mutations that confer drug resistance, their clonal emergence required prolonged dasatinib exposure. KD P-loop mutations predominated in mice receiving less intensive therapy, whereas high-dose treatment selected for T315I "gatekeeper" mutations resistant to all 3 Food and Drug Administration-approved BCR-ABL kinase inhibitors. The addition of dexamethasone and/or L-asparaginase to reduced-intensity dasatinib therapy improved long-term survival of the majority of mice that received all 3 drugs. Although non-tumor-cell-autonomous mechanisms can prevent full eradication of dasatinib-refractory ALL in this clinically relevant model, the emergence of resistance to BCR-ABL kinase inhibitors can be effectively circumvented by the addition of "conventional" chemotherapeutic agents with alternate antileukemic mechanisms of action.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Mutagênese/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pirimidinas/uso terapêutico , Tiazóis/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Inibidor p16 de Quinase Dependente de Ciclina/genética , Dasatinibe , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação de Sentido Incorreto/efeitos dos fármacos , Cromossomo Filadélfia/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Transplante IsogênicoRESUMO
Poly(ADP-ribose) polymerase (PARP)-1 (EC 2.4.2.30) is a nuclear enzyme that promotes the base excision repair of DNA breaks. Inhibition of PARP-1 enhances the efficacy of DNA alkylating agents, topoisomerase I poisons, and ionizing radiation. Our aim was to identify a PARP inhibitor for clinical trial from a panel of 42 potent PARP inhibitors (K(i), 1.4-15.1 nmol/L) based on the quinazolinone, benzimidazole, tricyclic benzimidazole, tricyclic indole, and tricyclic indole-1-one core structures. We evaluated chemosensitization of temozolomide and topotecan using LoVo and SW620 human colorectal cells; in vitro radiosensitization was measured using LoVo cells, and the enhancement of antitumor activity of temozolomide was evaluated in mice bearing SW620 xenografts. Excellent chemopotentiation and radiopotentiation were observed in vitro, with 17 of the compounds causing a greater temozolomide and topotecan sensitization than the benchmark inhibitor AG14361 and 10 compounds were more potent radiosensitizers than AG14361. In tumor-bearing mice, none of the compounds were toxic when given alone, and the antitumor activity of the PARP inhibitor-temozolomide combinations was unrelated to toxicity. Compounds that were more potent chemosensitizers in vivo than AG14361 were also more potent in vitro, validating in vitro assays as a prescreen. These studies have identified a compound, AG14447, as a PARP inhibitor with outstanding in vivo chemosensitization potency at tolerable doses, which is at least 10 times more potent than the initial lead, AG14361. The phosphate salt of AG14447 (AG014699), which has improved aqueous solubility, has been selected for clinical trial.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Neoplasias Colorretais/radioterapia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Raios gama , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Dose Máxima Tolerável , Camundongos , Camundongos Nus , Poli(ADP-Ribose) Polimerases/metabolismo , Relação Estrutura-Atividade , Temozolomida , Inibidores da Topoisomerase I , Topotecan/farmacologiaRESUMO
The nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) facilitates DNA repair, and is, therefore, an attractive target for anticancer chemo- and radio-potentiation. Novel benzimidazole-4-carboxamides (BZ1-6) and tricyclic lactam indoles (TI1-5) with PARP-1 K(i) values of <10 nM have been identified. Whole cell PARP-1 inhibition, intrinsic cell growth inhibition, and chemopotentiation of the cytotoxic agents temozolomide (TM) and topotecan (TP) were evaluated in LoVo human colon carcinoma cells. The acute toxicity of the inhibitors was investigated in PARP-1 null and wild-type mice. Tissue distribution and in vivo chemopotentiation activity was determined in nude mice bearing LoVo xenografts. At a nontoxic concentration (0.4 micro M) the PARP-1 inhibitors potentiated TM-induced growth inhibition 1.0-5.3-fold and TP-induced inhibition from 1.0-2.1-fold. Concentrations of the PARP-1 inhibitors that alone inhibited cell growth by 50% ranged from 8 to 94 micro M. Maximum potentiation of TM activity was achieved at nongrowth inhibitory concentrations (=1 micro M) of potent PARP-1 inhibitors BZ5 and TI4. Whole cell PARP-1 inhibition by BZ3, BZ5, BZ6, TI1, and TI4 was confirmed by attenuation of DNA damage-induced NAD(+) depletion. Selected inhibitors (TI1, TI3, and TI4), in contrast to the benchmark compound PD128763, caused only mild hypothermia in both PARP-1 null and wild-type mice. Excellent distribution of BZ5, TI1, and TI3 into tumor tissue was observed, and TI3 enhanced TM antitumor activity in vivo. These studies have identified potent nontoxic PARP-1 inhibitors with structural modifications that promote aqueous solubility, tolerability, and tissue distribution. These compounds are important leads in the development of clinically viable PARP-1 inhibitors.
Assuntos
Dacarbazina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Antineoplásicos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Divisão Celular , Linhagem Celular Tumoral , Reparo do DNA , Dacarbazina/uso terapêutico , Dacarbazina/toxicidade , Relação Dose-Resposta a Droga , Humanos , Indóis/metabolismo , Cinética , Modelos Químicos , Poli(ADP-Ribose) Polimerases/genética , Temozolomida , Temperatura , Fatores de Tempo , Distribuição Tecidual , Topotecan/uso terapêutico , Topotecan/toxicidadeRESUMO
BACKGROUND: Poly(ADP-ribose) polymerase-1 (PARP-1) facilitates the repair of DNA strand breaks. Inhibiting PARP-1 increases the cytotoxicity of DNA-damaging chemotherapy and radiation therapy in vitro. Because classical PARP-1 inhibitors have limited clinical utility, we investigated whether AG14361, a novel potent PARP-1 inhibitor (inhibition constant <5 nM), enhances the effects of chemotherapy and radiation therapy in human cancer cell cultures and xenografts. METHODS: The effect of AG14361 on the antitumor activity of the DNA alkylating agent temozolomide, topoisomerase I poisons topotecan or irinotecan, or x-irradiation or gamma-radiation was investigated in human cancer cell lines A549, LoVo, and SW620 by proliferation and survival assays and in xenografts in mice by tumor volume determination. The specificity of AG14361 for PARP-1 was investigated by microarray analysis and by antiproliferation and acute toxicity assays in PARP-1-/- and PARP-1+/+ cells and mice. After intraperitoneal administration, the concentration of AG14361 was determined in mouse plasma and tissues, and its effect on PARP-1 activity was determined in tumor homogenates. All statistical tests were two-sided. RESULTS: AG14361 at 0.4 micro M did not affect cancer cell gene expression or growth, but it did increase the antiproliferative activity of temozolomide (e.g., in LoVo cells by 5.5-fold, 95% confidence interval [CI] = 4.9-fold to 5.9-fold; P =.004) and topotecan (e.g., in LoVo cells by 1.6-fold, 95% CI = 1.3-fold to 1.9-fold; P =.002) and inhibited recovery from potentially lethal gamma-radiation damage in LoVo cells by 73% (95% CI = 48% to 98%). In vivo, nontoxic doses of AG14361 increased the delay of LoVo xenograft growth induced by irinotecan, x-irradiation, or temozolomide by two- to threefold. The combination of AG14361 and temozolomide caused complete regression of SW620 xenograft tumors. AG14361 was retained in xenografts in which PARP-1 activity was inhibited by more than 75% for at least 4 hours. CONCLUSION: AG14361 is, to our knowledge, the first high-potency PARP-1 inhibitor with the specificity and in vivo activity to enhance chemotherapy and radiation therapy of human cancer.