RESUMEN
RNA modifications play crucial roles in regulating gene expression and cellular homeostasis. Modulating RNA modifications, particularly by targeting the enzymes responsible for their catalysis, has emerged as a promising therapeutic strategy. However, limitations, such as the lack of identified modifying enzymes and compensatory mechanisms, hinder targeted interventions. Chemical approaches independent of enzymatic activity offer an alternative strategy for RNA modification modulation. Here, we present the identification of 2-chloro-3,5-dinitrobenzoic acid as a highly effective photochemical deprenylase of i6A RNA. This method demonstrates exceptional selectivity towards i6A, converting its substituent into a "N-doped" ozonide, which upon hydrolysis releases natural adenine. We believe that this chemical approach will pave the way for a better understanding of RNA modification biology and the development of novel therapeutic modalities.
Asunto(s)
ARN , ARN/metabolismo , ARN/química , Nitrobenzoatos/química , Nitrobenzoatos/metabolismo , Procesos Fotoquímicos , Estructura MolecularRESUMEN
Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor, and due to its unique features, its management is certainly one of the most challenging ones among all cancers. N6-isopentenyladenosine (IPA) and its analog N6-benzyladenosine (N6-BA) are modified nucleosides endowed with potent antitumor activity on different types of human cancers, including GBM. Corroborating our previous finding, we demonstrated that IPA and N6-BA affect GBM cell line proliferation by modulating the expression of the F-box WD repeat domain-containing-7 (FBXW7), a tumor suppressor with a crucial role in the turnover of many proteins, such as SREBPs and Mcl1, involved in malignant progression and chemoresistance. Luciferase assay revealed that IPA-mediated upregulation of FBXW7 translates in transcriptional inactivation of its oncogenic substrates (Myc, NFkB, or HIF-1α). Moreover, downregulating MGMT expression, IPA strongly enhances the killing effect of temozolomide (TMZ), producing a favorable sensitizing effect starting from a concentration range much lower than TMZ EC50. Through DNA methyltransferase (DNMT) activity assay, analysis of the global DNA methylation, and the histone modification profiles, we demonstrated that the modified adenosines behave similar to 5-AZA-dC, known DNMT inhibitor. Overall, our results provide new perspectives for the first time, suggesting the modified adenosines as epigenetic tools able to improve chemo- and radiotherapy efficacy in glioblastoma and potentially other cancers.
RESUMEN
N6 -isopentenyladenosine (i6A), a modified adenosine monomer, is known to induce cell death upon its addition to the culture medium. However, the molecular fate of extracellularly added i6A has yet to be identified. Here we show that i6A addition to cell culture medium results in i6A incorporation into cellular RNA in several cell lines, including the 5-fluorouracil (5-FU)-resistant human oral squamous cell carcinoma cell line FR2-SAS and its parental 5-FU-sensitive cell line SAS. i6A was predominantly incorporated into 18S and 28S rRNAs, and i6A incorporation into total RNA was mostly suppressed by treating these cell lines with an RNA polymerase I (Pol I) inhibitor. i6A was incorporated into RNA even upon inactivation of TRIT1, the only cellular i6A-modifying enzyme. These results indicate that upon cellular uptake of i6A, it is anabolized to be used for Pol I transcription. Interestingly, at lower i6A concentrations, the cytotoxic effect of i6A was substantially more pronounced in FR2-SAS cells than in SAS cells. Moreover, in FR2-SAS cells, i6A treatment decreased the rate of cellular protein synthesis and increased intracellular protein aggregation, and these effects were more pronounced than in SAS cells. Our work provides insights into the molecular fate of extracellularly applied i6A in the context of intracellular nucleic acid anabolism and suggests investigation of i6A as a candidate for a chemotherapy agent against 5-FU-resistant cancer cells.
Asunto(s)
Antineoplásicos , Carcinoma de Células Escamosas , Neoplasias de la Boca , Línea Celular Tumoral , Fluorouracilo/metabolismo , Fluorouracilo/farmacología , Humanos , Isopenteniladenosina , ARN , ARN Ribosómico/metabolismoRESUMEN
BACKGROUND: Vasculogenic mimicry (VM) is a functional microcirculation pattern formed by aggressive tumor cells. Thus far, no effective drugs have been developed to target VM. Glioblastoma (GBM) is the most malignant form of brain cancer and is a highly vascularized tumor. Vasculogenic mimicry represents a means whereby GBM can escape anti-angiogenic therapies. METHODS: Here, using an in vitro tube formation assay on Matrigel, we evaluated the ability of N6-isopentenyladenosine (iPA) to interfere with vasculogenic mimicry (VM). RhoA activity was assessed using a pull-down assay, while the modulation of the adherens junctions proteins was analyzed by Western blot analysis. RESULTS: We found that iPA at sublethal doses inhibited the formation of capillary-like structures suppressing cell migration and invasion of U87MG, U343MG, and U251MG cells, of patient-derived human GBM cells and GBM stem cells. iPA reduces the vascular endothelial cadherin (VE-cadherin) expression levels in a dose-dependent manner, impairs the vasculogenic mimicry network by modulation of the Src/p120-catenin pathway and inhibition of RhoA-GTPase activity. CONCLUSIONS: Taken together, our results revealed iPA as a promising novel anti-VM drug in GBM clinical therapeutics.
Asunto(s)
Cateninas/metabolismo , Glioblastoma/tratamiento farmacológico , Isopenteniladenosina/farmacología , Neovascularización Patológica/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rhoA/metabolismo , Familia-src Quinasas/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Cateninas/genética , Línea Celular Tumoral , Glioblastoma/irrigación sanguínea , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Proteína de Unión al GTP rhoA/genética , Familia-src Quinasas/genéticaRESUMEN
Transfer RNA (tRNA) is the most diversely modified RNA. Although the strictly conserved purine position 37 in the anticodon stem-loop undergoes modifications that are phylogenetically distributed, we do not yet fully understand the roles of these modifications. Therefore, molecular dynamics simulations are used to provide molecular-level details for how such modifications impact the structure and function of tRNA. A focus is placed on three hypermodified base families that include the parent i6A, t6A, and yW modifications, as well as derivatives. Our data reveal that the hypermodifications exhibit significant conformational flexibility in tRNA, which can be modulated by additional chemical functionalization. Although the overall structure of the tRNA anticodon stem remains intact regardless of the modification considered, the anticodon loop must rearrange to accommodate the bulky, dynamic hypermodifications, which includes changes in the nucleotide glycosidic and backbone conformations, and enhanced or completely new nucleobase-nucleobase interactions compared to unmodified tRNA or tRNA containing smaller (m1G) modifications at the 37th position. Importantly, the extent of the changes in the anticodon loop is influenced by the addition of small functional groups to parent modifications, implying each substituent can further fine-tune tRNA structure. Although the dominant conformation of the ASL is achieved in different ways for each modification, the molecular features of all modified tRNA drive the ASL domain to adopt the functional open-loop conformation. Importantly, the impact of the hypermodifications is preserved in different sequence contexts. These findings highlight the likely role of regulating mRNA structure and translation.
Asunto(s)
Adenosina/análogos & derivados , Anticodón/química , Escherichia coli/genética , Procesamiento Postranscripcional del ARN , ARN de Transferencia de Lisina/química , ARN de Transferencia de Fenilalanina/química , Adenosina/metabolismo , Anticodón/genética , Anticodón/metabolismo , Emparejamiento Base , Secuencia de Bases , Escherichia coli/metabolismo , Isopenteniladenosina/química , Isopenteniladenosina/metabolismo , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Nucleósidos/química , Nucleósidos/metabolismo , ARN de Transferencia de Lisina/genética , ARN de Transferencia de Lisina/metabolismo , ARN de Transferencia de Fenilalanina/genética , ARN de Transferencia de Fenilalanina/metabolismoRESUMEN
N6 -isopentenyladenosine (i6 A) is an RNA modification found in cytokinins, which regulate plant growth/differentiation, and a subset of tRNAs, where it improves the efficiency and accuracy of translation. The installation and removal of this modification is mediated by prenyltransferases and cytokinin oxidases, and a chemical approach to selective deprenylation of i6 A has not been developed. We show that a selected group of oxoammonium cations function as artificial deprenylases to promote highly selective deprenylation of i6 A in nucleosides, oligonucleotides, and live cells. Importantly, other epigenetic modifications, amino acid residues, and natural products were not affected. Moreover, a significant phenotype difference in the Arabidopsis thaliana shoot and root development was observed with incubation of the cation. These results establish these small organic molecules as direct chemical regulators/artificial deprenylases of i6 A.
Asunto(s)
Óxidos N-Cíclicos/farmacología , Citocininas/metabolismo , Isopenteniladenosina/metabolismo , Piperidinas/farmacología , Prenilación/efectos de los fármacos , ARN/metabolismo , Arabidopsis/efectos de los fármacos , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/toxicidad , Citocininas/química , Epigénesis Genética/efectos de los fármacos , Humanos , Isopenteniladenosina/química , Células MCF-7 , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismo , Piperidinas/química , Piperidinas/toxicidad , Reguladores del Crecimiento de las Plantas/química , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Brotes de la Planta/efectos de los fármacos , ARN/químicaRESUMEN
N6-isopentenyladenosine has been shown to exert potent in vitro antitumor activity on different human cancers, including colorectal cancer. Although some potential biochemical targets have been identified, its precise mechanism of action remains unclear. We found that N6-isopentenyladenosine affects colorectal cancer proliferation in in vitro models carrying different mutational status of FBXW7 and TP53 genes, and in HCT116 xenografts in SCID mice, by increasing the expression of the well-established tumor suppressor FBXW7, a component of the SCF-E3 ubiquitin ligase complex that promotes degradation of various oncoproteins and transcription factors, such as c-Myc, SREBP and Mcl1. Corroborating our previous studies, we identified for the first time the FBXW7/SREBP/FDPS axis as a target of the compound. Pull down of ubiquitinated proteins, immunoprecipitation and luciferase assays, reveal that through the increase of FBXW7/c-Myc binding, N6-isopentenyladenosine induces the ubiquitination of c-Myc, inhibiting its transcriptional activity. Moreover, in FBXW7- and TP53-wild type cells, N6-isopentenyladenosine strongly synergizes with 5-Fluorouracil to inhibit colon cancer growth in vitro. Our results provide novel insights into the molecular mechanism of N6-isopentenyladenosine, revealing its multi-targeting antitumor action, in vitro and in vivo. Restoring of FBXW7 tumor-suppressor represents a valid therapeutic tool, enabling N6-isopentenyladenosine as optimizable compound for patient-personalized therapies in colorectal cancer.
RESUMEN
N6-isopentenyladenosine is an anti-proliferative and pro-apoptotic atypical nucleoside for normal and tumor cells. Considering the role of angiogenesis in various diseases, we investigated the cytotoxic effect of N6-isopentenyladenosine on human microvascular endothelial cells, protagonists in angiogenesis. Our results show that N6-isopentenyladenosine induced a significant reduction of cell viability, upregulated p21 and promoted caspase-3 cleavage in a dose dependent manner leading to apoptotic cell death as detected by FACS analysis. To understand structure-function relationship of N6-isopentenyladenosine, we investigated the effect of some N6-isopentenyladenosine analogs. Our results suggest that N6-isopentenyladenosine and some of its derivatives are potentially novel angiostatic agents and might be associated with other anti-angiogenic compounds for a better outcome.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Endotelio Vascular/citología , Isopenteniladenosina/farmacología , Inhibidores de la Angiogénesis/administración & dosificación , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Células Endoteliales/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Isopenteniladenosina/administración & dosificaciónRESUMEN
OBJECTIVE: N6-isopentenyladenosine (iPA) is an intermediate of the mevalonate pathway that exhibits various anti-cancer effects. However, studies on its anti-inflammatory activity are scarce and underlying molecular mechanisms are unknown. Therefore, we aimed to investigate the ability of iPA to exert anti-inflammatory effects in the human cystic fibrosis (CF) cell model of exacerbated inflammation. MATERIALS AND METHODS: TNFα-stimulated CF cells CuFi-1 and its normal counterpart NuLi-1 were pre-treated with increasing concentrations of iPA and cell viability and proliferation were assessed by MTT and BrdU assays. The effect of iPA on IL-8 and RANTES secretion was determined by ELISA, and the activation and expression of signaling molecules and selenoproteins were studied by Western blot. To assess the direct effect of iPA on NFκB activity, luciferase assay was performed on TNFα-stimulated HEK293/T cells transfected with a NFκB reporter plasmid. RESULTS: We demonstrated for the first time that iPA prevents IL-8 and RANTES release in TNFα-stimulated CF cells and this effect is mediated by increasing the expression of the direct NFκB inhibitor IκBα and decreasing the levels of STAT3. Consistent with this, we showed that iPA inhibited TNFα-mediated NFκB activation in HEK/293T cells. Finally, we also found that iPA improved the levels of glutathione peroxidase 1 and thioredoxin reductase 1 only in CF cells suggesting its ability to maintain sufficient expression of these anti-oxidant selenoproteins. CONCLUSIONS: Our findings indicate that iPA can exert anti-inflammatory activity especially in the cases of excessive inflammatory response as in CF.
Asunto(s)
Antiinflamatorios/farmacología , Fibrosis Quística/metabolismo , Isopenteniladenosina/farmacología , FN-kappa B/metabolismo , Factor de Transcripción STAT3/metabolismo , Línea Celular , Supervivencia Celular , Quimiocina CCL5/metabolismo , Fibrosis Quística/enzimología , Glutatión Peroxidasa/metabolismo , Células HEK293 , Humanos , Inflamación/metabolismo , Interleucina-8/metabolismo , Isopenteniladenosina/toxicidad , FN-kappa B/antagonistas & inhibidores , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
N6-Isopentenyladenosine, a member of the family of plant hormones, possesses anti-cancer activities on a number of cancer cell lines. However, its mode of action in cervical cancer cell remains poorly understood. Our computational docking studies showed that N6-Isopentenyladenosine could bind with the really interesting new gene domain of tumor necrosis factor receptor-associated factor 6, which is an ubiquitination E3 ligase. Tumor necrosis factor receptor-associated factor 6-mediated ubiquitination is known to activate both protein kinase B (also known as AKT) and transforming growth factor ß-activated kinase 1, and the really interesting new gene domain comprises the core of the ubiquitin ligase catalytic domain. First, we evaluated the effects of iPA on cervical cancer cell line HeLa using MTT and flow cytometry. Second, we examined the effects of iPA on activation of tumor necrosis factor receptor-associated factor 6-mediated downstream targets using western blot or immunoprecipitation. iPA could reduce HeLa cell proliferation through apoptosis, and such anti-cancer activity is associated with inhibitions of both AKT and transforming growth factor ß-activated kinase 1 signaling pathways. In addition, suppression of the anti-apoptotic protein Bcl-2 and elevation of the pro-apoptotic protein Bax were also observed. Anti-proliferation properties of iPA are likely due to its binding at the really interesting new gene domain of tumor necrosis factor receptor-associated factor 6 and loss of AKT and transforming growth factor ß-activated kinase 1 activities as a result of functional modulations of tumor necrosis factor receptor-associated factor 6. These results support the emerging notion that tumor necrosis factor receptor-associated factor 6 could serve as a viable target for developing new cancer therapeutics.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Isopenteniladenosina/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Factor 6 Asociado a Receptor de TNF/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Simulación del Acoplamiento Molecular , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Ubiquitinación , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
N(6)-isopentenyladenosine (i(6)A), a naturally occurring modified nucleoside, inhibits the proliferation of human tumor cell lines in vitro, but its mechanism of action remains unclear. Treatment of MCF7 human breast adenocarcinoma cells with i(6)A or with three synthetic analogs (allyl(6)A, benzyl(6)A, and butyl(6)A) inhibited growth and altered gene expression. About 60% of the genes that were differentially expressed in response to i(6)A treatment were also modulated by the analogs, and pathway enrichment analysis identified the NRF2-mediated oxidative stress response as being significantly modulated by all four compounds. Luciferase reporter gene assays in transfected MCF7 cells confirmed that i(6)A activates the transcription factor NRF2. Assays for cellular production of reactive oxygen species indicated that i(6)A and analogs had antioxidant effects, reducing basal levels and inhibiting the H2O2- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced production in MCF7 or dHL-60 (HL-60 cells induced to differentiate along the neutrophilic lineage) cell lines, respectively. In vivo, topical application of i(6)A or benzyl(6)A to mouse ears prior to TPA stimulation lessened the inflammatory response and significantly reduced the number of infiltrating neutrophils. These results suggest that i(6)A and analogs trigger a cellular response against oxidative stress and open the possibility of i(6)A and benzyl(6)A being used as topical anti-inflammatory drugs.
Asunto(s)
Antioxidantes/administración & dosificación , Isopenteniladenosina/análogos & derivados , Isopenteniladenosina/administración & dosificación , Factor 2 Relacionado con NF-E2/metabolismo , Otitis/tratamiento farmacológico , Administración Tópica , Animales , Antioxidantes/farmacología , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Peróxido de Hidrógeno/efectos adversos , Isopenteniladenosina/farmacología , Células MCF-7 , Ratones , Otitis/inducido químicamente , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/efectos adversosRESUMEN
Endogenous cytokinin (CK) levels of in vitro-cultured and greenhouse-acclimatized 'Williams' bananas treated with six aromatic CKs were quantified using UPLC-MS/MS. The underground parts had higher endogenous CK levels than the aerial parts. Control plantlets had more isoprenoid CKs while the aromatic-type CKs were predominant in all other regenerants. Following acclimatization of the control and 10 µM CK regenerants, there was a rapid decline in both isoprenoid and aromatic CK in the greenhouse-grown plants. Apart from the control and 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine (MemTTHP) treatment with higher level of isoprenoid CK, aromatic CK remain the predominant CK-type across all CK treatments. The most abundant CK forms were meta-topolin (mT) and benzyladenine (BA) in the micropropagated and acclimatized plants, respectively. Micropropagated plantlets had cis-Zeatin (cZ) as the major isoprenoid CK-type which was in turn replaced by isopentenyladenine (iP) upon acclimatization. On a structural and functional basis, 9-glucoside, a deactivation/detoxicification product was the most abundant and mainly located in the underground parts (micropropagation and acclimatization). The results establish the wide variation in metabolic products of the tested aromatic CKs during micropropagation and acclimatization. The findings are discussed with the possible physiological roles of the various CK constituents on the growth and development of banana plants.
Asunto(s)
Citocininas/metabolismo , Citocininas/farmacología , Musa/efectos de los fármacos , Musa/metabolismo , Citocininas/química , Relación Dosis-Respuesta a Droga , Ambiente Controlado , Glucósidos/química , Glucósidos/metabolismo , Glucósidos/farmacología , Hidrocarburos Aromáticos/química , Hidrocarburos Aromáticos/metabolismo , Hidrocarburos Aromáticos/farmacología , Isopenteniladenosina/química , Isopenteniladenosina/metabolismo , Isopenteniladenosina/farmacología , Musa/química , Componentes Aéreos de las Plantas/química , Componentes Aéreos de las Plantas/efectos de los fármacos , Componentes Aéreos de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Raíces de Plantas/química , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Terpenos/química , Terpenos/metabolismo , Terpenos/farmacología , Técnicas de Cultivo de Tejidos/métodos , Zeatina/química , Zeatina/metabolismo , Zeatina/farmacologíaRESUMEN
Hutchinson-Gilford progeria syndrome is caused by mutations in the lamin A/C gene that lead to expression of a truncated, permanently farnesylated prelamin A variant called progerin. The accumulation of progerin at the nuclear envelope causes mis-shapen nuclei and results in progeroid syndromes. Previous studies in cells from individuals with Hutchinson-Gilford progeria syndrome have shown that blocking of farnesylation of prelamin A ameliorates the nuclear shape abnormalities. Here we observed that an inhibitor of farnesyl diphosphate synthase, N6-isopentenyladenosine, impeded the farnesylation of prelamin A, causing a decrease in the frequency of nuclear shape abnormalities and redistribution of prelamin A away from the inner nuclear envelope. A combination of lovastatin and N6-isopentenyladenosine significantly improved nuclear shape in fibroblast cell lines from atypical progeria patients. These findings establish a paradigm for ameliorating the most obvious cellular pathology in lamin-related progeroid syndromes, and suggest a potential strategy for treating children with Hutchinson-Gilford progeria syndrome.