RESUMO
Periodontal ligament stem cells (PDLSCs) show plasticity towards the adipogenic lineage; however, little has been done on the participation of epigenetic mechanisms. Histone acetylation is a dynamic process, though balanced by histone acetyltransferases (HATs) and histone deacetylases (HDACs) activities. This process can be halted by HDACs inhibitors, such as trichostatin A (TSA) and valproic acid (VPA). This study aimed to determine the role of HDACs class I in adipogenic differentiation of PDL cells. PDLSCs were treated with TSA at concentrations of 100, 200, and 250 nM, or VPA at 1, 4 and 8 mM. Cell viability was assessed using MTT assays. Gene expression of pluripotency markers (NANOG, OCT4, SOX2), HAT genes (p300, GCN5), and HDACs genes (HDAC1-3) was analyzed by RT-qPCR. Adipogenic differentiation was evaluated via oil red O staining, and acetylation of histone H3 lysine 9 (H3K9ac) was examined by Western blot. VPA treatment resulted in a 60% reduction in cell proliferation, compared to a 50% when using TSA. Cell viability was not affected by either inhibitor. Furthermore, both TSA and VPA induced adipogenic differentiation, through an increase in the deposition of lipid droplets and in GCN5 and p300 expression were observed. Western blot analysis showed that TSA increased H3K9ac levels on adipogenic differentiation of PDLSCs. These findings highlight the potential of HDAC inhibitors as a tool for modulating H3K9 acetylation status and thus influencing adipogenic differentiation of PDLCs.
Assuntos
Adipogenia , Diferenciação Celular , Sobrevivência Celular , Inibidores de Histona Desacetilases , Ligamento Periodontal , Ácido Valproico , Humanos , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Ácido Valproico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Ácidos Hidroxâmicos/farmacologia , Células Cultivadas , Histonas/metabolismo , Proliferação de Células/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
BACKGROUND: There is a need for novel treatments for neuroblastoma, despite the emergence of new biological and immune treatments, since refractory pediatric neuroblastoma is still a medical challenge. Phyto cannabinoids and their hemisynthetic derivatives have shown evidence supporting their anticancer potential. The aim of this research was to examine Phytocannabinoids or hemisynthetic cannabinoids, which reduce the SHSY-5Y, neuroblastoma cell line's viability. METHODS: Hexane and acetyl acetate extracts were produced starting with Cannabis sativa L. as raw material, then, 9-tetrahidrocannabinol, its acid counterpart and CBN were isolated. In addition, acetylated derivatives of THC and CBN were synthesized. The identification and purity of the chemicals was determined by High Performance Liquid Chromatography and 1H y 13C Magnetic Nuclear Resonance. Then, the capacity to affect the viability of SHSY-5Y, a neuroblastoma cell line, was examined using the resazurin method. Finally, to gain insight into the mechanism of action of the extracts, phytocannabinoids and acetylated derivatives on the examined cells, a caspase 3/7 determination was performed on cells exposed to these compounds. RESULTS: The structure and purity of the isolated compounds was demonstrated. The extracts, the phytocannabinoids and their acetylated counterparts inhibited the viability of the SHSY 5Y cells, being CBN the most potent of all the tested molecules with an inhibitory concentration of 50 percent of 9.5 µM. CONCLUSION: Each of the evaluated molecules exhibited the capacity to activate caspases 3/7, indicating that at least in part, the cytotoxicity of the tested phytocannabinoids and their hemi-synthetic derivatives is mediated by apoptosis.
Assuntos
Canabinoides , Cannabis , Caspase 3 , Sobrevivência Celular , Neuroblastoma , Extratos Vegetais , Humanos , Cannabis/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Linhagem Celular Tumoral , Neuroblastoma/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 3/efeitos dos fármacos , Canabinoides/farmacologia , Canabinoides/química , Caspase 7/metabolismo , Apoptose/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Cromatografia Líquida de Alta PressãoRESUMO
The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.
Assuntos
Dano ao DNA , Replicação do DNA , Proteína do Grupo de Complementação A da Anemia de Fanconi , Histonas , Histonas/metabolismo , Humanos , Replicação do DNA/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Mitomicina/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Vorinostat/farmacologia , Ácidos Hidroxâmicos/farmacologiaRESUMO
AIM: To evaluate the potential use of Cephaeline as a therapeutic strategy to manage mucoepidermoid carcinomas (MEC) of the salivary glands. MATERIAL AND METHODS: UM-HMC-1, UM-HMC-2, and UM-HMC-3A MEC cell lines were used to establish the effects of Cephaeline over tumor viability determined by MTT assay. In vitro wound healing scratch assays were performed to address cellular migration while immunofluorescence staining for histone H3 lysine 9 (H3k9ac) was used to identify the acetylation status of tumor cells upon Cephaeline administration. The presence of cancer stem cells was evaluated by the identification of ALDH enzymatic activity by flow cytometry and through functional assays using in vitro tumorsphere formation. RESULTS: A single administration of Cephaeline resulted in reduced viability of MEC cells along with the halt on tumor growth and cellular migration potential. Administration of Cephaeline resulted in chromatin histone acetylation as judged by the increased levels of H3K9ac and disruption of tumorspheres formation. Interestingly, ALDH levels were increased in UM-HMC-1 and UM-HMC-3A cell lines, while UM-HMC-2 showed a reduced enzymatic activity. CONCLUSION: Cephaeline has shown anti-cancer properties in all MEC cell lines tested by regulating tumor cells' viability, migration, proliferation, and disrupting the ability of cancer cells to generate tumorspheres.
Assuntos
Carcinoma Mucoepidermoide , Acetilação/efeitos dos fármacos , Carcinoma Mucoepidermoide/metabolismo , Linhagem Celular Tumoral , Emetina/análogos & derivados , Emetina/farmacologia , Histonas/metabolismo , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologiaRESUMO
Evaluate the biological action of valproic acid in the acetylation of histones and in the methylation of tumor suppressor genes via oral rinse in patients with a previous history of head and neck squamous cell carcinoma (HNSCC). Forty-two active or former smokers were included in this randomized, double-blind, placebo-controlled trial. Oral rinse samples were collected prior to treatment with valproic acid or placebo and after 90 days of treatment. The methylation status of five tumor suppressor genes and histone acetylation were evaluated by pyrosequencing and ELISA techniques, respectively. Differences between the 90-day and baseline oral rinse acetylation and methylation results were analyzed by comparing groups. Thirty-four patients were considered for analysis. The mean percentage adherence in the valproic and placebo groups was 93.4 and 93.0, respectively (p = 0.718). There was no statistically significant difference between groups when comparing the medians of the histone acetylation ratio and the methylation ratio for most of the studied genes. A significant reduction in the DCC methylation pattern was observed in the valproic group (p = 0.023). The use of valproic acid was safe and accompanied by good therapeutic adherence. DCC methylation was lower in the valproic acid group than in the placebo group.
Assuntos
Acetilação/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Ácido Valproico/uso terapêutico , Método Duplo-Cego , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Histonas/metabolismo , Humanos , Masculino , Estudos Prospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismoRESUMO
The cellular damage caused by redox imbalance is involved in the pathogenesis of many cardiovascular diseases. Besides, redox imbalance is related to the alteration of protein acetylation processes, causing not only chromatin remodeling but also disturbances in so many processes where protein acetylation is involved, such as metabolism and signal transduction. The modulation of acetylases and deacetylases enzymes aids in maintaining the redox homeostasis, avoiding the deleterious cellular effects associated with the dysregulation of protein acetylation. Of note, regulation of protein acetylation has shown protective effects to ameliorate cardiovascular diseases. For instance, HDAC inhibition has been related to inducing cardiac protective effects and it is an interesting approach to the management of cardiovascular diseases. On the other hand, the upregulation of SIRT protein activity has also been implicated in the relief of cardiovascular diseases. This review focuses on the major protein acetylation modulators described, involving pharmacological and bioactive compounds targeting deacetylase and acetylase enzymes contributing to heart protection through redox homeostasis.
Assuntos
Acetilação/efeitos dos fármacos , Doenças Cardiovasculares/enzimologia , Coração/fisiologia , Animais , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Miocárdio/metabolismo , Oxirredução , Substâncias Protetoras/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Hypoxia is associated with tumor aggressiveness and poor prognosis, including breast cancer. Low oxygen levels induces global genomic hypomethylation and hypermethylation of specific loci in tumor cells. DNA methylation is a reversible epigenetic modification, usually associated with gene silencing, contributing to carcinogenesis and tumor progression. Since the effects of DNA methyltransferase inhibitor are context-dependent and as there is little data comparing their molecular effects in normoxic and hypoxic microenvironments in breast cancer, this study aimed to understand the gene expression profiles and molecular effects in response to treatment with DNA methyltransferase inhibitor in normoxia and hypoxia, using the breast cancer model. For this, a cDNA microarray was used to analyze the changes in the transcriptome upon treatment with DNA methyltransferase inhibitor (5-Aza-2'-deoxycytidine: 5-Aza-2'-dC), in normoxia and hypoxia. Furthermore, immunocytochemistry was performed to investigate the effect of 5-Aza-2'-dC on NF-κB/p65 inflammation regulator subcellular localization and expression, in normoxia and hypoxia conditions. We observed that proinflammatory pathways were upregulated by treatment with 5-Aza-2'-dC, in both conditions. However, treatment with 5-Aza-2'-dC in normoxia showed a greater amount of overexpressed proinflammatory pathways than 5-Aza-2'-dC in hypoxia. In this sense, we observed that the NF-κB expression increased only upon 5-Aza-2'-dC in normoxia. Moreover, nuclear staining for NF-κB and NF-κB target genes upregulation, IL1A and IL1B, were also observed after 5-Aza-2'-dC in normoxia. Our results suggest that 5-Aza-2'-dC induces a greater inflammatory change, at the molecular levels, in normoxic than hypoxic tumor microenvironment. These data may support further studies and expand the understanding of the DNA methyltransferase inhibitor effects in different tumor contexts.
Assuntos
Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/genética , Decitabina/farmacologia , Inflamação/genética , Acetilação/efeitos dos fármacos , Linhagem Celular Tumoral , Metilases de Modificação do DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Epigênese Genética/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Fator de Transcrição RelA/genética , Hipóxia Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genéticaRESUMO
Sodium valproate (VPA) is a classic anticonvulsive, a histone deacetylase inhibitor, and a chromatin remodeling inducer. When injected into specimens of Triatoma infestans, a vector of Chagas disease, VPA affects the chromatin supraorganization of chromocenter heterochromatin in only a few cells of the Malpighian tubules. To test whether this result was explained by the inaccessibility of all of the organ's cells to the drug, we investigated the nuclear phenotypes and global acetylation of lysine 9 in histone H3 (H3K9ac) in Malpighian tubules cultivated in vitro for 1-24 h in the presence of 0.05 mM-1 mM VPA. The present results revealed that the chromatin decondensation event in the chromocenter body, which was detected only under low VPA concentrations up to a 4-h treatment, was not frequent during organ culture, similar to the results for injected insects. Cultivation of T. infestans Malpighian tubules in vitro for 24 h revealed inadequate for cell preservation even in the absence of the drug. Immunofluorescence signals for H3K9ac following VPA treatment showed a slightly increased intensity in the euchromatin, but were never detected in the chromocenter bodies, except with great intensity at their periphery, where the 18S rDNA is located. In conclusion, when VPA affects the chromocenter heterochromatin in this animal cell model, it occurs through a pathway that excludes a classic global H3K9ac mark. Investigation of nonhistone proteins associated with histone methylation marks is still required to further explain the differential response of T. infestans chromatin to VPA.
Assuntos
Eucromatina/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Triatoma/efeitos dos fármacos , Ácido Valproico/farmacologia , Acetilação/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Heterocromatina/metabolismo , Túbulos de Malpighi/citologia , Túbulos de Malpighi/efeitos dos fármacos , Triatoma/citologiaRESUMO
Osteoporosis is characterized by decreased bone mass and adipocyte accumulation within the bone marrow that inhibits osteoblast maturation, leading to a high risk of fractures. Thus, we hypothesized that osteoblasts, besides being negatively affected by interacting with adipocytes, reduce the differentiation of neighboring osteoblasts through the same mechanisms that affect osteoblasts under osteoporotic conditions. We investigated the effect of osteoporosis on osteoblast differentiation and the effect of the conditioned medium of osteoblasts cocultured with adipocytes on the differentiation of other osteoblasts. Osteoporosis was induced by orchiectomy in rats and bone marrow mesenchymal stromal cells (MSCs) were differentiated into osteoblasts. Also, the bone marrow and adipose tissue MSCs were obtained from healthy rats and differentiated into osteoblasts and adipocytes, respectively. Messenger RNA expression, in situ alkaline phosphatase activity, and mineralization confirmed the inhibitory effect of osteoporosis on osteoblast differentiation. This harmful effect was mimicked by the in vitro model using the conditioned medium and it was demonstrated that osteoblasts keep the memory of the negative impact of interacting with adipocytes, revealing an unknown mechanism relevant to the osteoporotic bone loss. Finally, we showed the involvement of acetyl-histone 3 (AcH3) in bone homeostasis as its reduction induced by osteoporosis and conditioned medium impaired osteoblast differentiation. The AcH3 involvement was proved by treating osteoblasts with Trichostatin A that recovered the AcH3 expression and osteoblast differentiation capacity in both situations. Together, our findings indicated that AcH3 might be a target for future studies focused on epigenetic-based therapies to treat bone diseases.
Assuntos
Adipócitos/metabolismo , Diferenciação Celular , Regulação para Baixo , Histonas/metabolismo , Osteoblastos/metabolismo , Osteoporose/patologia , Acetilação/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Regulação para Baixo/efeitos dos fármacos , Masculino , Modelos Biológicos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteoporose/metabolismo , Ratos WistarRESUMO
Sirtuin 2 is a key enzyme in gene expression regulation that is often associated with tumor proliferation control and therefore is a relevant anticancer drug target. Anilinobenzamide derivatives have been discussed as selective sirtuin 2 inhibitors and can be developed further. In the present study, hologram and three-dimensional quantitative structure-activity relationship (HQSAR and 3D-QSAR) analyses were employed for determining structural contributions of a compound series containing human sirtuin-2-selective inhibitors that were then correlated with structural data from the literature. The final QSAR models were robust and predictive according to statistical validation (q2 and r2pred values higher than 0.85 and 0.75, respectively) and could be employed further to generate fragment contribution and contour maps. 3D-QSAR models together with information about the chemical properties of sirtuin 2 inhibitors can be useful for designing novel bioactive ligands.Communicated by Ramaswamy H. Sarma.