Identifying the changes in gene profiles regulating the amelioration of age-related oxidative damages in kidney tissue of rats by the intervention of adult-onset calorie restriction.

This study was initiated to investigate gene expression profiles that are involved in the molecular mechanisms regulating the amelioration of age-related oxidative damages in male Fischer-344 rats (12 months) through adult-onset calorie restriction (CR) intervention for 6 months. The adult-onset CR was initiated with 10 and 25% restriction for the first and second weeks, respectively, and then maintained at 40% throughout the experiment. The adult-onset CR significantly (p < 0.05) decreased urinary 8-isoprostane and protein carbonyl in kidney for the markers of lipid peroxidation and protein oxidation, respectively, in rats from the CR group when compared with control group. Based on Yu's and Melk's methods, the age-related renal pathological changes in the kidney of rats from CR group were retarded by adult-onset CR. Such changes could result from the decrease of plasminogen activation inhibition-1 and clusterin and the increase of kallikrein mRNA expressions significantly (p < 0.05) in the kidneys of rats from the CR group. They were further confirmed by quantitative RT-PCR. Moreover, inflammatory response pathway was down-regulated significantly (p < 0.05) in rats from the CR group, while fatty acid synthesis, mitochondrial fatty acid betaoxidation, glycolysis, and gluconeogenesis were considerably up-regulated in kidney tissue of rats. In conclusion, the adult-onset CR could retard the age-related oxidative damages and renal pathological changes due to variations in gene expressions and biological pathways.

Regulating the age-related oxidative damage, mitochondrial integrity, and antioxidative enzyme activity in Fischer 344 rats by supplementation of the antioxidant epigallocatechin-3-gallate.

In this study, epigallocatechin-3-gallate (EGCG) was examined for the first time for its anti-aging effect on middle-aged male Fischer 344 rats as a dietary supplement at 50 (low dose) and 500 (high dose) mg/kg/day over a 6-month period. Such levels of EGCG concentration were well-tolerated by rats without causing tissue damage or dysfunction in the liver and kidney, as evaluated by histopathological and biochemical observations. Compared to the rats in the low-dose and control groups, rats fed with high-dose EGCG showed a significant decline in the concentration of 8-hydroxy-2'-deoxyguanosine in the plasma while maintaining a better mitochondrial potential in the peripheral lymphocytes and preventing the deletion of ND4 region from mitochondrial DNA in the liver. The protective effects of high-dose EGCG against oxidative stress were comparable with the effects of caloric restriction, a well-established dietary intervention that retards aging. However, the supplementation of EGCG influenced merely the antioxidative enzyme activities and their gene expressions in rats, suggesting that EGCG may either function as an antioxidant itself or regulate other bioprocesses, including energy metabolism, biosynthesis, and stress response, as shown in the gene profiling analysis of microarray data. Thus, the present study provides preliminary information on the anti-aging property of EGCG in male Fischer 344 rats.

Effects of epigallocatechin-3-gallate on mitochondrial integrity and antioxidative enzyme activity in the aging process of human fibroblast.

Mitochondrial integrity and antioxidative enzyme activity are two of the determinants of intracellular reactive oxygen species (ROS) accumulation probably underlying the aging mechanism. In this study, epigallocatechin-3-gallate (EGCG) was examined for its antiaging effect on human diploid fibroblasts (HDF). EGCG was evaluated for its cytotoxicity, and LC50 values were 78.0 and 84.4 microM for young and old HDF, respectively. HDF treated with EGCG at 25 and 50 microM for 24 h considerably increased catalase, superoxide dismutase (SOD)1, SOD2, and glutathione peroxidase gene expressions and their enzyme activities, thus protecting HDF against H2O2-induced oxidative damage, accompanied with decreased intracellular ROS accumulation and well-maintained mitochondrial potential. Moreover, HDF treated with EGCG at 12.5 microM for long term showed less intracellular ROS with higher mitochondrial potential, more intact mitochondrial DNA, much elevated antioxidative enzyme efficiency, and more juvenile cell status compared to those of the untreated group. Taken together, in this study we investigated the effects of EGCG in the regulation of mitochondrial integrity and antioxidative enzyme activity of HDF, suggesting that EGCG can be considered one of the possible antiaging reagents in the future.

Thapsigargin resistance in human prostate cancer cells.

BACKGROUND: Thapsigargin (TG) is a potent inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCAs). TG-based prodrugs are being developed for the treatment of prostate cancer (PC). To develop optimal TG-based therapeutics it is important to understand the mechanisms of resistance to TG that may potentially occur in cancer cells. METHODS: DU145/TG and PC3/TG cells were derived from human PC DU145 and PC3 cells, respectively, by incremental exposure to TG. Growth assays, Western blot analyses, cDNA microarrays, semiquantitative and real-time polymerase chain reaction (PCR), Northern blot analyses, and immunohistochemistry were used to study these cells. RESULTS: DU145/TG cells are 1100-fold and PC3/TG cells are 1350-fold resistant to TG. Although expression of both SERCA and p-glycoprotein can mediate TG resistance in hamster cells, neither is modulated in DU145/TG cells. In contrast, in PC3/TG cells, SERCA, and not p-glycoprotein, is significantly overexpressed but cannot by itself account for the 1350-fold resistance to TG in these cells. Several genes not previously identified to be altered by TG selection are modulated in DU145/TG and PC3/TG cells. Furthermore, the spectrum of genes modulated in DU145/TG cells are distinct from that in PC3/TG cells, even though both cells are of prostate origin and share the same TG-resistant phenotype. CONCLUSIONS: PC cells can adapt to SERCA inhibition by TG. However, they demonstrate cell type-specific plasticity with respect to gene expression upon TG selection. Further, previously not described mechanisms of resistance appear to be recruited in the TG-resistant PC cells, which provide a novel model to study mechanisms of resistance and adaptation in PC on TG-mediated dysregulation of Ca2+ homeostasis.

A Mediator-responsive form of metazoan RNA polymerase II.

RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene- and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to Mediator. Thus, in an in vitro assay with general transcription factors, Pol II lacking Gdown1 displays unfettered levels of activator-dependent transcription in the presence or absence of Mediator. In contrast, Pol II(G) is dramatically less efficient in responding to activators in the absence of Mediator yet is highly and efficiently responsive to activators in the presence of Mediator. Our results reveal a transcriptional control mechanism in which Mediator-dependent regulation is enforced by means of Gdown1, which likely restricts Pol II function only to be reversed by Mediator.