n-(n-(3-5-difluorophenacetyl)alanyl)phenylglycine-tert-butyl-ester and Colonic-Neoplasms

Notch is a signaling molecule which plays a role in angiogenesis and γ-secretase is required for processing of Notch. In this study, we investigated the effect of γ-secretase inhibitor (DAPT) on tumor angiogenesis in diet-induced obese mice.. 18 mice were divided into three groups; control, obese (diet-induced) and obese+DAPT. After 15 weeks, the obese mice were subjected for tumor induction of CT26 colon adenocarcinoma cells (5 x 105 cells). When the tumor size reached approximately 350 ± 50 mm3, half of the obese animals received DAPT (10mg/kg/day) subcutaneously. Blood samples were taken after 14 days and the tumors harvested for immunohistochemical staining and capillary density were reported as CD31 positive cells/mm2.. The obese animals had higher serum leptin and NO concentrations, while, serum VEGF and VEGFR-1 concentrations were not different compare to control group. Administration of DAPT in obese mice significantly reduced serum VEGFR-1 and leptin concentrations and increased serum NO level (p < 0.05). Capillary density in the tumors of obese animals was not different compare to control groups. DAPT administration could not alter capillary density in the tumors.. Administration of DAPT in obese mice altered serum angiogenic factors, however, it could not modulate tumor angiogenesis in diet-induced obese mice (Fig. 4, Ref. 26).

Topics: Adenocarcinoma; Amyloid Precursor Protein Secretases; Animals; Colonic Neoplasms; Dipeptides; Immunohistochemistry; Male; Mice; Mice, Obese; Neoplasms, Experimental; Neovascularization, Pathologic; Obesity

The Wnt and Notch1 signaling pathways play major roles in intestinal development and tumorigenesis. Sub-cellular localization of β-catenin has been implicated in colorectal carcinogenesis. However, the β-catenin and Notch intracellular domain (NICD) interaction has to be addressed. Immunohistochemistries of β-catenin, NICD, and dual immunofluorescence of β-catenin and NICD were analyzed in colorectal tissues and HT29 cell line. Moreover, real-time PCR analysis of CyclinD1, Hes1 and MUC2 was done in HT29 cells upon N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment. Dual staining emphasized the strong interaction of β-catenin and NICD in adenoma and adenocarcinoma than in normal tissues. Hes1 transcript levels were decreased 1.5- and 7.1-fold in 12.5 and 25 µM DAPT-treated HT29 cells. CyclinD1 transcript levels decreased 1.2- and 1.6-fold, and MUC2 transcript level increased 4.3- and 7.5-fold in 12.5 and 25 µM DAPT-treated HT29 cells. The results of this study showed that the sub-cellular localization of β-catenin converges with NICD inducing proliferation through the activation of CyclinD1 and Hes1. Moreover, the inhibition of Notch1 signaling by DAPT leads to the arrest of cell proliferation and induces apoptosis leading to the upregulation of MUC2, a secretory cell lineage marker.

Topics: Adenocarcinoma; Adenoma; Basic Helix-Loop-Helix Transcription Factors; beta Catenin; Cell Proliferation; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Dipeptides; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; HT29 Cells; Humans; Mucin-2; Protein Structure, Tertiary; Receptor, Notch1; Reference Values; Signal Transduction; Transcription Factor HES-1

Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell-derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice.. C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor difluorophenacetyl-l-alanyl-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9-/- mice and HCT116 cells that were stably transfected with MMP-9.. Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown by increased numbers of tumors and level of dysplasia compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21WAF1/Cip1, Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP-9-/- mice on γ-radiation-induced damage of DNA.. MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.

Topics: Amyloid Precursor Protein Secretases; Animals; Apoptosis; Azoxymethane; Caspase 3; Colitis; Colon; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cytokines; Dextran Sulfate; Dipeptides; Disease Models, Animal; DNA Damage; Enzyme Inhibitors; Fibroblasts; Gamma Rays; HCT116 Cells; Humans; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptor, Notch1; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Tumor Suppressor Protein p53

Cyclooxygenase-2 (COX-2) is a key factor in the development of colorectal cancer, and non-steroidal anti-inflammatory drugs (NSAIDs) have anti-colorectal cancer activity. However, the potential molecular mechanism of the COX-2 selective inhibitor effect on proliferation and apoptosis of colon cancer cells is unclear. In this study, we have demonstrated for the first time that the Delta1/Notch1 signal transduction pathway mediates the COX-2 selective inhibitor effect on colorectal cancer cells, and we reveal the mechanism of the Notch1 pathway in terms of regulating the proliferation and apoptosis of colorectal cancer cells.. Colon cancer cell lines HT-29 and SW480 were treated with NS-398 (a COX-2 selective inhibitor) and DAPT (a gamma-secretase inhibitor). The colormetric MTT cell proliferation assay and flow cytometry were used to measure cell proliferation and apoptosis. Reverse transcriptase (RT)-PCR and ELISA analyses were used to detect the levels of COX-2 mRNA expression and prostaglandin E2 (PGE2) concentration from the two cell lines, respectively. The expression of the Notch1, Delta1, ICN, Hes1 and NF-kappaB2 proteins was measured by Western blot. Immunohistochemistry results showed that Notch1 was expressed mainly in the cytoplasm and ICN mainly in the nucleus. COX-2 mRNA was highly expressed in HT-29 cells but not in SW480 cells. Both COX-2 mRNA expression and PGE2 concentration decreased in HT-29 cells treated with NS-398; however, PGE2 levels did not change in SW480 cells treated with NS-398. NS-398 and DAPT inhibited cell proliferation and induced apoptosis in a dose time-dependent manner accompanied by significantly decreased Notch1 activity (P < 0.01), and resulted in a significant down-regulation of Hes1 and NF-kappaB2 (P < 0.01).. Our results show that the selective COX-2 inhibitor may inhibit the proliferation and induce apoptosis in colon cancer cells through the COX-2-dependent pathway (HT-29) by decreasing the COX-2 mRNA/PGE2 levels and the activity of the COX-2-independent pathway (SW480). The Notch1 signal pathway mediates the effects of the COX-2 inhibitor on the proliferation and apoptosis of colon cancer cells. This may be a new target of the selective COX-2 inhibitor effect on colon cancer.

Topics: Amyloid Precursor Protein Secretases; Antineoplastic Agents; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Cell Proliferation; Colonic Neoplasms; Colorimetry; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Dipeptides; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Homeodomain Proteins; HT29 Cells; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Membrane Proteins; NF-kappa B p52 Subunit; Nitrobenzenes; Receptor, Notch1; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sulfonamides; Time Factors; Transcription Factor HES-1