Evaluation of cell death caused by triterpene glycosides and phenolic substances from Cimicifuga racemosa extract in human MCF-7 breast cancer cells

We previously reported that the antiproliferative effect of an isopropanolic-aqueous extract of black cohosh (iCR) on MCF-7 estrogen-responsive breast cancer cell line was due to the induction of apoptosis. Here we address the question to what extent apoptosis induction can be ascribed to one of the two major fractions of iCR, the triterpene glycosides (TTG) or the cinnamic acid esters (CAE). Furthermore, as black cohosh is routinely administered orally, we studied whether its pharmacological effects would withstand simulated liver metabolism. The antiproliferative activity of TTG and CAE as well as of rat liver microsomal S9 fraction-pretreated iCR on MCF-7 cells were investigated by WST-1 assay. The features of cell death induced were tested for apoptosis by flow cytometry (light scatter characteristics, Annexin V binding). Irrespective of S9-pretreatment, 72 h iCR treatment induced a dose-dependent down regulation of cell proliferation with the same IC50 of 55.3 microg/ml dry residue which corresponds to 19.3 microg/ml TTG and 2.7 microg/ml CAE. The degree of apoptotic MCF-7 cells was also comparable. Both, isolated TTG and CAE fractions inhibited cell growth, the IC50 being 59.3 microg/ml and 26.1 microg/ml, respectively. Interestingly, whereas IC50 and apoptosis induction correspond well for the whole extract, TTG and CAE fractions induced apoptosis at concentrations (25 and 5 microg/ml) well below those required for significant growth inhibition. Observation of this study firstly showed that the cell death induced by iCR withstood a metabolic activation system. In addition, TTG and CAE compounds significantly contributed to its apoptotic effect, CAE being the more potent inhibitor of proliferation and apoptosis inducer.     

Anticancer effect of a new benzophenanthridine isolated from Zanthoxylum madagascariense (Rutaceline)

Fractionation of the cyclohexane extract from the stem bark powder of Zanthoxylum madagascariense led to the isolation of a new benzophenanthridine-type alkaloid, hydrochloride of 2,3-methylendioxy-8-hydroxy- 7-methoxy-benzo[C]phenanthridine (Rutaceline), characterized on the basis of its spectral data. Rutaceline was evaluated for its antiproliferative capacity on the human colorectal adenocarcinoma (Caco-2) and the African green monkey kidney (Vero) cell lines. The 50% inhibition of cell growth (IC50) obtained after 24 h incubation was similar for both cells lines (110-115 microg/ml, i.e. 269-281 microM), but at 48 h the IC50 value for the Caco-2 cells was lower than for the Vero cells (20 microg/lml, i.e. 49 microM versus 90 microg/ml, i.e. 220 microM) indicating a higher cell growth inhibitory effect on the colon adenocarcinoma cells. At the respective IC50 concentrations, Rutaceline did not significantly induce apoptosis but induced cell cycle arrest in the GO/G1 phase, as well as a decrease of cells in S phase. Rutaceline also induced DNA fragmentation in both cell lines, as revealed by agarose gel electrophoresis, and a dose-dependent clastogenic effect in both cell lines as revealed by the Comet assay.     

Serum effect on cellular uptake of spermidine, spergualin, 15-deoxyspergualin, and their metabolites by L5178Y cells

Spergualin (SG) and 15-deoxyspergualin (DSG) were more slowly incorporated into L5178Y cells than spermidine. SG and DSG inhibited carrier-mediated transport of [3H]spermidine competitively with inhibition constants of 0.67 mM and 0.45 mM, respectively. Addition of calf serum stimulated uptake of [3H]spermidine into the cells in a serum concentration-dependent manner. The effect was not observed when horse serum was used in place of calf serum. Preincubation of spermidine in calf serum for 1 hour before addition to cells remarkably decreased cellular incorporation of tritium. Three amine oxidase inhibitors, aminoguanidine, 3-hydroxybenzyloxyamine, and semicarbazide, inhibited stimulation of uptake of [3H]spermidine by calf serum and the decrease of it by preincubation in calf serum. So we propose that cellular incorporation or binding of products generated by oxidation of spermidine by amine oxidase in calf serum was much faster than that of spermidine itself and they were unstable and transformed quickly to unincorporable or non-binding substances if cellular targets were not present. Effect of amine oxidase inhibitors on cytotoxic activity of SG and DSG were determined in low and high concentrations of calf serum. In the presence of 10% calf serum in the basal medium, cytotoxicity to L5178Y cells by SG and DSG was suppressed at high drug concentrations (above 10 micrograms/ml) and enhanced at low drug concentrations (below 2.5 micrograms/ml) by amine oxidase inhibitors. In the presence of 0.5% calf serum suppression of cytotoxicity at high drug concentrations by amine oxidase inhibitors was also observed, but enhancement at low drug concentrations was obscure. These data may suggest the existence of two kinds of cytotoxic mechanism of SG and DSG, one dependent on and one independent of amine oxidase in serum.     

Antiproliferative and antioxidant properties of an enzymatic hydrolysate from brown alga, Ecklonia cava.

The potential antiproliferative and antiradical activities of an enzymatic extract of Ecklonia cava together with its crude polysaccharide (CpoF) and crude polyphenolic fractions (CphF) were evaluated in vitro. Tested extracts showed strong selective cell proliferation inhibition on all cancer cell lines tested, especially CphF extract, containing high polyphenol amount, showed 5.1 microg/ml of IC(50) value on murine colon cancer (CT-26) cell line. According to the nuclear staining experiment, antiproliferative effect of CphF was associated with apoptotic cell demise in CT-26. In addition, The CphF at 5 microg/ml scavenged 70% of DPPH radical, which is much higher than those of BHA and BHT at same concentration. Further more CphF exhibited interesting antiradical properties, expressed by its capacity to scavenge superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radical (OH()). In reducing power assay, CphF extract at 5 microg/ml was found to be as high as that of BHT at same concentration. Also, in total antioxidant assay the effect of CphF at 50 microg/ml was equivalent or slightly higher than those of commercial counterparts at 5 microg/ml concentration. Taken together, the CphF may be a promising alternative to synthetic substances as natural compound with high antiproliferative and antiradical activity.     

Comparison of embryotoxicity of ESBO and phthalate esters using an in vitro battery system.

Epoxidized soy bean oil (ESBO) and phthalate esters have been used as a plasticizer in polyvinyl chloride products. In this study, the embryotoxicity of ESBO and phthalate esters, namely, diethyl hexyl phthalate (DEHP), butylbenzyl phthalate (BBP) and dibutyl phthalate (DBP) was evaluated using short-term in vitro battery system, such as the whole embryo, midbrain and limb bud culture systems. Whole embryos at gestation day 9.5 were cultured for 48 h and the morphological scoring was measured. The cytotoxic effect and differentiation for mid-brain (MB) and limb bud (LB) cell were assessed by 50% inhibition concentration (IC(50)) with neutral red uptake and hematoxylin-stained foci (MB) or Alcian Blue staining (LB), respectively. In the whole embryo culture assay, ESBO (83, 250 and 750 microg/ml) exerted no toxic effect on growth and development of the embryo, whereas phthalate esters (1, 10, 100 microg/ml for DEHP, 10, 100, 1,000 microg/ml for BBP and DBP) inhibited growth and development dose dependently. In mid-brain and limb bud culture, the IC(50) of differentiation and cytotoxicity in BBP was 412.24 and 231. 76 microg/ml for mid-brain, and 40.13 and 182.38 microg/ml for limb bud, respectively. The IC(50) of differentiation and cytotoxicity in DBP was 27.47 and 44.53 microg/ml for mid-brain, and 21.21 and 25.54 microg/ml for limb bud cells, respectively. The lower IC(50) in both cells was obtained from DBP when compared to BBP. From these results, limb bud cells responded more sensitively to BBP and DBP than mid-brain cells. The IC(50) of limb bud cell differentiation and cytotoxicity in DBP is 1.9 and 7.1 less than that of BBP. However, any alteration in cytotoxicity and differentiation was observed with ESBO treatment. These studies suggested that ESBO is not embryotoxic; however, DEHP, BBP and DBP exhibit embryotoxic potential at high concentration.     

Antiproliferative constituents in the plant 8. Seeds of Rhynchosia volubilis.

The MeOH extract of the seeds of Rhynchosia volubilis (Leguminosae) showed antiproliferative activity against human gastric adenocarcinoma [MK-1, 50% growth inhibition (GI50): 25 microg/ml], human uterus carcinoma (HeLa, GI50: 30 microg/ml), and murine melanoma (B16F10, GI50: 8 microg/ml) cells. Bioactivity-guided fractionation resulted in the isolation of gallic acid methylester (1), gallic acid (2), 7-O-galloylcatechin (3), 1,6-di-O-galloylglucose (4), 1-O-galloylglucose (5), and trigalloylgallic acid (6), and their antiproliferative activity was estimated. All showed much stronger inhibition against B16F10 cell growth than against HeLa and MK-1 cell growth. Compound 2 and its tetramer (6) with a free carboxyl group showed higher activity than those which did not have a free carboxyl group. In relation to the gallic acid tetramer (6), two gallic acid dimers (ellagic acid and dehydrodigallic acid) and trimers (tergallic acid dilactone and flavogallonic acid dilactone) were tested for their activity, and compared with those of the isolates.     

Antioxidant and free radical-scavenging activity of Choto-san and its related constituents

The antioxidant properties of Choto-san and its related constituents such as Chotoko and Choto-san without Chotoko, and phenolic compounds contained in Chotoko such as epicatechin, caffeic, acid and quercetin were evaluated. In the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay, the scavenging activity of Chotoko (IC(50) 14.3 microg/ml) was found to be higher than that of Choto-san (IC(50) 206.2 microg/ml) and Choto-san without Chotoko (IC(50) 244.3 microg/ml). Epicatechin (IC(50) 10.4 microM), caffeic acid (IC(50) 13.8 microM), and quercetin (IC(50) 7.1 microM) also revealed scavenging activity against DPPH radicals. Choto-san (IC(50) 67.7 microg/ml) exhibited stronger inhibitory activity against superoxide anion formation than Choto-san without Chotoko (IC(50) 92.4 microg/ml) but weaker activity than Chotoko (IC(50) 18.3 microg/ml). The generation of superoxide anion was also inhibited by epicatechin (IC(50) 175.2 microM), caffeic acid (IC(50) 141.7 microM), and quercetin (IC(50) 18.7 microM). In a hydroxyl radical-scavenging experiment, Choto-san (IC(50) 2.4 mg/ml), Chotoko (IC(50) 2.2 mg/ml), Choto-san without Chotoko (IC(50) 2.8 mg/ml), epicatechin (IC(50) 3.9 mM), caffeic acid (IC(50) 3.6 mM), and quercetin (IC(50) 1.9 mM) exhibited activity. In NG108-15 cells, when added simultaneously with H(2)O(2) (500 microM), Choto-san (250 microg/ml), Chotoko (250 microg/ml), Choto-san without Chotoko (500 microg/ml), epicatechin (200 microM), caffeic acid (200 microM), and quercetin (200 microM) effectively protected cells from oxidative damage. In conclusion, the present results provide evidence that Choto-san acts as an antioxidant and cytoprotective agent against oxidative damage, which is due at least partly to the phenolic compounds contained in Chotoko.     

Apoptosis inducing activity of viscin, a lipophilic extract from Viscum album L

Detection of antiproliferative activity and bioactivity-guided fractionation of viscin, a lipophilic extract from Viscum album L., led to the isolation of betulinic acid, oleanolic acid and ursolic acid as active components. Viscin, betulinic acid, oleanolic acid and ursolic acid inhibited growth and induced apoptotic cell death in Molt4, K562 and U937 leukaemia cells. The growth inhibitory effect of viscin was more pronounced in Molt4 and U937 cells (IC50 (concentration that inhibited cell proliferation by 50%): 118 +/- 24 and 138 +/- 24 microg mL(-1)) than in K562 cells (IC50: 252 +/- 37 microg mL(-1)). Oleanolic acid was the least effective in all cell lines (7.5-45.5% inhibition at 10 microg mL(-1)) and ursolic acid the most active in Molt4 and U937 cells (81.8 and 97.8% inhibition, respectively, at 5 microg mL(-1)). A dose-dependent loss of membrane phospholipid asymmetry associated with apoptosis was induced in all cell lines as shown in flow cytometry by the externalization of phosphatidylserine and morphological changes in cell size and granularity. There were differences in individual cell lines' response towards the apoptosis-inducing effect of viscin, betulinic acid, oleanolic acid and ursolic acid. The triterpenoids beta-amyrin, beta-amyrinacetate, lupeol, lupeolacetate, beta-sitosterol and stigmasterol, and the fatty acids oleic acid, linoleic acid, palmitic acid and stearic acid were also present in the lipophilic extract.     

Byssochlamysol,a new antitumor steroid against IGF-1-dependent cells from Byssochlamys nivea. I. Taxonomy,fermentation, isolation and biological activity

A new antitumor steroid, byssochlamysol, was isolated from the mycelium of Byssochlamys nivea M#5187. Byssochlamysol inhibited IGF-1-dependent growth of MCF-7 human breast cancer cells with an IC50 of 20 ng/ml, whereas serum-dependent cell growth was not inhibited by less than 10 microg/ml of byssochlamysol. This substance induced apoptosis in IGF-1-dependent Colo320DM human colon cancer cells.     

Structure-activity relationship of oleanane disaccharides isolated from Akebia quinata versus cytotoxicity against cancer cells and NO inhibition

In order to further determine the nature of structure-activity relationship on the cytotoxicities of saponins with 1-->2 and 1-->3 linkages of disaccharides, we isolated guaianin N, collinsonidin, kalopanaxsaponin A and hederoside D(2) as disaccharides, and patrinia glycoside B-II as a trisaccharide, from the n-BuOH extract of Akebia quinata (Lardizabalaceae). Complete acid hydrolysis of the extract afforded oleanolic acid (1) and hederagenin (2). By sulforhodamine B (SRB) assay, kalopanaxsaponin A containing an alpha-L-rhap-(1-->2)-alpha-L-arap moiety exhibited distinctly higher cytotoxicity (IC(50) 1.8-2.7 microg/ml) against all of the tested cell lines than the other saponins (IC(50), 4-8 microg/ml). These results suggest that the alpha-L-rhap-(1-->2)-alpha-L-arap moiety has a unique structural significance in terms of its cell biochemistry, compared to those oleanane glycosides with other sugar linkages. On the other hand, kalopanaxsaponin A exhibited a significant inhibitory effect on nitric oxide production by lipopolysaccharide (LPS)-activated macrophage 264.7, whereas other saponins had weaker activities.     

Inhibition of prostaglandin E2 and leukotriene C4 in mouse peritoneal macrophages and thromboxane B2 production in human platelets by flavonoids from Stachys chrysantha and Stachys candida

Seven flavonoids of Stachys chrysantha and Stachys candida have been isolated. The structures of the compounds were elucidated by spectroscopic methods, particularly highfield NMR spectroscopy. The effects of the methanol extracts of these two endemic Greek Stachys sp. and their main flavonoids were examined on arachidonic acid (AA) metabolism in the cellular system (mouse peritoneal macrophages and human platelets). Their cytotoxicity on cells was also investigated. Most samples assayed did not exhibit any significant effect on prostaglandin E2 (PGE2)-release from calcium ionophore-stimulated mouse peritoneal macrophages. Only chrysoeriol-7-O-beta-(3'-E-p-coumaroyl)-glucopyranoside, at the highest non-cytotoxic dose (50 microM), inhibited the release of PGE2, but this effect is not statistically significant. The release of leukotriene C4 (LTC4) by mouse peritoneal macrophages stimulated with calcium ionophore was inhibited by a crude extract of S. chrysantha, with an IC50 value of 34.3 microg/ml. Xanthomicrol (IC50 = 29.2 microM) and chrysoeriol-7-O-beta-D-(3'-E-p-coumaroyl)-glucopyranoside (IC50 = 11.1 microM) also inhibited the release of LTC4, although it showed less potency than the reference compound nordihydroguaiaretic acid (NDGA) (IC50 = 2 microM). However, most samples assayed showed a significant effect on thromboxane B2 (TXB2)-release from calcium ionophore-stimulated human platelets, with inhibition percentages slightly lower than the reference drug ibuprofen (IC50 = 7 microM). The IC50 values are: crude extract of S. candida 23.3 microg/ml; crude extract of S. chrysantha 23.1 microg/ml; xanthomicrol 28.8 microM; calcycopterin 2.66 microM and chrysoeriol-7-O-beta-D-(3'-E-p-coumaroyl)-glucopyranoside 8.8 microM. Our results indicate that the selective inhibition of TX-synthase enzyme may be the primary target of action of most of these samples, and one of the mechanisms through which thus exert their antiinflammatory effects.     

ICM0301s, new angiogenesis inhibitors from Aspergillus sp. F-1491. I. Taxonomy, fermentation, isolation and biological activities

In the course of screening program for inhibitors of angiogenesis, novel substances designated as ICM0301A approximately H (1 approximately 8) were isolated from the culture broth of Aspergillus sp. F-1491. ICM0301s inhibited the growth of human umbilical vein endothelial cells (HUVECs) induced by basic fibroblast growth factor (bFGF) with IC50 values of 2.2 approximately 9.3 microg/ml. ICM0301A (1) showed significant anti-angiogenic activity at lower than 10 microg/ml in the angiogenesis model using rat aorta cultured in fibrin gel. ICM0301s showed very low cytotoxicity against various tumor cells. Furthermore, 1CM0301A did not show any toxic symptom in mice by intraperitoneal injection at 100 mg/kg.     

Inhibitory effect of Salvia miltiorrhia BGE on matrix metalloproteinase-9 activity and migration of TNF-alpha-induced human aortic smooth muscle cells

The migration and matrix metalloproteinases (MMPs) production of vascular smooth muscle cells (VSMC) may play a key role in the development of atherosclerosis. The Radix of Salvia miltiorrhiza Bunge (Labiatae) (SM), an eminent herb, is often included as an ingredient in various herbal remedies recommended for vascular therapies and has been used to treat vascular diseases for many centuries. In this study, we investigated the inhibitory effect of SM on TNF-alpha induced human aortic smooth muscle cells (HASMC) migration and MMP-9 activity. Various extracts prepared from stems of SM were tested for cytotoxic activity on HASMC using the XTT assay method. The ethanol extract (IC50 > 100 microg/ml), water extract (IC50 > 100 microg/ml) and chloroform (IC50 = 90 microg/ml) fraction exhibited weak cytotoxic activity. However, buthanol (IC50 = 80 microg/ml) and ethyl acetate (EtOAc; IC50 = 70 microg/ml) fraction exhibited strongly cytotoxic activity. Also, the extracts and fractions were investigated the inhibitory effects on MMP-9 activity using gelatin zymography. Gelatin zymography showed that the TNF-alpha-treated HASMC secreted MMP, probably including MMP-9, which may be involved in HASMC migration. The EtOAc fraction showed stronger inhibitory effect of proteolytic activity than other fractions. The EtOAc fraction was able to decrease the proteolytic activity of MMP-9 in a concentration-dependent manner on zymography. The Matrigel migration assay showed that SM effectively inhibited the TNF-alpha induced migration of HASMC as compared with the control group in a dose-dependent manner (IC50 = 65 microg/ml) and that the EtOAc fraction effectively inhibited the migration of HASMC, as compared with the control group in a dose-dependent manner. These results suggest that SM could be used as potential anti-atherosclerotic agent for anti-migration in TNF-alpha treated HASMC.     

Anti-tumor effect of Coriolus versicolor methanol extract against mouse B16 melanoma cells: in vitro and in vivo study.

Numerous studies have shown immunostimulatory and anti-tumor effects of water and standardized aqueous ethanol extracts derived from the medicinal mushroom, Coriolus versicolor, but the biological activity of methanol extracts has not been examined so far. In the present study we investigated the anti-tumor effect of C. versicolor methanol extract (which contains terpenoids and polyphenols) on B16 mouse melanoma cells both in vitro and in vivo.In vitro treatment of the cells with the methanol extract (25-1600 microg/ml) reduced melanoma cell viability in a dose-dependent manner. Furthermore, in the presence of the methanol extract (200 microg/ml, concentration IC(50)) the proliferation of B16 cells was arrested in the G(0)/G(1) phase of the cell cycle, followed by both apoptotic and secondary necrotic cell death. In vivo methanol extract treatment (i.p. 50 mg/kg, for 14 days) inhibited tumor growth in C57BL/6 mice inoculated with syngeneic B16 tumor cells. Moreover, peritoneal macrophages collected 21 days after tumor implantation from methanol extract-treated animals exerted stronger tumoristatic activity ex vivo than macrophages from control melanoma-bearing mice. Taken together, our results demonstrate that C. versicolor methanol extract exerts pronounced anti-melanoma activity, both directly through antiproliferative and cytotoxic effects on tumor cells and indirectly through promotion of macrophage anti-tumor activity.     

Synthesis, antiprotozoal and cytotoxic activities of new N-(3,4-dimethyl-5-isoxazolyl)-1,2-naphthoquinone-4-amino derivatives

Three derivatives of N-(3,4-dimethyl-5-isoxazolyl)-1,2-naphthoquinone-4-amino (1), a compound which exhibits significant activity against Trypanosoma cruzi and Plasmodium falciparum but with cytotoxicity toward murine L-6 cells, were synthesized with the aim of ameliorating its cytotoxicity. The in vitro antiprotozoal and cytotoxic activities of the synthesized compounds were evaluated against T. cruzi, Trypanosoma brucei rhodesiense, P. falciparum and murine L-6 cells. The hydroxymethyl (2) and the oxime (3) derivatives were active against T. cruzi, with IC50 values in a range comparable to those of 1 (IC50: 0.65 microg/ml) and benznidazole (IC50: 0.56 microg/ml) while the carboxymethyloxime (4) was inactive. Compounds 2 and 3 were cytotoxic toward L-6 cells, with IC50 values identical to that of 1 (IC50: 0.50 microg/ml). The results did not support the suggestion that 2 and 3 may be used as prodrugs of 1.     

Inhibitory effect on replicative DNA polymerases, human cancer cell proliferation, and in vivo anti-tumor activity by glycolipids from spinach

We succeeded in purifying a major glycolipids fraction (i.e., Fraction-II) in the class of monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG) from spinach using hydrophobic column chromatography. Fraction-II inhibited the activities of replicative DNA polymerases (pols) such as alpha, delta and epsilon, and mitochondrial pol gamma with IC(50) values of 43-79 microg/ml, but had no influence on the activity of repair-related pols beta and lambda. MGDG, DGDG, SQDG were purified from Fraction-II of spinach using silica gel column chromatography, and SQDG was the strongest inhibitor of mammalian pols in the three glycolipids. Therefore, SQDG and its related compounds were chemically synthesized, and the sulfate group and fatty acid moiety of the compound were suggested to be important for pol inhibition. These glycolipids showed no effect even on the activities of plant pols, prokaryotic pols and other DNA metabolic enzymes such as T4 polynucleotide kinase, T7 RNA polymerase and deoxyribonuclease I. Fraction-II also inhibited the proliferation of human cervix carcinoma (HeLa) cells with LD(50) values of 57 microg/ml, and could halt the cell cycle at the G1-phase, and subsequently induced severe apoptosis. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, Fraction-II was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that tumor necrosis with hemorrhage was significantly enhanced with Fraction-II in vivo. The spinach Fraction-II containing SQDG might be a potent anti-tumor compound, and may be a healthy food substance with anti-tumor activity.     

Endothelium-dependent relaxation induced by cathepsin G in porcine pulmonary arteries

Serine proteinases elicit profound cellular effects in various tissues mediated by activation of proteinase-activated receptors (PAR). In the present study, we investigated the vascular effects of cathepsin G, a serine proteinase that is present in the azurophil granules of leukocytes and is known to activate several cells that express PARs. In prostaglandin F2alpha (3 microM)-precontracted rings from porcine pulmonary arteries with intact endothelium, cathepsin G caused concentration-dependent relaxant responses (pEC(50)=9.64+/-0.12). The endothelium-dependent relaxant effect of cathepsin G could also be demonstrated in porcine coronary arteries (pEC(50)=9.23+/-0.07). In pulmonary arteries the cathepsin G-induced relaxation was inhibited after blockade of nitric oxide synthesis by L-NAME (200 microM) and was absent in endothelium-denuded vessels. Bradykinin- and cathepsin G-induced relaxant effects were associated with a 5.7 fold and 2.4 fold increase in the concentration of cyclic GMP, respectively. Compared with thrombin and trypsin, which also produced an endothelium-dependent relaxation in pulmonary arteries, cathepsin G was 2.5 and four times more potent, respectively. Cathepsin G caused only small homologous desensitization. In cathepsin G-challenged vessels, thrombin was still able to elicit a relaxant effect. The effects of cathepsin G were blocked by soybean trypsin inhibitor (IC(50)=0.043 microg ml(-1)), suggesting that proteolytic activity is essential for induction of relaxation. Recombinant acetyl-eglin C proved to be a potent inhibitor (IC(50)=0.14 microg ml(-1)) of the cathepsin G effect, whereas neither indomethacin (3 microM) nor the thrombin inhibitor hirudin (5 ATU ml(-1)) elicited any inhibitory activity. Due to their polyanionic structure defibrotide (IC(50)=0.11 microg ml(-1)), heparin (IC(50)=0.48 microg ml(-1)) and suramin (IC(50)=1.85 microg ml(-1)) diminished significantly the relaxation in response to the basic protein cathepsin G. In conclusion, like thrombin and trypsin, cathepsin G is able to induce endothelium-dependent vascular relaxation. It can be released from activated leukocytes at sites of vascular injury and inflammation and, therefore, sufficiently high concentrations might be reached locally in the vascular space to induce vasodilatation.     

An in vitro study of inhibitory activity of gossypol, a cottonseed extract, in human carcinoma cell lines.

Gossypol, a cottonseed extract, has been shown to have antiproliferative activity in a variety of cancer cell lines. The objective of this study was to determine the inhibitory effects of gossypol on cell proliferation. Five human carcinoma cell lines were evaluated including endometrial (RL95-2), ovarian (SKOV-3), medullary thyroid (TT), and adrenocortical (NCI-H295R and SW-13). Gossypol and the metabolite, apogossypol hexaacetate, were examined at concentrations up to 500 microg ml(-1) and the IC(50) was determined using the MTT assay. Gossypol and apogossypol hexaacetate produced a dose-dependent growth inhibition in all cellular lines examined. The IC(50) for gossypol ranged from 1.3 to 18.9 microM while the IC(50) for apogossypol hexaacetate ranged from 5.2 to 9.0 microM. The results indicate that gossypol possesses antiproliferative action toward human carcinoma cells in vitro. These investigations suggest that gossypol may have therapeutic potential for the treatment of cancer.     

Antimalarial activity of crambescidin 800 and synthetic analogues against liver and blood stage of Plasmodium sp

Structural features associated with the antimalarial activity of the marine natural product crambescidin 800 were studied using synthetic analogues of the related compound ptilomycalin A. The study suggests that the guanidine moiety is cytotoxic, whereas the spermidine-containing aliphatic chain increases activity. The most active analogue, compound 11, had in vitro activity against Plasmodium falciparum strain 3D7 (IC50=490 nM) that was stronger than the in vitro activity against murine L5178Y cells (IC50 = 8.5-59 microM). In vitro growth inhibition of liver stages of P. yoelii yoelii in mouse hepatocytes was observed (IC50 = 9.2 microM). The compound did not significantly prolong median survival time after a single subcutaneous administration of 80 mg/kg in P. berghei-infected mice. Compound 11 did not cause DNA fragmentation in an in vitro micronucleus assay.