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1.
Toxicol Lett ; 320: 19-27, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31778773

RESUMO

The deleterious effects of glucocorticoids on glucose homeostasis limit their clinical use. There is substantial evidence demonstrating that islet function impaired by long-term glucocorticoids exposure is a core defect in the progression of impaired glucose tolerance to diabetes. The activity of heat-shock protein (Hsp) 90 is required to maintain the hormone-binding activity and stability of glucocorticoid receptor (GR). In the present study, Hsp90 inhibition by 17-DMAG counteracted dexamethasone-mediated inhibition of glucose-stimulated insulin secretion in isolated rat islets as well as expressions of neuropeptide Y (NPY) and somatostatin receptor 3 (SSTR3), two negative regulators of insulin secretion. Like 17-DMAG, both the pan-histone deacetylase (HDAC) inhibitor TSA and HDAC6 inhibitor Tubacin exhibited a similar action in protecting islet function against dexamethasone-induced injury, along with the downregulation of NPY and SSTR3 expressions. The hyperacetylation of Hsp90 by TSA and Tubacin disrupted its binding ability to GR and blocked dexamethasone-elicited nuclear translocation of GR in INS-1 ß-cell lines. In addition, Tubacin treatment triggered the GR protein degradation through the ubiquitin-proteasome pathway. These findings suggest that Hsp90 acetylation by inhibiting HDAC6 activity may be a potential strategy to prevent the development of steroid diabetes mellitus via alleviating glucocorticoid-impaired islet function.


Assuntos
Anilidas/farmacologia , Benzoquinonas/farmacologia , Dexametasona/toxicidade , Glucocorticoides/toxicidade , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Acetilação , Animais , Linhagem Celular , Proteínas de Choque Térmico HSP90/metabolismo , Desacetilase 6 de Histona/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Ratos Sprague-Dawley , Via Secretória , Técnicas de Cultura de Tecidos
2.
Neurochem Res ; 44(11): 2643-2657, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31606837

RESUMO

Schwann cells (SCs) play an important role in producing myelin for rapid neurotransmission in the peripheral nervous system. Activation of the differentiation and myelination processes in SCs requires the expression of a series of transcriptional factors including Sox10, Oct6/Pou3f1, and Egr2/Krox20. However, functional interactions among several transcription factors are poorly defined and the important components of the regulatory network are still unknown. Until now, available evidence suggests that SCs require cAMP signaling to initiate the myelination program. Heat shock protein 90 (Hsp90) is known as a chaperone required to stabilize ErbB2 receptor. In recent years, it was reported that cAMP transactivated the ErbB2/ErbB3 signaling in SCs. However, the relationship between Hsp90 and cAMP-induced differentiation in SCs is undefined. Here we investigated the role of Hsp90 during cAMP-induced differentiation of SCs using Hsp90 inhibitor, geldanamycin and Hsp90 siRNA transfection. Our results showed that dibutyryl-cAMP (db-cAMP) treatment upregulated Hsp90 expression and led to nuclear translocation of Gab1/ERK, the downstream signaling pathway of the ErbB2 signaling mechanism in myelination. The expression of myelin-related genes and nuclear translocation of Gab1/ERK following db-cAMP treatment was inhibited by geldanamycin pretreatment and Hsp90 knockdown. These findings suggest that Hsp90 might play a role in cAMP-induced differentiation via stabilization of ErbB2 and nuclear translocation of Gab1/ERK in SCs.


Assuntos
Diferenciação Celular/fisiologia , Proteínas de Choque Térmico HSP90/fisiologia , Células de Schwann/fisiologia , Animais , Benzoquinonas/farmacologia , Bucladesina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP90/genética , Lactamas Macrocíclicas/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos Sprague-Dawley , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Células de Schwann/citologia , Regulação para Cima
3.
Exp Eye Res ; 189: 107821, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31589841

RESUMO

Posterior capsule opacification (PCO) is a common complication of cataract surgery, resulting from a combination of proliferation, migration, epithelial-mesenchymal transition (EMT) of residual capsular epithelial cells and fibrosis of myofibroblasts. HSP90 is known to regulate the proteostasis of cells under pathophysiological conditions. The role of HSP90 in PCO formation, however, is not clear. To do this, the lens epithelial cell lines and an ex vivo cultured rat capsular bag model were used to study the role of HSP90 in PCO formation. The expression of protein and mRNA was measured by immunoblotting and quantitative RT-PCR, and cell apoptosis was measured by TUNEL(TdT-mediated dUTP nick-end labeling). The cell proliferation was measured by cell viability assays. The results showed that 17-AAG (Tanespimycin), an inhibitor of HSP90, suppresses the proliferation of immortalized lens epithelial cell lines HLE-B3, SRA01/04, and mLEC, with IC50 values of 0.27, 0.27, and 0.49 µM, respectively. In an ex vivo cultured rat capsular model, the capsular residual epithelial cells resisted the stress of the capsulorhexis surgery and took 3-6 days to completely overlay the capsular posterior wall. During this process, heat shock factor 1 and its downstream targets HSP90, HSP25, αB-crystallin, and HSP40 were upregulated. Treatment with 17-AAG inhibited the viability of capsular residual epithelial cells and induced the cells apoptosis, characterized by increases in ROS levels, apoptotic DNA injury, and the activation of caspases 9 and 3. HSP90 participated in regulating both EGF receptor (EGFR) and TGF receptor (TGFR) signaling pathways. HSP90 was found to interact with the EGFR, such that inhibition of HSP90 by 17-AAG destabilized the EGFR protein and suppressed p-ERK1/2 and p-AKT levels. 17-AAG also inhibited the TGF-ß-induced phosphorylation of SMAD2/3 and ERK1/2 and the decrease in E-cadherin and ZO-1 expression. Accordingly, these data suggest that the induction of HSP90 protects capsular residual epithelial cells against capsulorhexis-induced stress and participates in regulating the processes of proliferation, EMT and migration of rat capsular residual epithelial cells, at least partly, through the EGFR and TGFR signaling pathways. Treatment with 17-AAG suppresses PCO formation and is therefore a potential therapeutic candidate for PCO prevention.


Assuntos
Benzoquinonas/farmacologia , Opacificação da Cápsula/tratamento farmacológico , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Cápsula Posterior do Cristalino/metabolismo , Animais , Western Blotting , Opacificação da Cápsula/metabolismo , Opacificação da Cápsula/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Proteínas de Choque Térmico HSP90/metabolismo , Cápsula Posterior do Cristalino/patologia , Ratos , Ratos Wistar , Transdução de Sinais
4.
Nat Commun ; 10(1): 3604, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399568

RESUMO

ROS1 gene rearrangement was observed in around 1-2 % of NSCLC patients and in several other cancers such as cholangiocarcinoma, glioblastoma, or colorectal cancer. Crizotinib, an ALK/ROS1/MET inhibitor, is highly effective against ROS1-rearranged lung cancer and is used in clinic. However, crizotinib resistance is an emerging issue, and several resistance mechanisms, such as secondary kinase-domain mutations (e.g., ROS1-G2032R) have been identified in crizotinib-refractory patients. Here we characterize a new selective ROS1/NTRK inhibitor, DS-6051b, in preclinical models of ROS1- or NTRK-rearranged cancers. DS-6051b induces dramatic growth inhibition of both wild type and G2032R mutant ROS1-rearranged cancers or NTRK-rearranged cancers in vitro and in vivo. Here we report that DS-6051b is effective in treating ROS1- or NTRK-rearranged cancer in preclinical models, including crizotinib-resistant ROS1 positive cancer with secondary kinase domain mutations especially G2032R mutation which is highly resistant to crizotinib as well as lorlatinib and entrectinib, next generation ROS1 inhibitors.


Assuntos
Crizotinibe/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptor trkB/antagonistas & inibidores , Benzamidas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenvolvimento de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Indazóis/farmacologia , Lactamas Macrocíclicas/farmacologia , Neoplasias Pulmonares/genética , Mutação/efeitos dos fármacos , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética
5.
Biomed Res ; 40(4): 169-178, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31413238

RESUMO

Migration of osteoblasts to the sites resorbed by osteoclasts is an essential step in bone remodeling. However, the exact mechanism of osteoblast migration is still not known. We have shown that platelet-derived growth factor (PDGF)-BB induces the migration of osteoblast-like MC3T3-E1 cells through the activation of p38 mitogen-activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK) and p44/p42 MAP kinase. Evidence is accumulating that heat shock protein 90 (HSP90) acts as a central regulator of proteostasis under stress conditions and physiological cell functions. In the present study, using transwell cell migration assay and wound-healing assay, we investigated the involvement of HSP90 in the PDGF-BB-stimulated migration of MC3T3-E1 cells, and the underlying signaling mechanism estimated by Western blot analyses. Onalespib, an HSP90 inhibitor, significantly reduced the PDGF-BB-stimulated migration evaluated by the two types of migration assays. The cell migration was also suppressed by geldanamycin, another type of HSP90 inhibitor. Onalespib markedly attenuated the PDGF-BB-elicited phosphorylation of p44/p42 MAP kinase without affecting that of p38 MAP kinase or JNK. In addition, the phosphorylation of p44/p42 MAP kinase by PDGF-BB was reduced by geldanamycin. Taken together, these results strongly suggest that HSP90 inhibitors suppress the PDGF-BB-induced osteoblast migration through the attenuation of p44/p42 MAP kinase activity.


Assuntos
Becaplermina/farmacologia , Benzamidas/farmacologia , Benzoquinonas/farmacologia , Movimento Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoindóis/farmacologia , Lactamas Macrocíclicas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteoblastos/metabolismo , Animais , Linhagem Celular , Proteínas de Choque Térmico HSP90/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos
6.
J Pharmacol Sci ; 140(3): 255-262, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31402210

RESUMO

Heat shock protein 90 (HSP90) antagonists are currently being evaluated as potential anticancer drugs. However, adverse effects related to these drugs, such as fatigue and pain, suggest that they affect neurons. Therefore, to understand the influence of HSP90 inhibitors on neurons, we investigated the effects of geldanamycin, an HSP90 antagonist, on nerve growth factor (NGF)-differentiated pheochromocytoma 12 (PC12) cells, particularly, on the expression and phosphorylation of proteins and kinases in the NGF pathway. Geldanamycin significantly inhibited NGF-induced neurite outgrowth and phosphorylation of Akt and extracellular signal-related kinase 1/2 in PC12 cells. Furthermore, geldanamycin inhibited the phosphorylation of collapsin response mediator protein 2 and the expression of cyclin-dependent kinase 5 in the presence of NGF, but did not significantly affect the expression of glycogen synthase kinase 3ß. These results suggest that geldanamycin influences microtubule-binding proteins and kinases relating to neurite outgrowth, thereby inducing neuronal impairment.


Assuntos
Neoplasias das Glândulas Suprarrenais/tratamento farmacológico , Benzoquinonas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Fator de Crescimento Neural/metabolismo , Neuritos/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Feocromocitoma/tratamento farmacológico , Neoplasias das Glândulas Suprarrenais/metabolismo , Animais , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Feocromocitoma/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos
7.
J Therm Biol ; 84: 1-7, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31466741

RESUMO

Heat can trigger testicular damage and impair fertility. Leydig cells produce testosterone in response to stimulation by luteinizing hormone (LH), which induces Ca2+ entry and K+ efflux through ion channels in their plasma membrane. Considering that mechanisms coordinating the Leydig cell responses to hyperthermic stress remain unclear; the present study analyzed the effects of heat stress (HS, 43°C, 15 min) and inhibition of Hsp90 on T-type calcium currents and voltage-dependent potassium currents (VKC) in mice Leydig cells. Results show that HS reduced the VKC steady state currents at +80 mV (45.3%) and maximum conductance (71.5%), as well as increased the activation time constant (31.7%) and the voltage for which half the channels are open (30%). Hsp90 inhibition did not change the VKC currents. T-type calcium currents were not affected by HS or Hsp90 inhibition. In conclusion, HS can slow the activation, reduce the currents and voltage dependence of the VKC, suggesting a possible role of these currents in the response to hyperthermic stress in Leydig cells.


Assuntos
Canais de Cálcio Tipo T/fisiologia , Proteínas de Choque Térmico HSP90/fisiologia , Resposta ao Choque Térmico/fisiologia , Células Intersticiais do Testículo/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Animais , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Temperatura Alta , Lactamas Macrocíclicas/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos
8.
Life Sci ; 232: 116672, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31336120

RESUMO

AIMS: Obesity is not only associated with metabolic diseases but is also a symptom of menopause in women. To date, there are no effective drugs for the management of obesity, and it is important to find new agents with fewer side effects, for the treatment of obesity. This study aimed to determine the anti-obesity effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein 90 (Hsp90) inhibitor, and its underlying mechanism in rats with ovariectomy-induced obesity. MAIN METHODS: Ovariectomy (Ovx) rats were treated with 17-DMAG (1 mg kg-1, intraperitoneally) for eight weeks from one week after surgery. The body weight, food intake, locomotor activity, adipogenic- and autophagy-related protein expression in white adipose tissue (WAT) and plasma triglyceride (TG) levels were measured in sham and Ovx rats. KEY FINDINGS: Compared with sham rats, Ovx rats showed increased weight gain, food intake, WAT mass, TG levels, adipogenic protein expression, and decreased locomotor activity. Furthermore, autophagy-related proteins and Foxo3a of WAT were significantly increased in Ovx rats. However, with the exclusion of increased food intake, the changes induced by Ovx were all reversed in 17-DMAG-treated Ovx rats. In addition, the expression of Hsp70 and phosphorylation of Akt increased in 17-DMAG-treated Ovx rats. SIGNIFICANCE: These results suggest that 17-DMAG significantly ameliorated obesity induced by Ovx, and this phenomenon is accompanied by the downregulation of adipogenic-related and autophagy-related proteins as well as the upregulation of Akt-phosphorylation and Hsp70 expression. Therefore, 17-DMAG may be a potential agent for preventing or treating obesity in postmenopausal women.


Assuntos
Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Obesidade/etiologia , Obesidade/prevenção & controle , Ovariectomia/efeitos adversos , Adipogenia , Tecido Adiposo/metabolismo , Animais , Autofagia , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Locomoção/efeitos dos fármacos , Ratos
9.
PLoS One ; 14(6): e0218189, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31181115

RESUMO

In this study, we report antifungal activity of auroramycin against Candida albicans, Candida tropicalis, and Cryptococcus neoformans. Auroramycin, a potent antimicrobial doubly glycosylated 24-membered polyene macrolactam, was previously isolated and characterized, following CRISPR-Cas9 mediated activation of a silent polyketide synthase biosynthetic gene cluster in Streptomyces rosesporous NRRL 15998. Chemogenomic profiling of auroramycin in yeast has linked its antifungal bioactivity to vacuolar transport and membrane organization. This was verified by disruption of vacuolar structure and membrane integrity of yeast cells with auroramycin treatment. Addition of salt but not sorbitol to the medium rescued the growth of auroramycin-treated yeast cells suggesting that auroramycin causes ionic stress. Furthermore, auroramycin caused hyperpolarization of the yeast plasma membrane and displayed a synergistic interaction with cationic hygromycin. Our data strongly suggest that auroramycin inhibits yeast cells by causing leakage of cations from the cytoplasm. Thus, auroramycin's mode-of-action is distinct from known antifungal polyenes, reinforcing the importance of natural products in the discovery of new anti-infectives.


Assuntos
Antifúngicos/farmacologia , Lactamas Macrocíclicas/farmacologia , Polienos/farmacologia , Leveduras/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candida tropicalis/efeitos dos fármacos , Cátions/metabolismo , Cryptococcus neoformans/efeitos dos fármacos , Citoplasma/metabolismo , Vacúolos/metabolismo
10.
Free Radic Res ; 53(6): 618-628, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31185751

RESUMO

With a significant increase in the incidence of system invasive fungal infections, the limited antifungal drugs and increased frequency of cross-resistance make it necessary to explore new and effective therapeutic strategies. Combination drug therapy has become one widely used choice to alleviate this problem. Geldanamycin (GdA), as an inhibitor of Hsp90, displayed broad antifungal activity when combined with fluconazole. However, due to its cytotoxicity, the dose and duration of GdA is limited. In this study, we observed the effect of fluconazole plus GdA on Candida tropicalis resistant to azoles and amphotericin B. The results showed that this synergism led to a decrease in growth and survival rate. In addition, fluconazole combined with GdA caused mitochondrial depolarisation, disruption of plasma membrane integrity and multinucleated morphology. However, the supplement of a reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), rescued the above phenotypes. This study indicated that the oxidative stress mediated by fluconazole plus GdA played an important role in the antifungal activity, and targeting oxidative stress might extend target choices to treat fungal infections.


Assuntos
Antifúngicos/farmacologia , Benzoquinonas/farmacologia , Candida tropicalis/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Fluconazol/farmacologia , Lactamas Macrocíclicas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Anfotericina B/farmacologia , Antifúngicos/química , Azóis/farmacologia , Candida tropicalis/crescimento & desenvolvimento , Candida tropicalis/metabolismo , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos
11.
Mol Pharmacol ; 96(2): 168-179, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31175180

RESUMO

Molecular chaperone heat shock protein 90 (HSP90) is involved in oncogenic signaling pathways including epithelial-mesenchymal transition (EMT), a key process in tumor initiation, progression, metastasis, and chemoresistance. The molecular mechanisms underlying the involvement of HSP90 in EMT are still under investigation. In this study, we identified a previously unrecognized role of HSP90 in cooperating with signal transducer and activator of transcription 3 (STAT3) to regulate TWIST1 transcription in cancer cells. The HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin suppressed TWIST1 mRNA expression and promoter activity in epithelial ovarian cancer, renal clear cell cancer, and nasopharyngeal cancer cell lines. The interactions between HSP90 and transcription factors were visualized in cancer cell lines and tumor tissues using proximity ligation assays. Our findings reveal that HSP90 promotes the binding of STAT3 to the TWIST1 promoter, leading to the transcription of TWIST1. The inhibition of HSP90 downregulates STAT3 activity and TWIST1 transcription, thereby suppressing EMT and potentially inhibiting tumor progression, metastasis, and chemoresistance in different types of cancers. SIGNIFICANCE STATEMENT: Our study provides new evidence that HSP90 promotes EMT through enhancing TWIST1 transcription, which can be suppressed by HSP90 inhibitors. The HSP90 inhibitor inhibits EMT, thus potentially slowing down tumor growth, invasion, dissemination, metastasis, and drug resistance. These findings will hopefully pave the way for new therapeutic opportunities to target EMT and metastasis using HSP90 inhibitors.


Assuntos
Benzoquinonas/farmacologia , Neoplasias Renais/genética , Lactamas Macrocíclicas/farmacologia , Neoplasias Nasofaríngeas/genética , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Fator de Transcrição STAT3/metabolismo , Proteína 1 Relacionada a Twist/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Análise Serial de Tecidos , Transcrição Genética/efeitos dos fármacos
12.
Int J Mol Sci ; 20(10)2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31108847

RESUMO

Of the three interleukin-22 binding protein (IL-22BP) isoforms produced by the human IL22RA2 gene, IL-22BPi2 and IL-22BPi3 are capable of neutralizing IL-22. The longest isoform, IL-22BPi1, does not bind IL-22, is poorly secreted, and its retention within the endoplasmic reticulum (ER) is associated with induction of an unfolded protein response (UPR). Therapeutic modulation of IL-22BPi2 and IL-22BPi3 production may be beneficial in IL-22-dependent disorders. Recently, we identified the ER chaperones GRP94 and cyclophilin B in the interactomes of both IL-22BPi1 and IL-22BPi2. In this study, we investigated whether secretion of the IL-22BP isoforms could be modulated by pharmacological targeting of GRP94 and cyclophilin B, either by means of geldanamycin, that binds to the ADP/ATP pocket shared by HSP90 paralogs, or by cyclosporin A, which causes depletion of ER cyclophilin B levels through secretion. We found that geldanamycin and its analogs did not influence secretion of IL-22BPi2 or IL-22BPi3, but significantly enhanced intracellular and secreted levels of IL-22BPi1. The secreted protein was heterogeneously glycosylated, with both high-mannose and complex-type glycoforms present. In addition, cyclosporine A augmented the secretion of IL-22BPi1 and reduced that of IL-22BPi2 and IL-22BPi3. Our data indicate that the ATPase activity of GRP94 and cyclophilin B are instrumental in ER sequestration and degradation of IL-22BPi1, and that blocking these factors mobilizes IL-22BPi1 toward the secretory route.


Assuntos
Benzoquinonas/farmacologia , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Lactamas Macrocíclicas/farmacologia , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Ciclofilinas/química , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Glicosilação , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Monócitos/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteólise , Receptores de Interleucina/química , Receptores de Interleucina/genética
13.
Org Biomol Chem ; 17(21): 5269-5278, 2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31089638

RESUMO

Mutasynthetic supplementation of the AHBA blocked mutant strain of S. hygroscopicus, the geldanamycin producer, with 21 aromatic and heteroaromatic amino acids provided new nonquinoid geldanamycin derivatives. Large scale (5 L) fermentation provided four new derivatives in sufficient quantity for full structural characterisation. Among these, the first thiophene derivative of reblastatin showed strong antiproliferative activity towards several human cancer cell lines. Additionally, inhibitory effects on human heat shock protein Hsp90α and bacterial heat shock protein from H. pylori HpHtpG were observed, revealing strong displacement properties for labelled ATP and demonstrating that the ATP-binding site of Hsps is the target site for the new geldanamycin derivatives.


Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Proteínas de Choque Térmico/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Benzoquinonas/química , Benzoquinonas/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Choque Térmico/metabolismo , Helicobacter pylori/química , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/isolamento & purificação , Estrutura Molecular , Streptomyces/química , Relação Estrutura-Atividade
14.
Oncol Res ; 27(7): 835-847, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30982499

RESUMO

Recently, novel therapeutic strategies have been designed with the aim of killing cancer stem-like cells (CSCs), and considerable interest has been generated in the development of specific therapies that target stemness-related marker of CSCs. In this study, nonsteroidal anti-inflammatory drugs (NSAIDs) significantly potentiated Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-mediated cytotoxicity through apoptotic and autophagic cell death induction, but COX-2-inhibitory function was not required for NSAID-induced autophagy in CD44-overexpressing human chronic myeloid leukemia K562 (CD44highK562) cells. Importantly, we found that treatment with NSAIDs resulted in a dose-dependent increase in LC3-II level and decrease in p62 level and simultaneous reduction in multiple stemness-related markers including CD44, Oct4, c-Myc, and mutant p53 (mutp53) in CD44highK562 cells, suggesting that NSAIDs could induce autophagy, which might mediate degradation of stemness-related marker proteins. Activation of AMPK and inhibition of Akt/mTOR/p70S6K/4EBP1 participated in NSAID-induced autophagy in CD44highK562 cells. In addition, treatment of CD44highK562 cells with NSAIDs inhibited expression of HSF1/Hsps, which resulted in suppression of 17-AAG-induced activation of Hsp70, leading to reversal of 17-AAG resistance and sensitization of CD44highK562 cells to 17-AAG by NSAIDs. In conclusion, combining NSAIDs with Hsp90 inhibitor may offer one of the most promising strategies for eradication of CD44-overexpressing CSCs.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Receptores de Hialuronatos/metabolismo , Lactamas Macrocíclicas/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Receptores de Hialuronatos/biossíntese , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/patologia
15.
Int J Pharm ; 564: 281-292, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-30999048

RESUMO

HSP90 inhibitors have the potential to treat many types of cancer due to the dependence of tumor cells on HSP90 for cell growth and proliferation. The Cullin-5 (Cul5) E3 ubiquitin ligase is required for HSP90 inhibitors to induce client protein degradation and subsequent cell death. Cul5 is expressed at low levels in breast cancer cells compared to patient matched controls. This observed low Cul5 expression may play a role in the reported decreased efficacy of 17-AAG and related HSP90 inhibitors as a monotherapy. We have developed a method for delivery of 17-AAG plus Cul5 DNA to cells via gold nanoparticles (AuNPs). Delivery of AuNPs containing Cul5 DNA increases the sensitivity of Cul5 deficient AU565 cells to 17-AAG. Characterization of AuNPs by UV-vis spectrum, TEM, gel electrophoresis assay and 1H NMR indicate attachment of both 17-AAG and DNA payload as well as AuNP stability. Studies in Cul5 deficient AU565 cells reveal that delivery of Cul5 and 17-AAG together increase cytotoxicity. Our results provide evidence that delivery of DNA with drug may serve as a method to sensitize drug resistant tumor cells.


Assuntos
Benzoquinonas/farmacologia , Neoplasias da Mama/terapia , Proteínas Culina/genética , DNA/administração & dosagem , Ouro/administração & dosagem , Lactamas Macrocíclicas/farmacologia , Nanopartículas Metálicas/administração & dosagem , Linhagem Celular , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos
16.
Exp Parasitol ; 200: 61-66, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30946841

RESUMO

Anthelmintic and in particular macrocyclic lactone (ML) resistance is a widespread problem in trichostrongyloid parasitic nematodes, yet mechanisms of ML resistance are still poorly understood. In the absence of target-site changes in resistant parasite field populations, increased drug extrusion and xenobiotic metabolism have been implicated in modification of susceptibility to MLs. In addition to P-glycoproteins, cytochrome P450 monooxygenases (CYPs) were considered to be involved in ML resistance. CYPs are highly divergent in nematodes with about 80 genes in the model organism Caenorhabditis elegans. Using larval development assays in the C. elegans model, piperonyl butoxide (PBO) and a temperature-sensitive variant of the emb-8 cytochrome reductase were used for chemical and genetic ablation of CYP activity. Additionally, a loss-of-function variant of cyp-14A5 was characterized to determine whether increased expression of this CYP in an ivermectin (IVM)-tolerant C. elegans line might be related to the phenotype. In a preliminary experiment with PBO, susceptibility to 5 nM IVM was synergistically increased by PBO. However, effects of genetic ablation of CYP activity on the EC50 values were small (1.5-fold decrease) for IVM and not significant for moxidectin (MOX). However, due to the steep concentration-response-curves, there were again strong differences between the wild-type and the CYP deficient genotype at individual IVM but not MOX concentrations. Although these results suggest small but significant effects on the susceptibility level of C. elegans to IVM, the cyp14A5 gene proposed by a previous study as candidate was ruled out since it was neither IVM/MOX inducible nor did a strain with a loss-of-function allele show increased susceptibility to either drug. In conclusion, the effect of the CYP system on IVM susceptibility in C. elegans is at best low while effects on MOX susceptibility were not detected. The previously suggested candidate cyp14A5 could be excluded to be involved in ML metabolism.


Assuntos
Anti-Helmínticos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Lactonas/farmacologia , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/farmacologia , Relação Dose-Resposta a Droga , Ivermectina/farmacologia , Modelos Logísticos , Macrolídeos/farmacologia , Butóxido de Piperonila/farmacologia , Reação em Cadeia da Polimerase em Tempo Real
17.
Mar Drugs ; 17(4)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30978939

RESUMO

Four new ansamycins, named divergolides T-W (1-4), along with two known analogs were isolated from the fermentation broth of the mangrove-derived actinomycete Streptomyces sp. KFD18. The structures of the compounds, including the absolute configurations of their stereogenic carbons, were determined by spectroscopic data and single-crystal X-ray diffraction analysis. Compounds 1-4 showed cytotoxic activity against the human gastric cancer cell line SGC-7901, the human leukemic cell line K562, the HeLa cell line, and the human lung carcinoma cell line A549, with 1 being the most active while compounds 5 and 6 were inactive against all the tested cell lines. Compounds 1 and 3 showed very potent and specific cytotoxic activities (IC50 2.8 and 4.7 µM, respectively) against the SGC-7901 cells. Further, the apoptosis-inducing effect of 1 and 3 against SGC-7901 cells was demonstrated by two kinds of staining methods for the first time.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Streptomyces/química , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/isolamento & purificação , Estrutura Molecular , Áreas Alagadas
18.
Daru ; 27(1): 111-119, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30835081

RESUMO

OBJECTIVE: The present study evaluated the anti-cancer effects of irradiation (Ir) alone, Ir after heat shock protein 90 inhibitor; 17-allylamino-17-demethoxygeldanamycin (17-AAG) and gold nanoparticle (GNP) treatments in human colorectal cancer cell line (HCT-116), with the targeting of related mechanisms. METHODS: Water-soluble tetrazolium salt-1 assay was utilized to study the cytotoxic effects of 17-AAG, GNP, Ir in single and combination cases on the cell viability of HCT-116 cells. The cells were examined with DNA fragmentation electrophoresis and evaluated for apoptosis induction. Caspase-3 expression as a critical apoptosis element in protein level was detected by western blotting. RESULTS: Treatment with 17-AAG in a dose dependent manner for 24 h inhibited the cellular viability of HCT-116 cells. GNP at a dose of 70 µM had the lowest cytotoxic effects and was thus selected for combination treatment studies. Based on the results, GNP at a dose of 70 µM did not have a significant effect on cellular viability of HCT-116. In contrast, the evaluation of double and triple combinations, GNP with Ir (2 Gy of 6 MV X-ray radiation) and 17-AAG in double combinations induced significant cytotoxicity. Both DNA damage pattern and caspase-3 protein upregulation were present in Ir,GNP/17-AAG,GNP and Ir,17-AAG combinations compared to single treatments. Furthermore, in the three combination of GNP,Ir,17-AAG, radiosensitization effects (increased caspase-3 expression) occurred with a minimum concentration of 17-AAG. CONCLUSION: According to the results of this study, 17-AAG as chemotherapeutic agent in combination with Ir and GNP exerts noticeable anti-cancer effects, inhibited cell viability, and increased apoptosis occurrence by upregulating caspase-3 expression. It is suggested that these combinations should be more evaluated as a promising candidate for colorectal cancer treatment. Graphical abstract Anti-cancer effects of chemotherapeutic agent; 17-AAG, in combined with gold nanoparticles and irradiation in human colorectal cancer cells.


Assuntos
Benzoquinonas/farmacologia , Caspase 3/metabolismo , Neoplasias Colorretais/metabolismo , Ouro/farmacologia , Lactamas Macrocíclicas/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiorradioterapia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/radioterapia , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HCT116 , Humanos , Nanopartículas Metálicas , Regulação para Cima
19.
Biochem Biophys Res Commun ; 511(2): 260-265, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30791979

RESUMO

Lung cancer causes the highest number of cancer-related deaths worldwide. Resistance to therapy is a major clinical issue contributing to the poor prognosis of lung cancer. In recent years, targeted therapy has become a concept where subgroups of non-small cell lung cancer (NSCLC) with genetically altered receptor tyrosine kinases are targeted by tyrosine kinase inhibitors (TKIs). One such subgroup harbors a gene fusion of echinoderm microtubule-associated protein-like 4 (EML4) with anaplastic lymphoma kinase (ALK). Although most NSCLC patients with EML4-ALK fusions initially respond to ALK TKI-therapy they eventually develop resistance. While ALK kinase domain mutations contribute to ALK TKI-refractoriness, they are only present in a fraction of all ALK TKI-resistant tumors. In this study we sought to explore a possible involvement of microRNAs (miRNAs) in conferring resistance to ALK TKIs in ALK TKI-refractory NSCLC cell lines. We subjected our ALK TKI-refractory cancer cells along with parental cancer cells to systematic miRNA expression arrays. Furthermore, ALK TKI-refractory cancer cells were exposed to a synthetic miRNA inhibitory Locked Nucleic Acid (LNA)-library in the presence of ALK TKIs Crizotinib or Lorlatinib. The outcome of the combined approaches uncovered miR-100-5p to confer resistance to Crizotinib and Lorlatinib in EML4-ALK NSCLC cells and to be a potential therapeutic target in drug resistance.


Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Neoplasias Pulmonares/tratamento farmacológico , MicroRNAs/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/farmacologia , Serina Endopeptidases/genética , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Crizotinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lactamas Macrocíclicas/farmacologia , Neoplasias Pulmonares/genética
20.
J Clin Invest ; 129(2): 744-758, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30640172

RESUMO

Goblet cell metaplasia, a disabling hallmark of chronic lung disease, lacks curative treatments at present. To identify novel therapeutic targets for goblet cell metaplasia, we studied the transcriptional response profile of IL-13-exposed primary human airway epithelia in vitro and asthmatic airway epithelia in vivo. A perturbation-response profile connectivity approach identified geldanamycin, an inhibitor of heat shock protein 90 (HSP90) as a candidate therapeutic target. Our experiments confirmed that geldanamycin and other HSP90 inhibitors prevented IL-13-induced goblet cell metaplasia in vitro and in vivo. Geldanamycin also reverted established goblet cell metaplasia. Geldanamycin did not induce goblet cell death, nor did it solely block mucin synthesis or IL-13 receptor-proximal signaling. Geldanamycin affected the transcriptome of airway cells when exposed to IL-13, but not when exposed to vehicle. We hypothesized that the mechanism of action probably involves TGF-ß, ERBB, or EHF, which would predict that geldanamycin would also revert IL-17-induced goblet cell metaplasia, a prediction confirmed by our experiments. Our findings suggest that persistent airway goblet cell metaplasia requires HSP90 activity and that HSP90 inhibitors will revert goblet cell metaplasia, despite active upstream inflammatory signaling. Moreover, HSP90 inhibitors may be a therapeutic option for airway diseases with goblet cell metaplasia of unknown mechanism.


Assuntos
Benzoquinonas/efeitos adversos , Células Caliciformes/metabolismo , Proteínas de Choque Térmico HSP90 , Interleucina-13/metabolismo , Interleucina-17/metabolismo , Lactamas Macrocíclicas/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Animais , Benzoquinonas/farmacologia , Células Caliciformes/patologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Masculino , Metaplasia , Camundongos
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