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.

Effects of Dietary Cystine and Tyrosine Supplementation on Melanin Synthesis in the Pacific Oyster (Crassostrea gigas).

Melanogenesis is a multistep process to produce melanin for dark pigmentation in skin coloration. Previous studies in vertebrates demonstrated that cystine and tyrosine amino acids are involved in the melanin synthesis. However, very little is known about the melanogenesis in bivalve. In this study, cystine supplementation for 30 days significantly upregulated the expression of CgB-aat1, CgCbs and CgTyr and pheomelanin content in the Pacific oyster Crassostrea gigas. Transmission electron microscope (TEM) results revealed more melanosomes in the connective tissue and melanin granules were secreted in epithelium of mantle. In contrast, tyrosine supplementation had no clear effect on melanogenesis except the gene expression changes of CgB-aat1 and CgCbs. In addition, prolonged supplementation of cystine or tyrosine for 60 days had a negative impact on melanogenesis. Indeed, after 60 days, expression of most of the melanin synthesis-related genes under study was decreased, and melanin content was significantly reduced, indicating that cystine and tyrosine might inhibit production of eumelanin and pheomelanin, respectively. In addition, in vitro analysis using primary cell culture from mantle tissue indicated that incubation with cystine, tyrosine, or B-AAT1 polypeptide, CBS/TYR recombinant proteins induced the increase of CgB-aat1 and CgCbs expression in a dose-dependent manner, suggesting the presence of a regulatory network in response to cystine and tyrosine amino acids intakes in pheomelanin synthesis-related gene expression. Taken together, these data indicate that cystine-CgB-aat1-CgCbs-CgTyr axis is a potential regulator of the pheomelanin biosynthesis pathway, and thus plays an important role in the mantle pigmentation in C. gigas. This work provides a new clue for selective cultivation of oyster strains with specific shell colors in bivalve breeding.

Contribution of NRF2 to sulfur metabolism and mitochondrial activity.

NF-E2-related factor 2 (NRF2) plays a crucial role in the maintenance of cellular homeostasis by regulating various enzymes and proteins that are involved in the redox reactions utilizing sulfur. While substantial impacts of NRF2 on mitochondrial activity have been described, the precise mechanism by which NRF2 regulates mitochondrial function is still not fully understood. Here, we demonstrated that NRF2 increased intracellular persulfides by upregulating the cystine transporter xCT encoded by Slc7a11, a well-known NRF2 target gene. Persulfides have been shown to play an important role in mitochondrial function. Supplementation with glutathione trisulfide (GSSSG), which is a form of persulfide, elevated the mitochondrial membrane potential (MMP), increased the oxygen consumption rate (OCR) and promoted ATP production. Persulfide-mediated mitochondrial activation was shown to require the mitochondrial sulfur oxidation pathway, especially sulfide quinone oxidoreductase (SQOR). Consistently, NRF2-mediated mitochondrial activation was also dependent on SQOR activity. This study clarified that the facilitation of persulfide production and sulfur metabolism in mitochondria by increasing cysteine availability is one of the mechanisms for NRF2-dependent mitochondrial activation.

Glutamine, proline, and isoleucine support maturation and fertilisation of bovine oocytes.

Successful in-vitro production of bovine embryos relies on meiotic maturation of oocytes in vitro (IVM) before they can be fertilised. High levels of IVM are currently achieved using a complex medium that contains all 20 common amino acids, namely TCM199, but can also be achieved using a simple inorganic salt solution containing non-essential amino acids, proline, and glutamine. Further simplification of the amino acid content of medium used for IVM could lead to a more defined medium that provides reproducible IVM. The aim of this study was, therefore, to determine the minimal amino acid requirements for bovine oocyte nuclear maturation, as measured by progression to metaphase II (MII) of meiosis. Supplementation of a simple medium composed of inorganic salts (M1 medium) with multiple amino-acid combinations showed that M1 containing glutamine, proline, and isoleucine resulted in nuclear maturation comparable to that of TCM199 (57.4 ± 3.4% vs 67% ± 1.7%, respectively) but was reduced when cystine (Cys2) to that seen with M1 alone (38.0 ± 2.2%). Viability of oocytes matured in this simplified medium was equal to those matured in TCM199 since the same proportion of zygotes with 2 pronuclei were observed following fertilisation in medium containing no amino acids (33.9 ± 6.5% vs 33.3 ± 3.6%, respectively). Addition of glutamine, proline and isoleucine to fertilisation medium also increased the proportion of zygotes but did not increase blastocyst development rates. Thus, a defined medium containing only glutamine, proline and isoleucine is sufficient for oocyte maturation and successful fertilisation.

A novel treatment strategy to prevent Parkinson's disease: focus on iron regulatory protein 1 (IRP1).

We propose that neural damage in Parkinson's disease (PD) is due to dysregulation of iron utilization rather than to high iron levels per se. Iron deposits are associated with neuronal cell death in substantia nigra (SN) resulting in PD where high levels of iron in SNs are due to dysregulation of iron utilization. Cytosolic aconitase (ACO1) upon losing an iron-sulfur cluster becomes iron regulatory protein 1 (IRP1). Rotenone increases levels of IRP1 and induces PD in rats. An increase in iron leads to inactivation of IRP1. We propose a novel treatment strategy to prevent PD. Specifically in rats given rotenone by subcutaneous injections, iron, from iron carbonyl from which iron is slowly absorbed, given three times a day by gavage will keep iron levels constant in the gut whereby iron levels and iron utilization systematically can be tightly regulated. Rotenone adversely affects complex 1 iron-sulfur proteins. Iron supplementation will increase iron-sulfur cluster formation switching IRP1 to ACO1. With IRP1 levels kept constantly low, iron utilization will systematically be tightly regulated stopping dysregulation of complex 1 and the neural damage done by rotenone preventing PD.

Nutritional and Pharmaceutical Applications of Under-Explored Knottin Peptide-Rich Phytomedicines.

Phytomedicines reportedly rich in cystine knot peptides (Knottins) are found in several global diets, food/herbal supplements and functional foods. However, their knottin peptide content has largely been unexplored, notably for their emerging dual potentials at both the food and medicine space. The nutritional roles, biological targets and mechanism(s) of activity of these knotted peptides are largely unknown. Meanwhile, knottins have recently been unveiled as emerging peptide therapeutics and nutraceuticals of primary choice due to their broad spectrum of bioactivity, hyper stability, selective toxicity, impressive selectivity for biomolecular targets, and their bioengineering applications. In addition to their potential dietary benefits, some knottins have displayed desirable limited toxicity to human erythrocytes. In an effort to appraise what has been accomplished, unveil knowledge gaps and explore the future prospects of knottins, an elaborate review of the nutritional and pharmaceutical application of phytomedicines rich in knottins was carried out. Herein, we provide comprehensive data on common dietary and therapeutic knottins, the majority of which are poorly investigated in many food-grade phytomedicines used in different cultures and localities. Findings from this review should stimulate scientific interest to unveil novel dietary knottins and knottin-rich nutraceutical peptide drug candidates/leads with potential for future clinical application.

Cystine/Glutamine Mixture Supplementation Attenuated Fatigue during Endurance Exercise in Healthy Young Men by Enhancing Fatty Acid Utilization.

Exercise-induced fatigue is a multi-origin physical and mental phenomenon. Efforts to diminish the above predisposition may contribute to endurance, along with athletic well-being, while development of nutritional strategies to optimize condition and exercise performance are essential issues for athletes and trainers. Dietary amino acids are being discussed for their specific health-promoting properties beyond their role as building blocks of proteins. Glutamine, along with cysteine, are two kinds of amino acids that are reported extensively for their anti-oxidation, anti-inflammation, and immune-regulation properties, and are promising in sport applications. In the present study, we designed a randomized, placebo-controlled, crossover trial to examine effects of 7-day supplementation of cystine/glutamine mixture (Cys2/Gln) on self-reporting fatigue index (ratings of perceived exertion, RPE), energy metabolism, and inflammation. We also employed a C2C12 myotube model to examine the capacity of cystine for fatty acid utilization. Cys2/Gln supplementation alleviated fatigue by decreasing RPE and enhanced fatty acid oxidation during a 60 min endurance exercise in human trials, while cystine increased fatty acid utilization in C2C12 myotubes by enhancing mitochondrial respiration. In summary, Cys2/Gln supplementation exerts positive effects on ameliorating exercise-induced fatigue, mechanisms of which can be attributed to enhancement of fatty acid utilization.

d-Cysteine supplementation partially protects against ferroptosis induced by xCT dysfunction via increasing the availability of glutathione.

Glutathione (GSH) is synthesized from three amino acids and the overall process is highly dependent on the availability of l-cysteine (l-Cys). GSH serves as an essential cofactor for glutathione peroxidase 4 (Gpx4), which reduces phospholipid hydroperoxides. The inactivation of Gpx4 or an insufficient supply of l-Cys results in the accumulation of lipid hydroperoxides, eventually leading to iron-dependent cell death, ferroptosis. In this study, we investigated the anti-ferroptotic properties of d-cysteine (d-Cys) under conditions of dysfunction in cystine transporter, xCT. l-Cys supplementation completely rescued ferroptosis that had been induced by the erastin-mediated inhibition of xCT in Hepa 1-6 cells. Upon d-Cys supplementation, the erastin-treated cells remained completely viable for periods of up to 24 h but eventually died after 48 h. d-Cys supplementation suppressed the production of lipid peroxides, thereby ferroptosis. The addition of d-Cys sustained intracellular Cys and GSH levels to a certain extent. When Hepa 1-6 cells were treated with a combination of buthionine sulfoximine and erastin, the anti-ferroptotic effect of d-Cys was diminished. These collective results indicate that, although d-Cys is not the direct source of GSH, d-Cys supplementation protects cells from ferroptosis in a manner that is dependent on GSH synthesis via stimulating the uptake of l-Cys.

Cystine reduces mitochondrial dysfunction in C2C12 myotubes under moderate oxidative stress induced by H2O2.

Moderate oxidative stress induces temporal impairment in mitochondrial ATP production. As glutathione (GSH) content is reduced to eliminate oxidative stress by oxidation-reduction reaction, intracellular GSH content is crucial for maintaining mitochondrial function under oxidative stress. GSH precursors such as N-acetyl cysteine (NAC) and cysteine are known to suppress oxidative stress based on the supply of cysteine residues being rate-limiting for GSH synthesis. However, it remains unclear whether cystine (Cys2) can suppress mitochondrial dysfunction under oxidative stress conditions. Therefore, we examined whether Cys2 could attenuate mitochondrial dysfunction under moderate oxidative stress without scavenging reactive oxygen species (ROS) in the medium. C2C12 myotubes were incubated for 120 min in a Cys2-supplemented medium and subsequently exposed to hydrogen peroxide (H2O2). Heme oxygenase-1 (HO-1) gene expression, intracellular cysteine and GSH content, intracellular ATP level, and maximal mitochondrial respiration were assessed. Cys2 treatment significantly increased GSH content in a dose-dependent manner under oxidative stress. Cys2 treatment significantly decreased HO-1 expression induced by H2O2 exposure. In addition, maximal mitochondrial respiration rate was decreased by H2O2 exposure, but improved by Cys2 treatment. In conclusion, Cys2 treatment mitigates oxidative stress-induced mitochondrial dysfunction by maintaining GSH content under moderate oxidative stress without scavenging ROS in the medium.

A quantitative metabolomics assay targeting 14 intracellular metabolites associated with the methionine transsulfuration pathway using LC-MS/MS in breast cancer cells.

The methionine transsulfuration pathway plays an important role in some fundamental biological processes, such as redox and methylation reactions. However, quantitative analysis of the majority of intracellular metabolites is rather challenging. In this study, we developed a simple, fast and reliable method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous detection of 14 methionine-related metabolites, including methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), homocysteine (HCY), cystathionine (Cysta), cysteine (CYS), glutathione (GSH), dimethylglycine (DMG), betaine, serine, folic acid (FA), dihydrofolic acid (DHF), tetrahydrofolic acid (THF) and 5-methyltetrahydrofolic acid (5-MTHF), in MCF-7 and MDA-MB-231 breast cancer cells. By taking advantage of a surrogate matrix, the linearity, sensitivity, precision, accuracy, stability, matrix effect, recovery, dilution integrity and carryover of the established method were evaluated and validated. This method enabled the precise measurement of methionine-related metabolites both in cells and in the medium and was successfully applied to profile these metabolites involved in the methionine transsulfuration pathway. The data showed that cystine deprivation or excessive supplementation with cystine had a marked impact on methionine metabolism, in addition to its effects on intracellular CYS and GSH levels, indicating that the methionine transsulfuration pathway was dependent on intracellular cystine levels. The established method provides a reliable way to target metabolomics for the quantitative determination of intracellular metabolites in the methionine transsulfuration pathway, which can greatly facilitate the understanding of the mechanisms involved in methylation and redox homeostasis in cellular metabolomic studies.

Modulation of Human Hydrogen Sulfide Metabolism by Micronutrients, Preliminary Data.

BACKGROUND: Hydrogen sulfide (H2S) is a pivotal gasotransmitter networking with nitric oxide (NO) and carbon monoxide (CO) to regulate basic homeostatic functions. It is released by the alternative pathways of transulfuration by the enzymes Cystathionine Beta Synthase (CBS) and Cystathionine Gamma Lyase (CSE), and by Cysteine AminoTransferase (CAT)/ 3-Mercaptopyruvate Sulfur Transferase (3MPST). A non-enzymatic, intravascular release is also in place. We retrospectively investigated the possibility to modulate the endogenous H2S release and signaling in humans by a dietary manipulation with supplemented micronutrients (L-cystine, Taurine and pyridoxal 5-phopsphate/P5P). METHODS: Patients referring for antiaging purposes underwent a 10-day supplementation. Blood was collected at baseline and after treatment and the metabolome was investigated by mass spectrometry to monitor the changes in the metabolites reporting on H2S metabolism and related pathways. RESULTS: Data were available from 6 middle aged subjects (2 women). Micronutrients increased 3-mercaptopyruvate (P = .03), reporting on the activity of CAT that provides the substrate for H2S release within mitochondria by 3MPST, decreased lanthionine (P = .024), reporting the release of H2S from CBS, and had no significant effect of H2S release from CSE. This is compatible with a homeostatic balancing. We also recorded a strong increase of reporters of H2S-induced pathways including 5-MethylTHF (P = .001) and SAME (P = .022), reporting on methylation capacity, and of BH4 (P = .021) and BH2 (P = .028) reporting on nitric oxide metabolism. These activations may be explained by the concomitant induction of non-enzymatic release of H2S. CONCLUSIONS: Although the current evidences are weak and will need to be confirmed, the effect of micronutrients was compatible with an increase of the H2S endogenous release and signaling within the control of homeostatic mechanisms, further endorsing the role of feeding in health and disease. These effects might result in a H2S boosting effect in case of defective activity of pathologic origin, which should be checked in duly designed clinical trials.

The effects of the oral supplementation of L-Cystine associated with reduced L-Glutathione-GSH on human skin pigmentation: a randomized, double-blinded, benchmark- and placebo-controlled clinical trial.

BACKGROUND: Glutathione has become a potential skin-lightening ingredient after the discovery of its anti-melanogenic properties. Various mechanisms of action have been considered to explain this property, one of them being the skewing of the melanin synthesis pathway toward the production of lighter pheomelanin instead of darker eumelanin, consequently producing a lightening effect. AIMS: To evaluate the skin lightening and anti-dark spot effects of oral supplementation with L-Cystine associated with L-Glutathione as compared to placebo and benchmark. METHODS: Effects of this L-Cystine-L-Glutathione oral combination were investigated in a 12-week randomized, double-blind, parallel-group, benchmark- and placebo-controlled trial involving 124 Asian female subjects. Women were randomly allocated into 4 equal groups (500 mg L-Cystine and 250 mg L-Glutathione, 250 mg reduced L-Glutathione, 500 mg L-Cystine, or a placebo, daily). Skin color was measured at baseline, after 6 and 12 weeks by spectrophotometry. Size and color of facial dark spots were determined from digital photographs. RESULTS: A significant skin lightening was observed after 12 weeks of oral supplementation with L-Cystine associated with L-Glutathione. This combination also induced a significant reduction in the size of facial dark spots after 6 and 12 weeks. It is noteworthy that the observed effects were not only significantly better than those obtained with placebo, but also with L-Cystine alone or L-Glutathione alone. CONCLUSION: The daily oral administration of 500 mg L-Cystine and 250 mg L-Glutathione during 12 weeks was a safe treatment to effectively lighten the skin and reduce the size of facial dark spots of Asian women.

A mix of functional amino acids and grape polyphenols promotes the growth of piglets, modulates the gut microbiota in vivo and regulates epithelial homeostasis in intestinal organoids.

Weaning is a challenging period for gut health in piglets. Previous studies showed that dietary supplementations with either amino acids or polyphenols promote piglet growth and intestinal functions, when administered separately. Thus, we hypothesized that a combination of amino acids and polyphenols could facilitate the weaning transition. Piglets received during the first two weeks after weaning a diet supplemented or not with a mix of a low dose (0.1%) of functional amino acids (L-arginine, L-leucine, L-valine, L-isoleucine, L-cystine) and 100 ppm of a polyphenol-rich extract from grape seeds and skins. The mix of amino acids and polyphenols improved growth and feed efficiency. These beneficial effects were associated with a lower microbiota diversity and a bloom of Lactobacillaceae in the jejunum content while the abundance of Proteobacteria was reduced in the caecum content. The mix of amino acids and polyphenols also increased the production by the caecum microbiota of short-chain fatty acids (butyrate, propionate) and of metabolites derived from amino acids (branched-chain fatty acids, valerate, putrescine) and from polyphenols (3-phenylpropionate). Experiments in piglet jejunum organoids revealed that the mix of amino acids and polyphenols upregulated the gene expression of epithelial differentiation markers while it reduced the gene expression of proliferation and innate immunity markers. In conclusion, the supplementation of a mix of amino acids and polyphenols is a promising nutritional strategy to manage gut health in piglets through the modulation of the gut microbiota and of the epithelial barrier.

Use of low dosage amino acid blends to prevent stress-related piglet diarrhea.

Weaning is a challenging period for piglets associated with reduced feed intake, impairment of gut integrity, and diarrhea. Previous studies demonstrate that supplementation with single functional amino acids (AA) promote piglets' performance due to the improvement of intestinal health. Thus, we hypothesized that a combination of functional AA provided beyond the postulated requirement for growth could facilitate the weaning transition. Ninety piglets, initially stressed after weaning by 100 min overland transport, received a control diet or the same diet supplemented with a low-dosed (0.3%) mixture of AA (AAB-1: L-arginine, L-leucine, L-valine, L-isoleucine, L-cystine; AAB-2: L-arginine, L-leucine, L-valine, L-isoleucine, L-cystine, and L-tryptophan) for 28 days. Fecal consistency was ranked daily, growth performance was assessed weekly. On days 1 and 14 of the trial, blood samples were collected from a subset of 10 piglets per group to assess concentrations of insulin-like growth factor 1. After 28 days of feeding, tissues were obtained from the same piglets to analyze gut morphology and relative mRNA expression of genes related to gut function. Even if the stress response as indicated by rectal temperature was not different between the groups, pigs supplemented with AAB-2 showed firmer feces after weaning and less days with diarrhea compared to control. Furthermore, the jejunal expression of the MUC-2 gene was reduced (P < 0.05) in group AAB-2. Both AA mixtures increased crypt depth in the duodenum. Collectively, the given results indicate that 0.3% extra AA supplementation might alleviate postweaning diarrhea but did not alter growth performance of weanling piglets.

Influence of Redox Imbalances on the Transposition of Insertion Sequences in Deinococcus geothermalis.

The transposition of insertion sequence elements was evaluated among different Deinococcus geothermalis lineages, including the wild-type, a cystine importer-disrupted mutant, a complemented strain, and a cystine importer-overexpressed strain. Cellular growth reached early exponential growth at OD600 2.0 and late exponential growth at OD600 4.0. Exposing the cells to hydrogen peroxide (80-100 mM) resulted in the transposition of insertion sequences (ISs) in genes associated with the carotenoid biosynthesis pathway. Particularly, ISDge7 (an IS5 family member) and ISDge5 (an IS701 family member) from the cystine importer-disrupted mutant were transposed into phytoene desaturase (dgeo_0524) via replicative transposition. Further, the cystine importer-overexpressed strain Δdgeo_1985R showed transposition of both ISDge2 and ISDge5 elements. In contrast, IS transposition was not detected in the complementary strain. Interestingly, a cystine importer-overexpressing strain exhibited streptomycin resistance, indicating that point mutation occurred in the rpsL (dgeo_1873) gene encoding ribosomal protein S12. qRT-PCR analyses were then conducted to evaluate the expression of oxidative stress response genes, IS elements, and low-molecular-weight thiol compounds such as mycothiol and bacillithiol. Nevertheless, the mechanisms that trigger IS transposition in redox imbalance conditions remain unclear. Here, we report that the active transposition of different IS elements was affected by intracellular redox imbalances caused by cystine importer deficiencies or overexpression.

Cyclotides Isolated From Violet Plants of Cameroon Are Inhibitors of Human Prolyl Oligopeptidase.

Traditional medicine and the use of herbal remedies are well established in the African health care system. For instance, Violaceae plants are used for antimicrobial or anti-inflammatory applications in folk medicine. This study describes the phytochemical analysis and bioactivity screening of four species of the violet tribe Allexis found in Cameroon. Allexis cauliflora, Allexis obanensis, Allexis batangae and Allexis zygomorpha were evaluated for the expression of circular peptides (cyclotides) by mass spectrometry. The unique cyclic cystine-rich motif was identified in several peptides of all four species. Knowing that members of this peptide family are protease inhibitors, the plant extracts were evaluated for the inhibition of human prolyl oligopeptidase (POP). Since all four species inhibited POP activity, a bioactivity-guided fractionation approach was performed to isolate peptide inhibitors. These novel cyclotides, alca 1 and alca 2 exhibited IC50 values of 8.5 and 4.4 µM, respectively. To obtain their amino acid sequence information, combinatorial enzymatic proteolysis was performed. The proteolytic fragments were evaluated in MS/MS fragmentation experiments and the full-length amino acid sequences were obtained by de novo annotation of fragment ions. In summary, this study identified inhibitors of the human protease POP, which is a drug target for inflammatory or neurodegenerative disorders.

The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae.

Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 µM Sulfasalazine (SS) and 0.5 mM CysS + 500 µM SS (CysS + SS). After 1 h of incubation at 37 °C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 °C under 5% CO2. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 × 105 sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean ± SEM) tended to increase in CysS group (44.0 ± 12.3) respect CTR (40.8 ± 10.8) while decreased in SS group (32.4 ± 7.8) (p < 0.01). Moreover, CysS + SS group showed a lower binding rate (32.0 ± 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation.

Cystine supplementation rebalances the redox homeostasis of microenvironment in non-small cell lung cancer cells and reverses their resistance to docetaxel.

Continuous docetaxel (DTX) treatment of non-small cell lung cancer induces development of drug resistance, but the mechanism is poorly understood. In this study we performed metabolomics analysis to characterize the metabolic patterns of sensitive and resistant A549 non-small cell lung cancer cells (A549/DTX cells). We showed that the sensitive and resistant A549 cells exhibited distinct metabolic phenotypes: the resistant cells were characterized by an altered microenvironment of redox homeostasis with reduced glutathione and elevated reactive oxygen species (ROS). DTX induction reprogrammed the metabolic phenotype of the sensitive cells, which acquired a phenotype similar to that of the resistant cells: it reduced cystine influx, inhibited glutathione biosynthesis, increased ROS and decreased glutathione/glutathione disulfide (GSH/GSSG); the genes involved in glutathione biosynthesis were dramatically depressed. Addition of the ROS-inducing agent Rosup (25, 50 µg/mL) significantly increased P-glycoprotein expression and reduced intracellular DTX in the sensitive A549 cells, which ultimately acquired a phenotype similar to that of the resistant cells. Supplementation of cystine (1.0 mM) significantly increased GSH synthesis, rebalanced the redox homeostasis of A549/DTX cells, and reversed DTX-induced upregulation of P-glycoprotein, and it markedly improved the effects of DTX and inhibited the growth of A549/DTX in vitro and in vivo. These results suggest that microenvironmental redox homeostasis plays a key role in the acquired resistance of A549 cancer cells to DTX. The enhancement of GSH synthesis by supplementary cystine is a promising strategy to reverse the resistance of tumor cells and has potential for translation in the clinic.

Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy

The cystine/glutamate antiporter SLC7A11 (also commonly known as xCT) functions to import cystine for glutathione biosynthesis and antioxidant defense and is overexpressed in multiple human cancers. Recent studies revealed that SLC7A11 overexpression promotes tumor growth partly through suppressing ferroptosis, a form of regulated cell death induced by excessive lipid peroxidation. However, cancer cells with high expression of SLC7A11 (SLC7A11

Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy.

The cystine/glutamate antiporter SLC7A11 (also commonly known as xCT) functions to import cystine for glutathione biosynthesis and antioxidant defense and is overexpressed in multiple human cancers. Recent studies revealed that SLC7A11 overexpression promotes tumor growth partly through suppressing ferroptosis, a form of regulated cell death induced by excessive lipid peroxidation. However, cancer cells with high expression of SLC7A11 (SLC7A11high) also have to endure the significant cost associated with SLC7A11-mediated metabolic reprogramming, leading to glucose- and glutamine-dependency in SLC7A11high cancer cells, which presents potential metabolic vulnerabilities for therapeutic targeting in SLC7A11high cancer. In this review, we summarize diverse regulatory mechanisms of SLC7A11 in cancer, discuss ferroptosis-dependent and -independent functions of SLC7A11 in promoting tumor development, explore the mechanistic basis of SLC7A11-induced nutrient dependency in cancer cells, and conceptualize therapeutic strategies to target SLC7A11 in cancer treatment. This review will provide the foundation for further understanding SLC7A11 in ferroptosis, nutrient dependency, and tumor biology and for developing novel effective cancer therapies.