Cladosporol A, a new peroxisome proliferator-activated receptor γ (PPARγ) ligand, inhibits colorectal cancer cells proliferation through ß-catenin/TCF pathway inactivation.

BACKGROUND: Cladosporol A, a secondary metabolite from Cladosporium tenuissimum, exhibits antiproliferative properties in human colorectal cancer cells by modulating the expression of some cell cycle genes (p21(waf1/cip1), cyclin D1). METHODS: PPARγ activation by cladosporol A was studied by overexpression and RNA interference assays. The interactions between PPARγ and Sp1 were investigated by co-immunoprecipitation and ChIp assays. ß-Catenin subcellular distribution and ß-catenin/TCF pathway inactivation were analyzed by western blot and RTqPCR, respectively. Cladosporol A-induced ß-catenin proteasomal degradation was examined in the presence of the specific inhibitor MG132. RESULTS: Cladosporol A inhibits cell growth through upregulation of p21(waf1/cip1) gene expression mediated by Sp1-PPARγ interaction. Exposure of HT-29 cells to cladosporol A causes ß-catenin nuclear export, proteasome degradation and reduced expression of its target genes. Upon treatment, PPARγ also activates E-cadherin gene at the mRNA and protein levels. CONCLUSION: In this work we provide evidence that PPARγ mediates the anti-proliferative action of cladosporol A in colorectal cancer cells. Upon ligand activation, PPARγ interacts with Sp1 and stimulates p21(waf1/cip1) gene transcription. PPARγ activation causes degradation of ß-catenin and inactivation of the downstream target pathway and, in addition, upregulates E-cadherin expression reinforcing cell-cell interactions and a differentiated phenotype. GENERAL SIGNIFICANCE: We elucidated the molecular mechanisms by which PPARγ mediates the anticancer activity of cladosporol A.

Cladosporol a stimulates G1-phase arrest of the cell cycle by up-regulation of p21(waf1/cip1) expression in human colon carcinoma HT-29 cells.

Cladosporols, purified and characterized as secondary metabolites from Cladosporium tenuissimum, display an antifungal activity. In this study, we tested the antiproliferative properties of cladosporol A, the main isoform of this metabolite family, against human cancer cell lines. By assessing cell viability, we found that cladosporol A inhibits the growth of various human colon cancers derived cell lines (HT-29, SW480, and CaCo-2) in a time- and concentration-dependent manner, specifically of HT-29 cells. The reduced cell proliferation was due to a G1-phase arrest, as assessed by fluorescence activated cell sorting analysis on synchronized HT-29 cells, and was associated with an early and robust over-expression of p21(waf1/cip1) , the well-known cyclin-dependent kinases inhibitor. This suggests that the drug may play a role in the control of cancer cell proliferation. Consistently, cyclin D1, cyclin E, CDK2, and CDK4 proteins were reduced and histone H1-associated CDK2 kinase activity inhibited. In addition to p21(waf1/cip1) , exposure to 20 µM cladosporol A caused a simultaneous increase of pERK and pJNK, suggesting that this drug activates a circuit that integrates cell cycle regulation and the signaling pathways both involved in the inhibition of cell proliferation. Finally, we showed that the increase of p21(waf1/cip1) expression was generated by a Sp1-dependent p53-independent stimulation of its gene transcription as mutagenesis of the Sp1 binding sites located in the p21 proximal promoter abolished induction. To our knowledge, this is the first report showing that cladosporol A inhibits colon cancer cell proliferation by modulating p21(waf1/cip1) expression.

Secondary mould metabolites of Cladosporium tenuissimum, a hyperparasite of rust fungi.

Investigation of the extracts of a culture of Cladosporium tenuissimum, a known hyperparasite of several rust fungi, gave rise to the isolation of cladosporols B-E (2-5). Their structure and stereochemistry were elucidated on the basis of 1H and 13C NMR evidence and CD measures. Cladosporols 1-5 were active in inhibiting the urediniospore germination of the bean rust agent Uromyces appendiculatus.

The proteome of exudates from germinating Lupinus albus seeds is secreted through a selective dual-step process and contains proteins involved in plant defence.

The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre-formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post-extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual-step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.

A polyphasic approach for the characterization of endophytic Alternaria strains isolated from grapevines.

A polyphasic approach was set up and applied to characterize 20 fungal endophytes belonging to the genus Alternaria, recovered from grapevine in different Italian regions. Morphological, microscopical, molecular and chemical investigations were performed and the obtained results were combined in a pooled cluster analysis. Following morphological analyses, all strains were grouped according to their three-dimensional sporulation pattern on PCA and to the colony characteristics on different substrates. After DNA extraction, all strains were analyzed by RAPD-PCR and the resulting profiles were subjected to cluster analysis. The metabolites extracted from the 20 Alternaria endophytes were analyzed by a HPLC and the resulting metabolite profiles were subjected to multivariate statistic analyses. In comparison with reference 'small-spored' Alternaria species, the 20 strains were segregated into two morphological groups: one belonging to the A. arborescens species-group and a second to the A. tenuissima species-group. RAPD analysis also showed that grapevine endophytes belonged to either the A. arborescens or the A. tenuissima species-group and that they were molecularly distinct from strains belonging to A. alternata. Chemotaxonomy gave the same grouping: the grapevine endophytic strains belong to A. arborescens or A. tenuissima species-groups producing known metabolites typical of these species-groups. Interestingly, the 20 grapevine endophytes were able to produce also a number of unknown metabolites, whose characterization could be useful for a more precise segregation of the two species-groups. The results show how complementary morphological, molecular and chemical data can clarify relationships among endophyte species-groups of low morphological divergence.

Mating behavior of a Northern Italian population of Fusarium verticillioides associated with maize.

Fusarium verticillioides, the most common causal organism of Fusarium stalk and ear rot of maize in Northern Italy, produces important mycotoxins such as fumonisins. Reproductive biology of F. verticillioides has been widely studied in numerous maize growing areas, but up to now no information is available on the mating behavior and genetic structure of this plant pathogen in Italy. Mating type and female fertility distribution and effective population number, N ( e ), were assessed for a population of 181 F. verticillioides strains isolated from three fields located in Lombardia region (Northern Italy) during 2007-2008 maize growing season. The ratio of MAT-1:MAT-2 was significantly different from the theoretical 1:1 ratio expected in an idealized population in which individuals mate at random. The frequency of hermaphroditic strains was 20 % of the total population. N ( e ) for mating type was 89 % of the count (total population) and the N ( e ) for male or hermaphrodite status was 55 %. The number of isolates that can function as the female parent limited N ( e ) in the examined population. Under equilibrium cycle, assuming that female fertility has been lost due to selection and mutation rate during asexual reproduction, sexual reproduction needed to occur only once per 40 to 118 asexual generations to maintain this level of sexual fertility.

Enhancement of a pentacyclic tyrosine kinase inhibitor production in Cladosporium cf. cladosporioides by Cladosporol.

The binaphthyl derivative cladosporol 3 was supplied from 60 to 200 mg l(-1) to shaken cultures of Cladosporium cf. cladosporioides. Compared to blank, fungal biomass was not affected by adding cladosporol till 100 mg l(-1): it rather increased at higher ratios between 150 and 200 mg l(-1). The production of the major pentacyclic metabolite 1, a cytokine production and tyrosine kinase inhibitor, was enhanced tenfold when cladosporol was supplied at the highest ratio (200 mg l(-1)) to shaken growing cultures of the fungus. The bioconversion of cladosporol to cladosporol D through reductive cleavage of the epoxide group was also observed. Interest in this kind of metabolites lies in their potential activity vs DNA topoisomerase I.

Histological studies on the mycoparasitism of Cladosporium tenuissimum on urediniospores of Uromyces appendiculatus.

Interactions between the mycoparasite Cladosporium tenuissimum and the bean rust Uromyces appendiculatus were studied through light and electron microscopy in vitro at the host-parasite interface. Urediniospore germination decreased on contact with ungerminated C. tenuissimum conidia, possibly due to antibiosis mechanisms. C. tenuissimum grew towards the bean rust spores and coiled around their germ tubes. Penetration of the urediniospores occurred either enzymatically and/or mechanically, through appressorium or infection cushion structures, from which a thin penetrating hypha was generated. Enzyme production by the mycoparasite was suggested by the loosening of the matricial components of the spore wall, which sometimes left chitin fibrils visible. Mycoparasite hyphae grew within the host spore, emptied its content, and emerged profusely forming conidiophores and conidia. C. tenuissimum was able to grow on media containing laminarin, suggesting the ability of producing glucanases, but not when chitin was used as the sole carbon source. Conidia that had been grown on a sugar-rich medium, filtered, and extracted with organic solvents, were found to contain cladosporol and related compounds. Complete control of the bean rust disease was achieved by application of C. tenuissimum culture filtrates but not by conidial suspensions. This is the first report of parasitism by C. tenuissimum on U. appendiculatus. These investigations provide additional observations on a genus besides Melampsora and Cronartium from which this fungus has been isolated and tested to date. The possible role of environmental factors for the exploitation of this organism as a biocontrol agent is also mentioned.

Cryphonectric acid and other minor metabolites from a hypovirulent strain of Cryphonectria parasitica.

Investigations carried out on secondary metabolites produced in culture by a hypovirulent strain of Cryphonectria parasitica allowed the isolation of several compounds which were characterized by NMR analysis and derivatization reactions. The most abundant metabolite was a new compound, called cryphonectric acid (1). Other metabolites were diaporthin, the only known phytotoxic compound isolated from both virulent and hypovirulent strains of C. parasitica, (+)-orthosporin, and L-p-hydroxyphenyllactic acid (HOPLA). Root growth activity of the purified compounds was evaluated both on tomato seedlings and maize subapical segments.