Anibal Guerrero

Effects and mode of action of 1,4-naphthoquinones isolated from Calceolaria sessilis on tumoral cells and Trypanosoma parasites

The naphthoquinones 2-hydroxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (CS-1), (-)-2,3,3-trimethyl-2-3-dihydronaphtho[2,3-b]furan-4,9-quinone (CS-3), and 2-acetoxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (CS-5) isolated from Calceolaria sessilis were tested against Trypanosoma cruzi epimastigotes, the TA3 tumor cell line and the methotrexate-resistant subline TA3-MTX-R. Naphthoquinone CS-3 was the most active; the 50% culture growth inhibition (I50) on T. cruzi (Tulahuén and LQ strain and DM28c clone) was at concentrations ranging from 2.1 to 5.2 mumolar. Also CS-3 inhibited TA3 and TA3-MTX-R culture growth with an I50 of 2.1 and 3.8 mumolar, respectively. Naphthoquinone CS-3 inhibited the respiration of the tumor cells by interfering with the electron transport at some point between NADH and ubiquinone. The respiration of T. cruzi was not inhibited by naphthoquinone CS-3. Naphthoquinone CS-3 produced a temporary increase of oxygen consumption in T. cruzi and tumor cells, suggesting the generation and participation of free radicals.

Inhibition of tumoral cell respiration and growth by nordihydroguaiaretic acid

The effects of nordihydroguaiaretic acid (NDGA), best known as an inhibitor of lipoxygenase activities, on the culture growth, oxygen consumption, ATP level, viability, and redox state of some electron carriers of intact TA3 and 786A ascites tumor cells have been studied. NDGA inhibited the respiration rate of these two tumor cell lines by preventing electron flow through the respiratory chain. Consequently, ATP levels, cell viability and culture growth rates were decreased. NDGA did not noticeably inhibit electron flow through both cytochrome oxidase and ubiquinone-cytochrome b-c1 complex. Also, the presence of NDGA changed to redox state of NAD(P)+ to a more reduced level, and the redox states of ubiquinone, cytochrome b and cytochromes c + c1 changed to a more oxidized level. These observations suggest that the electron transport in the tumor mitochondria was inhibited by NDGA at the NADH-dehydrogenase-ubiquinone level (energy-conserving site 1). As a consequence, mitochondrial ATP synthesis would be interrupted. This event could be related to the cytotoxic effect of NDGA.

Quinacrine and copper, compounds with anticonceptive and antineoplastic activity

Changes in the evolution of a malignant transplantable tumor in mice to whom quinacrine, copper and zinc were supplied in drinking water are reported. Male AJ mice were inoculated in the right thigh with 1,000,000 TA3 or TA3 MTXR tumoral cells. Three experiments were designed with different types of tumors and different schedules of quinacrine and cations administered in drinking water. The animals that received quinacrine or quinacrine plus copper in drinking water had significantly smaller tumors, and some groups had a high rate of complete tumor regression (up to 60%). Quinacrine and copper have synergistic antineoplastic activity. Zinc salts do not improve the antitumoral effect of quinacrine. The relevant fact of this experiment lies in the fact that a large number of women using IUDs with copper could occasionally be treated with quinacrine.

Effects of betamethasone, sulindac and quinacrine drugs on the inflammatory neoangiogenesis response induced by polyurethane sponge implanted in mouse

In this study, we showed the effect of the betamethasone, sulindac and quinacrine alone or combined, on the inflammatory angiogenesis promoted by polyurethane sponge on mice. The main finding reported here is that the formation of new blood vessels was strongly inhibited by low concentration of betamethasone, sulindac or quinacrine, whether alone or in combination. It is known that steroidal anti-inflammatory drugs inhibit the enzymes required for the production of prostaglandins through a nuclear glucocorticoid receptor (GR) mediated mechanism. This mechanism may occur in endothelial cells as well. Considering that activity of cyclo-oxigenases 1 and 2 is inhibited by sulindac, and that these enzymes are located in the stromal tissue, we propose that the anti-angiogenic effect of these agents may occur via inhibition of both COX isoforms. On the other hand, quinacrine inhibited PLA2 activity, and we propose here that the anti-angiogenic effect occurs via inhibition of the enzyme PLA2. The potentiated effect of the association of betamethasone, sulindac and quinacrine may have some therapeutic benefit in the control of pathological angiogenesis. Further studies are required to validate these propositions.

Effects of Steroidal and Non Steroidal Drugs on the Neovascularization Response Induced by Tumoral TA3 Supernatant on CAM from Chick Embryo

Angiogenesis, the development of new blood vessels from the existing vascular network, may result as a consequence of the increase or decrease of proangiogenic or antiangiogenic factors, respectively. The tumor itself could up-regulate the production of angiogenic factors. Recently, we established that the steroidal drug betamethasone in low concentration inhibit the neovascularization promoted by TA3 Ts on CAM of chick embryos. We describe here the effects of the non-steroidal drug ketoprofen, alone or in association with betamethasone, on the angiogenesis promoted by TA3 Ts on CAM. The main finding reported here is that the formation of new blood vessels is strongly inhibited by low concentrations of ketoprofen. The association of both drugs produced a synergistic effect, significantly decreasing tumoral supernatant angiogenesis. It is known that steroidal anti-inflammatory drugs inhibit the enzymes required for the production of prostaglandins through a nuclear GR mediated mechanism. This may operate as a general mechanism in endothelial cells as well. Considering that the induction of COX 1 and COX2 are inhibited by ketoprofen, and that these enzymes are located in the stromal compartment of the CAM, we propose that its antiangiogenic effect may occur via inhibition of the two COX isoforms. In fact, we found that ketoprofen induced apoptosis in both the stromal fibroblast and endothelial cells. The potentiated effect of the combination of betamethasone and ketoprofen may have some therapeutic projections in the control of pathological angiogenesis.

Effect of sulfated β-cyclodextrin, a water soluble cycloamylose, on the promotion and/or inhibition of angiogenesis

Previous studies have reported that sulfated β-cyclodextrin, a naturally occurring cycloamylose built up from six to eight glucopyranose units, when administered alone promotes angiogenesis, but administered with an angiostatic steroid inhibits angiogenesis in the cick embryo bioassay. In our experiments sulfated β-cyclodextrin has been shown to posses many properties unrelated to its classical functions in the promotion and inhibition of angiogenesis that were not previously described. We studied the angiogenic and angiostatic properties of β-cyclodextrin in a subcutaneosus plastic sponge model in mice. We realized two set of experiments. In each set mice were randomized into five groups (n= 5 mice). The first group was treated with sulfated β-cyclodextrin (200 ng), the second group was treated with sulfated β-cyclodextrin (2000 ng), the third group received unsubstituted β-cyclodextrin (2000 ng), the fourth group was treated with sulfated β-cyclodextrin (20 000 ng) and the last group was used as a control group. In all groups compounds were administered intraperitonally 4 days after subcutaneous implantation of a sterile polyvinyl sponge on day 0, controls were not treated. Cyclodextrin administered alone at low drug concentration (200 ng) promoted angiogenesis and increased the development of venules in the sponge matrix. However, cyclodextrin administered at high drug concentration (2000 and 20 000 ng) reduced the vessel index in the sponge and areas of microhemorraghes were observed. From our results we propose that β-cyclodextrin contains both a promoter and an inhibitor of angiogenesis and that the activation of both is drug concentration dependent.

Antiangiogenic effect of betamethasone on the chick cam stimulated by TA3 tumor supernatant

Tumor growth is the result of combined cell proliferation overwhelming cell death and neoangiogenesis. This report shows CAM angiogenesis promoted by TA3 tumor supernatant with or without low dosis of betamethasone (Minimal antiangiogenic concentration: beta-MAAC). Methylcellulose discs instilled with 10 microliters of beta-MAAC (0.08 microgram/ml), 10 microliters of tumor supernatant (TA3ts), 5 microliters beta-MAAC + 5 microliters TA3ts, and 10 microliters of PBS as control were implanted in host chick eggs. On day 12, the grafts were removed, photographed and fixed. Sections were stained in parallel, one and three with hematoxylin-eosin, and section two by the Tunel method. The number of vessels was evaluated in a microscopic field of the CAM (2250 micron 2). The results show that beta-MAAC produced a significant inhibition of neovascularization in comparison to that observed in controls (P < 0.0025; Student t-Test). Discs instilled with TA3ts produced an intense stimulation of angiogenesis in contrast, when discs were instilled with 5 microliters of beta-MAAC + 5 microliters of TA3ts the angiogenesis was significantly inhibited (P < 0.001). The results show that effective antiangiogenic doses of betamethasone are in the range of 10(-7) M, (probably a genomic mediated action) and that this effect of low concentration may have clinical applications.

Inhibitory effect of vanillin-like compounds on respiration and growth of adenocarcinoma TA3 and its multiresistant variant TA3-MTX-R

The effects of some imine and amine derivatives of vanillin on the respiration rate of mouse mammary adenocarcinoma TA3 line, its multiresistant variant TA3-MTX-R line and mouse hepatocytes, together with their respective mitochondrial fractions, are described. These derivatives inhibit respiration in both tumour cell lines more effectively than vanillin in the absence or presence of the uncoupler CCCP. Since both types of derivatives block the electron flow, mainly through the NADH-CoQ span, they behave as oxidative phosphorylation inhibitors. Thus, they prevent ATP synthesis and alter cellular processes requiring energy, which would lead to cellular death. Amine derivatives of vanillin present a similar effect on both tumour cell lines, being amine C the most efficient inhibitor. Moreover, mouse hepatocytes are about 4-fold less sensitive to amine C than tumour cells. These amine derivatives are better inhibitors than the corresponding imines; probably because they should interact better with the respiratory chain reaction site.

Microscopic and histochemical study of odontoclasts in physiologic resorption of teeth of the polyphyodont lizard, Liolaemus gravenhorsti

Using tartrate‐resistant acid phosphatase (TRAP), we examined the cytodifferentiation of odontoclast cells in resorbing areas of dental tissues during the replacement of teeth in a polyphyodont lizard, Liolaemus gravenhorsti. We also report, by means of Lectin‐HRP histochemistry, the distribution pattern of some specific sugar residues of TRAPase‐positive cells. For detection of TRAPase activity, the azo dye‐coupling technique was used. Lectin binding sites were demonstrated by means of specific HRP‐lectins. The process of tooth resorption was divided into four stages: 1) preresorption—the wall of the dental pulp is covered with an odontoblast layer, and no TRAP‐positive cells are in the dental pulp; 2) early resorption—TRAP‐positive multinucleate odontoclasts are present on the dental wall, but the rest of the pulp surface is still covered with an odontoblast layer; 3) later resorption—the entire surface of the pulp chamber is lined with multinucleate odontoclasts; and 4) final resorption—the tooth has been totally resorbed. Odontoclasts are usually detached from the resorbed surface, and show signs of degeneration. Of the six lectins used, PNA, ECA, and UEA‐1 bind to multinucleated but not mononuclear cells. All the remaining lectins, BS‐1, RCA120, and LTA showed no binding to any cells of the teeth. The significance of saccharidic moieties such as acetyl‐galactosamine, acetyl‐glucosamine, and fucose sugar residues is difficult to ascertain. Perhaps these oligosaccharides might be borne on molecules associated with odontoclastic resorption or associated with multinucleation of odontoclasts after attachment to the dentine surface. J. Morphol. 242:295–309, 1999.

Quinacrine: sclerosing agent of the utero-tubal junction in women, with anticarcinogenic actions in transplanted tumors in mice.

Quinacrine, an acridine derivative that was in widespread use as an anti‐malarial, has been shown to have both sclerosant and anticarcinogenic actions. The sclerosant action of quinacrine has been used to produce occlusion of Fallopian tube in both experimental animals and women, and several clinical studies are reviewed. Both actions of quinacrine are potentiated by steroidal and non‐steroidal antiprostaglandins as well as by ionic copper. Combinations of quinacrine with antiprostaglandin drugs, and also with copper, improved the efficacy of quinacrine when used for female sterilization and reduced side effects. A review of the experimental and epidemiological evidence suggests that quinacrine has no carcinogenic effects.