Effects of bistheonellide A,an Actin-polymerization Inhibitor,on Chinese Hamster V79 Cells and on IL-8 Production in PMA-stimulated HL-60 Cells

Bioassay-guided isolation from the ethanol extract of a marine sponge Theonella sp. collected in Palau yielded bistheonellide A, which strongly inhibited the colony formation of Chinese hamster V79 cells (EC₅₀ = 6.8 nM). Bistheonellide A is an actin-polymerization inhibitor and was suggested to control cytokine production. Therefore, we attempted to detect an effect of this compound on IL-8 production in PMA-stimulated HL-60 cells. Interestingly, bistheonellide A did not modulate the production of IL-8 under cytotoxic concentrations as determined by LDH analysis. Although the correlation between the inhibition of microtubule assembly and the stimulation of IL-8 production has been observed for several compounds, the polymerization of actin was not related to an IL-8 production in the case of bistheonellide A. It will be suggested that the actin polymerization is not involved in the IL-8 production system.     

Effects of melophlins on colony formation of Chinese hamster V79 cells and IL-8 production in PMA-stimulated HL-60 cells

We have recently isolated four new melophlins P (1), Q (2), R (3), and S (4) together with seven known melophlins A (5), D (6), E (7), G (8), H (9), I (10), and O (11) from two marine sponges of the genus Melophlus collected in Palau. In this study, the influence of these compounds on the colony formation of Chinese hamster V79 cells and the production of IL-8 in PMA-stimulated HL-60 cells were examined. These 11 compounds did not show any effect on IL-8 production. The EC₅₀ values of compounds 2, 3, 4, 5, 7, 9, 10, and 11 against V79 cells were 44.0, 13.3, 16.7, 27.2, 19.8, 8.5, 23.1, and 9.6 μM, respectively. The linear-chain-type compounds (1, 6, and 8) were not active against V79 cells at 50 μM. Although the growth inhibitory activity of these melophlins was not remarkable, some structure-activity relationships of these compounds against V79 and murine leukemia L1210 cells were observed.     

Effects of Lissoclibadins and Lissoclinotoxins,Isolated from a Tropical Ascidian Lissoclinum cf. badium,on IL-8 production in a PMA-stimulated Promyelocytic Leukemia Cell Line

The effects of seven compounds 1–7, isolated from a tropical ascidian Lissoclinum cf. badium, on IL-8 production in PMA-stimulated HL-60 cells were examined. Lissoclibadins 2 (2) and 3 (3) and lissoclinotoxin F (5) increased the IL-8 production in a dose-dependent manner. Compounds 2 and 5 are structural isomers possessing dimeric structures of trans and cis-orientations, respectively, and showed a very similar activity on the induction of IL-8 levels. Compound 3 and lissoclinotoxin E (4) are also structural isomers having dimeric trans and cis-structures, respectively, but 4 did not induce the IL-8 production. Lissoclibadin 1 (1, trimeric compound) and two monomeric compounds (6 and 7) did not increase the IL-8 level. Therefore, the differences in their structures remarkably affected the IL-8 production activity, the inhibition of cell proliferation, and the survival of HL-60 cells. Lissoclibadin 2 was the most interesting compound of the seven metabolites tested in this study.     

Briarane Diterpenoids from the Gorgonian Dichotella gemmacea

Seven new briarane diterpenoids, gemmacolides AS-AY (1–7), were isolated together with ten known analogues (8–17) from the South China Sea gorgonian Dichotella gemmacea. The structures of the new compounds were elucidated by the detailed analysis of spectroscopic data and comparison with reported data. The absolute configuration of compounds was determined based on electronic circular dichroism (ECD) experiments and genetic correlations as well. Compounds 15 and 16 were reported for the first time for the gorgonian. In the preliminary in vitro bioassays, compound 5 showed potential growth inhibitory activity against MG63 cells.     

Capgermacrenes A and B,Bioactive Secondary Metabolites from a Bornean Soft Coral,Capnella sp.

Two new bicyclogermacrenes, capgermacrenes A (1) and B (2), were isolated with two known compounds, palustrol (3) and litseagermacrane (4), from a population of Bornean soft coral Capnella sp. The structures of these metabolites were elucidated based on spectroscopic data. Compound 1 was found to inhibit the accumulation of the LPS-induced pro-inflammatory IL-1β and NO production by down-regulating the expression of iNOS protein in RAW 264.7 macrophages.     

Apoptogenic Metabolites in Fractions of the Benthic Diatom Cocconeis scutellum parva

Benthic diatoms of the genus Cocconeis contain a specific apoptogenic activity. It triggers a fast destruction of the androgenic gland in the early post-larval life of the marine shrimp Hippolyte inermis, leading to the generation of small females. Previous in vitro investigations demonstrated that crude extracts of these diatoms specifically activate a dose-dependent apoptotic process in human cancer cells (BT20 breast carcinoma) but not in human normal lymphocytes. Here, a bioassay-guided fractionation has been performed to detect the apoptogenic compound(s). Various HPLC separation systems were needed to isolate the active fractions, since the apoptogenic metabolite is highly active, present in low amounts and is masked by abundant but non-active cellular compounds. The activity is due to at least two compounds characterized by different polarities, a hydrophilic and a lipophilic fraction. We purified the lipophilic fraction, which led to the characterization of an active sub-fraction containing a highly lipophilic compound, whose molecular structure has not yet been identified, but is under investigation. The results point to the possible medical uses of the active compound. Once the molecular structure has been identified, the study and modulation of apoptotic processes in various types of cells will be possible.     

High-Throughput Screening of a Marine Compound Library Identifies Anti-Cryptosporidium Activity of Leiodolide A

Cryptosporidium sp. are apicomplexan parasites that cause significant morbidity and possible mortality in humans and valuable livestock. There are no drugs on the market that are effective in the population most severely affected by this parasite. This study is the first high-throughput screen for potent anti-Cryptosporidium natural products sourced from a unique marine compound library. The Harbor Branch Oceanographic Institute at Florida Atlantic University has a collection of diverse marine organisms some of which have been subjected to medium pressure liquid chromatography to create an enriched fraction library. Numerous active compounds have been discovered from this library, but it has not been tested against Cryptosporidium parvum. A high-throughput in vitro growth inhibition assay was used to test 3764 fractions in the library, leading to the identification of 23 fractions that potently inhibited the growth of Cryptosporidium parvum. Bioassay guided fractionation of active fractions from a deep-sea sponge, Leiodermatium sp., resulted in the purification of leiodolide A, the major active compound in the organism. Leiodolide A displayed specific anti-Cryptosporidium activity at a half maximal effective concentration of 103.5 nM with selectivity indexes (SI) of 45.1, 11.9, 19.6 and 14.3 for human ileocecal colorectal adenocarcinoma cells (HCT-8), human hepatocellular carcinoma cells (Hep G2), human neuroblastoma cells (SH-SY5Y) and green monkey kidney cells (Vero), respectively. The unique structure of leiodolide A provides a valuable drug scaffold on which to develop new anti-Cryptosporidium compounds and supports the importance of screening natural product libraries for new chemical scaffolds.     

Seven New and Two Known Lipopeptides as well as Five Known Polyketides: The Activated Production of Silent Metabolites in a Marine-Derived Fungus by Chemical Mutagenesis Strategy Using Diethyl Sulphate

AD-2-1 is an antitumor fungal mutant obtained by diethyl sulfate mutagenesis of a marine-derived Penicillium purpurogenum G59. The G59 strain originally did not produce any metabolites with antitumor activities in MTT assays using K562 cells. Tracing newly produced metabolites under guidance of MTT assay and TLC analysis by direct comparison with control G59 extract, seven new (1–7) and two known (8–9) lipopeptides were isolated together with five known polyketides 10–14 from the extract of mutant AD-2-1. Structures of the seven new compounds including their absolute configurations were determined by spectroscopic and chemical evidences and named as penicimutalides A–G (1–7). Seven known compounds were identified as fellutamide B (8), fellutamide C (9), 1′-O-methylaverantin (10), averantin (11), averufin (12), nidurufin (13), and sterigmatocystin (14). In the MTT assay, 1–14 inhibited several human cancer cell lines to varying extents. All the bioassays and HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses demonstrated that the production of 1–14 in the mutant AD-2-1 was caused by the activated production of silent metabolites in the original G59 fungal strain. Present results provided additional examples for effectiveness of the chemical mutagenesis strategy using diethyl sulphate mutagenesis to discover new compounds by activating silent metabolites in fungal isolates.     

Preclinical Pharmacology of BA-TPQ,a Novel Synthetic Iminoquinone Anticancer Agent

Marine natural products and their synthetic derivatives represent a major source of novel candidate anti-cancer compounds. We have recently tested the anti-cancer activity of more than forty novel compounds based on an iminoquinone makaluvamine scaffold, and have found that many of the compounds exert potent cytotoxic activity against human cancer cell lines. One of the most potent compounds, BA-TPQ [(11,12),7-(benzylamino)-1,3,4,8-tetrahydropyrrolo[4,3,2-de]quinolin-8(1H)-one], was active against a variety of human cancer cell lines, and inhibited the growth of breast and prostate xenograft tumors in mice. However, there was some toxicity noted in the mice following administration of the compound. In order to further the development of BA-TPQ, and in a search for potential sites of accumulation that might underlie the observed toxicity of the compound, we accomplished preclinical pharmacological studies of the compound. We herein report the in vitro and in vivo pharmacological properties of BA-TPQ, including its stability in plasma, plasma protein binding, metabolism by S9 enzymes, and plasma and tissue distribution. We believe these studies will be useful for further investigations, and may be useful for other investigators examining the use of similar compounds for cancer therapy.     

Protective Effect of Flavonoids from a Deep-Sea-Derived Arthrinium sp. against ox-LDL-Induced Oxidative Injury through Activating the AKT/Nrf2/HO-1 Pathway in Vascular Endothelial Cells

Oxidized low-density lipoprotein (ox-LDL)-induced oxidative injury in vascular endothelial cells is crucial for the progression of cardiovascular diseases, including atherosclerosis. Several flavonoids have been shown cardiovascular protective effects. Recently, our research group confirmed that the novel flavonoids isolated from the deep-sea-derived fungus Arthrinium sp., 2,3,4,6,8-pentahydroxy-1-methylxanthone (compound 1) and arthone C (compound 2) effectively scavenged ROS in vitro. In this study, we further investigated whether these compounds could protect against ox-LDL-induced oxidative injury in endothelial cells and the underlying mechanisms. Our results showed that compounds 1 and 2 inhibited ox-LDL-induced apoptosis and adhesion factors expression in human umbilical vein vascular endothelial cells (HUVECs). Mechanistic studies showed that these compounds significantly inhibited the ROS level increase and the NF-κB nuclear translocation induced by ox-LDL. Moreover, compounds 1 and 2 activated the Nrf2 to transfer into nuclei and increased the expression of its downstream antioxidant gene HO-1 by inducing the phosphorylation of AKT in HUVECs. Importantly, the AKT inhibitor MK-2206 2HCl or knockdown of Nrf2 by RNA interference attenuated the inhibition effects of these compounds on ox-LDL-induced apoptosis in HUVECs. Meanwhile, knockdown of Nrf2 abolished the effects of the compounds on ox-LDL-induced ROS level increase and the translocation of NF-κB to nuclei. Collectively, the data showed that compounds 1 and 2 protected endothelial cells against ox-LDL-induced oxidative stress through activating the AKT/Nrf2/HO-1 pathway. Our study provides new strategies for the design of lead compounds for related cardiovascular diseases treatment.     

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.     

Inhibitory Effect of N,N-Didesmethylgrossularine-1 on Inflammatory Cytokine Production in Lipopolysaccharide-Stimulated RAW 264.7 Cells

N,N-Didesmethylgrossularine-1 (DDMG-1), a compound with a rare α-carboline structure, was isolated from an Indonesian ascidian Polycarpa aurata as responsible for the observed inhibitory activity against TNF-α production in lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells. DDMG-1 inhibited the mRNA level of mTNF-α, IκB-α degradation, and binding of NF-κB to the target DNA site in LPS-stimulated RAW 264.7 cells. Moreover, DDMG-1 had an inhibitory effect on the production of IL-8, which is produced in CD14⁺-THP-1 cells stimulated by LPS. DDMG-1 is thus a promising drug candidate lead compound for the treatment of chronic inflammatory diseases, such as rheumatoid arthritis.     

Prolamin,a rice protein,augments anti-leukaemia immune response

Rice (Oryza sativa), an important cereal as a staple food worldwide, can be used in gluten-free diets. This study aimed to isolate and characterize the active rice proteins and assess the anti-leukaemia response via in vitro and ex vivo experiments. Temperature-stable protein-containing extracts augmented this effect. Proteomic analysis identified various protein spots known with functions involving metabolism-related, transport, storage, antioxidation, development, and disease-resistance proteins. Among these, storage proteins were the most abundant. To avoid masking the other relatively scanty rice storage proteins, albumin, globulin, glutelin and prolamin were separated and quantified. The human peripheral blood mononuclear cells-conditioned medium (PBMC-CM) prepared from prolamin treatment showed an increment in production of tumor necrosis factor-α. Human leukaemia U937 cells cultured in the presence of prolamin-prepared PBMC-CM were inhibited in growth capacity and were triggered differentiation toward monocytes. Neutralization of prolamin by polyclonal antibody attenuated its activity. Rice prolamin, undetectable by wheat gliadin-specific antibody, has greater anti-leukaemia activity than wheat proteins glutenin and gliadin. In conclusion, rice prolamin is effective in activating human anti-leukaemia immunity and may not induce unwanted inflammatory diseases.     

Cubitanoids and Cembranoids from the Soft Coral Sinularia nanolobata

Two new cubitanoids, nanoculones A and B (1 and 2), and three new cembranoids, nanolobols A–C (3–5), as well as six known compounds, calyculone I (6), sinulariuol A (7), sinulariols C, D, H, and J (8–11), were isolated from the soft coral Sinularia nanolobata, collected off the coast of the eastern region of Taiwan. Their structures were elucidated on the basis of extensive spectroscopic analysis. Cytotoxicity of compounds 1–11 was evaluated. The nitric oxide (NO) inhibitory activity of selected compounds was further measured by assay of lipopolysaccharide (LPS)-stimulated NO production in activated RAW264.7 cells. The results showed that none of 1–11 exhibited cytotoxicity against the tested cancer cell lines, whereas compound 8 was found to significantly reduce NO production.     

Anti-Inflammatory Polyketides from an Alga-Derived Fungus Aspergillus ochraceopetaliformis SCSIO 41020

A new linear polyketide, named aspormisin A (1), together with five known polyketides (2–6), were isolated from the alga-derived fungus Aspergillus ochraceopetaliformis SCSIO 41020. Their structures were elucidated through a detailed comprehensive spectroscopic analysis, as well as a comparison with the literature. An anti-inflammatory evaluation showed that compounds 2, 5, and 6 possessed inhibitory activity against the excessive production of nitric oxide (NO) and pro-inflammatory cytokines in LPS-treated RAW 264.7 macrophages in a dose-dependent manner without cytotoxicity. Further studies revealed that compound 2 was active in blocking the release of pro-inflammatory cytokines (IL-6, MCP-1, and TNF-α) induced by LPS both in vivo and in vitro. Our findings provide a basis for the further development of linear polyketides as promising anti-inflammatory agents.     

Inhibition of A549 Lung Cancer Cell Migration and Invasion by Ent-Caprolactin C via the Suppression of Transforming Growth Factor-β-Induced Epithelial—Mesenchymal Transition

The epithelial–mesenchymal transition (EMT) of cancer cells is a crucial process in cancer cell metastasis. An Aquimarina sp. MC085 extract was found to inhibit A549 human lung cancer cell invasion, and caprolactin C (1), a new natural product, α-amino-ε-caprolactam linked to 3-methyl butanoic acid, was purified through bioactivity-guided isolation of the extract. Furthermore, its enantiomeric compound, ent-caprolactin C (2), was synthesized. Both 1 and 2 inhibited the invasion and γ-irradiation-induced migration of A549 cells. In transforming growth factor-β (TGF-β)-treated A549 cells, 2 inhibited the phosphorylation of Smad2/3 and suppressed the EMT cell marker proteins (N-cadherin, β-catenin, and vimentin), as well as the related messenger ribonucleic acid expression (N-cadherin, matrix metalloproteinase-9, Snail, and vimentin), while compound 1 did not suppress Smad2/3 phosphorylation and the expression of EMT cell markers. Therefore, compound 2 could be a potential candidate for antimetastatic agent development, because it suppresses TGF-β-induced EMT.     

Toxicity of quinones against two-spotted spider mite and three species of aphids in laboratory and greenhouse conditions

Toxicities of the eight quinones were evaluated through leaf dip bioassays conducted against Tetranychus urticae, Myzus persicae, Myzocallis walshii, and Illinoia liriodendri. Based on LC₅₀ values, plumbagin (LC₅₀ = 0.001%) was the most active compound against T. urticae and ubiquinone Q₀ (LC₅₀ = 0.005%), plumbagin (LC₅₀ = 0.010%), and dibromothymoquinone (LC₅₀ = 0.012%) were the most active compounds against M. persicae. The most active compounds against M. walshii were juglone (LC₅₀ = 0.011%) and ubiquinone Q₀ (LC₅₀ = 0.019%), whereas dibromothymoquinone (LC₅₀ = 0.030%), plumbagin (LC₅₀ = 0.033%) and ubiquinone Q₀ (LC₅₀ = 0.058%) were the most toxic to I. liriodendri. Ecotrol (positive control) was the least toxic compound (LC₅₀ = 0.39%) against T. urticae and M. persicae (LC₅₀ = 0.447%). Although the majority of the compounds tested were toxic to all four test species in residual bioassays, there was little overlap among the test species in terms of susceptibility to the compounds and interspecific differences were observed. Regarding structure-activity relationships for quinones, the addition of a hydroxyl group resulted in a significant increase in the toxicity of the 1,4-naphthoquinones, and those possessing a methyl group exhibited the highest levels of activity in T. urticae. The bromine atom at the 2- and 5-positions of the benzoquinone ring is crucial to the toxicity of the compounds against I. liriodendri. Toxicity was greatly affected not only by the number of hydroxyl groups, but also by their positions in the ring in the case of M. walshii. Juglone and plumbagin as residual toxins in the laboratory also reduced the population of two-spotted spider mites compared to EcoTrol™ (positive control) and the negative control in the greenhouse experiment. Some quinones tested may have potential as commercial insecticides and miticides, or alternatively, could serve as lead compounds for the development of more potent crop protection agents.