Studies with Human-Induced Pluripotent Stem Cells Reveal That CTNS Mutations Can Alter Renal Proximal Tubule Differentiation.

Cystinosis is an autosomal recessive disease resulting from mutations in ctns, which encodes for cystinosin, a proton-coupled cystine transporter that exports cystine from lysosomes. The major clinical form, infantile cystinosis, is associated with renal failure due to the malfunctioning of the renal proximal tubule (RPT). To examine the hypothesis that the malfunctioning of the cystinotic RPT arises from defective differentiation, human-induced pluripotent stem cells (hiPSCs) were generated from human dermal fibroblasts from an individual with infantile cystinosis, as well as a normal individual. The results indicate that both the cystinotic and normal hiPSCs are pluripotent and can form embryoid bodies (EBs) with the three primordial germ layers. When the normal hiPSCs were subjected to a differentiation regime that induces RPT formation, organoids containing tubules with lumens emerged that expressed distinctive RPT proteins, including villin, the Na+/H+ Exchanger (NHE) isoform 3 (NHE3), and the NHE Regulatory Factor 1 (NHERF1). The formation of tubules with lumens was less pronounced in organoids derived from cystinotic hiPSCs, although the organoids expressed villin, NHE3, and NHERF1. These observations can be attributed to an impairment in differentiation and/or by other defects which cause cystinotic RPTs to have an increased propensity to undergo apoptosis or other types of programmed cell death.

Exploring the Contribution of the Transporter AGT1/rBAT in Cystinuria Progression: Insights from Mouse Models and a Retrospective Cohort Study.

More than 20 years have passed since the identification of SLC3A1 and SLC7A9 as causative genes for cystinuria. However, cystinuria patients exhibit significant variability in the age of lithiasis onset, recurrence, and response to treatment, suggesting the presence of modulatory factors influencing cystinuria severity. In 2016, a second renal cystine transporter, AGT1, encoded by the SLC7A13 gene, was discovered. Although it was discarded as a causative gene for cystinuria, its possible effect as a modulatory gene remains unexplored. Thus, we analyzed its function in mouse models of cystinuria, screened the SLC7A13 gene in 34 patients with different lithiasic phenotypes, and functionally characterized the identified variants. Mice results showed that AGT1/rBAT may have a protective role against cystine lithiasis. In addition, among the four missense variants detected in patients, two exhibited a 25% impairment in AGT1/rBAT transport. However, no correlation between SLC7A13 genotypes and lithiasis phenotypes was observed in patients, probably because these variants were found in heterozygous states. In conclusion, our results, consistent with a previous study, suggest that AGT1/rBAT does not have a relevant effect on cystinuria patients, although an impact in patients carrying homozygous pathogenic variants cannot be discarded.

Mechanism of Cell Death by Combined Treatment with an xCT Inhibitor and Paclitaxel: An Alternative Therapeutic Strategy for Patients with Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian carcinoma that responds poorly to chemotherapy. Glutathione (GSH) is a primary antioxidant, which protects cells against reactive oxygen species (ROS). High levels of GSH are related to chemotherapeutic resistance. The glutamine/cystine transporter xCT is essential for intracellular GSH synthesis. However, whether xCT inhibition can overcome the resistance to chemotherapeutic agents in OCCC remains unclear. This study demonstrated that combined treatment with paclitaxel (PTX) and the xCT inhibitor sulfasalazine (SAS) significantly enhanced cytotoxicity more than the individual drugs did in OCCC cells. Treatment with PTX and SAS induced apoptosis more effectively than did individual drug treatments in the cells with significant generation of ROS. Moreover, combined treatment with PTX and SAS induced ferroptosis in the cells with low expression of glutathione peroxidase (GPx4), high levels of intracellular iron and significant lipid ROS accumulation. Therefore, our findings provide valuable information that the xCT inhibitor might be a promising therapeutic target for drug-resistant OCCC. The strategy of combined administration of PTX and SAS can potentially be used to treat OCCC and help to develop novel therapeutic methods.

Insights into the molecular basis for substrate binding and specificity of the fungal cystine transporter CgCYN1.

Cystine transporters are a clinically important class of transporters found in bacteria, pathogenic fungi and mammalian cells. Despite their significance, very little is known about the mechanism of substrate recognition and transport. We have carried out studies on the plasma membrane Candida glabrata cystine transporter, CgCYN1 a member of the amino acid-polyamine-organocation (APC) transporter superfamily. A homology model of CgCYN1 was generated by using crystal structures of three known bacterial APC transporters followed by further refinement using molecular dynamics simulations. This revealed a possible translocation channel lined by TMD1, TMD3, TMD6, TMD8 and TMD10 helices. In silico docking studies with cystine along with comparison with other known cystine permeases and closely related lysine permeases allowed prediction of amino acid residues specifically involved in cystine binding. To validate this model a total of 19 predicted residues were subjected to site directed mutagenesis and functionally evaluated by growth on cystine and the analogues cystathionine and seleno-dl-cystine. Biochemical evaluation by radioactive uptake assays confirmed that these mutants showed reduced cystine uptake. Detailed kinetic analysis studies for the transport defective mutants revealed the involvement of residue G255 from the conserved FAYGGTE motif of TMD 6, and T339, S340 and H347 (all from TMD 8) in cystine binding. The implications of these findings on the homologous mammalian cystine transporter, XcT are also discussed.

Addressing Chirality in the Structure and Synthesis of [18 F]5-Fluoroaminosuberic Acid ([18 F]FASu).

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 [18 F]-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, [18 F]5-fluoroaminosuberic acid ([18 F]FASu), 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 18 FASu with fully known configuration. Our study serves as an example of a systematic method to identify and characterize amino acid tracers with chiral centers.

A simple route to [11C]N-Me labeling of aminosuberic acid for proof of feasibility imaging of the x(C)⁻ transporter.

Oxidative stress has been implicated in a variety of conditions, including cancer, heart failure, diabetes, neurodegeneration and other diseases. A potential biomarker for oxidative stress is the cystine/glutamate transporter, system x(C)(-). L-Aminosuberic acid (L-ASu) has been identified as a system x(C)(-) substrate. Here we report a facile method for [(11)C]N-Me labeling of L-ASu, automation of the radiochemical process, and preliminary PET imaging with EL4 tumor bearing mice. The results demonstrate uptake in the tumor above background, warranting further studies on the use of radiolabeled analogs of L-ASu as a PET imaging agent for system x(C)(-).

Serum lncRNAs TUG1, H19, and NEAT1 and their target miR-29b/SLC3A1 axis as possible biomarkers of preeclampsia: Potential clinical insights.

To date, the epigenetic signature of preeclampsia (PE) is not completely deciphered. Oxidative stress-responsive long non-coding RNAs (lncRNAs) are deregulated in preeclamptic placenta; however, their circulating profiles and diagnostic abilities are still unexplored. We investigated serum redox-sensitive lncRNAs TUG1, H19, and NEAT1, and their target miR-29b/cystine/neutral/dibasic amino acids transporter solute carrier family 3, member 1 (SLC3A1) as potential non-invasive biomarkers of PE risk, onset, and severity. We recruited 82 patients with PE and 78 healthy pregnant women. We classified PE patients into early-onset (EOPE) and late-onset (LOPE) subgroups at a cut-off 34 gestational weeks and into severe and mild PE subgroups by blood pressure and proteinuria criteria. Bioinformatics analysis was employed to select lncRNAs/microRNA/target gene interactions. Serum H19, NEAT1, and SLC3A1 mRNA expression were reduced, meanwhile miR-29b levels were elevated, whereas there was no significant difference in TUG1 levels between PE patients and healthy pregnancies. Serum H19 levels were lower, whereas miR-29b levels were higher in EOPE versus LOPE. Serum miR-29b and H19 levels were higher in severe versus mild PE. ROC analysis identified serum H19, NEAT1, miR-29b, and SLC3A1 as potential diagnostic markers, with H19 (AUC = 0.818, 95%CI = 0.744-0.894) and miR-29b (AUC = 0.82, 95%CI = 0.755-0.885) were superior discriminators. Only H19 and miR-29b discriminated EOPE and severe PE cases. In multivariate logistic analysis, miR-29b and H19 were associated with EOPE, using maternal age and gestational age as covariates, while miR-29b was associated with severe PE, using maternal age as covariate. Studied markers were correlated with clinical and ultrasound data in the overall PE group. Serum H19 and TUG1 were negatively correlated with albuminuria in EOPE and LOPE, respectively. NEAT1 and SLC3A1 were correlated with ultrasound data in EOPE. Likewise, TUG1, miR-29b, and SLC3A1 showed significant correlations with ultrasound data in LOPE. Conclusively, this study configures SLC3A1 expression as a novel potential serum biomarker of PE and advocates serum H19 and miR-29b as biomarkers of EOPE and miR-29b as a biomarker of PE severity.

Blocking xCT and PI3K/Akt pathway synergized with DNA damage of Riluzole-Pt(IV) prodrugs for cancer treatment.

Cancer treatment requires the participation of multiple targets/pathways, and single approach is hard to effectively curb the proliferation and metastasis of carcinoma cells. In this work, we conjugated FDA-approved riluzole and platinum(II) drugs into a series of unreported riluzole-Pt(IV) compounds, which were designed to simultaneously target DNA, the solute carrier family 7 member 11 (SLC7A11, xCT), and the human ether a go-go related gene 1 (hERG1), to exert synergistic anticancer effect. Among them, c,c,t-[PtCl2(NH3)2(OH)(glutarylriluzole)] (compound 2) displayed excellent antiproliferative activity with IC50 value of 300-times lower than that of cisplatin in HCT-116, and optimal selectivity index between carcinoma and human normal liver cells (LO2). Mechanism studies indicated that compound 2 released riluzole and active Pt(II) species after entering cells to exhibit a prodrug behavior against cancer, which obviously increased DNA-damage and cell apoptosis, as well as suppressed metastasis in HCT-116. Compound 2 persisted in the xCT-target of riluzole and blocked the biosynthesis of glutathione (GSH) to trigger oxidative stress, which could boost the killing to cancer cells and reduce Pt-drug resistance. Meanwhile, compound 2 significantly inhibited invasion and metastasis of HCT-116 cells by targeting hERG1 to interrupt the phosphorylation of phosphatidylinositide 3-kinases/proteinserine-threonine kinase (PI3K/Akt), and reverse epithelial-mesenchymal transformation (EMT). Based on our results, the riluzole-Pt(IV) prodrugs studied in this work could be regarded as a new class of very promising candidates for cancer treatment compared to traditional platinum drugs.

Simple Fluorescence Assay for Cystine Uptake via the xCT in Cells Using Selenocystine and a Fluorescent Probe.

The cystine/glutamate antiporter (xCT) is a crucial transporter that maintains cellular redox balance by regulating intracellular glutathione synthesis via cystine uptake. However, no robust and simple method to determine the cystine uptake activity of xCT is currently available. We have developed a method to measure the xCT activity via the reaction of selenocysteine and fluorescein O,O'-diacrylate (FOdA). Selenocystine, a cystine analogue, is transported into cells through xCT on the cell membrane. The amount of the transported selenocystine was then determined by a reaction using tris(2-carboxyethyl)phosphine (TCEP) and FOdA in a weak acidic buffer at pH 6. Using this method, the cystine uptake activity of xCT in various cells and the inhibitory efficiency of xCT inhibitors, were evaluated.

Cystine transporter expression is a marker to identify a subpopulation of canine adipose-derived stem cells.

Adipose-derived stem cells (ADSCs) are promising cell sources for regenerative medicine due to the simplicity of their harvest and culture; however, their biological properties are not completely understood. Moreover, recent murine and human studies identified several functional subpopulations of ADSCs varying in differentiation potential; however, there is a lack of research on canine ADSCs. Cystine transporter (xCT) is a stem cell marker in gastric and colon cancers that interacts with CD44 to enhance cystine uptake from the cell surface and subsequently accelerates intercellular glutathione levels. In this study, we identified a ~5% functional subpopulation of canine ADSCs with xCT+ expression (xCTHi). Compared with those of the xCT- subpopulation (xCTLo), the xCTHi subpopulation showed a significantly higher proliferation rate, higher expression of conventional stem cell markers (SOX2, KLF4, and c-Myc), and higher expression of adipogenic markers (FABP4 and PPARγ). By contrast, the xCTLo subpopulation showed significantly higher expression of osteogenic markers (BMP1 and SPP) than xCTHi cells. These results suggest xCT as a candidate marker for detecting a functional subpopulation of canine ADSCs. Mechanistically, xCT could increase the adipogenic potential while decreasing the osteogenic differentiation potential, which could serve as a valuable target marker in regenerative veterinary medicine.

The Effect of Chirality on the Application of 5-[18F]Fluoro-Aminosuberic Acid ([18F]FASu) for Oxidative Stress Imaging.

PURPOSE: The cystine transporter, system xC-, plays a crucial role in sustaining redox homeostasis and is reported to be overexpressed in several cancer subtypes. 5-[18F]Fluoroaminosuberic acid ([18F]FASu) is a novel positron emission tomography (PET) tracer, which exhibits specific uptake via system xC-. [18F]FASu synthesis by the commonly used Kryptofix 2.2.2/K2CO3-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 2S-[18F]FASu from chiral precursors. Our preliminary results indicated preferential uptake of the 2S-isomer by tumor cells compared to 2R-[18F]FASu. Few studies have investigated the biodistribution of chiral 18F-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 (2S,5R/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 (2S,5R/S-) displaying similar image contrast as compared to the 5- position diastereomers and the 2S,5S-[18F]FASu 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,5R/S-, 2S,5S-, and 2S,5R- 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 2S-[18F]FASu can be used to noninvasively image system xC- in a variety of cancers, either as the racemic mixture (2S,5R/S-) or optically pure form. Furthermore, this work shows potential utility of [18F]FASu for detection of glioblastoma and prostate cancer.

In silico characterization of residues essential for substrate binding of human cystine transporter, xCT.

xCT is a sodium-independent amino acid antiporter that imports L-cystine and exports L-glutamate in a 1:1 ratio. It is a component of heterodimeric amino acid transporter system Xc- working at the cross-roads of maintaining neurological processes and regulating antioxidant defense. The transporter has 12 transmembrane domains with intracellular N- and C-termini, and like other transporter proteins can undergo various conformational changes while switching the ligand accessibilities from intracellular to extracellular site. In the present study, we generated two homology models of human xCT in two distinct conformations: inward-facing occluded state and outward-facing open state. Our results indicated the substrate translocation channel composed of transmembrane helices TMs 1, 3, 6, 8, and 10. We docked anionic L-cystine and L-glutamate within the cavities to assess the two distinct binding scenarios for xCT as antiporter. We also assessed the interactions between the ligands and transporter and observed that ligands bind to similar residues within the channel. Using MM-PBSA/MM-GBSA approach, we computed the binding energies of these ligands to different conformational states. Cystine and glutamate bind xCT with favorable binding energies, with more favorable binding observed in inward occluded state than in outward open state. We further computed the residue-wise decomposition of these binding energies and identified the residues as essential for substrate binding/permeation. Filtering the residues that form favorable energetic contributions to the ligand binding in both the states, our studies suggest T56, A60, R135, A138, V141, Y244, A247, F250, S330, L392, and R396 as critical residues for ligand binding as well as ligand transport for any conformational state adopted by xCT during its transport cycle. .Graphical Abstract.

Non-invasive Use of Positron Emission Tomography to Monitor Diethyl maleate and Radiation-Induced Changes in System xC- Activity in Breast Cancer.

PURPOSE: The system xC- transporter is upregulated in cancer cells in response to oxidative stress (OS). 5-[18F]fluoroaminosuberic acid ([18F]FASu) has been reported as a novel positron emission tomography (PET) imaging agent, targeting system xC-. The goal of this study was to evaluate the utility of [18F]FASu in monitoring cellular response to diethyl maleate (DEM) and radiation-induced OS fluctuations. PROCEDURES: [18F]FASu uptake by breast cancer cells was studied in correlation to OS biomarkers: glutathione (GSH) and reactive oxygen species (ROS), as well as transcriptional and translational levels of xCT (the functional subunit of xC-). System xC- inhibitor, sulfasalazine (SSZ), and small interfering RNA (siRNA) knockdown were used as negative controls. Radiotracer uptake was evaluated in three breast cancer models: MDA-MB-231, MCF-7, and ZR-75-1, at two-time points (1 h and 16 h) following OS induction. In vivo [18F]FASu imaging and biodistribution were performed using MDA-MB-231 xenograft-bearing mice at 16 and 24 h post-radiation treatment. RESULTS: [18F]FASu uptake was positively correlated to intracellular GSH and SLC7A11 expression levels, and radiotracer uptake was induced both by radiation treatment and by DEM at time points longer than 3 h. In an in vivo setting, there was no statistically significant uptake difference between irradiated and control tumors. CONCLUSION: [18F]FASu is a specific system xC- PET radiotracer and as such it can be used to monitor system xC- activity due to OS. As such, [18F]FASu has the potential to be used in therapy response monitoring by PET. Further optimization is required for in vivo application.

Disrupting the 'Warburg effect' re-routes cancer cells to OXPHOS offering a vulnerability point via 'ferroptosis'-induced cell death.

The evolution of life from extreme hypoxic environments to an oxygen-rich atmosphere has progressively selected for successful metabolic, enzymatic and bioenergetic networks through which a myriad of organisms survive the most extreme environmental conditions. From the two lethal environments anoxia/high O2, cells have developed survival strategies through expression of the transcriptional factors ATF4, HIF1 and NRF2. Cancer cells largely exploit these factors to thrive and resist therapies. In this review, we report and discuss the potential therapeutic benefit of disrupting the major Myc/Hypoxia-induced metabolic pathway, also known as fermentative glycolysis or "Warburg effect", in aggressive cancer cell lines. With three examples of genetic disruption of this pathway: glucose-6-phosphate isomerase (GPI), lactate dehydrogenases (LDHA and B) and lactic acid transporters (MCT1, MCT4), we illuminate how cancer cells exploit metabolic plasticity to survive the metabolic and energetic blockade or arrest their growth. In this context of NRF2 contribution to OXPHOS re-activation we will show and discuss how, by disruption of the cystine transporter xCT (SLC7A11), we can exploit the acute lethal phospholipid peroxidation pathway to induce cancer cell death by 'ferroptosis'.

Lipocalin 2 attenuates iron-related oxidative stress and prolongs the survival of ovarian clear cell carcinoma cells by up-regulating the CD44 variant.

Ovarian clear cell carcinoma (CCC) arises from ovarian endometriosis. Intra-cystic fluid contains abundant amounts of free iron, which causes persistent oxidative stress, a factor that has been suggested to induce malignant transformation. However, the mechanisms linking oxidative stress and carcinogenesis in CCC currently remain unclear. Lipocalin 2 (LCN2), a multifunctional secretory protein, functions as an iron transporter as well as an antioxidant. Therefore, we herein examined the roles of LCN2 in the regulation of intracellular iron concentrations, oxidative stress, DNA damage, and antioxidative functions using LCN2-overexpressing (ES2), and LCN2-silenced (RMG-1) CCC cell lines. The results of calcein staining indicated that the up-regulated expression of LCN2 correlated with increases in intracellular iron concentrations. However, a DCFH-DA assay and 8OHdG staining revealed that LCN2 reduced intracellular levels of reactive oxygen species and DNA damage. Furthermore, the expression of LCN2 suppressed hydrogen peroxide-induced apoptosis and prolonged cell survival, suggesting an antioxidative role for LCN2. The expression of mRNAs and proteins for various oxidative stress-catalyzing enzymes, such as heme oxygenase (HO), superoxide dismutase (SOD), and glutathione peroxidase, was not affected by LCN2, whereas the intracellular concentration of the potent antioxidant, glutathione (GSH), was increased by LCN2. Furthermore, the expression of xCT, a cystine transporter protein, and CD44 variant 8-10 (CD44v), a stem cell marker, was up-regulated by LCN2. Although LCN2 increased intracellular iron concentrations, LCN2-induced GSH may catalyze and override oxidative stress via CD44v and xCT, and subsequently enhance the survival of CCC cells in oxidative stress-rich endometriosis.

Potential of sulfasalazine as a therapeutic sensitizer for CD44 splice variant 9-positive urogenital cancer.

Cancer stem-like cells (CSCs) with high expression of CD44 splice variant (CD44v) have an enhanced capacity for intracellular reduced glutathione synthesis and defense against reactive oxygen species, resulting in resistance to various therapeutic stresses. Sulfasalazine (SSZ), a drug used in the treatment of rheumatoid arthritis (RA), inhibits glutamate-cystine transport, and suppressed CD44v-dependent tumor growth and increased sensitivity to cytotoxic drugs in an in vivo study. Here, we present two cases of CD44v9-positive urogenital cancer with concomitant treatment with SSZ for RA. Patient 1 was a 62-year-old man who had received SSZ for RA beginning 2 months before the diagnosis of urinary bladder cancer. Although he had multiple metastases to the bladder, abdominal, left cervical and left axillary lymph nodes, and brain, complete response with multidisciplinary therapy was maintained for more than 2 years. Patient 2 was a 74-year-old man with castration-resistant prostate cancer who was diagnosed with RA during chemotherapy and a gradual increase in prostate-specific antigen (PSA) level. When SSZ was added, his PSA value (ng/mL) decreased from 12.93 to 5.58 in only 2 weeks and then quickly rebounded, whereas levels of neuron-specific enolase, a neuroendocrine differentiator and CSC marker, remained almost unchanged. We therefore speculate that SSZ treatment may represent a new adjuvant treatment option for patients with CD44v9-positive urogenital cancer.

Redox status in mammalian cells and stem cells during culture in vitro: critical roles of Nrf2 and cystine transporter activity in the maintenance of redox balance.

Culturing cells and tissues in vitro has provided valuable insights into the molecular mechanisms regulating redox signaling in cells with implications for medicine. However, standard culture techniques maintain mammalian cells in vitro under an artificial physicochemical environment such as ambient air and 5% CO2. Oxidative stress is caused by the rapid oxidation of cysteine to cystine in culture media catalyzed by transition metals, leading to diminished intracellular cysteine and glutathione (GSH) pools. Some cells, such as fibroblasts and macrophages, express cystine transport activity, designated as system [Formula: see text], which enables cells to maintain these pools to counteract oxidative stress. Additionally, many cells have the ability to activate the redox sensitive transcription factor Nrf2, a master regulator of cellular defenses against oxidative stress, and to upregulate xCT, the subunit of the [Formula: see text] transport system leading to increases in cellular GSH. In contrast, some cells, including lymphoid cells, embryonic stem cells and iPS cells, express relatively low levels of xCT and cannot maintain cellular cysteine and GSH pools. Thus, fibroblasts have been used as feeder cells for the latter cell types based on their ability to supply cysteine. Other key Nrf2 regulated gene products include heme oxygenase 1, peroxiredoxin 1 and sequestosome1. In macrophages, oxidized LDL activates Nrf2 and upregulates the scavenger receptor CD36 forming a positive feedback loop to facilitate removal of the oxidant from the vascular microenvironment. This review describes cell type specific responses to oxygen derived stress, and the key roles that activation of Nrf2 and membrane transport of cystine and cysteine play in the maintenance and proliferation of mammalian cells in culture.

Potential use of the anti-inflammatory drug, sulfasalazine, for targeted therapy of pancreatic cancer.

Pancreatic cancer is an aggressive, drug-resistant disease; its first-line chemotherapeutic, gemcitabine, is only marginally effective. Intracellular depletion of glutathione, a major free-radical scavenger, has been associated with growth arrest and reduced drug resistance (chemosensitization) of cancer cells. In search of a new therapeutic approach for pancreatic cancer, we sought to determine whether specific inhibition of the plasma membrane x(c) (-) cystine transporter could lead to reduced uptake of cysteine, a key precursor of glutathione, and subsequent glutathione depletion. Sulfasalazine (approximately 0.2 mmol/L), an anti-inflammatory drug with potent x(c) (-)-inhibitory properties, markedly reduced l¹4C]-cystine uptake, glutathione levels, and growth and viability of human MIA PaCa-2 and PANC-1 pancreatic cancer cells in vitro. These effects were shown to result primarily from inhibition of cystine uptake mediated by the x(c) (-) cystine transporter and not from inhibition of nuclear factor kappaB activation, another property of sulfasalazine. The efficacy of gemcitabine could be markedly enhanced by combination therapy with sulfasalazine both in vitro and in immunodeficient mice carrying xenografts of the same cell lines. No major side effects were observed in vivo.The results of the present study suggest that the x(c) (-) transporter plays a major role in pancreatic cancer by sustaining or enhancing glutathione biosynthesis, and as such, represents a potential therapeutic target. Sulfasalazine, a relatively nontoxic drug approved by the U.S. Food and Drug Administration, may, in combination with gemcitabine, lead to more effective therapy of refractory pancreatic cancer.