Chemical Constituents and Anticancer Activities of Marine-Derived Fungus Trichoderma lixii.

The fungal genus Trichoderma is a rich source of structurally diverse secondary metabolites with remarkable pharmaceutical properties. The chemical constituents and anticancer activities of the marine-derived fungus Trichoderma lixii have never been investigated. In this study, a bioactivity-guided investigation led to the isolation of eleven compounds, including trichodermamide A (1), trichodermamide B (2), aspergillazine A (3), DC1149B (4), ergosterol peroxide (5), cerebrosides D/C (6/7), 5-hydroxy-2,3-dimethyl-7-methoxychromone (8), nafuredin A (9), and harzianumols E/F (10/11). Their structures were identified by using various spectroscopic techniques and compared to those in the literature. Notably, compounds 2 and 5-11 were reported for the first time from this species. Evaluation of the anticancer activities of all isolated compounds was carried out. Compounds 2, 4, and 9 were the most active antiproliferative compounds against three cancer cell lines (human myeloma KMS-11, colorectal HT-29, and pancreas PANC-1). Intriguingly, compound 4 exhibited anti-austerity activity with an IC50 of 22.43 µM against PANC-1 cancer cells under glucose starvation conditions, while compound 2 did not.

Oxy-Polybrominated Diphenyl Ethers from the Indonesian Marine Sponge, Lamellodysidea herbacea: X-ray, SAR, and Computational Studies.

Polybrominated diphenyl ether (PBDE) compounds, derived from marine organisms, originate from symbiosis between marine sponges and cyanobacteria or bacteria. PBDEs have broad biological spectra; therefore, we analyzed structure and activity relationships of PBDEs to determine their potential as anticancer or antibacterial lead structures, through reactions and computational studies. Six known PBDEs (1-6) were isolated from the sponge, Lamellodysdiea herbacea; 13C NMR data for compound 6 are reported for the first time and their assignments are confirmed by their theoretical 13C NMR chemical shifts (RMSE < 4.0 ppm). Methylation and acetylation of 1 (2, 3, 4, 5-tetrabromo-6-(3', 5'-dibromo-2'-hydroxyphenoxy) phenol) at the phenol functional group gave seven molecules (7-13), of which 10, 12, and 13 were new. New crystal structures for 8 and 9 are also reported. Debromination carried out on 1 produced nine compounds (1, 2, 14, 16-18, 20, 23, and 26) of which 18 was new. Debromination product 16 showed a significant IC50 8.65 ± 1.11; 8.11 ± 1.43 µM against human embryonic kidney (HEK293T) cells. Compounds 1 and 16 exhibited antibacterial activity against Gram-positive Staphylococcus aureus and Gram-negative Klebsiella pneumoniae with MID 0.078 µg/disk. The number of four bromine atoms and two phenol functional groups are important for antibacterial activity (S. aureus and K. pneumoniae) and cytotoxicity (HEK293T). The result was supported by analysis of frontier molecular orbitals (FMOs). We also propose possible products of acetylation and debromination using analysis of FMOs and electrostatic charges and we confirm the experimental result.

3-(Phenethylamino)demethyl(oxy)aaptamine as an anti-dormant mycobacterial substance: Isolation, evaluation and total synthesis.

3-(Phenethylamino)demethyl(oxy)aaptamine (1) was re-discovered from the marine sponge of Aaptos sp. as an anti-dormant mycobacterial substance through the bioassay-guided separation. Compound 1 showed potent anti-microbial activity against Mycobacterium bovis BCG with a minimum inhibitory concentration of 0.75 µg/mL under both aerobic conditions and hypoxic conditions inducing dormant state. Compound 1 was also effective against pathogenic M. tuberculosis strains including clinical multidrug-resistant strains. Furthermore, the successful total syntheses of 1 and its analog 3-aminodemethyl(oxy)aaptamine (2) afford sufficient quantities for further biological studies.

Mitochondrial Targeting in an Anti-Austerity Approach Involving Bioactive Metabolites Isolated from the Marine-Derived Fungus Aspergillus sp.

The tumor microenvironment is a nutrient-deficient region that alters the cancer cell phenotype to aggravate cancer pathology. The ability of cancer cells to tolerate nutrient starvation is referred to as austerity. Compounds that preferentially target cancer cells growing under nutrient-deficient conditions are being employed in anti-austerity approaches in anticancer drug discovery. Therefore, in this study, we investigated physcion (1) and 2-(2',3-epoxy-1',3',5'-heptatrienyl)-6-hydroxy-5-(3-methyl-2-butenyl) benzaldehyde (2) obtained from a culture extract of the marine-derived fungus Aspergillus species (sp.), which were isolated from an unidentified marine sponge, as anti-austerity agents. The chemical structures of 1 and 2 were determined via spectroscopic analysis and comparison with authentic spectral data. Compounds 1 and 2 exhibited selective cytotoxicity against human pancreatic carcinoma PANC-1 cells cultured under glucose-deficient conditions, with IC50 values of 6.0 and 1.7 µM, respectively. Compound 2 showed higher selective growth-inhibitory activity (505-fold higher) under glucose-deficient conditions than under general culture conditions. Further analysis of the mechanism underlying the anti-austerity activity of compounds 1 and 2 against glucose-starved PANC-1 cells suggested that they inhibited the mitochondrial electron transport chain.

Target Identification of the Marine Natural Products Dictyoceratin-A and -C as Selective Growth Inhibitors in Cancer Cells Adapted to Hypoxic Environments.

Hypoxia-adapted cancer cells in tumors contribute to the pathological progression of cancer. The marine spongean sesquiterpene phenols dictyoceratin-A (1) and -C (2) have been shown to induce hypoxia-selective growth inhibition in cultured cancer cells and exhibit in vivo antitumor effects. These compounds inhibit the accumulation of hypoxia-inducible factor-1α (HIF-1α), which is a drug target in hypoxia-adapted cancer cells, under hypoxic conditions. However, the target molecules of compounds 1 and 2, which are responsible for decreasing HIF-1α expression under hypoxic conditions, remain unclear. In this study, we synthesized probe molecules for compounds 1 and 2 to identify their target molecules and found that both compounds bind to RNA polymerase II-associated protein 3 (RPAP3), which is a component of the R2TP/Prefoldin-like (PEDL) complex. In addition, RPAP3-knockdown cells showed a phenotype similar to that of compound-treated cells.

Biakamides A-D, Unique Polyketides from a Marine Sponge, Act as Selective Growth Inhibitors of Tumor Cells Adapted to Nutrient Starvation.

Biakamides A-D, novel unusually unique polyketides, were isolated from an Indonesian marine sponge (Petrosaspongia sp.) with a constructed bioassay using PANC-1 human pancreatic cancer cells. Through detailed analyses of the one- and two-dimensional NMR spectra of biakamides, planar chemical structures possessing a terminal thiazole, two N-methyl amides, a chloromethylene, and a substituted butyryl moiety were obtained. After elucidation of the configuration of the secondary alcohol moiety in biakamides A and B, the absolute stereostructures of the two secondary methyl groups in biakamides A-D were determined by the asymmetric total syntheses of all possible stereoisomers from the optically pure monoprotected 2,4-dimethyl-1,5-diol. Biakamides A-D showed selective antiproliferative activities against PANC-1 cells cultured under glucose-deficient conditions in a concentration-dependent manner. The primary mode of action of biakamides was found to be inhibition of complex I in the mitochondrial electron transport chain.

N-Methylniphatyne A, a New 3-Alkylpyridine Alkaloid as an Inhibitor of the Cancer Cells Adapted to Nutrient Starvation, from an Indonesian Marine Sponge of Xestospongia sp.

In the course of searching for selective growth inhibitors of the cancer cells adapted to nutrient starvation, a new 3-alkylpyridine alkaloid named N-methylniphatyne A (1) was isolated from an Indonesian marine sponge of Xestospongia sp. The chemical structure of 1 was determined on the basis of the spectroscopic analysis and comparison with the synthesized 1 and its analogues. Compound 1 showed the cytotoxic activity against PANC-1 cells under the condition of glucose starvation with IC50 value of 16 µM, whereas no growth-inhibition was observed up to 100 µM under the general culture conditions.

A STUDY OF THE ASSOCIATION BETWEEN SELENIUM AND CARDIOVASCULAR DISEASE IN LAMPUNG, INDONESIA.

Selenium deficient areas have been associated with a higher prevalence of cardiovascular disease in some countries. In this study, we investigated the correlation between cardiovascular disease prevalence and selenium concentration in paddy soil and rice grains, the main staple food in Lampung, Indonesia. Paddy soil and rice samples (n(s) = 35) from eight regencies (n(d) = 8) in Lampung were analyzed for selenium content. The prevalences of heart disease, stroke, and hypertension in those regencies were obtained from the Ministry of Health of Indonesia. The Shapiro-Wilk's test was used to examine the data distribution. The Pearson's correlation was used to examine the correlation between cardiovascular disease prevalence and selenium concentration in the paddy soil and rice grains. Heart disease prevalence was negatively correlated with the selenium concentration in the paddy soil (r = -0.77, p = 0.02) and rice grain (r = -0.71, p = 0.05). A negative correlation was seen for stroke prevalence and selenium concentration in paddy soil (r = -0.76, p = 0.02). Hypertension prevalence was negatively correlated with the selenium concentration in the rice grains (r = -0.83, p = 0.01). These findings suggest that the selenium concentration in paddy soil and rice grains in the Lampung area may play a role in the fact the area has the lowest cardiovascular disease prevalence in Indonesia. Keywords: selenium, cardiovascular diseases, paddy soil, rice grain, Indonesia

Identification of Antiviral Agents Targeting Hepatitis B Virus Promoter from Extracts of Indonesian Marine Organisms by a Novel Cell-Based Screening Assay.

The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.

Identification of the target protein of agelasine D, a marine sponge diterpene alkaloid, as an anti-dormant mycobacterial substance.

One of the major reasons for the wide epidemicity of tuberculosis and for the necessity for extensive chemotherapeutic regimens is that the causative agent, Mycobacterium tuberculosis, has an ability to become dormant. Therefore, new lead compounds that are anti-bacterial against M. tuberculosis in both active and dormant states are urgently needed. Marine sponge diterpene alkaloids, agelasines B, C, and D, from an Indonesian marine sponge of the genus Agelas were rediscovered as anti-dormant-mycobacterial substances. Based on the concept that the transformants over-expressing targets of antimicrobial substances confer drug resistance, strains resistant to agelasine D were screened from Mycobacterium smegmatis transformed with a genomic DNA library of Mycobacterium bovis BCG. Sequence analysis of the cosmids isolated from resistant transformants revealed that the responsible gene was located in the genome region between 3475.051 and 3502.901 kb. Further analysis of the transformants over-expressing the individual gene contained in this region indicated that BCG3185c (possibly a dioxygenase) might be a target of the molecule. Moreover, agelasine D was found to bind directly to recombinant BCG3185c protein (KD 2.42 µm), based on surface plasmon resonance (SPR). This evidence strongly suggests that the BCG3185c protein is the major target of agelasine D, and that the latter is the anti-mycobacterial substance against dormant bacilli.

Marine spongian sesquiterpene phenols, dictyoceratin-C and smenospondiol, display hypoxia-selective growth inhibition against cancer cells.

In the course of our search for hypoxia-selective growth inhibitors against cancer cells, a sesquiterpene phenol, dictyoceratin-C (1), was isolated from the Indonesian marine sponge of Dactylospongia elegans under the guidance of the constructed bioassay. Dictyoceratin-C (1) inhibited proliferation of human prostate cancer DU145 cells selectively under hypoxic condition in a dose-dependent manner at the concentrations ranging from 1.0 to 10 µM. The subsequent structure-activity relationship study using nine sesquiterpene phenol/quinones (2-10), which were isolated from marine sponge, was executed. We found that smenospondiol (2) also exhibited the similar hypoxia-selective growth inhibitory activity against DU145 cells, and the para-hydroxybenzoyl ester moiety would be important for hypoxia-selective growth inhibitory activity of 1. In addition, the mechanistic analysis of dictyoceratin-C (1) revealed that the 10 µM of 1 inhibited accumulation of Hypoxia-Inducible Factor-1α under hypoxic condition.

Stylissamide X, a new proline-rich cyclic octapeptide as an inhibitor of cell migration, from an Indonesian marine sponge of Stylissa sp.

A new proline-rich cyclic octapeptide named stylissamide X (1) was isolated from an Indonesian marine sponge of Stylissa sp. as an inhibitor of cell migration from the guidance of wound-healing assay. The chemical structure of stylissamide X (1) was determined on the basis of spectroscopic analysis, and stereostructure of the amino acids were deduced by Marfey's method. Compound 1 showed inhibitory activity against migration of HeLa cells in the ranges of 0.1-10 µM concentration through both wound-healing assay and chemotaxicell chamber assay, while the cell viability was maintained more than 75% up to 10 µM concentration of 1.

Anti-Alopecia Activity of Alkaloids Group from Noni Fruit against Dihydrotestosterone-Induced Male Rabbits and Its Molecular Mechanism: In Vivo and In Silico Studies.

Androgenic alopecia (AA) is a condition that most commonly affects adult men and is caused by an increase in the hormone dihydrotestosterone (DHT) in the hair follicles. Anti-alopecia drugs should be discovered for hair follicles to enter the anagen growth phase. Therefore, this study evaluated the hair growth-promoting activity of Noni fruit's water, ethyl acetate, n-hexane fractions, and sub-fractions from the active fraction in the alopecia male white rabbit model. The Matias method was modified by inducing rabbits using DHT for 17 days, followed by topical application of Noni fruit solution for 21 days. Meanwhile, hair growth was evaluated by histological observation of the follicular density and the anagen/telogen (A/T) ratio in skin tissue. In the first stage, five groups of male white rabbits were studied to obtain the active fraction; DHT+Minoxidil as standard, DHT+vehicle (NaCMC 1%), DHT+FW, DHT+FEA, and DHT+FH. The FEA as the active fraction was followed by open-column chromatography separation (DCM:Methanol) with a gradient of 10% to produce sub-fractions. In the second stage, the six main sub-fraction groups of male rabbits studied were DHT+FEA-1 to DHT+FEA-6. The follicular density of groups FEA-3 was 78.00 ± 1.52 compared with 31.55 ± 1.64 and 80.12 ± 1.02 in the Vehicle and Minoxidil groups. Additionally, group FEA-3 showed large numbers of anagen follicles with an A/T ratio of 1.64/1 compared to the vehicle group of 1/1.50 and 1.39/1 for Minoxidil control. Group FEA-3 was identified by LC-MS/MS-QTOF, followed by molecular docking to the androgen receptor (PDB: 4K7A), causing alopecia. The results showed that three alkaloid compounds with skeleton piperazine and piperidine, namely (compounds 2 (−4.99 Kcal/mol), 3 (−4.60 Kcal/mol), and 4 (−4.57 Kcal/mol)) had a binding affinity similar to Minoxidil, with also has alkaloid skeleton piperidine−pyrimidine (−4.83 Kcal/mol). The dynamic behavior showed the stability of all androgen receptor compounds with good RMSD, SMSF, and SASA values after being studied with 100 ns molecular dynamics (MD) simulations. This study produced a common thread in discovering a class of alkaloid compounds as inhibitors of androgen receptors that cause alopecia.

Asteltoxin inhibits extracellular vesicle production through AMPK/mTOR-mediated activation of lysosome function.

Cancer cells secrete aberrantly large amounts of extracellular vesicles (EVs) including exosomes, which originate from multivesicular bodies (MVBs). Because EVs potentially contribute to tumor progression, EV inhibitors are of interest as novel therapeutics. We screened a fungal natural product library. Using cancer cells engineered to secrete luciferase-labeled EVs, we identified asteltoxin, which inhibits mitochondrial ATP synthase, as an EV inhibitor. Low concentrations of asteltoxin inhibited EV secretion without inducing mitochondrial damage. Asteltoxin attenuated cellular ATP levels and induced AMPK-mediated mTORC1 inactivation. Consequently, MiT/TFE transcription factors are translocated into the nucleus, promoting transcription of lysosomal genes and lysosome activation. Electron microscopy analysis revealed that the number of lysosomes increased relative to that of MVBs and the level of EVs decreased after treatment with asteltoxin or rapamycin, an mTORC1 inhibitor. These findings suggest that asteltoxin represents a new type of EV inhibitor that controls MVB fate.

Fungicide Activity of Culture Extract from Kocuria palustris 19C38A1 against Fusarium oxysporum.

Secondary metabolites of actinomycetes are a potential source of bioactive compounds in the agricultural sector. This study aimed to determine the fungicidal properties of extracts of marine organism-derived actinomycetes. Actinomycetes were isolated from marine organisms using agar media with 1% colloidal chitin in artificial seawater. Then, the isolates were cultured on liquid media with 1% colloidal chitin in artificial seawater under static conditions for 14 days. The culture was extracted, the fungicide properties were evaluated using the microtiter 96-well plate method, and the influence of inhibition was visualized using apotome and SEM. Finally, the active extract was analyzed using LCMSMS. In the present study, 19 actinomycetes were isolated from marine organisms, and the isolates were examined with regard to their antifungal activities. Of these nineteen isolates, the isolate 19C38A1 was picked out from the rest. Hence, it showed significant control towards F. oxysporum. The prospective strain 19C38A1 was determined to be Kocuria palustris 19C38A1. The extract 19C38A1 was shown to cause damage to cell integrity, indicated by the shrinking form, and inhibited germination in the F. oxysporum; subsequently, the chemical characteristics of the compound produced by the potential isolate 19C38A1 indicated the presence of benzimidazole compounds in the active fraction of C38BK2FA. These results indicate that actinomycetes derived from marine organisms near the coast of Oluhuta, Tomini Bay, Gorontalo, related to strain 19C38A1, are not widely known as sources of valuable fungicides. This preliminary information is important, as it can be used as a basis for further development in the search for fungicides derived from marine actinomycetes.

DedA protein relates to action-mechanism of halicyclamine A, a marine spongean macrocyclic alkaloid, as an anti-dormant mycobacterial substance.

A macrocyclic alkaloid, halicyclamine A, was re-discovered from an Indonesian marine sponge of Haliclona sp. 05A08 as an anti-dormant mycobacterial substance. To clarify action-mechanism of halicyclamine A, halicyclamine A-resistant strains were screened from the transformants of Mycobacterium smegmatis with the genomic DNA library of M. bovis BCG, which were constructed in the multi-copy shuttle cosmid pYUB145. Sequencing analysis of the cosmids isolated from the halicyclamine A-resistant transformants revealed that the responsible gene was involved in the genome region between 2920.549 kb and 2933.210 kb. Further experiments using the transformants over-expressing individual gene contained in the responsible region were executed, and the transformant, which over-expressed BCG2664 gene assigned as dedA gene, was found to become halicyclamine A-resistant. This evidence strongly suggested that DedA protein correlates with the action-mechanism of halicyclamine A as an anti-dormant mycobacterial substance.

Secalonic acid D as a selective cytotoxic substance on the cancer cells adapted to nutrient starvation.

Cancer cells adapted to the microenvironment in tumor such as hypoxic and nutrient-starved conditions are now paid much attention as the therapeutic target of cancer. In the course of search for selective cytotoxic substances against cancer cells adapted to nutrient starvation, xanthone derivative of secalonic acid D (1) was isolated from culture extract of marine-derived Penicillium oxalicum. Compound 1 showed cytotoxic activity on the human pancreatic carcinoma PANC-1 cells adapted to glucose-starved conditions with IC50 value of 0.6 µM, whereas IC50 value of compound 1 against PANC-1 cells under general culture conditions was calculated to be more than 1000 µM. Further study indicated that compound 1 inhibited the Akt signaling pathway under glucose-starved conditions, and slightly affected the induction of glucose-regulated protein 78 (GRP78), and these effects would be mediated by the uncoupling action of compound 1 on the mitochondria.

Selective cytotoxicity of epidithiodiketopiperazine DC1149B, produced by marine-derived Trichoderma lixii on the cancer cells adapted to glucose starvation.

The core of solid tumors is characterized by hypoxia and a nutrient-starved microenvironment and has gained much attention as targets of anti-cancer drugs. In the course of search for selective growth inhibitors against the cancer cells adapted to nutrient starvation, epidithiodiketopiperazine DC1149B (1) together with structurally related compounds, trichodermamide A (2) and aspergillazine A (3), were isolated from culture extract of marine-derived Trichoderma lixii. Compounds 1 exhibited potent selective cytotoxic activity against human pancreatic carcinoma PANC-1 cells cultured under glucose-starved conditions with IC50 values of 0.02 µM. The selective index of the compound 1 was found to be 35,500-fold higher for cells cultured under glucose-starved conditions than those under the general culture conditions. The mechanistic analysis indicated that compound 1 inhibited the response of the ER stress signaling. In addition, these effects of compound 1 could be mediated by inhibiting complex II in the mitochondrial electron transport chain.

Selective cytotoxicity of marine-derived fungal metabolite (3S,6S)-3,6-dibenzylpiperazine-2,5-dione against cancer cells adapted to nutrient starvation.

The cancer cells that are adapted to the hypoxic and nutrient-starved conditions of the tumor microenvironment have become a key target for anticancer therapies. In the course of search for selective cytotoxic substances against cancer cells adapted to nutrient starvation, (3S,6S)-3,6-dibenzylpiperazine-2,5-dione (1) was isolated from culture extract of marine-derived Paecilomyces formous 17D47-2. Compound 1 showed cytotoxic activity on the human pancreatic carcinoma PANC-1 cells adapted to glucose-starved conditions with IC50 value of 28 µM, whereas no effect was observed against PANC-1 cells under general culture conditions up to 1000 µM. Further studies on the mechanism of the selective cytotoxicity of 1 against the glucose-starved PANC-1 cells suggest that it may function via uncoupling of mitochondrial oxidative phosphorylation.

Mycobacterium smegmatis alters the production of secondary metabolites by marine-derived Aspergillus niger.

It is generally accepted that fungi have a number of dormant gene clusters for the synthesis of secondary metabolites, and the activation of these gene clusters can expand the diversity of secondary metabolites in culture. Recent studies have revealed that the mycolic acid-containing bacterium Tsukamurella pulmonis activates dormant gene clusters in the bacterial genus Streptomyces. However, it is not clear whether the mycolic acid-containing bacteria activate dormant gene clusters of fungi. We performed co-culture experiments using marine-derived Aspergillus niger with Mycobacterium smegmatis, a mycolic acid-containing bacteria. The co-cultivation resulted in the production of a pigment by A. niger and increased cytotoxic activity of the extract against human prostate cancer DU145 cells. An analysis of secondary metabolites in the extract of the co-culture broth revealed that the increase in cytotoxic activity was caused by the production of malformin C (1), and that TMC-256A1 (2), desmethylkotanin (3), and aurasperone C (4) were selectively produced under co-culture conditions. In addition, further study suggested that direct interaction between the two microorganisms was necessary for the production of the pigment and the cytotoxic compound malformin C (1) from A. niger. Given the biological activities of malformin C, including cytotoxic activity, our approach for increasing the production of bioactive secondary metabolites has important practical applications and may facilitate structural analyses of novel bioactive compounds.