RESUMEN
Pancracine, a montanine-type Amaryllidaceae alkaloid (AA), is one of the most potent compounds among natural isoquinolines. In previous studies, pancracine exhibited cytotoxic activity against diverse human cancer cell lines in vitro. However, further insight into the molecular mechanisms that underlie the cytotoxic effect of pancracine have not been reported and remain unknown. To fill this void, the cell proliferation and viability of cancer cells was explored using the Trypan Blue assay or by using the xCELLigence system. The impact on the cell cycle was determined by flow cytometry. Apoptosis was evaluated by Annexin V/PI and by quantifying the activity of caspases (-3/7, -8, and -9). Proteins triggering growth arrest or apoptosis were detected by Western blotting. Pancracine has strong antiproliferative activity on A549 cells, lasting up to 96 h, and antiproliferative and cytotoxic effects on MOLT-4 cells. The apoptosis-inducing activity of pancracine in MOLT-4 cells was evidenced by the significantly higher activity of caspases. This was transmitted through the upregulation of p53 phosphorylated on Ser392, p38 MAPK phosphorylated on Thr180/Tyr182, and upregulation of p27. The pancracine treatment negatively altered the proliferation of A549 cells as a consequence of an increase in G1-phase accumulation, associated with the downregulation of Rb phosphorylated on Ser807/811 and with the concomitant upregulation of p27 and downregulation of Akt phosphorylated on Thr308. This was the first study to glean a deeper mechanistic understanding of pancracine activity in vitro. Perturbation of the cell cycle and induction of apoptotic cell death were considered key mechanisms of pancracine action.
Asunto(s)
Adenocarcinoma del Pulmón/patología , Proliferación Celular/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Leucemia/patología , Neoplasias Pulmonares/patología , Células A549 , Alcaloides/aislamiento & purificación , Alcaloides/farmacología , Amaryllidaceae/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Células Hep G2 , Compuestos Heterocíclicos de 4 o más Anillos/aislamiento & purificación , Humanos , Células MCF-7RESUMEN
Plants of the Amaryllidaceae family are promising therapeutic tools for human diseases and have been used as alternative medicines. The specific secondary metabolites of this plant family, called Amaryllidaceae alkaloids (AA), have attracted considerable attention due to their interesting pharmacological activities. One of them, galantamine, is already used in the therapy of Alzheimer's disease as a long acting, selective, reversible inhibitor of acetylcholinesterase. One group of AA is the montanine-type, such as montanine, pancracine and others, which share a 5,11-methanomorphanthridine core. So far, only 14 montanine-type alkaloids have been isolated. Compared with other structural-types of AA, montanine-type alkaloids are predominantly present in plants in low concentrations, but some of them display promising biological properties, especially in vitro cytotoxic activity against different cancerous cell lines. The present review aims to summarize comprehensively the research that has been published on the Amaryllidaceae alkaloids of montanine-type.
Asunto(s)
Alcaloides de Amaryllidaceae/química , Amaryllidaceae/química , Antineoplásicos Fitogénicos/química , Antiprotozoarios/química , Inhibidores de la Colinesterasa/química , Nootrópicos/química , Amaryllidaceae/metabolismo , Alcaloides de Amaryllidaceae/aislamiento & purificación , Alcaloides de Amaryllidaceae/farmacología , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Antiprotozoarios/aislamiento & purificación , Antiprotozoarios/farmacología , Línea Celular Tumoral , Inhibidores de la Colinesterasa/aislamiento & purificación , Inhibidores de la Colinesterasa/farmacología , Galantamina/química , Galantamina/aislamiento & purificación , Galantamina/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/aislamiento & purificación , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Concentración 50 Inhibidora , Isoquinolinas/química , Isoquinolinas/aislamiento & purificación , Isoquinolinas/farmacología , Nootrópicos/aislamiento & purificación , Nootrópicos/farmacología , Fenantridinas/química , Fenantridinas/aislamiento & purificación , Fenantridinas/farmacología , Extractos Vegetales/química , Metabolismo SecundarioRESUMEN
Bersavine is the new bisbenzylisoquinoline alkaloid isolated from the Berberis vulgaris L.(Berberidaceae) plant. The results of cytotoxicity screening 48 h post-treatment showed thatbersavine considerably inhibits the proliferation and viability of leukemic (Jurkat, MOLT-4), colon(HT-29), cervix (HeLa) and breast (MCF-7) cancer cells with IC50 values ranging from 8.1 to 11 µM.The viability and proliferation of leukemic Jurkat and MOLT-4 cells were decreased after bersavinetreatment in a time- and dose-dependent manner. Bersavine manifested concentration-dependentantiproliferative activity in human lung, breast, ovarian and hepatocellular carcinoma cell linesusing a xCELLigence assay. Significantly higher percentages of MOLT-4 cells exposed to bersavineat 20 µM for 24 h were arrested in the G1 phase of the cell cycle using the flow cytometry method.The higher percentage of apoptotic cells was measured after 24 h of bersavine treatment. Theupregulation of p53 phosphorylated on Ser392 was detected during the progression of MOLT-4 cellapoptosis. Mechanistically, bersavine-induced apoptosis is an effect of increased activity ofcaspases, while reduced proliferation seems dependent on increased Chk1 Ser345 phosphorylationand decreased Rb Ser807/811 phosphorylation in human leukemic cells.
Asunto(s)
Alcaloides , Antineoplásicos Fitogénicos , Apoptosis/efectos de los fármacos , Berberis/química , Citotoxinas , Fase G1/efectos de los fármacos , Leucemia/tratamiento farmacológico , Alcaloides/química , Alcaloides/aislamiento & purificación , Alcaloides/farmacología , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Citotoxinas/química , Citotoxinas/aislamiento & purificación , Citotoxinas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Células HT29 , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Leucemia/metabolismo , Leucemia/patología , Células MCF-7RESUMEN
The increasing risk of radiation exposure underlines the need for novel radioprotective agents. Hence, a series of novel 1-(2-hydroxyethyl)piperazine derivatives were designed and synthesized. Some of the compounds protected human cells against radiation-induced apoptosis and exhibited low cytotoxicity. Compared to the previous series of piperazine derivatives, compound 8 exhibited a radioprotective effect on cell survival in vitro and low toxicity in vivo. It also enhanced the survival of mice 30 days after whole-body irradiation (although this increase was not statistically significant). Taken together, our in vitro and in vivo data indicate that some of our compounds are valuable for further research as potential radioprotectors.
Asunto(s)
Piperazinas/química , Piperazinas/farmacología , Protectores contra Radiación/química , Protectores contra Radiación/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Humanos , Dosis Máxima Tolerada , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Piperazinas/administración & dosificación , Piperazinas/efectos adversos , Radiación Ionizante , Protectores contra Radiación/administración & dosificación , Protectores contra Radiación/efectos adversos , Relación Estructura-Actividad , Análisis de SupervivenciaRESUMEN
We report the design, synthesis and biological evaluation of 17 novel 8-aryl-2-morpholino-3,4-dihydroquinazoline derivatives based on the standard model of DNA-PK and PI3K inhibitors. Novel compounds are sub-divided into two series where the second series of five derivatives was designed to have a better solubility profile over the first one. A combination of in vitro and in silico techniques suggested a plausible synergistic effect with doxorubicin of the most potent compound 14d on cell proliferation via DNA-PK and poly(ADP-ribose) polymerase-1 (PARP-1) inhibition, while alone having a negligible effect on cell proliferation.
Asunto(s)
Antineoplásicos/farmacología , Doxorrubicina/farmacología , Inhibidores Enzimáticos/farmacología , Morfolinas/farmacología , Quinazolinonas/farmacología , Animales , Animales no Consanguíneos , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Diseño de Fármacos , Sinergismo Farmacológico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/toxicidad , Femenino , Células HT29 , Humanos , Ratones , Morfolinas/síntesis química , Morfolinas/toxicidad , Proteínas Nucleares/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Quinazolinonas/síntesis química , Quinazolinonas/toxicidadRESUMEN
Analysis of Exhaled breath condensate (EBC) is a re-discovered approach to monitoring the course of the disease and reduce invasive methods of patient investigation. However, the major disadvantage and shortcoming of the EBC is lack of reliable and reproducible standardization of the method. Despite many articles published on EBC, until now there is no clear consensus on whether the analysis of EBC can provide a clue to diagnosis of the diseases. The purpose of this paper is to investigate our own method, to search for possible standardization and to obtain our own initial experience. Thirty healthy volunteers provided the EBC, in which we monitored the density, pH, protein, chloride and urea concentration. Our results show that EBC pH is influenced by smoking, and urea concentrations are affected by the gender of subjects. Age of subjects does not play a role. The smallest coefficient of variation between individual volunteers is for density determination. Current limitations of EBC measurements are the low concentration of many biomarkers. Standardization needs to be specific for each individual biomarker, with focusing on optimal condensate collection. EBC analysis has a potential become diagnostic test, not only for lung diseases.
Asunto(s)
Pruebas Respiratorias/métodos , Cloruros/metabolismo , Proteínas/metabolismo , Urea/metabolismo , Adulto , Factores de Edad , Anciano , Biomarcadores/análisis , Biomarcadores/metabolismo , Cloruros/análisis , Femenino , Voluntarios Sanos , Humanos , Concentración de Iones de Hidrógeno , Masculino , Persona de Mediana Edad , Proyectos Piloto , Proteínas/análisis , Estándares de Referencia , Valores de Referencia , Factores Sexuales , Fumar/metabolismo , Manejo de Especímenes , Urea/análisisRESUMEN
Four diruthenium trithiolato chlorambucil conjugates have been prepared via Steglich esterification from chlorambucil and the corresponding trithiolato precursors. All conjugates are highly cytotoxic towards human ovarian A2780 and A2780cisR cancer cell lines with IC50 values in the nanomolar range. The conjugates exhibit selectivity towards A2780 cells as compared to non-cancerous HEK293 cells, while being only slightly selective for RF24 and A2780cisR cells. In vivo, the conjugate [10]BF4 suppressed the growth of a solid Ehrlich tumor in immunocompetent NMRI mice but did not prolong their overall survival. The reactivity of the chlorambucil conjugates with glutathione, a potential target of the dinuclear ruthenium motive, and with the 2-deoxyguanosine 5'-monophosphate (dGMP-a model target of chlorambucil) was studied by mass spectrometry and NMR spectroscopy. The conjugates did not show catalytic activity for the oxidation of glutathione nor binding to nucleotides, indicating that glutathione oxidation and DNA alkylation are not key mechanisms of action. Four highly cytotoxic diruthenium trithiolato chlorambucil conjugates have been prepared. All conjugates exhibit selectivity towards A2780 cells as compared to HEK293 cells, while being only slightly active in RF24 and A2780cisR cells. In vivo, the best candidate suppressed the growth of a solid Ehrlich tumor in immunocompetent NMRI mice but did not prolong their overall survival.
Asunto(s)
Antineoplásicos/farmacología , Clorambucilo/química , Monoterpenos/química , Compuestos Organometálicos/farmacología , Rutenio/química , Compuestos de Sulfhidrilo/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cimenos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ratones , Conformación Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Relación Estructura-ActividadRESUMEN
In the present study, we investigated the anticancer action of the trithiolato arene ruthenium complex, [(η-p-MeC6H4Pr)2Ru2(µ-S-p-C6H4OH)3]Cl, named diruthenium-2, both in vitro and in vivo. The mechanism of antiproliferative, cytotoxic, and DNA-damaging activity, and the effect on expressions of cell cycle regulatory proteins were investigated using a WST-1-based proliferation assay, lactate dehydrogenase leakage assay, comet assay, flow cytometry, and western blot analysis. In-vivo anticancer activity was evaluated using Ehrlich tumor-bearing NMRI mice. Diruthenium-2 inhibited the growth of all cancer cell lines used, the most sensitive being gastric (AGS), breast cancer (BT-549, MCF-7, MDA-MB-231), and leukemic (HL-60, MOLT-4) cells. In MCF-7 cells, it caused a G1/S cell cycle arrest, along with an increase in the expression of protein p21 and cyclin B1. We also observed increased levels of MRN complex proteins, which, together with the results from the comet assay, indicate the formation of DNA double-strand breaks. In tumor-bearing mice, diruthenium-2 at doses of 3 and 5 mg/kg inhibits the growth of solid Ehrlich tumor, although weaker than cisplatin. However, it did not prolong the post-therapeutic survival. Our results suggest the in-vitro potential of diruthenium-2 should be further evaluated in studies using other in-vivo models.
Asunto(s)
Antineoplásicos/farmacología , Complejos de Coordinación/farmacología , Rutenio/farmacología , Animales , Antineoplásicos/química , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Complejos de Coordinación/química , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células HL-60 , Humanos , Leucemia/tratamiento farmacológico , Células MCF-7 , Ratones , Rutenio/química , Neoplasias Gástricas/tratamiento farmacológico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Boldine is an aporphine alkaloid widely consumed in the folk medicine of some regions. Its anticancer potential has been shown but not yet elucidated. We compared the antitumor effect of orally and parenterally applied boldine in mice bearing solid Ehrlich tumor. We also explored the effects of boldine on breast adenocarcinoma MCF-7 cells in vitro. Repeated i.âp. injections of 30, 60, or 90 mg boldine/kg, either alone or combined with doxorubicin, slowed tumor growth in vivo. The latter two doses also prolonged the post-therapeutic survival of the mice. When fed food supplemented with boldine at a dose of 90 mg/kg, the tumor-bearing mice survived significantly longer, but there was no effect on tumor size. Interestingly, continuous p.âo. administration did not produce detectable levels of boldine in plasma or tissue samples, in contrast to high but short-lived concentrations after i.âp. injections. There was neither antagonism nor synergism between boldine and doxorubicin, except a possible synergism of i.âp. boldine 90 mg/kg combined with doxorubicin when compared with doxorubicin alone.Boldine was cytotoxic to MCF-7 cells and reduced their viability and proliferation in vitro. Exposure to boldine decreased bromodeoxyuridine incorporation and histone H3 phosphorylation but did not induce apoptosis. Boldine treatment resulted in p38, ERK, and JNK activation in the mitogen-activated protein kinase pathway in a dose-dependent manner. Since bioavailability in mice seems to be different from that reported in rats, pharmacokinetic studies in humans are needed to evaluate the role of boldine in the beneficial effects of Boldo infusions.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antioxidantes/uso terapéutico , Aporfinas/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Animales , Antioxidantes/farmacología , Aporfinas/farmacología , Doxorrubicina , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Células MCF-7 , Ratones , FitoterapiaRESUMEN
Evidence gathered in various studies points to the fact that haemanthamine, an isoquinoline alkaloid, has multiple medicinally interesting characteristics, including antitumor, antileukemic, antioxidant, antiviral, anticonvulsant and antimalarial activity. This work presents, for the first time, a universal LC-MS/MS method for analysis of haemanthamine in plasma, bile and urine which has been verified in a pilot pharmacokinetic experiment on rats. Chromatographic separation was performed on a pentafluorophenyl core-shell column in gradient elution mode with a mobile phase consisting of acetonitrile-methanol-ammonium formate buffer. A sample preparation based on liquid-liquid extraction with methyl tert-butyl ether was employed with ambelline used as an internal standard. Quantification was performed using LC-MS-ESI(+) in Selected Reaction Monitoring mode. The method was validated according to the European Medicines Agency guideline in a concentration range of 0.1-10 µmol/L in plasma, bile and urine. The concentration-time profiles of haemanthamine in plasma, bile and urine after a single i.v. bolus of 10 mg/kg have been described for the first time. The presented study addresses the lack of information on haemanthamine pharmacokinetics and also introduces a new universal method of haemanthamine analysis in complex biological matrices. Copyright © 2015 John Wiley & Sons, Ltd.
Asunto(s)
Alcaloides de Amaryllidaceae/farmacocinética , Bilis/metabolismo , Fenantridinas/farmacocinética , Espectrometría de Masas en Tándem/métodos , Alcaloides de Amaryllidaceae/sangre , Alcaloides de Amaryllidaceae/orina , Animales , Límite de Detección , Fenantridinas/sangre , Fenantridinas/orina , Proyectos Piloto , Ratas , Reproducibilidad de los ResultadosRESUMEN
Myofibroblast expansion is a critical event in the pathogenesis of liver fibrosis. The activation of hepatic stellate cells (HSC) to myofibroblast (MFB) results in the enhanced production of extracellular matrix (ECM). In this study, we explored the effect of acidic fibroblast growth factor (FGF-1) treatment on a transforming growth factor (TGF-ß1) induced MFB conversion. We used HSC-T6 cell line, which represents well-established model of activated HSC. These cells strongly expressed α-smooth muscle actin (α-SMA) and fibronectin (FN-EDA) after stimulation with TGF-ß1, which is a stimulus for MFB differentiation and ECM production. FGF-1 reduced proteins expression to levels comparable with untreated cells. Mild repression of secreted gelatinases was seen in culture media after FGF-1 treatment. The exposure of cells to collagen gel leads to changes in cell morphology and in expression of MFB markers. Lack of α-SMA in cells embedded to collagen gel was detected. When stimulated with TGF-ß1, the cells increased expression of FN-EDA, but not α-SMA. Although the cells on plastic and in collagen gel show different properties, FGF-1 reduced expression of FN-EDA in both conditions. Disrupting TGF-ß1 signalling pathway represents a potential strategy for the treatment of fibrosis. We showed that FGF-1 could antagonize signals initiated by TGF-ß1.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Factor 1 de Crecimiento de Fibroblastos/farmacología , Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/fisiología , ARN Mensajero/análisis , Factor de Crecimiento Transformador beta/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Línea Celular , Colágeno/farmacología , Fibronectinas/genética , Fibronectinas/metabolismo , Miofibroblastos , Ratas , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Primary cilia act as physical-chemical sensors and their functions include the perception of the extracellular milieu, regulation of organogenesis, and cell polarity. In general, these cells are monociliated and the single cilium possesses diverse receptors and channels which are involved in morphogenesis and growth signaling, and are, therefore, important for cell proliferation and differentiation. In this study, we used an in vitro model of C2C12 myoblasts to evaluate the effect of DNA damage induced by gamma ionizing radiation on primary cilia incidence. A significantly higher number of ciliated cells were observed after 1 day post-irradiation with 2-20 Gy when compared with non-irradiated cells. After 3 days post-irradiation, the cilia incidence in cells had decreased slightly when treated with 2, 6, and 10 Gy, although an increase in incidence rate was observed in cells treated with 20 Gy. Multi-ciliated cells were also detected in myoblasts irradiated with 10 and 20 Gy but not in non-irradiated cells or after low irradiation (2-6 Gy). Irradiation also caused a dose-dependent decrease in cell viability and proliferation and corresponding cell cycle arrest. Furthermore, an activation of caspases 3/7, 8, and 9 was observed after higher radiation (10 and 20 Gy) with increased apoptosis. Together, our results show that irradiation by gamma rays promotes myoblast ciliogenesis, with pronounced effects observed after 3 days post-irradiation. We conclude that irradiation doses of 10 and 20 Gy are sufficient to induce cell death and are responsible for the formation of multiple cilia originating from multiple basal bodies.
Asunto(s)
Cilios/efectos de la radiación , Mioblastos/efectos de los fármacos , Apoptosis/efectos de la radiación , Caspasas/metabolismo , Diferenciación Celular/efectos de la radiación , Línea Celular , Proliferación Celular/efectos de la radiación , Centrosoma/efectos de la radiación , Roturas del ADN de Doble Cadena , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Humanos , Mioblastos/citología , Mioblastos/metabolismo , Radiación IonizanteRESUMEN
Terminally-differentiated cells cease to proliferate and acquire specific sets of expressed genes and functions distinguishing them from less differentiated and cancer cells. Mature granulocytes show lobular structure of cell nuclei with highly condensed chromatin in which HP1 proteins are replaced by MNEI. These structural features of chromatin correspond to low level of gene expression and the loss of some important functions as DNA damage repair, shown in this work and, on the other hand, acquisition of a new specific function consisting in the release of chromatin extracellular traps in response to infection by pathogenic microbes. Granulocytic differentiation is incomplete in myeloid leukemia and is manifested by persistence of lower levels of HP1γ and HP1ß isoforms. This immaturity is accompanied by acquisition of DDR capacity allowing to these incompletely differentiated multi-lobed neutrophils of AML patients to respond to induction of DSB by γ-irradiation. Immature granulocytes persist frequently in blood of treated AML patients in remission. These granulocytes contrary to mature ones do not release chromatin for NETs after activation with phorbol myristate-12 acetate-13 and do not exert the neutrophil function in immune defence. We suggest therefore the detection of HP1 expression in granulocytes of AML patients as a very sensitive indicator of their maturation and functionality after the treatment. Our results show that the changes in chromatin structure underlie a major transition in functioning of the genome in immature granulocytes. They show further that leukemia stem cells can differentiate ex vivo to mature granulocytes despite carrying the translocation BCR/ABL.
Asunto(s)
Diferenciación Celular , Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Granulocitos/patología , Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/patología , Neutrófilos/patología , Western Blotting , Proliferación Celular , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Técnica del Anticuerpo Fluorescente , Granulocitos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Acetato de TetradecanoilforbolRESUMEN
Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a part of their anticancer treatment. The objectives of the current study were to investigate the effects of the small molecule inhibitors of Wee1 kinase II (681641) and Rad51 (RI-1) on cell cycle progression, DNA double-strand breaks repair and apoptosis following ionizing radiation exposure in human leukemic T-cells Jurkat and MOLT-4. Pre-treatment with the Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to irradiation, however combining both inhibitors together resulted in a further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24h upon irradiation. MOLT-4 cells were less affected by inhibitors application prior to ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4 cells.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Daño del ADN/efectos de la radiación , Leucemia de Células T/enzimología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Recombinasa Rad51/antagonistas & inhibidores , Reparación del ADN , Humanos , Células Jurkat , Leucemia de Células T/genética , Leucemia de Células T/patología , Inhibidores de Proteínas Quinasas/farmacología , Radiación IonizanteRESUMEN
DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)-triggered by radiation-induced double strand breaks-is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.
Asunto(s)
Rayos gamma , Inhibidores de Proteínas Quinasas/farmacología , Proteoma/metabolismo , Pirazinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Sulfonas/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiaciónRESUMEN
Oxidative DNA damage markers (8OHdG, comet assay, gammaH2AX) are becoming widely used in clinical cardiology research. To conduct this review of DNA damage in relation to hypertension in humans, we used databases (e.g. PubMed, Web of Science) to search for English-language publications up to June 30, 2022 and the terms: DNA damage, comet assay, gammaH2AX, 8OHdG, strand breaks, and arterial hypertension. Exclusion criteria were: children, absence of relevant controls, extra-arterial hypertensive issues, animal, cell lines. From a total of 79526, 15 human studies were selected. A total of 902 hypertensive patients (pts): (comet: N=418 pts; 8OHdG: N=484 pts) and 587 controls (comet: N=203; 8OHdG: N=384) were included. DNA damage was significantly higher in hypertensive pts than healthy controls (comet 26.6±11.0 vs 11.7±4.07 arbitrary units /A.U./; P<0.05 and="" 8ohdg="" 13="" 1="" 4="" 12="" vs="" 6="" 97="" 2="" 67="" ng="" mg="" creatinine="" i=""> P<0.05) confirmed with meta-analysis for both. Greater DNA damage was observed in more adverse cases (concentric cardiac hypertrophy 43.4±15.4 vs 15.6±5.5; sustained/untreated hypertension 31.4±12.1 vs 14.2±5/35.0±5.0 vs 25.0 ±5.0; non-dippers 39.2±15.5 vs 29.4±11.1 A.U.; elderly 14.9±4.5 vs 9.3±4.1 ng/mg creatinine; without carvedilol 9.1±4.2 vs 5.7±3.9; with coronary heart disease 0.5±0.1 vs 0.2±0.1 ng/mL) (P<0.05) confirmed with meta-analysis. DNA damage correlated strongly positively with serum glycosylated haemoglobin (r=0.670; P<0.05) and negatively with total antioxidant status (r=-0.670 to -0.933; P<0.05). This is the first systematic review with meta-analysis showing that oxidative DNA damage was increased in humans with arterial hypertension compared to controls.
Asunto(s)
Daño del ADN , Hipertensión , Niño , Animales , Humanos , Anciano , 8-Hidroxi-2'-Desoxicoguanosina , Creatinina , Ensayo CometaRESUMEN
The increasing threat of nuclear incidents and the widespread use of ionizing radiation (IR) in medical treatments underscore the urgent need for effective radiation countermeasures. Despite the availability of compounds such as amifostine, their clinical utility is significantly limited by adverse side effects and logistical challenges in administration. This study focuses on the synthesis and evaluation of novel piperazine derivatives as potential radioprotective agents, with the aim of overcoming the limitations associated with current countermeasures. We designed, synthesized, and evaluated a series of 1-(2-hydroxyethyl)piperazine derivatives. The compounds were assessed for cytotoxicity across a panel of human cell lines, and for their radioprotective effects in the MOLT-4 lymphoblastic leukemia cell line and in peripheral blood mononuclear cells (PBMCs) exposed to gamma radiation. The radioprotective efficacy was further quantified using the dicentric chromosome assay (DCA) to measure DNA damage mitigation. Among the synthesized derivatives, compound 6 demonstrated the most significant radioprotective effects in vitro, with minimal cytotoxicity across the tested cell lines. Compound 3 also showed notable efficacy, particularly in reducing dicentric chromosomes, thus indicating its potential to mitigate DNA damage from IR. Both compounds exhibited superior safety profiles and effectiveness compared to amifostine, suggesting their potential as more viable radioprotective agents. This study highlights the development of novel piperazine derivatives with promising radioprotective properties. Compound 6 emerged as the leading candidate, offering an optimal balance between efficacy and safety, with compound 3 also displaying significant potential. These findings support the further development and clinical evaluation of these compounds as safer, and more effective radiation countermeasures.
RESUMEN
We compared the effects of inhibitors of kinases ATM (KU55933) and ATR (VE-821) (incubated for 30 min before irradiation) on the radiosensitization of human promyelocyte leukaemia cells (HL-60), lacking functional protein p53. VE-821 reduces phosphorylation of check-point kinase 1 at serine 345, and KU55933 reduces phosphorylation of check-point kinase 2 on threonine 68 as assayed 4 h after irradiation by the dose of 6 Gy. Within 24 h after gamma-irradiation with a dose of 3 Gy, the cells accumulated in the G2 phase (67 %) and the number of cells in S phase decreased. KU55933 (10 µM) did not affect the accumulation of cells in G2 phase and did not affect the decrease in the number of cells in S phase after irradiation. VE-821 (2 and 10 µM) reduced the number of irradiated cells in the G2 phase to the level of non-irradiated cells and increased the number of irradiated cells in S phase, compared to irradiated cells not treated with inhibitors. In the 144 h interval after irradiation with 3 Gy, there was a considerable induction of apoptosis in the VE-821 group (10 µM). The repair of the radiation damage, as observed 72 h after irradiation, was more rapid in the group exposed solely to irradiation and in the group treated with KU55933 (80 and 77 % of cells, respectively, were free of DSBs), whereas in the group incubated with 10 µM VE-821, there were only 61 % of cells free of DSBs. The inhibition of kinase ATR with its specific inhibitor VE-821 resulted in a more pronounced radiosensitizing effect in HL-60 cells as compared to the inhibition of kinase ATM with the inhibitor KU55933. In contrast to KU55933, the VE-821 treatment prevented HL-60 cells from undergoing G2 cell cycle arrest. Taken together, we conclude that the ATR kinase inhibition offers a new possibility of radiosensitization of tumour cells lacking functional protein p53.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Leucemia Promielocítica Aguda/patología , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Sulfonas/farmacología , Apoptosis/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Células HL-60 , Humanos , Morfolinas/farmacología , Pironas/farmacologíaRESUMEN
Activated hepatic stellate cells (HSC) are a major source offibrous proteins in cirrhotic liver. Inducing or accelerating their apoptosis is a potential way of liver fibrosis treatment. Extracellular matrix (ECM) surrounding cells in tissue affects their differentiation, migration, proliferation and function. Type I collagen is the main ECM component in fibrotic liver. We have examined how this protein modifies apoptosis of normal rat HSC induced by gliotoxin, cycloheximide and cytochalasin D in vitro and spontaneous apoptosis of HSC isolated from CCl4-damaged liver. We have found that type I collagen gel enhances HSC apoptosis regardless of the agent triggering this process.
Asunto(s)
Apoptosis/efectos de los fármacos , Colágeno Tipo I/farmacología , Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática/patología , Animales , Tetracloruro de Carbono , Técnicas de Cultivo de Célula , Cicloheximida , Citocalasina D , Modelos Animales de Enfermedad , Gliotoxina , Células Estrelladas Hepáticas/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
A series of molybdenum(II) compounds [(η5 -Cp')Mo(CO)2 (L2 )][BF4 ] (Cp'=C5 H4 (CH2 )2 SPh, C9 H6 (CH2 )2 OMe, L2= N,N-chelating ligand) have been synthesized and characterized by spectroscopic and analytical methods including X-ray crystallography. The inâ vitro assay on human leukemia cells MOLT-4 has shown that the substitution in the π-ligand in combination with suitable N,N-chelating ligand can lead to species with cytotoxicity considerably higher than reported to cisplatin. Unusually high activity was observed for compounds bearing phenanthroline ligands [{η5 -C9 H6 (CH2 )2 OMe}Mo(CO)2 (3,4,7,8-Me4 phen)][BF4 ] (IC50 =0.7±0.3â µM) and [{η5 -C9 H6 (CH2 )2 OMe}Mo(CO)2 (4,7-Ph2 phen)][BF4 ] (IC50 values 0.8±0.4â µM).