The relationship between Taxol and (+)-discodermolide: synthetic analogs and modeling studies.

BACKGROUND: During the past decade, Taxol has assumed an important role in cancer chemotherapy. The search for novel compounds with a mechanism of action similar to that of Taxol, but with greater efficacy particularly in Taxol-resistant cells, has led to the isolation of new natural products. One such compound, (+)-discodermolide, although structurally distinct from Taxol, has a similar ability to stabilize microtubules. In addition, (+)-discodermolide is active in Taxol-resistant cell lines that overexpress P-glycoprotein, the multidrug-resistant transporter. Interestingly, (+)-discodermolide demonstrates a profound enhancement of the initiation process of microtubule polymerization compared to Taxol. RESULTS: The synthesis of (+)-discodermolide analogs exploiting our highly efficient, triply convergent approach has permitted structure-activity relationship (SAR) studies. Small changes to the (+)-discodermolide structure resulted in a dramatic decrease in the ability of all four discodermolide analogs to initiate tubulin polymerization. Two of the analogs also demonstrated a decrease in total tubulin polymerization, while a change in the olefin geometry at the C8 position produced a significant decrease in cytotoxic activity. CONCLUSIONS: The availability of (+)-discodermolide and the analogs, and the resultant SAR analysis, have permitted an exploration of the similarities and differences between (+)-discodermolide and Taxol. Docking of the X-ray/solution structure of (+)-discodermolide into the Taxol binding site of beta-tubulin revealed two possible binding modes (models I and II). The preferred pharmacophore model (I), in which the C19 side chain of (+)-discodermolide matches with the C2 benzoyl group of Taxol and the delta-lactone ring of (+)-discodermolide overlays with the C13 side chain of Taxol, concurred with the results of the SAR analysis.

Taxol and discodermolide represent a synergistic drug combination in human carcinoma cell lines.

Recently, three natural products have been identified, the epothilones, eleutherobin, and discodermolide, whose mechanism of action is similar to that of Taxol in that they stabilize microtubules and block cells in the mitotic phase of the cell cycle. In this report, we have compared and contrasted the effects of these new agents in Taxol-sensitive and -resistant cell lines. We also have taken advantage of a human lung carcinoma cell line, A549-T12, that was isolated as a Taxol-resistant cell line and found to require low concentrations of Taxol (2-6 nM) for normal cell division. This study then examined the ability of these new compounds to substitute for Taxol in sustaining the growth of A549-T12 cells. Immunofluorescence and flow cytometry have both indicated that the epothilones and eleutherobin, but not discodermolide, can substitute for Taxol in this Taxol-dependent cell line. In A549-T12 cells, the presence of Taxol significantly amplified the cytotoxicity of discodermolide, and this phenomenon was not observed in combinations of Taxol with either the epothilones or eleutherobin. Median effect analysis using the combination index method revealed a schedule-independent synergistic interaction between Taxol and discodermolide in four human carcinoma cell lines, an effect that was not observed between Taxol and epothilone B. Flow cytometry revealed that concurrent exposure of A549 cells to Taxol and discodermolide at doses that do not induce mitotic arrest caused an increase in the hypodiploid population, thereby indicating that a possible mechanism for the observed synergy is the potentiation of apoptosis. Our results suggest that Taxol and discodermolide may constitute a promising chemotherapeutic combination.

Regulation of electrolyte transport by nitric oxide in the mouse cecum.

The effect and role of nitric oxide (NO) in the regulation of ion transport in the mouse cecum were investigated. L-arginine, used to increase NO production, increased short-circuit current (Isc), a measure of ion transport, in a concentration-dependent manner with a maximal increase of 193.8+/-65.5 microA/cm2. This increase was not changed in Cl-- or HCO3--free buffers, but was significantly decreased in Na+-free buffer. Using immunohistochemistry, the constitutive form of nitric oxide synthase was found not to be different in the inflamed cecum. The inducible form of the enzyme, however, which was absent in the cecum of normal mice, was present in high levels in the cecum of the colitic mouse. These results suggest that NO causes an increase in Na+ absorption. The increased levels of inducible NO synthase in the inflamed cecum suggest a role for NO in the pathophysiology of inflammatory bowel disease.

Morphological factors influencing transepithelial conductance in a rabbit model of ileitis.

BACKGROUND & AIMS: Infection of rabbits with coccidia (Eimeria magna) causes chronic ileal inflammation and diarrhea. Inflamed ileum also shows decreased transmural conductance. The aim of this study was to characterize morphological factors known to affect paracellular permeability that may alter transmural conductance in inflamed ileum. METHODS: Ileal mucosa was mounted in Ussing chambers for study of [3H]mannitol and [3H]inulin fluxes. Light and electron microscopy were used for morphometric studies. Alterations in the zonula occludens of epithelial cells were evaluated in freeze-fracture replicas. RESULTS: Inflamed ileum showed diminished paracellular fluxes. Inoculated rabbits showed marked lymphoplasmocytic infiltration and villus blunting in ileum. Villus linear junctional density was unaffected. However, total villus apical surface area per square centimeter of tissue was reduced in inflamed ileum, causing a diminished total villus linear junctional pathway per square centimeter of apical surface. Villus zonula occludens strand number was reduced in inflamed ileum, whereas the frequency of both villus and crypt lateral surface extrajunctional strands increased. CONCLUSIONS: Chronic inflammation exerts a profound effect on ileal paracellular permeability. Morphological data suggest that this effect may be caused in part by alterations in inflamed ileal mucosal structure and tight junctional organization and density, particularly on villi.

Identification of protein kinase C-alpha, epsilon and zeta in rabbit ileal enterocytes.

Activation of protein kinase C (PKC) has been shown to regulate electrolyte transport in rabbit small intestine. We investigated the types of PKC isoforms in rabbit ileal villus and crypt cells and the potential for phorbol 12-myristate 13-acetate (PMA) to induce translocation of the PKC from the cytosol to the plasma membrane. Our results indicate that there are at least three PKC isoforms, alpha, epsilon and zeta, are present in both villus and crypt enterocytes. Acute treatment with PMA resulted in translocation of the PKC-alpha from the cytosol to the membrane fraction in both cell types. Prolonged exposure of the villus cells to PMA resulted in a significant progressive decrement in PKC-alpha, suggesting down regulation. Since PMA treatment results in translocation, this isoform may be involved in the regulation of electrolyte transport in the rabbit ileum.