Ji Hyeon Lee

Suppression of apoptosis by calorie restriction in aged kidney.

Programmed cell death by apoptosis is regarded as an organisms protective mechanism against the accumulation of defective cells. Apoptotic activity is shown to be elevated in most aged tissues, and its intracellular regulation is intricately manipulated by mitochondria. In this study, to determine the progression of apoptosis during aging, we investigated the expression of several key apoptosis-related markers in kidney of 12- and 24-month-old rats. Mitochondrial damage was detected by lipid peroxidation and Western blot analysis in several target apoptotic proteins in aged rat kidney. Our results showed that the expression levels of a pro-apoptotic Bax protein, was significantly enhanced at the age of 24 months, while an anti-apoptotic protein, Bcl-2, was reduced in the aged rat kidney. We also found that the cytosolic cytochrome c level was significantly increased in the aged kidney. However, these age-related changes were reversed by calorie restriction (CR), exhibiting its modulatory action on apoptotic activity. Furthermore, caspase-3 activation was markedly increased in kidney of 24-month-old rats fed ad libitum (AL), as indicated by the cleaved, active form of caspase-3 (17-19 kDa), which we found was replaced with the procaspase (32 kDa) in the CR rats of both age groups. We also found that a cleaved active form (85 kDa) of poly (ADP-ribose) polymerase (116 kDa inactivated form), which serves as a nuclear substrate for active caspase-3, was increased in aged AL kidney and was blunted by CR. In addition, to investigate the oxidative status in aged kidney, we measured and compared the malondialdehyde (MDA) and 4-hydroxynonenal (HNE) levels in aged AL and CR rat kidneys. Our results showed increased MDA and HNE levels in aged AL rats, while these levels were markedly lower in CR rats, even at 24 months. These results indicate that the kidneys of rats fed ad libitum are under the influence of high oxidative stress compared to CR rats. Thus, our present data strongly suggest that the apoptotic activity observed in the aged kidney is likely modulated by the age-related oxidative status, and reversed by CR as a result of its anti-oxidative and anti-aging actions.

The effect of lipopolysaccharide on enhanced inflammatory process with age: Modulation of NF-κB

Oxidative stress is thought to be a causative factor for age-related damage in a wide variety of cellular constituents that can lead to dysfunction and various pathological conditions, including the inflammatory process. At the molecular level, the redox-sensitive transcription factor, NF-κB plays a key role in the regulation of the inflammatory process, along with cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). We studied the mechanism underlying the modulation of the inflammatory reaction with age by investigating NF-κB activation and the expression of COX-2, iNOS, and cytokines genes in hepatic tissues isolated from young and old rats. We expanded our investigation of these factors in rats injected with the inflammatory activator, lipopolysaccharide (LPS). Data showed that NF-κB activity was up-regulated with age and was further enhanced by LPS injection, indicating an increased susceptibility and sensitivity to the inflammatory stimulus with age. To explore further the molecular events leading to NF-κB activation, we investigated the inhibitory component of NF-κB complex, IκB. Cytosolic IκBα, but not IκBβ, was significantly decreased in both old and LPS-treated rats, signifying the enhanced migration of cytosolic NF-κB complex into the nucleus following dissociation from the inhibitor. The appearance of the polypeptide, p65, as determined in the nucleus, corresponded with the change in IκBα, providing further supporting evidence for the molecular process involved in NF-κB activation. Our additional investigation of two proinflammatory-related enzymes, COX-2 and iNOS, and three cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α, clearly showed aged-related increases, in corroboration with the NF-κB activation. Our results demonstrated that LPS injection caused the enhanced gene expression of inducible proinflammatory proteins, COX-2 and iNOS through NF-κB activation.

Activity of crude extract ofRubus crataegifolius roots as a potent apoptosis inducer and DNA topoisomerase I inhibitor

The effects of methanol extract ofRubus crategifolius roots and its solvent fractions were investigated on the proliferation of MCF-7 human breast carcinoma cells. The methanol extract inhibited the proliferation of MCF-7 cells in a concentration dependent manner. Moreover, their methanol soluble (W-M) fraction had the greatest inhibitory effect on the growth of MCF-7 cells. To evaluate whether the W-M fraction affects on the cell cycle of MCF-7 cells, cells treated with this fraction were analyzed with flow cytometry. The W-M fraction increased G0/G1 phase after 24 h-treatment and induced apoptosis after 48 h-treatment. The hallmark of apoptosis, DNA fragmentation, also appeared by W-M fraction after 48 h-treatment. Furthermore the methanol extract and its W-M fraction inhibited the activity of the topoisomerase I enzyme in the relaxation assay. From these results, their W-M fraction as well as methanol extract ofR. crategifolius roots are necessary for further studies as a potent inhibitor of the growth of cancer cells.

All-trans retinoic acid induced differentiation of rat mammary epithelial cells cultured in serum-free medium

Retinoids are applied to not only cancer prevention but also cancer chemotherapy by stimulating differentiation of cells. We studied differentiation inducing effect of all-trans retinoic acid (ATRA) by studying proportion of high dense fractions of stem-like cells and the size of S phase fraction in cell cycle. From mammary organoids obtained from 7- to 8-week old F344 female rat mammary gland, we cultured rat mammary epithelial cells (RMEC) and treated physiological doses of 10−6, 10−7, and 10−8 M ATRA from the first day and then cultured for 4, 7, and 14 days. After that, immunostaining was performed using peanut agglutinin (PNA) and anti-Thy-1.1 monoclonal antibody (Thy-1.1) that can be used as markers of differentiation. We separated four different cell subpopulations by flow cytometry: cells negative to both reagents (B-), PNA-positive cells (PNA+), Thy-1.1-positive cells (Thy-1.1+), and cells positive to both reagents (B+). We observed continuous decreases of high dense fractions of stem-like cells (PNA+ subpopulations) for 14 days and as much decreases as high doses of ATRA, which were thought to be proportional to doses of ATRA. We labeled RMEC with bromodeoxyuridine and investigated cell cycle fractions that went through S phase. We observed a tendency of decrease of S phase fraction with time in culture, which is though to be related to continuous decreases of PNA+ subpopulations and inhibitory role of ATRA on cell cycle. These results suggest that physiological doses of ATRA could stimulate differentiation of RMEC and convert stem-like RMEC to differentiated cells in SFM for a relatively long period of 14 days.