Inhibition study of protein kinase C-iota mediated signaling pathways in diffuse midline glioma by ICA-1S inhibitor and temozolomide

The Diffuse Midline Glioma (DMG) is a subtype of glial carcinoma that develops in the brain, primarily affecting the pons, midbrain, and thalamus. This malignant and fast-growing cancer affects both children and young adults. At present, pediatric DMG is an incurable childhood brain cancer. Interpreting the regulation of cell signaling pathways in pediatric DMG is crucial for targeting tumor cell migration and metastasis. These pathways must be understood comprehensively for effective treatment of this incurable primary brain tumor. Our goal is to investigate the role of atypical Protein Kinase C-iota (PKC-ι) expression levels in DMG cells, given that the kinase is over-expressed in most cancer cells. We aim to understand the PKC-ι-mediated cellular pathways involved in migration, invasion, and apoptosis in DMG (D1008) cells. Moreover, our study focuses on the inhibition of PKC-ι by the inhibitor ICA-1S (5-Amino-1-[(1R,2S,3R,4R)-2,3-dihydroxy-4[(phosphonooxy)methyl]cyclopentyl]-1H-imidazole-4-carboxamide) in combination with the FDA-approved glioblastoma treatment drug named temozolomide (TMZ) in the DMG D1008 cells. In this study, the effect of in vitro combination treatment with ICA-1S and TMZ on D-1008 cells is being investigated using cell viability assays, western blot analysis, scratch and wound healing assays, immunoprecipitation, flow cytometry, immunofluorescence, and small interfering RNA (siRNA) technology. The dose-response curve suggests that the ICA-1S and TMZ combination treatment is viable, as indicated by the half-maximal inhibitory concentrations (IC50). The Western blot analysis suggests that, in ICA-1S-treated DMG D-1008 cells, the expression of phosphorylated PKC-ι and total PKC-ι is reduced compared to untreated control cells. Moreover, our findings indicate that the inhibition of PKC-ι in D-1008 cells with ICA-1S leads to a significant increase in apoptotic markers, such as cleaved PARP and P53, compared to dual inhibition. The immunoprecipitation experiments in a previous study demonstrated that dual inhibition with ICA-1S and TMZ decreased the invasion of glioma cell lines by lowering the phosphorylation of Focal Adhesion Kinase (FAK) and paxillin in the PKC-ι/FAK/Paxillin pathway. In D-1008 cells, the complex between PKC-ι and FAK was not observed in immunoprecipitation, indicating a distinction in the cellular invasion pathway. The results from scratch & wound healing assays, immunoprecipitation, flow cytometry, immunofluorescence, and siRNA techniques will also be reported in this study. The objective is to establish the correlation between PKC-ι expression levels in D-1008 cells and the effects on PKC-ι-related cell signaling pathways when inhibited by the PKC-ι inhibitor ICA-1S and the drug TMZ as part of a new personalized treatment approach for this pediatric glioma.