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This study investigated the agonistic activity of magnesium lithospermate B (1), isolated from Salvia miltiorrhiza, on peroxisome proliferator-activated receptor (PPARβ/δ) and the expressions of collagen genes (COL1A1 and COL3A1) and transforming growth factor-β1 (TGF-β1) in models of skin aging. The action of compound 1as a PPARβ/δ agonist was determined by reporter gene assay, immunostaining, and Western blotting. To determine the antiaging effects of compound 1on skin, aged Sprague–Dawley rat skin and ultraviolet B (UVB)-irradiated human skin fibroblasts were used. The results show that 1presented a marked enhancement of both nuclear protein levels and activity of PPARβ/δ in fibroblasts. In addition, 1prevented downregulation of PPARβ/δ activity in aged rat skin and UVB-induced fibroblasts. Furthermore, 1increased the expressions of COL1A1, COL3A1, and TGF-β1 in vivoand in a cell culture system. Therefore, the present study shows that compound 1prevents collagen degradation in aged rat skin and UVB-exposed fibroblasts through PPARβ/δ activation. The therapeutic and cosmetic applications of compound 1need further investigation.

Brominated flame retardants (BFRs) are present in many consumer products ranging from fabrics to plastics and electronics. Wide use of flame retardants can pose an environmental hazard, which makes it important to determine the mechanism of their toxicity. In the present study, dose-dependent toxicity of tetrabromobisphenol A (TBBPA), a flame retardant, was examined in male prepubertal rats (postnatal day 18) treated orally with TBBPA at 0, 125, 250 or 500 mg/kg for 30 days. There were no differences in body weight gain between the control and TBBPA-treated groups. However, absolute and relative liver weights were significantly increased in high dose of TBBPA-treated groups. TBBPA treatment led to significant induction of CYP2B1 and constitutive androstane receptor (CAR) expression in the liver. In addition, serum thyroxin (T4) concentration was significantly reduced in the TBBPA treated group. These results indicate that repeated exposure to TBBPA induces drug-metabolising enzymes in rats through the CAR signaling pathway. In particular, TBBPA efficiently produced reactive oxygen species (ROS) through CYP2B1 induction in rats. We measured 8-hydroxy-2’-deoxyguanosine (8-OHdG), a biomarker of DNA oxidative damage, in the kidney, liver and testes of rats following TBBPA treatment. As expected, TBBPA strongly induced the production of 8-OHdG in the testis and kidney. These observations suggest that TBBPA-induced target organ toxicity may be due to ROS produced by metabolism of TBBPA in Sprague-Dawley rats.

β-lapachone, a quinone compound obtained from the bark of the lapacho tree (Tabebuia avellanedae), was reported to have anti-inflammatory and anti-cancer activities. In this study, we investigated novel functions of β-lapachone in terms of anti-metastasis and anti-invasion abilities using human hepatocarcinoma cell lines, HepG2 and Hep3B. β-lapachone dose-dependently inhibited cell viability and migration of both HepG2 and Hep3B cells, as determined by methylthiazoletetrazolium (MTT) assay and wound healing assay. RT-PCR and Western blot data revealed that β-lapachone dramatically increased the levels of protein, as well as mRNA expression of early growth response gene-1 (Egr-1) and throbospondin-1 (TSP-1) at an early point in time, and then decreased in a time-dependent manner. In addition, down-regulation of Snail and up-regulation of E-cadherin expression were observed in β-lapachone-treated HepG2 and Hep3B cells, and this the associated with decreased invasive ability as measured by matrigel invasion assay. Taken together, our results strongly suggest that β-lapachone may be expected to inhibit the progression and metastasis of hepatoma cells, at least in part by inhibiting the invasive ability of the cells viaup-regulation of the expression of the Egr-1, TSP-1, and E-cadherin.

Abstract: Apoptosis‐inducing activity of synthetic CDCA derivatives, HS‐1199 and HS‐1200, on gastric cancer cell line SNU‐1 cells was explored. CDCA derivatives demonstrated various apoptosis hallmarks, such as mitochondrial changes, activation of caspase, DNA fragmentation, and nuclear condensation. Importantly, the orphan receptor Nur77 (TR3) was shown to translocate from the nucleus to mitochondria at the early time points after CDCA derivatives treatment. These data support the theory that CDCA derivatives‐induced apoptosis of SNU‐1 gastric cancer cell lines is mediated by mitochondria and caspase, and, at least in part, by Nur77.

IL-3 deprivation has been reported to induce apoptosis of bone marrow-derived mast cells. In order to evaluate this type of cell death further, we employed trypan blue and propidium iodide stainings, photometric enzyme immunoassay, fluorescence measurement of caspase-3, DNA electrophoresis, flow cytometry and transmission electron microscopy. In this experiment, although several evidences supporting apoptosis were demonstrated some findings were not consistent with typical apoptosis. On the other hand, electron microscopical observation demonstrated that most cells from all the time phases after IL-3 deprivation showed the morphology of typical oncosis, i.e. cell swelling, disintegration of ultrastructure and subsequent karyolysis. Only a small number of cells from the later time phases showed apoptotic morphology. We here suggest that BMMCs undergo both apoptosis and oncosis after IL-3 deprivation and that the dominant type of prelethal change is oncosis in all time phases, although apoptosis also plays a partial role in the late time phases.

Rat mammary glands contain a subpopulation of clonogenic epithelial cells with large proliferation and differentiation potentials. When transplanted, the clonogens in monodispersed rat mammary epithelial cell suspensions give rise to either alveolar units (AUs) or ductal units (DUs) depending on the nature of the hormonal milieu in the graft recipient. Clonogens are also the primary cells of origin of mammary cancer following exposure to ionizing radiation or chemical carcinogens. Given the other stem cell characteristics of mammary clonogens, it would be expected that the primary AUs and DUs to which they give rise when grafted and hormonally stimulated (a) would be derived from the same clonogenic cell subpopulation, (b) would contain all of the functionally differentiated cell types of homologous parts of comparably stimulated mammary glands in situ,and (c) would also contain clonogen subpopulations capable when subtransplanted of giving rise to secondary AUs and DUs of similar cell composition. The current experiments were designed to test these expectations. The data are discussed in the context of results of previous studies with this and other experimental models. The results further support the conclusion that rat mammary clonogens are multipotent mammary stem cells.

Although previous studies demonstrated that genistein-induced apoptosis of various cell types including RPE-J cells, the involvement of mitochondrial events in such types of apoptosis has not been demonstrated to date. In this investigation of genistein-induced apoptosis of RPE-J cells, genistein induced the reduction of the mitochondrial membrane potential and the release of cytochrome c to cytosol. A mitochondrial permeability transition pore (PTP) blocker bongkrekic acid prevented the reduction of the mitochondrial membrane potential and cytochrome c release, and consequently abolished caspase-3 activation, nuclear condensation, and DNA fragmentation. On the other hand, zVAD-fmk did not inhibit the mitochondrial event such as the reduction of the mitochondrial membrane potential and cytochrome c release although it prevented caspase-3 activation, nuclear condensation, and DNA fragmentation. Taken together, genistein induces apoptosis of RPE-J cells by opening the mitochondrial PTP, and the mitochondrial event in this type of apoptosis is caused independently of caspase.

Ursolic acid (UA), a pentacyclic triterpene acid, has been reported to exhibit anti-tumor activity. In this study, we investigated the pro-apoptotic effect of UA on HepG2 human hepatoblastoma cells. Treatment with UA decreased the viability of HepG2 cells in a concentration- and time-dependent manner. Furthermore, 30 μM of UA induced DNA fragmentation and subdiploid cells and enhanced the release of cytochrome c and the activation of caspase-3. These results suggest that UA induces cell death through apoptosis, which may be mediated by cytochrome c-dependent caspase-3 activation. In addition, cell-cycle analysis revealed that UA-treated cells were arrested predominantly in the G0 and G1 phases with a concomitant decrease in the cell population of S phase. Moreover, expression of p21^WAF1, a cell-cycle regulator, was increased by UA, indicating that p21^WAF1 might mediate UA-induced cell-cycle arrest. However, UA markedly inhibited SV40 DNA replication in the initiation stage in vitro and significantly reduced the DNA cleaving of topoisomerase I and the ssDNA binding activity of replication protein A. These results indicate that the inhibition of DNA replication by UA may result from blockade of the establishment of the replication fork during initiation stage, consequently contributing to UA-induced cell-cycle arrest. Taken together, we suggest that UA-induced cell-cycle arrest may be mediated by inhibition of DNA replication and the increase of p21^WAF1 expression, which induces the release of cytochrome c and the activation of caspase-3, leading to apoptosis of HepG2 cells. Int. J. Cancer 87:629–636, 2000. © 2000 Wiley-Liss, Inc.

IL-3 deprivation has been reported to induce apoptosis of bone marrow-derived mast cells. In order to evaluate this type of cell death further, we employed trypan blue and propidium iodide stainings, photometric enzyme immunoassay, fluorescence measurement of caspase-3, DNA electrophoresis, flow cytometry and transmission electron microscopy. In this experiment, although several evidences supporting apoptosis were demonstrated some findings were not consistent with typical apoptosis. On the other hand, electron microscopical observation demonstrated that most cells from all the time phases after IL-3 deprivation showed the morphology of typical oncosis, i.e. cell swelling, disintegration of ultrastructure and subsequent karyolysis. Only a small number of cells from the later time phases showed apoptotic morphology. We here suggest that BMMCs undergo both apoptosis and oncosis after IL-3 deprivation and that the dominant type of prelethal change is oncosis in all time phases, although apoptosis also plays a partial role in the late time phases.

We studied the growth of rat mammary epithelial cell (RMEC) subpopulations cultured in Matrigel. Mammary glands were removed, minced, and enzymatically dispersed. Mammary organoids (ductal and endbud fragments) were collected, enzymatically monodispersed, filtered, and labeled with fluorescein isothiocyanate-peanut agglutinin (PNA) and phycoerythrin-anti-Thy-1.1 monoclonal antibody. By flow cytometry, we separated four RMEC subpopulations: 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+). The ultrastructural characteristics of sorted cells were examined by transmission electron microscopy. Thy-1.1+ cells appeared to be myoepithelial cells with actin filaments. PNA+, B-, and B+ cells were morphologically similar. Sorted RMEC subpopulations, unsorted mixed cells, and undispersed organoids were seeded in Matrigel and cultured in either a complete hormone medium (CHM) with 10% fetal bovine serum, progesterone, estradiol, hydrocortisone, insulin, and prolactin or a serum-free medium containing EGF, insulin, hydrocortisone, and human transferrin. Several types of colonies were observed: stellate, ductal, webbed, squamous, and lobuloductal colonies. At the immunocytochemical and electron micrograph levels, casein proteins were predominantly localized near the apical surfaces of the cells or in the lumina of ductal or lobuloductal colonies. Laminin and collagen IV were detected only in the basal lamina of ductal structures cultured in CHM. RMEC and subpopulations from immature virgin rats developed the capacity to synthesize and secrete lipid and casein when grown in Matrigel in serum medium or under defined serum-free conditions.

A cell separation method was developed for studies of the growth kinetics of rat mammary epithelial cell (RMEC) subpopulations in grafts and in culture in vitro. By flow cytometry of RMEC stained with fluorescein isothiocyanate-peanut agglutinin and phycoerythrin-anti-Thy-1.1 monoclonal antibody, we could distinguish four cell subpopulations from primary cultures of 7- to 8-week-old F344 female rat mammary glands: both negative (B-), PNA+, Thy-1.1+, and both positive (B+). We studied the growth patterns of these subpopulations in vitrofor 1 to 14 days in complete hormone medium (CHM) containing 10% fetal bovine serum and prolactin, 17β-estradiol, cortisol, progesterone, and insulin. The fractions of PNA+ and B- cells steadily decreased with time in culture. The fraction of Thy-1.1+ cells steadily increased with time in culture. There were small numbers of B+ cells. We grafted RMEC in hyperprolactinemic recipient rats. The mean numbers of transplanted cells required to produce at least one alveolar unit in 50% of the graft sites (AD50values) are inversely related to the clonegenie fractions. AD50s of RMEC are as follows: unsorted mammary cells cultured in CHM for 1 to 4 days, ∼220; unsorted cells cultured in CHM for 7 days, ∼550; sorted PNA+ RMEC from outgrowths of 3-day cultures, ∼82; B+, ∼350; B-, ∼545; Thy-1.1+, ∼9372. We conclude that the PNA+ cell subpopulation includes most of the clonegenic cells. Thy-1.1+ cells appear to he terminally differentiated; they are likely to be myoepithelial cells.

The primary factors in determining beef quality grades are the amount and distribution of intramuscular fat percentage (IMFAT). Texture analysis was applied to ultrasound B-mode images from ribeye muscle of live beef cattle to predict its IMFAT. We used wavelet transform (WT) for multiresolutional texture analysis and second-order statistics using a gray-level co-occurrence matrix (GLCM) technique. Sets of WT-and GLCM-based texture features were calculated from ultrasonic images from 207 animals and linear regression methods were used for IMFAT prediction. WT-based features included energy ratios, central moments of wavelet-decomposed subimages and wavelet edge density. The regression model using WT features provided a root mean square error (RMSE) of 1.44 for prediction of IMFAT using validation images, while that of GLCM features provided an RMSE of 1.90. The prediction models using the WT features showed potential for objective quality evaluation in the live animals.

Multiresolutional texture analysis techniques using wavelet transforms have been used for predicting percentage intramuscular fat (IMFAT) of beef ribeye muscle. Ultrasound B-mode images have been collected from 207 live beef animals and digitized through frame grabber connected to a personal computer. Since fat has acoustic properties that are appreciably different from that of muscle, transmitted ultrasound is reflected from the interface between fat and muscle. As fat deposits increase, the speckle content of ultrasound B-mode images also increases. Since the speckle alters the texture of the image, IMFAT can be estimated using texture analysis methods. The Haar wavelet was used as a basis function for generating the fast wavelet transform and several features were calculated from the 2-D wavelet decomposed ultrasound images. Significant features included energy ratios, second and fourth order central moments. Correlation coefficients of the features with the actual IMFAT were calculated to select features for predicting IMFAT. Linear regression (LR) models were used as a tool for predicting IMFAT from the selected features.