RESUMEN
Investigating the bioactivity of traditional medical remedies under the controlled conditions of a laboratory is an option to find additional applications, novel formulations or lead structures for the development of new drugs. The present work analysed the antineoplastic activity of increasing polar extracts of the rainforest plant Critonia morifolia (Asteraceae) that has been successfully used as traditional remedy to treat various inflammatory conditions in the long-lasting medical tradition of the Central American Maya, which was here also confirmed in vitro. The apolar petroleum ether extract exhibited the most potent antiproliferative and proapoptotic effects in HL60 cells and triggered down-regulation of Cdc25C and cyclin D1 within 30 min followed by the inhibition of c-Myc expression and the onset of caspase-3 activation within 2 h. Subsequent to these very rapid molecular responses Chk2 and H2AX became phosphorylated (γH2AX) after 4 h. Analysis of the cell cycle distribution showed an accumulation of cells in the G2-M phase within 8 h and after 24 h in S-phase. This was temporally paralleled by the down-regulation of Cdc25A, Cdc25B, Wee1 and Akt. Therefore, the attenuation of cell cycle progression in the G2-M phase was consistent with the known role of Chk2 for G2-M arrest and with the role of Cdc25B in S-phase progression. These findings suggest the presence of two distinct active principles in the petroleum ether extract of C. moriflia. These facilitated the strong apoptotic response evidenced by the rapid activation of caspase-3 that was later enforced by the inhibition of the survival kinase Akt. Importantly, the efficient down-regulation of Akt, which is successfully tested in current clinical trials, is a unique property of C. morifolia.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Asteraceae/química , Proteínas de Ciclo Celular/metabolismo , Extractos Vegetales/farmacología , Alcanos/química , Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Solventes/química , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismoRESUMEN
Several lines of evidence suggest that besides antioxidant also prooxidant properties are crucially involved in cytotoxic and protective activities of the major green tea catechin epigallocatechin-3-gallate (EGCG) in vitro (Elbling et al., 2011). Furthermore recent data suggest that EGCG induces oxidative stress also in vivo (Li et al., 2010). Here we set out to identify factors modulating cellular effects of EGCG in vitro. Using the HaCat keratinocytes model, we demonstrate that the cytotoxic, genotoxic and signal-activating effects of EGCG are significantly dependent on the ratio of cell number to working volume. Treatment with identical EGCG concentrations at altered experimental settings resulted in IC(50) values differing up to orders of magnitude and could even exert contradictory effects. This effect was based on cell-mediated clearance of autooxidation-derived H(2)O(2) from the supernatant. In order to estimate EGCG/H(2)O(2) concentrations equally effective under different settings, we have rationally derived and experimentally verified a simple algorithm relating concentration, working volume, cell number and - indirectly - exposure time. Algorithm application resulted in similar H(2)O(2) clearance curves from cell supernatants as well as comparable EGCG/H(2)O(2) effects at different settings. Our results demonstrate the importance of standardized experimental settings when investigating cytotoxic and/or beneficial effects of autooxidizing compounds.
Asunto(s)
Catequina/análogos & derivados , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Queratinocitos/efectos de los fármacos , Mutágenos/toxicidad , Pruebas de Toxicidad/métodos , Algoritmos , Western Blotting , Catequina/toxicidad , Recuento de Células , Técnicas de Cultivo de Célula , Línea Celular , Supervivencia Celular/efectos de los fármacos , Interpretación Estadística de Datos , Relación Dosis-Respuesta a Droga , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Factores de Tiempo , Pruebas de Toxicidad/normasRESUMEN
The beneficial health effects of (-)-epigallocatechin-3-gallate (EGCG), the main catechin of green tea, have been attributed to complex interactions with a focus on antioxidative properties. Susceptibility to autoxidation and production of cytotoxic reactive oxygen species (ROS), mostly H(2)O(2), have been suggested to occur in vitro but also in vivo. In this study, we address whether autoxidation-derived H(2)O(2) may be involved in the cytoprotective effects of EGCG. To that end we investigated keratinocyte-derived HaCat and HL-60 promyelocytic leukemia cells with significantly different sensitivities to H(2)O(2) (IC(50) 117.3 versus 58.3 µM, respectively) and EGCG (134.1 versus 84.1 µM). HaCat cells significantly resisted cytotoxicity and DNA damage based on enhanced H(2)O(2) clearance, improved DNA repair, and reduced intracellular ROS generation. Cumulative versus bolus EGCG and H(2)O(2) treatment and H(2)O(2) pretreatment before subsequent high-dose EGCG and vice versa significantly reduced DNA damage and cytotoxicity in HaCat cells only. Addition of catalase abolished the protective activities of low-dose H(2)O(2) and EGCG. In summary, our data suggest that autoxidative generation of low-dose H(2)O(2) is a significant player in the cell-type-specific cytoprotection mediated by EGCG and support the hypothesis that regular green tea consumption can contribute as a pro-oxidant to increased resistance against high-dose oxidative stressors.
Asunto(s)
Antioxidantes/farmacología , Catequina/análogos & derivados , Citoprotección , Queratinocitos/efectos de los fármacos , Estrés Oxidativo , Apoptosis/efectos de los fármacos , Catalasa/farmacología , Catequina/farmacología , Línea Celular , Daño del ADN/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/metabolismo , Queratinocitos/metabolismo , Queratinocitos/patología , Oxidación-Reducción/efectos de los fármacosRESUMEN
Avemar (MSC) is a nontoxic fermented wheat germ extract demonstrated to have antitumor effects. Avemar has the potential to significantly improve the survival rate in patients suffering from malignant colon tumors. We studied its effects in the HT-29 human colon carcinoma cell line. Avemar had an inhibiting effect on colonies of HT-29 cells with an IC50 value of 118 microg/ml (7 days of incubation); this value could be decreased to 100 and 75 microg/ml in the presence of vitamin C. In the cell line examined, Avemar induced both necrosis and apoptosis, as demonstrated by Hoechst/propidium iodide staining. The incubation of cells with 3200 microg/ml Avemar for 24 hrs caused necrosis in 28% and the induction of apoptosis in 22% of the cells. Avemar inhibited the cell-cycle progression of HT-29 cells in the G1 phase of the cell cycle. In addition, Avemar inhibited the activity of the key enzyme of de novo DNA synthesis, ribonucleotide reductase. In addition, we determined the effects of Avemar on the activity of cyclooxygenase-1 and -2. Both enzymes were significantly inhibited by Avemar with IC50 values of 100 and 300 microg/ml, respectively. We outline new explanations for its antitumor activity, which might serve as the basis for further studies using Avemar.