The inhibition of phosphatidylinositol 3-kinase by quercetin and analogs.

Phosphatidylinositol (PtdIns) 3-kinase is an enzyme involved in cellular responses to growth factors. Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyrano-4-one), a naturally occuring bioflavinoid, was found to inhibit PtdIns 3-kinase with an IC50 of 1.3 micrograms/ml (3.8 microM); inhibition appears to be directed towards the ATP binding site of the kinase. Analogs of quercetin were also investigated as PtdIns 3-kinase inhibitors, with the most potent compounds exhibiting IC50's in the range of 1.7-8.4 micrograms/ml (5-19 microM). In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 greater than 30 micrograms/ml). These findings suggest that flavinoids may serve as potent inhibitors of PtdIns 3-kinase. Furthermore, the enzyme is much more sensitive to substituents at the 3-position of the flavinoid ring than are other protein and PtdIns kinases, suggesting that specific inhibitors of PtdIns 3-kinase can be developed to explore the biological role of the enzyme in cellular proliferation and growth factor response.     

Structural and functional uncoupling of the enzymatic and angiogenic inhibitory activities of tissue inhibitor of metalloproteinase-2 (TIMP-2): loop 6 is a novel angiogenesis inhibitor

Tissue inhibitors of metalloproteinases (TIMPs) regulate tumor growth, progression, and angiogenesis in a variety of experimental cancer models and in human malignancies. Results from numerous studies have revealed important differences between TIMP family members in their ability to inhibit angiogenic processes in vitro and angiogenesis in vivo despite their universal ability to inhibit matrix metalloproteinase (MMP) activity. To address these differences, a series of structure-function studies were conducted to identify and to characterize the anti-angiogenic domains of TIMP-2, the endogenous MMP inhibitor that uniquely inhibits capillary endothelial cell (EC) proliferation as well as angiogenesis in vivo. We demonstrate that the COOH-terminal domain of TIMP-2 (T2C) inhibits the proliferation of capillary EC at molar concentrations comparable with those previously reported for intact TIMP-2, while the NH2-terminal domain (T2N), which inhibits MMP activity, has no significant anti-proliferative effect. Interestingly, although both T2N and T2C inhibited embryonic angiogenesis, only T2C resulted in the potent inhibition of angiogenesis driven by the exogenous addition of angiogenic mitogen, suggesting that MMP inhibition alone may not be sufficient to inhibit the aggressive neovascularization characteristic of aberrant angiogenesis. We further mapped the anti-proliferative activity of T2C to a 24-amino acid peptide corresponding to Loop 6 of TIMP-2 and show that Loop 6 is a potent inhibitor of both embryonic and mitogen-stimulated angiogenesis in vivo. These findings demonstrate that TIMP-2 possesses two distinct types of anti-angiogenic activities which can be uncoupled from each other, the first represented by its MMP-dependent inhibitory activity which can inhibit only embryonic neovascularization and the second represented by an MMP-independent activity which inhibits both normal angiogenesis and mitogen-driven angiogenesis in vivo. In addition, we report, for the first time, the discovery of Loop 6 as a novel and potent inhibitor of angiogenesis.     

Screening of crude drug extracts for prolyl endopeptidase inhibitory activity.

Prolyl endopeptidase (PEP, EC 3.4.21.26) is an enzyme to play a role in metabolism of proline-containing neuropeptides, such as vasopressin, substance P and thyrotropin-releasing hormone (TRH), which were suggested to be involved with learning and memory processes. Then, specific inhibitor of PEP is expected to have antiamnesic effects, and thus we screened forty-six water- and methanol-extracts from crude drugs selected on the basis of traditional Chinese medicine theory, for Flavobacterium prolyl endopeptidase inhibition. Among them, the water-extracts of Rhodiola sacra (IC50, 0.77 microgram/ml) and the methanol-extracts of Lycopodium clavatum (IC50, 1.3 micrograms/ml), Paeonia lactiflora var. trichocarpa (IC50, 5.7 micrograms/ml), Paeonia veitchii (IC50, 2.4 micrograms/ml) and Rhodiola sacra (IC50, 0.67 microgram/ml) showed strong inhibitory activity. In addition, we also examined the PEP inhibitory activity of eleven compounds from Salvia deserta, and found that in addition to a catechol group alpha-hydroxy-para-quinone group may be related to the PEP inhibition.     

A metalloproteinase inhibitor as an inhibitor of neovascularization.

Metalloproteinases and their endogenous inhibitors are key components of an enzyme system which is important in a number of fundamental biochemical and cellular processes. Our recent work has focused on the role of a particular metalloproteinase, collagenase, and the role of an endogenous inhibitor of this enzyme in the control of neovascularization. The proteolytic degradation of extracellular matrix components by capillary endothelial cells (EC) has been shown to be one of the key prerequisites of the angiogenic process. As part of a study of the effect(s) of the inhibition of collagenase on neovascularization, we have recently reported the purification, characterization and partial NH2-terminal sequence of a cartilage-derived inhibitor (CDI) of angiogenesis in vivo and in vitro. Evidence is presented which suggests that one means of controlling deregulated vascular growth characteristic of a number of "angiogenic diseases" may be at the level of the control of metalloproteinase activity.     

Endothelial proliferation, migration, and differentiation are blunted by conditionally expressed protein kinase C pseudosubstrate peptides

Peptides based on the pseudosubstrate (PS) sequence of conventional protein kinase C isoenzymes (alpha, beta, gamma) specifically inhibit PKC activity in permeabilized cells, but whether PS can be used to study the role of PKC in the proliferation or migration of intact endothelial cells (EC) and angiogenesis is unknown. Peptides based on the PKCeta pseudosubstrate (etaPS) sequence were 3.5- to 8-fold more potent in inhibiting the PKCalpha, delta, epsilon, or eta kinase activity than was the peptide based on the PKCalpha pseudosubstrate (alphaPS) sequence. Thus, etaPS was conditionally overexpressed in intact EC and compared to alphaPS. Serum-induced growth of EC expressing etaPS was significantly slower than that of control EC. etaPS EC demonstrated slower rate of serum stimulated migration than that of either control or alphaPS EC. Expression of either etaPS or alphaPS produced slower rates of PMA induced EC migration, as compared to control EC. In an in vitro three-dimensional assay in which EC organize into capillary tubules, the EC that expressed etaPS formed fewer such tubules. This study shows that pseudosubstrate inhibitors derived from PKCeta are more potent both in vitro and in vivo than one based on the conventional isoenzyme PKCalpha. These data further support a role for PKC in proliferation and migration of intact EC, and angiogenesis.     

Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2-/-) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2+/+ and bcl-2-/- mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bcl-2-/- retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2-/- mice compared with bcl-2+/+ mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2-/- retinal EC. Mechanistically, bcl-2-/- cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from bcl-2-/- mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis.     

Antioxidant activity of brahma rasayana

Free oxygen radical scavenging activity of brahma rasayana (BR) was studied by in vitro and in vivo models. Addition of aqueous extract of BR was found to scavenge the lipid peroxides already present in rat liver homogenate (IC50 700 micrograms/ml) and inhibit the lipid peroxide generated by Fe(2+)-ascorbate (IC50 2600 micrograms/ml) and Fe(3+)-ADP-ascorbate system (IC50 1200 micrograms/ml). BR was found to scavenge the hydroxyl radical generated by Fenton reaction (IC50 7400 micrograms/ml) and superoxide generated by photoreduction of riboflavin (IC50 180 micrograms/ml). BR was also found to inhibit the nitric oxide radical generated in vitro from sodium nitroprusside (IC50 5.5 micrograms/ml). Oral administration of BR (50 mg/dose/animal) was found to inhibit the PMA induced superoxide generation in mice peritoneal macrophages. Oral administration of BR; 10 and 50 mg/dose/animal was also found to inhibit the nitrite production in peritoneal macrophages and percentage inhibition was 25.2% and 37.8% respectively. These results indicate significant antioxidant activity of BR in vitro and in vivo.     

The discovery of a potent and selective lethal factor inhibitor for adjunct therapy of anthrax infection

A potent and selective anthrax LF inhibitor 40, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, was identified through SAR study of a high throughput screen lead. It has an IC50 of 54 nM in the enzyme assay and an IC50 of 210 nM in the macrophage cytotoxicity assay. Compound 40 is also effective in vivo in several animal model studies.     

Inhibition of alpha-fetoprotein synthesis in hepatocytes of adult mice by dextran sulfate in vivo and in vitro

Embryospecific serum protein alpha-fetoprotein (AFP) is known to be synthesized in the adult liver only during regeneration and development of hepatocellular carcinomas. It was shown that collagenase digestion of hepatic tissue followed by monolayer cell cultivation was a powerful inducer of AFP synthesis, more potent than the liver regeneration in vivo. The treatment of hepatocytes in culture with 50-100 micrograms/ml of dextran sulphate caused a remarkable inhibition of cell proliferation, formation of cord-like multicellular structures and reduction of AFP synthesis. Mouse liver regeneration after CCL4 poisoning was accompanied by a 1000-fold increase in blood AFP levels. Blood AFP levels and the content of AFP-positive cells in the liver tissue were maximum on the 3rd-4th day after poisoning. Injections of 50 micrograms of dextran sulphate per g body weight 3-5 h after poisoning and 24 and 48 h later caused nearly tenfold reduction in AFP blood level and a decrease in the content of AFP-positive cells in the liver on the 3rd day of regeneration.     

Equilibrium Binding Analysis of Neural Cell Adhesion Molecule Binding to Heparin

The kinetics of neural cell adhesion molecule (NCAM) binding to heparin were studied in a heparin-Sepharose-based solid-phase binding assay. The observed binding is time dependent and saturable. A binding constant of 5.2 +/- 1.4 X 10(-8) M is observed for binding of newborn rat NCAM to heparin. This is approximately 25 times lower than the binding constant determined for newborn rat NCAM homophilic binding. Both Scatchard and Hill plot analyses suggest the presence of only one binding site. Fab' fragments of antibodies to rat NCAM significantly inhibit binding, a result indicating that a specific site on NCAM is involved in binding to heparin. The binding is inhibited by heparin (IC50, approximately 5 micrograms/ml), whereas chondroitin sulfate is a less potent inhibitor (IC50, approximately 15 micrograms/ml).     

Effects of retinol and dexamethasone on cytokine-mediated control of metalloproteinases and their inhibitors by human articular chondrocytes and synovial cells in culture

Human articular chondrocytes in culture produced large amounts of specific mammalian collagenase, gelatinase and proteoglycanase when exposed to dialysed supernatant medium derived from cultured human blood mononuclear cells (mononuclear cell factor) or to conditioned medium, partially purified by fractionation with ammonium sulphate (60–90% fraction), from cultures of human synovial tissue (synovial factor). Human chondrocytes and synovial cells also released into culture medium an inhibitor of collagenase of apparent molecular weight about 30 000, which appeared to be similar to the tissue inhibitor of metalloproteinases synthesised by tissues in culture. The amounts of free collagenase inhibitor were reduced in culture media from chondrocytes or synovial cells exposed to mononuclear cell factor or synovial factor. While retinol inhibited the production of collagenase brought about by mononuclear cell factor or synovial factor, it restored the levels of inhibitor, which were reduced in the presence of mononuclear cell factor or synovial factor. Dexamethasone markedly reduced the production of collagenase by synovial cells, while only partially inhibiting factor-stimulated collagenase production by chondrocytes. Addition of puromycin as an inhibitor of protein synthesis reduced the amounts of both collagenase and inhibitor to control or undetectable levels.     

Interleukin-18 acts as an angiogenesis and tumor suppressor.

Interleukin-18 (IL-18), also called interferon-gamma (IFN-gamma)-inducing factor, has recently been characterized as a potent IFN-gamma-inducing cytokine. We now report that IL-18 is a novel antiangiogenic and antitumor cytokine. In vitro, IL-18 specifically inhibits fibroblast growth factor-2-stimulated proliferation of capillary endothelial cells. In vivo, IL-18 is sufficiently potent to suppress the fibroblast growth factor-induced corneal neovascularization by systemic administration in mice. This cytokine also inhibits embryonic angiogenesis in the chick chorioallantoic membrane assay. Systemic and intralesional administrations of IL-18 produce a significant suppression of the growth of murine T241 fibrosarcoma in syngeneic C57Bl6/J and immunodeficient SCID mice. The antitumor effect appears to be potent because an average of >75% inhibition of primary tumor growth was observed at a dose of 50 microg/kg/day. In cell culture, murine T241 fibrosarcoma cells are insensitive to recombinant IL-18 at concentrations that significantly inhibit endothelial cell proliferation. Immunohistochemical studies of tumor tissues reveal hypovascularization of the IL-18-treated tumors. These results suggest that IL-18 may participate in the regulation of a switch of tumor angiogenesis.-Cao, R., Farnebo, J., Kurimoto, M., Cao, Y. Interleukin-18 acts as an angiogenesis and tumor suppressor.     

Inhibition of angiogenesis in vitro and in ovo with an inhibitor of cellular protein kinases, MDL 27032.

Protein kinase C (PKC) was implicated as an important positive regulator of angio-genesis by studies showing that tumor promoting phorbol esters, which activate PKC, stimulate angiogenesis both in vitro and in vivo. Therefore, inhibitors of PKC might be expected to block angiogenesis. MDL 27032 [4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone], an inhibitor of cellular protein kinases, prevented capillary-like tube formation by human umbilical vein endothelial cells (HUVEC) on basement membrane preparations, an in vitro model for angiogenic activity. MDL 27032 had an IC50 = 50 microM, whereas MDL 27044, the 4-methyl analog of MDL 27032, was less effective (IC50 greater than 100 microM). This selectivity was reflected in the relative abilities of the two compounds to inhibit PKC and protein kinase A (PKA) activity prepared from HUVEC, and also to inhibit the basic fibroblast growth factor stimulated proliferation of HUVEC. MDL 27032 (0.3 microgram/egg) also significantly inhibited neovascularization in yolk sac membranes of developing chick embryos, whereas MDL 27044 added at concentrations up to 3 micrograms/egg was not inhibitory when compared with vehicle treated controls. Adhesion of HUVEC to individual extracellular matrix proteins, including laminin, fibronectin, and fibrinogen, but not to the mixture of matrix components or collagen type I and IV, was inhibited after treatment with MDL 27032. These studies suggest that MDL 27032, may have potential as an anti-angiogenic agent because it disrupts both formation of tube-like structures by HUVEC on Matrigel and normal neovascularization in ovo. This inhibition may in part be due to altered cellular interactions with the extracellular matrix.     

Inhibitory effect of mitoxantrone on activity of protein kinase C and growth of HL60 cells.

Mitoxantrone, a new anthraquinone, showed inhibitory an effect on protein kinase C (PKC) activity. Its IC50 value was 4.4 micrograms/ml (8.5 microM), which is much lower than those of the well-known anthracyclines daunorubicin and doxorubicin, the IC50 values of which are more than 100 micrograms/ml (> 170 microM). Kinetic studies demonstrated that mitoxantrone inhibited PKC in a competitive manner with respect to histone H1, and its Ki value was 6.3 microM (Ki values of daunorubicin and doxorubicin were 0.89 and 0.15 mM, respectively), and in a non-competitive manner with respect to phosphatidylserine and ATP. Inhibition of phosphorylation by mitoxantrone was observed with various substrates including S6 peptide, myelin basic protein and its peptide substrate derived from the amino-terminal region. Their IC50 values were 0.49 microgram/ml (0.95 microM), 1.8 micrograms/ml (3.5 microM), and 0.82 microgram/ml (1.6 microM), respectively. Mitoxantrone did not markedly inhibit the activity of cyclic AMP-dependent protein kinase, casein kinase I or casein kinase II, at concentrations of less than 10 micrograms/ml. On the other hand, brief exposure (5 min) of HL60 cells to mitoxantrone caused the inhibition of cell growth with an IC50 value of 52 ng/ml (0.1 microM). In HL60 cells, most of the PKC activity (about 90%) was detected in the cytosolic fraction. When HL60 cells exposed to 10 micrograms/ml mitoxantrone for 5 min were observed with fluorescence microscopy, the fluorescence elicited from mitoxantrone was detected in the extranuclear area. These results indicated that mitoxantrone is a potent inhibitor of PKC, and this inhibition may be one of the mechanisms of antitumor activity of mitoxantrone.     

Antioxidant and anticarcinogenic activity of Lycovin--an indigenous herbal preparation

Aqueous extract of Lycovin has been found to be a potent inhibitor of lipid peroxide formation, (IC50 = 500 micrograms/ml) and scavenger of hydroxyl radical (IC50 = 44 micrograms/ml) and superoxide radical (IC50 = 30 micrograms/ml) in vitro. Lycovin syrup 1.5 ml and 7.5 ml/kg body wt administered orally, reduced the development of sarcoma induced by 20 MC by 35% and 70% respectively. Lycovin syrup was also found to inhibit the hepatocarcinogenesis induced by NDEA. The tumour incidence was 100% in the control group, while none of the drug treated animals developed tumour. Liver weight, gamma-glutamyl transpeptidase (GGT), GSH-S-transferase (GST), reduced glutathione, (GSH) and aniline-4-hydroxylase in liver were elevated in NDEA alone treated animals. The serum parameters indicative of liver injury such as bilirubin, lipid peroxides, alkaline phosphatase and glutamate pyruvate transaminase were also elevated by NDEA administration. These elevated parameters were significantly reduced in animals treated with Lycovin syrup along with NDEA in a dose dependent manner. Even though the exact mechanism of action is not known at present, the observed anticarcinogenic activity may be due to the inhibition of P.450 enzyme activity and subsequent inhibition of the production of the ultimate carcinogen as well as scavenging of oxygen free radicals during promotion of the transformed cell.     

Leishmania mexicana: enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals

A major difference between the metabolism of Leishmania species amastigotes and cultured promastigotes was found in the area of CO2 fixation and phosphoenolpyruvate metabolism. Malate dehydrogenase (EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were at much higher activities in amastigotes than promastigotes of both L. m. mexicana and L. donovani, whereas the reverse was true of pyruvate kinase (EC 2.7.1.40). Pyruvate carboxylase (EC 6.4.1.1) and malic enzyme (carboxylating) (EC 1.1.1.40) could not be detected in L. m. mexicana amastigotes. Promastigotes of L. m. mexicana had a high NAD-linked glutamate dehydrogenase activity in comparison to amastigotes, whereas NADP-linked glutamate dehydrogenase activity was detected only in amastigotes. Leishmania m. mexicana culture promastigotes were killed in vitro by the trivalent antimonial Triostam (LD50, 20 micrograms/ml) and the trivalent arsenical melarsen oxide (LD50, 20 micrograms/ml), but they were unaffected by Pentostam. Neither antimonial drug significantly inhibited leishmanial hexokinase (EC 2.7.1.2), phosphofructokinase (EC 2.7.1.11), pyruvate kinase, malate dehydrogenase or phosphoenolpyruvate carboxykinase, whereas melarsen oxide was a potent inhibitor of all the enzymes tested except phosphoenolpyruvate carboxykinase.     

Experimental studies on the pathogenesis of ochronotic arthropathy. The effects of homogentisic acid on adult and fetal articular chondrocyte morphology, proliferative capacity and synthesis of proteoglycans in vitro

Lapine adult and fetal articular chondrocytes in monolayer culture were employed as an experimental model for studying the effects of homogentisic acid on chondrocyte morphology, proliferation and synthesis of proteoglycans. Concentrations of homogentisic acid in the range from 0.1 to 100 micrograms/ml were investigated and a cytotoxic effect was detected with concentrations of 5 micrograms/ml and above. Thus, with adult articular chondrocytes this effect was seen between 36 and 48 h of subculture with 5 micrograms/ml or more of homogentisic acid present from the beginning of subculture. In fetal articular chondrocyte cultures a concentration of 1 microgram/ml elicited a statistically significant reduction (13%) in cell proliferation without altering sulphated macromolecular synthesis. Experiments with 3H-thymidine autoradiography to measure the pulse labeling index (LI) in fetal chondrocytes in vitro showed that the 5 micrograms/ml concentration of homogentisic acid present for 21 h from the beginning of subculture gave a LI of 2%, compared with 25% for controls. Thus, homogentisic acid can reduce chondrocyte proliferative capacity before morphological alterations can be detected by light microscopy. No significant alterations in the synthesis of proteoglycans were detected at concentrations below the cytotoxic level. The homogentisic acid-induced cytotoxicity and reduction of proliferation in chondrocytes represents a possible pathway by which cartilage is damaged in ochronotic arthropathy.     

Insulin stimulates secretion of a collagenase inhibitor by Swarm rat chondrosarcoma chondrocytes

Swarm rat chondrosarcoma chondrocytes produce an inhibitor of collagenase similar to that found in bovine articular chondrocytes and extracts of bovine scapular cartilage. These cells synthesize normal levels of cartilage type proteoglycans when cultured in serum free medium with insulin. Collagen synthesis is also increased when insulin is added to chondrosarcoma chondrocytes. We have demonstrated that insulin stimulates collagenase inhibitor production by these chondrocytes. Enhancement of inhibitory activity occurs over the range of 10 to 1000 ng/ml. A 3.2 fold stimulation was observed at a concentration of 1 microgram/ml. There was a lag period of 24 to 48 hours before the insulin effect became evident. Latent or active collagenase was not detectable under these conditions. These results suggest that the hormone insulin controls the levels of collagen in this tumor by stimulating synthesis of collagen and inhibitors of collagenase.     

Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene

The effect of isoflavones on the growth of the human breast carcinoma cell lines, MDA-468 (estrogen receptor negative), and MCF-7 and MCF-7-D-40 (estrogen receptor positive), has been examined. Genistein is a potent inhibitor of the growth of each cell line (IC50 values from 6.5 to 12.0 micrograms/ml), whereas biochanin A and daidzein are weaker growth inhibitors (IC50 values from 20 to 34 micrograms/ml). The isoflavone beta-glucosides, genistin and daidzin, have little effect on growth (IC50 values greater than 100 micrograms/ml). The presence of the estrogen receptor is not required for the isoflavones to inhibit tumor cell growth (MDA-468 vs MCF-7 cells). In addition, the effects of genistein and biochanin A are not attenuated by overexpression of the multi-drug resistance gene product (MCF-7-D40 vs MCF-7 cells).