A Novel Role of Silibinin as a Putative Epigenetic Modulator in Human Prostate Carcinoma.

Silibinin, extracted from milk thistle (Silybum marianum L.), has exhibited considerable preclinical activity against prostate carcinoma. Its antitumor and chemopreventive activities have been associated with diverse effects on cell cycle, apoptosis, and receptor-dependent mitogenic signaling pathways. Here we hypothesized that silibinin's pleiotropic effects may reflect its interference with epigenetic mechanisms in human prostate cancer cells. More specifically, we have demonstrated that silibinin reduces gene expression levels of the Polycomb Repressive Complex 2 (PRC2) members Enhancer of Zeste Homolog 2 (EZH2), Suppressor of Zeste Homolog 12 (SUZ12), and Embryonic Ectoderm Development (EED) in DU145 and PC3 human prostate cancer cells, as evidenced by Real Time Polymerase Chain Reaction (RT-PCR). Furthermore immunoblot and immunofluorescence analysis revealed that silibinin-mediated reduction of EZH2 levels was accompanied by an increase in trimethylation of histone H3 on lysine (Κ)-27 residue (H3K27me3) levels and that such response was, in part, dependent on decreased expression levels of phosphorylated Akt (ser473) (pAkt) and phosphorylated EZH2 (ser21) (pEZH2). Additionally silibinin exerted other epigenetic effects involving an increase in total DNA methyltransferase (DNMT) activity while it decreased histone deacetylases 1-2 (HDACs1-2) expression levels. We conclude that silibinin induces epigenetic alterations in human prostate cancer cells, suggesting that subsequent disruptions of central processes in chromatin conformation may account for some of its diverse anticancer effects.

Spiroscytalin, a new tetramic acid and other metabolites of mixed biogenesis from Scytalidium cuboideum.

Spiroscytalin (1), a new tetramic acid that possesses an uncommon spiro-ring fusion between a polyketide-derived octalin ring system and a 2,4-pyrrolidinedione, along with two known compounds, leporin B (2) and purpactin A (3), were isolated from a solid phase culture of the fungus Scytalidium cuboideum (MSX 68345). The molecular connectivity of 1-3 was determined using NMR spectroscopy and mass spectrometry. The relative configurations of 1 and 2 were determined by NOESY experiments. The absolute configuration of 1 was determined by electronic circular dichroism (ECD) via a combination of experimental measurements and computational calculations. While leporin B was known, it displayed activities that had not been reported previously, including cytotoxicity against three human tumor cell lines and antibacterial activity against Candida albicans and Staphylococcus aureus.

Enhanced bioactivity of silybin B methylation products.

Flavonolignans from milk thistle (Silybum marianum) have been investigated for their cellular modulatory properties, including cancer chemoprevention and hepatoprotection, as an extract (silymarin), as partially purified mixtures (silibinin and isosilibinin), and as pure compounds (a series of seven isomers). One challenge with the use of these compounds in vivo is their relatively short half-life due to conjugation, particularly glucuronidation. In an attempt to generate analogues with improved in vivo properties, particularly reduced metabolic liability, a semi-synthetic series was prepared in which the hydroxy groups of silybin B were alkylated. A total of five methylated analogues of silybin B were synthesized using standard alkylation conditions (dimethyl sulfate and potassium carbonate in acetone), purified using preparative HPLC, and elucidated via spectroscopy and spectrometry. Of the five, one was monomethylated (3), one was dimethylated (4), two were trimethylated (2 and 6), and one was tetramethylated (5). The relative potency of all compounds was determined in a 72 h growth-inhibition assay against a panel of three prostate cancer cell lines (DU-145, PC-3, and LNCaP) and a human hepatoma cell line (Huh7.5.1) and compared to natural silybin B. Compounds also were evaluated for inhibition of both cytochrome P450 2C9 (CYP2C9) activity in human liver microsomes and hepatitis C virus infection in Huh7.5.1 cells. The monomethyl and dimethyl analogues were shown to have enhanced activity in terms of cytotoxicity, CYP2C9 inhibitory potency, and antiviral activity (up to 6-fold increased potency) compared to the parent compound, silybin B. In total, these data suggested that methylation of flavonolignans can increase bioactivity.

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 (Marfey's 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.

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.

Waol A, trans-dihydrowaol A, and cis-dihydrowaol A: polyketide-derived γ-lactones from a Volutella species.

An organic extract of a filamentous fungus (MSX 58801), identified as a Volutella sp. (Hypocreales, Ascomycota), displayed moderate cytotoxic activity against NCI-H460 human large cell lung carcinoma. Bioactivity-directed fractionation led to the isolation of three γ-lactones having the furo[3,4-b]pyran-5-one bicyclic ring system [waol A (1), trans-dihydrowaol A (2), and cis-dihydrowaol A (3)]. The structures were elucidated using a set of spectroscopic and spectrometric techniques; the absolute configuration of 2 was established via a modified Mosher's ester method. Compounds 1 and 2 were evaluated for cytotoxicity against a human cancer cell panel.

Indole alkaloids from two cultured cyanobacteria, Westiellopsis sp. and Fischerella muscicola.

Chemical investigation of two cultured cyanobacteria, Westiellopsis sp. (SAG strain number 20.93) and Fischerella muscicola (UTEX strain number LB1829) led to the isolation of three hapalindole-type alkaloids, namely hapalindole X (1), deschloro hapalindole I (2), and 13-hydroxy dechlorofontonamide (3), along with ten known indole alkaloids (hapalindoles A, C, G, H, I, J, and U, hapalonamide H, anhydrohapaloxindole A, and fischerindole L) and fischerellins A and B. The structures were determined by a combination of spectroscopic analyses mainly based on 1D and 2D NMR and HRESIMS data. Selected compounds were evaluated for cytotoxicity and exhibited weak to moderate cytotoxicity against HT-29, MCF-7, NCI-H460, SF268, and IMR90 cells. All compounds, except hapalindole C, were evaluated for 20S proteasome inhibition and displayed either weak or no inhibition at 25 µg/mL. Selected compounds were also evaluated for antimicrobial activity, and hapalindoles X (1) and A, and hapalonamide H showed potent activity against both Mycobacterium tuberculosis and Candida albicans with MIC values ranging from 0.6 to 2.5 µM.

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 Marfey's 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.

Nitrile-Containing Fischerindoles from the Cultured Cyanobacterium Fischerella sp.

Chemical investigation of the cultured cyanobacterium Fischerella sp. (SAG strain number 46.79) led to the isolation of four nitrile-containing indole alkaloids, namely 12-epi-fischerindole I nitrile (1), deschloro 12-epi-fischerindole I nitrile (2), 12-epi-fischerindole W nitrile (3), and deschloro 12-epi-fischerindole W nitrile (4) along with a known metabolite hapalosin. The structures were determined by detailed spectroscopic analyses on the basis of 1D and 2D NMR and HRESIMS data. All isolates were evaluated for cytotoxicity against human cancer cells and for 20S proteasome inhibition. Deschloro 12-epi-fischerindole I nitrile (2) was found to be weakly cytotoxic against HT-29 cells with an ED(50) value of 23 µM. Hapalosin showed weak cytotoxicity against HT-29 and MCF-7 cells with ED(50) values of 22 and 27 µM, respectively, as well as moderate 20S proteasome inhibition with an IC(50) value of 12 µM. Compounds 1-4 all contain a nitrile moiety instead of the isonitrile found in all fischerindoles reported to date. Compounds 3 and 4 also display a new carbon skeleton, in which a six-membered ring replaces the five-membered ring normally found in fischerindole-type alkaloids.

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.

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.

Thielavin B methyl ester: a cytotoxic benzoate trimer from an unidentified fungus (MSX 55526) from the Order Sordariales.

As part of our ongoing investigation of filamentous fungi for anticancer leads, an active fungal extract was identified from the Mycosynthetix library (MSX 55526; from the Order Sordariales). Bioactivity-directed fractionation yielded the known ergosterol peroxide (2) and 5α,8α-epidioxyergosta-6,9(11),22-trien-3ß-ol(3), and a new benzoate trimer, termed thielavin B methyl ester (1). The structure elucidation of 1 was facilitated by the use of HRMS coupled to an APPI (atmospheric pressure photoionization) source. Compound 1 proved to be moderately active against a panel of three cancer cell lines.

Obionin B: An o-pyranonaphthoquinone decaketide from an unidentified fungus (MSX 63619) from the Order Pleosporales.

A fungal extract (MSX 63619), from the Mycosynthetix library of over 50,000 fungi, displayed promising cytotoxicity against a human tumor cell panel. Bioactivity-directed fractionation led to the isolation of an o-pyranonaphthoquinone decaketide, which we termed obionin B (1). The structure of 1 was deduced via spectroscopic and spectrometric techniques. The IC(50) value of 1 was moderate, ranging from 3 to 13 µM, depending on the cell line tested.

Two flavonolignans from milk thistle (Silybum marianum) inhibit CYP2C9-mediated warfarin metabolism at clinically achievable concentrations.

Milk thistle (Silybum marianum) is a popular herbal product used for hepatoprotection and chemoprevention. Two commercially available formulations are the crude extract, silymarin, and the semipurified product, silibinin. Silymarin consists of at least seven flavonolignans, of which the most prevalent are the diastereoisomers silybin A and silybin B; silibinin consists only of silybin A and silybin B. Based on a recent clinical study showing an interaction between a silymarin product and the CYP2C9 substrate losartan, the CYP2C9 inhibition properties of silybin A and silybin B and corresponding regioisomers, isosilybin A and isosilybin B, were evaluated using human liver microsomes (HLMs), recombinant CYP2C9 (rCYP2C9) enzymes, and the clinically relevant probe, (S)-warfarin. Silybin B was the most potent inhibitor in HLMs, followed by silybin A, isosilybin B, and isosilybin A (IC(50) of 8.2, 18, 74, and >100 microM, respectively). Next, silybin A and silybin B were selected for further characterization. As with HLMs, silybin B was more potent than silybin A toward rCYP2C9 1 (6.7 versus 12 microM), rCYP2C9 2 (9.3 versus 19 microM), and rCYP2C9 3 (2.4 versus 9.3 microM). Using a matrix of five substrate (1-15 microM) and six inhibitor (1-80 microM) concentrations and HLMs, both diastereoisomers inhibited (S)-warfarin 7-hydroxylation in a manner described best by a mixed-type inhibition model (K(i) values of 4.8 and 10 microM for silybin B and silybin A, respectively). These observations, combined with the high systemic silibinin concentrations (>5-75 microM) achieved in a phase I study involving prostate cancer patients, prompt clinical evaluation of a potential warfarin-milk thistle interaction.

Isosilybin A induces apoptosis in human prostate cancer cells via targeting Akt, NF-κB, and androgen receptor signaling.

Prostate cancer (PCA) is the second most malignancy in American men. Advanced stage PCA cells possess unlimited replication potential as well as resistance to apoptosis. Therefore, targeting survival mechanisms and activating apoptotic machinery in PCA cells using nontoxic phytochemicals is suggested as an attractive strategy against this deadly malignancy. In the present study, we assessed the effect of one such botanical agent, namely isosilybin A, on apoptotic machinery and key members of cell survival signaling [Akt, NF-κB, and androgen receptor (AR)] in different PCA cells. Results showed that isosilybin A (90-180 µM) treatment significantly induces apoptotic death by activating both extrinsic (increased level of DR5 and cleaved caspase 8) and intrinsic pathways (caspase 9 and 3 activation) of apoptosis in three different human PCA cell lines namely 22Rv1, LAPC4, and LNCaP. Further, isosilybin A treatment decreased the levels of phospho-Akt (serine-473), total Akt, and the nuclear levels of NF-κB constituents (p50 and p65). Isosilybin A treatment also decreased the AR and PSA level in 22Rv1, LAPC4, and LNCaP cells. Employing pan-caspase inhibitor (Z-VAD.fmk), we confirmed that isosilybin A-mediated decreased AR is independent of caspases activation. Temporal kinetics analysis showed that the primary effect of isosilybin A is on AR, as decrease in AR was evident much earlier (4 h) relative to caspase activation and apoptosis induction (12 h). Overall, our results demonstrated that isosilybin A activates apoptotic machinery in PCA cells via targeting Akt-NF-κB-AR axis; thereby, indicating a promising role for this phytochemical in the management of clinical PCA.

Large-scale isolation of flavonolignans from Silybum marianum extract affords new minor constituents and preliminary structure-activity relationships.

The gram-scale isolation of the major flavonolignan diastereoisomers from milk thistle ( Silybum marianum) extract provided an entree into the isolation of two related analogues that are present in extremely minute quantities. The isolation and structure elucidation of these two new compounds, which we have termed isosilybin C and isosilybin D due to their structural similarities to isosilybin A and isosilybin B, respectively, afforded a preliminary analysis of structure-activity relationships toward prostate cancer growth, survival, and apoptotic endpoints.

Design, synthesis, and biological evaluation of antibody-drug conjugates comprised of potent camptothecin analogues.

Antibody-drug conjugates (ADCs) were prepared with potent camptothecin analogues attached to monoclonal antibodies (mAbs) via dipeptide or glucuronide-based linkers. Aniline-containing camptothecin analogues were employed to provide a site of linker attachment via carbamate bonds that would be stable in circulation. The camptothecin analogues, 7-butyl-10-amino-camptothecin and 7-butyl-9-amino-10,11-methylenedioxy-camptothecin, are generally 10-1000 times more potent than camptothecin. Dipeptide and glucuronide drug linkers were employed containing self-immolative spacers that release drug following lysosomal degradation upon ADC internalization into antigen-positive cell lines. The camptothecin drug linkers were conjugated to three antibodies: chimeric BR96, chimeric AC10, and humanized 1F6, which bind to the Lewis-Y antigen on carcinomas, CD30 on hematologic malignancies, and CD70 present on hematologic malignancies and renal cell carcinoma, respectively. ADCs bearing the potent camptothecin analogue, 7-butyl-9-amino-10,11-methylenedioxy-camptothecin, were highly potent and immunologically specific on a panel of cancer cell lines in vitro, and efficacious at well-tolerated doses in a renal cell carcinoma xenograft model.

Discovery of anticancer agents of diverse natural origin.

A collaborative multidisciplinary research project is described in which new natural product anticancer drug leads are obtained from a diverse group of organisms, constituted by tropical plants, aquatic cyanobacteria, and filamentous fungi. Information is provided on how these organisms are collected and processed. The types of bioassays are indicated in which crude extracts of these acquisitions are tested. Progress made in the isolation of lead bioactive secondary metabolites from three tropical plants is discussed.

Bioactive constituents of the stem bark of Mitrephora glabra.

Bioactivity-guided fractionation of the stem bark of Mitrephora glabra yielded nine compounds, comprising three ent-kaurenoids (1-3), five polyacetylenic acids/esters (4-8), and one aporphine alkaloid, liriodenine (9). The structures of the six new compounds (1-3, 5, 7, and 8) were determined by spectroscopic data interpretation. All compounds were evaluated for their inhibitory activities against a panel of cancer cell lines and a battery of microorganisms.

Identifying the differential effects of silymarin constituents on cell growth and cell cycle regulatory molecules in human prostate cancer cells.

Prostate cancer (PCa) is the leading cause of cancer-related deaths in men; urgent measures are warranted to lower this deadly malignancy. Silymarin is a known cancer chemopreventive agent, but the relative anticancer efficacy of its constituents is still unknown. Here, we compared the efficacy of 7 pure flavonolignan compounds isolated from silymarin, namely silybin A, silybin B, isosilybin A, isosilybin B, silydianin, isosilydianin, silychristin and isosilychristin, in advanced human PCa PC3 cells. Silybin A, silybin B, isosilybin A, isosilybin B, silibinin and silymarin strongly inhibited the colony formation by PC3 cells (p < 0.001), while silydianin, silychristin and isosilychristin had marginal effect (p < 0.05). Using cell growth and death assays, we identified isosilybin B as the most effective isomer. FACS analysis for cell cycle also showed that silybin A, silybin B, isosilybin A, isosilybin B, silibinin and silymarin treatment resulted in strong cell cycle arrest in PC3 cells after 72 hr of treatment, while the effect of silydianin, silychristin and isosilychristin was marginal (if any). Western blot analysis also showed the differential effect of these compounds on the levels of cell cycle regulators-cyclins (D, E, A and B), CDKs (Cdk2, 4 and Cdc2), CDKIs (p21 and p27) and other cell cycle regulators (Skp2, Cdc25A, B, C and Chk2). This study provided further evidence for differential anticancer potential among each silymarin constituent, which would have potential implications in devising better formulations of silymarin against prostate and other cancers.