Leishmania infantum: Antiproliferative effect of recombinant plant cystatins on promastigotes and intracellular amastigotes estimated by direct counting and real-time PCR

Two recombinant barley cystatins, HvCPI5 and HvCPI6, have been tested in vitro against promastigotes and intracellular amastigotes of Leishmania infantum in the J774 monocytic cell line. Toxicity of cystatins for J774 cells was also determined. In addition, a comparison between direct counts of intracellular amastigotes and quantitation of burden by Q-PCR was carried out. Low concentrations (2 μM) from both cystatins were unable to inhibit promastigote replication. HvCPI5 was toxic for mammalian cells; 0.1 μM reduced by more than 50% the cell viability. On the contrary, HvCPI6 did not exhibit any toxicity for J774 cells up to 6 μM and inhibited the intracellular amastigote multiplication. Dose-response analysis showed that 4.8 μM HvCPI6 reduced by >90% the intracellular parasite load and had an approximate IC₅₀ value of 1.5 μM. Comparable results were obtained by direct counting of intracellular amastigotes and Q-PCR. Results point towards the direct inhibition of amastigote multiplication by HvCPI6 and the interest of this recombinant cystatin in the chemotherapy of leishmaniasis.     

In vitro and in vivo leishmanicidal activity of Dysoxylum binectariferum and its fractions against Leishmania donovani

The leishmanicidal effect of crude ethanolic extract of stem bark of Dysoxylum binectariferum and its fractions has been investigated against Leishmania donovani, the causative agent of visceral leishmaniasis. Ethanolic extract was lethal to promastigotes as well as amastigote forms in macrophage system at the concentration of 100 microg/ml. Chloroform fraction significantly inhibited promastigote multiplication and was also active against amastigotes in infected J774A.1 macrophages at 100 microg/ml. Hexane fraction was moderately active and the other fractions were inactive against both the forms. When tested in vivo in hamsters, ethanolic extract was toxic at 500 mg/kg whereas exhibited marginal activity (67.7+/-5.3%) at 250 mg/kg x 5, p.o. on day 7 post treatment (p.t.) which increases slightly (69+/-4.7) by day 30 p.t. Chloroform and n-hexane fractions exhibited 64.3+/-4% and 47.8+/-4.6% parasite inhibition at the dose of 100 mg/kg x 5 p.o., respectively. The pure compound, rohitukine, obtained from chloroform fraction showed weaker in vitro activity and was ineffective in infected hamsters. The lead potential of this plant need further investigations.     

Taxol protects against calcium-mediated death of differentiated rat pheochromocytoma cells

Elevated levels of intraneuronal calcium may contribute to neuronal death in both Alzheimer's disease and stroke. In part, this neuronal death may be due to calcium-induced disruption of microtubules and inhibition of axonal transport. Taxol stabilizes microtubules to disaggregation. To determine whether taxol could protect against calcium-mediated neuron cell death, a test system was established using a nerve growth factor-differentiated rat pheochromocytoma cell line (PC12 cells). PC12 cells were cultured with nerve growth factor to induce a neuronal phenotype. After 15 days, the cells were exposed to taxol, the calcium ionophore, A23187, or taxol plus ionophore for up to 24 h. Taxol alone reduced cell survival in a concentration dependent manner. At a concentration of 50 nM survival was reduced to between 63% and 84% of control after 4 h of exposure. The ionophore (1 μM) variably reduced cell survival to between 10 and 55% at 4h. However, when tacol was added to the ionophore the cell survival was significantly increased by 1.5 to 4-fold. The protective effect of taxol lasted up to 24h. We conclude that taxol has a protective effect on calcium-mediated neurotoxicity. Drugs targeting underlying cellular mechanisms involved in calcium-mediated neuronal death may lead to successful therapy for Alzheimer's disease and stroke.     

Taxol binds to cellular microtubules

Taxol is a low molecular weight plant derivative which enhances microtubule assembly in vitro and has the unique ability to promote the formation of discrete microtubule bundles in cells. Tritium-labeled taxol binds directly to microtubules in vitro with a stoichiometry approaching one (Parness, J., and S. B. Horwitz, 1981, J. Cell Biol. 91:479-487). We now report studies in cells on the binding of [3H]taxol and the formation of microtubule bundles. [3H]Taxol binds to the macrophagelike cell line, J774.2, in a specific and saturable manner. Scatchard analysis of the specific binding data demonstrates a single set of high affinity binding sites. Maximal binding occurs at drug concentrations which produce maximal growth inhibition. Conditions which depolymerize microtubules in intact and extracted cells as determined by tubulin immunofluorescence inhibit the binding of [3H]taxol. This strongly suggests that taxol binds specifically to cellular microtubules. Extraction with 0.1% Nonidet P-40 or depletion of cellular ATP by treatment with 10 mM NaN3 prevents the characteristic taxol-induced bundle formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there must be specific cellular mechanisms which are required for bundle formation, in addition to the direct binding of taxol to cytoplasmic microtubules.     

Isolation of a taxol-resistant Leishmania donovani promastigote mutant that exhibits a multidrug-resistant phenotype

We raised a strain of Leishmania donovani in the laboratory that was resistant to 500 nM taxol. The IC50 of the wild-type strain for taxol was 35 nM and that of the taxol-resistant strain (T-500) was 1 microM. The T-500 strain exhibited a Mdr phenotype; it was also resistant to other unrelated drugs like vinblastine, adriamycin and the commonly used antimonial drugs pentostam and glucantime. Verapamil (20 nM), a calcium channel blocker, was found to reverse the resistance of T-500 to taxol. Acquired resistance to taxol has been reported to be mediated by alterations involving tubulin in cancer cells. Thus polymerisation assays with tubulin fractions in wild-type versus taxol-resistant cells (T-500) were performed in vitro. The tubulin fraction from T-500 was more resistant to in vitro polymerisation than the tubulin isolated from the wild-type, suggesting that this is one means by which the parasite may acquire resistance to taxol.     

Abietane diterpenoids and triterpenoic acids from Salvia cilicica and their antileishmanial activities

Bioguided-fractionation of an acetone extract of the roots of Salvia cilicica (Lamiaceae) led to isolation of two new diterpenes, 7-hydroxy-12-methoxy-20-nor-abieta-1,5(10),7,9,12-pentaen-6,14-dione and abieta-8,12-dien-11,14-dione (12-deoxy-royleanone), together with oleanolic acid, ursolic acid, ferruginol, inuroyoleanol and cryptanol. Their structures were determined spectroscopically, which included HREIMS and 2D NMR spectroscopic analysis. The new abietane derivatives showed appreciable in vitro antileishmanial activity against intracellular amastigote forms of both Leishmania donovani (IC(50) values of 170 and 120 nM, respectively) and Leishmania major (IC(50) values of 290 and 180 nM, respectively). The triterpenoic acids were found to be potently active against amastigote (IC(50) values of 7-120 nM) and moderately active against promastigote stages (IC(50) values of 51-137 nM) of the two Leishmania species.     

Taxol-induced mitotic block triggers rapid onset of a p53-independent apoptotic pathway.

BACKGROUND: At therapeutic concentrations, the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis, similar to the mechanism of cell death induced by other antineoplastic agents. However, taxol has shown efficacy against drug-refractory cancers, raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. MATERIALS AND METHODS: The kinetics and IC50 of mitotic (M) block, aberrant mitosis, and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). RESULTS: Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or, for those cells progressing through aberrant mitosis, arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast, G1-arrested, multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase, both wt p53-expressing and p53-null MEFs responded similarly to taxol, showing rapid onset of DNA nicking and apoptosis. However, p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive, and remained viable. CONCLUSIONS: Taxol induces two forms of cell cycle arrest, which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway, whereas G1-block and the resulting slow (3-5 days) apoptotic pathway are p53 dependent.     

Antileishmanial and Cytotoxic Effects of Essential Oil and Methanolic Extract of Myrtus communis L.

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were α-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC₅₀ values for essential oil and methanolic extract was 8.4 and 28.9 μg/ml against promastigotes, respectively. These values were 11.6 and 40.8 μg/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC₅₀ values of 88.3 and 44.6 μg/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.     

Taxol suppresses dynamics of individual microtubules in living human tumor cells

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30-100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.     

In vitro evaluation of newly synthesised [1,2,4]triazolo[1,5a]pyrimidine derivatives against Trypanosoma cruzi, Leishmania donovani and Phytomonas staheli

The antiprotozoal activity of newly synthesised compounds, all [1,2,4]triazolo [1,5a]pyrimidine derivatives, was tested against the protozoan parasites Trypanosoma cruzi, Leishmania donovani and Phytotmonas staheli. Six of these compounds significantly inhibited in vitro cell growth of the epimastigote forms of T. cruzi, and the promastigote forms of L. donovani and P. staheli. Some of the compounds reached complete growth inhibition at 1 microg/ml for 48 h of parasite/drug interaction. None of the compounds tested showed significant toxicity against cells of Aedes albopictus, mouse macrophages J-774A.1 and Lycopersicum esculentum at dosages five times greater than used against parasites.     

Cytotoxic cardenolides from Streptocaulon griffithii

A new cardenolide, 3-O-(beta-glucopyranosyl)acovenosigenin A (1), was isolated from the roots of Streptocaulon griffithii, together with eight known cardenolides, compounds 2-9. All compounds showed significant in vitro inhibition of the proliferation of the human gastrointestinal cancer cell line HGC-27, with IC50 values in the range of ca. 20-564 nM, taxol being used as positive control. Compound 1 was also found to be strongly cytotoxic against other human tumor cell lines, including A549, MCF-7, and Hela cells.     

Acivicin: a highly active potential chemotherapeutic agent against visceral leishmaniasis

Acivicin, a chlorinated amino acid antibiotic, is found to be remarkably effective in killing both the vector and the host form of the parasitic protozoa, Leishmania donovani, the causative agent for visceral leishmaniasis or Kala-azar. The ED50 (50 nM) for the pathogenic amastigote form in in vitro screening system is significantly lower than the reported values for other drugs under trial. The drug irreversibly inactivates both in vitro and in vivo carbamyl phosphate synthetase II, the first enzyme of the pyrimidine biosynthetic pathway. The irreversible inactivation of this sensitive target enzyme and lack of effective reversal by glutamine makes acivicin a preferred candidate for potential chemotherapy against increasing number of Kala-azar cases that are reported to be unresponsive to pentavalent antimonials.     

Taxol inhibits stimulation of cell DNA synthesis by human cytomegalovirus

The microtubule (MT)-stabilizing drug, taxol, inhibited human cytomegalovirus (CMV)-initiated cell DNA synthesis by up to 100% in serum-arrested mouse embryo (ME) fibroblasts that were abortively infected by CMV. Taxol concentrations known to increase MT polymerization and to stabilize existing MTs (10 to 20 micrograms/ml) blocked CMV-stimulated cell DNA synthesis, while taxol concentrations of 2.5 micrograms/ml, or less, did not. Taxol maximally inhibited CMV initiation of cell DNA synthesis when added 3 h after virus infection and inhibited this initiation by greater than 50% when added up to 12 h after CMV infection. Control experiments suggest that taxol specifically inhibited CMV-stimulated cell DNA synthesis. Pretreatment of CMV stock with taxol did not reduce the stimulatory effect of CMV on cell DNA synthesis and taxol had no detectable effect on CMV-specific early protein synthesis. Moreover, taxol did not appear to alter thymidine pool sizes, affect cell viability, or compromise the DNA synthetic machinery in CMV-infected cells. Since taxol increases tubulin polymerization and inhibits MT disassembly, these results suggest that dynamic changes in MTs or in the pool of free tubulin subunits are necessary for CMV to stimulate cell entry into a proliferative cycle.     

Human cytochrome c enters murine J774 cells and causes G1 and G2/M cell cycle arrest and induction of apoptosis

Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c(551) from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c(551), the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G(1) to S phase, as well as at the G(2)/M phase at higher concentrations. Unlike P. aeruginosa cytochrome c(551) which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G(2)/M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 degrees C, suggesting energy requirement in the entry process.     

红豆杉悬浮细胞放大培养的细胞生长与紫杉醇合成动力学

研究了在Murashige&skoog s(MS)和 6 2号两种不同的培养基中 ,红豆杉细胞悬浮细胞从摇瓶到 1 0L机械通气搅拌式反应器放大培养过程中细胞生长与紫杉醇合成动力学 .结果表明 :尽管在不同的培养条件下 ,细胞生长曲线均呈现“S”型 .紫杉醇在延迟期与指数生长期中基本上没有积累 ,而且随着培养规模的增大 ,紫杉醇的含量逐渐降低 .进一步对各级放大培养的细胞生长 ,比生长率与胞内外紫杉醇合成量进行分析 ,发现MS利于细胞生长但不利于紫杉醇合成 ,而 6 2号则相反 .根据此文的结果 ,提出了红豆杉细胞培养条件的优化和大规模细胞培养生产紫杉醇应采取的策略     

Hsp70 protects macrophages infected with Salmonella choleraesuis against TNF-α-induced cell death

Hsp70 plays an important role in cytoprotection against tumor necrosis factor (TNF) α-mediated cytotoxicity. To investigate the role of Hsp70 in cytoprotein during Salmonella infection, we examined endogenous Hsp70 induction and TNF-α production in a monocyte/macrophage line, J774A.1, after infection with a virulent strain of Salm.choleraesuis RF-1 carrying a 50 kb virulent plasmid or the plasmid-cured avirulent strain 31N-1. Intracellular bacteria progressively increased in J774A.1 cells phagocytosing avirulent 31N-1 bacteria, whereas such progressive growth was not evident in J774A.1 cells phagocytosing avirulent 31N-1 bacteria. On the contrary, J774A.1 cells infected with virulent RF-1 bacteria expressed less Hsp70 than those infected with avirulent 31N-1 bacteria. The level of TNF-α production by J774A.1 infected with virulent RF-1 was much the same as that by J774A.1 infected with avirulent 31N-1. J774A.1 infected with virulent RF-1 died spontaneously; death was inhibited by the addition of anti-TNF-α mAb. Although the frequency of dead J774A.1 with hypodiploid DNA content increased only marginally after infection with avirulent 31N-1, treatment with Hsp70 anti-sense oligonucleotide resulted in a dramatic increase of dead cells in the infected macrophages. Taken together, these results suggest that Hsp70 induced macrophages plays an important role in host defense against Salmonella infection by protecting the macrophages against TNF α-induced cell death. Furthermore, cell death due to impaired endogenous Hsp synthesis in the phagocytes implies a novel pathogenic mechanism for virulence of Salm. choleraesuis RF-1.     

Inhibitory effect of taxol, a microtubule stabilizing agent, on induction of DNA synthesis is dependent upon cell lines and growth factors.

Growth-arrested rat fibroblasts, 3Y1, and human diploid fibroblasts, TIG-1, were induced to synthesize DNA by stimulation with various agents such as fetal bovine serum (FBS), epidermal growth factor (EGF), colcemid, or colchicine. Taxol, a microtubule-stabilizing agent, blocked the induction of DNA synthesis after stimulation with colcemid or colchicine in both cell lines. Taxol inhibited the induction of DNA synthesis after stimulation with FBS or EGF in TIG-1, but did not in 3Y1. 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis in TIG-1, which was reduced only partly by taxol. Taxol stabilized or polymerized microtubules in both cell lines. These results indicate that the inhibitory effect of taxol on the induction of DNA synthesis varied among cell lines and among growth factors, and suggest that signal transduction processes may be differentiated by taxol sensitivity. In TIG-1 cells, when taxol was added within 6 h, about halfway into the initiation of DNA synthesis after the addition of FBS or EGF, the inhibition of DNA synthesis still occurred. Taxol did not inhibit the induction of c-fos and c-myc genes by FBS or EGF stimulation. Colchicine itself did not induce these genes in TIG-1. Thus, taxol appeared to inhibit the induction of DNA synthesis not by blockage in the early transduction process of the growth signal from the cell surface to nuclei but by blockage in processes operating in the mid- or late-prereplicative phase.     

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37 masculineC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.     

Effect of the anti-microtubule compound tubulozole on Leishmania protozoan parasites in vitro

Abstract For the cis and trans stereoisomers of the synthetic anti-microtubule compound tubulozole, at micromolar concentrations, tubulozole-C is cytotoxic to mammalian cells whereas tubulozole-T is not. The effect of tubulozoles on the parasitic protozoan Leishmania was tested. For the promastigote stage of L. mexicana amazonensis , both isomers inhibited parasite growth. For the amastigote stage of L. mexicana amazonensis and L. major , within murine J774 macrophage line as host cells in vitro, tubulozole-T reduced the infective index. Despite the observation of macrophage cytotoxicity of tubulozole-T, this compound may be a potentially useful and novel anti-leishmanial drug.