midostaurin and Astrocytoma

Protein kinase C (PKC) designates a family of kinases that regulate many essential functions including cell growth and differentiation. The tight regulation of PKC activity is crucial for maintaining normal cellular proliferation and excessive activity leads to abnormal or uncontrolled cell growth. Recent reports indicate that malignant glioma cell lines express 100 to 1000-fold higher PKC activity when compared to non-neoplastic astrocytes. This high activity correlates well with the proliferation of tumor cells in vitro. We recently reported on the anti-proliferative properties of selective PKC inhibitors on the growth of U-373MG human astrocytoma cell line, and their ability to block mitogen-activated protein (MAP) kinase pathway activated by substance P (SP) neuropeptide receptor signaling via a PKC-dependent mechanism. Therefore, inhibiting PKC activity by selective PKC inhibitors may present a promising approach for improving astroglial brain tumor therapy. For this purpose, we constructed a high throughput model cell system to evaluate the efficacy of PKC inhibitors. This system is based on the measurement of light production in U-373MG cells stably transfected with the luciferase reporter gene whose expression depends on the transcriptional activation of GAL4-Elk1 fusion protein by enzyme components of the MAP kinase pathway and the upstream activation of PKC (PKC activation-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity). In brief, we have demonstrated that the PKC activator 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced luciferase activity in this cell system is mediated via the MAP kinase pathway and can be blocked in the presence of MEK1 selective inhibitors (PD 098059 or U0126). We also demonstrated that TPA-induced luciferase activity in U-373MG stable clones can be blocked by PKC inhibitors (CGP 41251, Go 6976, and GF 109203X) in a concentration dependent manner. In contrast, epidermal growth factor (EGF)-induced luciferase activity, which is independent of PKC activation (Ras-->Raf-1-->MEK1-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity) can only be blocked using a selective EGF receptor inhibitor (AG 1478). In conclusion, we have constructed a model cell system for the high throughput screening and identification of PKC inhibitors potentially active against astrocytoma cells in culture.

Topics: Antineoplastic Agents; Astrocytoma; Carbazoles; Cell Division; DNA-Binding Proteins; Drug Screening Assays, Antitumor; Enzyme Inhibitors; ets-Domain Protein Elk-1; Humans; Indoles; Potassium Channels; Protein Kinase C; Proto-Oncogene Proteins; Staurosporine; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

The substance P (SP) receptor (NK-1 subtype) is widely expressed in primary human astrocytomas and glioblastomas and many brain tumor-derived cell lines. SP receptor activation stimulates the mitogen-activated protein (MAP) kinase pathway and the expression of immediate-early genes (e.g., c-Fos and c-Myc), resulting in an increase in DNA synthesis in human astrocytoma U-373 MG cells. In this study, we investigated the role of protein kinase C (PKC) in SP receptor activation of the MAP kinase pathway. SP peptide, epidermal growth factor, and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the tyrosine phosphorylation of the Erk1 and Erk2 MAP kinases in a concentration-dependent manner in U-373 MG cells. Pretreatment of the cells with PKC inhibitors, CGP 41251 or tamoxifen, inhibited tyrosine phosphorylation of Erk1 and Erk2 MAP kinases induced by low concentrations of SP or TPA and significantly attenuated phosphorylation at high concentrations of SP or TPA. The inhibitory effect exhibited by tamoxifen on SP-induced MAP kinase activation is similar to that exhibited by the selective PKC inhibitor CGP 41251, suggesting that the PKC enzyme is the in situ target for both inhibitors. Furthermore, SP-induced c-Fos phosphoprotein expression is inhibited by CGP 41251 or tamoxifen with similar efficacy. Importantly, neither CGP 41251 nor tamoxifen has any detectable effect on the MAP kinase activation by epidermal growth factor, consistent with the ability of this growth factor to activate the MAP kinase pathway by a PKC-independent mechanism. Prolonged treatment with TPA resulted in down-regulation of PKC and selective inhibition of TPA- and SP-induced Erk1 and Erk2 tyrosine phosphorylation in U-373 MG cells. Consistent with the in situ results, CGP 41251 and tamoxifen significantly inhibited endogenous PKC enzymatic activity from U-373 MG cells in vitro. In contrast to CGP 41251 and tamoxifen, Gö 6976, a highly selective inhibitor for PKC alpha and PKC beta 1 isozymes, did not inhibit SP- or TPA-induced tyrosine phosphorylation of Erk1 and Erk2 MAP kinases; rather, it inhibited a signaling pathway leading to the phosphorylation of cAMP-responsive element binding protein in U-373 MG cells. To investigate whether selective PKC isozyme(s) are involved in the activation of the MAP kinase pathway by SP, we determined the expression of PKC isozymes in U-373 MG cells. We found that U-373 MG cells express nine different PKC isozymes (alpha, beta I,

Topics: Astrocytoma; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cyclic AMP Response Element-Binding Protein; Enzyme Activation; Enzyme Inhibitors; Humans; Indoles; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins c-fos; Staurosporine; Substance P; Tamoxifen; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrosine