Acetone Fraction of the Red Marine Alga Laurencia papillosa Reduces the Expression of Bcl-2 Anti-apoptotic Marker and Flotillin-2 Lipid Raft Marker in MCF-7 Breast Cancer Cells.

Marine macroalgae have attracted much attention in recent years as a valuable source of bioactive metabolites. The cytotoxic potential of the Laurencia papillosa red alga collected from the Lebanese coast has been investigated on human breast cancer cells MCF-7. The crude extract of Laurencia papillosa (L. papillosa) was fractionated by column chromatography using a series of increasingly polar solvents (methylene chloride, acetone and methanol). Cytotoxicity of the crude extract and fractions was determined by MTT assay in MCF-7 cells. Apoptosis was detected by annexin V/propidium iodide assay and by measurement of Bcl-2 expression. Flotillin-2 expression was examined using RT-qPCR and Western blot. The crude extract, and the fractions of CH2Cl2 and acetone exhibited a dose-dependent cytotoxic effect on MCF-7 cells. Apoptosis was specifically induced by one of the acetone fractions having the highest cytotoxicity. It has been demonstrated by an increase in late phase apoptotic cell populations, and a decrease in Bcl-2 anti-apoptotic marker expression on mRNA and protein levels in a dose- and time- dependent manner. Furthermore, this active fraction decreased Flotillin-2 expression associated with cancer progression. Our data suggest that L. papillosa is an important source of cytotoxic metabolites. Further studies are needed for the chemical characterization of the metabolite associated with observed biological activities.

4-cholesten-3-one decreases breast cancer cell viability and alters membrane raft-localized EGFR expression by reducing lipogenesis and enhancing LXR-dependent cholesterol transporters.

BACKGROUND: The alteration of lipid metabolism in cancer cells is recognized as one of the most important metabolic hallmarks of cancer. Membrane rafts defined as plasma membrane microdomains enriched in cholesterol and sphingolipids serve as platforms for signaling regulation in cancer. The main purpose of this study was to evaluate the effect of the cholesterol metabolite, 4-cholesten-3-one, on lipid metabolism and membrane raft integrity in two breast cancer cell lines, MCF-7 and MDA-MB-231. Its ability to reduce cell viability and migration has also been investigated. METHODS: RT-qPCR was performed to evaluate the expression of enzymes involved in lipogenesis and cholesterol synthesis, and ABCG1 and ABCA1 transporters involved in cholesterol efflux. Its effect on cell viability and migration was studied using the MTT assay, the wound healing assay and the Transwell migration assay, respectively. The effect of 4-cholesten-3-one on membrane rafts integrity was investigated by studying the protein expression of flotillin-2, a membrane raft marker, and raft-enriched EGFR by western blot. RESULTS: Interestingly, we found that 4-cholesten-3-one treatment decreased mRNA expression of different enzymes including ACC1, FASN, SCD1 and HMGCR. We further demonstrated that 4-cholesten-3-one increased the expression of ABCG1 and ABCA1. We also found that 4-cholesten-3-one decreased the viability of MCF-7 and MDA-MB-231 cells. This effect was neutralized after treatment with LXR inverse agonist or after LXRß knockdown by siRNA. As a result, we also demonstrated that 4-cholesten-3-one disrupts membrane rafts and cell migration capacity. CONCLUSION: Our results show that 4-cholesten-3-one exerts promising antitumor activity by altering LXR-dependent lipid metabolism in breast cancer cells without increasing lipogenesis.

Marine natural products and related compounds as anticancer agents: an overview of their clinical status.

Marine ecosystems constitute a huge reservoir of biologically active secondary metabolites. Consequently during the last past few decades, several marine-derived molecules have been approved for anticancer treatment or are under clinical trials. This review reports the present state of the art of the sixteen molecules approved or currently on the clinical pipeline for anticancer chemotherapy. The molecules are classified according to their current status in the phase (approved / phase IV / phase III / phase II / phase I) and data are updated to April 2012.

Search for hydrophilic marine fungal metabolites: a rational approach for their production and extraction in a bioactivity screening context.

In the search for bioactive natural products, our lab screens hydrophobic extracts from marine fungal strains. While hydrophilic active substances were recently identified from marine macro-organisms, there was a lack of reported metabolites in the marine fungi area. As such, we decided to develop a general procedure for screening of hydrophobic metabolites. The aim of this study was to compare different processes of fermentation and extraction, using six representative marine fungal strains, in order to define the optimized method for production. The parameters studied were (a) which polar solvent to select, (b) which fermentation method to choose between solid and liquid cultures, (c) which raw material, the mycelium or its medium, to extract and (d) which extraction process to apply. The biochemical analysis and biological evaluations of obtained extracts led to the conclusion that the culture of marine fungi by agar surface fermentation followed by the separate extraction of the mycelium and its medium by a cryo-crushing and an enzymatic digestion with agarase, respectively, was the best procedure when screening for hydrophilic bioactive metabolites. During this development, several bioactivities were detected, confirming the potential of hydrophilic crude extracts in the search for bioactive natural products.

Enhancement of domoic acid neurotoxicity on Diptera larvae bioassay by marine fungal metabolites.

Peptaibols are small linear fungal peptides which are produced in the marine environment. They exhibit neurotoxicity by forming pores in neuronal membranes. This work describes their combine effect with domoic acid, a neurotoxic phycotoxin, on Diptera larvae. The Acute toxicity bioassay on this biological model was tested with a panel of different toxins (microbial, algal or fungal). It allowed the discrimination of neurotoxins and non-neurotoxic toxins, and an evaluation of the toxicity level (MED and ED(50)) which were correlated with published LD(50) in mice for neurotoxins tested. The highest activities on this test were found for Na(+) channel blockers tetrodotoxin (ED(50) = 0.026 mg/kg) and saxitoxin (ED(50) = 0.18 mg/kg). Domoic acid was less active with an ED(50) = 7.6 mg/kg. For synergism study, longibrachin-A-I, a 20-mer peptaibol isolated from cultures of a marine-derived strain of Trichoderma longibrachiatum Rifai was chosen. Bioassay results confirmed its neuroactivity. Its level of toxicity (ED(50) = 270 mg/kg) was lower than those of phycotoxins tested but higher than mycotoxin ones. Injected together, longibrachin-A-I and domoic acid exhibited an increase of their activities. With doses of longibrachin-A-I below its Minimal Effective Dose (MED), the synergism factor which expresses the enhancement of domoic acid toxicity could reach 34.5. Both domoic acid and longibrachin-A-I are acting on ion channels and pores in neuronal membranes which contribute to the intake of Ca(2+) into cells.

New Trichobrachins, 11-residue peptaibols from a marine strain of Trichoderma longibrachiatum.

A marine strain of Trichoderma longibrachiatum isolated from blue mussels (Mytilus edulis) was investigated for short peptaibol production. Various 11-residue peptaibols, obtained as microheterogenous mixtures after a chromatographic fractionation, were identified by positive mass spectrometry fragmentation (ESI-IT-MS(n), CID-MS(n) and GC/EI-MS). Thirty sequences were identified, which is the largest number of analogous sequences so far observed at once. Twenty-one sequences were new, and nine others corresponded to peptaibols already described. These peptaibols belonged to the same peptidic family based on the model Ac-Aib-xxx-xxx-xxx-Aib-Pro-xxx-xxx-Aib-Pro-xxol. They were named trichobrachin A when the residue in position 2 was an Asn, and trichobrachin C when it was a Gln. Major chromatographic sub-fractions, corresponding to purified peptaibols, were assayed for their cytotoxic activity. Trichobrachin A-IX and trichobrachin C exhibited the highest activities. There was an exponential relation between their relative hydrophobicity and their cytotoxicity on KB cells.

A new and rapid bioassay for the detection of gliotoxin and related epipolythiodioxopiperazines produced by fungi.

Gliotoxin is an immunosuppressive cytotoxin produced by numerous environmental or pathogenic fungal species. For this reason, it is one of the mycotoxins which must be systematically searched for in samples for biological control. In this study, a new, rapid and sensitive method for detecting gliotoxin has been developed. This bioassay is based on the induction of morphological changes in cultured cells (human KB cell line) by gliotoxin. Interpretation of the assay can be carried out after 1 h of incubation, either by direct microscopic observation, or with an automated microplate-reader at 630 nm. The limit of detection is 18-20 ng of gliotoxin in the well, depending on the used observation method. A high degree of specificity of the detection is brought about by the ability of the reducing reactant dithiothreitol to inhibit the biological activities of epipolythiodioxopiperazines (ETPs), such as gliotoxin, by reducing their polysulfide bridge. The bioassay allows a rapid primary screening of samples and a semi-quantitative evaluation of the gliotoxin concentration in extracts. The method has been used to study the gliotoxin production by different fungal strains, allowing to highlight 3 strains of Aspergillus fumigatus producing gliotoxin in various extracts.