IDENTIFICATION AND QUANTIATION OF CERAMIDES AND THE EFFECT OF ANTI-CANCER DRUGS ON THEIR LEVELS IN GLIOBLASTOMA USING MASS SPECTROMETERY

Abstract

Glioblastoma (GBM) is one of the most common and aggressive forms of brain tumor. Prior studies from our lab found that carmofur is a promising candidate for suppressing GBM proliferation and inducing tumor cell death. Previous studies have identified acid ceramidase (ASAH1) as a potential therapeutic target for this disease. Carmofur, an ASAH1 inhibitor, has shown to increase ceramide levels in glioblastoma leading to apoptosis. Ceramides are lipid signaling molecules central to the sphingolipid pathway which can influence and regulate cellular processes of inflammation, differentiation, cell growth and apoptosis. A more thorough understanding of ceramide species may facilitate improved chemotherapeutic treatments in the future.An LC–MS/MS–based analytical method was developed to quantitatively measure multiple ceramide species present in biological samples. The developed method was sensitive, selective, and robust with a linear range of 1 ng/mL to 1000 ng/mL. C18, C20, and C23 ceramides were identified as important ceramide targets for glioblastoma treatment. A practical and reproducible ceramide extraction protocol was developed by systematic evaluation of all steps of sample preparation. Application of the optimized method yielded a recovery of over 75%, representing a 15-fold improvement over the initial protocol. Along with ceramides, high concentrations of galactosylceramide (GalCer) were found in glioblastoma cells. Unlike normal ceramides, elevated levels of GalCer have been linked to inhibiting apoptosis and increasing drug resistance in cancer cells. GalCer concentrations did not significantly rise when treated with carmofur. Gallium maltolate (GaM), an anticancer agent that disrupts iron homeostasis, did not substantially elevate ceramide levels and was identified as a potential dual-therapeutic candidate with carmofur for glioblastoma treatment. Lastly, imaging mass spectrometry (MALDI-TOF-MS) was used to understand the spatial distribution of ceramides and GalCer species present in rat brain tissue. The ceramides were found to be highly concentrated in the peripheral gray matter along the edge of the brain tissue, while the interior tissue shows clustering of ceramides toward the center of the brain.