Reengineering an Azaphilone Biosynthesis Pathway in <i>Aspergillus nidulans</i> To Create Lipoxygenase Inhibitors

Sclerotiorin, an azaphilone polyketide, is a bioactive natural product known to inhibit 15-lipoxygenase and many other biological targets. To readily access sclerotiorin and analogs, we developed a 2–3 step semisynthetic route to produce a variety of azaphilones starting from an advanced, putative azaphilone intermediate (<b>5</b>) overproduced by an engineered strain of <i>Aspergillus nidulans</i>. The inhibitory activities of the semisynthetic azaphilones against 15-lipoxygenase were evaluated with several compounds displaying low micromolar potency

Anti-inflammatory Activity of 8‑Hydroxydaidzein in LPS-Stimulated BV2 Microglial Cells via Activation of Nrf2-Antioxidant and Attenuation of Akt/NF-κB-Inflammatory Signaling Pathways, as Well As Inhibition of COX‑2 Activity

It was demonstrated that isoflavones can cross the blood–brain barrier, making them desirable candidate agents for the prevention of neurological symptoms. 8-Hydroxydaidzein (8-OHD, 4′,7,8-trihydoxyisoflavone) is an isoflavone found only in fermented soy food. Current results showed that 8-OHD inhibited LPS-stimulated production of nitric oxide (NO) and proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, by inhibiting gene expression in BV2 microglial cells. Moreover, 8-OHD markedly quenched reactive oxygen species (ROS) and activated NF-E2-related factor 2 (Nrf2) so as to upregulate expression of Phase II enzymes, including heme oxygenase (HO)-1, NAD­(P)H quinone dehydrogenase 1 (NQO1), and the modifier subunit of glutamate cysteine ligase (GCLM). 8-OHD also suppressed LPS-stimulated phosphorylation of Akt and NF-κB-p65. The anti-inflammatory activity of 8-OHD was attenuated by the HO-1 inhibitor zinc protoporphyrin (Znpp) but augmented by the PI3K/Akt inhibitor LY294002. 8-OHD also diminished LPS-induced prostaglandin E₂ (PGE₂) production without affecting cyclooxygenase (COX)-2 expression. In vitro assay shows that 8-OHD displayed mixed-type inhibition of COX-2 with an IC₅₀ of 8.9 ± 1.2 μM. These data suggest that the anti-inflammatory activity of 8-OHD may be associated with the activation of Nrf2/HO-1 and attenuation of Akt/NF-κB signaling pathways as well as inhibition of COX-2 enzyme activity. In conclusion, 8-OHD, a potent Nrf2 activator, Akt/NF-κB activation suppressor, and COX-2 enzyme inhibitor, may have health-promoting effects for mitigating microglia activation and preventing neuroinflammation

Expression of pre-S2Δ large surface protein increased ER stress and NF-κB, ERK, and Akt phosphorylation in Huh-7 cells.

<p>(A) (B) Cells were maintained in FBS–supplemented DMEM and cell lysates were obtained by RIPA lysis buffer. The cell lysates were determined by Western blotting using antibodies specific for GRP78, NF-κB p65, p-Ser276 p65, p-Ser311 p65, ERK, p-ERK, Akt, p-AKTand β-actin.</p

Expression of Bcl-2 contributes to 5-fluorouracil resistance in Huh-7 pre-S2Δ cells.

<p>(A) Huh-7 V, Huh-7 pre-S2Δ and Huh-7 pre-S cells (1×10³) were seeded in 6-well plates. Cells were treated with 5-fluorouracil with or without Bcl-2 inhibitor, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028977#s2" target="_blank">Materials and Methods</a>, and then incubated for another 10 days, and the colony formation ability of three cell lines were evaluated using a crystal violet assay determined by crystal violet in response to 5-fluorouracil. (B) A quantitative measure of colony formation was determined by crystal violet and the number of colonies in the graphs was representative of three independent experiments (lower panel). Data represent the mean ± SD (n = 3). Significant differences (**, <i>P</i><0.01 and ***, <i>P</i><0.001) between the control and experimental group are marked with an asterisks. (C) The morphologic changes after a 72-hour 0.25 mM 5-fluorouracil with or without Bcl-2 inhibitor treatment of Huh-7 pre-S2Δ cells. (D) The effect of 5-fluorouracil or combination with Bcl-2 inhibitor on caspase-3 activity in Huh-7 pre-S2Δ cell line. Enzymatic activity of caspase-3 was determined by caspase-3 antibody, as described under “materials and methods”. (E) The relative caspase-3 activities, normalized to DMSO control, at the indicated 0.25 mM 5-fluorouracil or with 5 µM Bcl-2 inhibitor. <i>Columns</i>, mean of three independent experiments; bars, SD (n = 3). Significant differences (***, <i>P</i><0.001) between the control and experimental group are marked with an asterisks. (F) Both apoptosis and necrosis were involved in 5-Fluorouracil or combination with Bcl-2 inhibitor-induced cell death. Huh-7 pre-S2Δ cells were treated with 5-fluorourail with or without Bcl-2 inhibitor and cells were analyzed by annexin V assay. (G) (H) The ratio of apoptotic and necrotic cells, normalized to DMSO control, at the indicated 0.25 mM 5-fluorouracil or with 5 µM Bcl-2 inhibitor. <i>Columns</i>, mean of three independent experiments; bars, SD (n = 3). Significant differences (*, <i>P</i><0.05; ***, <i>P</i><0.001) between the control and experimental group are marked with an asterisks. (I) Downregulation of Bcl-2 family expression by combination 5-fluorouracil with Bcl-2 inhibitor. Cells were treated with 5-fluorourail with or without Bcl-2 inhibitor and cell lysates were obtained by RIPA lysis buffer. The cell lysates were determined by Western blotting using antibodies specific for Bcl-2, Bcl-xL, Mcl-1, Bax, Bak and β-actin. (J) The effect of 5-fluorouracil or combination with Bcl-2 inhibitor on Akt, ERK and NF-κB phosphorylation in Huh-7 pre-S2Δ cell line. The total lysates were analyzed by Western blotting using ERK, p-ERK, Akt, p-Akt, p65, p-ser²⁷⁶-p65, p-ser³¹¹-p65 and β-actin antibodies.</p

Expression of HBV pre-S2Δ large surface protein in Huh-7 cells.

<p>Schematic representation of various lengths of HBV surface protein. (A) The pre-S represents full length (384 a.a). S represents C-terminal of pre-S2Δ (158–384 a.a.) Numbers in each construct correspond to amino acid positions. The arrow represents the point mutation of pre-S2 start codon from ATG to ATA. Gray box indicates pre-S2 deletion region. (B) Huh-7 cells were transfected with pIRES-pre-S2Δ-HA (pre-S2Δ), pIRES-pre-S-HA (pre-S), or pIRES vector control (V). The expression of pre-S2Δ protein and pre-S in Huh-7 cells were analyzed by Western blotting with antibody for HA. β-actin was used as a loading control.</p

HBV pre-S2Δ large surface protein increases cell growth and colony formation.

<p>(A) The effect of HBV large surface protein on Huh-7 growth rate was determined and cells were maintained in FBS–supplemented DMEM for 4 days, and the number of cells was assessed by trypan blue staining assay. (B) Huh-7 stable cell lines were analyzed for colony formation ability by colony formation assay, and colony formation was scored after 7 days. The number of colonies in the graphs was representative of three independent experiments (lower panel). Data represent the mean ± SD (n = 3). Significant differences (*, <i>P</i><0.05 and **, <i>P</i><0.01) between the control and experimental group are marked with an asterisks.</p

Expression of pre-S2Δ contributes to 5-fluorouracil treatment in Huh-7 cells.

<p>(A) The morphologic changes after a 96-hour 0.1 mM, 0.25 mM and 0.5 mM 5-fluorouracil treatment of Huh-7 V, Huh-7 pre-S2Δ, and Huh-7 Pre-S cells. The cells were followed by photography under phase-contrast magnification. (B) Cytotoxicity of 5-fluorouracil to Huh-7 V, Huh-7 pre-S2Δ and Huh-7 pre-S cells were analyzed by trypan blue staining assay. <i>Columns</i>, mean of three independent experiments; bars, SD (n = 3). Significant differences (*, <i>P</i><0.05) between the control and experimental group are marked with an asterisks (C) Cell viability after treatment with 5-FU. Huh-7 V, Huh-7 pre-S2Δ and Huh-7 pre-S cells (1×10³) were seeded in 6-well plate, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028977#s2" target="_blank">Materials and Methods</a>. Cells were then incubated for another 10 days, and the colony formation ability of three cell lines were evaluated using a crystal violet assay determined by crystal violet in response to 5-fluorouracil. (D) A quantitative measure of colony formation was determined by crystal violet and the number of colonies in the graphs was representative of three independent experiments (lower panel). Columns, mean; bars, SD (n = 3). Significant differences (**, <i>P</i><0.01 and ***, <i>P</i><0.001) between the control and experimental group are marked with an asterisks. (E) Flow cytometric analysis the effect of 5-fluorouracil on increasing caspase-3 activity in Huh7 V, Huh-7 pre-S2Δ, and Huh-7 pre-S cells, increased caspase-3 activity was observed in Huh7 V, Huh-7 pre-S2Δ, and Huh-7 pre-S cells, after 72-h exposure to 5-Fluorouracil. (F) The relative caspase-3 activities, normalized to DMSO control, at the indicated 0.25 mM of 5-fluorouracil. <i>Columns</i>, mean of three independent experiments; bars, SD (n = 3). Significant differences (*, <i>P</i><0.05) between the control and experimental group are marked with an asterisks.</p

Elevated Bcl-2 expression in HBV pre-S2Δ cells.

<p>(A) Total RNA was isolated from Huh-7 -V, Huh-7 pre-S2Δ, and Huh-7 pre-S cells and then subjected to RT-PCR analysis with Bcl-2 and G3APDH specific primers. (B) For Huh-7 -V, Huh-7 pre-S2Δ, and Huh-7 pre-S cells, the relative levels of Bcl-2 mRNA were quantified. The data represent the mean of Bcl-2 mRNA expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (**, <i>P</i><0.01) between the control and experimental group are marked with an asterisks. (C) The cell lysates collected from three stable clones, and cell lysates were analyzed by Western blotting analysis with antibodies for hemagglutinin (HA) tag, Bcl-2, and β-actin. (D) The levels of Bcl-2 protein in the graphs were representative of three independent experiments (lower panel). Columns, mean; bars, SD (n = 3). Significant differences (**, <i>P</i><0.01) between the control and experimental group are marked with an asterisks. (E) Furthermore, expression level of Bcl-2 was determined by using Western blotting in three individual pre-S2Δ stable cell lines. (F) Expression level of Bcl-2 was enhanced by pre-S2Δ in the immortalized human hepatocyte cell line. Total cell lysate from NeHep and NeHep-pre-S2Δ were determined by Western blotting analysis with Bcl-2, pre-S, and β-actin specific antibosies.</p

The pre-S2Δ large surface protein induced expression of Bcl-2 family proteins.

<p>(A) Total cell lysate was isolated from Huh-7 -V, Huh-7 pre-S2Δ, and Huh-7 pre-S cells and then subjected to Western blotting analysis with Bcl-xL, Mcl-1, Bax, and Bad specific antibodies. (B) For Huh-7 -V, Huh-7 pre-S2Δ, and Huh-7 pre-S cells, the relative levels of Bcl-xL, Mcl-1, Bax, and Bad protein were quantified. The data represent the mean of Bcl-xL, Mcl-1, Bax, and Bad expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (*, <i>P</i><0.05) between the control and experimental group are marked with an asterisks.</p

GSK3β signaling pathway does not involve in p53 expression through ER stress.

<p>Effects of GSK-3β inhibitor, lithium chloride, on p53 expression in ER stress. MCF-7 cells were incubated 1 µg/ml Brefeldin A with or without lithium chloride as time indicated. The total lysates were subjected to immunoblotting with antibodies against anti-p53, anti-GSK-3β, p-ser⁹-GSK-3β, and α-tubulin.</p