RESUMEN
In the adult mouse brain, the subventricular zone (SVZ) underlying the lateral ventricles harbours a population of quiescent neural stem cells, which can be activated (aNSCs) to initiate proliferation and generate a neurogenic lineage consisting of transit amplifying progenitors (TAPs), neuroblasts (NBs) and newborn neurons. This process is markedly reduced during aging. Recent studies suggest that the aged SVZ niche decreases the pool of proliferating neural/stem progenitor cells (NSPCs), and hence adult neurogenesis, by causing transcriptomic changes that promote NSC quiescence. The transcription factors that mediate these changes, however, remain unclear. We previously found that the homeobox gene Dbx2 is upregulated in NSPCs of the aged mouse SVZ and can inhibit the growth of NSPC cultures. Here, we further investigate its role as a candidate transcriptional regulator of neurogenic decline. We show that Dbx2 expression is downregulated by Epidermal Growth Factor receptor signaling, which promotes NSPC proliferation and decreases in the aged SVZ. By means of transgenic NSPC lines overexpressing Dbx2, we also show that this gene inhibits NSPC proliferation by hindering the G2/M transition. Furthermore, we exploit RNA sequencing of transgenic NSPCs to elucidate the transcriptomic networks modulated by Dbx2. Among the top hits, we report the downregulation of the molecular pathways implicated in cell cycle progression. Accordingly, we find that Dbx2 function is negatively correlated with the transcriptional signatures of proliferative NSPCs (aNSCs, TAPs and early NBs). These results point to Dbx2 as a transcription factor relaying the anti-neurogenic input of the aged niche to the NSPC transcriptome.
Asunto(s)
Genes Homeobox , Neurogénesis , Animales , Ratones , Envejecimiento/genética , División Celular , Neurogénesis/genética , Neuronas , Factores de TranscripciónRESUMEN
Macroautophagy/autophagy has been shown to exert a dual role in cancer i.e., promoting cell survival or cell death depending on the cellular context and the cancer stage. Therefore, development of potent autophagy modulators, with a clear mechanistic understanding of their target action, has paramount importance in both mechanistic and clinical studies. In the process of exploring the mechanism of action of a previously identified cytotoxic small molecule (SM15) designed to target microtubules and the interaction domain of microtubules and the kinetochore component NDC80/HEC1, we discovered that the molecule acts as a potent autophagy inhibitor. By using several biochemical and cell biology assays we demonstrated that SM15 blocks basal autophagic flux by inhibiting the fusion of correctly formed autophagosomes with lysosomes. SM15-induced autophagic flux blockage promoted apoptosis-mediated cell death associated with ROS production. Interestingly, autophagic flux blockage, apoptosis induction and ROS production were rescued by genetic or pharmacological inhibition of OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) or by expressing an O-GlcNAcylation-defective mutant of the SNARE fusion complex component SNAP29, pointing to SNAP29 as the molecular target of SM15 in autophagy. Accordingly, SM15 was found to enhance SNAP29 O-GlcNAcylation and, thereby, inhibit the formation of the SNARE fusion complex. In conclusion, these findings identify a new pathway in autophagy connecting O-GlcNAcylated SNAP29 to autophagic flux blockage and autophagosome accumulation, that, in turn, drives ROS production and apoptotic cell death. Consequently, modulation of SNAP29 activity may represent a new opportunity for therapeutic intervention in cancer and other autophagy-associated diseases.
Asunto(s)
Autofagosomas , Autofagia , Autofagosomas/metabolismo , Autofagia/fisiología , Macroautofagia , Especies Reactivas de Oxígeno/metabolismo , Lisosomas/metabolismo , Proteínas SNARE/metabolismo , ApoptosisRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest. METHODS: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin-factors involved in the control of cell cycle progression. RESULTS: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines. CONCLUSIONS: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.
Asunto(s)
Glioblastoma/metabolismo , Glioblastoma/patología , Mitosis , Receptor Muscarínico M2/metabolismo , Huso Acromático/metabolismo , Acetilación/efectos de los fármacos , Arecolina/análogos & derivados , Arecolina/farmacología , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Humanos , Metafase/efectos de los fármacos , Sirtuina 2/metabolismo , Imagen de Lapso de Tiempo , Tubulina (Proteína)/metabolismoRESUMEN
Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.
Asunto(s)
Mutación , Células-Madre Neurales/citología , Neuroglía/citología , Neuronas/patología , Proteína FUS de Unión a ARN/metabolismo , Médula Espinal/citología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Ratones , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/metabolismo , Proteína FUS de Unión a ARN/genética , Médula Espinal/embriología , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
Adipose-derived stem cells (ASCs) are an attractive source for regenerative medicine as they can be easily isolated, rapidly expandable in culture and show excellent in vitro differentiation potential. Acetylcholine (ACh), one of the main neurotransmitters in central and peripheral nervous systems, plays key roles in the control of several physiological processes also in non-neural tissues. As demonstrated in our previous studies, ACh can contribute to the rat ASCs physiology, negatively modulating ASCs proliferation and migration via M2 muscarinic receptor (mAChR) activation. In the present work we show that rat ASCs also express α7 nicotinic receptors (nAChRs). In particular, we have investigated the effects mediated by the selective activation of α7 nAChRs, which causes a reduction of ASC proliferation without affecting cell survival and morphology, and significantly promotes cell migration via upregulation of the CXCR4 expression. Interestingly, the activation of the α7 nAChR also upregulates the expression of M2 mAChR protein, indicating a cooperation between muscarinic and nicotinic receptors in the inhibition of ASC proliferation.
Asunto(s)
Movimiento Celular/fisiología , Proliferación Celular/fisiología , Células Madre Mesenquimatosas/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Tejido Adiposo/citología , Animales , Bungarotoxinas/farmacología , Fumaratos/farmacología , Masculino , Ratas Sprague-Dawley , Compuestos de Espiro/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Receptor Nicotínico de Acetilcolina alfa 7/antagonistas & inhibidoresRESUMEN
One of the major limits of chemotherapy is depending on the ability of the cancer cells to elude and adapt to different drugs. Recently, we demonstrated how the activation of the M2 muscarinic receptor could impair neuroblastoma cell proliferation. In the present paper, we investigate the possible effects mediated by the preferential M2 receptor agonist arecaidine propargyl ester (APE) on drug resistance in two neuroblastoma cell lines, SK-N-BE and SK-N-BE(2C), a sub-clone presenting drug resistance. In both cell lines, we compare the expression of the M2 receptor and the effects mediated by the M2 agonist APE on cell cycle, demonstrating a decreased percentage of cells in S phase and an accumulation of SK-N-BE cells in G1 phase, while the APE treatment of SK-N-BE(2C) cells induced a block in G2/M phase. The withdrawal of the M2 agonist from the medium shows that only the SK-N-BE(2C) cells are able to rescue cell proliferation. Further, we demonstrate that the co-treatment of low doses of APE with doxorubicin or cisplatin significantly counteracts cell proliferation when compared with the single treatment. Analysis of the expression of ATP-binding cassette (ABC) efflux pumps demonstrates the ability of the M2 agonist to downregulate their expression and that this negative modulation may be dependent on N-MYC decreased expression induced by the M2 agonist. Our data demonstrate that the combined effect of low doses of conventional drugs and the M2 agonist may represent a new promising therapeutic approach in neuroblastoma treatment, in light of its significant impact on drug resistance and the possible reduction in the side effects caused by high doses of chemotherapy drugs.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Arecolina/análogos & derivados , Neuroblastoma/tratamiento farmacológico , Receptor Muscarínico M2/agonistas , Transportadoras de Casetes de Unión a ATP/genética , Arecolina/administración & dosificación , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Doxorrubicina/administración & dosificación , Resistencia a Antineoplásicos , Expresión Génica/efectos de los fármacos , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patología , Receptor Muscarínico M2/genéticaRESUMEN
Exposure of the developing or adult brain to ionizing radiation (IR) can cause cognitive impairment and/or brain cancer, by targeting neural stem/progenitor cells (NSPCs). IR effects on NSPCs include transient cell cycle arrest, permanent cell cycle exit/differentiation, or cell death, depending on the experimental conditions. In vivo studies suggest that brain age influences NSPC response to IR, but whether this is due to intrinsic NSPC changes or to niche environment modifications remains unclear. Here, we describe the dose-dependent, time-dependent effects of X-ray IR in NSPC cultures derived from the mouse foetal cerebral cortex. We show that, although cortical NSPCs are resistant to low/moderate IR doses, high level IR exposure causes cell death, accumulation of DNA double-strand breaks, activation of p53-related molecular pathways and cell cycle alterations. Irradiated NSPC cultures transiently upregulate differentiation markers, but recover control levels of proliferation, viability and gene expression in the second week post-irradiation. These results are consistent with previously described in vivo effects of IR in the developing mouse cortex, and distinct from those observed in adult NSPC niches or in vitro adult NSPC cultures, suggesting that intrinsic differences in NSPCs of different origins might determine, at least in part, their response to IR.
Asunto(s)
Corteza Cerebral/citología , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de la radiación , Animales , Biomarcadores/metabolismo , Ciclo Celular/efectos de la radiación , Muerte Celular/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Histonas/metabolismo , Cinética , Ratones , Modelos Biológicos , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de la radiación , Rayos XRESUMEN
Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.
Asunto(s)
Ciclo Celular/fisiología , Proliferación Celular/fisiología , Glioblastoma/patología , Receptor Muscarínico M2/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Neoplasias Encefálicas/genética , División Celular/fisiología , Línea Celular Tumoral , Receptores ErbB/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Células Madre Neoplásicas/patología , Receptor Muscarínico M2/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Glioblastoma multiforme (GBM) is the most malignant brain tumor. Hypoxic condition is a predominant feature of the GBM contributing to tumor growth and resistance to conventional therapies. Hence, the identification of drugs able to impair GBM malignancy and aggressiveness is considered of great clinical relevance. Previously, we demonstrated that the activation of M2 muscarinic receptors, through the agonist arecaidine propargyl ester (Ape), arrests cell proliferation in GBM cancer stem cells (GSCs). In the present work, we have characterized the response of GSCs to hypoxic condition showing an upregulation of hypoxia-inducible factors and factors involved in the regulation of GSCs survival and proliferation. Ape treatment in hypoxic conditions is however able to inhibit cell cycle progression, causing a significant increase of aberrant mitosis with consequent decreased cell survival. Additionally, qRT-PCR analysis suggest that Ape downregulates the expression of stemness markers and miR-210 levels, one of the main regulators of the responses to hypoxic condition in different tumor types. Our data demonstrate that Ape impairs the GSCs proliferation and survival also in hypoxic condition, negatively modulating the adaptive response of GSCs to hypoxia.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Oxígeno/metabolismo , Receptor Muscarínico M2/metabolismo , Neoplasias Encefálicas/genética , Hipoxia de la Célula , Glioblastoma/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Receptor Muscarínico M2/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Resveratrol and its natural stilbene-containing derivatives have been extensively investigated as potential chemotherapeutic agents. The synthetic manipulation of the stilbene scaffold has led to the generation of new analogues with improved anticancer activity and better bioavailability. In the present study we investigated the anticancer activity of a novel trimethoxystilbene derivative (3,4,4'-trimethoxylstilbene), where two methoxyl groups are adjacent on the benzene ring (ortho configuration), and compared its activity to 3,5,4'-trimethoxylstilbene, whose methoxyl groups are in meta configuration. RESULTS: We provide evidence that the presence of the two methoxyl groups in ortho configuration renders 3,4,4'-trimethoxystilbene more efficient than the meta isomer in inhibiting cell proliferation and producing apoptotic death in colorectal cancer cells. Confocal microscopy of α- and γ-tubulin staining shows that the novel compound strongly depolymerizes the mitotic spindle and produces fragmentation of the pericentrosomal material. Computer assisted docking studies indicate that both molecules potentially interact with γ-tubulin, and that 3,4,4'-trimethoxystilbene is likely to establish stronger interactions with the protein. CONCLUSIONS: These findings demonstrate the ortho configuration confers higher specificity for γ-tubulin with respect to α-tubulin on 3,4,4' trimethoxystilbene, allowing it to be defined as a new γ-tubulin inhibitor. A strong interaction with γ-tubulin might be a defining feature of molecules with high anticancer activity, as shown for the 3,4,4' isomer.
RESUMEN
In previous studies, we found that the orthosteric muscarinic agonist arecaidine propargyl ester (APE) (100⯵M) induced a decreased cell proliferation and severe apoptosis in glioblastoma cancer stem cells (GSCs). In this report, we have investigated the effects mediated by hybrid (orthosteric/allosteric) muscarinic agonists P-6-Iper and N-8-Iper on GSCs survival. At variance with APE, the agonist N-8-Iper inhibited cell growth in a dose dependent manner and also impaired cell survival at low doses. The inhibitory effects of the N-8-Iper action appear to be mediated by M2 receptor activation, since they were strongly reduced by co-administration of the selective M2 receptor antagonist methoctramine as well as upon M2 receptor silencing. Moreover, analysis of the expression of phosphorylated histone H2AX (γ-H2AX) indicated that the treatment with N-8-Iper produced a decreased cell survival by induction of DNA damage. The ability of N-8-Iper to produce a cytotoxic effect and apoptosis at low doses indicates that this muscarinic agonist is a suitable probe in a putative therapeutic intervention on glioblastoma through M2 receptor activation.
Asunto(s)
Glioblastoma/patología , Agonistas Muscarínicos/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Receptor Muscarínico M2/agonistas , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Receptor Muscarínico M2/metabolismoRESUMEN
Mesenchymal stem cells (MSCs), also known as stromal mesenchymal stem cells, are multipotent cells, which can be found in many tissues and organs as bone marrow, adipose tissue and other tissues. In particular MSCs derived from Adipose tissue (ADSCs) are the most frequently used in regenerative medicine because they are easy to source, rapidly expandable in culture and excellent differentiation potential into adipocytes, chondrocytes, and other cell types. Acetylcholine (ACh), the most important neurotransmitter in Central nervous system (CNS) and peripheral nervous system (PNS), plays important roles also in non-neural tissue, but its functions in MSCs are still not investigated. Although MSCs express muscarinic receptor subtypes, their role is completely unknown. In the present work muscarinic cholinergic effects were characterized in rat ADSCs. Analysis by RT-PCR demonstrates that ADSCs express M1-M4 muscarinic receptor subtypes, whereas M2 is one of the most expressed subtype. For this reason, our attention was focused on M2 subtype. By using the selective M2 against Arecaidine Propargyl Ester (APE) we performed cell proliferation and migration assays demonstrating that APE causes cell growth and migration inhibition without affecting cell survival. Our results indicate that ACh via M2 receptors, may contribute to the maintaining of the ADSCs quiescent status. These data are the first evidence that ACh, via muscarinic receptors, might contribute to control ADSCs physiology.
Asunto(s)
Acetilcolina/metabolismo , Tejido Adiposo/citología , Células Madre Mesenquimatosas/citología , Receptor Muscarínico M2/genética , Acetilcolina/antagonistas & inhibidores , Tejido Adiposo/metabolismo , Animales , Arecolina/análogos & derivados , Arecolina/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratas , Receptor Muscarínico M1/genética , Receptor Muscarínico M2/agonistas , Activación Transcripcional/efectos de los fármacosRESUMEN
Natural compounds are extensively studied for their potential use in traditional and non-traditional medicine. Several natural and synthetic Resveratrol analogues have shown interesting biological activities in the field of cancer chemoprevention. In the present study, we have focused on the ability of Resveratrol and two methoxylated derivatives (Trimethoxystilbene and Pterostilbene) to inhibit human cancer cell growth particularly analyzing their ability to interfere with tubulin dynamics at mitosis. We show that Trimethoxystilbene, differently from Resveratrol and Pterostilbene, alters microtubule polymerization dynamics in HeLa cells specifically inducing multipolar spindles and mitotic arrest coupled to a reduction of cell growth and an increase in apoptotic death by mitotic catastrophe. This work demonstrates that the structural modification of Rsv causes substantial changes in the mechanism of action of the derivatives. The presence of three extra methyl groups renders Trimethoxy very efficient in impairing cell proliferation by inducing mitotic catastrophe in cancer cells. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Mitosis/efectos de los fármacos , Neoplasias/genética , Estilbenos/farmacología , Tubulina (Proteína)/metabolismo , Animales , Células CHO , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cricetulus , Células HeLa , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Resveratrol , Estilbenos/química , Tubulina (Proteína)/efectos de los fármacosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Xanthium strumarium L. is a member of the Asteraceae family popularly used with multiple therapeutic purposes. Whole extracts of this plant have shown anti-mitotic activity in vitro suggesting that some components could induce mitotic arrest in proliferating cells. AIM OF THE SUDY: Aim of the present work was to characterize the anti-mitotic properties of the X. strumarium whole extract and to isolate and purify active molecule(s). MATERIALS AND METHODS: The capacity of the whole extract to inhibit mitotic progression in mammalian cultured cells was investigated to identify its anti-mitotic activity. Isolation of active component(s) was performed using a bioassay-guided multistep separation procedure in which whole extract was submitted to a progressive process of fractionation and fractions were challenged for their anti-mitotic activity. RESULTS: Our results show for the first time that X. strumarium whole extract inhibits assembly of the mitotic spindle and spindle-pole separation, thereby heavily affecting mitosis, impairing the metaphase to anaphase transition and inducing apoptosis. The purification procedure led to a fraction with an anti-mitotic activity comparable to that of the whole extract. Chemical analysis of this fraction showed that its major component was xanthatin. CONCLUSIONS: The present work shows a new activity of X. strumarium extract, i.e. the alteration of the mitotic apparatus in cultured cells that may be responsible for the anti-proliferative activity of the extract. Anti-mitotic activity is shown to be mainly exerted by xanthatin.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Furanos/química , Furanos/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Huso Acromático/efectos de los fármacos , Xanthium/química , Animales , Apoptosis/efectos de los fármacos , Células CHO , Línea Celular , CricetulusRESUMEN
Resveratrol (3,4',5-trihydroxystilbene; RSV) acts on cancer cells in several ways, inducing cell cycle delay and apoptotic death, and enhancing ionizing radiation (IR)-mediated responses. However, fewer studies have examined RSV effects on normal cells. We have treated human lymphocytes in vitro with RSV, either alone or combined with IR, to evaluate its potential use as a radioprotector. We measured the effects of RSV on induction of DNA damage, repair kinetics, and modulation of histone deacetylase activity.
Asunto(s)
Radiación Ionizante , Estilbenos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Citometría de Flujo , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/efectos de la radiación , ResveratrolRESUMEN
Various naturally occurring stilbene-like compounds that are related to resveratrol (RSV) possess some of the beneficial effects of the parent molecule and provide even further benefits. Therefore, a series of methoxylated analogues of RSV were prepared with the aim of increasing antitumour and proapoptotic activity. In a previous article, we studied two methoxy-derivatives, pterostilbene (PTERO) and trimethoxystilbene (TRIMETHOXY), in which the first was formed by the substitution of two hydroxyl groups with two methoxy groups (trans-3,5-dimethoxy-4'-hydroxystilbene) and the second was formed by the replacement of all three OH groups with methoxy groups (trans-3,5,4'-trimethoxystilbene). Both methoxy-derivatives showed stronger antioxidant activity when compared with RSV. In the present article, we focused on the analysis of the ability of RSV and its two methoxylated derivatives to protect proliferating non-tumoural cells from the damage induced by ionising radiation (IR). First we showed that the methoxy derivatives, contrary to their parental compound, are unable to affect topoisomerase enzyme and consequently are not clastogenic per se Second we showed that both PTERO and TRIMETHOXY more efficiently reduce the chromosome damage induced by IR. Furthermore, TRIMETHOXY, but not PTERO, causes a delay in cell proliferation, particularly in mitosis progression increasing the number of cells in metaphase at the expense of prophases and ana/telophases.
Asunto(s)
Daño del ADN , Radiación Ionizante , Estilbenos/farmacología , Animales , Células CHO , Proliferación Celular/efectos de los fármacos , Cricetulus/genética , Cricetulus/fisiología , ADN/efectos de la radiación , Mitosis/efectos de los fármacos , Resveratrol , Estilbenos/toxicidad , Inhibidores de Topoisomerasa/farmacologíaRESUMEN
The p38/MAPK-activated kinase 2 (MK2) pathway is involved in a series of pathological conditions (inflammation diseases and metastasis) and in the resistance mechanism to antitumor agents. None of the p38 inhibitors entered advanced clinical trials because of their unwanted systemic side effects. For this reason, MK2 was identified as an alternative target to block the pathway but avoiding the side effects of p38 inhibition. However, ATP-competitive MK2 inhibitors suffered from low solubility, poor cell permeability, and scarce kinase selectivity. Fortunately, non-ATP-competitive inhibitors of MK2 have been already discovered that allowed circumventing the selectivity issue. These compounds showed the additional advantage to be effective at lower concentrations in comparison to the ATP-competitive inhibitors. Therefore, although the significant difficulties encountered during the development of these inhibitors, MK2 is still considered as an attractive target to treat inflammation and related diseases to prevent tumor metastasis and to increase tumor sensitivity to chemotherapeutics.
Asunto(s)
Ensayos Clínicos como Asunto , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas , Cristalización , Cristalografía por Rayos X , Humanos , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
Glioblastomas are the most common brain tumors in humans. Previously, we demonstrated that the muscarinic receptor agonist, arecaidine propargyl ester, via M2 receptors, inhibits cell proliferation in a time and dose-dependent manner and induces a severe apoptosis in human U251 and U87 glioblastoma cell lines. In order to clarify the mechanisms causing apoptosis after arecaidine treatment, we analyzed the ability of arecaidine to induce oxidative stress. By dichloro-dihydro-fluorescein diacetate (DCFDA) staining, we demonstrated that arecaidine increased the intracellular ROS levels. ROS accumulation was completely counteracted by the ROS scavenger, N-acetyl-l-cysteine (NAC). Apoptotic cell death appeared directly correlated to ROS production since NAC was able to counteract this effect. Although there was an up-regulation of some detoxifying enzyme expression such as superoxide dismutase (MnSOD) and sirtuin-1 (SIRT1), the cytotoxic effect caused by arecaidine treatment caused DNA damage, as demonstrated by the increase of histone γ-H2AX positive cells, and chromosomal aberrations. These effects were mediated by M2 receptor activation; in fact after silencing of M2 receptors by siRNA, the increase of γ-H2AX positive cells was abolished. In conclusion, in addition to a cytostatic effect previously described, in the present study we have better characterized the mechanisms causing the cytotoxic effects and the apoptotic cell death in glioblastoma cells after M2 receptor activation. These data allow to consider this receptor a new interesting therapeutic tool for the glioblastoma treatment.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Glioblastoma/metabolismo , Agonistas Muscarínicos/farmacología , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Acetilcisteína/farmacología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Humanos , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismoRESUMEN
Xanthium strumarium L. is a member of the Asteraceae commonly used in Cuba, mainly as diuretic. Some toxic properties of this plant have also been reported and, to date, very little is known about its genotoxic properties. The present work aims was to evaluate the potential cytotoxic and genotoxic risk of whole extract from Xanthium strumarium L. whole extract of aerial parts. No positive response was observed in a battery of four Salmonella typhimurium strains, when exposed to concentrations up to 5 mg/plate, with and without mammalian metabolic activation (liver microsomal S9 fraction from Wistar rats). In CHO cells, high concentrations (25-100 µg/mL) revealed significant reduction in cell viability. Results from sister chromatid exchanges, chromosome aberrations, and comet assay showed that X. strumarium extract is genotoxic at the highest concentration used, when clear cytotoxic effects were also observed. On the contrary, no increase in micronuclei frequency in bone marrow cells was observed when the extract was orally administered to mice (100, 500, and 2000 mg/Kg doses). The data presented here constitute the most complete study on the genotoxic potential of X. strumarium L. and show that the extract can induce in vitro DNA damage at cytotoxic concentrations.
Asunto(s)
Daño del ADN/efectos de los fármacos , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Extractos Vegetales/administración & dosificación , Animales , Células CHO , Cricetulus , Ratones , Extractos Vegetales/química , Ratas , Salmonella typhimurium/efectos de los fármacos , Xanthium/químicaRESUMEN
The molecular mechanism(s) mediating long-term adverse effects of dichlorvos, a widely used insecticide, are still unclear. Our work uncovered a new cellular effect of dichlorvos in cultured human cells, i.e. its capacity to induce extremely aberrant mitotic spindles with monopolar microtubule arrays that were associated with hypercondensed chromosomes and pyknotic chromatin masses. Monopolar spindles produced by dichlorvos treatment were characterized by the delocalization of the depolymerizing kinesin Kif2a from spindle poles. Dichlorvos-induced spindle monopolarity could be reversed by promoting microtubule stabilization through chemical treatment or by inhibiting the depolymerizing function of the kinesin MCAK at kinetochores. These findings demonstrate that dichlorvos inhibits the depolymerizing activity of Kif2a at centrosomes and thereby disrupts the balance of opposing centrosomal and kinetochore forces controlling spindle bipolarity during prometaphase. Dichlorvos-induced defects in spindle bipolarity may be responsible for the previously reported induction of aneuploidy by this chemical. Collectively, these results indicate that environmental chemicals, such as dichlorvos, may promote chromosome instability by interfering with the cell division machinery.