Isolation, Structure Determination, and Semisynthesis of Diphenazine Compounds from a Deep-Sea-Derived Strain of the Fungus <i>Cystobasidium laryngis</i> and Their Biological Activities

Phenazostatins E–J (1–6), six new diphenazine derivatives, were isolated from the EtOAc extract of the culture broth of a strain of Cystobasidium laryngis derived from deep-sea sediments of the Indian Ocean Ridge. The structures of 1–6 were elucidated based on the HRESIMS and 1D and 2D NMR spectra. The absolute configurations of 1–6, except for 3 and 6, were determined by modified Mosher’s method, ECD data analysis, and calculations of optical rotation values. The absolute configurations of 3 and 6 were identified by chemical derivatization and comparing the specific rotation values with those of semisynthetic 3 obtained by the oxidation of 1 and saphenic acid (7). Phenazostatin J (6) was semisynthesized using saphenic acid (7) to prepare additional material for biological testing. During the purification of semisynthetic 6, a side product 9 was obtained from the reaction mixture along with 6. Compounds 1–6, along with previously reported 7 and 8, were assessed for anti-neuroinflammatory activity in LPS-induced BV-2 microglia cells. Compound 6 exhibited the highest anti-neuroinflammatory effect with an IC50 value of 0.30 μM, but it showed cytotoxicity at higher concentrations than 1.0 μM. Accordingly, cytotoxicities of 1–9 were evaluated against six human cancer cell lines. Among tested compounds, 6 and 9 showed potent cytotoxicity (IC50 values: 7.7–72 nM). Especially, 6 exhibited the strongest cytotoxicity with an IC50 value of 7.7 nM against the NUGC-3 (stomach) cell line, displaying 19-fold stronger activity than the positive control, adriamycin

Aspersterols A–D, Ergostane-Type Sterols with an Unusual Unsaturated Side Chain from the Deep-Sea-Derived Fungus<i>Aspergillus unguis</i>

Four previously undescribed ergostane-type sterols, aspersterols A–D (1–4), were isolated from a deep-sea-derived fungus, Aspergillus unguis IV17-109. The structures of the new compounds were determined by extensive analyses of their spectroscopic data, pyridine-induced deshielding effect, Mosher’s method, and electronic circular dichroism calculations. The key feature of these sterols is the presence of a rare unsaturated side chain with conjugated double bonds at Δ17 and Δ22. The absolute configuration of C-24 in the side chain was determined by hydrogenation and comparing 13C NMR chemical shifts of the hydrogenated products with literature values. In addition, aspersterol A (1) is the second representative of anthrasteroids with a hydroxy group at the C-2 position. Compound 1 showed cytotoxicity against six cancer cell lines, with GI50 values of 3.4 ± 0.3 to 4.5 ± 0.7 μM, while 2–4 showed anti-inflammatory activity, with IC50 values ranging from 11.6 ± 1.6 to 19.5 ± 1.2 μM

Cytotoxic Psammaplysin Analogues from a <i>Suberea</i> sp. Marine Sponge and the Role of the Spirooxepinisoxazoline in Their Activity

Seventeen bromotyrosine-derived metabolites, including eight new compounds, were isolated from a Micronesian sponge of the genus <i>Suberea</i>. Four of the new compounds were psammaplysin derivatives (<b>10</b>–<b>13</b>), and the other four were ceratinamine derivatives (<b>14</b>–<b>17</b>). Of the compounds obtained, the psammaplysins exhibited cytotoxicity against human cancer cell lines (GI₅₀ values down to 0.8 μM), while the ceratinamine and moloka’iamine analogues showed almost no activity. These results suggest that the spirooxepinisoxazoline ring system is a requirement for cytotoxicity and, therefore, may serve as an attractive molecular scaffold for the development of a potent anticancer agent

Lanostane Triterpenes Isolated from <i>Antrodia heteromorpha</i> and Their Inhibitory Effects on RANKL-Induced Osteoclastogenesis

Two new spiro-lanostane triterpenoids, antrolactones A and B (<b>1</b> and <b>2</b>), along with polyporenic acid C (<b>3</b>), were isolated from an EtOAc-soluble extract of <i>Antrodia heteromorpha</i> culture medium, and the chemical structures of the new compounds were elucidated by application of NMR, MS, and ECD spectroscopic techniques. All isolated compounds exhibited inhibitory effects on receptor activator of nuclear factor-kappaB ligand-induced osteoclastogenesis

The effects of miR-6734 were abolished by p21 siRNA in HCT-116 cells.

Cells were transfected with mock, dsCon or miR-6734 at 10 nmol/L and transfected simultaneously with p21 siRNA (siP21, 10 nmol/L) for p21 knockdown. (A) The mRNA expression of p21 was analyzed by qPCR after 72 h. (B) Viable cells were measured using cell proliferation kit (XTT) after 72 h. (C) The treated cells were stained with propidium iodide and annexin V-FITC after 72 h and apoptotic cells were measured by flow cytometry. The percentage of early and late apoptotic cells were calculated using the WinMDI program. (D) Culture supernatants were collected after 72 h, and the activity of caspase-3/7 was determined via caspase-Glo 3/7 assay kit. (E) Cell cycle analysis was performed after 48 h and the percentage of cells in G0/G1 and S phases were calculated using the ModFit program. Data are presented as mean ± S.D. of triplicate experiments. Statistical significance was analyzed by one-way ANOVA and Dunnett’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001 versus miR-6734). (F) Protein levels of p21, cyclin-A, p-Rb, caspase 3, PARP and GAPDH in the total cell lysates were determined after 72 h by Western Immunoblot analysis.</p

Histone modification of p21 promoter by miR-6734.

Chromatin immunoprecipitation (ChIP) assays were performed by using antibodies against IgG, di-methyl-histone-H3-lysine 9 (H3K9me2), acetyl-histone-H2B (H2Bac) and acetyl-histone-H3 (H3ac) to pull down associated DNA. The precipitated DNA was amplified by RT-PCR using primer sets specific to miR-6734 target sites (-360/-260) of p21 gene promoter. Input DNA was amplified as a loading control. DNA pulled down in the IgG antibody served to identify background amplification.</p

miR-6734 induced p21 expression and inhibits proliferation and survival in HCT-116 colon cancer cells.

(A,B) HCT-116 cells were transfected with mock, dsCon and dsP21-322 at 10 nmol/L or the indicated concentrations of miR-6734 for 72 h. The mRNA expression of p21 was determined by qPCR and the protein expression of p21 was determined by Western Immunoblot analysis. (C) Cells were transfeced with 10 nmol/L miR-6734 for the indicated lengths of time. The mRNA expression of p21 and β-actin was assessed by RT-PCR. (D) Cell proliferation was measured from day 1 to 4 after treatment with the indicated concentrations of miR-6734 using the Cell proliferation kit (XTT). (E,F) The treated cells were seeded in 6-well plates at a density of 2,500 cells per well. Colony formation was analyzed on day 10 by staining the cells with crystal violet and the number of colonies was counted. Data are presented as mean ± S.D. of triplicate experiments. Statistical significance was analyzed by one-way ANOVA and Dunnett’s t-test (* p < 0.05; *** p < 0.001 versus mock).</p

miR-6734 targets p21 promoter and induces p21 gene expression.

<p>(A) Sequence of dsP21-322 and miR-6734 target site located at nucleotide -322 relative to the transcriptional start site in p21 promoter. (B) Five human cancer cells were transfected with miR-6734 at 10nmol/L for 72h. The mRNA expression of p21 was analyzed by qPCR. (C) HCT-116 cells were transfected with mock and miR-6734-5P inhibitor at 30 nmol/L for 72 h. The mRNA expression of p21 was analyzed by qPCR. Data are presented as mean ± S.D. of quadruplicate experiments. Statistical significance was analyzed by one-way ANOVA and Dunnett’s t-test (* p < 0.05; ** p < 0.01 versus mock). (D) HCT-116 cells were transfected with biotin-labeled miR-6734 and chromatin immunoprecipitation (ChIP) assays were performed by using antibodies against biotin to pull down associated DNA. The precipitated DNA was amplified by PCR using primer sets specific to miR-6734 target sites (-360/-260) of p21 promoter. Input DNA was amplified as a loading control. DNA pulled down by the anti-IgG antibody was served to identify background amplification. Duplicate samples were analyzed for each treatment.</p

<i>Xylodon flaviporus</i>-Derived Drimane Sesquiterpenoids That Inhibit Osteoclast Differentiation

The presence of excessive osteoclasts is a major factor in skeletal diseases. The present study aimed to discover osteoclast differentiation inhibitors from the basidiomycete Xylodon flaviporus. Seven new drimane sesquiterpenoids (1–7) and 7-ketoisodrimenin-5-ene (8) were obtained and characterized by various spectroscopic methods. The isolated compounds were evaluated for their inhibitory effects against receptor activator of nuclear factor-kappa-B ligand-induced osteoclastogenesis in mouse bone marrow macrophages. Compounds 1, 3, and 6 showed potent activities with IC50 values of 1.6, 0.9, and 2.1 μM, respectively, while 4, 5, and 7 exhibited relatively weak activities with IC50 values of 10.7, 10.1, and 8.5 μM, respectively

miR-6734 induces apoptosis in HCT-116 cells.

<p>HCT-116 cells were treated with mock, dsCon or the indicated concentrations of miR-6734 for 72 h. (A) Transfected cells were stained with propidium iodide and annexin V-FITC and apoptotic cells were measured by flow cytometry. (B) The percentage of early and late apoptotic cells were calculated using the WinMDI program. (C) Culture supernatants were collected, and the activity of caspase-3/7 was determined via caspase-Glo 3/7 assay kit. Data are presented as mean ± S.D. of triplicate experiments. Statistical significance was analyzed by one-way ANOVA and Dunnett’s t-test (** p < 0.01; *** p < 0.001 versus mock).</p