Effects of in vitro brevetoxin exposure on apoptosis and cellular metabolism in a leukemic T cell line (Jurkat)

Harmful algal blooms (HABs) of the toxic dinoflagellate, Karenia brevis, produce red tide toxins, or brevetoxins. Significant health effects associated with red tide toxin exposure have been reported in sea life and in humans, with brevetoxins documented within immune cells from many species. The objective of this research was to investigate potential immunotoxic effects of brevetoxins using a leukemic T cell line (Jurkat) as an in vitro model system. Viability, cell proliferation, and apoptosis assays were conducted using brevetoxin congeners PbTx-2, PbTx-3, and PbTx-6. The effects of in vitro brevetoxin exposure on cell viability and cellular metabolism or proliferation were determined using trypan blue and MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), respectively. Using MTT, cellular metabolic activity was decreased in Jurkat cells exposed to 5 – 10 μg/ml PbTx-2 or PbTx-6. After 3 h, no significant effects on cell viability were observed with any toxin congener in concentrations up to 10 μg/ml. Viability decreased dramatically after 24 h in cells treated with PbTx-2 or -6. Apoptosis, as measured by caspase-3 activity, was significantly increased in cells exposed to PbTx-2 or PbTx-6. In summary, brevetoxin congeners varied in effects on Jurkat cells, with PbTx-2 and PbTx-6 eliciting greater cellular effects compared to PbTx-3.     

Eunicellin-Based Diterpenoids,Hirsutalins N–R,from the Formosan Soft Coral Cladiella hirsuta

New eunicellin-type hirsutalins N–R (1–5), along with two known eunicellins, (6 and 7) were isolated from the soft coral Cladiella hirsuta. The structures of the metabolites were determined by extensive spectroscopic analysis. Cytotoxic activity of compounds 1–7 against the proliferation of a limited panel of cancer cell lines was measured. The in vitro anti-inflammatory activity of compounds 1–7 was evaluated by measuring their ability in suppressing superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils.     

Bioactive Cembranoids,Sarcocrassocolides P–R,from the Dongsha Atoll Soft Coral Sarcophyton crassocaule

New cembranoids, sarcocrassocolides P–R (1–3) and four known compounds (4–7) were isolated from the soft coral Sarcophyton crassocaule. The structures of the metabolites were determined by extensive spectroscopic analysis. Compounds 3–5 and 7 were shown to exhibit cytotoxicity toward a limited panel of cancer cell lines and all compounds 1–7 displayed potent in vitro anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells by inhibiting the expression of inducible nitric oxide synthase (iNOS) protein. Compound 7 also showed significant activity in reducing the accumulation of cyclooxygenase-2 (COX-2) protein in the same macrophage cells.     

Novel Adociaquinone Derivatives from the Indonesian Sponge Xestospongia sp.

Seven new adociaquinone derivatives, xestoadociaquinones A (1a), B (1b), 14-carboxy-xestoquinol sulfate (2) and xestoadociaminals A–D (3a, 3c, 4a, 4c), together with seven known compounds (5–11) were isolated from an Indonesian marine sponge Xestospongia sp. Their structures were elucidated by extensive 1D and 2D NMR and mass spectrometric data. All the compounds were evaluated for their potential inhibitory activity against eight different protein kinases involved in cell proliferation, cancer, diabetes and neurodegenerative disorders as well as for their antioxidant and antibacterial activities.     

Eunicellin-Based Diterpenoids,Hirsutalins S–V,from the Formosan Soft Coral Cladiella hirsuta

Four new eunicellin-type hirsutalins S–V (1–4), along with a known compound (–)-6α-hydroxy polyanthellin A (5), were isolated from the soft coral Cladiella hirsuta. The structures of the metabolites were determined by extensive spectroscopic analysis. Cytotoxity of compounds 1–5 against the proliferation of a limited panel of cancer cell lines was measured. Anti-inflammatory activity of compounds 1–5 was evaluated by measuring their ability in suppressing superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils.     

Screening of In-Vitro Anti-Inflammatory and Antioxidant Activity of Sargassum ilicifolium Crude Lipid Extracts from Different Coastal Areas in Indonesia

Sargassum brown seaweed is reported to exhibit several biological activities which promote human health, such as anticancer, antimicrobial, antidiabetic, anti-inflammatory, and antioxidant activity. This study aimed to investigate the anti-inflammatory and antioxidant activity of crude lipid extracts of Sargassum ilicifolium obtained from four different coastal areas in Indonesia, namely Awur Bay–Jepara (AB), Pari Island–Seribu Islands (PI), Sayang Heulang Beach–Garut (SHB), and Ujung Genteng Beach–Sukabumi (UGB). Results showed that treatment of RAW 264.7 macrophage cells with UGB and AB crude lipid extracts (12.5–50 µg/mL) significantly suppressed the nitric oxide production after lipopolysaccharide stimulation, both in pre-incubated and co-incubated cell culture model. The anti-inflammatory effect was most marked in the pre-incubated cell culture model. Both two crude lipid extracts showed 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and high ferric reducing antioxidant power, which were amounted to 36.93–37.87 µmol Trolox equivalent/g lipid extract and 681.58–969.81 µmol FeSO₄/g lipid extract, respectively. From this study, we can conclude that crude lipid extract of tropical S. ilicifolium can be further developed as a source of anti-inflammatory and antioxidant agent.     

Semi-Synthesis,Cytotoxic Evaluation,and Structure—Activity Relationships of Brefeldin A Derivatives with Antileukemia Activity

Brefeldin A (1), a potent cytotoxic natural macrolactone, was produced by the marine fungus Penicillium sp. (HS-N-29) from the medicinal mangrove Acanthus ilicifolius. Series of its ester derivatives 2–16 were designed and semi-synthesized, and their structures were characterized by spectroscopic methods. Their cytotoxic activities were evaluated against human chronic myelogenous leukemia K562 cell line in vitro, and the preliminary structure–activity relationships revealed that the hydroxy group played an important role. Moreover, the monoester derivatives exhibited stronger cytotoxic activity than the diester derivatives. Among them, brefeldin A 7-O-2-chloro-4,5-difluorobenzoate (7) exhibited the strongest inhibitory effect on the proliferation of K562 cells with an IC₅₀ value of 0.84 µM. Further evaluations indicated that 7 induced cell cycle arrest, stimulated cell apoptosis, inhibited phosphorylation of BCR-ABL, and thereby inactivated its downstream AKT signaling pathway. The expression of downstream signaling molecules in the AKT pathway, including mTOR and p70S6K, was also attenuated after 7-treatment in a dose-dependent manner. Furthermore, molecular modeling of 7 docked into 1 binding site of an ARF1–GDP-GEF complex represented well-tolerance. Taken together, 7 had the potential to be served as an effective antileukemia agent or lead compound for further exploration.     

Briarane Diterpenes from the South China Sea Gorgonian Coral,Junceella gemmacea

Four new briarane diterpenoids, junceellolides M–P (1–4), were isolated together with seven known analogs (5–11) from the South China Sea gorgonian, Junceella gemmacea. The structures of these compounds were elucidated by detailed spectroscopic analysis and comparison with the reported data. The absolute configuration of compounds 1–3 were determined based on an ECD experiment, while the absolute configuration of compound 4 was genetically determined. All the compounds were isolated for the first time from J. gemmacea. These compounds showed no growth inhibitory activity against A549, MG63 and SMMC-7721 cell lines in an in vitro bioassay.     

Antitumor and Antimicrobial Potential of Bromoditerpenes Isolated from the Red Alga,Sphaerococcus coronopifolius

Cancer and infectious diseases continue to be a major public health problem, and new drugs are necessary. As marine organisms are well known to provide a wide range of original compounds, the aim of this study was to investigate the bioactivity of the main constituents of the cosmopolitan red alga, Sphaerococcus coronopifolius. The structure of several bromoditerpenes was determined by extensive spectroscopic analysis and comparison with literature data. Five molecules were isolated and characterized which include a new brominated diterpene belonging to the rare dactylomelane family and named sphaerodactylomelol (1), along with four already known sphaerane bromoditerpenes (2–5). Antitumor activity was assessed by cytotoxicity and anti-proliferative assays on an in vitro model of human hepatocellular carcinoma (HepG-2 cells). Antimicrobial activity was evaluated against four pathogenic microorganisms: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. Compound 4 exhibited the highest antimicrobial activity against S. aureus (IC₅₀ 6.35 µM) and compound 5 the highest anti-proliferative activity on HepG-2 cells (IC₅₀ 42.9 µM). The new diterpene, sphaerodactylomelol (1), induced inhibition of cell proliferation (IC₅₀ 280 µM) and cytotoxicity (IC₅₀ 720 µM) on HepG-2 cells and showed antimicrobial activity against S. aureus (IC₅₀ 96.3 µM).     

New Trichothecenes Isolated from the Marine Algicolous Fungus Trichoderma brevicompactum

Eight trichothecenes, including four new compounds 1–4 and four known entities 5–8, together with one known cyclonerane (9) were isolated from the solid-state fermentation of Trichoderma brevicompactum NTU439 isolated from the marine alga Mastophora rosea. The structures of 1–9 were determined by 1D/2D NMR (nuclear magnetic resonance), MS (mass spectrometry), and IR (infrared spectroscopy) spectroscopic data. All of the compounds were evaluated for cytotoxic activity against HCT-116, PC-3, and SK-Hep-1 cancer cells by the SRB assay, and compound 8 showed promising cytotoxic activity against all three cancer cell lines with the IC₅₀ values of 3.3 ± 0.3, 5.3 ± 0.3, and 1.8 ± 0.8 μM, respectively. Compounds 1–2, 4–6, and 7–8 potently inhibited LPS-induced NO production, and compounds 5 and 8 showed markedly inhibited gelatinolysis of MMP-9 in S1 protein-stimulated THP-1 monocytes.     

New Scalarane Sesterterpenoids from the Formosan Sponge Ircinia felix

Five new scalarane sesterterpenoids, felixins A–E (1–5), were isolated from the Formosan sponge Ircinia felix. The structures of scalaranes 1–5 were elucidated on the basis of spectroscopic analysis. Cytotoxicity of scalaranes 1–5 against the proliferation of a limited panel of tumor cell lines was evaluated.     

Bioactive Steroids from the Formosan Soft Coral Umbellulifera petasites

Three new steroids, petasitosterones A and B (1 and 2) and a spirosteroid petasitosterone C (3), along with eight known steroids (4–11), were isolated from a Formosan marine soft coral Umbellulifera petasites. The structures of these compounds were elucidated by extensive spectroscopic analysis and comparison of spectroscopic data with those reported. Compound 3 is a marine steroid with a rarely found A/B spiro[4,5]decane ring system. Compounds 1–3 and 5 displayed inhibitory activity against the proliferation of a limited panel of cancer cell lines, whereas 2 and 5 exhibited significant anti-inflammatory activity to inhibit nitric oxide (NO) production. The inhibitory activities for superoxide anion generation and elastase release of compounds 1–11 were also examined to evaluate the anti-inflammatory potential, and 2–4 were shown to exhibit significant activities.     

BDDE-Inspired Chalcone Derivatives to Fight Bacterial and Fungal Infections

The growing number of infectious diseases around the world threatens the effective response of antibiotics, contributing to the increase in antibiotic resistance seen as a global health problem. Currently, one of the main challenges in antimicrobial drug discovery is the search for new compounds that not only exhibit antimicrobial activity, but can also potentiate the antimicrobial activity and revert antibiotics’ resistance, through the interference with several mechanisms, including the inhibition of efflux pumps (EPs) and biofilm formation. Inspired by macroalgae brominated bromophenol BDDE with antimicrobial activity, a series of 18 chalcone derivatives, including seven chalcones (9–15), six dihydrochalcones (16–18, and 22–24) and five diarylpropanes (19–21, and 25 and 26), was prepared and evaluated for its antimicrobial activity and potential to fight antibiotic resistance. Among them, chalcones 13 and 14 showed promising antifungal activity against the dermatophyte clinical strain of Trichophyton rubrum, and all compounds reversed the resistance to vancomycin in Enterococcus faecalis B3/101, with 9, 14, and 24 able to cause a four-fold decrease in the MIC of vancomycin against this strain. Compounds 17–24 displayed inhibition of EPs and the formation of biofilm by S. aureus 272123, suggesting that these compounds are inhibiting the EPs responsible for the extrusion of molecules involved in biofilm-related mechanisms. Interestingly, compounds 17–24 did not show cytotoxicity in mouse embryonic fibroblast cell lines (NIH/3T3). Overall, the results obtained suggest the potential of dihydrochalcones 16–18 and 22–24, and diarylpropanes 19–21, 25 and 26, as hits for bacterial EPs inhibition, as they are effective in the inhibition of EPs, but present other features that are important in this matter, such as the lack of antibacterial activity and cytotoxicity.     

Precursor-Directed Biosynthesis Mediated Amplification of Minor Aza Phenylpropanoid Piperazines in an Australian Marine Fish-Gut-Derived Fungus,Chrysosporium sp. CMB-F214

Chemical analysis of an M1 agar plate cultivation of a marine fish-gut-derived fungus, Chrysosporium sp. CMB-F214, revealed the known chrysosporazines A–D (11–14) in addition to a suite of very minor aza analogues 1–6. A microbioreactor (MATRIX) cultivation profiling analysis failed to deliver cultivation conditions that significantly improved the yields of 1–6; however, it did reveal that M2 agar cultivation produced the new natural product 15. A precursor-directed biosynthesis strategy adopting supplementation of a CMB-F214 M1 solid agar culture with sodium nicotinate enhanced production of otherwise inaccessible azachrysposorazines A1 (1), A2 (2), B1 (3), C1 (4), C2 (5) and D1 (6), in addition to four new chrysosporazines; chrysosporazines N–P (7–9) and spirochrysosporazine A (10). Structures inclusive of absolute configurations were assigned to 1–15 based on detailed spectroscopic and chemical analyses, and biosynthetic considerations. Non-cytotoxic to human carcinoma cells, azachrysosporazies 1–5 were capable of reversing doxorubicin resistance in P-glycoprotein (P-gp)-overexpressing human colon carcinoma cells (SW620 Ad300), with optimum activity exhibited by the C-2′ substituted analogues 3–5.     

Six New Diterpene Glycosides from the Soft Coral Lemnalia bournei

A chemical study on the extracts of soft coral Lemnalia bournei resulted in the isolation and identification of six new bicyclic diterpene glycosides including three new lemnaboursides E–G (1–3), and three new lemnadiolboursides A–C (4–6), along with three known lemnaboursides (7–9). Their structures were elucidated by detailed spectroscopic analysis, ECD analysis, chemical methods, and comparison with the literature data. Lemnadiolboursides A–C (4–6) contained a lemnal-1(10)-ene-7,12-diol moiety compared with the lemnaboursides. All these compounds were evaluated for antibacterial activity; cell growth inhibition of A549, Hela, HepG2, and CCRF-CEM cancer cell lines; and inhibition of LPS-induced NO production in RAW264.7 macrophages. The results indicated that compounds 1, 2, and 4–6 exhibited antibacterial activity against Staphylococcus aureus and Bacillus subtilis (MIC 4–16 μg/mL); compounds 1–9 displayed low cytotoxicity on the CCRF-CEM cell lines (IC₅₀ 10.44–27.40 µM); and compounds 1, 2, and 5 showed weak inhibition against LPS-induced NO production (IC₅₀ 21.56–28.06 μM).     

The Oligo Fucoidan Inhibits Platelet-Derived Growth Factor-Stimulated Proliferation of Airway Smooth Muscle Cells

In the pathogenesis of asthma, the proliferation of airway smooth muscle cells (ASMCs) is a key factor in airway remodeling and causes airway narrowing. In addition, ASMCs are also the effector cells of airway inflammation. Fucoidan extracted from marine brown algae polysaccharides has antiviral, antioxidant, antimicrobial, anticlotting, and anticancer properties; however, its effectiveness for asthma has not been elucidated thus far. Platelet-derived growth factor (PDGF)-treated primary ASMCs were cultured with or without oligo-fucoidan (100, 500, or 1000 µg/mL) to evaluate its effects on cell proliferation, cell cycle, apoptosis, and Akt, ERK1/2 signaling pathway. We found that PDGF (40 ng/mL) increased the proliferation of ASMCs by 2.5-fold after 48 h (p < 0.05). Oligo-fucoidan reduced the proliferation of PDGF-stimulated ASMCs by 75%–99% after 48 h (p < 0.05) and induced G₁/G₀ cell cycle arrest, but did not induce apoptosis. Further, oligo-fucoidan supplementation reduced PDGF-stimulated extracellular signal-regulated kinase (ERK1/2), Akt, and nuclear factor (NF)-κB phosphorylation. Taken together, oligo-fucoidan supplementation might reduce proliferation of PDGF-treated ASMCs through the suppression of ERK1/2 and Akt phosphorylation and NF-κB activation. The results provide basis for future animal experiments and human trials.     

Low Temperature and Cold Stress Significantly Increase Saxitoxins (STXs) and Expression of STX Biosynthesis Genes sxtA4 and sxtG in the Dinoflagellate Alexandrium catenella

Toxic dinoflagellate Alexandrium spp. produce saxitoxins (STXs), whose biosynthesis pathway is affected by temperature. However, the link between the regulation of the relevant genes and STXs’ accumulation and temperature is insufficiently understood. In the present study, we evaluated the effects of temperature on cellular STXs and the expression of two core STX biosynthesis genes (sxtA4 and sxtG) in the toxic dinoflagellate Alexandrium catenella Alex03 isolated from Korean waters. We analyzed the growth rate, toxin profiles, and gene responses in cells exposed to different temperatures, including long-term adaptation (12, 16, and 20 °C) and cold and heat stresses. Temperature significantly affected the growth of A. catenella, with optimal growth (0.49 division/day) at 16 °C and the largest cell size (30.5 µm) at 12 °C. High concentration of STXs eq were detected in cells cultured at 16 °C (86.3 fmol/cell) and exposed to cold stress at 20→12 °C (96.6 fmol/cell) compared to those at 20 °C and exposed to heat stress. Quantitative real-time PCR (qRT-PCR) revealed significant gene expression changes of sxtA4 in cells cultured at 16 °C (1.8-fold) and cold shock at 20→16 °C (9.9-fold). In addition, sxtG was significantly induced in cells exposed to cold shocks (20→16 °C; 19.5-fold) and heat stress (12→20 °C; 25.6-fold). Principal component analysis (PCA) revealed that low temperature (12 and 16 °C) and cold stress were positively related with STXs’ production and gene expression levels. These results suggest that temperature may affect the toxicity and regulation of STX biosynthesis genes in dinoflagellates.     

Enzyme-Assisted Extraction of Bioactive Material from Chondrus crispus and Codium fragile and Its Effect on Herpes simplex Virus (HSV-1)

Codium fragile and Chondrus crispus are, respectively, green and red seaweeds which are abundant along the North Atlantic coasts. We investigated the chemical composition and antiviral activity of enzymatic extracts of C. fragile (CF) and C. crispus (CC). On a dry weight basis, CF consisted of 11% protein, 31% neutral sugars, 0.8% sulfate, 0.6% uronic acids, and 49% ash, while CC contained 27% protein, 28% neutral sugars, 17% sulfate, 1.8% uronic acids, and 25% ash. Enzyme-assisted hydrolysis improved the extraction efficiency of bioactive materials. Commercial proteases and carbohydrases significantly improved (p ≤ 0.001) biomass yield (40%–70% dry matter) as compared to aqueous extraction (20%–25% dry matter). Moreover, enzymatic hydrolysis enhanced the recovery of protein, neutral sugars, uronic acids, and sulfates. The enzymatic hydrolysates exhibited significant activity against Herpes simplex virus (HSV-1) with EC₅₀ of 77.6–126.8 μg/mL for CC and 36.5–41.3 μg/mL for CF, at a multiplicity of infection (MOI) of 0.001 ID₅₀/cells without cytotoxity (1–200 μg/mL). The extracts obtained from proteases (P1) and carbohydrases (C3) were also effective at higher virus MOI of 0.01 ID₅₀/cells without cytotoxity. Taken together, these results indicate the potential application of enzymatic hydrolysates of C. fragile and C. crispus in functional food and antiviral drug discovery.     

In Vitro Anticancer and Cancer-Preventive Activity of New Triterpene Glycosides from the Far Eastern Starfish Solaster pacificus

Sea stars or starfish (class Asteroidea) and holothurians or sea cucumbers (class Holothuroidea), belonging to the phylum Echinodermata (echinoderms), are characterized by different sets of glycosidic metabolites: the steroid type in starfish and the triterpene type in holothurians. However, herein we report the isolation of eight new triterpene glycosides, pacificusosides D–K (1–3, 5–9) along with the known cucumarioside D (4), from the alcoholic extract of the Far Eastern starfish Solaster pacificus. The isolated new compounds are closely related to the metabolites of sea cucumbers, and their structures of 1–3 and 5–9 were determined by extensive NMR and ESIMS techniques. Compounds 2, 5, and 8 have a new type of tetrasaccharide chain with a terminal non-methylated monosaccharide unit. Compounds 3, 6, and 9 contain another new type of tetrasaccharide chain, having 6-O-SO₃-Glc as one of the sugar units. The cytotoxic activity of 1–9 against non-cancerous mouse epidermal cells JB6 Cl41 and human melanoma cell lines SK-MEL-2, SK-MEL-28, and RPMI-7951 was determined by MTS assay. Compounds 1, 3, 4, 6, and 9 showed potent cytotoxicity against these cell lines, but the cancer selectivity (SI > 9) was observed only against the SK-MEL-2 cell line. Compounds 1, 3, 4, 6, and 9 at the non-toxic concentration of 0.1 μM significantly inhibited neoplastic cell transformation of JB6 Cl41 cells induced by chemical carcinogens (EGF, TPA) or ionizing radiation (X-rays and UVB). Moreover, compounds 1 and 4 at the non-toxic concentration of 0.1 µM possessed the highest inhibiting activity on colony formation among the investigated compounds and decreased the colonies number of SK-MEL-2 cells by 64% and 70%, respectively. Thus, triterpene glycosides 1 and 4 can be considered as prospective cancer-preventive and anticancer-compound leaders.     

Parathyrsoidins A–D,Four New Sesquiterpenoids from the Soft Coral Paralemnalia thyrsoides

Four new nardosinane-type sesquiterpenoids, parathyrsoidins A–D (1–4) were isolated from the soft coral Paralemnalia thyrsoides. The structures of parathyrsoidins A–D (1–4) were determined by extensive spectral analysis and their cytotoxicity against selected cancer cell lines as well as antiviral activity against human cytomegalovirus (HCMV) were evaluated in vitro.