Isolation, synthesis, and biological activity of aphrocallistin, an adenine-substituted bromotyramine metabolite from the Hexactinellida sponge Aphrocallistes beatrix.
ABSTRACT: A new adenine-substituted bromotyrosine-derived metabolite designated as aphrocallistin (1) has been isolated from the deep-water Hexactinellida sponge Aphrocallistes beatrix. Its structure was elucidated on the basis of spectral data and confirmed through a convergent, modular total synthetic route that is amenable toward future analogue preparation. Aphrocallistin inhibits the growth of a panel of human tumor cell lines with IC(50) values ranging from 7.5 to >100 microM and has been shown to induce G1 cell cycle arrest in the PANC-1 pancreatic carcinoma cell line. Aphrocallistin has been fully characterized in the NCI cancer cell line panel and has undergone in vitro ADME pharmacological profiling.
Project description:In the present study, we profiled bacterial and archaeal communities from 13 phylogenetically diverse deep-sea sponge species (Demospongiae and Hexactinellida) from the South Pacific by 16S rRNA-gene amplicon sequencing. Additionally, the associated bacteria and archaea were quantified by real-time qPCR. Our results show that bacterial communities from the deep-sea sponges are mostly host-species specific similar to what has been observed for shallow-water demosponges. The archaeal deep-sea sponge community structures are different from the bacterial community structures in that they are almost completely dominated by a single family, which are the ammonia-oxidizing genera within the Nitrosopumilaceae. Remarkably, the archaeal communities are mostly specific to individual sponges (rather than sponge-species), and this observation applies to both hexactinellids and demosponges. Finally, archaeal 16s gene numbers, as detected by quantitative real-time PCR, were up to three orders of magnitude higher than in shallow-water sponges, highlighting the importance of the archaea for deep-sea sponges in general.
Project description:The ADME Core Panel assays 184 variants across 34 pharmacogenes, many of which are difficult to accurately genotype with standard multiplexing methods.We genotyped 326 frequently medicated individuals of European descent in Vanderbilt's biorepository linked to de-identified electronic medical records, BioVU, on the ADME Core Panel to assess quality and performance of the assay. We compared quality control metrics and determined the extent of direct and indirect marker overlap between the ADME Core Panel and the Illumina Omni1-Quad.We found the quality of the ADME Core Panel data to be high, with exceptions in select copy number variants and markers in certain genes (notably CYP2D6). Most of the common variants on the ADME panel are genotyped by the Omni1, but absent rare variants and copy number variants could not be accurately tagged by single markers.Our frequently medicated study population did not convincingly differ in allele frequency from reference populations, suggesting that heterogeneous clinical samples (with respect to medications) have similar allele frequency distributions in pharmacogenetics genes compared with reference populations.
Project description:Mitochondrial genomes (mtDNA) of numerous sponges have been sequenced as part of an ongoing effort to resolve the class-level phylogeny of the Porifera, as well as to place the various lower metazoan groups on the animal-kingdom tree. Most recently, the partial mtDNA of two glass sponges, class Hexactinellida, were reported. While previous phylogenetic estimations based on these data remain uncertain due to insufficient taxon sampling and accelerated rates of evolution, the mtDNA molecules themselves reveal interesting traits that may be unique to hexactinellids. Here we determined the first complete mitochondrial genome of a hexactinellid sponge, Aphrocallistes vastus, and compared it to published poriferan mtDNAs to further describe characteristics specific to hexactinellid and other sponge mitochondrial genomes.The A. vastus mtDNA consisted of a 17,427 base pair circular molecule containing thirteen protein-coding genes, divergent large and small subunit ribosomal RNAs, and a reduced set of 18 tRNAs. The A. vastus mtDNA showed a typical hexactinellid nucleotide composition and shared a large synteny with the other sequenced glass sponge mtDNAs. It also contained an unidentified open reading frame and large intergenic space region. Two frameshifts, in the cox3 and nad6 genes, were not corrected by RNA editing, but rather possessed identical shift sites marked by the extremely rare tryptophan codon (UGG) followed by the common glycine codon (GGA) in the +1 frame.Hexactinellid mtDNAs have shown similar trends in gene content, nucleotide composition, and codon usage, and have retained a large gene syntenty. Analysis of the mtDNA of A. vastus has provided evidence diagnostic for +1 programmed translational frameshifting, a phenomenon disparately reported throughout the animal kingdom, but present in the hexactinellid mtDNAs that have been sequenced to date.
Project description:BACKGROUND:Glass sponges (Class Hexactinellida) are important components of deep-sea ecosystems and are of interest from geological and materials science perspectives. The reconstruction of their phylogeny with molecular data has only recently begun and shows a better agreement with morphology-based systematics than is typical for other sponge groups, likely because of a greater number of informative morphological characters. However, inconsistencies remain that have far-reaching implications for hypotheses about the evolution of their major skeletal construction types (body plans). Furthermore, less than half of all described extant genera have been sampled for molecular systematics, and several taxa important for understanding skeletal evolution are still missing. Increased taxon sampling for molecular phylogenetics of this group is therefore urgently needed. However, due to their remote habitat and often poorly preserved museum material, sequencing all 126 currently recognized extant genera will be difficult to achieve. Utilizing morphological data to incorporate unsequenced taxa into an integrative systematics framework therefore holds great promise, but it is unclear which methodological approach best suits this task. RESULTS:Here, we increase the taxon sampling of four previously established molecular markers (18S, 28S, and 16S ribosomal DNA, as well as cytochrome oxidase subunit I) by 12 genera, for the first time including representatives of the order Aulocalycoida and the type genus of Dactylocalycidae, taxa that are key to understanding hexactinellid body plan evolution. Phylogenetic analyses suggest that Aulocalycoida is diphyletic and provide further support for the paraphyly of order Hexactinosida; hence these orders are abolished from the Linnean classification. We further assembled morphological character matrices to integrate so far unsequenced genera into phylogenetic analyses in maximum parsimony (MP), maximum likelihood (ML), Bayesian, and morphology-based binning frameworks. We find that of these four approaches, total-evidence analysis using MP gave the most plausible results concerning congruence with existing phylogenetic and taxonomic hypotheses, whereas the other methods, especially ML and binning, performed more poorly. We use our total-evidence phylogeny of all extant glass sponge genera for ancestral state reconstruction of morphological characters in MP and ML frameworks, gaining new insights into the evolution of major hexactinellid body plans and other characters such as different spicule types. CONCLUSIONS:Our study demonstrates how a comprehensive, albeit in some parts provisional, phylogeny of a larger taxon can be achieved with an integrative approach utilizing molecular and morphological data, and how this can be used as a basis for understanding phenotypic evolution. The datasets and associated trees presented here are intended as a resource and starting point for future work on glass sponge evolution.
Project description:Two new bromotyrosine derivatives, anomoian B (1) and aplyzanzine B (2), were isolated, respectively, from the organic extracts of a Verongida sponge belonging to the Hexadella genus and from a two-sponge association (Jaspis sp. and Bubaris sp.), both collected off the coast of Indonesia. The planar structure of 1 and 2 was determined by 1D and 2D NMR experiments and by high-resolution mass spectrometry, while their absolute stereochemistry was assigned by comparison with optical rotation values of known bromotyrosines and by chemical degradation. Both compounds showed moderate antiproliferative activity against a panel of different cancer cell lines. Their cytotoxic activity is facilitated through the induction of apoptosis, which is mediated neither by the generation of reactive oxygen species nor by the inhibition of histone deacetylases in these cell lines.
Project description:The first total synthesis of the marine bromotyrosine purpurealidin I (<b>1</b>) using trifluoroacetoxy protection group and its dimethylated analog (<b>29</b>) is reported along with 16 simplified bromotyrosine derivatives lacking the tyramine moiety. Their cytotoxicity was evaluated against the human malignant melanoma cell line (A-375) and normal skin fibroblast cells (Hs27) together with 33 purpurealidin-inspired simplified amides, and the structure?activity relationships were investigated. The synthesized simplified analogs without the tyramine part retained the cytotoxic activity. Purpurealidin I (<b>1</b>) showed no selectivity but its simplified pyridin-2-yl derivative (<b>36</b>) had the best improvement in selectivity (Selectivity index 4.1). This shows that the marine bromotyrosines are promising scaffolds for developing cytotoxic agents and the full understanding of the elements of their SAR and improving the selectivity requires further optimization of simplified bromotyrosine derivatives.
Project description:SS1P is an antimesothelin recombinant immunotoxin (RIT). Pancreatic ductal adenocarcinoma (PDAC) cell lines are resistant to SS1P, despite high mesothelin expression. The aim of this study is to examine whether combining SS1P and BH3-mimetic ABT-737 induces cell death in a panel of PDAC cell lines. ABT-737 binds and neutralizes several antiapoptotic BCL2 family proteins, but has a low affinity for the short-lived MCL1 and BCL2A1. SS1P inhibits protein synthesis, which has shown to downregulate MCL1. PDAC cell lines KLM-1, BxPc-3, and Panc 3.014 were resistant to SS1P or ABT-737 alone. Combining both compounds led to a significant increase in cell death. After 48 hours of treatment, cell death was observed in 92% of KLM-1, 55% of BxPc-3, and 23% of Panc 3.014 cells. Panc 3.014 had the highest number of mesothelin-binding sites (92×10(3)), followed by KLM-1 (58×10(3)) and BxPc-3 (3×10(3)). ABT-737 had no effect on SS1P internalization, but enhanced SS1P-induced protein synthesis inhibition significantly in KLM-1, to a lesser extent in BxPc-3, and very little in Panc 3.014. SS1P alone or in combination with ABT-737 downregulated MCL1 in KLM-1 and BxPc-3, but not in Panc 3.014. Similar observations were made for BCL2A1, which had the highest levels in Panc 3.014. Compared with KLM-1, Panc 3.014, and BxPc-3 also had lower proapoptotic BAK and a trend toward higher MCL1. Proapoptotic BAX was similar in KLM-1 and BxPc-3, but lower in Panc 3.014. In conclusion, combining SS1P with ABT-737 overcomes SS1P-resistance in PDAC, although to a variable extent. The efficacy of the combination is mainly associated with the RIT-associated inhibition of protein synthesis and the ability to downregulate MCL1 and BCL2A1, while levels of other key apoptotic proteins may also be important. Our data support the combination of an RIT and a BH3-mimetic, and identify factors that potentially limit the efficacy of such therapeutic approach.
Project description:The goal of our study was to determine the susceptibility of different pancreatic cell lines to clinically applicable photodynamic therapy (PDT). The efficacy of PDT of two different commercially available photosensitizers, verteporfin and sodium porfimer, was compared using a panel of four different pancreatic cancer cell lines, PANC-1, BxPC-3, CAPAN-2, and MIA PaCa-2, and an immortalized non-neoplastic pancreatic ductal epithelium cell line, HPNE. The minimum effective concentrations and dose-dependent curves of verteporfin and sodium porfimer on PANC-1 were determined. Since pancreatic cancer is known to have significant stromal components, the effect of PDT on stromal cells was also assessed. To mimic tumor-stroma interaction, a co-culture of primary human fibroblasts or human pancreatic stellate cell (HPSCs) line with PANC-1 was used to test verteporfin-PDT-mediated cell death of PANC-1. Two cytokines (TNF-? and IL-1?) were used for stimulation of primary fibroblasts (derived from human esophageal biopsies) or HPSCs. The increased expression of smooth muscle actin (?-SMA) confirmed the activation of fibroblasts or HPSC upon treatment with TNF-? and IL-1?. Cell death assays showed that both sodium porfimer- and verteporfin-mediated PDT-induced cell death in a dose-dependent manner. However, verteporfin-PDT treatment had a greater efficiency with 60??×?? lower concentration than sodium porfimer-PDT in the PANC-1 incubated with stimulated fibroblasts or HPSC. Moreover, activation of stromal cells did not affect the treatment of the pancreatic cancer cell lines, suggesting that the effects of PDT are independent of the inflammatory microenvironment found in this two-dimensional culture model of cancers.
Project description:Two new bromotyrosine alkaloids, ceratinadins E (<b>1</b>) and F (<b>2</b>), were isolated from an Okinawan marine sponge <i>Pseudoceratina</i> sp. as well as a known bromotyrosine alkaloid, psammaplysin F (<b>3</b>). The gross structures of <b>1</b> and <b>2</b> were elucidated on the basis of spectroscopic data. The absolute configurations of <b>1</b> and <b>2</b> were assigned by comparison of the NMR and ECD data with those of a known related bromotyrosine alkaloid, psammaplysin A (<b>4</b>). Ceratinadins E (<b>1</b>) and F (<b>2</b>) are new bromotyrosine alkaloids possessing an 8,10-dibromo-9-methoxy-1,6-dioxa-2-azaspiro[4.6]undeca-2,7,9-trien-4-ol unit with two or three 11-<i>N</i>-methylmoloka'iamine units connected by carbonyl groups, respectively. Ceratinadin E (<b>1</b>) exhibited antimalarial activities against a drug-resistant and a drug-sensitive strains of <i>Plasmodium falciparum</i> (K1 and FCR3 strains, respectively).
Project description:Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various types of cancer cells without damaging normal cells. However, in terms of pancreatic cancer, not all cancer cells are sensitive to TRAIL. In this study, we examined a panel of human pancreatic cancer cell lines for TRAIL sensitivity and investigated the effects of Bcl-2 family inhibitors on their response to TRAIL. Both ABT-263 and ABT-737 inhibited the function of Bcl-2, Bcl-xL, and Bcl-w. Of the nine pancreatic cancer cell lines tested, six showed no or low sensitivity to TRAIL, which correlated with protein expression of Bcl-xL. ABT-263 significantly sensitized four cell lines (AsPC-1, Panc-1, CFPAC-1, and Panc10.05) to TRAIL, with reduced cell viability and increased apoptosis. Knockdown of Bcl-xL, but not Bcl-2, by siRNA transfection increased the sensitivity of AsPC-1 and Panc-1 cells to TRAIL. ABT-263 treatment had no effect on protein expression of Bcl-2, Bcl-xL, or c-FLIPs. In Panc-1 cells, ABT-263 increased the surface expression of death receptor (DR) 5; the NF-?B pathway, but not endoplasmic reticulum stress, participated in the increase. In xenograft mouse models, the combination of TRAIL and ATB-737 suppressed the in vivo tumor growth of AsPC-1 and Panc-1 cells. These results indicate that Bcl-xL is responsible for TRAIL resistance in human pancreatic cancer cells, and that Bcl-2 family inhibitors could represent promising reagents to sensitize human pancreatic cancers in DR-targeting therapy.