Role of Luteolin as Potential New Therapeutic Option for Patients with Glioblastoma through Regulation of Sphingolipid Rheostat.

Glioblastoma (GBM) is the most aggressive brain tumor, still considered incurable. In this study, conducted on primary GBM stem cells (GSCs), specifically selected as the most therapy-resistant, we examined the efficacy of luteolin, a natural flavonoid, as an anti-tumoral compound. Luteolin is known to impact the sphingolipid rheostat, a pathway regulated by the proliferative sphingosine-1-phosphate (S1P) and the proapoptotic ceramide (Cer), and implicated in numerous oncopromoter biological processes. Here, we report that luteolin is able to inhibit the expression of SphK1/2, the two kinases implicated in S1P formation, and to increase the expression of both SGPL1, the lyase responsible for S1P degradation, and CERS1, the ceramide synthase 1, thus shifting the balance toward the production of ceramide. In addition, luteolin proved to decrease the expression of protumoral signaling as MAPK, RAS/MEK/ERK and PI3K/AKT/mTOR and cyclins involved in cell cycle progression. In parallel, luteolin succeeded in upregulation of proapoptotic mediators as caspases and Bcl-2 family and cell cycle controllers as p53 and p27. Furthermore, luteolin determined the shutdown of autophagy contributing to cell survival. Overall, our data support the use of luteolin as add-on therapy, having demonstrated a good ability in impairing GSC viability and survival and increasing cell sensitivity to TMZ.

Testing calpain inhibition in tumor endothelial cells: novel targetable biomarkers against glioblastoma malignancy.

Introduction: Glioblastoma IDH-wildtype (GBM) is the most malignant brain tumor in adults, with a poor prognosis of approximately 15 months after diagnosis. Most patients suffer from a recurrence in <1 year, and this renders GBM a life-threatening challenge. Among molecular mechanisms driving GBM aggressiveness, angiogenesis mediated by GBM endothelial cells (GECs) deserves consideration as a therapeutic turning point. In this scenario, calpains, a family of ubiquitously expressed calcium-dependent cysteine proteases, emerged as promising targets to be investigated as a novel therapeutic strategy and prognostic tissue biomarkers. Methods: To explore this hypothesis, GECs were isolated from n=10 GBM biopsies and characterized phenotypically by immunofluorescence. The expression levels of calpains were evaluated by qRT-PCR and Western blot, and their association with patients' prognosis was estimated by Pearson correlation and Kaplan-Meier survival analysis. Calpain targeting efficacy was assessed by a time- and dose-dependent proliferation curve, MTT assay for viability, caspase-3/7 activity, migration and angiogenesis in vitro, and gene and protein expression level modification. Results: Immunofluorescence confirmed the endothelial phenotype of our primary GECs. A significant overexpression was observed for calpain-1/2/3 (CAPN) and calpain-small-subunits-1/2 (CAPNS1), whereas calpastatin gene, the calpain natural inhibitor, was reported to be downregulated. A significant negative correlation was observed between CAPN1/CAPNS1 and patient overall survival. GEC challenging revealed that the inhibition of calpain-1 exerts the strongest proapoptotic efficacy, so GEC mortality reached the 80%, confirmed by the increased activity of caspase-3/7. Functional assays revealed a strong affection of in vitro migration and angiogenesis. Gene and protein expression proved a downregulation of MAPK, VEGF/VEGFRs, and Bcl-2, and an upregulation of caspases and Bax-family mediators. Conclusion: Overall, the differential expression of calpains and their correlation with patient survival suggest a novel promising target pathway, whose blockade showed encouraging results toward precision medicine strategies.