Antitumor activity of the isodon diterpenoids: Structural requirements for the activity

Summary A significant antitumor activity of oridonin (1) and lasiokaurin (2), the kaurene-type diterpenoids ofIsodon species, was shown by their i.p. injection to the test mice inoculated by Ehrlich ascites carcinoma. Enmein (8), compounds9 and3 were also active under larger dose. Subsequently, the relationship between their chemical structure and antitumor activity was investigated, and the activity of oridonin (1) and lasiokaurin (2) was rationalized in terms of their structural feature.     

Effect of oridonin, a Rabdosia diterpenoid, on radiosensitization with misonidazole

The radiosensitization brought about by oridonin, one of the Rabdosia diterpenoids, alone or in combination with misonidazole, was investigated in Chinese hamster V79 cells. The enhancement ratio of 1.92 was obtained when 0.01 mM oridonin and 1 mM misonidazole were administered to hypoxic cells under radiation. The enhancement ratios of oridonin and misonidazole for hypoxic cells were 1.16 and 1.59 respectively. Hence, a supra-additive effect was obtained by the combined treatment with these two drugs. Under aerobic conditions, no effect of 0.01 mM oridonin on the radiosensitization caused by misonidazole was observed.     

Altered brain cholinergic enzymes activity in the genetically obese rat

Summary Genetically obese male Zucker rats (fa/fa) and their lean littermates (Fa/-) were used in this experiment. Fourteen-week-old obese and lean littermates were sacrificed and choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) enzymes were assayed in specific brain regions. The assays of these enzymes indicate that obese animals and a significantly lower ChAT activity in the cerebellum, pons, and cerebral cortex and a significant increase in ChAT activity in the thalamus and hypothalamus. Meanwhile, the cerebral cortex, cerebellum, midbrain, thalamus and hypothalamus of the obese animals showed significantly higher AChE activity than their lean littermates. It was concluded from this study that obesity may be associated with changes in the enzymes of the brain cholinergic system.This work was supported by a grant from the National Aeronautics and Space Administration (NAG 2-411), a grant from the National Institutes of Health (NIH RR 0811), and a grant from the Division of Research Resources, National Institutes of Health (NIH Grant RR 03020).     

Effect of stress on choline acetyltransferase activity of the brain and the adrenal of the rat

Choline acetyltransferase (ChAT) activity was determined in cerebral cortex, hypothalamus, hippocampus, cerebellum, medulla oblongata, midbrain and adrenal gland of rats exposed to acute or chronic stress. The exposure of animals to acute immobilization and cold stress (4°C) for one hour resulted in a significant decline of ChAT activity in all brain regions examined except for the medulla oblongata. Moreover, the exposure to acute stress resulted in significant increase of the same enzyme in the adrenal gland. However, chronic exposure of animals to cold stress (4°C) for 7 days resulted in no significant changes of ChAT activity in all tissues examined except for a decline in the midbrain and an increase in the medulla oblongata. The administration of corticosterone (2.0 mg/kg) 1 h prior to sacrificing caused an effect similar to that of acute stress on ChAT activity in all brain regions except for the hypothalamus and the cerebellum. It was concluded from this experiment that stress-induced changes in the ChAT activity of specific brain regions might be mediated by the adrenal steroids.This work was supported by a grant from the National Aeronautics and Space Administration (NSG 2183 and NAG 2-411), a grant from the National Institutes of Health (NIH RR 0811) and a grant from the Division of Research Resources (NIH grant RR 03020).     

Unscheduled CDK1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells

Cyclin-dependent kinase 1 (CDK1) is a major component of the cell cycle progression engine. Recently, several investigations provided evidence demonstrating that unscheduled CDK1 activation may also be involved in apoptosis in cancerous cells. In this article, we demonstrate that X-ray irradiation induced G1 arrest in MOLT-4 lymphocytic leukemia cells, the arrest being accompanied by reduction in the activity of CDK2, but increased CDK1 activity and cell apoptosis in the G1 phase. Interestingly, this increase in CDK1 and apoptosis by ionizing radiation was prevented by pretreatment with the CDK1 inhibitor, roscovitine, suggesting that CDK1 kinase activity is required for radiation-induced apoptotic cell death in this model system. Furthermore, cyclin B1 and CDK1 were detected co-localizing and associating in G1 phase MOLT-4 cells, with the cellular lysates from these cells revealing a genotoxic stress-induced increase in CDK1 phosphorylation (Thr-161) and dephosphorylation (Tyr-15), as analyzed by postsorting immunoprecipitation and immunoblotting. Finally, X-irradiation was found to increase Bcl-2 phosphorylation in G1 phase cells. Taken together, these novel findings suggest that CDK1 is activated by unscheduled accumulation of cyclin B1 in G1 phase cells exposed to X-ray, and that CDK1 activation, at the wrong time and in the wrong phase, may directly or indirectly trigger a Bcl-2-dependent signaling pathway leading to apoptotic cell death in MOLT-4 cells. Received 30 March 2006; received after revision 23 June 2006; accepted 24 August 2006 J. Wu and Y. Feng contributed equally to this work.     

Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

The protein kinase D (PKD) family of proteins are important regulators of tumor growth, development, and progression. CRT0066101, an inhibitor of PKD, has antitumor activity in multiple types of carcinomas. However, the effect and mechanism of CRT0066101 in bladder cancer are not understood. In the present study, we show that CRT0066101 suppressed the proliferation and migration of four bladder cancer cell lines in vitro. We also demonstrate that CRT0066101 blocked tumor growth in a mouse flank xenograft model of bladder cancer. To further assess the role of PKD in bladder carcinoma, we examined the three PKD isoforms and found that PKD2 was highly expressed in eight bladder cancer cell lines and in urothelial carcinoma tissues from the TCGA database, and that short hairpin RNA (shRNA)-mediated knockdown of PKD2 dramatically reduced bladder cancer growth and invasion in vitro and in vivo, suggesting that the effect of the compound in bladder cancer is mediated through inhibition of PKD2. This notion was corroborated by demonstrating that the levels of phospho-PKD2 were markedly decreased in CRT0066101-treated bladder tumor explants. Furthermore, our cell cycle analysis by flow cytometry revealed that CRT0066101 treatment or PKD2 silencing arrested bladder cancer cells at the G2/M phase, the arrest being accompanied by decreases in the levels of cyclin B1, CDK1 and phospho-CDK1 (Thr161) and increases in the levels of p27^Kip1 and phospho-CDK1 (Thr14/Tyr15). Moreover, CRT0066101 downregulated the expression of Cdc25C, which dephosphorylates/activates CDK1, but enhanced the activity of the checkpoint kinase Chk1, which inhibits CDK1 by phosphorylating/inactivating Cdc25C. Finally, CRT0066101 was found to elevate the levels of Myt1, Wee1, phospho-Cdc25C (Ser216), Gadd45α, and 14-3-3 proteins, all of which reduce the CDK1-cyclin B1 complex activity. These novel findings suggest that CRT0066101 suppresses bladder cancer growth by inhibiting PKD2 through induction of G2/M cell cycle arrest, leading to the blockade of cell cycle progression.     

Host immune rejection of TA3 ascites carcinoma cells following administration of a water soluble carbodiimide.

Regression of TA3 ascites carcinoma tumors occurred following i.p. injection of 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide-HC1. An immunological mechanism of drug action was indicated by the fact that no significant antitumor activity was demonstrable in mice that had previously received an immunosuppressive 700 rad dose of 60Co radiation.     

Comparative platelet anti-aggregant activity of D-cysteinolic acid analogues

D-Cysteinolic acid (1) analogues with an S-C-C-N skeleton showed increased platelet anti-aggregant activity in the following order: 2-aminoethanesulfonic acids, thiazolidines, 2-aminoethanethiols and 2-aminoethyl disulfides. Methyl substitutions at the 2-position potentiated the activity. Of these analogues, bis [(R)-2-aminopropyl] disulfide was the most potent inhibitor of platelet aggregation, with about 600-fold the activity of (1).     

Effects of ovarian steroids on superoxide dismutase activity in the rat brain

The levels of manganese superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase (CuZnSOD) were determined in appropriate subcellular fractions prepared from whole brain homogenates of cycling and long-term (3 week) ovariectomized (OVX) Wistar rats, and were compared to the levels found in corresponding samples prepared from OVX rats treated with progesterone (P) or estradiol 17B-benzoate (EB). The activity of both SODs was steady during the estrous cycle, except at proestrus, when MnSOD activity was elevated significantly. Bilateral ovariectomy resulted three weeks later in an increase of the MnSOD activity even higher than that recorded at proestrus. High post-castration MnSOD activity was lowered profoundly by exogenous P (2 mg) or EB (0.5 g), given s.c. to OVX animals 2 h or 24 h before sacrifice. Neither removal of the ovaries nor the hormone treatments affected the activity of CuZnSOD. These results suggest suppressive effects of ovarian steroids on MnSOD activity in the rat brain.     

Multispecies-compatible antitumor effects of a cross-species small-interfering RNA against mammalian target of rapamycin

Successful development of sequence-specific siRNA (small interfering RNA)-based drugs requires an siRNA design that functions consistently in different organisms. Utilizing the CAPSID program previously developed by our group, we here designed siRNAs against mammalian target of rapamycin (mTOR) that are entirely complementary among various species and investigated their multispecies-compatible gene-silencing properties. The mTOR siRNAs markedly reduced mTOR expression at both the mRNA and protein levels in human, mouse, and monkey cell lines. The reduction in mTOR expression resulted in inactivation of both mTOR complex I and II signaling pathways, as confirmed by reduced phosphorylation of p70S6K (70-kDa ribosomal protein S6 kinase), 4EBP1 (eIF4E-binding protein 1), and AKT, and nuclear accumulation of FOXO1 (forkhead box O1), with consequent cell-cycle arrest, proliferation inhibition, and autophagy activation. Moreover, interfering with mTOR activity in vivo using mTOR small-hairpin RNA-expressing recombinant adeno-associated virus led to significant antitumor effects in xenograft and allograft models. Thus, the present study demonstrates that cross-species siRNA successfully silences its target and readily produces multispecies-compatible phenotypic alterations-antitumor effects in the case of mTOR siRNA. Application of cross-species siRNA should greatly facilitate the development of siRNA-based therapeutic agents.     

Bactericidal activity againstPseudomonas aeruginosa is acquired by cultured human monocyte-derived macrophages after uptake of myeloperoxidase

Myeloperoxidase (MPO) is an enzyme located within polymorphonuclear neutrophils capable of producting cytotoxic oxidant species that are particularly active against bacteria with polysaccharide capsules.Pseudomonas aeruginosa (10⁶ bacteria per 1 ml) are killed within 1 h in vitro by a MPO/H₂O₂/Cl^– system (48 mU=132 ng of MPO). The question arose as to whether human macrophages would acquire cytotoxic activity when loaded with this enzyme. Monocytes were therefore isolated from human blood and cultured for up to ten days to induce maturation to macrophages. These cells lost endogenous MPO within five days while H₂O₂ production in response to stimulation by phorbol myristate acetate (10^–6M) decreased to 23% within ten days. On the other hand, their capacity to take up exogenous MPO increased fourfold from day three to day ten. Human macrophages cultured from eight days (when both H₂O₂ production and MPO uptake were sufficient) were therefore used to study the effects of MPO uptake on cytocidal activity againstPseudomonas aeruginosa. After a 1 h MPO loading period, macrophages (5×10⁵ cells per ml) were incubated in the presence of bacteria (0.5 to 2×10⁶ bacteria per ml) for 2 h at 37°C. At a bacteria/macrophage ratio of 1, only 34.8±7.0% of bacteria survived (compared to killing by non-loaded macrophages), while 74.4±9.3% survived at a ratio of 4. From these results, we conclude that loading macrophages with exogenous MPO could enhance their microbicidal activity, suggesting a potentially useful therapeutic application.     

Superoxide anion scavenging effect and superoxide dismutase activity ofGinkgo biloba extract

Summary Ginkgo biloba extract is known to be efficient in diseases associated with free radical generation. The purpose of this work was to study, under in vitro conditions, the action ofGinkgo biloba extract (Gbe) against superoxide anion ( ), which is directly or indirectly implicated in cell damage.Gbe appears to have both an scavenging effect and also a superoxide dismutase activity. Its antiradical effect was demonstrated by low temperature electron spin resonance and in a non-enzymatic system (phenazine methosulfate-NADH), and its enzymatic activity was shown by polarographic determination.     

Aragusterol C: A novel halogenated marine steroid from an Okinawan sponge,Xestospongia sp., possessing potent antitumor activity

A novel chlorinated steroid, aragusterol C, was isolated from an Okinawan marine sponge of the genusXestospongia. The compound strongly inhibited the proliferation of KB cells in vitro, and also showed potent in vivo antitumor activity against L1210 cells in mice. The complete structure of aragusterol C was determined by spectroscopic analysis and X-ray crystallographic analysis.     

Role of phospholipids in the binding activity of vasoactive intestinal peptide receptors

Phospholipase digestion of rat intestinal epithelial cell membranes was performed in order to study the influence of membrane phospholipids on the binding activity of VIP receptors. Phospholipases A2 and C strongly (ED50 congruent to 4 X 10(-2) and 4 X 10(-1) micrograms/ml, respectively) and rapidly reduced 125I-VIP binding to membranes whereas phospholipase D was ineffective. This suggests an important role of both hydrophobic and hydrophilic groups of phospholipids on VIP receptor binding activity.     

Potentiation of antifungal activity of sesquiterpene dialdehydes against Candida albicans and two other fungi.

The antifungal activity of two drimane sesquiterpene dialdehydes, polygodial (1) and warburganal (2), alone and in combination with several other substances, was examined against three fungi, Candida albicans, Saccharomyces cerevisiae and Pityrosporum ovale employing a broth dilution method. Anethole significantly synergized the activity of the two sesquiterpenoids against C. albicans and S. cerevisiae; however, it had only an additive effect against P. ovale. By contrast, two antioxidants, ascorbic acid (vitamin C) and BHA (butylated hydroxyanisole), noticeably enhanced the activity of the sesquiterpenoids against P. ovale, but had no effect against C. albicans and S. cerevisiae.     

Establishment of serum based medium essentially free of proteolytic activity for the culture of mouse pancreatic islets

Summary The present study demonstrates that a) serum based culture medium degrades¹²⁵I insulin; b) heat in-activation of serum (1 h, 56°C) inhibits its proteolytic activity leading to the recovery of more insulin secreted by islets cultured in the presence of high glucose concentration alone or with glucagon; c) aprotinin also favoured the accumulation of secreted insulin by protecting the hormone from a residual degradative capacity of the heat treated serum.This work was supported by a grant (No. 71 5 426-2) from the INSERM and by the CNRS.These results have been presented at the V International Congress of Endocrinology. Hamburg, July 18–24, 1976.Acknowledgments. We should like to thank Mrs E. Gammelgard and K. Christensen for their technical assistance.     

Oyster mushroom (Pleurotus ostreatus) reduces the activity of 3-hydroxy-3-methylglutaryl CoA reductase in rat liver microsomes

The effect of dried oyster mushroom (Pleurotus ostreatus) on cholesterol (C) content in serum, in lipoproteins and in liver, and on the activity of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase in liver microsomes, was studied in male rats (strain Wistar, initial body weight 75 g) fed on low-cholesterol (9 mg/100 g) and high-cholesterol (0.3%) diets. Addition of 5% oyster mushroom to both diets reduced significantly the C-content in serum (by 30%), in very-low- and low-density lipoproteins (in a 11 ratio to the decrease of total serum C) and in liver (by 50%), as well as the activity of HMG-CoA reductase (by more than 30%).     

Binding of matrilysin-1 to human epithelial cells promotes its activity

Matrix metalloproteinase-7 (MMP-7, matrilysin- 1) modulates crucial biological events by processing many epithelial cell surface-associated effectors. We addressed MMP-7 interaction with human epithelial cells and its resulting activity. In human endometrium, a model of controlled tissue remodeling, proMMP-7 was diffusely immunolocalized inside epithelial cells, whereas MMP-7 delineated their entire plasma membrane. Endometrial explants preferentially retained active MMP-7, but not proMMP-7. Endometrial epithelial cells and carcinoma cells from various tissues bound active MMP-7. Endometrial carcinoma-derived Ishikawa cells showed high affinity (K_D of ~2.5 nM) and capacity (~260 000 sites per cell) for MMP-7. MMP-7 binding decreased by extracting membrane sterols or interfering with heparan sulfate proteoglycans, and was abrogated by tissue inhibitors of metalloproteinase-2 (TIMP-2) or synthetic MMP inhibitors. Bound MMP-7 not only remained fully active towards a macromolecular substrate but also became resistant to TIMP-2. We conclude that MMP-7-selective targeting to the plasma membrane of epithelial cells promotes its activity by conferring resistance to TIMP-2. A. Berton, C. Selvais: These authors contributed equally to this work. P. J. Courtoy, E. Marbaix, H. Emonard: These authors contributed equally to the supervision of this work. Received 20 September 2006; received after revision 30 November 2006; accepted 18 January 2007     

Phagocytic activity of the reticuloendothelial system in the rat after administration of an anticholinesterasic pesticide, carbaryl (author's transl)]

The effects of 4 carbaryl doses (0.375, 0.75, 1.50 and 3 mg/100 g) on the reticuloendothelial system (RES) phagocytic activity were studied 1 h after their administration to male rats. Carbaryl reduced RES phagocytic activity. Results showed a dose-dependent drop in RES phagocytic activity. Carbaryl might act as an inhibitor of phagocytes by saturing them to greater or lesser degree, depending on the dose administered.     

Superoxide anion scavenging effect and superoxide dismutase activity of Ginkgo biloba extract

Ginkgo biloba extract is known to be efficient in diseases associated with free radical generation. The purpose of this work was to study, under in vitro conditions, the action of Ginkgo biloba extract (Gbe) against superoxide anion (O2-.), which is directly or indirectly implicated in cell damage. Gbe appears to have both an O2-. scavenging effect and also a superoxide dismutase activity. Its antiradical effect was demonstrated by low temperature electron spin resonance and in a non-enzymatic system (phenazine methosulfate-NADH), and its enzymatic activity was shown by polarographic determination.