Decrease In Glutathione Levels Research Articles

Neuroprotective effects of Paederia foetida Linn. on scopolamine-induced cognitive impairment in rats

Background and Aim: Alzheimer’s disease (AD) poses a significant health-care challenge, often linked to cognitive decline caused by oxidative stress. This study investigated the potential neuroprotective effects of the Paederia foetida leaf extract (PFE) in rats that exhibited scopolamine-induced dementia mimicking AD. Materials and Methods: Forty-two male rats were treated with either donepezil (0.5 mg/kg) or PFE at doses of 250, 500, and 1000 mg/kg for 14 days before and 14 days after the beginning of Alzheimer’s-like symptoms after 14 consecutive days of scopolamine administration. Behavioral tests, including the open-field test for locomotor activity and the Morris water maze task for learning and memory assessment, were conducted. Neuronal cell counts and biochemical assays were performed to further analyze outcomes. Results: All groups exhibited normal locomotor activity. The scopolamine group displayed longer escape latency times, reduced time in the target quadrant, decreased number of surviving neurons, and increased malondialdehyde and decreased glutathione levels compared with the control group. However, pre-treatment with 1000 mg/kg PFE notably mitigated the neurotoxic effects of scopolamine. Conclusion: The neuroprotective properties of PFE are highlighted, suggesting its potential as a promising treatment strategy for AD. Keywords: Alzheimer’s disease, neuroprotection, oxidative stress, Paederia foetida, scopolamine.

Quercetin protects against sepsis-associated encephalopathy by inhibiting microglia-neuron crosstalk via the CXCL2/CXCR2 signaling pathway

BackgroundSepsis-associated encephalopathy (SAE) is a common brain lesion associated with severe sepsis, for which ferroptosis is a key driving factor. Thus, suppressing ferroptosis may be an effective strategy for treating SAE. Quercetin (QUE) is a natural flavonoid with antioxidant and anti-inflammatory properties. However, its role on ferroptosis in SAE remains unclear. PurposeThis study aimed to investigate the mechanism underlying the therapeutic effect of QUE on cecal ligation perforation (CLP)-induced SAE. MethodsIn vivo and in vitro SAE models were established using CLP and lipopolysaccharide (LPS), respectively. Both models underwent pre-treatment with QUE. ResultsQUE attenuated CLP-induced symptoms, including temperature changes, neurological severity scores, learning and memory dysfunction, inflammatory cytokine release, and microglia activation in SAE mice, and inhibited LPS-induced microglia recruitment and chemotaxis. Bioinformatics analysis revealed that the C-X-C motif chemokine ligand 2 (CXCL2)/C-X-C motif chemokine receptor 2 (CXCR2) axis may play a key role in QUE-mediated protection against SAE. Moreover, QUE significantly inhibited LPS-induced CXCL2 up-regulation and protein secretion from microglia. Recombinant mouse-derived CXCL2 (rmCXCL2) promoted inflammatory cytokine secretion, NF-κB/NLRP3 signaling activation, and microglia recruitment and chemotaxis. Furthermore, rmCXCL2 induced ferroptosis in mouse hippocampal neurons, as evidenced by elevated malondialdehyde levels, decreased glutathione levels, excessive iron uptake, and altered ferroptosis-related protein expression. The CXCR2 antagonist SB225002 effectively reversed the effects of rmCXCL2. Importantly, in vivo experiments further demonstrated that the therapeutic effect of QUE on SAE was inhibited by rmCXCL2. ConclusionThis study demonstrates that CXCL2 secreted by activated microglia mediates microglia self-activation and induces hippocampal neuronal ferroptosis via CXCR2 and that QUE exerts neuroprotective effects on SAE by blocking interactions between microglia and neurons via CXCL2/CXCR2 pathway inhibition.

N-acetyl Cysteine Overdose Induced Acute Toxicity and Hepatic Microvesicular Steatosis by Disrupting GSH and Interfering Lipid Metabolisms in Normal Mice.

N-acetyl cysteine (NAC) is a versatile drug used in various conditions, but the limitations and toxicities are not clear. The acute toxicity and toxicological mechanisms of an intraperitoneal injection of NAC in normal mice were deciphered. The LD50 for male and female BALB/cByJNarl mice were 800 mg/kg and 933 mg/kg. The toxicological mechanisms of 800 mg/kg NAC (N800) were investigated. The serum biomarkers of hepatic and renal indices dramatically increased, followed by hepatic microvesicular steatosis, renal tubular injury and necrosis, and splenic red pulp atrophy and loss. Thus, N800 resulted in mouse mortality mainly due to acute liver, kidney, and spleen damages. The safe dose (275 mg/kg) of NAC (N275) increased hepatic antioxidant capacity by increasing glutathione levels and catalase activity. N275 elevated the hepatic gene expressions of lipid transporter, lipid synthesis, β-oxidation, and ketogenesis, suggesting a balance between lipid production and consumption, and finally, increased ATP production. In contrast, N800 increased hepatic oxidative stress by decreasing glutathione levels through suppressing Gclc, and reducing catalase activity. N800 decreased the hepatic gene expressions of lipid transporter, lipid synthesis, and interferred β-oxidation, leading to lipid accumulation and increasing Cyp2E1 expression, and finally, decreased ATP production. Therefore, NAC doses are limited for normal individuals, especially via intraperitoneal injection or similar means.

Antioxidant and anti-inflammatory activity by modulating IL-6 as a potential mechanism in the nephroprotective and hepatoprotective properties of Tribulus terrestris

Background and purpose: Carboplatin, a second-generation platinum-containing compound is associated with renal tubular injury and hepatic damage in cancer patients. Tribulus terrestris (TT) is widely used in Indian traditional medicine for its anti-inflammatory properties. The present study aimed to evaluate TT's beneficial effects against liver and kidney damage induced by carboplatin. Experimental approach: An in-vivo study was conducted on thirty rats. All the groups, except the control, received intraperitoneal carboplatin 90 mg/kg on day 5; the three treatment groups received TT extract (1 g/kg, 1.25, and 1.5 g/kg) for 14 days. Serum and tissue parameters for liver functions, kidney functions, oxidative stress, and inflammatory marker interleukin 6 were measured along with histopathological assessment. Findings/Results: TT at 1.5 g/kg on day 14 significantly reduced creatinine and aspartate transaminase levels compared to the carboplatin group. The increase in malondialdehyde levels and decrease in glutathione levels was significantly reversed in the groups treated with TT 1.25 and 1.5 g/kg. Interleukin 6 showed a significant decrease in treatment groups when compared to the carboplatin group. Carboplatin distorted hepatic architecture and caused diffused inflammatory cell infiltration in the peritubular interstitial spaces in the kidney. The histopathological evaluation confirmed that TT extract ameliorated hepatic and kidney damage by restoring to normal architecture. Conclusion and implications: Aqueous extract of TT demonstrated a therapeutic effect against nephrotoxicity and hepatotoxicity caused by carboplatin. The observed benefits can be attributed to its anti-inflammatory action and antioxidant properties.

Taurine exhibits antioxidant, anti-inflammatory, and antiapoptotic effects against pyraclostrobin exposure in rats.

Pyraclostrobin, a strobilurin-derived fungicide, causes oxidative stress and DNA damage in the organism. Taurine plays an important role in metabolic processes such as osmoregulatory, cytoprotective, and antioxidant effects. The study aimed to investigate the protective effect of taurine in Sprague Dawley male rats exposed to pyraclostrobin. The rats were separated into 6 groups and were found 8 animals in each group. Rats were given 30mg/kg pyraclostrobin and pyraclostrobin together with three different taurine concentrations (50, 100, and 200mg/kg) via oral gavage for 28days. While pyraclostrobin increased biochemical parameters, lipid peroxidation, and DNA damage, it decreased glutathione levels and enzyme activities of catalase and superoxide dismutase. Pyraclostrobin increased apoptotic, proinflammatory, and CYP2E1 mRNA expression levels, whereas antiapoptotic gene Bcl-2 mRNA expression levels decreased in liver tissue. Additionally, pyraclostrobin caused histopathological alterations in tissues. Taurine in a dose-dependent manner reversed the changes caused by pyraclostrobin. As a result, taurine exhibited a cytoprotective effect by showing antioxidant, anti-inflammatory, and antiapoptotic activities against oxidative damage caused by pyraclostrobin.

Embryonic ethanol exposure induces oxidative stress and inflammation in zebrafish model: A dose-dependent study

Alcohol, or ethanol, is a major contributor to detrimental diseases and comorbidities worldwide. Alcohol use during pregnancy intervenes the developing embryos leading to morphological changes, neurocognitive defects, and behavioral changes known as fetal alcohol spectrum disorder (FASD). Zebrafish have been used as a model to study FASD; however, the mechanism and the impact of ethanol on oxidative stress and inflammation in the zebrafish FASD model remain unexplored. Hence, we exposed zebrafish embryos to different concentrations of ethanol (0 %, 0.5 %, 1.0 %, 1.25 %, and 1.5 % ethanol (v/v)) at 4–96 hours post-fertilization (hpf) to study and characterize the ethanol concentration for the FASD model to induce oxidative stress and inflammation. Here, we studied the survival rate and developmental toxicity parameters at different time points and measured oxidative stress, reactive oxygen species (ROS) generation, apoptosis, and pro-inflammatory gene expression in zebrafish larvae. Our findings indicate that ethanol causes various developmental abnormalities, including decreased survival rate, spontaneous tail coiling, hatching rate, heart rate, and body length, associated with increased malformation. Further, ethanol exposure induced oxidative stress by increasing lipid peroxidation and nitric oxide production and decreasing glutathione levels. Subsequently, ethanol increased ROS generation, apoptosis, and pro-inflammatory gene (TNF-α and IL-1β) expression in ethanol exposed larvae. 1.25 % and 1.5 % ethanol had significant impacts on zebrafish larvae in all studied parameters. However, 1.5 % ethanol showed decreased survival rate and increased malformations. Overall, 1.25 % ethanol is the ideal concentration to study the oxidative stress and inflammation in the zebrafish FASD model.

Biliary atresia: insights into mechanisms using a toxic model of the disease including Wnt and Hippo signaling pathways and microtubules.

Mechanisms underlying bile duct injury in biliary atresia (BA) remain unclear and mechanisms of bile duct repair are unknown. This study aimed to explore the roles of microtubule instability and Wnt and Hippo signaling pathways in a biliatresone-induced BA model. Using primary murine neonatal cholangiocytes in both 2D and 3D cultures, and ex-vivo extra hepatic bile ducts (EHBD) which also has peri-cholangiocyte area, we analyzed injury and recovery processes. Injury was induced by the toxin biliatresone and recovery was induced by toxin wash-out. Microtubule stabilizer paclitaxel prevented biliatresone-induced injury, both to cholangiocytes as well as reduced periductal αSMA stain, this process is mediated by decreased glutathione levels. RhoU and Wnt11 (Wnt signaling) and Pard6g and Amotl1 (Hippo signaling) are involved in both injury and recovery processes, with the latter acting upstream to Wnt signaling. Early stages of biliatresone-induced EHBD injury in cholangiocytes and periductal structures are reversible. Wnt and Hippo signaling pathways play crucial roles in injury and recovery, providing insights into BA injury mechanisms and potential recovery avenues. Microtubule stabilization prevents cholangiocyte injury and lumen obstruction in a toxic model of biliary atresia (biliatresone induced). Early stages of biliatresone-induced injury, affecting both cholangiocytes and periductal structures, are reversible. Both Wnt and Hippo signaling pathways play a crucial role in bile duct injury and recovery, with a noted interplay between the two. Understanding mechanisms of cholangiocyte recovery is imperative to unveil potential therapeutic avenues.

Metformin-induced oxidative stress inhibits LNCaP prostate cancer cell survival.

Preclinical and clinical studies over the past several decades have indicated the potential value of metformin, a widely utilized treatment for Type 2 diabetes, in prostate cancer therapy. Notably, these studies demonstrated metformin's pleiotropic effects on several molecular and metabolic pathways, such as androgen signaling, cell cycle, and cellular bioenergetics. In this study we investigated the role of metformin in regulating intracellular redox status and cell survival in LNCaP prostate cancer cells. The cytotoxic effects of metformin with or without the presence of SBI0206965 (AMPK inhibitor) on LNCaP cells were determined using MTT and trypan blue exclusion assays. Seahorse XP extracellular analysis, Liquid Chromatography/ Mass Spectrophotometry (LC/MS), and 2,7- and Dichlorofluoresin diacetate (DCFDA) assay were used to assess the effects of metformin on cellular bioenergetics, redox status, and redox-related metabolites. mRNA expression and protein concentration of redox-related enzymes were measured using Real Time-qPCR and ELISA assay, respectively. Independently of AMP-activated protein kinase, metformin exhibited a dose- and time-dependent inhibition of LNCaP cell survival, a response mitigated by glutathione or N-acetylcysteine (ROS scavengers) treatment. Notably, these findings were concomitant with a decline in ATP levels and the inhibition of oxidative phosphorylation. The results further indicated metformin's induction of reactive oxygen species, which significantly decreased glutathione levels and the ratio of reduced to oxidized glutathione, as well as the transsulfuration metabolite, cystathionine. Consistent with an induction of oxidative stress condition, metformin increased mRNA levels of the master redox transcription factor Nrf-2 (nuclear factor erythroid-derived 2-like), as well as transsulfuration enzymes cystathionine beta-synthase and cystathionase and GSH synthesis enzymes γ-glutamylcysteine synthetase and glutathione synthetase. Our findings highlight multiple mechanisms by which metformin-induced formation of reactive oxygen species may contribute to its efficacy in prostate cancer treatment, including promotion of oxidative stress, Nrf2 activation, and modulation of redox-related pathways, leading to its anti-survival action.

Assessment of the Antioxidant and Hypolipidemic Properties of Salicornia europaea for the Prevention of TAFLD in Rats.

Halophyte species represent valuable reservoirs of natural antioxidants, and, among these, Salicornia europaea stands out as a promising edible plant. In this study, young and old S. europaea leaves were compared for the content of bioactive compounds and antioxidant activity to assess changes in different growth phases; then, the potential protective effects against low-dose CCl4-induced toxicant-associated fatty liver disease (TAFLD) were investigated by administering an aqueous suspension of young leaves to rats daily for two weeks. Quantification of total and individual phenolic compounds and in vitro antioxidant activity assays (DPPH, FRAP, and ORAC) showed the highest values in young leaves compared to mature ones. Salicornia treatment mitigated CCl4-induced hepatic oxidative stress, reducing lipid peroxidation and protein carbonyl levels, and preserving the decrease in glutathione levels. Electronic paramagnetic resonance (EPR) spectroscopy confirmed these results in the liver and evidenced free radicals increase prevention in the brain. Salicornia treatment also attenuated enzymatic disruptions in the liver's drug metabolizing system and Nrf2-dependent antioxidant enzymes. Furthermore, histopathological examination revealed reduced hepatic lipid accumulation and inflammation. Overall, this study highlights Salicornia's potential as a source of bioactive compounds with effective hepatoprotective properties capable to prevent TAFLD.

Effect of RNF113A deficiency on oxidative stress-induced NRF2 pathway

ABSTRACT The ring finger protein 113A (RNF113A) serves as an E3 ubiquitin ligase and a subunit of the spliceosome. Mutations in the RNF113A gene are associated with X-linked trichothiodystrophy (TTD). However, the cellular roles of RNF113A remain largely unknown. In this study, we performed transcriptome profiling of RNF113A knockout (KO) HeLa cells using RNA sequencing and revealed the upregulation of NRF2 pathway-associated genes. Further analysis confirmed that the KO of RNF113A promotes nuclear localization of the NRF2 protein and elevates the mRNA levels of NRF2 target genes. RNF113A KO cells showed high levels of intracellular reactive oxygen species (ROS) and decreased resistance to cell death following H2O2 treatment. Additionally, RNF113A KO cells more sensitively formed stress granules (SGs) under arsenite-induced oxidative stress. Moreover, RNF113A KO cells exhibited a decrease in glutathione levels, which could be attributed to a reduction in GLUT1 expression levels, leading to decreased glucose uptake reactions and lower intracellular glucose levels. These alterations potentially caused a reduction in ROS scavenging activity. Taken together, our findings suggest that the loss of RNF113A promotes oxidative stress-mediated activation of the NRF2 pathway, providing novel insights into RNF113A-associated human diseases.

Evaluation of Herbal Composite for Activity Against Anti-cancer Drug-induced Nephrotoxicity in Wistar Rats

Background: Nephrotoxicity is among the significant side effects and a dose-restriction mechanism for cisplatin treatment. According to recent studies, Oxidative stress and inflammation are the causative agents for acute renal failure on Cisplatin. Curcumin and resveratrol are considered to have potent anti-inflammatory and antioxidant effects. The current investigation aimed to determine whether a composite of curcumin and resveratrol attenuates nephrotoxicity caused by Cisplatin in Wistar rats. Method: The herbal compound was administered together with 5 mg/kg of cisplatin daily for 10 days with doses of 100 and 400 mg/kg. To evaluate renal damage, serum creatinine, nitrogen in blood urea, Creatinine, and levels of nitrite were measured. The levels of renal malondialdehyde, decreased glutathione levels, catalase, and also the superoxide dismutase enzyme were measured to quantify renal oxidative stress. Result: Significant oxidative stress, severe inflammation, and nephrotoxicity were all seen after a single dosage of cisplatin. In Wistar rats, a herbal composite treatment significantly reduced inflammation, improved renal function in a dose-dependent manner, reduced oxidative stress, and provided protection from the nephrotoxicity caused by Cisplatin. Conclusion: The herbal composite of curcumin - and - resveratrol was found to act as a therapeutic adjutant against Cisplatin nephrotoxicity.

Insight into sodium selenite-induced metabolic reprogramming in response to main seleno-amino acid enrichment in Saccharomyces cerevisiae using metabolomics

Conversion of inorganic selenium (Se) to its organic form by Saccharomyces cerevisiae (S. cerevisiae) has attracted significant attention, for organic Se has higher nutritional value with less negative effects. However, the mechanism of transformation and accumulation of organic Se at the metabolic level remains unclear. We investigated the metabolic response to sodium selenite supplementation in culture medium at 0, 5, 15, and 45 mg/L in two S. cerevisiae strains using an ultra-high performance liquid chromatography-high resolution mass spectrometry-based untargeted method. In total, 455 metabolites were detected, and a separation trend was clearly observed between Se-treated groups and controls. In depth, metabolic pathways involving amino acid, nucleotide metabolism and tricarboxylic acid cycle were significantly affected. Furthermore, high inorganic-Se supplementation effectively decreased the seleno-amino acid proportion in total Se, inhibited cell growth, and decreased glutathione levels. These results provide novel insights into the Se-enrichment process of S. cerevisiae.

Individual and Dual Cytotoxicity of the Combination of Passiflora Caerulea Leaf Extract and Titanium Oxide Nanoparticles against A549, U937, and Hela Cells

Background: The study addresses the underexplored realm of cytotoxicity evaluation involving binary mixtures of Passiflora caerulea leaf extracts and Titanium nanoparticles (TiO2NPs). The focus lies on understanding the combined effects of these components on cancer cells (A549, U937, and HeLa). Methods: In vitro cytotoxicity assessments were employed to evaluate the toxicity of P. caerulea leaf extracts, TiO2NPs, and their combination. The study utilized MTT, NRU, and LDH assays to measure cellular viability. Additionally, reactive oxygen species (ROS) and glutathione levels were assessed alongside the aforementioned assays. Results: The toxicity percentage exhibited dose-dependent behavior for P. caerulea leaf extracts, TiO2NPs, and their combination. Interestingly, when P. caerulea leaf extract and TiO2NPs were combined, the reduction in cell viability was noticeably more than when the exposures were made separately. Moreover, the production of ROS was higher in the combined toxicity scenario, and a more pronounced decrease in glutathione levels was observed compared to individual exposures. Conclusion: The findings suggest that the combined effects of P. caerulea leaf extract and TiO2NPs induce greater cytotoxicity compared to their impacts. This underscores the potential for synergistic cytotoxicity in combined exposure scenarios, warranting further exploration of combined effects in future studies.

Analysis of cantharidin-induced kidney injury and the protective mechanism of resveratrol in mice determined by liquid chromatography/mass spectrometry-based metabonomics.

Cantharidin (CTD) is the main active component in the traditional Chinese medicine Mylabris and an effective anti-tumor agent. However, it is relatively toxic and exhibits nephrotoxicity, which limits its clinical use. However, its toxic mechanism is not clear. The toxic effects of CTD exposure on the kidney and the protective effect of resveratrol (RES) were studied in a mouse model, by determination of serum biochemical and renal antioxidant indicators, histopathological and ultrastructural observation, and metabonomics. After CTD exposure, serum uric acid, creatinine, and tissue oxidative stress indicators increased, and the renal glomerular and tubular epithelial cells showed clear pathological damage. Ultrastructure observation revealed marked mitochondrial swelling, endoplasmic reticulum dilation, and the presence of autophagy lysosomes in glomerular epithelial cells. RES ameliorated the renal injury induced by CTD. Metabonomics analysis indicated that CTD can induce apoptosis and oxidative damage in kidney cells, mainly by disrupting sphingolipid and glutathione metabolism, increasing sphingosine and sphingomyelin levels, and decreasing glutathione levels. RES counteracts these effects by regulating renal cell proliferation, the inflammatory response, oxidative stress, and apoptosis, by improving the levels of phosphatidylcholine (PC), LysoPC, and lysophosphatidyl glycerol in the glycerophospholipid metabolism pathway, thereby reducing CTD-induced nephrotoxicity. The mechanisms of CTD-induced renal injury and the protective effect of RES were revealed by metabonomics, providing a basis for evaluating clinical treatment regimens to reduce CTD-induced nephrotoxicity.

Kaempferol mitigates reproductive dysfunctions induced by Naja nigricollis venom through antioxidant system and anti-inflammatory response in male rats

Naja nigricollis Venom (NnV) contains complex toxins that affects various vital systems functions after envenoming. The venom toxins have been reported to induce male reproductive disorders in envenomed rats. This present study explored the ameliorative potential of kaempferol on NnV-induced male reproductive toxicity. Fifty male wistar rats were sorted randomly into five groups (n = 10) for this study. Group 1 were noted as the control, while rats in groups 2 to 5 were injected with LD50 of NnV (1.0 mg/kg bw; i.p.). Group 2 was left untreated post envenomation while group 3 was treated with 0.2 ml of polyvalent antivenom. Groups 4 and 5 were treated with 4 and 8 mg/kg of kaempferol, respectively. NnV caused substantial reduction in concentrations of follicle stimulating hormone, testosterone and luteinizing hormone, while sperm motility, volume and counts significantly (p < 0.05) decreased in envenomed untreated rats. The venom enhanced malondialdehyde levels and substantially decreased glutathione levels, superoxide dismutase and glutathione peroxidase activities in the testes and epididymis of envenomed untreated rats. Additionally, epididymal and testicular myeloperoxidase activity and nitric oxide levels were elevated which substantiated severe morphological defects noticed in the reproductive organs. However, treatment of envenomed rats with kaempferol normalized the reproductive hormones with significant improvement on sperm functional parameters. Elevated inflammatory and oxidative stress biomarkers in testis and epididymis were suppressed post kaempferol treatment. Severe histopathological lesions in the epididymal and testicular tissues were ameliorated in the envenomed treated groups. Results highlights the significance of kaempferol in mitigating reproductive toxicity induced after snakebite envenoming.

RAGE induces physiological activation of NADPH oxidase in neurons and astrocytes and neuroprotection.

The transmembrane receptor for advanced glycation end products (RAGE) is a signaling receptor for many damage- and pathogen-associated molecules. Activation of RAGE is associated with inflammation and an increase in reactive oxygen species (ROS) production. Although several sources of ROS have been previously suggested, how RAGE induces ROS production is still unclear, considering the multiple targets of pathogen-associated molecules. Here, using acute brain slices and primary co-culture of cortical neurons and astrocytes, we investigated the effects of a range of synthetic peptides corresponding to the fragments of the RAGE V-domain on redoxsignaling. We found that the synthetic fragment (60-76) of the RAGE V-domain induces activation of ROS production in astrocytes and neurons from the primary co-culture and acute brain slices. This effect occurred through activation of RAGE and could be blocked by a RAGE inhibitor. Activation of RAGE by the synthetic fragment stimulates ROS production in NADPH oxidase (NOX). This RAGE-induced NOX activation produced only minor decreases in glutathione levels and increased the rate of lipid peroxidation, although it also reduced basal and β-amyloid induced cell death in neurons and astrocytes. Thus, specific activation of RAGE induces redox signaling through NOX, which can be a part of a cell protective mechanism.

Regulatory network of ferroptosis and autophagy by targeting oxidative stress defense using sulfasalazine in triple-negative breast cancer

AimsThe cellular defense system against oxidative stress is important for the survival ability and sensitization in chemotherapy; however, the regulatory mechanisms remain unknown in triple-negative breast cancer (TNBC) cells. This study aimed to investigate the relationship between ferroptosis and autophagy by targeting the defense of oxidative stress through the cystine transporter (xCT) using sulfasalazine (SASP), which is a widely employed xCT inhibitor. Main methodsWe analyzed the cell death process of SASP in human TNBC cells, and examined the effects of SASP on tumor progression by using xenograft mouse model. Key findingsTNBC cells demonstrated a high defense capacity against reactive oxidative species through xCT. SASP significantly attenuated oxidative stress resistance in MDA-MB-231, which is a generally used model cell as TNBC, through decreased glutathione levels, causing a marked iron-dependent ferroptotic cell death induction. Moreover, autophagy was required to trigger efficient SASP-induced ferroptosis at the early stage of cell death. Tamoxifen, which is currently in clinical use as the gold standard for endocrine therapy of estrogen receptor-positive breast cancer, was a beneficial tool as an autophagy regulator under ferroptotic cell death by SASP. Additionally, SASP suppressed tumor growth and metastasis progression through total glutathione reduction in the primary tumor, indicating high anticancer activity against TNBC without liver injury in vivo. SignificanceWe revealed that SASP can efficiently induce ferroptosis associated with autophagy and that an understanding of the mechanism of cell death regulation by SASP is a promising new strategy for TNBC therapy and drug repositioning.

Monitoring of bone matrix acidification by TRAP and ERK biomarkers in the chronic hypercholesterolemia male rats

Background: Hypercholesterolemia is frequently linked to an elevated risk of cardiovascular diseases, including heart attacks and strokes. Additionally, it could be connected to a higher susceptibility to osteoporosis. Hypercholesterolemia can stimulate differentiation and activity of osteoclasts, leading to enhanced bone reabsorption and a subsequent net loss of bone tissue. Aim: The purpose of this study was to examine the influence of a high cholesterol diet on osteoporosis in male rats with differences in biological and oxidative indicators in hypercholesterolemia diet in male rat. Method: The samples in this study were twenty male rats, ranging between 1.5 and 2 months, were separated into two groups. In one group, 10 rats were fed a regular diet, while in another group, 10 rats were fed a high cholesterol diet (2%) over the course of 8 weeks. Samples of blood were obtained at the last stage of the experiment. to calculate physiological and biological markers including ERK, TRAP, hormones, MDA, and GSH. Results: The results of this study demonstrated a decrease in glutathione levels, an increase in extracellular signal-regulated kinase levels (ERK), no significant change in serum TRAP levels, an increase in Malondialdehyde levels in the blood, and elevated levels of parathyroid hormone, Calcitonin, and Vitamin D in the cholesterol group. Conclusion: An increased oxidative stress, altered signaling, and disruptions in calcium/bone metabolism associated with cholesterol-related condition and monitoring biomarker ERK can provide valuable information about disease progression.

Imatinib induces ferroptosis in gastrointestinal stromal tumors by promoting STUB1-mediated GPX4 ubiquitination

Imatinib (IM) has significantly improved the prognosis of gastrointestinal stromal tumor (GIST) patients, but some patients still have primary resistance to IM, and approximately half of patients develop acquired drug resistance within 2 years of treatment, necessitating exploration of new treatment strategies. Targeting ferroptosis as a novel approach to tumor treatment has gained attention. Yet, there is limited research on ferroptosis in GIST, and the underlying mechanism remains unclear. In this study, we revealed that IM increased lipid reactive oxygen species and intracellular Fe2+ levels, and decreased glutathione levels in GIST. This effect could be partially inhibited by Ferrostatin-1. Additionally, knocking down STUB1 and overexpressing GPX4 reversed the IM-induced ferroptosis effect. Moreover, STUB1 was identified as a novel E3 ubiquitin ligase of GPX4, promoting the ubiquitination at site K191 of GPX4. The combination of the GPX4 inhibitor RSL3 and IM synergistically induces ferroptosis, inhibiting GIST proliferation both in vivo and in vitro. Furthermore, STUB1 and GPX4 expression serve as independent prognostic factors for GIST. In conclusion, This study is the first to demonstrate that IM induces ferroptosis by promoting STUB1-mediated GPX4 ubiquitination in GIST, and the combination of RSL3 and IM emerges as a promising therapeutic strategy for GIST.

Abstract A103: Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile

Abstract The tumor suppressor p53 is mutated (mt-p53) in over 50% of human cancers causing gain-of-function oncogenic effects, including metabolic changes that reduce tumor responsiveness to radio/chemotherapy. Common hot-spot mutations within the DNA-binding domain can be categorized as conformational (R175H) or DNA binding (R248W). NSC59984 has been characterized as a small molecule that targets mt-p53 for degradation and restores wt-p53 signaling. Using esophageal adenocarcinoma cells and CRISPR generated isogenic cell lines bearing matching hot-spot p53 mutations, we aim to understand how the molecular features of mt-p53 affect drug efficiency and enable the development of targeted therapies to limit cancer cell growth. We found that NSC59984 covalently modifies p53 by Michael addition at cysteine residues 124 and 229, which promote interactions that would stabilize the protein/DNA complex leading to increased p53 transcriptional activity. In cells, the effects of NSC59984 were substantially greater in cells harboring the R248W mutation compared with the R175H mutation. Treatment with NSC59984 reduced proliferation and increased apoptosis via the intrinsic mitochondrial pathway. It also induced changes in OXPHOS, ATP level, mitochondrial membrane potential, glycolysis, and lactate production. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. NSC59984 treatment increased G6PD activity, total NADPH levels, and expression of TIGAR. Knockout of TIGAR partially removed the antiproliferative effects of the drug and reduced G6PD levels in the p53-R248W cells. Incorporation of [13C6] into cellular metabolites suggests that p53-regulated transcription of TIGAR increased utilization of the pentose phosphate pathway and inhibited glycolysis at the fructose-6-P fructose-1,6-bisphosphate junction, supported by an increase in Hexokinase 2 and a decrease of phosphofructokinase-1. Thermal proteome profiling identified TIGAR as an additional reaction target of NSC59984, suggesting increased involvement in modulating these metabolic effects. Combining currently available therapeutic metabolic inhibitors with NSC59984 enhanced the antiproliferative effects in cells harboring p53-R248W creating a therapeutic window when compared to the wt-p53 expressing cells. This suggests these combinations could be used in a clinically relevant setting. Overall, this work has identified a distinctive mode of action for p53 reactivation resulting in not only transcriptional activity, but also a unique effect on cellular energetics. This study shows evidence of variation in responsiveness of different mt-p53 forms and allows the development of specific therapeutics directed to individuals for patient-centered precision medicine. Importantly, we have shown that targeting p53 signaling has significant effects on the metabolic profiles of cancer cells rendering them more vulnerable to neoadjuvant therapy. Citation Format: Kate Brown, Lisa Jenkins, Dan Crooks, Deborah Surman, Sharlyn Mazur, Yuan Xu, Bhargav Arimilli, Ye Yang, Andrew Lane, Stewart Durell, Teresa Fan, David Schrump, Marston Marston, Taylor Ripley, Ettore Appella, Gaelyn Lyons, Andrew Perciaccante, Jerry Dinan, Marco Robello, Herman Nikolayevskiy, Robert O’Connor, Daniel Appella. Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A103.