Inhibition of motility by NEO100 through the calpain-1/RhoA pathway

Abstract

OBJECTIVEGlioblastoma (GBM) is the most aggressive type of brain tumor with a high rate of tumor recurrence, and it often develops resistance over time to current standard of care chemotherapy. Its highly invasive nature plays an essential role in tumor progression and recurrence. Glioma stem cells (GSCs) are a subpopulation of glioma cells highly resistant to treatments and are considered responsible for tumor recurrence.METHODSPatient-derived populations of GSCs were analyzed by western blot, MTT, and cytoplasmic calcium labeling to determine the cytotoxicity of NEO100. High-performance liquid chromatography was used to evaluate the levels of NEO100 in the cell culture supernatants. The effects of the compound on GSC motility were studied using Boyden chamber migration, 3D spheroid migration and invasion assays, and an mRNA expression PCR array. A RhoA activation assay, western blot, and immunofluorescence techniques were employed to confirm the signaling pathways involved. Intracranial implantation of GSCs in athymic mice was used to evaluate the effects of NEO100 in vivo on tumor progression and overall survival.RESULTSHere, the authors show how NEO100, a highly purified good manufacturing practices–quality form of perillyl alcohol, is cytotoxic for different subtypes of GSCs, regardless of the mechanisms of DNA repair present. At doses similar to the IC50 (half maximal inhibitory concentration) values, NEO100 induces ER stress and activates apoptotic pathways in all GSC populations tested. At subcytotoxic doses in the micromolar range, NEO100 blocks migration and invasion of GSCs. These results correlate with a decrease in calpain-1 expression and an increase in RhoA activation, leading to enhanced contractility of the GSCs. In addition, NEO100 blocks the activation of the kinases Src, p42/44 MAPK, Akt, and Stat3, all related to cell proliferation and migration. Intranasal administration of NEO100 in mice with GSC-derived intracranial tumors led to a decrease in tumor progression and a 32% increase in overall survival. Immunostaining studies showed that NEO100 induces apoptosis and reduces GSC invasion in vivo.CONCLUSIONSNEO100 could have significant value targeting GSCs and could be used for GBM therapy as either monotherapy or a coadjuvant therapy during temozolomide rest cycles.