<i>N</i>‑Hydroxycinnamide Derivatives of Osthole Presenting Genotoxicity and Cytotoxicity against Human Colon Adenocarcinoma Cells in Vitro and in Vivo

Osthole is extracted from the Chinese herbs <i>Cnidium monnieri</i> and <i>Angelica pubescens</i>, and it was found to have antitumor activity in vitro and in vivo. A series of osthole derivatives have been synthesized, and the <i>N</i>-hydroxycinnamide derivatives of osthole, WJ1376-1 and WJ1398-1 were found to have the greatest potential against human colon adenocarcinoma cells. In contrast to the parental osthole, both WJ1376-1 and WJ1398-1 were found to induce multinucleation and polyploidy by microscopic observation and flow cytometry. WJ1376-1 and WJ1398-1 significantly activated ataxia telangiectasia and rad3 related (ATR) kinase, which triggered activation of the checkpoint kinase 2 (Chk2) signaling pathway and then down regulated Cdc25 phosphatase and Cdc2/cyclin B kinase activities. WJ1376-1 and WJ1398-1 also inhibited the phosphorylation of Aurora A kinase, which is associated with important processes during mitosis. The presence of a “comet” DNA fragment and phosphorylation of p53 at Ser 15 clearly indicated that DNA damage occurred with WJ1376-1 and WJ1398-1 treatment. WJ1376-1 and WJ1398-1 ultimately induced apoptosis as evidenced by the upregulation of Bad and activation of caspases-3, -7, and -9. Furthermore, WJ1376-1 and WJ1398-1 also showed a great effect in attenuating tumor growth without affecting the body weight of xenograft nude mice. Taken together, these results suggest that the toxic activities of WJ1376-1 and WJ1398-1 were dissimilar to that of the parental osthole, which can induce cell polyploidy and G₂/M cell cycle arrest in colon adenocarcinoma cells and may provide a potential therapeutic target for colon cancer treatment in the future

Impaired long-chain fatty acid (LCFA) β-oxidation and induction of reactive oxygen species (ROS)-dependent pro-inflammatory cytokines in cells infected with Japanese encephalitis virus (JEV).

<p>(A and B) A549 cells infected with JEV (multiplicity of infection [MOI] = 10) for 5 h were replenished with serum-free medium for 1 h, then treated with 200 μM palmitate conjugated to bovine serum albumin (BSA) (PA-BSA) or BSA control. (A) Real-time oxygen consumption rate (OCR) measured from 6 to 24 h post-infection (hpi). The OCR before PA-BSA or BSA treatment was set to 100%. (B) The area under the curve (AUC) OCR compared to that for mock cells treated with BSA (n = 3 per group). (C and D) A549 cells infected with JEV (MOI = 5 and 0.1) for 5 h were changed to medium without serum (C) or with serum (10% FBS) (D) for 1 h. Cells were then treated with PA-BSA or BSA for 18 h before Western blot analysis of protein levels of JEV NS3 and actin in cell lysates and virus titration in culture supernatants by plaque-forming assay (n = 3). (E and F) A549 cells were treated with N-acetylcysteine (NAC) 1 h before JEV (MOI = 10) infection and after virus adsorption. At 5 hpi, cells were incubated with serum-free medium for 1 h before treatment with PA-BSA or BSA for 18 h. RT-qPCR analysis of the relative mRNA levels of interleukin 6 (IL-6) (E) and tumor necrosis factor α (TNF-α) (F) (n = 3). Data are mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant.</p

JEV NS5 interacts with mitochondrial trifunctional protein (MTP), the enzyme complex involved in LCFA β-oxidation.

<p>(A) Western blot analysis of protein levels of the indicated proteins in A549 cells with JEV infection (MOI = 10) or NS5-Flag overexpression. (B) Western blot analysis of V5-tag, Flag-tag, and actin in HEK293T cells co-transfected with HADHα-V5-His plus the indicated plasmids expressing Flag-tagged JEV viral proteins for 24 h, then immunoprecipitated with anti-Flag affinity gel. WCL, whole-cell lysates. (C) Immunoprecipitation (IP) analysis with control IgG or anti-Flag affinity gel in A549, GFP-A549 and NS5-Flag-A549 cells. The protein bands identified as HADHα and HADHβ are indicated by arrows. (D) IP with anti-Flag affinity gel and Western blot analysis with the indicated antibodies in HEK293T cells co-transfected with vector control or NS5-Flag plus HADHα-V5-His or HADHβ-HA for 24 h. (E) IP analysis with nickel beads and Western blot analysis with the indicated antibodies in HEK293T cells adsorbed with JEV for 3 h, then transfected with vector control, HADHα-V5-His or HADHβ-V5-His for 24 h.</p

Subcellular localization of JEV NS5.

<p>(A-C) Cellular lysates of HEK293T cells infected with JEV (MOI = 5) or transfected with NS5-Flag for 24 h underwent Qproteome Mitochondria Isolation (A) or biochemical fractionation as outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004750#ppat.1004750.s005" target="_blank">S5B and S5C Fig</a>, respectively (B and C). Western blot analysis of indicated proteins in cytosolic and crude mitochondrial fractions. C, cytosolic fraction; H, heavy membrane fraction/crude mitochondrial fraction; L, light microsomal membrane fraction. (D) The crude mitochondrial fraction isolated from HEK293T cells infected with JEV (MOI = 3) or transfected with NS5-Flag for 24 h was treated with Proteinase K (100 μg/ml) for 30 min on ice. The reactants were developed by Western blot analysis with antibodies against NS5 or the indicated mitochondrial proteins. (E) Confocal microscopy of pEYFP-Mito-NS5-A549 cells stained with anti-Flag plus Alexa Fluor 568 goat anti-rabbit and anti-HADHα plus Alexa Fluor 647 goat anti-mouse antibody. (F) Confocal microscopy of pEYFP-Mito-NS5-A549 cells transfected with HADHβ-HA for 24 h and stained with anti-Flag plus Alexa Fluor 568 goat anti-rabbit and anti-HA plus Alexa Fluor 647 goat anti-mouse antibody.</p

The recombinant JEV with NS5-M19A mutation is less able to block LCFA β-oxidation and induces less cytokine expression.

<p>(A and B) A549 cells infected with JEV-WT or JEV-NS5-M19A (MOI = 10) for 5 h were changed to serum-free medium for 1 h, then incubated with PA-BSA or BSA control. (A) Real-time OCR was measured from 6 to 24 h post-infection. The OCR before PA-BSA or BSA treatment was set to 100%. (B) The AUC OCR with PA-BSA and BSA (n = 3). (C and D) A549 cells infected with the indicated JEV (MOI = 10) for 5 h were incubated with serum-free medium for 1 h before treatment with PA-BSA or BSA for 18 h. RT-qPCR analysis of relative mRNA levels of IL-6 (C) and TNF-α (D) (n = 3). (E-G) A549 cells were infected with JEV-WT or JEV-NS5-M19A (MOI = 10) for 24 h in serum (10% FBS)-containing medium. RT-qPCR analysis of relative mRNA levels of JEV RNA (E), IL-6 (F) and TNF-α (G) (n = 3). Data are mean±SD.*P < 0.05, **P < 0.01 and ***P < 0.001.</p

Japanese Encephalitis Virus Nonstructural Protein NS5 Interacts with Mitochondrial Trifunctional Protein and Impairs Fatty Acid β-Oxidation

<div><p>Infection with Japanese encephalitis virus (JEV) can induce the expression of pro-inflammatory cytokines and cause acute encephalitis in humans. β-oxidation breaks down fatty acids for ATP production in mitochondria, and impaired β-oxidation can induce pro-inflammatory cytokine expression. To address the role of fatty-acid β-oxidation in JEV infection, we measured the oxygen consumption rate of mock- and JEV-infected cells cultured with or without long chain fatty acid (LCFA) palmitate. Cells with JEV infection showed impaired LCFA β-oxidation and increased interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) expression. JEV nonstructural protein 5 (NS5) interacted with hydroxyacyl-CoA dehydrogenase α and β subunits, two components of the mitochondrial trifunctional protein (MTP) involved in LCFA β-oxidation, and NS5 proteins were detected in mitochondria and co-localized with MTP. LCFA β-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells. Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP. The recombinant JEV with NS5-M19A mutation (JEV-NS5-M19A) was less able to block LCFA β-oxidation and induced lower levels of IL-6 and TNF-α than wild-type JEV. Moreover, mice challenged with JEV-NS5-M19A showed less neurovirulence and neuroinvasiveness. We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA β-oxidation and inducing cytokine expression by association with MTP.</p></div

NS5 with mutation on residue 19 (M19A) showed reduced binding ability with MTP.

<p>(A) Western blot analysis of indicated proteins in HEK293T cells transfected with the plasmids expressing wild type (WT)-, mutated-NS5-Flag or vector control for 24 h after immunoprecipitation with anti-Flag affinity gel. Band densities were quantified by use of MetaMorph (Molecular Devices). (B and F) HEK293T cells transfected with NS5-Flag or NS5-M19A-Flag (B) or infected with wild-type JEV (JEV-WT) or JEV-NS5-M19A (MOI = 5) (F) for 24 h underwent Qproteome Mitochondria Isolation. Western blot analysis of indicated proteins in mitochondrial and cytosolic fractions. C, cytosolic fraction; H, heavy membrane fraction/crude mitochondrial fraction. (C and D) JEV-NS5-M19A mutant virus was generated by using a JEV infectious clone. (C) Plaque morphology of wild type JEV (JEV-WT) and JEV-NS5-M19A mutant in BHK-21 cells. (D) A549 cells were infected with JEV-WT or JEV-NS5-M19A (MOI = 0.1) for the indicated times. Western blot analysis of protein levels of NS3 and actin. Plaque-forming assay of virus titration in culture supernatants (n = 3). Data are mean±SD. (E) IP analysis with V5 or HA affinity gel and Western blot analysis with the indicated antibodies in HEK293T cells adsorbed with JEV for 1 h, then transfected with HADHα-V5-His or HADHβ-HA for 24 h.</p

N-terminus of NS5 is essential for its interaction with MTP.

<p>(A) Schematic diagram and properties of full-length and truncated NS5 constructs. (B and C) IP—Western and Western blot analysis with anti-Flag affinity gel and the indicated antibodies for Flag-tag, V5-tag and HA-tag in HEK293T cells co-transfected with full-length and truncated NS5-Flag plus HADHα-V5-His (B) or HADHβ-HA (C) for 24 h.</p

Reduced neurovirulence of NS5-M19A—mutated JEV in challenged mice.

<p>(A) Survival in C57BL/6 mice infected with 0.2, 2 or 20 plaque-forming units (PFU) of JEV-WT or JEV-NS5-M19A by an intracerebral (i.c.) injection. The animal number (<i>n</i>) and survival rate for each group are shown. (B-D) RT-qPCR of relative JEV RNA (B), IL-6 (C), and TNF-α (D) mRNA levels in brain tissues of mice inoculated with JEV-WT or JEV-NS5-M19A (20 PFU) (n = 3). Data are mean±SD.*P < 0.05.</p

Reduced neuroinvasiveness of NS5-M19A—mutated JEV in challenged mice.

<p>(A) Survival in C57BL/6 mice infected with 10³ or 10⁵ PFU JEV-WT or JEV-NS5-M19A by an intraperitoneal (i.p.) plus i.c. route. The animal number (<i>n</i>) and survival rate for each group are shown. (B) Plaque-forming assay of virus titers in brain tissues of mice inoculated with JEV-WT or JEV-NS5-M19A (10⁵ PFU) (n = 3). (C-E) RT-qPCR of relative JEV RNA (C), IL-6 (D), and TNF-α (E) mRNA levels in brain tissues (n = 3). (F) ELISA of IL-6 protein levels in the sera samples (n = 3). Data are mean±SD.*P < 0.05, ***P < 0.001.</p