Triacetin-based acetate supplementation as a chemotherapeutic adjuvant therapy in glioma

Abstract

Cancer is associated with epigenetic (i.e., histone hypoacetylation) and metabolic (i.e., aerobic glycolysis) alterations. Levels of N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate, are reduced in glioma; yet, few studies have investigated acetate as a potential therapeutic agent. This preclinical study sought to test the efficacy of the food additive Triacetin (glyceryl triacetate, GTA) as a novel therapy to increase acetate bioavailability in glioma cells. The growth-inhibitory effects of GTA, compared to the histone deacetylase inhibitor Vorinostat (SAHA), were assessed in established human glioma cell lines (HOG and Hs683 oligodendroglioma, U87 and U251 glioblastoma) and primary tumor-derived glioma stem-like cells (GSCs), relative to an oligodendrocyte progenitor line (Oli-Neu), normal astrocytes, and neural stem cells (NSCs) in vitro. GTA was also tested as a chemotherapeutic adjuvant with temozolomide (TMZ) in orthotopically grafted GSCs. GTA-induced cytostatic growth arrest in vitro comparable to Vorinostat, but, unlike Vorinostat, GTA did not alter astrocyte growth and promoted NSC expansion. GTA alone increased survival of mice engrafted with glioblastoma GSCs and potentiated TMZ to extend survival longer than TMZ alone. GTA was most effective on GSCs with a mesenchymal cell phenotype. Given that GTA has been chronically administered safely to infants with Canavan disease, a leukodystrophy due to ASPA mutation, GTA-mediated acetate supplementation may provide a novel, safe chemotherapeutic adjuvant to reduce the growth of glioma tumors, most notably the more rapidly proliferating, glycolytic and hypoacetylated mesenchymal glioma tumors.

Keywords: aspartoacylase; epigenetics; glioblastoma; glioma; glyceryl triacetate; metabolism; oligodendroglioma; triacetin.

Figure 1

ASPA expression is decreased in glioma tumors. (a) Quantitative real-time PCR revealed decreased ASPA mRNA expression in recurrent grade III oligodendroglioma, anaplastic astrocytoma and GBM. n = 4. Refer to Supplementary Fig. 2 for analysis of REMBRANDT and TCGA datasets. (b) Western blot (25 μg crude protein homogenate, normalized to actin) densitometric analysis revealed that ASPA expression was decreased in grade II (OII) and grade III (OIII) oligodendroglioma, anaplastic astrocytoma (AA) and glioblastoma (GBM) tumors, but similar to ASPA mRNA, ASPA protein was most significantly decreased in recurrent grade III (ReO) oligodendroglioma relative to normal (N) brain (pathologically normal tissue from patients undergoing surgery for epilepsy). n = 6 normal, 10 GBM, and 4 all others, with 2 representative protein samples shown. (c) Dual-label immunohistochemistry using normal human cerebral cortex (i.e., post-mortem brain) revealed that ASPA was more abundantly expressed in CNPase-positive oligodendrocytes within the corpus callosum (WM) than the overlying isocortex. ASPA expression was also detected within the cortical grey matter (GM, arrowheads) by GFAP-positive astrocytes. Immunohistochemistry using two independent tissue samples confirmed the western blot results that GBM and grade III (GIII) oligodendroglioma tumors possess significantly fewer ASPA immunoreactive cells. Scale bar = 100 μm (left panel), 50 μm (right panel). *p < 0.05, #p ≤ 0.001, ##p ≤ 0.0001.

Figure 2

Characterization of GSC genetic profile by whole genome cytogenetic analysis and PCR. (a) Principal component analysis (PCA) of SNP raw intensity data from GeneChip® Human Mapping 250K Nsp Arrays revealed that the established GBM cell lines U87 and U251 share similar gene amplifications/deletions to the proneural GSCs (GBM44, GBM8, and GBM2). The oligodendroglioma-derived cells (grade II OG33 and grade III OG35 GSCs and the HOG established oligodendroglioma cell line) were more similar to mesenchymal GBM GSCs (GBM12, GBM9, and GBM34). The Hs683 cell line, which was derived from a GBM tumor, but shares features of oligodendroglioma tumors, failed to cluster with either tumor type. (b) PCR was performed with a panel of well-accepted markers of proneural (e.g., CD133, Notch1, SOX2, PDGRFα, Nestin, and Olig2) and mesenchymal (e.g., BCL2A1, WT1, CD44, and CD44v6) glioma phenotypes. Although this analysis is non-quantitative, these markers display distinct bimodal expression patterns. Similar to STR profiling (Supplementary Table 1), PCR profiling confirms that GBM12, GBM34, and GBM9 GSCs exhibit a mesenchymal signature, while GBM8, GBM44, and GBM2 GSCs exhibit a proneural signature. In keeping with their oligodendroglial origin, OG33 and OG35 GSCs express PDGFRα and NG2 (not shown), but otherwise exhibit a mesenchymal signature.

Figure 3

GTA induces G₀ growth arrest of established glioma cell lines and primary tumor-derived GSCs in vitro. (a) Cell cycle profile of PI-labeled cells in growth/stem cell medium after 24 hours of 1 μM SAHA or 0.25% GTA treatment. GTA induced G₀ growth arrest of all glioma cells, except U87, U251 and GBM8 GSCs, without affecting Oli-Neu OPCs or astrocytes and promoted neural stem cell (NSC) expansion. In contrast, SAHA significantly reduced proliferation of glioma and normal cells equally. (b) GSCs (50,000 cells per well of 24 well plate) were cultured in SCM in the absence or presence of 0.25% GTA or 1 μM SAHA for 5 days with medium replenished every 48 hours. While GTA-mediated growth reduction was largely cytostatic, SAHA-mediated growth reduction did not promote differentiation (except in GBM8 GSCs), but was more cytocidal. *p < 0.05, **p ≤ 0.01, #p ≤ 0.001, ##p < 0.0001. n ≥ 3 independent experiments. Scale bar = 200 μm.

Figure 4

GTA-mediated growth reduction of established glioma cell lines and primary tumor-derived GSCs in vitro is not due to the promotion of differentiation. GSCs were dissociated and plated (10,000 cells per well of 24 well dish) in the absence or presence of 0.25% GTA or 1 μM SAHA in SCM (a) or DM (b). Growth dynamics were assessed using unbiased trypan blue exclusion based cytometry over 5 days, with medium replenished every 48 hours. (a) GTA reduced cell growth dynamics comparable to that of SAHA, except that proneural GBM GSCs (GBM8, GBM44, GBM2) were unresponsive in SCM. (b) When treated in DM, GTA was as or more effective than SAHA, particularly on oligodendroglioma-derived GSCs. (c, d) GSCs were cultured in DM for 3 days, fixed, and stained for markers of mature oligodendrocytes (CNPase, myelin basic protein [MBP]) and astrocytes (GFAP). Oli-Neu cells were used as a positive control. (c) OG33 and OG35 cells expressed CNPase, but failed to express MBP. (d) The proneural GSCs (GBM8, GBM44, GBM2) differentiated into GFAP-positive astrocytes, CNPase-positive oligodendrocytes, and Tuj1-positive neurons (not shown). In contrast, the mesenchymal GSCs (GBM12, GBM34, GBM9) failed to express GFAP, CNPase, or TuJ1 even when cultured for up to 7 days. *p < 0.05, **p ≤ 0.01, ^#p ≤ 0.001, ^##p ≤ 0.0001. Scale bar = 100 μm.

Figure 5

GTA enhances TMZ chemotherapeutic efficacy on orthotopically engrafted oligodendroglioma-derived GSCs. (a) Images and photon flux (p/cm²/s/sr) of representative mice imaged longitudinally throughout the study. OG35 GSCs (2,500 cells) expressing luciferase were engrafted in the striatum of athymic mice. After 3 days, mice were injected with luciferin (150 mg/kg, i.p.), imaged using the Xenogen imaging system, and randomized to a treatment group: 1) vehicle treated mice received daily oral suspension, 2) daily GTA (5.0 g/kg) with 10% Ora-Sweet to mask GTA’s bitterness, 3) TMZ (20mg/kg) on days 5, 7, 9, 11, 13 with oral suspension on alternate days, 4) GTA/TMZ with GTA administered daily starting at day 3 (2 days prior to TMZ) and TMZ on days 5, 7, 9, 11, 13. Treatment was administered by oral gavage until mice displayed neurological signs or weight loss of 20% the pre-surgical weight. Days when imaging failed to detect photon flux are indicated by a negative sign (e.g., 10-). Mean glucose levels were not different between the treatment groups. (b) Low and high magnification hematoxylin and eosin (H & E) stained sections of representative orthotopic tumors from each treatment group failed to reveal oligodendroglioma histological features, rather a preponderance of undifferentiated cells was observed. Immunohistochemical analysis of tumors failed to detect discernible differences in ASPA expression in the four treatment groups (not shown). Scale bar = 1 mm (low mag), 100 μm (high mag). (c) The study was negatively biased by assigning mice with the greatest flux on day 3 to the GTA/TMZ group (Initial Flux). Although GTA/TMZ treated mice started with greater flux, the rate of bioluminescence increase was reduced in GTA/TMZ treated mice relative to TMZ alone treated mice (Flux Slope). Terminal tumor volume (i.e., day of euthanasia), determined by unbiased stereology was only reduced in GTA/TMZ treated mice relative to vehicle treated mice (left bar graph). However, when taking into account the increased survival of TMZ and GTA/TMZ treated mice (i.e., tumor volume/survival day), the tumor volume of TMZ alone treated mice was reduced relative to vehicle treated mice and the tumor volume of GTA/TMZ treated mice was reduced relative to GTA alone treated mice (right bar graph). GTA/TMZ tumor volume did not differ from TMZ alone tumor volume (p = 0.068). (d) Kaplan-Meier analysis showed that GTA alone did not increase survival, but TMZ increased survival relative to vehicle and GTA/TMZ survival was greater than TMZ alone (upper panel). Survival of mice administered GTA for 2 days prior to TMZ (i.e., primed, Figs. 5a–c) was compared to GTA and TMZ both starting on day 5 (i.e., concurrent) and GTA administered after termination of TMZ (i.e., salvage). Only the primed therapy was associated with increased survival relative to TMZ alone, suggesting that GTA should be presented prior to TMZ to exert its maximal therapeutic effect (lower panel). *p < 0.05, **p ≤ 0.01, #p ≤ 0.001 unless otherwise indicated symbols represent significance relative to vehicle treated mice. n = 6 vehicle, 6 GTA, 10 TMZ, 10 GTA/TMZ.

Figure 6

GTA alone increases survival of mice orthotopically engrafted with GBM-derived GSCs. (a) Images and photon flux (p/cm²/s/sr) of representative mice engrafted with GBM12 GSCs (2,500 cells) imaged longitudinally throughout the study. Mice were treated with the “primed” combination GTA/TMZ therapy where GTA was started on post-surgical day 3 and TMZ started on post-surgical day 5. Days when imaging failed to detect photon flux are indicated by a negative sign (e.g., 10-). (b) Low and high magnification H & E stained sections of representative orthotopic tumors from each treatment group. Immunohistochemical analysis of tumors failed to detect discernible differences in ASPA expression among the four treatment groups (not shown). Scale bar = 1 mm (low mag), 200 μm (high mag). (c) Mean glucose levels were not different between the treatment groups. Bioluminescent flux and end tumor volume (not shown) of TMZ and GTA/TMZ treated mice were reduced relative to vehicle and GTA treated mice. Although GTA/TMZ did not reduce bioluminescent flux or end tumor volume greater than TMZ alone, GTA alone increased survival relative to vehicle treated mice and, in conjunction with TMZ, increased survival greater than TMZ alone. ##p ≤ 0.0001. n = 7 vehicle, 7 GTA, 6 TMZ, 8 GTA/TMZ.