Taxol, a microtubule stabilizing agent, blocks the replication of Trypanosoma cruzi.

Abstract

Taxol, an experimental antitumor agent and stabilizer of microtubules, inhibits in vitro replication of the human pathogenic hemoflagellate Trypanosoma cruzi. Micromolar concentrations of the drug prevent the completion of cell division in these organisms but allow the multiplication ofcell organelles such as the nucleus, kinetoplast, and flagellum. The result is the formation of motile organisms that have extra organelles but cannot fully replicate. Division proceeds to a relatively fixed locus on the long axis of the organism, suggesting the presence of a specific affected structure or function at this site. It is postulated that taxol produces these effects by stabilizing a portion of the microtubular cytoskeleton of T. cruzi.Trypanosoma cruzi is the causative agent of South American trypanosomiasis or Chagas disease. Infection with this organism may lead to severe chronic disruption ofthe autonomic nervous system in 20-40% of those infected and is fatal in 5-10% ofclinically ill patients (1, 2). Although many agents have been tested, there is currently no curative drug for this disease in man. T. cruzi is a eukaryote and presumably shares many metabolic pathways with human cells. Novel approaches will therefore be needed to identify drugs that have good therapeutic indices in this disease. In view of the prominent subsurface array of microtubules in Trypanosoma, we hypothesized that taxol, a drug affecting the stabilization ofmicrotubules, might have a specific deleterious effect on these organisms.Taxol is an experimental antitumor drug that was isolated from the plant Taxus brevifolia (3). Studies in our laboratory have shown that the drug has the unusual capacity to promote the assembly ofmicrotubules in a cell-free system (4). Taxol also stabilizes microtubules, protecting them from the depolymerizing effects of low temperatures, both in cells and in a cell-free system (5). The drug has an unusual chemical structure and is a potent inhibitor of the replication of mammalian cells in culture (5). We have found that taxol in micromolar amounts produces unique morphologic changes in T. cruzi growing in culture and results in cessation of their replication. MATERIALS AND METHODSTrypanosomes. The Brazil strain of T. cruzi was originally obtained from William Hanson (University of Georgia, Athens, GA). Trypanosomes were cultured in LIT growth medium [2% liver infusion broth (wt/vol, Difco)/0.5% tryptose (Difco)/ 0.4% NaCl/0.046% KCV0.8% Na2HPOJO.2% glucose/0.4% hemin (Sigma)]. Just before use, the medium was supplemented with 5% (vol/vol) fetal calfserum (GIBCO). The organisms were suspended in 20 ml ofLIT medium in a 125-ml Erlenmeyer flask and incubated at 270C on a shaker platform. During its life cycle, T. cruzi assumes several forms having varying degrees of infectivity for the human host. The epimastigote or insect form is so named because its kinetoplast is situated anterior to the nucleus. This is the form that T. cruzi assumes when the organism is cultured in LIT medium. Replication ofepimastig...

Histone deacetylase inhibitors and paclitaxel cause synergistic effects on apoptosis and microtubule stabilization in papillary serous endometrial cancer cells

“…Similarly, Blagosklonny et al (26) showed that acetylation of Lys 40 of a-tubulin occurs soon after TSA administration and parallels induction of apoptosis. Paclitaxel has also been shown to bind to and stabilize microtubules, eventually precipitating apoptosis (24,25). Our data show that the TSA/paclitaxel combination has the most pronounced effects on microtubule acetylation, cell apoptosis, and inhibition of tumor growth in a mouse xenograft model.…”

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“…A [ 3 H]thymidine incorporation assay was used to assess compound activity, and it was performed in two ways; (i) parasites were treated with compounds for 48 h, the compounds were removed by washing, and the [ 3 H]thymidine assay was performed for 48 h; (ii) parasites were treated with the compound for 48 h, the compound was removed by washing, fresh medium wash was added, the parasites were incubated for an additional 4 days (recovery experiment), and then the [ 3 H]thymidine assay was performed for 48 h. Specifically, the epimastigote form of T. cruzi (Brazil strain [31]) was grown in a 25-ml flask in 10 ml liver infusion tryptose (LIT) broth at 28°C with agitation at 80 rpm (orbital shaker; Forma Scientific, CA). The LIT medium contained 20 g LIT broth (Difco Laboratories, Detroit, MI), 5 g tryptose (Difco), 4 g NaCl, 0.4 g KCl, 8 g Na 2 HPO 4 , and 2 g of dextrose (Gibco, Burlington, ON, Canada) in 1 liter of distilled water (pH 7.4) and was filter sterilized.…”