Imaging 6-Phosphogluconolactonase Activity in Brain Tumors In Vivo Using Hyperpolarized δ-[1-13C]gluconolactone

Abstract

IntroductionThe pentose phosphate pathway (PPP) is essential for NADPH generation and redox homeostasis in cancer, including glioblastomas. However, the precise contribution to redox and tumor proliferation of the second PPP enzyme 6-phosphogluconolactonase (PGLS), which converts 6-phospho-δ-gluconolactone to 6-phosphogluconate (6PG), remains unclear. Furthermore, non-invasive methods of assessing PGLS activity are lacking. The goal of this study was to examine the role of PGLS in glioblastomas and assess the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive imaging.MethodsTo interrogate the function of PGLS in redox, PGLS expression was silenced in U87, U251 and GS2 glioblastoma cells by RNA interference and levels of NADPH and reduced glutathione (GSH) measured. Clonogenicity assays were used to assess the effect of PGLS silencing on glioblastoma proliferation. Hyperpolarized δ-[1-13C]gluconolactone metabolism to 6PG was assessed in live cells treated with the chemotherapeutic agent temozolomide (TMZ) or with vehicle control. 13C 2D echo-planar spectroscopic imaging (EPSI) studies of hyperpolarized δ-[1-13C]gluconolactone metabolism were performed on rats bearing orthotopic glioblastoma tumors or tumor-free controls on a 3T spectrometer. Longitudinal 2D EPSI studies of hyperpolarized δ-[1-13C]gluconolactone metabolism and T2-weighted magnetic resonance imaging (MRI) were performed in rats bearing orthotopic U251 tumors following treatment with TMZ to examine the ability of hyperpolarized δ-[1-13C]gluconolactone to report on treatment response.ResultsPGLS knockdown downregulated NADPH and GSH, elevated oxidative stress and inhibited clonogenicity in all models. Conversely, PGLS expression and activity and steady-state NADPH and GSH were higher in tumor tissues from rats bearing orthotopic glioblastoma xenografts relative to contralateral brain and tumor-free brain. Importantly, [1-13C]6PG production from hyperpolarized δ-[1-13C]gluconolactone was observed in live glioblastoma cells and was significantly reduced by treatment with TMZ. Furthermore, hyperpolarized δ-[1-13C]gluconolactone metabolism to [1-13C]6PG could differentiate tumor from contralateral normal brain in vivo. Notably, TMZ significantly reduced 6PG production from hyperpolarized δ-[1-13C]gluconolactone at an early timepoint prior to volumetric alterations as assessed by anatomical imaging.ConclusionsCollectively, we have, for the first time, identified a role for PGLS activity in glioblastoma proliferation and validated the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive in vivo imaging of glioblastomas and their response to therapy.

Sedoheptulose Kinase SHPK Expression in Glioblastoma: Emerging Role of the Nonoxidative Pentose Phosphate Pathway in Tumor Proliferation

“…To understand which cellular pathways were related to the different expressions of SHPK in GBMs and what this increased flux in the PPP of the S7P intermediate led to, we conducted a functional enrichment analysis. In agreement with that described in the literature [ 16 , 21 , 35 , 36 , 37 , 41 , 42 ], increased SHPK mRNA expression and a subsequent S7P flux in the PPP triggered a number of pathways involved in cell proliferation. In contrast, GBMs characterized by a low level of SHPK mRNA showed active physiological cell signaling pathways and had no differentially expressed genes involved in extracellular matrix remodeling and proliferation pathways.…”

“…Previous studies have examined the relative gene expression of enzymes involved in the oxidative phase of the PPP, finding an overall increase in glycolytic and PPP genes driving ATP production and excess nucleotides resulting in uncontrolled proliferation in GBM cells [ 36 , 41 , 42 ]. In addition, inhibitors of the oxidative branch of the PPP have been studied, the findings of which indicate increased radiosensitivity in human gliomas [ 43 ].…”

Molecular and metabolic alterations of 2,3-dihydroquinazolin-4(1H)-one derivatives in prostate cancer cell lines

“…Several essential pathways are utilized in cancer cells to maintain redox homeostasis and generate NADPH, including the PPP 39 and the antioxidant glutathione system 40 . PPP is a branch of glycolysis, that encompasses several oxidation and hydrolysis steps starting with the oxidation of glucose-6-phosphate and ending with the generation of ribulose-5-phosphate in concomitant reduction of NADP + to NADPH 39,41 . NADPH is a key element in maintaining GSH in its reduced form and thus opposing oxidative stress which promotes cancer growth 42 .…”

Identification and validation of PGLS as a metabolic target for early screening and prognostic monitoring of gastric cancer

“…Recently study has demonstrated that pentose phosphate pathway flux was up‐regulated in cancer cells. 20 …”

“…Moreover, by detecting hyperpolarized‐[1‐ 13 C] gluconolactone to form [1‐ 13 C] 6PG, they could significantly differentiate the glioblastoma tumor tissues from normal brain tissues in vivo. 20 …”