Semisynthesis of new aphidicolin derivatives with high activity against Trypanosoma cruzi.

Chagas disease continues to be a difficult disease to eradicate, largely because of the widespread populations it affects as well as the highly toxic effects of current therapies. Thus, the exploration of innovative scaffolds, ideally with distinct mechanisms of action, is urgently needed. The natural product aphidicolin and its effects on cell cycle division have been widely studied; it is a potent inhibitor of parasitic cells. In the present study, we report for the first time the semisynthesis of a series of aphidicolin derivatives, their unique structural features, and demonstration of their activity against Trypanosoma cruzi cells. Two demonstrated high potency and selectivity against parasitic amastigote cells, and thus show promise as new leads for Chagas disease treatment.

Toward a Cancer Drug of Fungal Origin.

Although fungi produce highly structurally diverse metabolites, many of which have served as excellent sources of pharmaceuticals, no fungi-derived agent has been approved as a cancer drug so far. This is despite a tremendous amount of research being aimed at the identification of fungal metabolites with promising anticancer activities. This review discusses the results of clinical testing of fungal metabolites and their synthetic derivatives, with the goal to evaluate how far we are from an approved cancer drug of fungal origin. Also, because in vivo studies in animal models are predictive of the efficacy and toxicity of a given compound in a clinical situation, literature describing animal cancer testing of compounds of fungal origin is reviewed as well. Agents showing the potential to advance to clinical trials are also identified. Finally, the technological challenges involved in the exploitation of fungal biodiversity and procurement of sufficient quantities of clinical candidates are discussed, and potential solutions that could be pursued by researchers are highlighted.

The anti-promyelocytic leukemia mode of action of two endophytic secondary metabolites unveiled by a proteomic approach.

As a result of a program to find antitumor compounds of endophytes from medicinal Asteraceae, the steroid (22E,24R)-8,14-epoxyergosta-4,22-diene-3,6-dione (a) and the diterpene aphidicolin (b) were isolated from the filamentous fungi Papulaspora immersa and Nigrospora sphaerica, respectively, and exhibited strong cytotoxicity against HL-60 cells. A proteomic approach was used in an attempt to identify the drugs' molecular targets and their respective antiproliferative mode of action. Results suggested that the (a) growth inhibition effect occurs by G2/M cell cycle arrest via reduction of tubulin alpha and beta isomers and 14-3-3 protein gamma expression, followed by a decrease of apoptotic and inflammatory proteins, culminating in mitochondrial oxidative damage that triggered autophagy-associated cell death. Moreover, the decrease observed in the expression levels of several types of histones indicated that (a) might be disarming oncogenic pathways via direct modulation of the epigenetic machinery. Effects on cell cycle progression and induction of apoptosis caused by (b) were confirmed. In addition, protein expression profiles also revealed that aphidicolin is able to influence microtubule dynamics, modulate proteasome activator complex expression, and control the inflammatory cascade through overexpression of thymosin beta 4, RhoGDI2, and 14-3-3 proteins. Transmission electron micrographs of (b)-treated cells unveiled dose-dependent morphological characteristics of autophagy- or oncosis-like cell death.

Increased systemic efficacy of aphidicolin encapsulated in liposomes.

Aphidicolin, a tetracyclic diterpene antibiotic produced by Cephalosporium aphidicola, is under investigation as anti-cancer drug. Because of its poor solubility in water, it cannot be administered directly in vivo. Systemic application of aphidicolin glycinate or aphidicolin gamma-cyclodextrin complexes resulted in tumour growth inhibition but not in cures. To improve the pharmacokinetics, a liposomal preparation of aphidicolin was developed and tested in neuroblastoma-bearing (UKF-NB-3) mice. The loading capacity of these liposomes was limited. Therefore, 4.5 mg aphidicolin/kg body weight was the maximum aphidicolin dose that could be applied as liposomal preparation in this approach. Comparison of effects on tumour growth exhibited by aphidicolin liposomes (4.5 mg aphidicolin/kg) given for 15 consecutive days to those of gamma-cyclodextrin inclusion complexes (15 mg aphidicolin/kg) revealed comparable tumour growth inhibition, although aphidicolin concentrations were approximately 3-fold lower. This shows that liposomal encapsulation is a promising strategy for the improvement of systemic anti-cancer activity of aphidicolin.

E2F-1 overexpression sensitizes colorectal cancer cells to camptothecin.

Topoisomerase I inhibitors have been shown to have clinical activity against human colorectal cancer. Previous studies showed that the cytotoxicity of camptothecin, a topoisomerase I inhibitor, occurs mainly in the S -phase of the cell cycle and is protectable by aphidicolin, an inhibitor of replicative DNA polymerase in some camptothecin-sensitive colorectal cells. Transcription factor E2F-1 regulates the G1/S transition, and recent studies have shown that E2F-1 potentiated the cytotoxicity of some cell-cycle-related drugs. Therefore, the present study was designed to investigate the effect of adenovirus-mediated E2F-1 gene transfer on chemosensitivity of colorectal cancer to camptothecin, in vitro and in vivo. Two human colorectal cancer cells, SW620 (mutant p53) and RKO (wild-type p53), were treated with camptothecin, alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ), or E2F-1 (Ad-E2F-1). E2F-1 overexpression was confirmed by Western blot analysis. Ad-E2F-1 gene transfer at low doses (less than the LD(20) dose) markedly increased the sensitivity of human colorectal cancer cells to camptothecin in vitro, which is because of induction of apoptosis. Aphidicolin did not have any protective effect on the Ad-E2F-1/camptothecin-mediated cytotoxicity. The level of topoisomerase I expression was not affected by combination treatment as well, suggesting that DNA replication and topoisomerase I activity may not account for the molecular mechanism of cell killing in response to Ad-E2F-1/camptothecin treatment. Fas and Fas ligand expression were not altered by treatment with camptothecin and/or Ad-E2F-1. Moreover, combination of camptothecin and Ad-E2F-1 has an additive antitumor effect in an in vivo nude mouse xenograft model. When combined with camptothecin, E2F-1 adenovirus therapy resulted in a 95.7% decrease in tumor size compared to control groups (P<.05). These results suggest a chemosensitization strategy that may have clinical utility in human colorectal cancer.

A single-nucleotide polymorphism in a herpesvirus DNA polymerase is sufficient to cause lethal neurological disease.

Epidemiological studies have shown that a single-nucleotide polymorphism in the equid herpesvirus type 1 DNA polymerase gene is associated with outbreaks of highly lethal neurological disease in horses. Reverse genetics experiments further demonstrated that a G(2254) A(2254) nucleotide mutation introduced in neurovirulent strain Ab4, which resulted in an asparagine for aspartic acid substitution (D(752) N(752)), rendered the virus nonneurovirulent in the equine. Here, we report that the nonneurovirulent strain equid herpesvirus type 1 strain NY03 caused lethal neurological disease in horses after mutation of A(2254) G(2254) (N(752) D(752)), thereby providing final proof that the D(752) allele in the viral DNA polymerase is necessary and sufficient for expression of the lethal neurovirulent phenotype in the natural host. Although virus shedding was comparable between the N(752) and D(752) variants, infection with the latter was accompanied by efficient establishment of prolonged cell-associated viremia in peripheral blood mononuclear cells and neurological disease in 2 of 6 animals.

Potentiation of in vitro ara-C cytotoxicity by ribonucleotide reductase inhibitors, cyclin-dependent kinase modulators and the DNA repair inhibitor aphidicolin in paediatric acute myeloid leukaemia.

To modulate in vitro cytarabine (ara-C) resistance we combined ara-C with six potential resistance modifiers in 10 paediatric acute myeloid leukaemia (AML) patient samples (methyl thiazol tetrazolium assay). Drug interactions were determined by median drug effect analysis. Co-incubation of ara-C/aphidicolin showed strong synergism. The combinations of ara-C/cladribine and ara-C/gemcitabine were synergistic. Nearly additive and moderately synergistic interactions were observed between ara-C/flavopiridol and ara-C/UCN-01. The combination of ara-C/decitabine was antagonistic. In conclusion, favourable interactions were observed between ara-C and aphidicolin, cladribine, gemcitabine and also with flavopiridol and UCN-01, supporting the evaluation of these combinations in clinical trials with AML patients.

Hydroxyurea significantly enhances tumor growth delay in vivo with herpes simplex virus thymidine kinase/ganciclovir gene therapy.

We have previously demonstrated with several cell lines in vitro that hydroxyurea (HU) synergistically enhances ganciclovir (GCV)-mediated cytotoxicity in bystander cells. In this study, we evaluated the role of DNA synthesis inhibition on enhanced bystander killing and assessed whether addition of HU would improve the efficacy of the HSV-TK/GCV system in vivo. Compared with GCV treatment alone, addition of HU resulted in increased DNA synthesis inhibition and delayed progression through S phase following removal of drug. In a xenograft tumor model, 1:10 and 1:1 mixtures of HSVtk- and LacZ-expressing SW620 cells were injected s.c. in the flanks of nude mice and treated i.p. (100 mg/kg GCV, 1500 mg/kg HU) daily for 5 days. Tumors from mice treated with GCV alone grew rapidly and increased to 10 times their initial size in 15.7 +/- 1.8 and 16.0 +/- 0.9 days for 1:10 and 1:1 mixtures, respectively. However, when both GCV and HU were administered in combination, a single complete tumor regression was observed in both the 1:10 and 1:1 groups. In the remaining mice treated with GCV/HU, it took 23.2 +/- 2.1 (1:10) and 26.4 +/- 3.8 days (1:1) to obtain a similar 10-fold increase in tumor size.

Treatment of drug-resistant human neuroblastoma cells with cyclodextrin inclusion complexes of aphidicolin.

Treatment failure in most neuroblastoma (NB) patients is related to primary and/or acquired resistance to conventional chemotherapeutic agents. Aphidicolin (APH), a tetracyclic diterpene, exhibits specific cytotoxic action against NB cells. The purpose of this study was to compare antitumoral efficacy of APH in parental NB cell lines and cell subclones that exhibit drug resistance to vincristine (VCR), doxorubicin (DOX) and cisplatin. Due to poor solubility of APH in water, gamma-cyclodextrin (gamma-CD) inclusion complexes of APH were used for systemic treatment of xenotransplanted parental and VCR-resistant UKF-NB-3 tumours. APH and its gamma-CD inclusion complexes inhibited growth of parental and drug-resistant NB cells at equimolar doses in vitro. Growth of VCR-sensitive and -resistant NB tumors was inhibited at equal doses in a dose-dependent fashion in vivo. These results indicate that the specific cytotoxic activity of APH against NB cells in vitro and in vivo is independent of cellular mechanisms facilitating drug resistance to conventional chemotherapeutic drugs. Hence, taking into account our previous findings that APH acts synergistically with VCR and DOX, APH might be an additive tool for the therapy of NB and is suitable for evaluation in clinical studies of NB treatment protocols.

[The inhibition of the proliferation and energy metabolism of Cloudman melanoma cells in vitro by aphidicolin and gamma radiation]. / Hemmung der Proliferation und des Energiestoffwechsels von Cloudman-Melanomzellen in vitro durch Aphidicolin und Gammastrahlung.

PURPOSE: The present investigation is concerned with the effect of aphidicolin (a tetracyclic diterpenoid) and gamma-ray [correction of X-ray] irradiation--alone or in combination--on cell proliferation, protein production, glucose consumption and lactate production of in vitro cultured Cloudman melanoma cells. METHODS: Monolayer cultures of Cloudman melanoma cells in exponential growth phase were irradiated with 2, 4 or 6 Gy (cobalt-60) or treated for 96 h with 0.1, 0.2 or 1.0 microgram/ml aphidicolin. Further, the combination of 0.1 or 0.2 micrograms/ml aphidicolin with the above mentioned radiation doses were investigated. Measurements were performed in regard to changes of cell number, protein content, glucose consumption and lactate production. RESULTS: Single treatments resulted in a dose-dependent inhibition of cell proliferation, protein production, glucose consumption and lactate production. Combined treatment with aphidicolin and irradiation caused an augmentation of the respective single effects. The inhibitory effects were more pronounced under serum-free than serum-containing culture conditions. Influence of aphidicolin and irradiation did not result in an immediate cell death, but in a state of unbalanced growth with enlarged cells (in part with an increased number of nuclei) and an increased cellular protein content. CONCLUSIONS: The combination of aphidicolin with gamma-ray [correction of X-ray] irradiation caused a stronger effect than the respective single treatments.