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1.
Cell Physiol Biochem ; 53(2): 400-412, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31403270

RESUMO

BACKGROUND/AIMS: Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. METHODS: We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. RESULTS: G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. CONCLUSION: Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Transportadores de Cassetes de Ligação de ATP/genética , Aminopiridinas/uso terapêutico , Anilidas/farmacologia , Benzodioxóis/uso terapêutico , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Leupeptinas/farmacologia , Lisossomos/metabolismo , Degeneração Macular/congênito , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , Transporte Proteico/efeitos dos fármacos
2.
Gene ; 710: 218-232, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31158448

RESUMO

Alterations in the global gene expression profile are considered to contribute to the various physiological and pathological changes during the course of ageing. Genes that code for the molecular components of the innate system are alter markedly as ageing occurs; and this may define the susceptibility of very young and very old individuals to reproductive tract infections. The expression pattern of genes that code for beta-defensins (effectors of innate immune response) in male reproductive tract tissues of different stages of ageing is not yet reported. Further, the induction of beta-defensins during endotoxin challenge and whether epigenetic modulators can influence the expression of these genes in different stages of ageing are not reported. We analyzed the basal mRNA levels of beta-defensins and defensin-like proteins (Sperm Associated Antigen 11 (SPAG11) family members), their induction during endotoxin challenge and modulation by epigenetic modifiers (Trichostatin A and Azacytidine) in the caput, cauda, testis, prostate and seminal vesicle of rats that represent early stage to late stages of life (20 day to 730 day old). We observed differential basal gene expression pattern in the male reproductive tract tissues and the induction by LPS was not consistent neither among the age groups not the tissues analyzed. Trichostatin A and Azacytidine also influenced antimicrobial gene expression and the pattern was not consistent in different tissues obtained from different age groups. Results of this study demonstrate that antimicrobial gene expression varies to a great extent during ageing and is strongly influenced by endotoxins and epigenetic modulators.


Assuntos
Envelhecimento/genética , Genitália Masculina/química , Glicopeptídeos/genética , beta-Defensinas/genética , Animais , Azacitidina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genitália Masculina/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Ratos , Ratos Wistar
3.
Mol Med Rep ; 19(6): 5251-5262, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059100

RESUMO

Keloids are benign fibrous overgrowths that occur as a result of abnormal wound healing following cutaneous injury. MicroRNAs (miRNAs/miRs) are short non­coding RNAs that serve critical roles in numerous important biological processes, such as cell proliferation, differentiation and apoptosis. However, their role in keloid development remains largely unknown. In the present study, the role of miR­30a­5p, a miRNA regulated by Trichostatin A (TSA), in apoptosis within cultured keloid fibroblasts was investigated. An MTT assay was used to detect the proliferation of cultured keloid fibroblasts treated with TSA. Cell apoptosis and cell cycle phases were analyzed using flow cytometry. In addition, an miRNA microarray was performed to compare expression profiles between cultured keloid fibroblasts treated with or without 1,000 nM TSA. Reverse transcription­quantitative polymerase chain reaction analysis was conducted to estimate miRNA expression levels. The direct target of miR­30a­5p was identified using a dual­luciferase reporter assay. Western blotting was employed to assess protein expression levels in keloid fibroblasts. The results demonstrated that TSA inhibited the proliferation of keloid fibroblasts in a time­ and dose­dependent manner. The miRNA microarray revealed alterations in the expression of numerous miRNA sequences in response to TSA when compared with controls. Notably, the expression of miR­30a­5p was downregulated in keloid tissues. In addition, overexpression of miR­30a­5p induced apoptosis by targeting B­cell lymphoma 2, which was similar to that observed in response to TSA. These results provide important information regarding a novel miR­30a­5p­mediated signaling pathway induced by TSA treatment, and suggest a potential use for TSA and miR­30a­5p as effective therapeutic strategies for keloids.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regiões 3' não Traduzidas , Antagomirs/metabolismo , Análise por Conglomerados , Colágeno/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Queloide/metabolismo , Queloide/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Pele/metabolismo , Pele/patologia
4.
Life Sci ; 228: 112-120, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31051152

RESUMO

AIMS: Cigarette smoking results in well-known negative reproductive consequences. However, the role of histone deacetylase 1 and 2 (HDAC1/2) in the structural changes of uterine tissues induced by cigarette smoke (CS) exposure and the therapeutic potential of trichostatin A (TSA), a HDAC inhibitor, have not been investigated. MAIN METHODS: Female mice were exposed to CS twice daily for 30 days and TSA was injected intraperitoneally into CS-exposed mice on alternate days in the TSA-treated group. Uteri in the estrus phase were weighed and uterine histomorphology and HDAC1 cell distribution were examined by HE and immunohistochemistry. Markers associated with macro-autophagy (Beclin-1), autophagic flux (increased LC3-II and a lack of p62 accumulation), autophagy inhibiting factor (mTOR, phosphorylated mTOR and its upstream IRS, phosphorylated IRS), HDAC1/2, FOXO1 and FOXO3 were assessed by Western blot. KEY FINDINGS: CS exposure decreased body weight and triggered uterine histomorphologic alterations, including a thinner myometrium and a reduced number of glandular and interstitial cells. HDAC1/2 were activated in uterine tissues after CS exposure and TSA effectively inhibited HDAC1/2 activation and attenuated the loss of body weight and uterine wet weight induced by CS exposure. TSA effectively restored the thickness of the myometrium and number of glandular and interstitial cells. TSA also restored the expression of markers of macro-autophagy (LC3-II and Beclin-1) and reduced phosphorylated mTOR, phosphorylated IRS, FOXO1 and FOXO3 activation. SIGNIFICANCE: TSA inhibited uterine histomorphologic alterations induced by CS exposure. The TSA effect might be associated with resumption of macro-autophagy via HDAC1/2 inhibition.


Assuntos
Fumar Cigarros/efeitos adversos , Fumar Cigarros/patologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Substâncias Protetoras/farmacologia , Útero/efeitos dos fármacos , Útero/patologia , Animais , Autofagia/efeitos dos fármacos , Fumar Cigarros/metabolismo , Feminino , Camundongos Endogâmicos C57BL , Útero/metabolismo
5.
J Enzyme Inhib Med Chem ; 34(1): 1062-1077, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31072216

RESUMO

Histone deacetylase 6 (HDAC6) is an attractive target for cancer therapeutic intervention. Selective HDAC6 inhibitors is important to minimise the side effects of pan inhibition. Thus, new class of hydroxamic acid-based derivatives were designed on structural basis to perform preferential activity against HDAC6 targeting solid tumours. Interestingly, 1-benzylbenzimidazole-2-thio-N-hydroxybutanamide 10a showed impressive preference with submicromolar potency against HDAC6 (IC50 = 510 nM). 10a showed cytotoxic activity with interesting profile against CCHE-45 at (IC50 = 112.76 µM) when compared to standard inhibitor Tubacin (IC50 = 20 µM). Western blot analysis of acetylated-α-tubulin verified the HDAC6 inhibiting activity of 10a. Moreover, the insignificant difference in acetylated-α-tubulin induced by 10a and Tubacin implied the on-target cytotoxic activity of 10a. Docking of 10a in the binding site of HDAC6 attributed the activity of 10a to π-π stacking with the amino acids of the hydrophobic channel of HDAC6 and capture of zinc metal in bidentate fashion. The therapeutic usefulness besides the on-target activity may define 10a as an interesting safe-lead inhibitor for future development.


Assuntos
Antineoplásicos/farmacologia , Carcinoma/tratamento farmacológico , Neoplasias do Plexo Corióideo/tratamento farmacológico , Desenho de Drogas , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Plexo Corióideo/metabolismo , Neoplasias do Plexo Corióideo/patologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Estrutura Molecular , Relação Estrutura-Atividade
6.
Bull Exp Biol Med ; 166(6): 719-721, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31020584

RESUMO

Pulmonary edema is the major factor of tissue hypoxia in acute lung injury. Disruption of cell-cell contacts and lung interstitium increases permeability of the vascular endothelium and alveolar epithelium, which leads to the development of pulmonary edema. Meprin metalloproteases cleave extracellular matrix proteins, thus aggravating pulmonary edema. Meprin inhibitor actinonin was administered to rats with LPS-induced acute lung injury. Damaged lungs looked spotted and had multiple hemorrhage focuses, protein concentration in lavage fluid was increased, and lung weight coefficient was high. Administration of meprin inhibitor actinonin considerably reduced protein content in the bronchoalvelolar lavage and lung coefficient; only solitary lung hemorrhages were seen after this treatment. Thus, inhibition of meprins potentially alleviates LPS-induced disorders in the lung tissue permeability and reduces pulmonary edema.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Edema Pulmonar/tratamento farmacológico , Tiopronina/antagonistas & inibidores , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/patologia , Animais , Líquido da Lavagem Broncoalveolar/química , Ácidos Hidroxâmicos/farmacologia , Lipopolissacarídeos/administração & dosagem , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/enzimologia , Edema Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Tiopronina/metabolismo
7.
Chem Biol Interact ; 306: 54-61, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30958996

RESUMO

In the present study, we investigated the p53-independent mechanism by which quercetin (Q) increased apoptosis in human lung cancer H1299 cells exposed to trichostatin A (TSA), a histone deacetylase inhibitor. We also investigated the role of Q in increasing the acetylation of histones H3 and H4 and the possible mechanism. Q at 5 µM significantly increased apoptosis by 88% in H1299 cells induced by TSA at 72 h. Q also significantly increased TSA-induced death receptor 5 (DR5) mRNA and protein expression as well as caspase-10/3 activities in H1299 cells. Transfection of DR5 siRNA into H1299 cells significantly diminished the enhancing effects of Q on TSA-induced apoptosis. Furthermore, TSA in combination with Q rather than TSA alone significantly increased p300 expression. Transfection of p300 siRNA in H1299 cells significantly diminished the increase of histone H3/H4 acetylation, DR5 protein expression, caspase-10/3 activity and apoptosis induced by Q. In addition, similar effects of Q were observed when Q was combined with vorinostat, another FDA-approved histone deacetylase inhibitor. These data suggest that the up-regulation of p300 expression, which in turn increases histone acetylation and DR5 expression, plays an important role in the enhancing effect of Q on TSA/vorinostat- induced apoptosis in H1299 cells.


Assuntos
Antineoplásicos/farmacologia , Proteína p300 Associada a E1A/genética , Ácidos Hidroxâmicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Quercetina/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteína p300 Associada a E1A/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Vorinostat/farmacologia
8.
Eur J Med Chem ; 173: 90-98, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30986574

RESUMO

As a group of biologically active compounds, polyether antibiotics (ionophores) show a broad spectrum of interesting pharmacological properties, ranging from anti-bacterial to anti-cancer activities. There is increasing evidence that ionophores, including salinomycin (SAL), and their semi-synthetic analogues are promising candidates for the development of drugs against parasitic diseases. Our previous studies have shown that esterification and amidation of the C1 carboxylate moiety of SAL provides compounds with potent activity against Trypanosoma brucei, protozoan parasites responsible for African trypanosomiasis. In this paper, we present the synthetic pathways, crystal structures and anti-trypanosomal activity of C1 esters, amides and hydroxamic acid conjugates of SAL, its C20-oxo and propargylamine analogues as well novel C1/C20 doubly modified derivatives. Evaluation of the trypanocidal and cytotoxic activity using bloodstream forms of T. brucei and human myeloid HL-60 cells revealed that the single-modified C20-oxo and propargylamine precursor molecules 10 and 16 were the most anti-trypanosomal and selective compounds with 50% growth inhibition (GI50) values of 0.037 and 0.035 µM, and selectivity indices of 252 and 300, respectively. Also the salicylhydroxamic acid conjugate of SAL (compound 9) as well as benzhydroxamic acid and salicylhydroxamic acid conjugates of 10 (compounds 11 and 12) showed promising trypanocidal activities with GI50 values between 0.032 and 0.035 µM but less favorable selectivities. The findings confirm that modification of SAL can result in derivatives with improved trypanocidal activity that might be interesting lead compounds for further anti-trypanosomal drug development.


Assuntos
Ácidos Hidroxâmicos/farmacologia , Salicilamidas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Células HL-60 , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Salicilamidas/síntese química , Salicilamidas/química , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/química , Células Tumorais Cultivadas
9.
Int J Mol Sci ; 20(5)2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30841499

RESUMO

Besides its key role in neural development, brain-derived neurotrophic factor (BDNF) is important for long-term potentiation and neurogenesis, which makes it a critical factor in learning and memory. Due to the important role of BDNF in synaptic function and plasticity, an in-house epigenetic library was screened against human neural progenitor cells (HNPCs) and WS1 human skin fibroblast cells using Cell-to-Ct assay kit to identify the small compounds capable of modulating the BDNF expression. In addition to two well-known hydroxamic acid-based histone deacetylase inhibitors (hb-HDACis), SAHA and TSA, several structurally similar HDAC inhibitors including SB-939, PCI-24781 and JNJ-26481585 with even higher impact on BDNF expression, were discovered in this study. Furthermore, by using well-developed immunohistochemistry assays, the selected compounds were also proved to have neurogenic potential improving the neurite outgrowth in HNPCs-derived neurons. In conclusion, we proved the neurogenic potential of several hb-HDACis, alongside their ability to enhance BDNF expression, which by modulating the neurogenesis and/or compensating for neuronal loss, could be propitious for treatment of neurological disorders.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Crescimento Neuronal , Benzimidazóis/farmacologia , Benzofuranos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Ácidos Hidroxâmicos/farmacologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo
10.
Int J Mol Sci ; 20(5)2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30866433

RESUMO

Trichostatin A (TSA), an antifungal antibiotic derived from Streptomyces, inhibits mammalian histone deacetylases, and especially, selectively inhibits class I and II histone deacetylase (HDAC) families of enzymes. TSA reportedly elicits an antiproliferative response in multifarious tumors. This study investigated the antitumor effects of TSA alone and in combination with paclitaxel when applied to two high-grade urothelial carcinoma (UC) cell lines (BFTC-905 and BFTC-909). Fluorescence-activated cell sorting, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay were used to assess TSA's cytotoxicity and effects on apoptosis induction. TSA induced synergistic cytotoxicity, when combined with paclitaxel (combination index < 1), resulted in concomitant suppression of paclitaxel-induced activation of phospho-extracellular signal-regulated kinase (ERK) 1/2. A xenograft nude mouse model confirmed that TSA enhances the antitumor effects of paclitaxel. These findings demonstrate that the administration of TSA in combination with paclitaxel elicits a synergistic cytotoxic response. The results of this study indicate that the chemoresistance of UC could be circumvented by combining HDAC inhibitors to target the ERK pathway.


Assuntos
Carcinoma de Células de Transição/tratamento farmacológico , Inibidores de Histona Desacetilases/administração & dosagem , Ácidos Hidroxâmicos/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Paclitaxel/administração & dosagem , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Paclitaxel/farmacologia , Resultado do Tratamento , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Int J Oncol ; 54(5): 1797-1808, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864703

RESUMO

Glioblastoma or grade IV astrocytoma is the most common and lethal form of glioma. Current glioblastoma treatment strategies use surgery followed by chemotherapy with temozolomide. Despite this, numerous glioblastoma cases develop resistance to temozolomide treatments, resulting in a poor prognosis for the patients. Novel approaches are being investigated, including the inhibition of histone deacetylase 6 (HDAC6), an enzyme that deacetylates α­tubulin, and whose overexpression in glioblastoma is associated with the loss of primary cilia. The aim of the present study was to treat glioblastoma cells with a selective HDAC6 inhibitor, tubastatin A, to determine if the malignant phenotype may be reverted. The results demonstrated a notable increase in acetylated α­tubulin levels in treated cells, which associated with downregulation of the sonic hedgehog pathway, and may hypothetically promote ciliogenesis in those cells. Treatment with tubastatin A also reduced glioblastoma clonogenicity and migration capacities, and accelerated temozolomide­induced apoptosis. Finally, HDAC6 inhibition decreased the expression of mesenchymal markers, contributing to reverse epithelial­mesenchymal transition in glioblastoma cells.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Temozolomida/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Desacetilase 6 de Histona/genética , Humanos , Fenótipo , Regulação para Cima/efeitos dos fármacos
12.
Int J Oncol ; 54(5): 1613-1624, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896789

RESUMO

The SLC5A8 gene encodes Na monocarboxylate transporter 1, which is epigenetically inactivated in various tumour types. This has been attributed to the fact that it prevents the entry of histone deacetylase (HDAC) inhibitors and favours the metabolic reprogramming of neoplastic cells. Nevertheless, its expression and regulation in cervical cancer (CC) have not been elucidated to date. The aim of the present study was to investigate whether SLC5A8 expression is silenced in CC and if epigenetic mechanisms are involved in its regulation. Using RNA and DNA from human CC cell lines and tumour tissues from patients with CC, the expression of SLC5A8 was analysed by reverse transcription polymerase chain reaction and the methylation status of its CpG island (CGI) by bisulphite­modified sequencing. Additionally, SLC5A8 reactivation was examined in the CC cell lines following treatment with DNA methylation (5­aza­2'­deoxycytidine) and HDAC inhibitors (trichostatin A and pyruvate). All the CC cell lines and a range of tumour tissues (65.5%) exhibited complete or partial loss of SLC5A8 transcription. The bisulphite­sequencing revealed that hypermethylation of the CGI within SLC5A8 first exon was associated with its downregulation in the majority of cases. The transporter expression was restored in the CC cell lines following exposure to 5­aza­2'­deoxycytidine alone, or in combination with trichostatin A or pyruvate, suggesting that DNA methylation and histone deacetylation contribute to its inhibition in a cell line­dependent manner. Together, the results of the present study demonstrate the key role of DNA hypermethylation in the repression of SLC5A8 in CC, as well as the involvement of histone deacetylation, at least partially. This allows for research focused on the potential function of SLC5A8 as a tumour suppressor in CC, and as a biomarker or therapeutic target in this malignancy.


Assuntos
Regulação para Baixo , Repressão Epigenética , Transportadores de Ácidos Monocarboxílicos/genética , Infecções por Papillomavirus/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Adulto , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA , Decitabina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Pessoa de Meia-Idade , Ácido Pirúvico/farmacologia , Análise de Sequência de DNA , Adulto Jovem
13.
BMC Cancer ; 19(1): 262, 2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30902084

RESUMO

BACKGROUND: Epithelial-mesenchymal transition (EMT) may be one of the reasons for the failure in some clinical trials regarding histone deacetylase inhibitors (HDACIs)-treated solid tumors. We investigated the effects of a pan-HDACI trichostatin A (TSA) on the proliferation and EMT of nasopharyngeal carcinoma (NPC) cells. METHODS: Poorly-differentiated NPC cell line CNE2 and undifferentiated C666-1 were treated with various concentrations of TSA, the cell viability was assessed by CCK-8 assay, the morphology was photographed, and the mRNA level of HDACs was assessed by semiquantitative PCR. After determination the cell cycle distributions, cells were subjected to western blotting analysis of cell cycle and EMT-associated genes expression. And the changes in migration ability were assessed by transwell migration assay and scratch wound healing assay. Finally, histone deacetylases activator ITSA-1 was used to assess the reverse of TSA-induced changes in NPC cells. RESULTS: TSA inhibited the proliferation of CNE2 and C666-1 cells in a concentration-dependent manner and arrested the cell cycle at G1 phases. TSA reduced PCNA, cyclin D1, cyclin E1, CDK2, p16 and p21 expressions and stimulated CDK6 levels. TSA stimulation for 48 h could effectively induce the EMT in CNE2 and C666-1 cells, which showed an increase of spindle-like cells and promoted expression of Vimentin and Snail1 expression in a concentration-dependent manner. Surprisingly, this short period of TSA treatment that induced EMT also impeded the migration ability of CNE2 and C666-1 cells. Interestingly, ITSA-1 rescued TSA-impeded CNE2 and C666-1 cells' proliferation, migration and HDACs expression, also re-induced the cells to turn into epithelial cell phenotypes. CONCLUSIONS: These results indicate that short-term stimulation of TSA effectively inhibits cell proliferation and induce EMT-like changes in NPC cells but not increase its invasion ability.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Ácidos Hidroxâmicos/uso terapêutico , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Fatores de Tempo
14.
Int J Oncol ; 54(5): 1759-1770, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30816432

RESUMO

Sunitinib is the most common primary molecular­targeted agent for metastatic clear cell renal cell carcinoma (ccRCC); however, intrinsic or acquired sunitinib resistance has become a significant problem in medical practice. The present study focused on microRNA (miR)­99a­3p, which was significantly downregulated in clinical sunitinib­resistant ccRCC tissues in previous screening analyses, and investigated the molecular network associated with it. The expression levels of miR­99a­3p and its candidate target genes were evaluated in RCC cells, including previously established sunitinib­resistant 786­o (SU­R­786­o) cells, and clinical ccRCC tissues, using reverse transcription­quantitative polymerase chain reaction. Gain­of­function studies demonstrated that miR­99a­3p significantly suppressed cell proliferation and colony formation in RCC cells, including the SU­R­786­o cells, by inducing apoptosis. Based on in silico analyses and RNA sequencing data, followed by luciferase reporter assays, ribonucleotide reductase regulatory subunit­M2 (RRM2) was identified as a direct target of miR­99a­3p in the SU­R­786­o cells. Loss­of­function studies using small interfering RNA against RRM2 revealed that cell proliferation and colony growth were significantly inhibited via induction of apoptosis, particularly in the SU­R­786­o cells. Furthermore, the RRM2 inhibitor Didox (3,4­dihydroxybenzohydroxamic acid) exhibited anticancer effects in the SU­R­786­o cells and other RCC cells. To the best of our knowledge, this is the first report demonstrating that miR­99a­3p directly regulates RRM2. Identifying novel genes targeted by tumor­suppressive miR­99a­3p in sunitinib­resistant RCC cells may improve our understanding of intrinsic or acquired resistance and facilitate the development of novel therapeutic strategies.


Assuntos
Carcinoma de Células Renais/genética , Resistencia a Medicamentos Antineoplásicos , Neoplasias Renais/genética , MicroRNAs/genética , Ribonucleosídeo Difosfato Redutase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Ribonucleosídeo Difosfato Redutase/metabolismo , Sunitinibe/farmacologia
15.
Planta ; 249(6): 1747-1760, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30820648

RESUMO

MAIN CONCLUSION: Siderophores are a driver of Pinus sylvestris root responses to metabolites secreted by pathogenic and mycorrhizal fungi. Structurally different siderophores regulate the uptake of Fe by microorganisms and may play a key role in the colonization of plants by beneficial or pathogenic fungi. Siderophore action, however, may be dependent on the distribution of Fe within cells. Here, the involvement of siderophores in determining the changes of organelle morphology and element composition of some cellular fractions of root cells in Pinus sylvestris to trophically diverse fungi was investigated. Changes in the morphology and concentrations of different elements within organelles of root cells in response to three structurally different siderophores were examined by transmission electron microscopy combined with energy-dispersive X-ray spectroscopy. Weak development of mitochondrial cristae and the deposition of backup materials in plastids occurred in the absence of Fe in the structures of triacetylfusarinine C and ferricrocin. In response to metabolites of both pathogenic and mycorrhizal fungi, Fe accumulated mainly in the cell walls and cytoplasm. Fe counts increased in all of the analyzed organelles in response to applications of ferricrocin and triacetylfusarinine C. Chelation of Fe within the structure of siderophores prevents the binding of exogenous Fe, decreasing the abundance of Fe in the cell wall and cytoplasm. The concentrations of N, P, K, Ca, Mn, Cu, Mg, and Zn also increased in cells after applications of ferricrocin and triacetylfusarinine C, while the levels of these elements decreased in the cell wall and cytoplasm when Fe was present within the structure of the siderophores. These results provide insight into the siderophore-driven response of plants to various symbionts.


Assuntos
Compostos Férricos/farmacologia , Ferricromo/análogos & derivados , Ácidos Hidroxâmicos/farmacologia , Ferro/metabolismo , Micorrizas/fisiologia , Pinus sylvestris/efeitos dos fármacos , Sideróforos/farmacologia , Núcleo Celular/ultraestrutura , Parede Celular/metabolismo , Citoplasma/metabolismo , Desferroxamina/química , Desferroxamina/farmacologia , Compostos Férricos/química , Ferricromo/química , Ferricromo/farmacologia , Fungos/fisiologia , Ácidos Hidroxâmicos/química , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Organelas/efeitos dos fármacos , Organelas/ultraestrutura , Pinus sylvestris/microbiologia , Pinus sylvestris/ultraestrutura , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/microbiologia , Raízes de Plantas/ultraestrutura , Sideróforos/metabolismo
16.
Int J Nanomedicine ; 14: 1335-1346, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30863064

RESUMO

Background: The efficacy of epigenetic drugs, such as histone deacetylase inhibitors, is often diminished by poor aqueous solubility resulting in limited bioavailability and a low therapeutic index. To overcome the suboptimal therapeutic index, we have developed a biocompatible starch nanoparticle formulation of CG-1521, a histone deacetylase inhibitor in preclinical development for hard-to-treat breast cancers, which improves its bioavailability and half-life. Methods: The physicochemical parameters (size, zeta potential, morphology, loading, and release kinetics) of these nanoparticles (CG-NPs) have been optimized and their cytotoxic and apoptotic capacities measured in MCF-7 breast cancer cell line. The mechanism of action of the encapsulated drug was compared with the free drug at molecular level. Results: We show that encapsulation of CG-1521 substantially reduces the release rate of drug and provides a significantly enhanced cytotoxic ability of nanoparticles compared with equivalent dose of free CG-1521. CG-NPs induced cell cycle arrest and significant apoptosis in MCF-7 cells in vitro. The biological action of encapsulated drug has the similar impact with free drug on gene expression. Conclusion: The findings suggest that encapsulation of CG-1521 into starch nanoparticles can improve drug delivery of histone deacetylase inhibitors for breast cancer therapy without interfering with the mechanism of action of the drug.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Nanopartículas/química , Amido/química , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Liberação Controlada de Fármacos , Feminino , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Cinética , Células MCF-7 , Nanopartículas/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solubilidade , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
17.
In Vivo ; 33(2): 425-432, 2019 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30804121

RESUMO

BACKGROUND/AIM: Oxidative stress caused by the production of excessive cellular reactive oxygen species (ROS) and high levels of nitric oxide contribute to several human pathologies. This study aimed to examine the anti-oxidant effects of fusigen, a compound produced from Aureobasidium melanogenum. MATERIALS AND METHODS: Extracts of A. melanogenum were selected as a source for the isolation of fusigen. The anti-oxidant, nitric oxide suppression, as well as the free radical scavenging activities of fusigen were tested in BEAS-2B human bronchial epithelial cell line (BEAS-2B cells) and human dermal papilla cells (DP cells) using specific fluorescence dyes and flow cytometry analysis. Cell viability was determined by the MTT assay. RESULTS: Fusigen did not exert cytotoxicity in the human normal BEAS-2B and DP cells at concentrations up to 100 µM. Fusigen decreased basal levels of cellular ROS, as well as the levels of ROS induced by hydrogen peroxide and ferrous ion enrichment. ROS decreasing effect was confirmed in DP cells. In addition, fusigen treatment suppressed intracellular NO levels in both BEAS-2B and DP cells. CONCLUSION: The optimal process of production of purified fusigen from A. melanogenum was determined. Fusigen exhibited a low cytotoxic effect and the potential to suppress ROS and NO. These results demonstrated that fusigen may be used for the treatment or prevention of human diseases.


Assuntos
Ascomicetos/química , Compostos Férricos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Brônquios/citologia , Brônquios/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Compostos Férricos/química , Humanos , Peróxido de Hidrogênio/química , Ácidos Hidroxâmicos/química , Óxido Nítrico/metabolismo , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo
18.
BMC Ophthalmol ; 19(1): 42, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717701

RESUMO

BACKGROUND: To compare the protective effects of the histone deacetylase inhibitors (HDACis) ß-hydroxybutyrate (ßOHB), trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) on human lens epithelial cells(HLECs) following ultraviolet-B (UVB) exposure. METHODS: HLECs were divided into subgroups: four HDACi groups, a control group, a UVB-treated group and a DMSO group (cells treated with DMSO and UVB irradiation). In the HDACi groups, HLECs were cultured with different concentrations of HDACis 12 h prior to UVB irradiation. The protective effects of the HDACis were evaluated by assessing apoptosis rates, cell activity and expression levels of genes associated with apotosis (caspase-3, Bcl-2, BAX, SOD1, FOXO3A and MT2). The levels of superoxide dismutase (SOD), reactive oxygen species (ROS), malondialdehyde (MDA) and total antioxidant capacity (T-AOC) were detected in order to evaluate oxidative stress. RESULTS: The results showed that SAHA (1 µmol/L, 2 µmol/L) and TSA (0.2 µmol/L) had mild protective effects on cell viability. ßOHB (4 mmol/L) and TSA (0.2 mol/L) demonstrated protective effects on BCL-2 expression. TSA (0.2 mol/L) showed protective effects on SOD1 expression. TSA (0.2 mol/L) and SAHA (1 µmol/L) suppressed BAX and caspase-3 expression. TSA (0.2 mol/L, 0.8 mol/L) and SAHA (1 µmol/L, 2 µmol/L) suppressed the expression of FOXO3A and MT2. SOD levels were increased after treatment with ßOHB (4 mmol/L), SAHA (8 µmol/L) and TSA (0.1 mol/L, 0.2 mol/L). T-AOC levels were increased in UVB-treated HLECs after treatment with SAHA (2 µmol/L). MDA levels decreased in UVB-treated HLECs following treatment with TSA (0.2 mol/L, 0.8 mol/L). ROS levels decreased in UVB-treated HLECs following treatment with ßOHB (4 mmol/L), SAHA (1 µmol/L, 2 µmol/L) and TSA (0.2 mol/L). Western blotting results demonstrated that SOD1 levels significantly increased in the ßOHB (4 mmol/L), SAHA (1 µmol/L, 2 µmol/L), TSA (0.1 mol/L, 0.2 mol/L) and VPA (5 mmol/L) groups. Only SAHA (1 µmol/L) had an anti-apoptotic effect on UVB-treated HLECs. CONCLUSIONS: Our findings indicate that low concentrations of HDACis (1 µmol/L of SAHA) mildly inhibit oxidative stress, thus protecting HLECs from oxidation. These results may suggest that there is a possibility to explore the clinical applications of HDACis for treatment and prevention of cataracts.


Assuntos
Antioxidantes/farmacologia , Células Epiteliais/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Raios Ultravioleta/efeitos adversos , Ácido 3-Hidroxibutírico/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos da radiação , Humanos , Ácidos Hidroxâmicos/farmacologia , Cristalino/citologia , Ácido Valproico/farmacologia , Vorinostat/farmacologia
19.
Drug Saf ; 42(2): 235-245, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30649740

RESUMO

Histone deacetylases (HDACs) are expressed at increased levels in cells of various malignancies, and the use of HDAC inhibitors has improved outcomes in patients with haematological malignancies (T-cell lymphomas and multiple myeloma). However, they are not as effective in solid tumours. Five agents are currently approved under various jurisdictions, namely belinostat, chidamide, panobinostat, romidepsin and vorinostat. These agents are associated with a range of class-related and agent-specific serious and/or severe adverse effects, notably myelosuppression, diarrhoea and various cardiac effects. Among the cardiac effects are ST-T segment abnormalities and QTc interval prolongation of the electrocardiogram, isolated cases of atrial fibrillation and, in rare instances, ventricular tachyarrhythmias. In order to improve the safety profile of this class of drugs as well as their efficacy in indications already approved and to further widen their indications, a large number of newer HDAC inhibitors with varying degrees of HDAC isoform selectivity have been synthesised and are currently under clinical development. Preliminary evidence from early studies suggests that they may be effective in non-haematological cancers as well when used in combination with other therapeutic modalities, but that they too appear to be associated with the above class-related adverse effects. As the database accumulates, the safety, efficacy and risk/benefit of the newer agents and their indications will become clearer.


Assuntos
Antineoplásicos/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Gastroenteropatias/induzido quimicamente , Gastroenteropatias/metabolismo , Inibidores de Histona Desacetilases/efeitos adversos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/efeitos adversos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Neoplasias/metabolismo , Sulfonamidas/efeitos adversos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico
20.
J Mol Histol ; 50(2): 167-178, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30671879

RESUMO

Peripheral nerves, which consist of an axon and a unique glial cell called a Schwann cell, transduce signals from the brain and spinal cord to target organs. Peripheral nerve degeneration leads to distal motor or sensory disorders such as diabetic neuropathy, Charcot-Marie-Tooth disease, and Gullain-Barré syndrome, with symptoms such as dysesthesia, speech impairment, vision change, erectile dysfunction, and urinary incontinence. Schwann cells play an important role in peripheral nerve degeneration. Therefore, revealing the characteristics of Schwann cells will be essential in understanding peripheral neurodegeneration-related diseases for which there is currently no effective treatment. Trichostatin A (TSA) is a noncompetitive, reversible inhibitor of class I and II histone deacetylases (HDACs). HDACs have been shown not only to deacetylate histones but also to target non-histone proteins involved in diverse signaling pathways. Recent studies have revealed that diverse HDAC subtypes regulate peripheral neurodegeneration. Thus, regulating HDAC levels could be an effective strategy for the development of drugs targeting peripheral nerve-related diseases. In fact, the use of TSA has been investigated for the treatment of many diseases, including degenerative diseases of the central nervous system; however, the effects of TSA on peripheral neurodegeneration have not yet been well established. In this study, we revealed the effect of TSA on the process of peripheral neurodegeneration. TSA successfully inhibited myelin fragmentation, axonal degradation, and trans-dedifferentiation and proliferation of Schwann cells, which are essential phenotypes in peripheral neurodegeneration. Therefore, TSA could be a potential drug for patients suffering from peripheral neurodegeneration-related diseases.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Degeneração Neural/prevenção & controle , Doenças do Sistema Nervoso Periférico/prevenção & controle , Axônios/efeitos dos fármacos , Axônios/metabolismo , Desdiferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Células de Schwann/patologia
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