Knockdown of caveolin-1 protein expression sensitizes HCT116 colon cancer cells to apoptosis.

<p>HCT116 colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. (<b>A</b>) Endogenous caveolin-1 expression was determined by immunoblotting analysis 24, 48 and 96 hours after transfection using an antibody probe specific for caveolin-1. Immunoblotting with anti-β-actin IgGs was done to show equal loading. (<b>B–D</b>) One day after transfection, HCT116 cells were treated with different concentrations of 5-FU for 48 hours. Untreated cells were used as control. In (<b>B</b>), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values represent mean ± SEM; *<i>P</i><0.001; ^#<i>P</i><0.005. In (<b>C</b>), cells were subjected to Annexin V staining and flow cytometry. Values represent mean. In (<b>D</b>), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3 and PARP. Immunoblotting with anti-β-actin IgGs was done to show equal loading.</p

Ku70 binds to the scaffolding domain of caveolin-1.

<p>(<b>A</b>) The consensus caveolin binding domain (CBD) is shown together with the caveolin binding domain of Ku70 (amino acids 471 to 478) and a mutant form of Ku70’s CBD in which aromatic residues were mutated to alanines. (<b>B</b>) Ponceau S staining of GST alone and GST-caveolin-1 fusion proteins. (<b>C</b>) GST-caveolin-1 fusion protein pull-down assays were performed using cell lysates from HCT116 cells transiently transfected with wild type Ku70-HA. A blot that is representative of two independent experiments is shown.</p

Chemotherapeutic drugs promote the Ku70-Cav-1 interaction.

<p>(<b>A</b>) HCT116 cells were treated with either etoposide or 5-FU for 24 hours. Untreated cells were used as control. Cell lysates were then immunoprecipitated with an antibody probe specific for caveolin-1 and immunoprecipitates subjected to immunoblotting analysis with anti-Ku70, anti-Bax and anti-caveolin-1 IgGs. (<b>B</b>) HCT116 colon cancer cells were transfected with HA-tagged wild type Ku70. One day after transfection, cells were treated with 5-FU for 24 hours. Untreated cells were used as control. Cells were then subjected to immunofluorescence analysis using antibody probes specific for the HA tag (green) and caveolin-1 (red). Nuclei were detected by DAPI staining (Blue). Yellow staining in the merged images shows co-localization between caveolin-1 and Ku70 only after 5-FU treatment (see arrows). Representative images are shown.</p

Knockdown of caveolin-1 protein expression enhances the dissociation of Bax from Ku70 and the activation of Bax induced by chemotherapeutic drugs.

<p>(A, C, D and E) HCT116 colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. One day after transfection, HCT116 cells were treated with either 5-FU (A, C and D) or etoposide (A and E) for 24 hours. In (B), untransfected cells were treated with 5-FU for 24 hours. In (A), cell lysates were immunoprecipitated with an antibody probe specific for Bax and immunoprecipitates subjected to Western blotting analysis with anti-Ku70 and anti-Bax IgGs. In (B), cell lysates were immunoprecipitated with either anti-caveolin-1 IgGs or anti-Bax IgGs. Immunoprecipitates were then subjected to immunoblotting analysis with antibody probes specific for caveolin-1, Ku70 and Bax. In (C and D), cell lysates were immunoprecipitated with a Bax (6A7) antibody and immunoprecipitates subjected to immunoblotting analysis with anti-Bax IgGs. A blot that is representative of three independent experiments is shown in (C), quantification of Bax levels pulled down by the Bax (6A7) antibody is shown in (D). Values in (D) represent mean ± SEM; *<i>P</i><0.001. In (E), mitochondrial fractions were isolated and expression of Bax in mitochondria was detected by immunoblotting analysis using an antibody probe specific for Bax.</p

Φ→A Ku70 fails to interact with caveolin-1, to protect against chemotherapeutic drug-induced apoptosis and to limit the 5-FU-induced dissociation of Bax from Ku70.

<p>HCT116 cells were transfected with either wild type Ku70-HA (WT Ku70) or Φ→A Ku70-HA (Φ→A Ku70) and treated with different concentrations of either 5-FU (for 24 h in A and E; for 48 h in C and D) or etoposide (for 24 h in C and D). Untreated cells were used as control. In (A), cell lysates were immunoprecipitated with an antibody probe specific for caveolin-1 and immunoprecipitates subjected to immunoblotting analysis with anti-HA, anti-Bax and anti-caveolin-1 IgGs. In (B), total expression of WT Ku70-HA and Φ→A Ku70-HA was detected by immunoblotting analysis with anti-HA IgGs before drug treatment. In (C), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values represent mean ± SEM; *<i>P</i><0.001. In (D), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3. Immunoblotting with anti-β-actin IgGs was done to show equal loading. In (E), cell lysates were immunoprecipitated with and antibody probe specific for Bax and immunoprecipitates subjected to Western blotting analysis with anti-HA and anti-Bax IgGs.</p

Caveolin-1 promotes long-term survival and inhibits apoptosis of colon cancer cells after treatment with chemotherapeutic drugs.

<p>HCT116 (A, B, E and F) and HT29 (C and D) colon cancer cells were transfected with siRNA directed against caveolin-1. Transfection with scrambled siRNA was used as control. One day after transfection, cells were treated with different concentrations of 5-FU for 48 h (A–D) or etoposide for 24 h (E and F). Untreated cells were used as control. In (A and B), cells were cultured for 7 days and stained with crystal violet. A crystal violet staining that is representative of three independent experiments is shown in (A), quantification of the number of clones after crystal violet staining is shown in (B). Values represent mean ± SEM; *<i>P</i><0.001; ^#<i>P</i><0.005. In (C and E), cells were stained with DAPI and the number of cells showing nuclear condensation was quantified. Values represent mean ± SEM; *<i>P</i><0.001; ^#<i>P</i><0.005. In (D and F), cells were subjected to immunoblotting analysis with antibody probes specific for cleaved caspase 3 and PARP. Immunoblotting with anti-β-actin IgGs was done to show equal loading.</p

Design of Potent and Proteolytically Stable Oxyntomodulin Analogs

Incretin-based peptides are effective therapeutics for treating type 2 diabetes mellitus (T2DM). Oxyntomodulin (OXM), a dual agonist of GLP-1R and GCGR, has shown superior weight loss and glucose lowering effects, compared to single GLP-1R agonists. To overcome the short half-life and rapid renal clearance of OXM, which limit its therapeutic potential, both lipid and PEG modified OXM analogs have been reported. However, these approaches often result in reduced potency or PEG-associated toxicity. Herein, we report a new class of cross-linked OXM analogs that show increased plasma stability and higher potency in activating both GLP-1R and GCGR. Moreover, the extended <i>in vivo</i> half-life results in superior antihyperglycemic activity in mice compared to the wild-type OXM

Rational Design of Dual Agonist-Antibody Fusions as Long-acting Therapeutic Hormones

Recent studies have suggested that modulation of two or more signaling pathways can achieve substantial weight loss and glycemic stability. We have developed an approach to the generation of bifunctional antibody agonists that activate leptin receptor and GLP-1 receptor. Leptin was fused into the complementarity determining region 3 loop of the light chain alone, or in combination with exendin-4 (EX4) fused at the N-terminus of the heavy chain of Herceptin. The antibody fusions exhibit similar or increased <i>in vitro</i> activities on their cognate receptors, but 50–100-fold longer circulating half-lives in rodents compared to the corresponding native peptides/proteins. The efficacy of the leptin/EX4 dual antibody fusion on weight loss, especially fat mass loss, was enhanced in <i>ob</i>/<i>ob</i> mice and <i>DIO</i> mice compared to the antibody fusion of either EX4 or leptin alone. This work demonstrates the versatility of this combinatorial fusion strategy for generating dual antibody agonists with long half-lives