Hseng-Kuang Hsu

Effects of nonsteroidal anti-inflammatory drugs and prostaglandins on osteoblastic functions

It has been reported that nonsteroidal anti-inflammatory drugs (NSAIDs) suppress bone repair and bone remodeling but only mildly inhibit bone mineralization at the earlier stage of the repair process. We proposed that the proliferation and/or the earlier stage of differentiation of osteoblasts may be affected by NSAIDs. This study was designed to investigate whether NSAIDs affect the proliferation and/or differentiation of osteoblasts and whether these effects are prostaglandin (PG) mediated. The effects of PGE1 and PGE2, indomethacin, and ketorolac on thymidine incorporation, cell count, intracellular alkaline phosphatase (ALP) activity, and Type I collagen content in osteoblast-enriched cultures derived from fetal calvaria were evaluated. The results showed that both PGs and NSAIDs inhibited DNA synthesis and cell mitosis in a time- and concentration-dependent manner. However, intracellular ALP activity and Type I collagen content were stimulated at an earlier stage of differentiation in osteoblasts. These results suggested that (i) the inhibitory effect of ketorolac on osteoblastic proliferation contributes to its suppressive effects on bone repair and remodeling in vivo; (ii) PGEs and NSAIDs may be involved in matrix maturation and biologic bone mineralization in the earlier stage of osteoblast differentiation; and (iii) the effects of ketorolac and indomethacin on cell proliferation and differentiation may not be through the inhibition of the synthesis of PGE1 or PGE2.

Extract from the Leaves of Toona sinensis Roemor Exerts Potent Antiproliferative Effect on Human Lung Cancer Cells

Recent study indicated that the components of Toona sinensis Roemor have potent anti-inflammatory and analgesic effects. These components have also been reported to inhibit the growth of boils in vivo. In this study, we investigated the effect of crude extract from the leaves of Toona sinensis Roemor on the proliferation of A549 lung cancer cells. We found that the extract effectively blocked cell cycle progression by inhibiting the expression of cyclin D1 and E in A549 cells. Additionally, incubation of the extract led to activation of caspase-3-like proteases and apoptotic cell death. Conversely, the extract did not show any significant cytotoxic effect on primarily cultured human foreskin fibroblasts or MRC-5 human lung fibroblasts. Therefore, antiproliferative action of the extract is specific for tumor cells. Our results suggest that the components of Toona sinensis Roemor have potent anticancer effects in vitro and identification of the useful components in the extract may lead to the development of a novel class of anticancer drugs.

ENHANCEMENT OF GLUCOSE UPTAKE IN 3T3-L1 ADIPOCYTES BY TOONA SINENSIS LEAF EXTRACT

The effects of substances extracted from Toona sinensis leaves, using 50% alcohol/water, on cellular [3H]‐2‐deoxyglucose uptake in differentiated cultured 3T3‐L1 adipocytes were investigated. Following treatment of cells with 0.001, 0.01, or 0.1 mg/mL extracts for 60 minutes, [3H]‐2‐deoxyglucose uptake increased from a basal value of 0.23 nmol/min/mg protein to 0.30, 0.33, and 0.38 nmol/min/mg protein, respectively. In insulin‐stimulated cells, cellular [3H]‐2‐deoxyglucose uptake was enhanced by Toona sinensis leaf extract from a basal value of 0.35 nmol/min/mg protein to 0.41, 0.46, and 0.52 nmol/min/mg protein, respectively. Cellular glucose uptake was also enhanced by Toona sinensis leaf extract after incubation of cells with 20 mM glucose for 48 hours. Cellular glucose uptake with a combination of Toona sinensis leaf extract and insulin was significantly inhibited by pretreatment of cells with the protein synthesis inhibitor cycloheximide and the protein kinase C inhibitor calphostin C in normal‐, medium‐ and high‐glucose media. However, the glucose uptake‐enhancing effect of Toona sinensis leaf extract was not diminished by cycloheximide and calphostin C in the absence of insulin. These results indicate that enhancement of cellular glucose uptake by Toona sinensis leaf extract in basal and insulin‐stimulated 3T3‐L1 adipocytes may be mediated by distinct mechanisms.

Blockage of N-Methyl-D-Aspartate Receptors Decreases Testosterone Levels and Enhances Postnatal Neuronal Apoptosis in the Preoptic Area of Male Rats

Sexual dimorphism has been found in the preoptic area of the hypothalamus (POA), a major site of glutamate actions via N-methyl-D-aspartate (NMDA) receptors. The sexually dimorphic nucleus of the preoptic area (SDN-POA) of male rats exhibits about seven-fold greater nuclear volume than that of females. A naturally occurring neonatal neuronal apoptosis, that can be prevented by testosterone, may contribute to this sexual difference in SDN-POA nuclear volume. Since activation of NMDA receptors in the POA induces GnRH secretion, it may be involved in both elevation of serum testosterone and prevention of neuronal death in the SDN-POA. In the present study, protein expression of NMDA receptors in the POA of male and female fetuses was quantified on the day preceding the fetal testosterone peak (embryonic day 16; ED 16). Rats were then distributed in four groups: (1) untreated males, (2) untreated females, (3) males pretreated with MK-801 (a noncompetitive NMDA receptor antagonist), and (4) females pretreated with MK-801. Serum levels of testosterone were estimated on the afternoon of ED 18. Expression of Bcl-2 and Bax, as well as neuronal apoptosis in SDN-POA, were observed on postnatal day 8. The results showed that (1) expression of NMDA receptors in the POA of male fetuses was higher than that of females on ED 16; (2) levels of testosterone were lower in MK-801 pretreated male fetuses than in intact males on ED 18; (3) expression of Bcl-2 in the POA of MK-801 pretreated male rats was significantly less than that of control males; (4) the apoptotic incidence in the SDN-POA of MK-801 pretreated male rats was significantly greater than in control males, while there was no significant difference in apoptotic incidence in the SDN-POA between MK-801 pretreated and intact females. These results suggest that the NMDA receptor is highly expressed in prenatal male fetuses, and that it might play an important role in the elevation of testosterone levels. Moreover, activation of NMDA receptors may protect SDN-POA neurons from naturally occurring neuronal death, by modulating testosterone and/or Bcl-2 expression.

Toona sinensis Roem (Meliaceae) leaf extract alleviates hyperglycemia via altering adipose glucose transporter 4

In this study we tested the effects of Toona sinensis leaf extracted with water (TSL1) on alloxan-induced (50 mg/kgBwt, i.v.) diabetic Long-Even rats. Diabetic rats given TS leaf with water (TSL1), with 50% alcohol (TSL3) or with H2O extract (TSL5) showed lower levels of plasma glucose. Normal rats given Glibenclamide (GC) had lower levels of plasma glucose, but TSL1 administration showed no significant effect on plasma glucose. By contrast, TSL1 or GC given to alloxan-induced diabetic rats showed a 40% reduction in plasma glucose compared to diabetic rats. Diabetic rats had lower levels of insulin. Interestingly, TSL1 or GC given to diabetic rats showed improvements in plasma insulin levels. Diabetic rats had lower expressions of glucose transporter 4 (GLUT4) mRNA (RT-PCR) and GLUT4 protein (Western blot) in brown and white adipose tissues; in contrast, diabetic rats given TSL1 or GC showed a significant increase in both GLUT4 mRNA and protein levels. Moreover, the expressions of GLUT4 mRNA in red and white muscles were not significantly different among diabetic rats, diabetic rats given TSL1 or GC, and the normal rats. Compared to diabetic rats, diabetic rats given TSL1 or GC had lower levels of GLUT4 protein in white muscle but not in red muscle. Conclusively, T. sinensis Roem (Meliaceae) leaf possesses the hypoglycemia effect underlying an increment of insulin to mediate the adipose glucose transporter 4 mechanism.

G protein and adenylate cyclase complex-mediated signal transduction in the rat heart during sepsis.

Changes in the protein level of various subunits of GTP-binding protein and the activity of adenylate cyclase in the rat heart during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The protein levels of various subunits of GTP-binding protein were determined by Western blot analysis. The activity of adenylate cyclase was measured based on the rate of formation of cAMP from [&agr;-32P]ATP. The results show that protein levels of G&agr;s and G&bgr; remained stable during the early and the late phases of sepsis. The protein levels of G&agr;i-2 and G&agr;i-3 remained relatively unaltered during the early phase of sepsis, but they were increased by 46.5% (P < 0.05) and 61.3% (P < 0.01), respectively, during the late phase of sepsis. The basal adenylate cyclase activity remained unchanged during the early phase while it was decreased by 25.7% (P < 0.05) during the late phase of sepsis. The isoproterenol-stimulated adenylate cyclase activity was unchanged during early sepsis while it was decreased by 44.6% (P < 0.01) during late sepsis. These data demonstrate that during the late hypodynamic phase of sepsis, myocardial G&agr;i-2 and G&agr;i-3 protein levels were increased and the increases were coupled with a reduction in adenylate cyclase activity. Because GTP-binding proteins mediate sympathetic control of cardiac function, the present findings may have a pathophysiological significance in contributing to the understanding of the pathogenesis of cardiac dysfunction during the late stage of sepsis.

Effects of Long-Term Testosterone Replacement on Copulatory Activity in Old Male Rats

Various parameters of copulatory behavior were examined in intact and castrated testosterone-(T-)treated male rats of various ages ranging from 3 to 27 months. Serum T levels were controlled by subcutaneous implantation of different sizes of T-filled Silastic capsules at the time of castration. In intact male rats, scores of various parameters of copulatory behavior declined gradually from middle age and no ejaculation was observed in any rats over 22 months of age. When serum T levels were maintained at 0.78-0.87 ng/ml for 6 months from 4 months of age, these rats showed the same copulatory activity as intact rats, except that fewer rats exhibited ejaculation at 8 and 10 months of age and showed decreased total mount frequency at 10 months of age. Even when serum T levels were maintained at 2.95-3.53 ng/ml for a period of 6 months from 18 months of age, copulatory activity still declined gradually and no rats over 22 months of age ejaculated. However, long-term elevation of serum T level could prevent further decline of intromission frequency in old age. These results indicate that the copulatory activity of male rats in response to circulatory T declined with advancing age and that prolonged low levels of serum T had only a minor role in the onset of decreased responsiveness of neural male sexual behavior.

Suppression of Protein Kinase Cα Triggers Apoptosis Through Down-regulation of Bcl-xl in a Rat Hepatic Epithelial Cell Line

Inactivation of protein kinase C (PKC)&agr; plays an important role in modulating hepatic failure and/or apoptosis during sepsis. To determine whether and how PKC&agr; inactivation mediates the apoptosis, PKC&agr; was suppressed by antisense treatment or transiently transfection in Clone-9 rat hepatic epithelial cell line. Apoptosis was evaluated by cell survival rate, poly-adenyl ribonuclease polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling stain. The expressions of PKC&agr; and Bcl-xL were quantified by Western blot analysis after antisense treatment. In the transfection studies, cells were co-transfected with green fluorescent protein cDNA as a transfection marker. The expressions of PKC&agr; and Bcl-xL were detected by immunohistochemical staining with second antibody conjugated with Texas red. Apoptosis was evaluated by tetramethyl-rhodamine labeling of DNA strand breaks and immunostaining of 85-kDa fragment of PARP. The results showed that cytosolic and membrane-associated PKC&agr; were decreased by 54.5% and 41.4%, respectively, after PKC&agr; antisense treatment. The apoptotic incidence and percentage of PARP cleavage were significantly increased, whereas protein expression of Bcl-xL was decreased after PKC&agr;-antisense treatment. In the transfection studies, the results showed that most of the cells expressing green fluorescent protein revealed less PKC&agr; and Bcl-xL protein contents and more in situ PARP cleavage and DNA strand breaks. These findings indicated that decrease of PKC&agr; declines the Bcl-xL content and leads to the vulnerability of apoptosis in hepatic epithelial cells. Taken together, our data provide evidence that suppression of PKC&agr; plays a critical role in triggering caspase-dependent apoptosis, which may act through modulating the Bcl-xL expression.

The Decrease Of Pkcα Is Associated With Hepatic Apoptosis At Early And Late Phases Of Polymicrobial Sepsis

The present study investigates the relationship between the PKC-alpha and hepatic apoptosis during sepsis. Cecal ligation and puncture- (CLP) induced animal model of polymicrobial sepsis was used, with early and late sepsis referring to those animals sacrificed at 9 and 18 h, respectively, after CLP. The expressions of PKCalpha and Bcl-2 family proteins as well as poly(ADP-ribose) polymerase (PARP) cleavage were quantified to evaluate the possible factors involved in the hepatic cell death during sepsis. The apoptosis of hepatocytes under septic condition or hepatocytes treated with PKCalpha antisense was evaluated by gel electrophoresis and/or flow cytometry after Annexin-V-Fluos and propidium iodide staining. The results indicated that (1) the protein expression of membrane-associated PKCalpha was decreased at early (P < 0.05) and late (P < 0.01) sepsis; (2) the protein expressions of Bcl-2 and Bcl-xL were decreased, whereas Bax expression was increased at late sepsis; (3) the percentage of PARP cleavage was increased at early (P < 0.05) and late (P < 0.01) sepsis; (4) severe DNA fragmentation was observed at late sepsis; (5) the apoptotic cell population was increased at early and late sepsis; and (6) the percentage of apoptotic cell population in PKCalpha antisense-treated cells was significantly higher than that in untreated cells. These results suggest that inactivation of PKCalpha may play an important role in modulating hepatic apoptosis during sepsis and the apoptosis is closely associated with the alterations of Bcl-2 family proteins.

Antiproliferative and antitumorigenic activity of Toona sinensis leaf extracts in lung adenocarcinoma.

Toona sinensis is a traditional Chinese herb, and the extracts of T. sinensis leaf possess a variety of biological functions. This study attempted to test the antiproliferative effect of TSL-1 (a bioactive fraction of T. sinensis) in H441 cells (lung adenocarcinoma). The data showed that the antiproliferative effect of TSL-1 on H441 cells is prominent using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TSL-1-induced apoptosis was confirmed by cell morphology, sub-G(1) peak accumulation, cleavage of poly(ADP)-ribose polymerase, and propidium iodide-annexin V double staining. Furthermore, decreased Bcl-2 accompanied by increased Bax (in western blotting) was found with TSL-1 treatment of H441 cells. TSL-1 treatment-induced G(1) arrest was concurrent with the down-regulation of protein levels of cyclin D1 and cyclin-dependent kinase 4 in H441 cells. Peroral and intraperitoneal administrations of TSL-1 were performed to evaluate the therapeutic efficacy, and peroral administration of TSL-1 was also used to elucidate the therapeutic efficacy in the H441 cell xenograft model in vivo. The data revealed that TSL-1 treatment inhibited H441 tumor growth in both therapeutic and preventive experiments. Taken together, these results demonstrate that TSL-1 possesses the capability of preventing and alleviating lung cancer proliferation in vitro and in vivo with proven nephrological and hepatic safety and has the potential to be developed as an anti-lung cancer drug.