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. 2017 Apr 11;8(15):24753-24761.
doi: 10.18632/oncotarget.15800.

Acid ceramidase is a novel drug target for pediatric brain tumors

Ninh B Doan  1   2 , Ha S Nguyen  2 , Andrew Montoure  2 , Mona M Al-Gizawiy  3 , Wade M Mueller  2 , Shekar Kurpad  2 , Scott D Rand  3 , Jennifer M Connelly  4 , Christopher R Chitambar  4 , Kathleen M Schmainda  3   5 , Shama P Mirza  1   6   7
Affiliations

Acid ceramidase is a novel drug target for pediatric brain tumors

Ninh B Doan et al. Oncotarget. .

Abstract

Pediatric brain tumors are the most common solid tumors in children and are also a leading culprit of cancer-related fatalities in children. Pediatric brain tumors remain hard to treat. In this study, we demonstrated that medulloblastoma, pediatric glioblastoma, and atypical teratoid rhabdoid tumors express significant levels of acid ceramidase, where levels are highest in the radioresistant tumors, suggesting that acid ceramidase may confer radioresistance. More importantly, we also showed that acid ceramidase inhibitors are highly effective at targeting these pediatric brain tumors with low IC50 values (4.6-50 μM). This data suggests acid ceramidase as a novel drug target for adjuvant pediatric brain tumor therapies. Of these acid ceramidase inhibitors, carmofur has seen clinical use in Japan since 1981 for colorectal cancers and is a promising drug to undergo further animal studies and subsequently a clinical trial as a treatment for pediatric patients with brain tumors.

Keywords: acid ceramidase inhibitors; carmofur; glioblastoma; medulloblastoma; pediatric glioblastoma.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors disclose no potential conflicts of interest.

Figures

Figure 1
Figure 1. Patient derived pediatric brain tumor cells express high levels of ASAH1
(A) Western blot of 4 patient derived pediatric brain tumors and 1 adult glioblastoma cell line (U87) are shown. The blot was overexposed to show the faint ASAH1 band from SJGBM2 cells. (B) Quantitation of the relative ASAH1 expression level was performed with ImageJ. CHLA200 cells express many folds higher level of ASAH1 than other tumors.
Figure 2
Figure 2. Pediatric brain tumor cells are highly sensitive to ASAH1 inhibitors: OE and carmofur
MTT assays of SJGBM2 (A), CHLA259 (B), CHLA200 (C), and CHLA266 (D) were performed with carmofur and OE. Results are expressed as means and ± s.e.m (N = 3).
Figure 3
Figure 3. Pediatric brain tumor cells treated with carmofur underwent apoptosis as demonstrated by the Annexin-V-Alexa-488 conjugate staining
Microscopy studies pediatric brain tumors are shown. Top left panel, SJGBM2; top right panel, CHLA266; bottom left panel, CHLA259; bottom right panel, CHLA200; control cells imaged with the brightlight (A), with the fluorescent light (B) vs cells treated (12 hrs) with 50 μM carmofur imaging with the brightlight (C), with the fluorescent light (D). Brightlight imaging of control live cells (blue arrows) and death cells (black arrows) are shown. Whereas a large number of apoptotic cells stained with Annexin-V-Alexa-488 (white arrows) were observed under fluorescent imaging when treated with carmofur, very little to no staining was seen in control untreated cells (B).
Figure 4
Figure 4. Pediatric brain tumors were more sensitive to carmofur than temozolomide
Cell survival studies using MTT assays of various pediatric brain tumors are shown for control cells vs cells treated with 50 μM of carmofur and 100 μM of TMZ. Results are expressed as means and ± s.e.m (N = 3).
Figure 5
Figure 5. A high level of ASAH1 is associated with a lower level of its substrates and a higher level of its endproducts, ceramides and sphingosines, respectively
Cell pellets were prepared and lipids were extracted for mass spectrometry as described in Materials and Methods. Intracellular ceramide and sphingolipid levels are from CHLA266 (black bars) and SJGBM2 (gray bars) cells. These levels were adjusted for equal loading based on the phosphate level. Results shown are mean ± SD of three replicates. *P < 0.05.
Figure 6
Figure 6. Treatment of SJGBM2 cells with carmofur resulted in intracellular accumulation of ceramides
Cell pellets were prepared and lipids were extracted for mass spectrometry as described in Materials and Methods. Intracellular ceramide and sphingolipid levels are from SJGBM2 cells (black bars) and SJGBM2 cells treated with 50 μM carmofur (gray bars). These levels were adjusted for equal loading based on the phosphate level. Results shown are mean ± SD of three replicates. *P < 0.05.
Figure 7
Figure 7. The schematic diagram of the metabolic ceramide pathway is shown
ASAH1 converts ceramide into sphingosine, which is subsequently metabolized to sphingosine-1-phosphate by SPHK1 or 2. Ceramide is a tumor suppressor, promoting apoptosis and chemo/radio-sensitization. Sphingosine-1-phosphate is a tumor promoter, enhancing survival, angiogenesis, and proliferation.
See this image and copyright information in PMC

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