RESUMO
Mechanosensory hair cells of the inner ears and lateral line of vertebrates display heightened vulnerability to environmental insult, with damage resulting in hearing and balance disorders. An important example is hair cell loss due to exposure to toxic agents including therapeutic drugs such as the aminoglycoside antibiotics such as neomycin and gentamicin and antineoplastic agents. We describe two distinct cellular pathways for aminoglycoside-induced hair cell death in zebrafish lateral line hair cells. Neomycin exposure results in death from acute exposure with most cells dying within 1 hour of exposure. By contrast, exposure to gentamicin results primarily in delayed hair cell death, taking up to 24 hours for maximal effect. Washout experiments demonstrate that delayed death does not require continuous exposure, demonstrating two mechanisms where downstream responses differ in their timing. Acute damage is associated with mitochondrial calcium fluxes and can be alleviated by the mitochondrially-targeted antioxidant mitoTEMPO, while delayed death is independent of these factors. Conversely delayed death is associated with lysosomal accumulation and is reduced by altering endolysosomal function, while acute death is not sensitive to lysosomal manipulations. These experiments reveal the complexity of responses of hair cells to closely related compounds, suggesting that intervention focusing on early events rather than specific death pathways may be a successful therapeutic strategy.
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There are no approved therapeutics for the prevention of hearing loss and vestibular dysfunction from drugs like aminoglycoside antibiotics. While the mechanisms underlying aminoglycoside ototoxicity remain unresolved, there is considerable evidence that aminoglycosides enter inner ear mechanosensory hair cells through the mechanoelectrical transduction (MET) channel. Inhibition of MET-dependent uptake with small molecules or modified aminoglycosides is a promising otoprotective strategy. To better characterize mammalian ototoxicity and aid in the translation of emerging therapeutics, a biomarker is needed. In the present study we propose that neonatal mice systemically injected with the aminoglycosides G418 conjugated to Texas Red (G418-TR) can be used as a histologic biomarker to characterize in vivo aminoglycoside toxicity. We demonstrate that postnatal day 5 mice, like older mice with functional hearing, show uptake and retention of G418-TR in cochlear hair cells following systemic injection. When we compare G418-TR uptake in other tissues, we find that kidney proximal tubule cells show similar retention. Using ORC-13661, an investigational hearing protection drug, we demonstrate in vivo inhibition of aminoglycoside uptake in mammalian hair cells. This work establishes how systemically administered fluorescently labeled ototoxins in the neonatal mouse can reveal important details about ototoxic drugs and protective therapeutics.
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Hearing and balance deficits have been reported during and following treatment with the antimalarial drug chloroquine. However, experimental work examining the direct actions of chloroquine on mechanoreceptive hair cells in common experimental models is lacking. This study examines the effects of chloroquine on hair cells using two common experimental models: the zebrafish lateral line and neonatal mouse cochlear cultures. Zebrafish larvae were exposed to varying concentrations of chloroquine phosphate or hydroxychloroquine for 1 h or 24 h, and hair cells assessed by antibody staining. A significant, dose-dependent reduction in the number of surviving hair cells was seen across conditions for both exposure periods. Hydroxychloroquine showed similar toxicity. In mouse cochlear cultures, chloroquine damage was specific to outer hair cells in tissue from the cochlear basal turn, consistent with susceptibility to other ototoxic agents. These findings suggest a need for future studies employing hearing and balance monitoring during exposure to chloroquine and related compounds, particularly with interest in these compounds as therapeutics against viral infections including coronavirus.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Cloroquina/análogos & derivados , Células Ciliadas Auditivas/efeitos dos fármacos , Hidroxicloroquina/toxicidade , Sistema da Linha Lateral/efeitos dos fármacos , Animais , Antivirais/toxicidade , Células Cultivadas , Cloroquina/toxicidade , Células Ciliadas Auditivas/citologia , Larva/efeitos dos fármacos , Camundongos , Modelos Animais , Ototoxicidade , Peixe-ZebraRESUMO
On-target, off-tissue toxicity limits the systemic use of drugs that would otherwise reduce symptoms or reverse the damage of arthritic diseases, leaving millions of patients in pain and with limited physical mobility. We identified cystine-dense peptides (CDPs) that rapidly accumulate in cartilage of the knees, ankles, hips, shoulders, and intervertebral discs after systemic administration. These CDPs could be used to concentrate arthritis drugs in joints. A cartilage-accumulating peptide, CDP-11R, reached peak concentration in cartilage within 30 min after administration and remained detectable for more than 4 days. Structural analysis of the peptides by crystallography revealed that the distribution of positive charge may be a distinguishing feature of joint-accumulating CDPs. In addition, quantitative whole-body autoradiography showed that the disulfide-bonded tertiary structure is critical for cartilage accumulation and retention. CDP-11R distributed to joints while carrying a fluorophore imaging agent or one of two different steroid payloads, dexamethasone (dex) and triamcinolone acetonide (TAA). Of the two payloads, the dex conjugate did not advance because the free drug released into circulation was sufficient to cause on-target toxicity. In contrast, the CDP-11R-TAA conjugate alleviated joint inflammation in the rat collagen-induced model of rheumatoid arthritis while avoiding toxicities that occurred with nontargeted steroid treatment at the same molar dose. This conjugate shows promise for clinical development and establishes proof of concept for multijoint targeting of disease-modifying therapeutic payloads.
Assuntos
Artrite Experimental , Corticosteroides , Animais , Artrite Experimental/tratamento farmacológico , Cartilagem , Humanos , Peptídeos , Ratos , EsteroidesRESUMO
Genetic ablation as well as pharmacological inhibition of sirtuin 2 (SIRT2), an NAD+-dependent protein deacylase, have therapeutic effects in various cancers and neurodegenerative diseases. Previously, we described the discovery of a dual SIRT1/SIRT2 inhibitor called cambinol (IC50 56 and 59 µM, respectively), which showed cytotoxic activity against cancer cells in vitro and a marked anti-proliferative effect in a Burkitt lymphoma mouse xenograft model. A number of recent studies have shown a protective effect of SIRT1 and SIRT3 in neurodegenerative and metabolic diseases as well as in certain cancers prompting us to initiate a medicinal chemistry effort to develop cambinol-based SIRT2-specific inhibitors devoid of SIRT1 or SIRT3 modulating activity. Here we describe potent cambinol-based SIRT2 inhibitors, several of which show potency of ~600 nM with >300 to >800-fold selectivity over SIRT1 and 3, respectively. In vitro, these inhibitors are found to be toxic to lymphoma and epithelial cancer cell lines. In particular, compounds 55 (IC50 SIRT2 0.25 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) and 56 (IC50 SIRT2 0.78 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) showed apoptotic as well as strong anti-proliferative properties against B-cell lymphoma cells.
Assuntos
Antineoplásicos/uso terapêutico , Descoberta de Drogas , Linfoma de Células B/tratamento farmacológico , Sirtuína 2/antagonistas & inibidores , Acetilação , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Sirtuína 2/metabolismo , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismoRESUMO
The demographic profile of the biomedical workforce in the U.S. does not reflect the population at large, raising concerns that there will be insufficient trained researchers in the future, and the scope of research interests will not be sufficiently broad. To diversify and expand the pool of researchers trained to conduct research on cancer and cancer health disparities, a series of training activities to recruit and train primarily Hispanic students at both the undergraduate and graduate level were developed. The strengths of both a Hispanic Serving Institution and an NIH-designated Comprehensive Cancer Center were leveraged to develop appropriate research training and professional development activities. The career progression of the participants and degree completion rates was tracked, along with persistent interest in biomedical research in general and cancer and cancer health disparities research in particular for these underrepresented individuals. Finally, this report demonstrates that these training activities increased general knowledge about cancer among participants.
Assuntos
Pesquisa Biomédica , Escolha da Profissão , Grupos Minoritários , Prática Associada , Pesquisa Biomédica/educação , Estudos Transversais , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Grupos Minoritários/educação , Recursos HumanosRESUMO
Aminoglycoside (AG) antibiotics are widely used to prevent life-threatening infections, and cisplatin is used in the treatment of various cancers, but both are ototoxic and result in loss of sensory hair cells from the inner ear. ORC-13661 is a new drug that was derived from PROTO-1, a compound first identified as protective in a large-scale screen utilizing hair cells in the lateral line organs of zebrafish larvae. Here, we demonstrate, in zebrafish larvae and in mouse cochlear cultures, that ORC-13661 provides robust protection of hair cells against both ototoxins, the AGs and cisplatin. ORC-13661 also prevents both hearing loss in a dose-dependent manner in rats treated with amikacin and the loading of neomycin-Texas Red into lateral line hair cells. In addition, patch-clamp recordings in mouse cochlear cultures reveal that ORC-13661 is a high-affinity permeant blocker of the mechanoelectrical transducer (MET) channel in outer hair cells, suggesting that it may reduce the toxicity of AGs by directly competing for entry at the level of the MET channel and of cisplatin by a MET-dependent mechanism. ORC-13661 is therefore a promising and versatile protectant that reversibly blocks the hair cell MET channel and operates across multiple species and toxins.
Assuntos
Antibacterianos/toxicidade , Antineoplásicos/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Ototoxicidade/prevenção & controle , Substâncias Protetoras/farmacologia , Tiofenos/farmacologia , Ureia/análogos & derivados , Amicacina/toxicidade , Aminoglicosídeos/toxicidade , Animais , Técnicas de Cultura de Células , Células Cultivadas , Cisplatino/toxicidade , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Ciliadas Auditivas/metabolismo , Humanos , Microscopia Intravital , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/metabolismo , Masculino , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Ototoxicidade/etiologia , Técnicas de Patch-Clamp , Substâncias Protetoras/uso terapêutico , Ratos , Tiofenos/uso terapêutico , Imagem com Lapso de Tempo , Ureia/farmacologia , Ureia/uso terapêutico , Peixe-ZebraRESUMO
Metabolic pathways play important roles in proliferation and differentiation of malignant cells. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr), a precursor in purine biosynthesis and a well-established activator of AMP-activated protein kinase (AMPK), induces widespread metabolic alterations and is commonly used for dissecting the role of metabolism in cancer. We have previously reported that AICAr promotes differentiation and inhibits proliferation of myeloid leukemia cells. Here, using metabolic assays, immunoblotting, flow cytometry analyses, and siRNA-mediated gene silencing in leukemia cell lines, we show that AICAr-mediated differentiation was independent of the known metabolic effects of AMPK, including glucose consumption, but instead depends on the activation of the DNA damage-associated enzyme checkpoint kinase 1 (Chk1) induced by pyrimidine depletion. LC/MS/MS metabolomics analysis revealed that AICAr increases orotate levels and decreases uridine monophosphate (UMP) levels, consistent with inhibition of UMP synthesis at a step downstream of dihydroorotate dehydrogenase (DHODH). AICAr and the DHODH inhibitor brequinar had similar effects on differentiation markers and S-phase arrest, and genetic or pharmacological Chk1 inactivation abrogated both of these effects. Our results delineate an AMPK-independent effect of AICAr on myeloid leukemia differentiation that involves perturbation of pyrimidine biosynthesis and activation of the DNA damage response network.
Assuntos
Diferenciação Celular , Quinase 1 do Ponto de Checagem/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Pirimidinas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Di-Hidro-Orotato Desidrogenase , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/fisiopatologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Ribonucleosídeos/genética , Ribonucleosídeos/metabolismo , Pontos de Checagem da Fase S do Ciclo CelularRESUMO
Understanding genetic and cellular bases of adult form remains a fundamental goal at the intersection of developmental and evolutionary biology. The skin pigment cells of vertebrates, derived from embryonic neural crest, are a useful system for elucidating mechanisms of fate specification, pattern formation, and how particular phenotypes impact organismal behavior and ecology. In a survey of Danio fishes, including the zebrafish Danio rerio, we identified two populations of white pigment cells-leucophores-one of which arises by transdifferentiation of adult melanophores and another of which develops from a yellow-orange xanthophore or xanthophore-like progenitor. Single-cell transcriptomic, mutational, chemical, and ultrastructural analyses of zebrafish leucophores revealed cell-type-specific chemical compositions, organelle configurations, and genetic requirements. At the organismal level, we identified distinct physiological responses of leucophores during environmental background matching, and we showed that leucophore complement influences behavior. Together, our studies reveal independently arisen pigment cell types and mechanisms of fate acquisition in zebrafish and illustrate how concerted analyses across hierarchical levels can provide insights into phenotypes and their evolution.
Assuntos
Plasticidade Celular/genética , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Genética Populacional/métodos , Melanóforos/fisiologia , Mutação/genética , Crista Neural/fisiologia , Fenótipo , Pigmentação/genética , Transcriptoma/genéticaRESUMO
Nitazoxanide (NTZ) is an FDA-approved anti-protozoal drug that inhibits several bacteria and viruses as well. However, its effect on poxviruses is unknown. Therefore, we investigated the impact of NTZ on vaccinia virus (VACV). We found that NTZ inhibits VACV production with an EC50 of ~2⯵M, a potency comparable to that reported for several other viruses. The inhibitory block occurs early during the viral life cycle, prior to viral DNA replication. The mechanism of viral inhibition is likely not due to activation of intracellular innate immune pathways, such as protein kinase R (PKR) or interferon signaling, contrary to what has been suggested to mediate the effects of NTZ against some other viruses. Rather, our finding that addition of exogenous palmitate partially rescues VACV production from the inhibitory effect of NTZ suggests that NTZ impedes adaptations in cellular metabolism that are needed for efficient completion of the VACV replication cycle.
Assuntos
Antivirais/farmacologia , Tiazóis/farmacologia , Vaccinia virus/efeitos dos fármacos , Vaccinia virus/fisiologia , Replicação Viral/efeitos dos fármacos , Células Cultivadas , Fibroblastos/virologia , Humanos , NitrocompostosRESUMO
Hearing loss is a major public health concern with no pharmaceutical intervention for hearing protection or restoration. Using zebrafish neuromast hair cells, a robust model for mammalian auditory and vestibular hair cells, we identified a urea-thiophene carboxamide, 1 (ORC-001), as protective against aminoglycoside antibiotic (AGA)-induced hair cell death. The 50% protection (HC50) concentration conferred by 1 is 3.2 µM with protection against 200 µM neomycin approaching 100%. Compound 1 was sufficiently safe and drug-like to validate otoprotection in an in vivo rat hearing loss model. We explored the structure-activity relationship (SAR) of this compound series to improve otoprotective potency, improve pharmacokinetic properties and eliminate off-target activity. We present the optimization of 1 to yield 90 (ORC-13661). Compound 90 protects mechanosensory hair cells with HC50 of 120 nM and demonstrates 100% protection in the zebrafish assay and superior physiochemical, pharmacokinetic, and toxicologic properties, as well as complete in vivo protection in rats.
Assuntos
Aminoglicosídeos/efeitos adversos , Perda Auditiva/induzido quimicamente , Perda Auditiva/prevenção & controle , Segurança , Tiofenos/química , Tiofenos/farmacologia , Ureia/química , Administração Oral , Animais , Avaliação Pré-Clínica de Medicamentos , Ratos , Relação Estrutura-Atividade , Tiofenos/administração & dosagem , Tiofenos/efeitos adversos , Peixe-ZebraRESUMO
HYPOTHESIS: The zebrafish lateral line can be used to identify small molecules that protect against cisplatin-induced hair cell death. BACKGROUND: Cisplatin is a commonly used chemotherapeutic agent, which causes hearing loss by damaging hair cells of the inner ear. There are currently no FDA-approved pharmacologic strategies for preventing this side effect. The zebrafish lateral line has been used successfully in the past to study hair cell death and protection. METHODS: In this study, we used the zebrafish lateral line to screen a library of 10,000 small molecules for protection against cisplatin-induced hair cell death. Dose-response relationships for identified protectants were determined by quantifying hair cell protection. The effect of each protectant on uptake of a fluorescent cisplatin analog was also quantified. RESULTS: From this screen, we identified 2 compounds exhibiting dose-dependent protection: cisplatin hair cell protectant 1 and 2 (CHCP1 and 2). CHCP1 reduced the uptake of a fluorescent cisplatin analog, suggesting its protective effects may be due to decreased cisplatin uptake. CHCP2 did not affect uptake, which suggests an intracellular mechanism of action. Evaluation of analogs of CHCP2 revealed 3 additional compounds that significantly reduced cisplatin-induced hair cell death, although none exceed the effectiveness or potency of the parent compound. CONCLUSION: The zebrafish lateral line was used to identify 2 small molecules that protected against cisplatin-induced hair cell death.
Assuntos
Antineoplásicos/toxicidade , Morte Celular/efeitos dos fármacos , Cisplatino/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Perda Auditiva/prevenção & controle , Sistema da Linha Lateral/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Perda Auditiva/induzido quimicamente , Peixe-ZebraRESUMO
UNLABELLED: TMPRSS2 is an androgen-regulated cell-surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority of human prostate cancer metastases. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries, we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-MET receptor tyrosine kinase signaling, and initiated a proinvasive epithelial-to-mesenchymal transition phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment. SIGNIFICANCE: The vast majority of prostate cancer deaths are due to metastasis. Loss of TMPRSS2 activity dramatically attenuated the metastatic phenotype through mechanisms involving the HGF-c-MET axis. Therapeutic approaches directed toward inhibiting TMPRSS2 may reduce the incidence or progression of metastasis in patients with prostate cancer.
Assuntos
Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Serina Endopeptidases/metabolismo , Androgênios/metabolismo , Animais , Linhagem Celular Tumoral , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Masculino , Camundongos Knockout , Camundongos SCID , Biblioteca de Peptídeos , Proteólise , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Androgênicos/metabolismo , Microambiente TumoralRESUMO
Sirtuins are a family of NAD(+)-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC50 ≈ 50 µM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and 8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class may be predominantly due to SIRT2 inhibition.
Assuntos
Antineoplásicos/síntese química , Isoxazóis/síntese química , Naftalenos/farmacologia , Pirazolonas/síntese química , Pirimidinonas/farmacologia , Sirtuínas/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Descoberta de Drogas , Isoxazóis/farmacologia , Espectroscopia de Ressonância Magnética , Pirazolonas/farmacologia , Relação Estrutura-AtividadeRESUMO
The development of effective therapies inhibiting prostate cancer progression and metastasis may substantially impact prostate cancer mortality and potentially reduce the rates of invasive treatments by enhancing the safety of active surveillance strategies. Hepsin (HPN) is a cell surface serine protease amplified in a subset of human sarcomas (7.2%), as well as in ovarian (10%), lung adeno (5.4%), lung squamous cell (4.5%), adenoid cystic (5%), breast (2.6%), uterine (1.7%) and colon (1.4%) carcinomas. While HPN is not amplified in prostate cancer, it is one of the most prominently overexpressed genes in the majority of human prostate tumors and genetic experiments in mice indicate that Hepsin promotes prostate cancer metastasis, particularly metastasis to the bone marrow. We report here the development, analysis and animal trial of the small-molecule Hepsin inhibitor HepIn-13. Long-term exposure to HepIn-13 inhibited bone, liver and lung metastasis in a murine model of metastatic prostate cancer. These findings indicate that inhibition of Hepsin with small-molecule compounds could provide an effective tool for attenuation of prostate cancer progression and metastasis.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias Ósseas/prevenção & controle , Proteínas de Membrana/antagonistas & inibidores , Naftalenos/farmacocinética , Naftalenos/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Inibidores de Serina Proteinase/uso terapêutico , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Administração Oral , Animais , Disponibilidade Biológica , Neoplasias Ósseas/secundário , Células HEK293 , Meia-Vida , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Modelos Moleculares , Naftalenos/farmacologia , Neoplasias da Próstata/metabolismo , Pirimidinas/farmacologia , Inibidores de Serina Proteinase/farmacocinéticaRESUMO
The majority of hearing loss is caused by the permanent loss of inner ear hair cells. The identification of drugs that modulate the susceptibility to hair cell loss or spur their regeneration is often hampered by the difficulties of assaying for such complex phenomena in mammalian models. The zebrafish has emerged as a powerful animal model for chemical screening in many contexts. Several characteristics of the zebrafish, such as its small size and external location of sensory hair cells, uniquely position it as an ideal model organism for the study of hair cell toxicity, protection, and regeneration. We have used this model to screen for drugs that affect each of these aspects of hair cell biology and have identified compounds that affect each of these processes. The identification of such drugs and drug-like compounds holds promise in the future ability to stem hearing loss in the human population.
RESUMO
SIRT1 is a mammalian NAD(+)-dependent histone deacetylase implicated in metabolism, development, aging and tumorigenesis. Prior studies that examined the effect of enterocyte-specific overexpression and global deletion of SIRT1 on polyp formation in the intestines of APC(+/min) mice, a commonly used model for intestinal tumorigenesis, yielded conflicting results, supporting either tumor-suppressive or tumor-promoting roles for SIRT1, respectively. In order to resolve the controversy emerging from these prior in vivo studies, in the present report we examined the effect of SIRT1 deficiency confined to the intestines, avoiding the systemic perturbations such as growth retardation seen with global SIRT1 deletion. We crossed APC(+/min) mice with mice bearing enterocyte-specific inactivation of SIRT1 and examined polyp development in the progeny. We found that SIRT1-inactivation reduced total polyp surface (9.3 mm(2) vs. 23.3 mm(2), pâ=â0.01), average polyp size (0.24 mm(2) vs. 0.51 mm(2), pâ=â0.005) and the number of polyps >0.5 mm in diameter (14 vs. 23, pâ=â0.04), indicating that SIRT1 affects both the number and size of tumors. Additionally, tumors in SIRT1-deficient mice exhibited markedly increased numbers of cells undergoing apoptosis, suggesting that SIRT1 contributes to tumor growth by enabling survival of tumor cells. Our results indicate that SIRT1 acts as a tumor promoter in the APC(+/min) mouse model of intestinal tumorigenesis.
Assuntos
Carcinogênese/metabolismo , Neoplasias do Colo/enzimologia , Enterócitos/enzimologia , Sirtuína 1/genética , Animais , Carcinogênese/genética , Colo/patologia , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Deleção de Sequência , Sirtuína 1/metabolismo , Carga Tumoral , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização WntRESUMO
Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Células Ciliadas Auditivas/efeitos dos fármacos , Sistema da Linha Lateral/citologia , Mecanorreceptores/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Contagem de Células/métodos , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Ácido Egtázico/farmacologia , Embrião não Mamífero , Feminino , Corantes Fluorescentes , Proteínas de Fluorescência Verde/genética , Células Ciliadas Auditivas/metabolismo , Larva , Sistema da Linha Lateral/efeitos dos fármacos , Masculino , Microscopia de Fluorescência , Miosina VIIa , Miosinas/metabolismo , Quinina/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
We have previously published results from a screen of 1,040 FDA-approved drugs and bioactives (NINDS Custom Collection) for drugs that protect against neomycin-induced hair cell death (Ou et al., J Assoc Res Otolaryngol 10:191-203, 2009). Further evaluation of this drug library identified eight protective drugs that shared a common quinoline scaffold. These drugs were tested further in terms of their protection against other aminoglycosides, as well as their effect on aminoglycoside uptake. All of the eight quinolines that protected against neomycin were found to protect against short- and long-term gentamicin damage protocols. We then tested the structurally related compounds quinoline, isoquinoline, naphthalene, and indole for protective effects. Of these compounds, indole demonstrated a small but significant amount of protection against neomycin, while quinoline and isoquinoline partially protected against long-term gentamicin damage. We examined whether the protective activity of this group of compounds was related to known targets of the quinoline derivatives. The protective effects did not seem linked to either the cholinergic or histaminergic pathways that are regulated by some members of the quinoline family. However, all eight protective drugs were found to reduce the uptake of aminoglycosides into hair cells. Subsequent experiments suggest that reduction of uptake is the primary mechanism of protection among the quinoline drugs.
Assuntos
Aminoglicosídeos/toxicidade , Antibacterianos/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Quinolinas/farmacologia , Aminoglicosídeos/farmacocinética , Animais , Bactérias/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Gentamicinas/farmacocinética , Gentamicinas/toxicidade , Células Ciliadas Auditivas/patologia , Neomicina/toxicidade , Peixe-ZebraRESUMO
Loss of mechanosensory hair cells in the inner ear accounts for many hearing loss and balance disorders. Several beneficial pharmaceutical drugs cause hair cell death as a side effect. These include aminoglycoside antibiotics, such as neomycin, kanamycin and gentamicin, and several cancer chemotherapy drugs, such as cisplatin. Discovering new compounds that protect mammalian hair cells from toxic insults is experimentally difficult because of the inaccessibility of the inner ear. We used the zebrafish lateral line sensory system as an in vivo screening platform to survey a library of FDA-approved pharmaceuticals for compounds that protect hair cells from neomycin, gentamicin, kanamycin and cisplatin. Ten compounds were identified that provide protection from at least two of the four toxins. The resulting compounds fall into several drug classes, including serotonin and dopamine-modulating drugs, adrenergic receptor ligands, and estrogen receptor modulators. The protective compounds show different effects against the different toxins, supporting the idea that each toxin causes hair cell death by distinct, but partially overlapping, mechanisms. Furthermore, some compounds from the same drug classes had different protective properties, suggesting that they might not prevent hair cell death by their known target mechanisms. Some protective compounds blocked gentamicin uptake into hair cells, suggesting that they may block mechanotransduction or other routes of entry. The protective compounds identified in our screen will provide a starting point for studies in mammals as well as further research discovering the cellular signaling pathways that trigger hair cell death.