RESUMEN
In this study, we utilized an in vitro model consisting of human malignant melanoma as well as non-tumorigenic immortalized keratinocyte cells with the aim of characterizing the therapeutic effectiveness of the clinical epigenetic drug Tazemetostat alone or in combination with various isothiocyanates. In doing so, we assessed markers of cell viability, apoptotic induction, and expression levels of key proteins capable of mediating the therapeutic response. Our data indicated, for the first time, that Tazemetostat caused a significant decrease in viability levels of malignant melanoma cells in a dose- and time-dependent manner via the induction of apoptosis, while non-malignant keratinocytes were more resistant. Moreover, combinatorial treatment protocols caused a further decrease in cell viability, together with higher apoptotic rates. In addition, a significant reduction in the Polycomb Repressive Complex 2 (PRC2) members [e.g., Enhancer of Zeste Homologue 2 (EZH2), Embryonic Ectoderm Development (EED), and suppressor of zeste 12 (SUZ12)] and tri-methylating lysine 27 at Histone 3 (H3K27me3) protein expression levels was observed, at least partially, under specific combinatorial exposure conditions. Reactivation of major apoptotic gene targets was determined at much higher levels in combinatorial treatment protocols than Tazemetostat alone, known to be involved in the induction of intrinsic and extrinsic apoptosis. Overall, we developed an optimized experimental therapeutic platform aiming to ensure the therapeutic effectiveness of Tazemetostat in malignant melanoma while at the same time minimizing toxicity against neighboring non-tumorigenic keratinocyte cells.
Asunto(s)
Benzamidas , Compuestos de Bifenilo , Histonas , Melanoma , Morfolinas , Piridonas , Humanos , Histonas/metabolismo , Complejo Represivo Polycomb 2/genética , Lisina/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , ApoptosisRESUMEN
Malignant melanoma is the most aggressive type of skin cancer with increasing incidence rates worldwide. On the other hand, watercress is a rich source of phenethyl isothiocyanate (PEITC), among others, which has been widely investigated for its anticancer properties against various cancers. In the present study, we evaluated the role of a watercress extract in modulating apoptotic induction in an in vitro model of human malignant melanoma consisting of melanoma (A375, COLO-679, COLO-800), non-melanoma epidermoid carcinoma (A431) and immortalized, non-tumorigenic keratinocyte (HaCaT) cells. Moreover, the chemical composition of the watercress extract was characterized through UPLC MS/MS and other analytical methodologies. In addition, cytotoxicity was assessed by the alamar blue assay whereas apoptosis was determined, initially, by a multiplex activity assay kit (measuring levels of activated caspases -3, -8 and -9) as well as by qRT-PCR for the identification of major genes regulating apoptosis. In addition, protein expression levels were evaluated by western immunoblotting. Our data indicate that the extract contains various phytochemicals (e.g. phenolics, flavonoids, pigments, etc.) while isothiocyanates (ITCs; especially PEITC) were the most abundant. In addition, the extract was shown to exert a significant time- and dose-dependent cytotoxicity against all malignant melanoma cell lines while non-melanoma and non-tumorigenic cells exhibited significant resistance. Finally, expression profiling revealed a number of genes (and corresponding proteins) being implicated in regulating apoptotic induction through activation of the intrinsic apoptotic cascade. Overall, our data indicate the potential of PEITC as a promising anti-cancer agent in the clinical management of human malignant melanoma.
RESUMEN
Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form.
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Hepatitis C Crónica , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Interferón-alfa/genética , Interferón-alfa/farmacología , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Aldehyde dehydrogenase 3A1 (ALDH3A1) oxidizes medium-chain aldehydes to their corresponding carboxylic acids. It is expressed at high rates in the human cornea, where it has been characterized as a multi-functional protein displaying various cytoprotective modes of action. Previous studies identified its association with the DNA damage response (DDR) pathway. Here, we utilized a stable transfected HCE-2 (human corneal epithelium) cell line expressing ALDH3A1, to investigate the molecular mechanisms underlying the cytoprotective role(s) of ALDH3A1. Our data revealed morphological differences among the ALDH3A1-expressing and the mock-transfected HCE-2 cells accompanied by differential expression of E-cadherin. Similarly, the ALDH3A1/HCE-2 cells demonstrated higher mobility, reduced proliferation, upregulation of ZEB1, and downregulation of CDK3, and p57. The expression of ALDH3A1 also affected cell cycle progression by inducing the sequestration of HCE-2 cells at the G2/M phase. Following 16 h cell treatments with either H2O2 or etoposide, a significantly lower percentage of ALDH3A1/HCE-2 cells were apoptotic compared to the respective treated mock/HCE-2 cells. Interestingly, the protective effect of ALDH3A1 expression under these oxidative and genotoxic conditions was accompanied by a reduced formation of γ-H2AX foci and higher levels of total and phospho (Ser15) p53. Finally, ALDH3A1 was found to be localized both in the cytoplasm and the nucleus of transfected HCE-2 cells. Its cellular compartmentalization was not affected by oxidant treatment, while the mechanism by which ALDH3A1 translocates to the nucleus remains unknown. In conclusion, ALDH3A1 protects cells from both apoptosis and DNA damage by interacting with key homeostatic mechanisms associated with cellular morphology, cell cycle, and DDR.
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Aldehído Deshidrogenasa , Peróxido de Hidrógeno , Humanos , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Córnea/metabolismo , Células Epiteliales/metabolismoRESUMEN
The biological activities of Zn(II) compounds have been extensively studied in recent years. In this study, the growth suppressive effect of Zn(II) 5,5-diethylbarbiturate complex on MCF-7 and MDA-MB-231 human breast cancer cells was determined by SRB and ATP viability assays and apoptosis-inducing effect by double staining method. Significant increase in cytokeratin 18 level, caspase 3/7 activity and annexin-V upregulation prove that Zn(II) complex has apoptotic effect in breast cancer cells. Intrinsic apoptosis pathway in MCF-7 cells and extrinsic apoptosis pathway in MDA-MB-231 cells was determined by Western blot (PARP, Cleave PARP, BAX, COX4, RIP, Caspase 8, Split Caspase 8, DR4 and B-Actin) and RT-PCR (PARP, Fas, Bcl-2, TNF10A, P53) analysis. No reduction of viability was found in MCF-710A healthy breast cells treated with Zn(II) complex. In breast cancer stem-like cells (MCF-7s), the Zn(II) complex was found to have a cytotoxic effect and to activate the apoptotic pathway. As a result, it was concluded that Zn(II) complex has anti-proliferative and apoptotic effects on breast cancer and breast cancer stem-like cells. Also this complex prevents the metastatic effect of cancer cells and does not effect to healthy cells so this complex has a specific effect on cancer cells. These findings might shed light on the discovery of new chemotherapeutic agents.
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Antineoplásicos , Neoplasias de la Mama , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Neoplasias de la Mama/patología , Caspasa 8/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Células MCF-7 , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Zinc/metabolismo , Zinc/farmacologíaRESUMEN
The human lifespan has been significantly increased due to scientific advancements in the management of disease; however, the health span of the aging population does not follow the same trend. Aging is the major risk factor for multimorbidity that is derived from the progressive loss of homeostasis, immunological and stem cell exhaustion, as well as exacerbated inflammation responses. Age-related diseases presenting with high frequencies include neurodegenerative, musculoskeletal, cardiovascular, metabolic diseases and cancer. These diseases can be co-morbid and are usually managed using a disease-specific approach that can eventually lead to polypharmacy, low medication adherence rates and undesired drug-drug interactions. Novel studies suggest targeting the shared biological basis of age-related diseases to retard the onset and manage their manifestations. Harvesting the anti-inflammatory and immunomodulatory capacity of probiotics to tackle the root cause of these diseases, could pose a viable alternative. In this article, a comprehensive review of the effects of probiotic supplementation on the molecular pathogenesis of age-related diseases, and the potential of probiotic treatments as preventative or alleviatory means is attempted. Furthermore, issues on the safety and efficiency of probiotic supplementation, as well as the pitfalls of current clinical studies are discussed, while new perspectives for systematic characterization of probiotic benefits on aged hosts are outlined.
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Probióticos , Anciano , Envejecimiento , Humanos , Probióticos/uso terapéutico , Factores de RiesgoRESUMEN
Melanoma is an aggressive skin cancer with increasing incidence rates globally. On the other hand, isothiocyanates are derived from cruciferous vegetables and are known to exert a wide range of anti-cancer activities including, among others, their ability to interact with the epigenome in order to supress cancer progression. The aim of this study was to determine the role of phenethyl and benzyl isothiocyanates in modulating histone acetylation and methylation as a potential epigenetic therapeutic strategy in an in vitro model of malignant melanoma. We report that both isothiocyanates induced cytotoxicity and influenced acetylation and methylation status of specific lysine residues on histones H3 and H4 by modulating the expression of various histone acetyltransferases, deacetylases and methyltransferases in malignant melanoma cells. Our data highlight novel insights on the interaction of isothiocyanates with components of the histone regulatory machinery in order to exert their anti-cancer action in malignant melanoma.
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Histonas/efectos de los fármacos , Isotiocianatos/farmacología , Melanoma/fisiopatología , Neoplasias Cutáneas/fisiopatología , Acetilación/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular , Epigénesis Genética , Humanos , Metilación/efectos de los fármacosRESUMEN
Melanoma is an aggressive and highly metastatic type of skin cancer where the design of new therapies is of utmost importance for the clinical management of the disease. Thus, we have aimed to investigate the mode of action by which a novel methylated analogue of L-Mimosine (e.g., L-SK-4) exerts its therapeutic potency in an in vitro model of malignant melanoma. Cytotoxicity was assessed by the Alamar Blue assay, oxidative stress by commercially available kits, ROS generation, caspase 3/7 activation and mitochondrial membrane depolarisation by flow cytometry, expression of apoptosis-related proteins by western immunoblotting and profiling of lipid biosynthesis by a metabolomic approach. Overall, higher levels of ROS, sphingolipids and apoptosis were induced by L-SK-4 suggesting that the compound's therapeutic potency is mediated through elevated ROS levels which promote the upregulation of sphingolipid (ceramide) biosynthesis thus leading to the activation of both extrinsic and intrinsic apoptosis, in an experimental model of malignant melanoma.
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Antineoplásicos/farmacología , Melanoma/tratamiento farmacológico , Mimosina/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Ceramidas/metabolismo , Ceramidas/farmacología , Citometría de Flujo , Humanos , Melanoma/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Metilación , Ratones , Mimosina/análogos & derivados , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
OBJECTIVE(S): Growing evidence supports that isothiocyanates exert a wide range of bioactivities amongst of which is their capacity to interact with the epigenetic machinery in various cancers including melanoma. Our aim was to characterise the effect of sulforaphane and iberin on histone acetylation and methylation as a potential anti-melanoma strategy. METHODS: We have utilised an in vitro model of malignant melanoma [consisting of human (A375, Hs294T, VMM1) and murine (B16F-10) melanoma cell lines as well as a non-melanoma (A431) and a non-tumorigenic immortalised keratinocyte (HaCaT) cell line] exposed to sulforaphane or iberin. Cell viability was evaluated by the Alamar blue assay whilst total histone deacetylases and acetyltransferases activities were determined by the Epigenase HDAC Activity/Inhibition and EpiQuik HAT Activity/Inhibition assay kits, respectively. The expression levels of specific histone deacetylases and acetyltransferases together with those of lysine acetylation and methylation marks were obtained by western immunoblotting. RESULTS: Overall, both sulforaphane and iberin were able to (1) reduce cell viability, (2) decrease total histone deacetylase activity and (3) modulate the expression levels of various histone deacetylases as well as acetyl and methyl transferases thus modulating the acetylation and methylation status of specific lysine residues on histones 3 and 4 in malignant melanoma cells. CONCLUSIONS: Our findings highlight novel insights as to how sulforaphane and iberin differentially regulate the epigenetic response in ways compatible with their anticancer action in malignant melanoma.
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Histonas , Melanoma , Acetilación , Animales , Línea Celular Tumoral , Epigénesis Genética , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Humanos , Isotiocianatos/farmacología , Melanoma/tratamiento farmacológico , Melanoma/genética , Metilación , Ratones , SulfóxidosRESUMEN
Surface active agents (SAAs), currently used in modern industry, are synthetic chemicals produced from non-renewable sources, with potential toxic impacts on humans and the environment. Thus, there is an increased interest for the identification and utilization of natural derived SAAs. As such, the marine environment is considered a promising source of biosurfactants with low toxicity, environmental compatibility, and biodegradation compared to their synthetic counterparts. MARISURF is a Horizon 2020 EU-funded project aiming to identify and functionally characterize SAAs, derived from a unique marine bacterial collection, towards commercial exploitation. Specifically, rhamnolipids produced by Marinobacter MCTG107b and Pseudomonas MCTG214(3b1) strains were previously identified and characterized while currently their toxicity profile was assessed by utilizing well-established methodologies. Our results showed a lack of cytotoxicity in in vitro models of human skin and liver as indicated by alamar blue and propidium iodide assays. Additionally, the use of the single gel electrophoresis assay, under oxidative stress conditions, revealed absence of any significant mutagenic/anti-mutagenic potential. Finally, both 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) cell-free assays, revealed no significant anti-oxidant capacity for neither of the tested compounds. Consequently, the absence of significant cytotoxicity and/or mutagenicity justifies their commercial exploitation and potential development into industrial end-user applications as natural and environmentally friendly biosurfactants.
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Bacterias/metabolismo , Queratinocitos/patología , Neoplasias/patología , Tensoactivos/efectos adversos , Tensoactivos/aislamiento & purificación , Apoptosis , Proliferación Celular , Humanos , Queratinocitos/efectos de los fármacos , Neoplasias/inducido químicamente , Pruebas de Toxicidad , Células Tumorales CultivadasRESUMEN
The anticancer activity of a series of novel synthesized, hydroxypyridone-based metal chelators (analogues of L-mimosine) was evaluated in an in vitro model of melanoma consisting of malignant melanoma (A375), non-melanoma epidermoid carcinoma (A431) and immortalized non-malignant keratinocyte (HaCaT) cells. More specifically, we have demonstrated that the L-enantiomer of a methylated analogue of L-mimosine (compound 22) can exert a potent anticancer effect in A375 cells when compared to either A431 or HaCaT cells. Moreover, we have demonstrated that this analogue has the ability to i) promote increased generation of reactive oxygen species (ROS), ii) activate both intrinsic and extrinsic apoptosis and iii) induce perturbations in cell cycle growth arrest. Our data highlights the potential of compound 22 to act as a promising therapeutic agent against an in vitro model of human malignant melanoma.
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Antineoplásicos/farmacología , Melanoma/tratamiento farmacológico , Mimosina/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Melanoma/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Metal-derived nanoparticles (Mt-NPs) are increasingly used in cosmetology due to their ultraviolet shielding (titanium dioxide [TiO2]), antioxidant (cerium dioxide [CeO2]), and biocidal (silver [Ag]) properties. In the absence of overt toxicity (i.e., cell death), Mt-NPs are considered safe for cosmetic applications. However, there is little understanding about the mechanisms involved in the survival of keratinocytes exposed to subtoxic levels of Mt-NPs. Human keratinocytes (HaCaT) were exposed subacutely to subtoxic concentrations (≤30 µg/mL, 48-72 h) of rutile (r) TiO2 (cylindrical), CeO2 (cubic) and Ag (spherical) with a core/hydrodynamic size of <50/<100 nm and >98% purity. Mt-NP uptake was indirectly quantified by changes in the light side scatter, where the kinetics (time/dose-response) suggested that the three types of Mt-NPs were similarly uptaken by keratinocytes. rTiO2 and CeO2, but not Ag-NPs, increased autophagy, whose inhibition prompted cell death. No increase in the steady-state levels of reactive oxygen species (ROS) was induced by exposure to any of the Mt-NPs tested. Interestingly, intracellular Ag-NP aggregates observed an increased far-red autofluorescence (≥740 nm em), which has been ascribed to their binding to thiol molecules such as glutathione (GSH). Accordingly, inhibition of GSH synthesis, but not the impairment of oxidized GSH recycling, sensitized keratinocytes to Ag-NPs suggesting that GSH homeostasis, and its direct scavenging of Ag-NPs, but not ROS, is essential for keratinocyte survival upon exposure to Ag-NP. rTiO2 and Ag, but not CeO2-NPs, compromised metabolic flux (glycolysis and respiration), but ATP levels were unaltered. Finally, we also observed that exposure to Mt-NPs sensitized keratinocytes to non-UV xenobiotic exposure (arsenite and paraquat). Our results demonstrate the differential contribution of autophagy and GSH homeostasis to the survival of human keratinocytes exposed to subtoxic concentrations of Mt-NPs and highlight the increased susceptibility of keratinocytes exposed to Mt-NPs to a second xenobiotic insult.
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Cerio/farmacología , Queratinocitos/efectos de los fármacos , Nanopartículas/química , Plata/farmacología , Titanio/farmacología , Xenobióticos/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cerio/química , Humanos , Queratinocitos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Tamaño de la Partícula , Plata/química , Propiedades de Superficie , Titanio/química , Xenobióticos/metabolismoRESUMEN
OBJECTIVE(S): Isothiocyanates (ITCs) are biologically active plant secondary metabolites capable of mediating various biological effects including modulation of the epigenome. Our aim was to characterize the effect of allyl isothiocyanate (AITC) on lysine acetylation and methylation marks as a potential epigenetic-induced anti-melanoma strategy. METHODS: Our malignant melanoma model consisted of (1) human (A375) and murine (B16-F10) malignant melanoma as well as of human; (2) brain (VMM1) and lymph node (Hs 294T) metastatic melanoma; (3) non-melanoma epidermoid carcinoma (A431) and (4) immortalized keratinocyte (HaCaT) cells subjected to AITC. Cell viability, histone deacetylases (HDACs) and acetyltransferases (HATs) activities were evaluated by the Alamar blue, Epigenase HDAC Activity/Inhibition and EpiQuik HAT Activity/Inhibition assay kits, respectively, while their expression levels together with those of lysine acetylation and methylation marks by western immunoblotting. Finally, apoptotic gene expression was assessed by an RT-PCR-based gene expression profiling methodology. RESULTS: AITC reduces cell viability, decreases HDACs and HATs activities and causes changes in protein expression levels of various HDACs, HATs, and histone methyl transferases (HMTs) all of which have a profound effect on specific lysine acetylation and methylation marks. Moreover, AITC regulates the expression of a number of genes participating in various apoptotic cascades thus indicating its involvement in apoptotic induction. CONCLUSIONS: AITC exerts a potent epigenetic effect suggesting its potential involvement as a promising epigenetic-induced bioactive for the treatment of malignant melanoma.
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Isotiocianatos/farmacología , Lisina/efectos de los fármacos , Lisina/metabolismo , Melanoma/metabolismo , Acetilación , Animales , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Isotiocianatos/metabolismo , Metilación , RatonesRESUMEN
Arsenic is a metalloid found in groundwater as a byproduct of soil/rock erosion and industrial and agricultural processes. This xenobiotic elicits its toxicity through different mechanisms, and it has been identified as a toxicant that affects virtually every organ or tissue in the body. In the central nervous system, exposure to arsenic can induce cognitive dysfunction. Furthermore, iAs has been linked to several neurological disorders, including neurodevelopmental alterations, and is considered a risk factor for neurodegenerative disorders. However, the exact mechanisms involved are still unclear. In this review, we aim to appraise the neurotoxic effects of arsenic and the molecular mechanisms involved. First, we discuss the epidemiological studies reporting on the effects of arsenic in intellectual and cognitive function during development as well as studies showing the correlation between arsenic exposure and altered cognition and mental health in adults. The neurotoxic effects of arsenic and the potential mechanisms associated with neurodegeneration are also reviewed including data from experimental models supporting epidemiological evidence of arsenic as a neurotoxicant. Next, we focused on recent literature regarding arsenic metabolism and the molecular mechanisms that begin to explain how arsenic damages the central nervous system including, oxidative stress, energy failure and mitochondrial dysfunction, epigenetics, alterations in neurotransmitter homeostasis and synaptic transmission, cell death pathways, and inflammation. Outlining the specific mechanisms by which arsenic alters the cell function is key to understand the neurotoxic effects that convey cognitive dysfunction, neurodevelopmental alterations, and neurodegenerative disorders.
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Intoxicación por Arsénico/etiología , Arsénico/toxicidad , Animales , Apoptosis/efectos de los fármacos , Intoxicación por Arsénico/genética , Autofagia/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Hipocampo/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Origanum species are plants rich in volatile oils that are mainly used for culinary purposes. In recent years, there has been a growing interest in the biological activities of their essential oils. Origanum onites L. is a plant mainly found in Greece, Turkey, and Sicily, whose oil is rich in carvacrol, a highly bioactive phytochemical. The aim of this study was to analyze the chemical composition of Origanum onites essential oil (OOEO), and investigate its potential anticancer effects in vitro and in vivo. GC/MS analysis identified carvacrol as OOEO's main constituent. In vitro antiproliferative activity was assayed with the sulforhodamine B (SRB) assay against human cancer cell lines from four tumor types. HT-29, a colorectal cancer cell line, was the most sensitive to the antiproliferative activity of OOEO. Wound-healing assay and Annexin V-PI staining were employed to investigate the antimigratory and the pro-apoptotic potential of OOEO, respectively, against human (HT-29) and murine (CT26) colon cancer cells. Notably, OOEO attenuated migration and induced apoptosis-related morphological changes in both cell lines. Prophylactic oral administration of the oil in a BALB/c experimental mouse model inhibited the growth of syngeneic CT26 colon tumors. As far as we know, this is the first report on the antitumor potential of orally administered OOEO.
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Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Aceites Volátiles/química , Aceites Volátiles/farmacología , Origanum/química , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fraccionamiento Químico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Ratones , Aceites Volátiles/aislamiento & purificación , Fitoquímicos/química , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacología , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The aim of the study was to characterize the chemical composition and biological properties of the essential oil from the plant Lippia citriodora grown in Greece. The essential oil volatiles were analyzed by gas chromatography-mass spectrometry GC-MS indicating citral as the major component. Τhe antimicrobial properties were assayed using the disk diffusion method and the minimum inhibitory and non-inhibitory concentration values were determined. Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, Saccharomyces cerevisiae, and Aspergillus niger were sensitive to Lippia citriodora oil, but not Escherichia coli, Salmonella Enteritidis, Salmonella typhimurium, and Pseudomonas fragi. Adversely, all microbes tested were sensitive to citral. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays were used to assess direct antioxidant activity, which proved to be weak for both agents, while comet assay was utilized to study the cytoprotective effects against H2O2-induced oxidative damage in Jurkat cells. Interestingly, the oil showed a more profound cytoprotective effect compared to citral. The antiproliferative activity was evaluated in a panel of cancer cell lines using the sulforhodamine B (SRB) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-S-(phenylamino) carbonyl-2-tetrazolium hydroxide (XTT) assays and both agents demonstrated potent antiproliferative activity with citral being more cytotoxic than the oil. Taken together, the essential oil of Lippia citriodora and its major component, citral, exert diverse biological properties worthy of further investigation.
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Lippia/química , Aceites Volátiles/química , Fitoquímicos/química , Aceites de Plantas/química , Antibacterianos/química , Antibacterianos/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Aceites Volátiles/farmacología , Fitoquímicos/farmacología , Aceites de Plantas/análisis , Aceites de Plantas/farmacologíaRESUMEN
Utilization of thermal therapy (hyperthermia) is defined as the application of exogenous heat induction and represents a concept that is far from new as it goes back to ancient times when heat was used for treating various diseases, including malignancies. Such therapeutic strategy has gained even more popularity (over the last few decades) since various studies have shed light into understanding hyperthermia's underlying molecular mechanism(s) of action. In general, hyperthermia is applied as complementary (adjuvant) means in therapeutic protocols combining chemotherapy and/or irradiation both of which can induce irreversible cellular DNA damage. Furthermore, according to a number of in vitro, in vivo and clinical studies, hyperthermia has been shown to enhance the beneficial effects of DNA targeting therapeutic strategies by interfering with DNA repair response cascades. Therefore, the continuously growing evidence supporting hyperthermia's beneficial role in cancer treatment can also encourage its application as a DNA repair mitigation strategy. In this review article, we aim to provide detailed information on how hyperthermia acts on DNA damage and repair pathways and thus potentially contributing to various adjuvant therapeutic protocols relevant to more efficient cancer treatment strategies.
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Daño del ADN , Reparación del ADN , Hipertermia Inducida/métodos , Neoplasias/genética , Neoplasias/terapia , Animales , HumanosRESUMEN
Silibinin, extracted from milk thistle (Silybum marianum L.), has exhibited considerable preclinical activity against prostate carcinoma. Its antitumor and chemopreventive activities have been associated with diverse effects on cell cycle, apoptosis, and receptor-dependent mitogenic signaling pathways. Here we hypothesized that silibinin's pleiotropic effects may reflect its interference with epigenetic mechanisms in human prostate cancer cells. More specifically, we have demonstrated that silibinin reduces gene expression levels of the Polycomb Repressive Complex 2 (PRC2) members Enhancer of Zeste Homolog 2 (EZH2), Suppressor of Zeste Homolog 12 (SUZ12), and Embryonic Ectoderm Development (EED) in DU145 and PC3 human prostate cancer cells, as evidenced by Real Time Polymerase Chain Reaction (RT-PCR). Furthermore immunoblot and immunofluorescence analysis revealed that silibinin-mediated reduction of EZH2 levels was accompanied by an increase in trimethylation of histone H3 on lysine (Κ)-27 residue (H3K27me3) levels and that such response was, in part, dependent on decreased expression levels of phosphorylated Akt (ser473) (pAkt) and phosphorylated EZH2 (ser21) (pEZH2). Additionally silibinin exerted other epigenetic effects involving an increase in total DNA methyltransferase (DNMT) activity while it decreased histone deacetylases 1-2 (HDACs1-2) expression levels. We conclude that silibinin induces epigenetic alterations in human prostate cancer cells, suggesting that subsequent disruptions of central processes in chromatin conformation may account for some of its diverse anticancer effects.
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Antineoplásicos/farmacología , Antioxidantes/farmacología , Proteína Potenciadora del Homólogo Zeste 2/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Complejo Represivo Polycomb 2/biosíntesis , Neoplasias de la Próstata/tratamiento farmacológico , Silimarina/farmacología , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epigénesis Genética , Histona Desacetilasa 1/biosíntesis , Histona Desacetilasa 2/biosíntesis , Histonas/metabolismo , Humanos , Masculino , Metilación/efectos de los fármacos , Proteínas de Neoplasias , Fosforilación/efectos de los fármacos , Complejo Represivo Polycomb 2/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Silibina , Factores de TranscripciónRESUMEN
Natural products, known for their medicinal properties since antiquity, are continuously being studied for their biological properties. In the present study, we analyzed the composition of the volatile preparations of essential oils of the Greek plants Ocimum basilicum (sweet basil), Mentha spicata (spearmint), Pimpinella anisum (anise) and Fortunella margarita (kumquat). GC/MS analyses revealed that the major components in the essential oil fractions, were carvone (85.4%) in spearmint, methyl chavicol (74.9%) in sweet basil, trans-anethole (88.1%) in anise, and limonene (93.8%) in kumquat. We further explored their biological potential by studying their antimicrobial, antioxidant and antiproliferative activities. Only the essential oils from spearmint and sweet basil demonstrated cytotoxicity against common foodborne bacteria, while all preparations were active against the fungi Saccharomyces cerevisiae and Aspergillus niger. Antioxidant evaluation by DPPH and ABTS radical scavenging activity assays revealed a variable degree of antioxidant potency. Finally, their antiproliferative potential was tested against a panel of human cancer cell lines and evaluated by using the sulforhodamine B (SRB) assay. All essential oil preparations exhibited a variable degree of antiproliferative activity, depending on the cancer model used, with the most potent one being sweet basil against an in vitro model of human colon carcinoma.
Asunto(s)
Mentha spicata/química , Ocimum basilicum/química , Aceites Volátiles/química , Aceites Volátiles/farmacología , Pimpinella/química , Rutaceae/química , Derivados de Alilbenceno , Anisoles/aislamiento & purificación , Anisoles/farmacología , Aspergillus niger/efectos de los fármacos , Bacterias/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Monoterpenos Ciclohexánicos , Ciclohexenos/aislamiento & purificación , Ciclohexenos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Microbiología de Alimentos , Humanos , Limoneno , Pruebas de Sensibilidad Microbiana , Monoterpenos/aislamiento & purificación , Monoterpenos/farmacología , Oxidación-Reducción/efectos de los fármacos , Aceites de Plantas/química , Aceites de Plantas/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Terpenos/aislamiento & purificación , Terpenos/farmacologíaRESUMEN
BACKGROUND: Nowadays, there has been an increased interest in essential oils from various plant origins as potential antimicrobial, antioxidant, and antiproliferative agents. This trend can be mainly attributed to the rising number and severity of food poisoning outbreaks worldwide along with the recent negative consumer perception against artificial food additives and the demand for novel functional foods with possible health benefits. Origanum dictamnus (dittany) is an aromatic, tender perennial plant that only grows wild on the mountainsides and gorges of the island of Crete in Greece. OBJECTIVE: The aim of the present study was to investigate the antimicrobial, antioxidant, and antiproliferative properties of O. dictamnus essential oil and its main components and assess its commercial potential in the food industry. DESIGN: O. dictamnus essential oil was initially analyzed by gas chromatography-mass spectrometry (GC-MS) to determine semi-quantitative chemical composition of the essential oils. Subsequently, the antimicrobial properties were assayed and the minimum inhibitory and non-inhibitory concentration values were determined. The antioxidant activity and cytotoxic action against the hepatoma adenocarcinoma cell line HepG2 of the essential oil and its main components were further evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and by the sulforhodamine B (SRB) assay, respectively. RESULTS: The main constituents of O. dictamnus essential oil identified by GC-MS analysis were carvacrol (52.2%), γ-terpinene (8.4%), p-cymene (6.1%), linalool (1.4%), and caryophyllene (1.3%). O. dictamnus essential oil and its main components were effective against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Listeria monocytogenes, Salmonella Enteritidis, Salmonella typhimurium, Saccharomyces cerevisiae, and Aspergillus niger. In addition, the estimated IC50 value for the DPPH radical scavenging activity for O. dictamnus essential oil was 0.045±0.0042% (v/v) and was mainly attributed to carvacrol. The EC50 value for the essential oil in the 72h SRB assay in HepG2 cells was estimated to be 0.0069±0.00014% (v/v). Among the individual constituents tested, carvacrol was the most bioactive compound and accounted for the observed antiproliferative activity of the essential oil. CONCLUSIONS: The results revealed that O. dictamnus essential oil is a noteworthy growth inhibitor against the microbes studied. It also possesses significant antioxidant activity and demonstrated excellent cytotoxicity against HepG2 cells. Taken together, O. dictamnus essential oil may represent an effective and inexpensive source of potent natural antimicrobial agents with health-promoting properties, which may be incorporated in food systems.