ent-Rosane and abietane diterpenoids as cancer chemopreventive agents

Two ent-rosane- (cuzcol, 1 and 6-dehydroxycuzcol, 2) and a abietatriene- (salvadoriol, 3) type diterpenoids have been isolated from Maytenus cuzcoina and Crossopetalum uragoga, respectively, along with five known diterpene compounds (4-8). Their stereostructures have been elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, and computational data. The absolute configuration of cuzcol was determined by application of Riguera ester procedure. This is the first instance of isolation of ent-rosane diterpenoids from species of the Celastraceae. The isolated diterpenes were found to be potent anti-tumour-promoter agents, and carnosol (7) also showed a remarkable chemopreventive effect in an in vivo two-stage carcinogenesis model.     

Does correlation of cellulase gene expression and cellulolytic activity in the gut of termite suggest synergistic collaboration of cellulases?

Termites play an important role in degradation of dead plant materials in nature. Over the last century, many researchers have investigated the mechanisms of their lignocellulose digesting system. A recent publication by Zhou et al. (Zhou, X., Smith, J.A., Oi, F.M., Koehler, P.G., Bennett, G.W., Scharf, M.E., 2007. Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite Reticulitermes flavipes. Gene 395, 29-39) dealt with the cellulolytic system of the flagellate-harboring termite R. flavipes and suggested "the presence of a single unified cellulose digestion system" in the termite, as an alternative hypothesis of a "dual (i.e. endogenous and symbiotic) cellulose digesting system" proposed by Nakashima et al. (Nakashima, K., Watanabe, H., Saitoh, H., Tokuda, G., Azuma, J.-I., 2002. Dual cellulose-digesting system of the wood-feeding termite, Coptotermes formosanus Shiraki. Insect Biochem. Mol. Biol. 32, 777-784). Here we show that their results actually support a dual cellulose digesting system rather than "a single unified cellulose digestion system". In addition, potential problems with their results are highlighted.     

Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma

We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.     

Phosphorylation of lipocortins in vitro by protein kinase C

Protein kinase C catalyzes the incorporation of about 1.1, 0.7 and 0.4 mole of phosphate per mole of Lipocortin-I (P35), Lipocortin-II (P36) and Lipocortin-85 (P36 oligomer) respectively. The phosphorylation is specific for protein kinase C and is dependent on the presence of both calcium and phospholipids. While Lipocortin-I is phosphorylated on threonine residues, Lipocortin-II and Lipocortin-85 are phosphorylated on serine residues. The substoichiometric phosphorylation of Lipocortin-85 appears to preclude the potential regulation of this protein by protein kinase C. The phosphorylation of Lipocortin-I on threonine residues and Lipocortin-II on serine residues suggests these proteins may be regulated by distinct phosphorylation-dephosphorylation reactions.     

p90 RSK-1 associates with and inhibits neuronal nitric oxide synthase

Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain.     

The effect of quercetin, a mutagenicity-enhancing agent, on the metabolism of 2-acetylaminofluorene with mammalian metabolic activation systems

The effect of quercetin as the comutagen on 2-acetylaminofluorene (AAF) was investigated. AAF was metabolized with mammalian metabolic systems (S9 mix) in the presence or absence of quercetin in vitro, and its metabolites were determined by high-performance liquid chromatography. In the presence of quercetin, the total metabolic rate of AAF decreased compared with that in the absence of quercetin, whereas the formation of N-hydroxy-AAF (N-OH-AAF) and 2-aminofluorene (AF) increased. Since the main metabolic pathway of AAF is aryl-hydroxylation, it is suggested that the decrease of total metabolic rate of AAF is due to the inhibition of aryl-hydroxylation by quercetin. From these results, it seems probable that the comutagenic effect of quercetin on AAF is due to the inhibition of aryl-hydroxylation (the detoxifying pathway) and the promotion of N-hydroxylation and deacetylation (the activating pathway) in the AAF metabolism with S9 mix.     

Mechanisms for the apoptosis of small cell lung cancer cells induced by anti-GD2 monoclonal antibodies: roles of anoikis

Anti-GD2 ganglioside antibodies could be a promising, novel therapeutic approach to the eradication of human small cell lung cancers, as anti-GD2 monoclonal antibodies (mAbs) induced apoptosis of small cell lung cancer cells in culture. In this study, we analyzed the mechanisms for the apoptosis of these cells by anti-GD2 mAbs and elucidated the mechanisms by which apoptosis signals were transduced via reduction in the phosphorylation levels of focal adhesion kinase (FAK) and the activation of a MAPK family member, p38, upon the antibody binding. Knock down of FAK resulted in apoptosis and p38 activation. The inhibition of p38 activity blocked antibody-induced apoptosis, indicating that p38 is involved in this process. Immunoprecipitation-immunoblotting analysis of immune precipitates with anti-FAK or anti-integrin antibodies using an anti-GD2 mAb revealed that GD2 could be precipitated with integrin and/or FAK. These results suggested that GD2, integrin, and FAK form a huge molecular complex across the plasma membrane. Taken together with the fact that GD2+ cells showed marked detachment from the plate during apoptosis, GD2+ small cell lung cancer cells seemed to undergo anoikis through the conformational changes of integrin molecules and subsequent FAK dephosphorylation.     

Cancer preventive agents 9. Betulinic acid derivatives as potent cancer chemopreventive agents

C-3 esterifications of betulinic acid (BA, 1) and its A-ring homolog, ceanothic acid (CA, 2), were carried out to provide sixteen terpenoids, 4–19, including nine new compounds (4–12). All synthesized compounds were evaluated in an in vitro antitumor-promoting assay using the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among them, compounds 4–6, 11–14, 16, and 17 displayed remarkable inhibitory effects of EBV-EA activation. BA analog 6, which contains a prenyl-like group, showed the most potent inhibitory effect (100%, 76%, 37%, and 11% inhibition of EBA activation at 1000, 500, 100, and 10 mol ratio/TPA, respectively, with IC₅₀ value of 285 mol ratio/32 pmol TPA). Compound 6 merits further development as a cancer preventive agent.     

An antitumor promoter from Moringa oleifera Lam.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(α- -rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(α- -rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), β-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6′-O-oleoyl-β- -glucopyranosyl)-β-sitosterol (7), and β-sitosterol-3-O-β- -glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.     

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.