Audrey F. Adcock

Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors

Three-dimensional (3D) cell culture systems have gained increasing interest in drug discovery and tissue engineering due to their evident advantages in providing more physiologically relevant information and more predictive data for in vivo tests. In this review, we discuss the characteristics of 3D cell culture systems in comparison to the two-dimensional (2D) monolayer culture, focusing on cell growth conditions, cell proliferation, population, and gene and protein expression profiles. The innovations and development in 3D culture systems for drug discovery over the past 5 years are also reviewed in the article, emphasizing the cellular response to different classes of anticancer drugs, focusing particularly on similarities and differences between 3D and 2D models across the field. The progression and advancement in the application of 3D cell cultures in cell-based biosensors is another focal point of this review.

Resorcylic acid lactones with cytotoxic and NF-κB inhibitory activities and their structure-activity relationships.

As part of our ongoing investigation of filamentous fungi for anticancer leads, an active fungal extract was identified from the Mycosynthetix library (MSX 63935; related to Phoma sp.). The initial extract exhibited cytotoxic activity against the H460 (human non-small cell lung carcinoma) and SF268 (human astrocytoma) cell lines and was selected for further study. Bioactivity-directed fractionation yielded resorcylic acid lactones (RALs) 1 (a new natural product) and 3 (a new compound) and the known RALs zeaenol (2), (5E)-7-oxozeaenol (4), (5Z)-7-oxozeaenol (5), and LL-Z1640-1 (6). Reduction of (5E)-7-oxozeaenol (4) with sodium borohydride produced 3, which allowed assignment of the absolute configuration of 3. Other known resorcylic acid lactones (7-12) were purchased and assayed in parallel for cytotoxicity with isolated 1-6 to investigate structure-activity relationships in the series. Moreover, the isolated compounds (1-6) were examined for activity in a suite of biological assays, including antibacterial, mitochondria transmembrane potential, and NF-κB. In the latter assay, compounds 1 and 5 displayed sub-micromolar activities that were on par with the positive control, and as such, these compounds may serve as a lead scaffold for future medicinal chemistry studies.

Peptaibols from two unidentified fungi of the order Hypocreales with cytotoxic, antibiotic, and anthelmintic activities

As part of an ongoing investigation of filamentous fungi for anticancer leads, an active culture was identified from the Mycosynthetix library (MSX 70741, of the order Hypocreales, Ascomycota). The fungal extract exhibited cytotoxic activity against the H460 (human nonsmall cell lung carcinoma) cell line, and bioactivity‐directed fractionation yielded peptaibols 1–12 and harzianums A (13) and B (14). Structure elucidation of 1–12 was facilitated by high‐resolution MS/MS using higher‐energy collisional dissociation and by high field NMR (950 MHz). The absolute configuration was determined by Marfeys analysis of the individual amino acids; the time required for such analysis was decreased via the development of a 10‐min ultra performance liquid chromatography method. The isolated peptaibols (1–12), along with three other peptaibols isolated and elucidated from a different fungus (MSX 57715) of the same order (15–17), were examined for activity in a suite of biological assays, including those for cytotoxic, antibacterial, and anthelmintic activities. Copyright

Cytotoxic epipolythiodioxopiperazine alkaloids from filamentous fungi of the Bionectriaceae

Bioactivity-directed fractionation of the organic extracts of two filamentous fungi of the Bionectriaceae, strains MSX 64546 and MSX 59553 from the Mycosynthetix library, led to the isolation of a new dimeric epipolythiodioxopiperazine alkaloid, verticillin H (1), along with six related analogs, Sch 52900 (2), verticillin A (3), gliocladicillin C (4), Sch 52901 (5), 11′-deoxyverticillin A (6) and gliocladicillin A (7). The structures of compounds 1–7 were determined by extensive NMR and HRMS analyses, as well as by comparisons to the literature. All compounds (1–7) were evaluated for cytotoxicity against a panel of human cancer cell lines, displaying IC50 values ranging from 1.2 μM to 10 nM. Compounds 1–5 were examined for activity in the NF-κB assay, where compounds 2 and 3 revealed activity in the sub-micromolar range. Additionally, compounds 1, 3 and 4 were tested for EGFR inhibition using an enzymatic assay, while compound 3 was examined against an overexpressing EGFR+ve cancer cell line.

Benzoquinones and Terphenyl Compounds As Phosphodiesterase-4B Inhibitors from a Fungus of the Order Chaetothyriales (MSX 47445)

Three bioactive compounds were isolated from an organic extract of an ascomycete fungus of the order Chaetothyriales (MSX 47445) using bioactivity-directed fractionation as part of a search for anticancer leads from filamentous fungi. Of these, two were benzoquinones [betulinan A (1) and betulinan C (3)], and the third was a terphenyl compound, BTH-II0204-207:A (2). The structures were elucidated using a set of spectroscopic and spectrometric techniques; the structure of the new compound (3) was confirmed via single-crystal X-ray diffraction. Compounds 1-3 were evaluated for cytotoxicity against a human cancer cell panel, for antimicrobial activity against Staphylococcus aureus and Candida albicans, and for phosphodiesterase (PDE4B2) inhibitory activities. The putative binding mode of 1-3 with PDE4B2 was examined using a validated docking protocol, and the binding and enzyme inhibitory activities were correlated.

Peptaibols, Tetramic Acid Derivatives, Isocoumarins, and Sesquiterpenes from a Bionectria sp. (MSX 47401)

An extract of the filamentous fungus Bionectria sp. (MSX 47401) showed both promising cytotoxic activity (>90% inhibition of H460 cell growth at 20 μg/mL) and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). A bioactivity-directed fractionation study yielded one new peptaibol (1) and one new tetramic acid derivative (2), and the fungus biosynthesized diverse secondary metabolites with mannose-derived units. Five known compounds were also isolated: clonostachin (3), virgineone (4), virgineone aglycone (5), AGI-7 (6), and 5,6-dihydroxybisabolol (7). Compounds 5 and 7 have not been described previously from natural sources. Compound 1 represents the second member of the peptaibol structural class that contains an ester-linked sugar alcohol (mannitol) instead of an amide-linked amino alcohol, and peptaibols and tetramic acid derivatives have not been isolated previously from the same fungus. The structures of the new compounds were elucidated primarily by high-field NMR (950 and 700 MHz), HRESIMS/MS, and chemical degradations (Marfeys analysis). All compounds (except 6) were examined for antibacterial and antifungal activities. Compounds 2, 4, and 5 showed antimicrobial activity against S. aureus and several MRSA isolates.

Cyclodepsipeptides, Sesquiterpenoids, and Other Cytotoxic Metabolites from the Filamentous Fungus Trichothecium sp. (MSX 51320)

Two new cyclodepsipeptides (1 and 2), two new sesquiterpenoids (3 and 4), and the known compounds guangomide A (5), roseotoxin S, and three simple trichothecenes were isolated from the cytotoxic organic extract of a terrestrial filamentous fungus, Trichothecium sp. The structures were determined using NMR spectroscopy and mass spectrometry. Absolute configurations of the cyclodepsipeptides were established by employing chiral HPLC, while the relative configurations of 3 and 4 were determined via NOESY data. The isolation of guangomide A was of particular interest, since it was reported previously from a marine-derived fungus.

Cytotoxic xanthone-anthraquinone heterodimers from an unidentified fungus of the order Hypocreales (MSX 17022).

Two new xanthone–anthraquinone heterodimers, acremoxanthone C (5) and acremoxanthone D (2), have been isolated from an extract of an unidentified fungus of the order Hypocreales (MSX 17022) by bioactivity-directed fractionation as part of a search for anticancer leads from filamentous fungi. Two known related compounds, acremonidin A (4) and acremonidin C (3) were also isolated, as was a known benzophenone, moniliphenone (1). The structures of these isolates were determined via extensive use of spectroscopic and spectrometric tools in conjunction with comparisons to the literature. All compounds (1–5) were evaluated against a suite of biological assays, including those for cytotoxicity, inhibition of the 20S proteasome, mitochondrial transmembrane potential and nuclear factor-κB.

Having excess levels of PCSK9 is not sufficient to induce complex formation between PCSK9 and the LDL receptor

Proprotein convertase subtilisin/kexin-9 (PCSK9) acts mainly by forming complexes with the LDL receptor at the cell surface, which are then degraded in the lysosome. Studies were performed to determine whether excess levels of PCSK9 was sufficient to induce PCSK9/LDL receptor complex formation in human hepatocyte-like C3A cells. It was demonstrated using ELISA that instead of considering the overall levels of PCSK9 protein that is produced in response to certain treatment, what is critical is how much PCSK9 is actually capable of forming complexes. Despite the high levels, most of the PCSK9 produced as a result of incubating cells with a medium supplemented with BD™ MITO+ serum extender (MITO+ medium) appeared to be inhibited by a secreted factor. Having lower levels of PCSK9/LDL receptor complexes did not prevent an increase in the degradation rate of LDL receptors in MITO+ medium as compared to fetal bovine serum (FBS) containing medium (Regular medium), an effect that did not correlate with an increase in protein levels of the inducible degrader of LDL receptors (IDOL), as demonstrated using Western blotting analysis. Additional studies are required to determine the exact mechanism(s) for the degradation of the LDL receptor and/or to identify the secreted inhibitor of PCSK9.

Three-Dimensional (3D) Cell Cultures in Cell-based Assays for in-vitro Evaluation of Anticancer Drugs

This study systematically investigated the cell proliferation rates, spheroid structures, cellular responses to different anti-cancer drugs, the expression of drug action-related proteins, and the possible correlations among these properties of 3D spheroids on Matrigel in comparison to 2D monolayer cells, using two cancer cell lines-the prostate cancer cell line, DU145, and the oral cancer cell line, CAL27. Compared to the traditional 2D-cultured cells, 3D-cultured CAL27 cells had enhanced proliferation by approximately 50-70% at various seeding cell densities, whereas 3D-cultured DU145 cells showed reduced proliferation at all tested seeding cell densities by 20-40%. In drug tests, the sensitivity of 3D-cultured DU145 cells relative to 2D-cultured cells showed an obvious drug action mechanism dependency in response to three anticancer drugs, Rapamycin, Docetaxel, and Camptothecin, whereas 3D-cultured CAL27 cells responded more sensitively than 2D-cultured cells to all three tested drugs, Docetaxel, Bleomycin, and Erlotinib, indicating the relative proliferation rate between 3D and 2D cultured cells may be a dominating factor in this case and mitigated the factor of drug action mechanism. The elevated expression of EGFR in 3D-cultured CAL27 was correlated with its more sensitive response to Erlotinib (acting through binding to EGRF) compared to 2D-cultured cells; Similarly, the expression of βIII tubulin in 3D-cultured DU145 cells was found to be increased and correlated with their higher resistance to Doxetaxel compared to 2D-cultured cells.