Anti-proliferative effects of ginsenosides extracted from mountain ginseng on lung cancer / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of three major ginsenosides from mountain ginseng as anticancer substance and explore the underlying mechanism involved in lung cancer.</p><p><b>METHODS</b>The inhibitory proliferation of lung cancer by major five ginsenosides (Rb1, Rb2, Rg1, Rc, and Re) was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Calculated 50% inhibition (IC50) values of five ginsenosides were determined and compared each other. Apoptosis by the treatment of single ginsenoside was performed by fluorescence-assisted cytometric spectroscopy. The alterations of apoptosis-related proteins were evaluated by Western blot analysis.</p><p><b>RESULTS</b>The abundance of ginsenosides in butanol extract of mountain ginseng (BX-MG) was revealed in the order of Rb1, Rg1, Re, Rc and Rb2. Among them, Rb1 was the most effective to lung cancer cell, followed by Rb2 and Rg1 on the basis of relative IC50 values of IMR90 versus A549 cell. The alterations of apoptotic proteins were confirmed in lung cancer A549 cells according to the administration of Rb1, Rb2 and Rg1. The expression levels of caspase-3 and caspase-8 were increased upon the treatment of three ginsenosides, however, the levels of caspase-9 and anti-apoptotic protein Bax were not changed.</p><p><b>CONCLUSION</b>Major ginsenosides such as Rb1, Rb2 and Rg1 comprising BX-MG induced apoptosis in lung cancer cells via extrinsic apoptotic pathway rather than intrinsic mitochondrial pathway.</p>

Activation of AMP-activated protein kinase stimulates the nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in human diploid fibroblasts

In addition to its well-known glycolytic activity, GAPDH displays multiple functions, such as nuclear RNA export, DNA replication and repair, and apoptotic cell death. This functional diversity depends on its intracellular localization. In this study, we explored the signal transduction pathways involved in the nuclear translocation of GAPDH using confocal laser scanning microscopy of immunostained human diploid fibroblasts (HDFs). GAPDH was present mainly in the cytoplasm when cultured with 10% FBS. Serum depletion by culturing cells in a serum-free medium (SFM) led to a gradual accumulation of GAPDH in the nucleus, and this nuclear accumulation was reversed by the re-addition of serum or growth factors, such as PDGF and lysophosphatidic acid. The nuclear export induced by the re-addition of serum or growth factors was prevented by LY 294002 and SH-5, inhibitors of phosphoinositide 3-kinase (PI3K) and Akt/protein kinase B, respectively, suggesting an involvement of the PI3K signaling pathway in the nuclear export of GAPDH. In addition, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), stimulated the nuclear translocation of GAPDH and prevented serum- and growth factor-induced GAPDH export. AMPK inhibition by compound C or AMPK depletion by siRNA treatment partially prevented SFM- and AICAR-induced nuclear translocation of GAPDH. Our data suggest that the nuclear translocation of GAPDH might be regulated by the PI3K signaling pathway acting mainly as a nuclear export signal and the AMPK signaling pathway acting as a nuclear import signal.

Alterations of epinephrine-induced gluconeogenesis in aging

The effects of glucagon and epinephrine on gluconeogenesis in young (4 month) and old (24 month) Fisher 344 rat hepatocytes were compared. In contrast to glucagon, which had a similar effect on gluconeogenesis in both young and old cells, epinephrine caused a smaller increase in gluconeogenesis in old rat hepatocytes than in young hepatocytes. beta2 adrenergic receptor (beta2-AR) expression slightly decreased in aged rat liver, and there were differences between young and old hepatocytes in their patterns of G protein coupled receptor kinases, which are involved in the activation of beta2-AR receptor signal desensitization. The major isoform of the kinase changed from GRK2 to GRK3 and the expression of beta-arrestin, which is recruited by the phosphorylated beta2-AR for internalization and degradation, increased in aged rat liver. GRK3 overexpression also decreased the glucose output from young rat hepatocytes. We conclude that an age-associated reduction in epinephrine-induced gluconeogenesis occurs through the epinephrine receptor desensitizing system.

Alterations of epinephrine-induced gluconeogenesis in aging

The effects of glucagon and epinephrine on gluconeogenesis in young (4 month) and old (24 month) Fisher 344 rat hepatocytes were compared. In contrast to glucagon, which had a similar effect on gluconeogenesis in both young and old cells, epinephrine caused a smaller increase in gluconeogenesis in old rat hepatocytes than in young hepatocytes. beta2 adrenergic receptor (beta2-AR) expression slightly decreased in aged rat liver, and there were differences between young and old hepatocytes in their patterns of G protein coupled receptor kinases, which are involved in the activation of beta2-AR receptor signal desensitization. The major isoform of the kinase changed from GRK2 to GRK3 and the expression of beta-arrestin, which is recruited by the phosphorylated beta2-AR for internalization and degradation, increased in aged rat liver. GRK3 overexpression also decreased the glucose output from young rat hepatocytes. We conclude that an age-associated reduction in epinephrine-induced gluconeogenesis occurs through the epinephrine receptor desensitizing system.

Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts

Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ia. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.

Adaptation of cAMP signaling system in SH-SY5Y neuroblastoma cells following expression of a constitutively active stimulatory G protein alpha, Q227L Gsalpha

Heterotrimeric GTP-binding proteins (G protein) are known to participate in the transduction of signals from ligand activated receptors to effector molecules to elicit cellular responses. Sustained activation of cAMP-G protein signaling system by agonist results in desensitization of the pathway at receptor levels, however it is not clear whether such receptor responses induce other changes in post-receptor signaling path that are associated with maintenance of AMP levels, i.e. cAMP-forming adenylate cyclase (AC), cAMP-degrading cyclic nucleotide phosphodiesterase (PDE) and cAMP-dependent protein kinase (PKA). Experiments were performed to determine the expression of AC, PDE, and PKA isoforms in SH-SY5Y neuroblastoma cells, in which cAMP system was activated by expressing a constitutively activated mutant of stimulatory G protein (Q227L Gsalpha). Expression of ACI mRNA was increased, but levels of ACVIII and ACIX mRNA were decreased. All of the 4 expressed isoforms of PDE (PDE1C, PDE2, PDE 4A, and PDE4B) were increased in mRNA expression; the levels of PKA RIalpha, RIbeta, and RIIbeta were increased moderately, however, those of RIIalpha and Calpha were increased remarkably. The activities of AC, PDE and PKA were also increased in the SH-SY5Y cells expressing Q227L Gsalpha. The similar changes in expression and activity of AC, PDE and PKA were observed in the SH-SY5Y cells treated with dbcAMP for 6 days. Consequently, it is concluded that the cAMP system adapts at the post-receptor level to a sustained activation of the system by differential expression of the isoforms of AC, PDE, and PKA in SH-SY5Y neuroblastoma. We also showed that an increase in cellular cAMP concentration might mediate the observed changes in the cAMP system.

Increased expression of Galphaq protein in the heart of streptozotocin-induced diabetic rats

Heart disease is one of the major cause of death in diabetic patients, but the thogenesis of diabetic cardio-myopathy remains unclear. In this experiment, to sess the significance of G protein signaling pathways in the pathogenesis of abetic cardiomyopathy, we analyzed the expression of G proteins and the tivities of second messenger dependent protein kinases: cAMP-dependent protein nase (PKA), DAG-mediated protein kinase C (PKC), and calmodulin dependent otein kinase II (CaM kinase II) in the streptozotocin induced diabetic rat art. The expression of Galphaq was increased by slightly over 10% (P<0.05) in abetic rat heart, while Galphas, Galphai, and Gbeta remained unchanged. The A activity in the heart did not change significantly but increased by 27%<0.01) in the liver. Insulin treatment did not restore the increased activity the liver. Total PKC activity in the heart was increased by 56% (P<0.01), and sulin treatment did not restore such increase. The CaM kinase II activity in e heart remained at the same level but was slightly increased in the liver 4% increase, P<0.05). These findings of increased expression of Galphaq in the reptozotocin-diabetic rat heart that are reflected by the increased level of C activity and insensitivity to insulin demonstrate that alteration of Galphaq y underlie, at least partly, the cardiac dysfunction that is associated with abetes. Copyright 2000 Academic Press.

Effects of overexpression of stimulatory G proteins on the expression of the other G proteins in COS-1 cells

To investigate the interaction of stimulatory GTP binding protein (G(s)) pathways with others, we overexpressed wild type alpha subunit of G(s) (G(s) alpha), constitutively activated R201E G(s) alpha, and dominant negative G226A G(s) alpha in COS-1 cells by transfection with DEAE-dextran, respectively, The expression of various G proteins in the transfected cells was analyzed after 72 h by quantitative Western blots, and cAMP production by stimulation with isoproterenol and forskolin was quantitated using cAMP binding proteins, The expression of Gs alpha increased about 5-fold in the transfected cells, with concomitant increase in the small forms. However, there was no significant alteration the in the level of the alpha subunit of inhibitory G protein (G(i)) and G(q), and the beta subunits of G proteins. The cAMP level without stimulation increased in the cells transfected with G(s) alpha regardless to the type of mutation, Treatment with either isoproterenol or forskolin resulted in comparable increase of the cAMP level in all the transfected cells, though the ratio to its respective basal level was smaller in the G(s) alpha-transfected cells, From this experiment, we found that the expression of the other G proteins and the signaling pathway producing cAMP did not change significantly by transiently expressing wild type, constitutively activated type, and dominant negative type of G(s) alpha. Analysis of the effects of long-term expression of Gs alpha would contribute to better understanding on how the G(s) alpha signaling system interacts with other signaling pathways and how it adapts to the changed environments.