Highly Efficient Synthesis of Mixed 3,3′-Bisindoles via Rh(II)-Catalyzed Three-Component Reaction of 3‑Diazooxindoles with Indoles and Ethyl Glyoxylate

A series of mixed 3,3′-bisindoles were efficiently synthesized via a Rh₂(OAc)₄-catalyzed three-component reaction of 3-diazooxindoles with indoles and ethyl glyoxylate in high yields with excellent diastereoselectivities. The product easily underwent further synthetic transformations and could be potentially applied to the total synthesis of (±)-gliocladin C and related natural alkaloids

Highly Efficient Synthesis of Mixed 3,3′-Bisindoles via Rh(II)-Catalyzed Three-Component Reaction of 3‑Diazooxindoles with Indoles and Ethyl Glyoxylate

A series of mixed 3,3′-bisindoles were efficiently synthesized via a Rh₂(OAc)₄-catalyzed three-component reaction of 3-diazooxindoles with indoles and ethyl glyoxylate in high yields with excellent diastereoselectivities. The product easily underwent further synthetic transformations and could be potentially applied to the total synthesis of (±)-gliocladin C and related natural alkaloids

Interaction of DcR3 with TRAIL.

<p>Biocore assays, flow cytometry, Western blot, and ELISA-like binding assays were performed to determine the physical interaction between DcR3 and TRAIL. (<b>A</b>) <b>Biacore analysis.</b> The binding curve of TRAIL to DcR3 was determined at different concentrations (1–2048 nM). The control <i>K_eq</i> of recombinant human LIGHT to DcR3 was 13.2 nM, while the <i>K_eq</i> of TRAIL to DcR3 was 52.8 nM. (<b>B</b>) <b>Flow cytometry for DcR3 with TRAIL on the cell surface.</b> Suspended AsPC-1 cells were incubated first with PBS, TRAIL (5 μg/ml) without (lane 2) or with DcR3 (lane 4), recombinant DcR3 (lane 3), or mouse anti-TRAIL MAb (1 μg/ml) with DcR3 (lane 5). Thereafter, 5 μg/ml of biotinylated anti-DcR3 was added to determine the binding of DcR3 on cell surface with flow cytometry. (<b>C and D</b>) <b>Flow cytometry for exposed TRAIL.</b> Gemcitabine was added to AsPC-1 (0–1000 nM) or MiaPaCa-2 (0–100 nM) cells to stimulate the expression of TRAIL. Cells were also treated with PBS, 10 nM of DcR3 siRNA (to reduce the binding and masking of TRAIL), or additional recombinant DcR3 (to block the accessing of anti-TRAIL) to detect TRAIL by flow cytometry. (<b>E and F</b>) <b>Colocalization of DcR3 and TRAIL.</b> AsPC-1 cells treated without (as control) or with 500 nM of gemcitabine (to enhance the expression of TRAIL) were stained with anti-TRAIL-PE and anti-DcR3-FITC. Doublestained cells were assessed with flow cytometry. (<b>G to L</b>) <b>Immunoprecipitation and Western blotting for DcR3-TRAIL complex.</b> 70-kDa DcR3-TRAIL complex in 100 μl of pure DcR3 and TRAIL mixture (<b>G</b> and <b>H</b>), lysate (<b>I</b> and <b>J</b>), or culture media (<b>K</b> and <b>L</b>) of AsPC-1 cells was captured with McAb anti-DcR3 (<b>G, I, K</b>) or anti-TRAIL (<b>H, J, L</b>) McAb and immobilized with 30 μl of protein A beads. The complex of McAb-DcR3-TRAIL was eluted with Laemmli buffer and subjected to Western blotting with biotinylated anti-TRAIL (<b>G,. </b><b>I, K</b>) or anti-DcR3 (<b>H, J, L</b>) followed by SA-HRP and ECL detector. (<b>M and N</b>) <b>Binding competition between free and immobilized forms of DcR3 and TRAIL. </b><b>M</b>: After 100 μl of 1 μg/ml recombinant TRAIL or FasL was immobilized on an ELISA plate, 1 μg/ml of DcR3 was added without (no competition group) or with 10 μg/ml of TRAIL or FasL (competition group) or boiled TRAIL or FasL (no function protein control), followed by biotinylated anti-DcR3 and SA-HRP and TMB substrate. <b>N</b>: DcR3 was immobilized on an ELISA plate. TRAIL and FasL were used as ligands without or with boiled or functional 10 μg/ml of TRAIL or FasL in the presence of DcR3, followed by biotinylated anti-TRAIL or anti-FasL, and SA-HRP and TMB substrate.</p

DcR3 siRNA enhanced gemcitabine-induced apoptosis.

<p>AsPC-1 or MiaPaCa-2 cells were transfected with 10 nM of control siRNA or DcR3 siRNA, respectively. After 24 hours, the cells were treated with gemcitabine (250 or 25 nM, respectively) for 24 hours. Cells were harvested and subjected to flow cytometry for the sub-G1 fraction (<b>A</b> and <b>C</b>) or Western blotting for cleaved PARP (<b>B</b> and <b>D</b>). The combination of DcR3 siRNA and gemcitabine significantly enhanced the proapoptotic effect.</p

DcR3 siRNA enhanced TRAIL-induced apoptosis.

<p>AsPC-1 or MiaPaCa-2 cells were transfected with 10 nM of control siRNA or DcR3 siRNA, respectively. After 24 hours, the cells were treated with recombinant FasL, LIGHT, or TRAIL (100 ng/ml for AsPC-1 and 20 ng/ml for MiaPaCa-2 cells) for 24 hours. Cells were harvested and subjected to flow cytometry for the sub-G1 fraction (<b>A</b> and <b>C</b>) or Western blotting for cleaved PARP (<b>B</b> and <b>D</b>). DcR3 siRNA significantly enhanced TRAIL-induced apoptosis (<i>P</i><0.05).</p

Biacore analysis for TRAIL or LIGHT binding to Recombinant DcR3.

<p><i>K_a</i>, association rate constant; <i>K_d</i>, dissociation rate constant; <i>K</i><b><i>_eq</i></b>, equilibrium constant.</p

DcR3 siRNA enhanced the antitumor effect of gemcitabine.

<p>Control siRNA and DcR3 siRNA-transfected AsPC-1 cells (2×10⁶/site) were subcutaneously injected into nude mice (8 mice/group) and allowed to establish for 7 days. Thereafter, the established tumors were treated with 100 mg/kg of gemcitabine or vehicle (PBS) via intravenous injection twice weekly for 4 weeks. (<b>A</b>) The tumor growth curve was calculated from tumor sizes measured twice weekly. The reduction of DcR3 enhanced the antitumor effect of gemcitabine. (<b>B</b>) Tumors were weighed at the end of the experiment. * <i>P</i><0.05, tumors in the gemcitabine-treated groups versus in untreated groups in vector-transfected AsPC-1 cells; $* <i>P</i><0.05, tumors in siRNA transfectants versus in vehicle transfectants with gemcitabine treatment. (<b>C</b>) The reduction of DcR3 in tumor tissues with different treatments determined with an ELISA. * <i>P</i><0.05, DcR3 in untreated tumors versus in gemcitabine-treated tumors formed by vector-transfected AsPC-1 cells; ** <i>P</i><0.05, DcR3 in tumor control siRNA versus in DcR3 siRNA-transfected AsPC-1 cells;$ <i>P</i><0.05, DcR3 in untreated tumors versus in gemcitabine-treated tumors formed by DcR3 siRNA-transfected AsPC-1 cells. (<b>D</b>) Western blotting was performed in tumor lysate (30 μg/sample) collected from each group and stained with primary antibodies of anti-TRAIL, anti-DcR3, anti-cleaved PARP, and anti-GAPDH (as loading control).</p