u-0126 and Central-Nervous-System-Neoplasms

The present study uses cell-based screening assays to assess the anticancer effects of targeting phosphatidylinositol 3-kinase-regulated integrin-linked kinase (ILK) in combination with small-molecule inhibitors of Raf-1 or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK). The objective was to determine if synergistic interactions are achievable through the use of agents targeting two key cell signaling pathways involved in regulating glioblastoma cancer. The phosphatidylinositol 3-kinase/protein kinase B (PKB)/Akt and the Ras/MAPK pathway were targeted for their involvement in cell survival and cell proliferation, respectively. The glioblastoma cell lines U87MG, SF-188, and U251MG were transiently transfected with an antisense oligonucleotide targeting ILK (ILKAS) alone or in combination with the Raf-1 inhibitor GW5074 or with the MEK inhibitor U0126. Dose and combination effects were analyzed by the Chou and Talalay median-effect method and indicated that combinations targeting ILK with either Raf-1 or MEK resulted in a synergistic interaction. Glioblastoma cells transfected with ILKAS exhibited reduced levels of ILK and phosphorylated PKB/Akt on Ser473 but not PKB/Akt on Thr308 as shown by immunoblot analysis. These results were confirmed using glioblastoma cells transfected with ILK small interfering RNA, which also suggested enhanced gene silencing when used in combination with U0126. U87MG glioblastoma cells showed a 90% (P < 0.05) reduction in colony formation in soft agar with exposure to ILKAS in combination with GW5074 compared with control colonies. A substantial increase in Annexin V-positive cells as determined by using fluorescence-activated cell sorting methods were seen in combinations that included ILKAS. Combinations targeting ILK and components of the Ras/MAPK pathway result in synergy and could potentially be more effective against glioblastoma cancer than monotherapy.

Topics: Butadienes; Cell Proliferation; Central Nervous System Neoplasms; Glioblastoma; Humans; Indoles; Mitogen-Activated Protein Kinases; Nitriles; Oligonucleotides, Antisense; Phenols; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; ras Proteins; Transfection

Malignant astrocytomas are characterized by extensive vascularization attributed to increased expression of the angiogenic cytokine vascular endothelial growth factor (VEGF). VEGF is elevated in astrocytomas under normal oxygen conditions and undergoes induction in hypoxic stress. Prior studies have shown that both the phosphatidylinositol 3'-kinase (PI3-kinase) and MEK1/2 (MAPK/ERK kinase 1/2) pathways promote proliferation of astrocytoma cells and growth of astrocytic tumors. Whether these pathways regulate growth by modulating angiogenesis as well as proliferation is not clear. In this study, pharmacologic inhibitors were used to specifically inhibit PI3-kinase and MEK1/2 activity in human malignant astrocytoma cell lines, and their effects on VEGF expression were determined. Northern blot analysis of VEGF messenger RNA (mRNA) from cells treated with inhibitors demonstrated cell line-specific responses. The PI3-kinase pathway regulated both the normoxic expression and hypoxic induction of VEGF in 2 cell lines, whereas MEK1/2 regulated only the normoxic expression in the same 2 lines. The third cell line showed no change in VEGF mRNA with inhibition of either of these 2 pathways. This study suggests that modulation of signaling pathways implicated in proliferation of astrocytoma cell lines may have varying effects in vivo depending on the role these pathways play in regulating tumor angiogenesis.

Topics: Astrocytoma; Blotting, Northern; Blotting, Western; Butadienes; Cell Hypoxia; Cell Line; Central Nervous System Neoplasms; Chromones; Endothelial Growth Factors; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase Kinases; Morpholines; Neovascularization, Pathologic; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); RNA, Messenger; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors