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

Trisubstituted-Imidazoles Induce Apoptosis in Human Breast Cancer Cells by Targeting the Oncogenic PI3K/Akt/mTOR Signaling Pathway

<div><p>Overactivation of PI3K/Akt/mTOR is linked with carcinogenesis and serves a potential molecular therapeutic target in treatment of various cancers. Herein, we report the synthesis of trisubstituted-imidazoles and identified 2-chloro-3-(4, 5-diphenyl-1H-imidazol-2-yl) pyridine (CIP) as lead cytotoxic agent. Naïve Base classifier model of <i>in silico</i> target prediction revealed that CIP targets RAC-beta serine/threonine-protein kinase which comprises the Akt. Furthermore, CIP downregulated the phosphorylation of Akt, PDK and mTOR proteins and decreased expression of cyclin D1, Bcl-2, survivin, VEGF, procaspase-3 and increased cleavage of PARP. In addition, CIP significantly downregulated the CXCL12 induced motility of breast cancer cells and molecular docking calculations revealed that all compounds bind to Akt2 kinase with high docking scores compared to the library of previously reported Akt2 inhibitors. In summary, we report the synthesis and biological evaluation of imidazoles that induce apoptosis in breast cancer cells by negatively regulating PI3K/Akt/mTOR signaling pathway.</p></div

A and B, MDA-MB-231 cells were treated with CIP (25 μM) for indicated time intervals (0, 12, 24, 36 and 48 h) and expression of apoptotic markers (PARP, Procaspase-3), antiapoptotic proteins (Bcl-2, Survivin, VEGF), cell cycle regulator (Cyclin D1) was profiled using western blot analysis.

<p>We observed the significant decline in the expression of PARP, procaspase-3, cyclin D1, VEGF, survivin and Bcl-2 proteins in the time-dependent manner without alteration in the levels of beta actin.</p

Trisubstituted-Imidazoles Induce Apoptosis in Human Breast Cancer Cells by Targeting the Oncogenic PI3K/Akt/mTOR Signaling Pathway - Fig 1

<p>A, Schematic representation for the synthesis of imidazole based small molecules. B, Breast cancer cells (2.5 X 10⁴/mL, MCF-7 & MDA-MB-231) were plated in triplicate, treated with indicated concentrations (0, 10, 25 and 50 μM) of CIP, and then subjected to MTT assay after 24, 48 and 72 h to analyse proliferation of cells. CIP suppresses the viability of various breast cancer cell lines in a dose- and time-dependent manner.</p

MDA-MB-231 cells were treated with CIP at indicated concentrations (0, 10, 15, 25 and 50 μM) for 8 h and levels of phospho-PDK, phospho-Akt, phospho-mTOR and PDK, Akt, mTOR was analysed using western blotting.

<p>We found that, CIP downregulated the phosphorylation of Akt, PDK and mTOR in a dose-dependent manner without any change in the expression levels of Akt, PDK and mTOR proteins.</p

<i>In silico</i> molecular docking studies of trisubstituted imidazoles against kinase domain of Akt2:

<p>Common binding poses of trisubstituted imidazoles towards the Akt2 kinase domain. The molecular surface of the protein is represented based on the surface polarity; green, pink and red colours show hydrophobic, polar and solvent exposed regions, respectively. For the sake of better visualisation of the binding pocket surface of Akt2, molecular surface was rendered in two panels (A and B) in which ligands were rotated 180 degrees.</p

MDA-MB-231 cells were suspended in serum-free DMEM and seeded into the Matrigel transwell chambers and were incubated with CIP (25 μM) for 8 h.

<p>The invading cells were fixed and stained with crystal violet solution and invaded cells were counted in five randomly selected areas. CIP significantly suppressed the cell invasion both in the presence and absence of CXCL12 suggesting that CIP interferes with invasive potential of breast cancer cells.</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