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Hyperpolarized ?-[1-13 C]gluconolactone as a probe of the pentose phosphate pathway.

ABSTRACT: The pentose phosphate pathway (PPP) is thought to be upregulated in trauma (to produce excess NADPH) and in cancer (to provide ribose for nucleotide biosynthesis), but simple methods for detecting changes in flux through this pathway are not available. MRI of hyperpolarized 13 C-enriched metabolites offers considerable potential as a rapid, non-invasive tool for detecting changes in metabolic fluxes. In this study, hyperpolarized ?-[1-13 C]gluconolactone was used as a probe to detect flux through the oxidative portion of the pentose phosphate pathway (PPPox ) in isolated perfused mouse livers. The appearance of hyperpolarized (HP) H13 CO3- within seconds after exposure of livers to HP-?-[1-13 C]gluconolactone demonstrates that this probe rapidly enters hepatocytes, becomes phosphorylated, and enters the PPPox pathway to produce HP-H13 CO3- after three enzyme catalyzed steps (6P-gluconolactonase, 6-phosphogluconate dehydrogenase, and carbonic anhydrase). Livers perfused with octanoate as their sole energy source show no change in production of H13 CO3- after exposure to low levels of H2 O2 , while livers perfused with glucose and insulin showed a twofold increase in H13 CO3- after exposure to peroxide. This indicates that flux through the PPPox is stimulated by H2 O2 in glucose perfused livers but not in livers perfused with octanoate alone. Subsequent perfusion of livers with non-polarized [1,2-13 C]glucose followed by 1 H NMR analysis of lactate in the perfusate verified that flux through the PPPox is indeed low in healthy livers and modestly higher in peroxide damaged livers. We conclude that hyperpolarized ?-[1-13 C]gluconolactone has the potential to serve as a metabolic imaging probe of this important biological pathway.

SUBMITTER: Moreno KX

PROVIDER: S-EPMC5502806 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Hyperpolarized δ-[1-13 C]gluconolactone as a probe of the pentose phosphate pathway.

Moreno Karlos X KX Harrison Crystal E CE Merritt Matthew E ME Kovacs Zoltan Z Malloy Craig R CR Sherry A Dean AD

NMR in biomedicine 20170308 6

The pentose phosphate pathway (PPP) is thought to be upregulated in trauma (to produce excess NADPH) and in cancer (to provide ribose for nucleotide biosynthesis), but simple methods for detecting changes in flux through this pathway are not available. MRI of hyperpolarized 13 C-enriched metabolites offers considerable potential as a rapid, non-invasive tool for detecting changes in metabolic fluxes. In this study, hyperpolarized δ-[1-13 C]gluconolactone was used as a probe ...[more]

PMID: 28272754

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Project description:BackgroundTelomere maintenance by telomerase reverse transcriptase (TERT) is essential for immortality in most cancers, including oligodendrogliomas. Agents that disrupt telomere maintenance such as the telomere uncapping agent 6-thio-2'-deoxyguanosine (6-thio-dG) are in clinical trials. We previously showed that TERT expression in oligodendrogliomas is associated with upregulation of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). We also showed that hyperpolarized δ-[1-13C]-gluconolactone metabolism to 6-phosphogluconate (6-PG) can be used to probe the PPP in glioblastomas. The goal of this study was to determine whether hyperpolarized 13C imaging using δ-[1-13C]-gluconolactone can monitor TERT expression and response to 6-thio-dG in oligodendrogliomas.MethodsWe examined patient-derived oligodendroglioma cells and orthotopic tumors to assess the link between TERT and hyperpolarized δ-[1-13C]-gluconolactone metabolism. We performed in vivo imaging to assess the ability of hyperpolarized δ-[1-13C]-gluconolactone to report on TERT and response to 6-thio-dG in rats bearing orthotopic oligodendrogliomas in vivo.ResultsDoxycycline-inducible TERT silencing abrogated 6-PG production from hyperpolarized δ-[1-13C]-gluconolactone in oligodendroglioma cells, consistent with the loss of G6PD activity. Rescuing TERT expression by doxycycline removal restored G6PD activity and, concomitantly, 6-PG production. 6-PG production from hyperpolarized δ-[1-13C]-gluconolactone demarcated TERT-expressing tumor from surrounding TERT-negative normal brain in vivo. Importantly, 6-thio-dG abrogated 6-PG production at an early timepoint preceding MRI-detectable alterations in rats bearing orthotopic oligodendrogliomas in vivo.ConclusionsThese results indicate that hyperpolarized δ-[1-13C]-gluconolactone reports on TERT expression and early response to therapy in oligodendrogliomas. Our studies identify a novel agent for imaging tumor proliferation and treatment response in oligodendroglioma patients.

Project description: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.

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Hyperpolarized ?-[1-13 C]gluconolactone as a probe of the pentose phosphate pathway.

Publications

Hyperpolarized δ-[1-<sup>13</sup> C]gluconolactone as a probe of the pentose phosphate pathway.

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