RESUMO
The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.
Assuntos
Retinopatia Diabética , Ácidos Hidroxâmicos , Edema Macular , Humanos , NF-kappa B/metabolismo , Retinopatia Diabética/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Edema Macular/metabolismo , Transdução de Sinais , Epitélio Pigmentado da Retina/metabolismo , Barreira Hematorretiniana/metabolismo , Junções Íntimas/metabolismo , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismo , Pigmentos da Retina/farmacologia , Pigmentos da Retina/uso terapêuticoRESUMO
Oncogenic BRAF mutations have been widely described in melanomas and promote tumour progression and chemoresistance. We previously provided evidence that the HDAC inhibitor ITF2357 (Givinostat) targets oncogenic BRAF in SK-MEL-28 and A375 melanoma cells. Here, we show that oncogenic BRAF localises to the nucleus of these cells, and the compound decreases BRAF levels in both the nuclear and cytosolic compartments. Although mutations in the tumour suppressor p53 gene are not equally frequent in melanomas compared to BRAF, the functional impairment of the p53 pathway may also contribute to melanoma development and aggressiveness. To understand whether oncogenic BRAF and p53 may cooperate, a possible interplay was considered in the two cell lines displaying a different p53 status, being p53 mutated into an oncogenic form in SK-MEL-28 and wild-type in A375 cells. Immunoprecipitation revealed that BRAF seems to preferentially interact with oncogenic p53. Interestingly, ITF2357 not only reduced BRAF levels but also oncogenic p53 levels in SK-MEL-28 cells. ITF2357 also targeted BRAF in A375 cells but not wild-type p53, which increased, most likely favouring apoptosis. Silencing experiments confirmed that the response to ITF2357 in BRAF-mutated cells depends on p53 status, thus providing a rationale for melanoma-targeted therapy.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Mutação , Linhagem Celular TumoralRESUMO
BACKGROUND: Histone deacetylases (HDAC) contribute to oncogenic program, pointing to their inhibitors as a potential strategy against cancers. We, thus, studied the mechanism of HDAC inhibitor ITF2357 in resistance of mutant (mut)-KRAS non-small cell lung cancer (NSCLC) to pemetrexed (Pem). METHODS: We first determined the expression of NSCLC tumorigenesis-related HDAC2 and Rad51 in NSCLC tissues and cells. Next, we illustrated the effect of ITF2357 on the Pem resistance in wild type-KARS NSCLC cell line H1299, mut-KARS NSCLC cell line A549 and Pem-resistant mut-KARS cell line A549R in vitro and in xenografts of nude mice in vivo. RESULTS: Expression of HDAC2 and Rad51 was upregulated in NSCLC tissues and cells. Accordingly, it was revealed that ITF2357 downregulated HDAC2 expression to diminish the resistance of H1299, A549 and A549R cells to Pem. HDAC2 bound to miR-130a-3p to upregulate its target gene Rad51. The in vitro findings were reproduced in vivo, where ITF2357 inhibited the HDAC2/miR-130a-3p/Rad51 axis to reduce the resistance of mut-KRAS NSCLC to Pem. CONCLUSION: Taken together, HDAC inhibitor ITF2357 restores miR-130a-3p expression by inhibiting HDAC2, thereby repressing Rad51 and ultimately diminishing resistance of mut-KRAS NSCLC to Pem. Our findings suggested HDAC inhibitor ITF2357 as a promising adjuvant strategy to enhance the sensitivity of mut-KRAS NSCLC to Pem.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Animais , Camundongos , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Pemetrexede/farmacologia , Pemetrexede/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Proteínas Proto-Oncogênicas p21(ras) , MicroRNAs/genética , MicroRNAs/metabolismo , Camundongos Nus , Linhagem Celular Tumoral , Proliferação de Células , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/farmacologiaRESUMO
As a type of central nervous system tumor, meningioma usually compresses the nerve center due to its local expansion, further causing neurological deficits. However, there are limited therapeutic approaches for meningiomas. ITF2357, a potent class I and II histone deacetylase inhibitor (HDACi), has been shown to inhibit cell proliferation, promote apoptosis, and block the cell cycle in various sarcoma cells, including glioblastoma and peripheral T-cell lymphoma. Here, we investigated the potential role of ITF2357 on meningioma cancer cells (IOMM-Lee cells). First, we demonstrated that the half-maximal inhibitory concentration (IC50) of ITF2357 was 1.842 µM by MTT assay. In addition, ITF2357 effectively inhibited the proliferation and colonization ability of IOMM-Lee cells. Flow cytometry analysis showed that ITF2357 induced G0/G1 and G2/M phase cell cycle arrest and cell apoptosis. Mechanically, the RNA sequencing data revealed that ITF2357 could affect the PI3K-Akt signaling pathway and the cell cycle progression. Furthermore, the expression levels of Akt, PI3K, p-Akt, and p-PI3K were determined by western blotting. Collectively, our data revealed that ITF2357 induces G0 G1 and G2/M phase arrest and apoptosis by inhibiting hyperactivation of the PI3K-Akt pathway, ultimately inhibiting cell viability and proliferation of meningioma cells, which developed a new approach to the treatment of meningioma.
Assuntos
Neoplasias Meníngeas , Meningioma , Humanos , Meningioma/tratamento farmacológico , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Pontos de Checagem do Ciclo Celular , Apoptose , Pontos de Checagem da Fase M do Ciclo Celular , Neoplasias Meníngeas/tratamento farmacológicoRESUMO
The histone deacetylase inhibitor (HDACi) Givinostat/ITF2357 provides neuroprotection in adult models of brain injury; however, its action after neonatal hypoxia-ischemia (HI) is still undefined. The aim of our study was to test the hypothesis that the mechanism of Givinostat is associated with the alleviation of inflammation. For this purpose, we analyzed the microglial response and the effect on molecular mediators (chemokines/cytokines) that are crucial for inducing cerebral damage after neonatal hypoxia-ischemia. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 60 min of hypoxia (7.6% O2). Givinostat (10 mg/kg b/w) was administered in a 5-day regimen. The effects of Givinostat on HI-induced inflammation (cytokine, chemokine and microglial activation and polarization) were assessed with a Luminex assay, immunohistochemistry and Western blot. Givinostat treatment did not modulate the microglial response specific for HI injury. After Givinostat administration, the investigated chemokines and cytokines remained at the level induced by HI. The only immunosuppressive effect of Givinostat may be associated with the decrease in MIP-1α. Neonatal hypoxia-ischemia produces an inflammatory response by activating the proinflammatory M1 phenotype of microglia, disrupting the microglia-neuron (CX3CL1/CX3CR1) axis and elevating numerous proinflammatory cytokines/chemokines. Givinostat/ITF2357 did not prevent an inflammatory reaction after HI.
Assuntos
Asfixia Neonatal , Hipóxia-Isquemia Encefálica , Animais , Animais Recém-Nascidos , Encéfalo , Carbamatos , Citocinas , Humanos , Ácidos Hidroxâmicos , Hipóxia , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/prevenção & controle , Recém-Nascido , Inflamação/tratamento farmacológico , Isquemia , RatosRESUMO
PURPOSE: Aberrant expression and activity of histone deacetylases (HDACs) sustain glioblastoma (GBM) onset and progression, and, therefore, HDAC inhibitors (HDACi) represent a promising class of anti-tumor agents. Here, we analyzed the effects of ITF2357 (givinostat), a pan-HDACi, in GBM models for its anti-neoplastic potential. METHODS: A set of GBM- and patient-derived GBM stem-cell lines was used and the ITF2357 effects on GBM oncophenotype were investigated in in vitro and in vivo xenograft models. RESULTS: ITF2357 inhibited HDAC activity and affected GBM cellular fate in a dose-dependent manner by inducing G1/S growth arrest (1-2.5 µM) or caspase-mediated cell death (≥ 2.5 µM). Chronic treatment with low doses (≤ 1 µM) induced autophagy-mediated cell death, neuronal-like phenotype, and the expression of differentiation markers, such as glial fibrillar actin protein (GFAP) and neuron-specific class III beta-tubulin (Tuj-1); this reduces neurosphere formation from patient-derived GBM stem cells. Autophagy inhibition counteracted the ITF2357-induced expression of differentiation markers in p53-expressing GBM cells. Finally, in in vivo experiments, ITF2357 efficiently passed the blood-brain barrier, so rapidly reaching high concentration in the brain tissues, and significantly affected U87MG and U251MG growth in orthotopic xenotransplanted mice. CONCLUSIONS: The present findings provide evidence of the key role played by HDACs in sustaining transformed and stem phenotype of GBM and strongly suggest that ITF2357 may have a clinical potential for the HDACi-based therapeutic strategies against GBM.
Assuntos
Carbamatos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Histona Desacetilases/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Apoptose , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Histone deacetylase inhibitors (HDACi) suppress cytokine production in immune and stromal cells of patients with rheumatoid arthritis (RA). Here, we investigated the effects of the HDACi givinostat (ITF2357) on the transcriptional and post-transcriptional regulation of inflammatory markers in RA fibroblast-like synoviocytes (FLS). METHODS: The effects of ITF2357 on the expression and messenger RNA (mRNA) stability of IL-1ß-inducible genes in FLS were analyzed using array-based qPCR and Luminex. The expression of primary and mature cytokine transcripts, the mRNA levels of tristetraprolin (TTP, or ZFP36) and other AU-rich element binding proteins (ARE-BP) and the cytokine profile of fibroblasts derived from ZFP36+/+ and ZFP36-/- mice was measured by qPCR. ARE-BP silencing was performed by small interfering RNA (siRNA)-mediated knockdown, and TTP post-translational modifications were analyzed by immunoblotting. RESULTS: ITF2357 reduced the expression of 85% of the analyzed IL-1ß-inducible transcripts, including cytokines (IL6, IL8), chemokines (CXCL2, CXCL5, CXCL6, CXCL10), matrix-degrading enzymes (MMP1, ADAMTS1) and other inflammatory mediators. Analyses of mRNA stability demonstrated that ITF2357 accelerates IL6, IL8, PTGS2 and CXCL2 mRNA degradation, a phenomenon associated with the enhanced transcription of TTP, but not other ARE-BP, and the altered post-translational status of TTP protein. TTP knockdown potentiated cytokine production in RA FLS and murine fibroblasts, which in the latter case was insensitive to inhibition by ITF2357 treatment. CONCLUSIONS: Our study identifies that regulation of cytokine mRNA stability is a predominant mechanism underlying ITF2357 anti-inflammatory properties, occurring via regulation of TTP. These results highlight the therapeutic potential of ITF2357 in the treatment of RA.
Assuntos
Artrite Reumatoide/metabolismo , Citocinas/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Sinoviócitos/efeitos dos fármacos , Animais , Artrite Reumatoide/imunologia , Células Cultivadas , Citocinas/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Sinoviócitos/metabolismo , Tristetraprolina/biossínteseRESUMO
Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/ß-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/terapia , Quimioterapia Combinada , Neoplasias Pulmonares/terapia , Evasão Tumoral/efeitos dos fármacos , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Imunoterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Camundongos , Linfócitos T/imunologia , Transcriptoma , Microambiente TumoralRESUMO
ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1ß and other cytokines at 25-100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC50 of 285 nm). Here we compared the production of IL-1ß, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1ß was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100-1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1ß and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1ß independent of inhibition of caspase-1 activity; however, synthesis of the IL-1ß precursor was reduced by 40% without significant decrease in IL-1ß mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1ß by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.