Resultative Compound Verb in Modern Chinese : A Comment on Imai(1985) and Lu(1986)

<p>A. API and DMO suppresses NF-κB DNA binding ability in HCT116 cells. HCT116 cells were treated with DMO and API at indicated doses, nuclear extracts were prepared, and 20 μg of the nuclear extract protein was used for the ELISA-based DNA-binding assay *p<0.05; **p<0.005). B & C. NF-κB responsive elements linked to a luciferase reporter gene were transfected with wild-type or dominant-negative IκB and transfected cancer cells were treated at indicated doses for 6 h and luciferase activity was measured as described in Materials and Methods section. All luciferase experiments were done in triplicate and repeated twice (*p<0.05; **p<0.005). D. API abrogates constitutive IκBα phosphorylation in dose-dependent manner in HCT116 cells. HCT116 cells were treated with different concentrations of API (0, 5, 10 and 20 μM) for 6 h and cytoplasmic extract was prepared. Lysates were resolved on SDS gel and electrotransferred to a nitrocellulose membrane and probed with anti-phospho-IκBα/IκBα. The blot was washed, exposed to HRP-conjugated secondary antibodies for 1 h, and finally examined by chemiluminescence. GAPDH was used as loading control.</p

Correction: Novel Synthetic Oxazines Target NF-kappa B in Colon Cancer In Vitro and Inflammatory Bowel Disease In Vivo (vol 11, e0163209, 2016)

10.1371/journal.pone.0175659PLOS ONE124e017565

Novel Synthetic Oxazines Target NF-κB in Colon Cancer In Vitro and Inflammatory Bowel Disease In Vivo.

Aberrant activation of nuclear factor kappa B (NF-κB) has been linked with the pathogenesis of several proinflammatory diseases including number of cancers and inflammatory bowel diseases. In the present work, we evaluated the anticancer activity of 1,2-oxazines derivatives against colorectal cancer cell lines and identified 2-((2-acetyl-6,6-dimethyl-4-phenyl-5,6-dihydro-2H-1,2-oxazin-3-yl)methyl)isoindoline-1,3-dione (API) as the lead anticancer agent among the tested compounds. The apoptosis inducing effect of API was demonstrated using flow cytometry analysis and measuring the caspase 3/7 activity in API treated cells. Based on the literature on inhibition of NF-κB by oxazines, we evaluated the effect of 1,2-oxazines against the ability of NF-κB binding to DNA, NF-κB-dependent luciferase expression and IκBα phosphorylation. We found that, API abrogate constitutive activation of NF-κB and inhibits IκBα phosphorylation in HCT116 cells. Our in silico analysis revealed the binding of oxazines to the hydrophobic cavity that present between the interface of p65 and IκBα. Given the relevance with aberrant activation of NF-κB in inflammation bowel disease (IBD), we evaluated the effect of API on dextran sulphate sodium-induced IBD mice model. The treatment of IBD induced mice with API decreased the myeloperoxidase activity in colonic extract, modulated the colon length and serum levels of pro- and anti-inflammatory cytokines such as TNF-α, IFN-γ, IL-6, IL-1β and IL-10. Furthermore, the histological analysis revealed the restoration of the distorted cryptic epithelial structure of colon in the API treated animals. In conclusion, we comprehensively validated the NF-κB inhibitory efficacy of API that targets NF-κB in in vitro colon cancer and an in vivo inflammatory bowel disease model

Novel Synthetic Oxazines Target NF-κB in Colon Cancer <i>In Vitro</i> and Inflammatory Bowel Disease <i>In Vivo</i> - Fig 1

<p>A. The lead compounds identified among the 1,2-oxazine derivatives tested decreased the cell proliferation of HCT116 cells in dose-dependent manner. B. Microscopic images to demonstrate the inhibition of cell proliferation (Magnification 4x). C. HCT116 cells were treated with API for 48 h and caspase 3/7 assay was performed. We observed a significant dose dependent increase in caspase 3/7 activity levels demonstrating the ability of API to induce apoptosis.</p

In silico interaction between the oxazines and IκBα/NF-κB complex.

<p>A. Representation of the native IκBα/NF-κB heterodimer and docked solution of tested oxazines (in stick representation). The sub-unit of p50 is represented in green, p65 in cyan, and IκBα in pink. B. Molecular docking of the lead structure API with the NF-κB heterodimer solution was shown. C. Interaction map lead compound API that bound with key amino acids of the crystal structure. Hydrogen bonding (black dots) between API oxygen atoms with Tyr251, and other Asp271, Arg246, His245 was shown.</p

Novel Synthetic Oxazines Target NF-κB in Colon Cancer <i>In Vitro</i> and Inflammatory Bowel Disease <i>In Vivo</i> - Fig 3

<p>A. API and DMO suppresses NF-κB DNA binding ability in HCT116 cells. HCT116 cells were treated with DMO and API at indicated doses, nuclear extracts were prepared, and 20 μg of the nuclear extract protein was used for the ELISA-based DNA-binding assay *p<0.05; **p<0.005). B & C. NF-κB responsive elements linked to a luciferase reporter gene were transfected with wild-type or dominant-negative IκB and transfected cancer cells were treated at indicated doses for 6 h and luciferase activity was measured as described in Materials and Methods section. All luciferase experiments were done in triplicate and repeated twice (*p<0.05; **p<0.005). D. API abrogates constitutive IκBα phosphorylation in dose-dependent manner in HCT116 cells. HCT116 cells were treated with different concentrations of API (0, 5, 10 and 20 μM) for 6 h and cytoplasmic extract was prepared. Lysates were resolved on SDS gel and electrotransferred to a nitrocellulose membrane and probed with anti-phospho-IκBα/IκBα. The blot was washed, exposed to HRP-conjugated secondary antibodies for 1 h, and finally examined by chemiluminescence. GAPDH was used as loading control.</p

Serum cytokine profile of control and experimental mice were estimated using murine mini ELISA development kits according to the manufacturer’s protocol.

<p>A. TNF-α. B. IFN-γ. C. IL-1β. D. IL-6. and E. IL-10. SZ-Sulfasalazine. Data are presented as mean ± S.E.M. * <i>p</i><0.05; **<i>p</i>< 0.01; ***<i>p</i><0.001.</p