Batsios Georgios, Taglang Céline, Cao Peng, Gillespie Anne Marie, Najac Chloé, Subramani Elavarasan, Wilson David M, Flavell Robert R, Larson Peder E Z, Ronen Sabrina M, Viswanath Pavithra
Department of Radiology and Biomedical Imaging, University of California, San Francisco, CA, United States.
Front Oncol. 2021 Apr 15;11:589570. doi: 10.3389/fonc.2021.589570. eCollection 2021.
The 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-C]gluconolactone for non-invasive imaging.
To 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-C]gluconolactone metabolism to 6PG was assessed in live cells treated with the chemotherapeutic agent temozolomide (TMZ) or with vehicle control. C 2D echo-planar spectroscopic imaging (EPSI) studies of hyperpolarized δ-[1-C]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-C]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-C]gluconolactone to report on treatment response.
PGLS 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-C]6PG production from hyperpolarized δ-[1-C]gluconolactone was observed in live glioblastoma cells and was significantly reduced by treatment with TMZ. Furthermore, hyperpolarized δ-[1-C]gluconolactone metabolism to [1-C]6PG could differentiate tumor from contralateral normal brain . Notably, TMZ significantly reduced 6PG production from hyperpolarized δ-[1-C]gluconolactone at an early timepoint prior to volumetric alterations as assessed by anatomical imaging.
Collectively, 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-C]gluconolactone for non-invasive imaging of glioblastomas and their response to therapy.
磷酸戊糖途径（PPP）对于癌症（包括胶质母细胞瘤）中的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）生成和氧化还原稳态至关重要。然而，PPP的第二种酶6-磷酸葡萄糖酸内酯酶（PGLS）将6-磷酸-δ-葡萄糖酸内酯转化为6-磷酸葡萄糖酸（6PG），其对氧化还原和肿瘤增殖的确切作用仍不清楚。此外，缺乏评估PGLS活性的非侵入性方法。本研究的目的是研究PGLS在胶质母细胞瘤中的作用，并评估使用超极化δ-[1-C]葡萄糖酸内酯探测PGLS活性用于非侵入性成像的效用。
为了探究PGLS在氧化还原中的功能，通过RNA干扰使U87、U251和GS2胶质母细胞瘤细胞中的PGLS表达沉默，并测量NADPH和还原型谷胱甘肽（GSH）水平。使用克隆形成试验评估PGLS沉默对胶质母细胞瘤增殖的影响。在用化疗药物替莫唑胺（TMZ）或溶剂对照处理的活细胞中评估超极化δ-[1-C]葡萄糖酸内酯向6PG的代谢。在3T光谱仪上对患有原位胶质母细胞瘤肿瘤的大鼠或无肿瘤对照进行超极化δ-[1-C]葡萄糖酸内酯代谢的二维回波平面光谱成像（EPSI）研究。在用TMZ治疗后，对患有原位U251肿瘤的大鼠进行超极化δ-[1-C]葡萄糖酸内酯代谢的纵向二维EPSI研究和T2加权磁共振成像（MRI），以检查超极化δ-[1-C]葡萄糖酸内酯报告治疗反应的能力。
在所有模型中，PGLS敲低下调了NADPH和GSH，升高了氧化应激并抑制了克隆形成。相反，相对于对侧脑和无肿瘤脑，患有原位胶质母细胞瘤异种移植物的大鼠肿瘤组织中的PGLS表达和活性以及稳态NADPH和GSH更高。重要的是，在活的胶质母细胞瘤细胞中观察到超极化δ-[1-C]葡萄糖酸内酯产生[1-C]6PG，并且用TMZ处理后显著降低。此外，超极化δ-[1-C]葡萄糖酸内酯向[1-C]6PG的代谢可以区分肿瘤与对侧正常脑。值得注意的是，通过解剖成像评估，在体积改变之前的早期时间点，TMZ显著降低了超极化δ-[1-C]葡萄糖酸内酯产生的6PG。
总体而言，我们首次确定了PGLS活性在胶质母细胞瘤增殖中的作用，并验证了使用超极化δ-[1-C]葡萄糖酸内酯探测PGLS活性用于胶质母细胞瘤及其治疗反应的非侵入性成像的效用。
