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Introduction
Acid ceramidase (ASAH1), a lysosomal cysteine amidase, helps metabolize ceramides into sphingosine and free fatty acids. Ceramides promote senescence and apoptosis, while sphingososine-1-phospate (Sph-1P), the immediate metabolite of sphingosine, promotes cell survival, proliferation, inflammation, and angiogenesis (1). As such, overexpression of ASAH1 confers resistance to apoptosis. Its levels have been shown to be elevated in several cancers, including breast (2), prostate (3,4), head and neck (5), colon, and melanoma (6). Moreover, downregulation or inhibition of ASAH1 may improve anticancer treatments (5,7,8).
Glioblastoma multiforme (GBM) is the most common primary, intracranial malignancy of the central nervous system. The standard treatment protocol, which involves surgical resection, concurrent radiation/temozolomide (TMZ), and adjuvant TMZ, still imparts a grim prognosis, where median overall survival (OS) is less than 15 months (9). Ultimately, all GBM develop resistance to standard treatment with recurrence or progression, where additional therapies yield a median survival of ~30 weeks (10–12). Though ASAH1 appears to be a promising therapeutic target in other tumors, no studies have explored its role in recurrent GBM.
It has been postulated that GBM cancer stem cells (CSCs) are the culprits that promote resistance to radiation, as CD133-carrying glioma cells are increased in proportion following ionizing radiation (13). Recent studies with prostate cancer suggest that upregulation of ASAH1 confers resistance to radiation by altering the sphingolipid metabolism pathway (14,15). This study examines whether ASAH1 plays a similar role in recurrent or irradiated GBM.
Materials and methods
Reagents and cells
Mouse antibody against ASAH1 (612302) was purchased from BD Biosciences (San Jose, CA, USA). Anti-actin, carmofur, temozolomide (TMZ), and N-oleoylethanolamine (OE), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HRP-conjugated goat anti-mouse IgG was supplied by R&D Systems, Inc. (Minneapolis, MN, USA). SDS-PAGE and western blot materials were obtained from Life Technologies, Inc. (Grand Island, NY, USA). Murine anti-Sph-1P monoclonal antibody, (LT1002) and humanized anti-Sph-1P monoclonal antibody (LT1009) were obtained from Lpath, Inc. (16,17).
Cells
The pediatric glioblastoma cell line (SJGBM2) was obtained from the Children's Oncology Group (COG) Cell Culture and Xenograft Repository. These cells were grown in Iscove's modified Dulbecco's medium supplemented with 20% fetal bovine serum, 4 mM L-glutamine, and 1X ITS (5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenous acid). The U87 glioblastoma cell...