The RAS family of proto-oncogenes are among the most commonly mutated genes in human cancers and predict poor clinical outcome. Several mechanisms underlying oncogenic RAS transformation are well ...documented, including constitutive signaling through the RAF-MEK-ERK proproliferative pathway as well as the PI3K-AKT prosurvival pathway. Notably, control of redox balance has also been proposed to contribute to RAS transformation. However, how homeostasis between reactive oxygen species (ROS) and antioxidants, which have opposing effects in the cell, ultimately influence RAS-mediated transformation and tumor progression is still a matter of debate and the mechanisms involved have not been fully elucidated. Here, we show that oncogenic KRAS protects fibroblasts from oxidative stress by enhancing intracellular GSH levels. Using a whole transcriptome approach,we discovered that this is attributable to transcriptional up-regulation of xCT, the gene encoding the cystine/glutamate antiporter. This is in line with the function of xCT, which mediates the uptake of cystine, a precursor for GSH biosynthesis. Moreover, our results reveal that the ETS-1 transcription factor downstream of the RAS-RAF-MEK-ERK signaling cascade directly transactivates the xCT promoter in synergy with the ATF4 endoplasmic reticulum stress-associated transcription factor. Strikingly, xCT was found to be essential for oncogenic KRAS-mediated transformation in vitro and in vivo bymitigating oxidative stress, as knockdown of xCT strongly impaired growth of tumor xenografts established from KRAS-transformed cells. Overall, this study uncovers a mechanism by which oncogenic RAS preserves intracellular redox balance and identifies an unexpected role for xCT in supporting RAS-induced transformation and tumorigenicity.
Purpose
The cystine transporter, system x
C
-
, plays a crucial role in sustaining redox homeostasis and is reported to be overexpressed in several cancer subtypes. 5-
18
FFluoroaminosuberic acid (
...18
FFASu) is a novel positron emission tomography (PET) tracer, which exhibits specific uptake
via
system x
C
-
.
18
FFASu synthesis by the commonly used Kryptofix 2.2.2/K
2
CO
3
-facilitated fluorination method results in four diastereomers, as a result of 2 chiral centers at positions 2- and 5- of the tracer. We recently reported the synthesis of the optically pure 2
S
-
18
FFASu from chiral precursors. Our preliminary results indicated preferential uptake of the 2
S
-isomer by tumor cells compared to 2
R
-
18
FFASu. Few studies have investigated the biodistribution of chiral
18
F-labeled amino acids. The aim of this study was to evaluate the imaging utility and biodistribution of the 5-position diastereomers as well as the racemic (2
S
,5
R/S
-) mixture in three different tumor models.
Procedures
In vitro
tracer uptake experiments and Western blotting were performed in breast cancer (MDA-MB-231), glioblastoma (U-87), and prostate (PC-3) cancer cell lines. PET imaging and biodistribution studies were conducted in xenograft-bearing immunocompromised Rag2M female mice.
Results
All three tracer conformations allowed for the visualization of tumor xenografts at 1 h (for U-87 and PC-3 tumors) or 2 h (in the case of MDA-MB-231 xenografts) post-injection, with the racemate (2
S
,5
R/S
-) displaying similar image contrast as compared to the 5- position diastereomers and the 2
S
,5
S
-
18
FFASu conformation exhibiting relatively higher contrast for imaging U-87 and PC-3 xenografts. Tumor uptake of the isomers was blocked by an excess of the non-radioactive standard, aminosuberic acid (ASu), confirming target specificity. All three isomers were excreted
via
the renal pathway. Biodistribution analyses showed that PC-3 tumors had the highest tracer uptake, and the accumulation (%ID/g) of the
2S,
5
R/S-
, 2
S
,5
S
-, and 2
S
,5
R
- isomers was 9.19 ± 1.14, 8.00 ± 1.41, and 7.16 ± 2.13 at 1 h post-injection, respectively. This gave corresponding tumor-to-muscle ratios of 33.68 ± 9.52, 31.42 ± 4.54, and 25.33 ± 4.97, respectively.
Conclusion
Our data suggest that pure 2
S
-
18
FFASu can be used to noninvasively image system x
C
-
in a variety of cancers, either as the racemic mixture (2
S
,5
R/S
-) or optically pure form. Furthermore, this work shows potential utility of
18
FFASu for detection of glioblastoma and prostate cancer.
An increasing number of positron emission tomography (PET) radiotracers are being developed that are modelled on various amino acids to better understand disease in a manner that is complementary to ...traditional glycolysis‐targeting 18F‐fluorodeoxyglucose. Since chiral centers are ubiquitous in amino acids, generating an optically pure radiolabeled amino acid is important for patient dose, image quality and understanding the physiology behaviour. Past studies on the radiosynthesis of amino acid radiotracers seldom address the impact of reaction conditions on their chirality. The amino acid PET tracer, 18F5‐fluoroaminosuberic acid (18FFASu), has two chiral centers at the 2‐ and 5‐positions and is being developed as a specific tracer for the cystine transporter (system xC−), a biomarker for oxidative stress. Herein we report a method for synthesizing pure 2S,5R/S‐FASu. We have resolved the 5‐position configuration by applying Mosher's method combined with 2D NMR, which has enabled the synthesis of 18FASu with fully known configuration. Our study serves as an example of a systematic method to identify and characterize amino acid tracers with chiral centers.
Heard it on the radio: Generating an optically pure radiolabeled amino acid is important for patient dose, image quality and understanding the physiology behaviour. A method for synthesizing optically pure FASu, an oxidative stress PET imaging tracer with 2‐ and 5‐position chiral centers, was developed. Cell uptake study showed only the 2S‐FASu was uptaken by the tumor cells.
An isocratic reversed-phase high-performance liquid chromatographic procedure was developed for the determination of minocycline in rat plasma and brain and applied to brain-to-blood (plasma) ...distribution studies. The procedure is based on isolation of the compound and the internal standard (either demeclocycline or tetracycline may be used) from plasma and brain constituents using the Oasis HLB cartridge, with satisfactory recovery and specificity, and separation on a Symmetry Shield RP8 (15 cm×4.6 mm, 3.5 μm) column coupled with a UV detector set at 350 nm. The assay was linear over a wide range, with a lower limit of quantification of 50 ng ml
−1 or g
−1, using 0.2 ml of plasma and about 200 mg of brain tissue. Precision and accuracy were acceptable. In the rat minocycline crossed the blood–brain barrier slowly, achieving mean brain concentrations between 30 and 40% of the equivalent systemic exposure, regardless of the dose and route of administration.
BACKGROUNDProstaglandin E2 (PGE2) acts via its EP4 receptor as a cytokine amplifier (e.g., interleukin IL-6) and induces the differentiation and expansion of inflammatory T-helper (Th) lymphocytes. ...These mechanisms play a key role in the onset and progression of rheumatoid arthritis (RA). We present the pharmacological characterisation of CR6086, a novel EP4 receptor antagonist, and provide evidence for its potential as a disease-modifying anti-rheumatic drug (DMARD).METHODSCR6086 affinity and pharmacodynamics were studied in EP4-expressing HEK293 cells by radioligand binding and cyclic adenosine monophosphate (cAMP) production, respectively. In immune cells, IL-6 and vascular endothelial growth factor (VEGF) expression were analysed by RT-PCR, and IL-23 and IL-17 release were measured by enzyme-linked immunosorbent assay (ELISA). In collagen-induced arthritis (CIA) models, rats or mice were immunised with bovine collagen type II. Drugs were administered orally (etanercept and methotrexate intraperitoneally) starting at disease onset. Arthritis progression was evaluated by oedema, clinical score and histopathology. Anti-collagen II immunoglobulin G antibodies were measured by ELISA.RESULTSCR6086 showed selectivity and high affinity for the human EP4 receptor (Ki = 16.6 nM) and functioned as a pure antagonist (half-maximal inhibitory concentration, 22 nM) on PGE2-stimulated cAMP production. In models of human immune cells in culture, CR6086 reduced key cytokine players of RA (IL-6 and VEGF expression in macrophages, IL-23 release from dendritic cells, IL-17 release from Th17 cells). In the CIA model of RA in rats and mice, CR6086 significantly improved all features of arthritis: severity, histology, inflammation and pain. In rats, CR6086 was better than the selective cyclooxygenase-2 inhibitor rofecoxib and at least as effective as the Janus kinase inhibitor tofacitinib. In mice, CR6086 and the biologic DMARD etanercept were highly effective, whereas the non-steroidal anti-inflammatory drug naproxen was ineffective. Importantly, in a study of CR6086/methotrexate, combined treatment greatly improved the effect of a fully immunosuppressive dose of methotrexate.CONCLUSIONSCR6086 is a novel, potent EP4 antagonist showing favourable immunomodulatory properties, striking DMARD effects in rodents, and anti-inflammatory activity targeted to immune-mediated inflammatory diseases and distinct from the general effects of cyclooxygenase inhibitors. These results support the clinical development of CR6086, both as a stand-alone DMARD and as a combination therapy with methotrexate. The proof-of-concept trial in patients with RA is ongoing.
High-performance liquid chromatography method coupled with tandem mass spectrometry was developed for the quantitative determination of I3,II8-biapigenin. The procedure includes solid-phase ...extraction and separation on an XTerra MS C18. The assay was linear over a wide range; precision and accuracy were acceptable. Biapigenin was present in mouse and rat plasma after a standardized
Hypericum perforatum extract. It was not detected in brain (<5
ng
g
−1), suggesting poor brain-to-blood permeability. Biapigenin concentrations were measurable in mice after intraperitoneal biapigenin (10
mg
kg
−1) but these amounted to about 2% of the equivalent systemic exposure, after correction for the contribution from residual blood.
An isocratic, reversed-phase high-performance liquid chromatographic procedure (HPLC) was developed for determination of the neuroprotective agent riluzole in mice plasma, brain and spinal cord. The ...procedure is based on isolation of the compound and the internal standard from plasma and central nervous system tissues using a Bakerbond spe™ C8 cartridge, with satisfactory recovery and specificity. Separation was on a C18 column, coupled with an UV detector at 263 nm. The assay was linear over a wide range, with a lower limit of quantification of 100
ng
ml
−1 or g
−1 using 0.1
ml of plasma and about 100
mg of brain tissue. The precision and accuracy were within the acceptable limits for an HPLC assay. The method is currently used to support pharmacological studies of the activity of riluzole when given in combination with other potential neuroprotective agents in an animal model of familiar amyotrophic lateral sclerosis (SOD1-G93A transgenic mice).
Previous structure–activity relationship studies in the search for a potent, noncompetitive α‐amino‐3‐(3‐hydroxy‐5‐methyl‐4‐isoxazolyl)propionic acid (AMPA) receptor antagonist led to ...2,3‐dimethyl‐6‐phenyl‐12H‐1,3dioxolo4,5‐himidazo1,2‐c2,3benzodiazepine (ZK 187638). However, the first synthesis had some drawbacks regarding reagents, processes, and overall yield, which furthermore decreased when the synthesis was scaled up. Therefore, we now report a new synthetic route for this compound which requires fewer steps and is suited for large‐scale production. This compound significantly relieved the symptoms of neuromuscular deficit in mnd mice, a model of neuronal ceroid lipofuscinosis with motor neuron dysfunction. After oral administration, the concentrations of the compound in the brain and spinal cord were about threefold higher than those in the plasma. In summary, this novel AMPA antagonist is accessible through an optimized synthetic route, has good neurobehavioral activity, oral bioavailability, and favorable brain penetration. This opens new possibilities for the treatment of devastating neurological diseases that are mediated by the AMPA receptor.
Two steps toward medicinal use of a novel 2,3‐benzodiazepine are reported: 1) Synthesis of ZK 187638 (1) was optimized to enable large‐scale production for clinical development. 2) Good brain availability and therapeutic efficacy were shown in a mouse model of neuronal ceroid lipofuscinosis, which is a fatal disease with unmet medical need.
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