RESUMEN
Resveratrol (R-3), a trihydroxy trans-stilbene from grape, inhibits multistage carcinogenesis in animal models. A resveratrol derivative 3,4,5,4'-tetrahydroxystilbene (R-4) exhibits potent growth inhibitory effect against transformed human cells. Here we report that 3,4,5,4'-tetramethoxystilbene (MR-4), converted from R-4, was more potent against cancer cell lines (WI38VA, IMR-90SV, HeLa, LNCaP, HT-29, and HepG2), but had almost no inhibitory effect on the growth of normal cells (WI38, IMR-90, BJ-T) at the concentrations tested. The IC50 value of MR-4 on the growth inhibition of transformed WI38VA human cells was 0.5 microM, as compared to the value of greater than 50 microM for the normal WI38 cells. Resveratrol, however, did not exhibit such clear differential effect and the IC50 value of R-3 for WI38VA cells was about 50 microM. The growth inhibitory effect of MR-4 correlated with the induction of apoptosis in the transformed cells. When normal WI38 cells and transformed WI38VA cells were compared, MR-4 induced increases of the Bax/Bcl-2 mRNA ratio, p53 and Bax protein level, activation of caspases, and DNA fragmentation in transformed, but not in normal cells. Further analysis revealed that MR-4 caused a rapid appearance of perinuclear aggregation of mitochondria in WI38VA but not in WI38 cells, suggesting that the mitochondria could serve as an early target of MR-4. R-3 also induced apoptosis and mitochondrial clustering but only at a much higher concentration, close to 500 microM. Taken together, the specific activation of the mitochondria-mediated apoptotic pathway could be a major reason for the striking differential growth inhibitory effect of MR-4.
Asunto(s)
Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Estilbenos/farmacología , División Celular/efectos de los fármacos , Línea Celular Transformada , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Humanos , Mitocondrias/ultraestructura , Resveratrol , Estilbenos/química , Células Tumorales CultivadasRESUMEN
The goal of this study was to evaluate morphofunctional changes in mitochondrial ultrastructure after platelet-derived growth factor application in fibroblasts as an indicator of mitochondrial activation in processes like wound healing. NRK-49F fibroblasts were synchronized, incubated with PDGF (platelet-derived growth factor) and studied by electron microscopy. Volume density (Vv), numerical density (Nv) and surface density (Sv) were measured by stereological analysis. Application of PDGF on NRK-49F caused an increase in mitochondrial volume density by 57% and surface area of cristae per mitochondrion by 65%. The numerical density of the mitochondria was decreased in the PDGF-treated cells by 23%, but at the same time their mean volume was increased. Furthermore, the mitochondria had a complex and highly variable shape both in control and PDGF-treated cells, possibly indicating the existence of a mitochondrial reticulum. The results demonstrated that biochemically active membrane systems in fibroblast mitochondria are enlarged as a direct effect of small doses of platelet-derived growth factor and support the concept that this factor and related peptides serve as mitogens for connective tissue forming cells. Thus, in mitogenic processes like wound healing, the high energy demand of fibroblasts is provided by the increase of the inner surface of mitochondria.
Asunto(s)
Mitocondrias/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Animales , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Microscopía Electrónica , Mitocondrias/ultraestructuraRESUMEN
The hypothesis of a common signal for heat shock (HS) and oxidative stress (OS) was analyzed in C6 cells with regard to the induction of heat shock proteins (Hsps). The synthesis rate and level of the strictly inducible Hsp68 was significantly higher after HS (44 degrees C) compared with OS (2 mm H2O2). This difference corresponded to higher and lower activation of the heat shock factor (HSF) by HS and OS, respectively. OS, on the other hand, showed stronger cytotoxicity compared with HS as indicated by drastic lipid peroxidation and inhibition of protein synthesis as well as of mitochondrial and endocytotic activity. Lactic dehydrogenase also revealed stronger inhibition of enzyme activity by OS than by HS as shown in cells and in vitro experiments. Conformational analysis of lactic dehydrogenase by the fluorophore 1-anilinonaphtalene-8-sulfonic acid, however, showed stronger exposure of hydrophobic domains after HS than after OS which correlates positively with the Hsp68 response. Treatment of cells with deoxyspergualin, which exhibits high affinity to Hsps, the putative inhibitors of HSF, strongly increased only OS-induced hsp68 expression. In conclusion, the results suggest that exposure of hydrophobic domains of cytosolic proteins represents the common first signal in the multistep activation pathway of HSF.
Asunto(s)
Proteínas HSP70 de Choque Térmico/biosíntesis , Respuesta al Choque Térmico , Estrés Oxidativo , Transducción de Señal , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción del Choque Térmico , Desnaturalización Proteica , Ratas , Factores de Transcripción , Células Tumorales CultivadasRESUMEN
The induction of Hsp68 by heat shock (HS) and oxidative stress (OS) involves different pathways in C6 rat glioma cells. The pathways were analyzed by specific inhibitors of signal transduction cascades. Quercetin (inhibitor of PLA(2) and lipoxygenase) inhibited only the OS-induced but not the HS-induced expression of Hsp68. Preincubation with quinacrine (inhibitor of PLA(2)) before stress also suppressed the expression of Hsp68 only after oxidative stress. Moreover, another inhibitor of lipoxygenase (alpha-tocopherol) exclusively suppressed OS-induced Hsp68 expression. This different regulation was confirmed by exposing the cells to arachidonic acid (AA) during stress which strongly increased the induction of Hsp68 only after OS. PGE(2) (metabolite of cyclooxygenase) and indomethacin (inhibitor of cyclooxygenase) had no influence on Hsp68 expression in response to both stressors. The results suggest that the induction of Hsp68 by oxidative stress is mainly transmitted by the lipoxygenase pathway in C6 rat glioma cells.