Comparison of Sample Tubes for the X-band EPR Measurement of an Aqueous Sample: Effects on Reproducibility of Signal Intensities

Reproducibility of the X-band EPR measurement of an aqueous solution sample was compared using three different types of sample tubes, and accuracy of quantitative performance was assessed. A PTEE tubing, a glass capillary and a quartz flat cuvette were compared to get a suitable condition for quantitative measurement. An accurate 0.1 mM water solution of TEMPOL was used as a standard sample. The TEMPOL solution was loaded into one of sample tube, and the sample tube was set in the TE-mode cavity of X-band EPR spectrometer. Two procedures below were tested for reproducibility of repeated measurements. 1) The sample tube in the cavity was washed or renewed every measurement, and measurements were repeated several times with fixed EPR parameters. 2) The sample tubes in the cavity were stayed and repeatedly measured several times. Next, EPR parameters such as sweep speed, time constant, modulation width, and/or microwave power were varied to seek optimum signal intensity. The PTFE tubing showed the best reproducibility when the measurements were repeated with staying the sample tube (the ratio of standard deviation to the averaged signal intensity; SD/AV = 0.0058). When the sample and sample tube was renewed every measurement, variation of signal intensity became larger (SD/AV = 0.0333). The glass capillary had the best reproducibility in both procedures 1 and 2 (SD/AV = 0.0104 and 0.0036, respectively). The signal reproducibility of the flat cuvette was relatively low (SD/AV = 0.0400 and 0.0690 for procedures 1 and 2, respectively). However, the flat cuvette gave the largest signal intensity when the loaded volume of the sample was the identical. In conclusion, the best quantitative performance of the X-band EPR spectroscopy for a liquid sample was obtained when the measurements are carried out with the capillary

Monitoring response to a clinically relevant IDH inhibitor in glioma—Hyperpolarized 13C magnetic resonance spectroscopy approaches

BackgroundMutant isocitrate dehydrogenase (IDHmut) catalyzes 2-hydroxyglutarate (2HG) production and is considered a therapeutic target for IDHmut tumors. However, response is mostly associated with inhibition of tumor growth. Response assessment via anatomic imaging is therefore challenging. Our goal was to directly detect IDHmut inhibition using a new hyperpolarized (HP) 13C magnetic resonance spectroscopy-based approach to noninvasively assess α-ketoglutarate (αKG) metabolism to 2HG and glutamate.MethodsWe studied IDHmut-expressing normal human astrocyte (NHAIDH1mut) cells and rats with BT257 tumors, and assessed response to the IDHmut inhibitor BAY-1436032 (n ≥ 4). We developed a new 13C Echo Planar Spectroscopic Imaging sequence with an optimized RF pulse to monitor the fate of HP [1-13C]αKG and [5-12C,1-13C]αKG with a 2.5 × 2.5 × 8 mm3 spatial resolution.ResultsCell studies confirmed that BAY-1436032-treatment leads to a drop in HP 2HG and an increase in HP glutamate detectable with both HP substrates. Data using HP [5-12C,1-13C]αKG also demonstrated that its conversion to 2HG is detectable without the proximal 1.1% natural abundance [5-13C]αKG signal. In vivo studies showed that glutamate is produced in normal brains but no 2HG is detectable. In tumor-bearing rats, we detected the production of both 2HG and glutamate, and BAY-1436032-treatment led to a drop in 2HG and an increase in glutamate. Using HP [5-12C,1-13C]αKG we detected metabolism with an signal-to-noise ratio of 23 for 2HG and 17 for glutamate.ConclusionsOur findings point to the clinical potential of HP αKG, which recently received FDA investigational new drug approval for research, for noninvasive localized imaging of IDHmut status

Mitoribosome sensitivity to HSP70 inhibition uncovers metabolic liabilities of castration-resistant prostate cancer.

The androgen receptor is a key regulator of prostate cancer and the principal target of current prostate cancer therapies collectively termed androgen deprivation therapies. Insensitivity to these drugs is a hallmark of progression to a terminal disease state termed castration-resistant prostate cancer. Therefore, novel therapeutic options that slow progression of castration-resistant prostate cancer and combine effectively with existing agents are in urgent need. We show that JG-98, an allosteric inhibitor of HSP70, re-sensitizes castration-resistant prostate cancer to androgen deprivation drugs by targeting mitochondrial HSP70 (HSPA9) to suppress aerobic respiration. Rather than impacting androgen receptor stability as previously described, JG-98's primary effect is inhibition of mitochondrial translation, leading to disruption of electron transport chain activity. Although functionally distinct from HSPA9 inhibition, direct inhibition of the electron transport chain with a complex I or II inhibitor creates a similar physiological state capable of re-sensitizing castration-resistant prostate cancer to androgen deprivation therapies. These data identify a significant role for HspA9 in mitochondrial ribosome function and highlight an actionable metabolic vulnerability of castration-resistant prostate cancer

Metabolic Reprogramming Associated with Aggressiveness Occurs in the G-CIMP-High Molecular Subtypes of IDH1mut Lower Grade Gliomas

BACKGROUND: Early detection of increased aggressiveness of brain tumors is a major challenge in the field of neuro-oncology because of the inability of traditional imaging to uncover it. IDH-mutant gliomas represent an ideal model system to study the molecular mechanisms associated with tumorigenicity because they appear indolent and non-glycolytic initially, but eventually a subset progresses towards secondary glioblastoma with a Warburg-like phenotype. The mechanisms and molecular features associated with this transformation are poorly understood. METHODS: We employed model systems for IDH1 mutant gliomas with different growth and proliferation rates in vivo and in vitro. We described the metabolome, transcriptome and epigenome of these models in order to understand the link between their metabolism and the tumor biology. To verify whether this metabolic reprogramming occurs in the clinic we analyzed TCGA data. RESULTS: We reveal that the aggressive glioma models have lost DNA methylation in the promoters of glycolytic enzymes, especially LDHA, and have increased mRNA and metabolite levels compared to the indolent model. We find that the acquisition of the high glycolytic phenotype occurs at the G-CIMP-high molecular subtype in patients and is associated with the worst outcome. CONCLUSION: We propose very early monitoring of lactate levels as a biomarker of metabolic reprogramming and tumor aggressiveness