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

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Glioblastoma (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.


Patient-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.


Here, the authors show how NEO100, a highly purified guanosine monophosphate–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.


NEO100 could have significant value targeting GSCs and could be used for GBM therapy as either monotherapy or a coadjuvant therapy during temozolomide rest cycles.

ABBREVIATIONS BER = base excision repair; ECM = extracellular matrix; ER = endoplasmic reticulum; GBM = glioblastoma; GSC = glioma stem cell; GST = glutathione S-transferase; GTP = guanosine triphosphate; HPLC = high-performance liquid chromatography; IC50 = half maximal inhibitory concentration; MMR = mismatch repair; PCR = polymerase chain reaction; POH = perillyl alcohol; TMZ = temozolomide; USC = University of Southern California.

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Article Information

Correspondence Thomas C. Chen: Keck School of Medicine, University of Southern California, Los Angeles, CA.

INCLUDE WHEN CITING Published online August 16, 2019; DOI: 10.3171/2019.5.JNS19798.

Disclosures Funding for this study was provided by NeOnc Technologies Inc. and by the Stop Cancer Foundation and Milken Family Foundation. T.C. Chen has ownership interest in NEO100, NeOnc Technologies Inc.

© AANS, except where prohibited by US copyright law.



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    NEO100 induces cytotoxicity in GSCs. A: Representative western blot images of basal expression of MSH2, MSH6, AAG, and MGMT DNA repair mechanisms in the 4 patient-derived GSC populations: USC02, USC04, USC08, and USC10. Actin was included as a loading control. B: Cell viability of GSCs at different doses of NEO100 after 48 hours of treatment. Data, expressed as percent cell viability relative to vehicle-treated cells from 3 independent experiments carried out in triplicate, are presented as mean ± SEM in dose-response curves. IC50 values were obtained with GraphPad Prism 5.0. C: Percentage of NEO100 remaining in the GSC supernatants after the indicated time points, measured via HPLC analysis. D: Cell viability of GSCs at different doses of NEO100 after 3 consecutive doses administered every 48 hours. Data are expressed as percent cell viability relative to vehicle-treated cells and as IC50 values. E: Representative western blot images of the proteins related to ER stress and apoptotic pathways GRP78, CHOP, ATF3, PARP, cleaved caspase 7 (Cl. C7), and P62 after 24 hours of treatment with 1.5 mM NEO100. Actin was included as a loading control. + = expressed; – = not expressed. F: Representative images of intracellular calcium labeled with Fluo-3 AM (green) in GSCs treated with either vehicle or 1.5 mM NEO100 for 24 hours. DAPI was used to stain all nuclei. Bar = 100 µm. Figure is available in color online only.

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    Subcytotoxic doses of NEO100 block GSC migration and invasion. A: Representative images of Boyden chamber migration and invasion assays performed with mesenchymal USC02 cells at different concentrations of NEO100. Bar = 200 µm. B and C: After 8 hours (migration) or 16 hours (invasion) of treatment with NEO100, the number of migrated (B) or invaded (C) USC02 cells per field was counted. The bar graphs represent the mean ± SEM of at least 3 independent experiments performed in triplicate. D and E: Representative images of the spheroid migration (D) and invasion (E) assays performed with proneural USC04 cells treated with vehicle or NEO100. After 72 hours, the area covered with cells was quantified, and represented as relative to the initial sphere size (0 hours). The bar graphs represent the average ± SEM of 3 independent experiments performed in sextuplicate. *p < 0.05; **p < 0.01; ***p < 0.001 (relative to vehicle-treated cells). Bar = 200 µm. Figure is available in color online only.

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    NEO100 regulates the expression pattern of several genes involved in cell motility. The human Cell Motility RT2 Profiler PCR Array was used to analyze the changes in the expression pattern of 84 genes related to cell motility in GSCs incubated with vehicle or 100 μM NEO100 for 24 hours. Data are expressed as fold change, relative to vehicle-treated cells (threshold = 2-fold change).

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    NEO100 impairs cell migration through activation of RhoA-dependent actomyosin contractility. A: Active GTP-bound RhoA from USC02 and USC04 treated with vehicle, 50 µM NEO100, or 100 µM NEO100 was immunoprecipitated and visualized by western blotting. Bar graphs show the GTP-RhoA/total RhoA ratio, expressed as fold change relative to vehicle-treated GSCs. **p < 0.01; ***p < 0.001 (relative to vehicle-treated cells). B and C: NEO100 increases cell contractility. Representative images of phase-contrast USC02 and USC04 cell cultures (B) and phalloidin-stained (red) filamentous actin (C). DAPI was used to stain all nuclei. Bar = 200 µm. D: NEO100 reduces the activation of Src, MAPK, Akt, and Stat3. Representative western blots of phosphorylated (P-) and total levels of each kinase. Actin was included as a loading control. E: Scheme summarizing the effects of NEO100 (red arrows) on calpain-1/RhoA signaling. Figure is available in color online only.

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    NEO100 decreases tumor progression and increases overall survival in an in vivo mesenchymal-GSC (USC02) orthotopic tumor model. A: Diagram showing the implantation and treatment schema. B: Kaplan-Meier survival analysis of mice treated with vehicle (black line), NEO100 7.2 mg/kg (blue line, HR 4.850), NEO100 9.6 mg/kg (yellow line, HR 9.621), and NEO100 14.4 mg/kg (red line, HR 8.612). *p < 0.05 (relative to vehicle-treated mice). C: Intranasal administration of POH delays tumor growth. Representative images of the luciferase-labeled USC02 GSC-derived tumors in the surviving animals at 7, 22, and 30 days postimplantation (DPI). D: NEO100 induces apoptosis and impairs GSC invasion in vivo. Representative images of brain sections stained for cleaved caspase 7 (upper) and the human marker TRA-1-85 (lower). Bar = 100 µm. Figure is available in color online only.




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