Cytotoxic potentiation of vinblastine and paclitaxel by L-canavanine in human cervical cancer and hepatocellular carcinoma cells.

BACKGROUND: The non-protein amino acid L-canavanine (L-CAV), found in several plants of the family Fabaceae is an antimetabolite which shows anticancer activity due to its ability to be incorporated into protein in the place of its analogue, L-arginine (L-ARG), leading to the alteration of the 3D conformation of newly synthesised proteins and usually a loss of their function. PURPOSE: In this study, the ability of L-CAV to potentiate the cytotoxicity of microtubule- targeting drugs used in the chemotherapy of cancer, vinblastine (VIN) and paclitaxel (PTX) was evaluated. MATERIAL AND METHODS: The following cancer cells grown in arginine-rich and arginine-free media were employed: HeLa, Hep G2 and SK-HEP-1. Drug combination experiment used a method based on the median-effect principle and mass-action law. RESULTS: We observed that L-CAV, which is hardly toxic alone, potentiated the cytotoxicity of VIN and PTX in HeLa and hepatocellular carcinoma cells. CONCLUSION: This is the first study showing the cytotoxic potentiation of microtubule-targeting drugs by L-CAV. The mechanism of synergy and animal studies need to be investigated further to see whether L-CAV might become an adjuvant in cancer treatment.

Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.

BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.

Interactions between phytochemical components of Sutherlandia frutescens and the antiretroviral, atazanavir in vitro: implications for absorption and metabolism.

PURPOSE: African traditional medicinal plants, such as Sutherlandia frutescens have the potential to interact pharmacokinetically with the protease inhibitor class of antiretrovirals, thereby impacting on their safety and efficacy. The effects of extracts and phytochemical components of Sutherlandia frutescens, on the in vitro absorption and metabolism of the protease inhibitor, atazanavir were thus investigated. METHODS: Aqueous and methanolic extracts of Sutherlandia frutescens were prepared by freeze-drying of hot water and methanol decoctions of Sutherlandia frutescens plant material respectively, whilst crude triterpenoid glycoside and flavonol glycoside fractions were isolated by solvent extraction and subsequent column chromatography. Atazanavir was quantitated in the absence or presence of these compounds as well as commercially available purported constituents of Sutherlandia frutescens, namely, L-canavanine, L-GABA and D-pinitol, after a one hour co-incubation in Caco-2 cell monolayers and human liver microsomes. RESULTS: The triterpenoid and flavonol glycoside fractions were found to be present in the aqueous and methanolic extracts of Sutherlandia frutescens and were shown to contain the sutherlandiosides and sutherlandins known to be present in Sutherlandia frutescens. The aqueous extract and D-pinitol significantly reduced atazanavir accumulation by Caco-2 cells, implying a decrease in atazanavir absorption, whilst the opposite was true for the triterpenoid glycoside fraction. Both the aqueous and methanolic extracts inhibited atazanavir metabolism in human liver microsomes, whilst enhanced atazanavir metabolism was exhibited by the triterpenoid glycoside fraction. CONCLUSIONS: The extracts and phytochemical components of Sutherlandia frutescens influenced the accumulation of atazanavir by Caco-2 cells and also affected ATV metabolism in human liver microsomes. These interactions may have important implications on the absorption and metabolism and thus the overall oral bioavailability of atazanavir.

Canavanine augments proapoptotic effects of arginine deprivation in cultured human cancer cells.

Arginine deprivation achieved by means of recombinant arginine-degrading enzymes is currently being developed as a novel anticancer enzymotherapy. In this study, we showed that arginine deprivation in vitro profoundly and selectively sensitized human cancer cells of different organ origin to low doses of canavanine, an arginine analogue of plant origin. In sensitive cancer cells arginine starvation led to the activation of caspase-9, caspase-3 and caspase-7, cleavage of reparation enzyme, polyADP ribosyl polymerase, and DNA fragmentation, which are the typical hallmarks of intrinsic apoptosis realized by the mitochondrial pathway. Co-administration of canavanine significantly accelerated and enhanced apoptotic manifestations induced by arginine deprivation. The augmentation of canavanine toxicity for cancer cells was observed when either a formulated arginine-free medium or complete medium supplemented with bovine arginase preparation was used. Cycloheximide efficiently rescued malignant cells from canavanine-induced cytotoxicity under arginine deprivation, suggesting that it results mainly from canavanine incorporation into newly synthesized proteins. Cancer cells sensitive or resistant to arginine deprivation alone were not capable of restoring their proliferation after 24 h of combined treatment, whereas pseudonormal cells retained such ability. Our data suggest that the incorporation of canavanine into anticancer treatment schemes based on artificially created arginine starvation could be a novel strategy in tumor enzymochemotherapy.

Impact of traditional medicinal plant extracts on antiretroviral drug absorption.

ETHNOPHARMACOLOGICAL RELEVANCE: [corrected] Traditional herbal medicines are often used for the treatment of different diseases in developing countries, especially in the rural areas where a lack of an efficient primary health care system is usually experienced. Many patients infected with the human immunodeficiency virus are taking traditional herbal medicines in conjunction with their modern antiretroviral medication and drug-herb interactions can occur in these cases. AIM OF THE STUDY: To investigate the effect of water extracts of two traditional medicinal plants, Hypoxis hemerocallidea and Sutherlandia frutescens as well as l-canavanine (a constituent of Sutherlandia frutescens) on the transport of nevirapine across human intestinal epithelial cells. MATERIALS AND METHODS: Nevirapine transport in the apical to basolateral and basolateral to apical directions across Caco-2 cell monolayers was determined alone (normal control) and in the presence of verapamil (positive control), water extracts of Hypoxis hemerocallidea and Sutherlandia frutescens and an aqueous solution of l-canavanine. The cumulative transport and apparent permeability coefficient (P(app)) values were calculated and compared. RESULTS: Nevirapine alone was substantially effluxed in the basolateral to apical direction across the intestinal epithelial cell monolayers, which was statistically significantly (p < or = 0.05) decreased by addition of verapamil, Hypoxis hemerocallidea extract and the l-canavinine solution. The effect of Sutherlandia frutescens on nevirapine transport was not statistically significantly different from the control. CONCLUSIONS: Hypoxis hemerocallidea and l-canavanine interact with the efflux of nevirapine across intestinal epithelial cells and therefore can potentially increase the bioavailability of this antiretroviral drug when taken concomitantly.

L-Canavanine made by Medicago sativa interferes with quorum sensing in Sinorhizobium meliloti.

Sinorhizobium meliloti is a gram-negative soil bacterium, capable of establishing a nitrogen-fixing symbiosis with its legume host, alfalfa (Medicago sativa). Quorum sensing plays a crucial role in this symbiosis, where it influences the nodulation process and the synthesis of the symbiotically important exopolysaccharide II (EPS II). S. meliloti has three quorum-sensing systems (Sin, Tra, and Mel) that use N-acyl homoserine lactones as their quorum-sensing signal molecule. Increasing evidence indicates that certain eukaryotic hosts involved in symbiotic or pathogenic relationships with gram-negative bacteria produce quorum-sensing-interfering (QSI) compounds that can cross-communicate with the bacterial quorum-sensing system. Our studies of alfalfa seed exudates suggested the presence of multiple signal molecules capable of interfering with quorum-sensing-regulated gene expression in different bacterial strains. In this work, we choose one of these QSI molecules (SWI) for further characterization. SWI inhibited violacein production, a phenotype that is regulated by quorum sensing in Chromobacterium violaceum. In addition, this signal molecule also inhibits the expression of the S. meliloti exp genes, responsible for the production of EPS II, a quorum-sensing-regulated phenotype. We identified this molecule as l-canavanine, an arginine analog, produced in large quantities by alfalfa and other legumes.

Evidence for an arginine exporter encoded by yggA (argO) that is regulated by the LysR-type transcriptional regulator ArgP in Escherichia coli.

The anonymous open reading frame yggA of Escherichia coli was identified in this study as a gene that is under the transcriptional control of argP (previously called iciA), which encodes a LysR-type transcriptional regulator protein. Strains with null mutations in either yggA or argP were supersensitive to the arginine analog canavanine, and yggA-lac expression in vivo exhibited argP(+)-dependent induction by arginine. Lysine supplementation phenocopied the argP null mutation in that it virtually abolished yggA expression, even in the argP+ strain. The dipeptides arginylalanine and lysylalanine behaved much like arginine and lysine, respectively, to induce and to turn off yggA transcription. Dominant missense mutations in argP (argPd) that conferred canavanine resistance and rendered yggA-lac expression constitutive were obtained. The protein deduced to be encoded by yggA shares similarity with a basic amino acid exporter (LysE) of Corynebacterium glutamicum, and we obtained evidence for increased arginine efflux from E. coli strains with either the argPd mutation or multicopy yggA+. The null yggA mutation abolished the increased arginine efflux from the argPd strain. Our results suggest that yggA encodes an ArgP-regulated arginine exporter, and we have accordingly renamed it argO (for "arginine outward transport"). We propose that the physiological function of argO may be either to prevent the accumulation to toxic levels of canavanine (which is a plant-derived antimetabolite) or arginine or to maintain an appropriate balance between the intracellular lysine and arginine concentrations.

The mechanism of L-canavanine cytotoxicity: arginyl tRNA synthetase as a novel target for anticancer drug discovery.

There is a clear need for agents with novel mechanisms of action to provide new therapeutic approaches for the treatment of pancreatic cancer. Owing to its structural similarity to L-arginine, L-canavanine, the beta-oxa-analog of L-arginine, is a substrate for arginyl tRNA synthetase and is incorporated into nascent proteins in place of L-arginine. Although L-arginine and L-canavanine are structurally similar, the oxyguanidino group of L-canavanine is significantly less basic than the guanidino group of L-arginine. Consequently, L-canavanyl proteins lack the capacity to form crucial ionic interactions, resulting in altered protein structure and function, which leads to cellular death. Since L-canavanine is selectively sequestered by the pancreas, it may be especially useful as an adjuvant therapy in the treatment of pancreatic cancer. This novel mechanism of cytotoxicity forms the basis for the anticancer activity of L-canavanine and thus, arginyl tRNA synthetase may represent a novel target for the development of such therapeutic agents.

Chloroquine stimulates nitric oxide synthesis in murine, porcine, and human endothelial cells.

Nitric oxide (NO) is a free radical involved in the regulation of many cell functions and in the expression of several diseases. We have found that the antimalarial and antiinflammatory drug, chloroquine, is able to stimulate NO synthase (NOS) activity in murine, porcine, and human endothelial cells in vitro: the increase of enzyme activity is dependent on a de novo synthesis of some regulatory protein, as it is inhibited by cycloheximide but is not accompanied by an increased expression of inducible or constitutive NOS isoforms. Increased NO synthesis is, at least partly, responsible for chloroquine-induced inhibition of cell proliferation: indeed, NOS inhibitors revert the drug-evoked blockage of mitogenesis and ornithine decarboxylase activity in murine and porcine endothelial cells. The NOS-activating effect of chloroquine is dependent on its weak base properties, as it is exerted also by ammonium chloride, another lysosomotropic agent. Both compounds activate NOS by limiting the availability of iron: their stimulating effects on NO synthesis and inhibiting action on cell proliferation are reverted by iron supplementation with ferric nitrilotriacetate, and are mimicked by incubation with desferrioxamine. Our results suggest that NO synthesis can be stimulated in endothelial cells by chloroquine via an impairment of iron metabolism.

L-canavanine and dexamethasone attenuate endotoxin-induced suppression of ischaemia-reperfusion arrhythmias.

The role of inducible nitric oxide synthase in the antiarrhythmic effects of Escherichia coli endotoxin was examined in an anaesthetised rat model of myocardial ischaemia (7 min occlusion) and reperfusion (7 min) arrhythmias by using its specific blocker L-canavanine (100 mg/kg) and dexamethasone (5 mg/kg), which inhibits its expression. Endotoxin (1 mg/kg) or its solvent saline was administered intraperitoneally 4 h before the occlusion of the left coronary artery and L-canavanine or dexamethasone was administered 1 h before endotoxin or saline injection. The mean arterial blood pressure of rats receiving endotoxin was significantly lower than that of saline-treated controls, and neither L-canavanine nor dexamethasone prevented the hypotension exerted by endotoxin. However, during both the occlusion and reperfusion periods, endotoxin significantly reduced the total number of ectopic beats (e.g., during reperfusion, saline: 1177 +/- 183, n = 11; endotoxin: 248 +/- 91, n = 9; P < 0.005) and the duration of ventricular tachycardia (e.g., during occlusion, saline: 30.9 +/- 5.7 s; endotoxin: 1.8 +/- 0.9 s; P < 0.0001) while L-canavanine or dexamethasone treatment abolished the reduction exerted by endotoxin. Therefore we conclude that endotoxin possesses significant antiarrhythmic (protectant) effects in this rat model of ischaemia-reperfusion arrhythmias, and that its mechanism appears to involve the inducible nitric oxide synthase since both L-canavanine and dexamethasone inhibited this phenomenon.

L-canavanine influences feed intake, plasma basic amino acid concentrations and kidney arginase activity in chicks.

L-Canavanine [2-amino-4-(guanidinooxy) butyric acid], a non-protein amino acid that is structurally analogous to arginine, has been proposed as a major antinutritional factor responsible for the toxic effects induced by raw Canavalia ensiformis (L.) seeds in chicks. We investigated the effects of L-canavanine on performance and select metabolic responses of growing chicks. Canavanine was added to a control diet, in an amount equivalent to that provided by 300 g raw canavalia seeds/kg diet (10 g free base canavanine/kg diet). Growth, plasma basic amino acids and kidney arginase, activity were measured. The incorporation of canavanine into a nutritionally balanced diet for growing chicks depressed feed intake and growth by approximately 25% (P < 0.01) compared with the control diet. Performance was unaffected by equimolar amounts of arginine. Canavanine exerted its growth-depressing effect exclusively by reducing feed intake, because this effect was not observed in a pair-feeding experiment. Chicks fed a diet containing 473 mmol canavanine sulfate/kg for 11 d were given an intracrop dose of 946 mmol of canavanine sulfate or arginine hydrochloride. In both cases, plasma histidine and lysine concentrations were significantly decreased compared with a placebo group dosed with water. Plasma arginine concentration was unaffected by the canavanine sulfate dose but, as expected, was significantly increased by the arginine hydrochloride dose. Free base canavanine significantly (P < 0.05) reduced kidney arginase activity. No overt toxic effects were observed at any point during the study. These data indicate that, although canavanine is not the principal antinutritional factor in Canavalia ensiformis seeds, its presence in the diet precludes optimum performance of chicks.

Regulation of enzymes involved in ornithine/arginine metabolism in the parasitic trypanosomatid Herpetomonas samuelpessoai.

The genetic regulation of enzymes involved in arginine and ornithine synthesis has been investigated in the parasitic trypanosomatid Herpetomonas samuelpessoai. The activities of two enzymes involved in arginine synthesis, ornithine carbamoyltransferase (OCTase) and argininosuccinate lyase (ASLase) were depressed whereas the enzyme citrulline hydrolase (CHase), which is involved in ornithine synthesis, was increased in arginine supplemented cultures of the parasites. The depression of OCTase activity in the presence of arginine was not due to feedback inhibition and CHase activity of uninduced cultures was not enhanced by exogeneous arginine. Studies of the kinetics of OCTase induction and repression revealed that arginine blocks OCTase synthesis but does not cause destruction of the enzyme. Ornithine, but not citrulline was found to counteract the arginine mediated repression of OCTase. Two classes of canavanine resistant mutants of H. samuelpessoai were isolated. One class was defective in arginine uptake whereas the other was affected in regulation of OCTase and ASLase which appear to be under coordinate control in H. samuelpessoai.

Reappraisal of L-canavanine as an anti-tumour agent.

L-canavanine, an analogue of L-arginine, is known to have a toxic effect on pathogens. In experiments performed on a specialised epithelial-like cell system which is conditionally transformable, L-canavanine appeared to exhibit (1) a reversible 'static' effect on exponentially growing cells at optimal concentration, and (2) a selectively toxic effect on proliferating cells. The capacity to recognise 'tumour cells' in a heterogeneous population of cells is of potential interest in cancer chemotherapy.

Mutagenicity of N-nitrosopiperazine derivatives in Saccharomyces cerevisiae.

The mutagenic properties of 8 N-nitrosopiperazines were examined in Saccharomyces cerevisiae. Forward mutations to canavanine resistance and reversions of his1-7 were induced by N'-methyl-N-nitrosopiperazine, dinitrosopiperazine, 2-methyldinitrosopiperazine, 2,5-dimethyldinitrosopiperazine, and 2,6-dimethyldinitrosopiperazine, in the presence of rat-liver homogenate. N-nitrosopiperazine, 2,3,5,6-tetramethyldinitrosopiperazine, and 4-benzoyl-3,5-dimethyldinitrosopiperazine were non-mutagenic.