sodium-oxybate and Colonic-Neoplasms

The insulin-like growth factor binding protein 3 (IGFBP-3) is the major circulating IGF binding protein, its function regulated by proteolytic cleavage. The fragments generated have recently been suggested to have IGF-independent biological activity. We have previously established that IGFBP-3 can potentiate apoptosis in colorectal epithelial cells, although its use as a therapeutic reagent may be limited by the fact that it is cleaved in the circulation. Therefore the aim of these experiments was to determine whether the 16-kDa proteolytic fragment (1-95IGFBP-3) would have IGF-independent pro-apoptotic activity in human colonic carcinoma derived cells. We report that the enforced expression of 1-95IGFBP-3 increased the induction of apoptosis by the naturally occurring short chain fatty acid sodium butyrate (NaBt) in the IGF non-responsive HT29 human colorectal carcinoma cell line. Furthermore, the addition of condition medium containing the secreted 1-95IGFBP-3 was as effective as the intact IGFBP-3 protein at potentiating apoptosis. Although not associated with changes in Bcl-2, Bcl-XL, Bax, Bad or Bak expression levels, we report that the expression of the pro-apoptotic 1-95IGFBP-3 fragment is associated with the inhibition of TNFalpha-induced NF-kappaB activity, similar to that reported for the full length IGFBP-3 protein. These results suggest that the 16-kDa 1-95IGFBP-3 fragment is as effective as an intact recombinant protein when used in combination with apoptosis inducing agents, and due to its relative stability in the circulation, it may be important for use as an adjuvant in the treatment of colorectal cancer.

Topics: Apoptosis; Carcinoma; Cell Line, Tumor; Chemotherapy, Adjuvant; Colonic Neoplasms; Culture Media, Conditioned; Humans; Insulin-Like Growth Factor Binding Protein 3; NF-kappa B; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; Sodium Oxybate; Tumor Necrosis Factor-alpha

UFT, a drug composed of uracil and tegafur at the molar ratio of 4:1, is an orally active agent for the treatment of a wide variety of malignant tumours. Using a murine dorsal air sac (DAS) assay, we have previously shown that UFT and its metabolites, gamma-hydroxybutyric acid (GHB) and 5-fluorouracil (5-FU), inhibited the angiogenesis induced by murine renal cell carcinoma. Here we report that UFT was more effective than other fluorinated pyrimidines such as 5-FU and doxifluridine (5'-DFUR) in blocking the angiogenic responses elicited by five human cancer cell lines which produced high levels of vascular endothelial growth factor (VEGF), but no detectable fibroblast growth factor-2 (FGF-2) in vitro. In contrast, UFT was unable to block the angiogenic response to one human gastric cancer cell line which produced both VEGF and FGF-2 in vitro. However, the production or secretion of VEGF by these cells was unaffected by GHB and 5-FU treatment. Interestingly, GHB suppressed the chemotactic migration and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by VEGF, without inhibiting their DNA synthesis. Since GHB did not affect the FGF-2-driven activities in HUVECs, its action appears to be VEGF-selective. On the other hand, 5-FU inhibited DNA synthesis and migration of HUVECs stimulated by both VEGF and FGF-2, and tube formation driven by VEGF, suggesting that 5-FU is cytotoxic to endothelial cells. The inhibitory effects of 5-FU, and especially those GHB, were reproduced under in vivo condition using the DAS assay. The VEGF-mediated angiogenesis was significantly inhibited by UFT, 5-FU, and especially by GHB. We propose that the selective inhibitory effects of GHB on VEGF-mediated responses of endothelial cells are involved in the anti-angiogenic activity of UFT.

Topics: Angiogenesis Inhibitors; Animals; Antimetabolites, Antineoplastic; Breast Neoplasms; Carcinoma; Carcinoma, Renal Cell; Chemotaxis; Colonic Neoplasms; DNA Replication; Drug Combinations; Endothelial Growth Factors; Endothelium, Vascular; Female; Fibroblast Growth Factor 2; Floxuridine; Fluorouracil; Humans; Intercellular Signaling Peptides and Proteins; Kidney Neoplasms; Lung Neoplasms; Lymphokines; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Neoplasm Proteins; Neovascularization, Pathologic; Prodrugs; Prostheses and Implants; Recombinant Proteins; Sodium Oxybate; Stomach Neoplasms; Tegafur; Tumor Cells, Cultured; Uracil; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Xenograft Model Antitumor Assays

Analysis of the glycogen synthase kinase-3beta (GSK-33) activity in several colon cancer cell lines suggested a correlation between comparatively low enzyme activity and moderate to high differentiation status. Treatment of LIM2537 cells, a poorly differentiated colon cancer cell line, with the potent differentiating agent sodium butyrate resulted in 34% reduction in GSK-3beta activity in the treated cells (P < 0.028, n = 3). Decreases in GSK-3beta activity were paralleled by stabilization of cytoplasmic beta-catenin, a hallmark of Wnt signaling. However, in contrast to Wnt signaling, expression of the beta-catenin/ TCF target genes c-myc and cyclin D1 did not appear to be increased in the sodium butyrate-treated cells. Interestingly, expression of membrane-bound beta-catenin was increased in the sodium butyrate-treated cells. This suggests that, in the context of cellular differentiation, increases in beta-catenin expression may be sequestered at the cell membrane and suggests that a possible role of sodium butyrate in promoting differentiation may be via increasing the levels of beta-catenin available for cell adhesion.

Topics: Alkaline Phosphatase; Animals; Axin Protein; beta Catenin; Cadherins; Calcium-Calmodulin-Dependent Protein Kinases; Cell Adhesion; Cell Differentiation; Cell Membrane; Colonic Neoplasms; Cyclin D1; Cytoskeletal Proteins; Fluorescent Antibody Technique, Indirect; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Humans; Immunoblotting; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Precipitin Tests; Protein Binding; Protein C; Proteins; Proto-Oncogene Proteins c-myc; Repressor Proteins; Signal Transduction; Sodium Oxybate; Time Factors; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured