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Colorectal cancer (CRC) is a frequent, worldwide tumor described for its huge complexity, including inter-/intra-heterogeneity and tumor microenvironment (TME) variability. Intra-tumor heterogeneity and its connections with metabolic reprogramming and epithelial–mesenchymal transition (EMT) were investigated with explorative shotgun proteomics complemented by a Random Forest (RF) machine-learning approach. Deep and superficial tumor regions and distant-site non-tumor samples from the same patients (n = 16) were analyzed. Among the 2009 proteins analyzed, 91 proteins, including 23 novel potential CRC hallmarks, showed significant quantitative changes. In addition, a 98.4% accurate classification of the three analyzed tissues was obtained by RF using a set of 21 proteins. Subunit E1 of 2-oxoglutarate dehydrogenase (OGDH-E1) was the best classifying factor for the superficial tumor region, while sorting nexin-18 and coatomer-beta protein (beta-COP), implicated in protein trafficking, classified the deep region. Down- and up-regulations of metabolic checkpoints involved different proteins in superficial and deep tumors. Analogously to immune checkpoints affecting the TME, cytoskeleton and extracellular matrix (ECM) dynamics were crucial for EMT. Galectin-3, basigin, S100A9, and fibronectin involved in TME–CRC–ECM crosstalk were found to be differently variated in both tumor regions. Different metabolic strategies appeared to be adopted by the two CRC regions to uncouple the Krebs cycle and cytosolic glucose metabolism, promote lipogenesis, promote amino acid synthesis, down-regulate bioenergetics in mitochondria, and up-regulate oxidative stress. Finally, correlations with the Dukes stage and budding supported the finding of novel potential CRC hallmarks and therapeutic targets.

Cancer is one of the most important problems of modern societies. Recently, studies have reported the anticancer properties of rosiglitazone related to its ability to bind peroxisome proliferator receptor γ (PPARγ), which has various effects on cancer and can inhibit cell proliferation. In this study, we investigated the effect of new 4-thiazolidinone (4-TZD) hybrids Les-4369 and Les-3467 and their effect on reactive oxygen species (ROS) production, metabolic activity, lactate dehydrogenase (LDH) release, caspase-3 activity, and gene and protein expression in human foreskin fibroblast (BJ) cells and lung adenocarcinoma (A549) cells. The ROS production and caspase-3 activity were mainly increased in the micromolar concentrations of the studied compounds in both cell lines. Les-3467 and Les-4369 increased the mRNA expression of PPARG, P53 (tumor protein P53), and ATM (ATM serine/threonine kinase) in the BJ cells, while the mRNA expression of these genes (except PPARG) was mainly decreased in the A549 cells treated with both of the tested compounds. Our results indicate a decrease in the protein expression of AhR, PPARγ, and PARP-1 in the BJ cells exposed to 1 µM Les-3467 and Les-4369. In the A549 cells, the protein expression of AhR, PPARγ, and PARP-1 increased in the treatment with 1 µM Les-3467 and Les-4369. We have also shown the PPARγ modulatory properties of Les-3467 and Les-4369. However, both compounds prove weak anticancer properties evidenced by their action at high concentrations and non-selective effects against BJ and A549 cells.

Chlorine (Cl2) exposure poses a significant risk to ocular health, with the cornea being particularly susceptible to its corrosive effects. Antioxidants, known for their ability to neutralize reactive oxygen species (ROS) and alleviate oxidative stress, were explored as potential therapeutic agents to counteract chlorine-induced damage. In vitro experiments using human corneal epithelial cells showed decreased cell viability by chlorine-induced ROS production, which was reversed by antioxidant incubation. The mitochondrial membrane potential decreased due to both low and high doses of Cl2 exposure; however, it was recovered through antioxidants. The wound scratch assay showed that antioxidants mitigated impaired wound healing after Cl2 exposure. In vivo and ex vivo, after Cl2 exposure, increased corneal fluorescein staining indicates damaged corneal epithelial and stromal layers of mice cornea. Likewise, Cl2 exposure in human ex vivo corneas led to corneal injury characterized by epithelial fluorescein staining and epithelial erosion. However, antioxidants protected Cl2-induced damage. These results highlight the effects of Cl2 on corneal cells using in vitro, ex vivo, and in vivo models while also underscoring the potential of antioxidants, such as vitamin A, vitamin C, resveratrol, and melatonin, as protective agents against acute chlorine toxicity-induced corneal injury. Further investigation is needed to confirm the antioxidants’ capacity to alleviate oxidative stress and enhance the corneal healing process.

The present study explores for the first time the effect of hyperbaric oxygen (HBO) on gingival mesenchymal stem cells’ (G-MSCs) gene expression profile, intracellular pathway activation, pluripotency, and differentiation potential under an experimental inflammatory setup. G-MSCs were isolated from five healthy individuals (n = 5) and characterized. Single (24 h) or double (72 h) HBO stimulation (100% O2, 3 bar, 90 min) was performed under experimental inflammatory [IL-1β (1 ng/mL)/TNF-α (10 ng/mL)/IFN-γ (100 ng/mL)] and non-inflammatory micro-environment. Next Generation Sequencing and KEGG pathway enrichment analysis, G-MSCs’ pluripotency gene expression, Wnt-/β-catenin pathway activation, proliferation, colony formation, and differentiation were investigated. G-MSCs demonstrated all mesenchymal stem/progenitor cells’ characteristics. The beneficial effect of a single HBO stimulation was evident, with anti-inflammatory effects and induction of differentiation (TLL1, ID3, BHLHE40), proliferation/cell survival (BMF, ID3, TXNIP, PDK4, ABL2), migration (ABL2) and osteogenic differentiation (p < 0.05). A second HBO stimulation at 72 h had a detrimental effect, significantly increasing the inflammation-induced cellular stress and ROS accumulation through HMOX1, BHLHE40, and ARL4C amplification and pathway enrichment (p < 0.05). Results outline a positive short-term single HBO anti-inflammatory, regenerative, and differentiation stimulatory effect on G-MSCs. A second (72 h) stimulation is detrimental to the same properties. The current results could open new perspectives in the clinical application of short-termed HBO induction in G-MSCs-mediated periodontal reparative/regenerative mechanisms.

Colorectal cancer (CRC) is a frequent, worldwide tumor described for its huge complexity, including inter-/intra-heterogeneity and tumor microenvironment (TME) variability. Intra-tumor heterogeneity and its connections with metabolic reprogramming and epithelial-mesenchymal transition (EMT) were investigated with explorative shotgun proteomics complemented by a Random Forest (RF) machine-learning approach. Deep and superficial tumor regions and distant-site non-tumor samples from the same patients (n = 16) were analyzed. Among the 2009 proteins analyzed, 91 proteins, including 23 novel potential CRC hallmarks, showed significant quantitative changes. In addition, a 98.4% accurate classification of the three analyzed tissues was obtained by RF using a set of 21 proteins. Subunit E1 of 2-oxoglutarate dehydrogenase (OGDH-E1) was the best classifying factor for the superficial tumor region, while sorting nexin-18 and coatomer-beta protein (beta-COP), implicated in protein trafficking, classified the deep region. Down- and up-regulations of metabolic checkpoints involved different proteins in superficial and deep tumors. Analogously to immune checkpoints affecting the TME, cytoskeleton and extracellular matrix (ECM) dynamics were crucial for EMT. Galectin-3, basigin, S100A9, and fibronectin involved in TME-CRC-ECM crosstalk were found to be differently variated in both tumor regions. Different metabolic strategies appeared to be adopted by the two CRC regions to uncouple the Krebs cycle and cytosolic glucose metabolism, promote lipogenesis, promote amino acid synthesis, down-regulate bioenergetics in mitochondria, and up-regulate oxidative stress. Finally, correlations with the Dukes stage and budding supported the finding of novel potential CRC hallmarks and therapeutic targets.

Activation of intravesical protease activated receptors-4 (PAR4) results in bladder pain through the release of urothelial macrophage migration inhibitory factor (MIF) and high mobility group box-1 (HMGB1). We aimed to identify HMGB1 downstream signaling events at the bladder that mediate HMGB1-induced bladder pain in MIF-deficient mice to exclude any MIF-related effects. We studied whether oxidative stress and ERK activation are involved by examining bladder tissue in mice treated with intravesical disulfide HMGB1 for 1 h and analyzed with Western blot and immunohistochemistry. HMGB1 intravesical treatment increased urothelium 4HNE and phospho-ERK1/2 staining, suggesting that HMGB1 increased urothelial oxidative stress and ERK activation. Furthermore, we examined the functional roles of these events. We evaluated lower abdominal mechanical thresholds (an index of bladder pain) before and 24 h after intravesical PAR4 or disulfide HMGB1. Intravesical pre-treatments (10 min prior) included: N-acetylcysteine amide (NACA, reactive oxygen species scavenger) and FR180204 (FR, selective ERK1/2 inhibitor). Awake micturition parameters (voided volume; frequency) were assessed at 24 h after treatment. Bladders were collected for histology at the end of the experiment. Pre-treatment with NACA or FR significantly prevented HMGB1-induced bladder pain. No significant effects were noted on micturition volume, frequency, inflammation, or edema. Thus, HMGB1 activates downstream urothelial oxidative stress production and ERK1/2 activation to mediate bladder pain. Further dissection of HMGB1 downstream signaling pathway may lead to novel potential therapeutic strategies to treat bladder pain.

Reactive oxygen species (ROS) are radical oxygen intermediates that serve as important second messengers in signal transduction. However, when the accumulation of these molecules exceeds the buffering capacity of antioxidant enzymes, oxidative stress and endothelial cell (EC) dysfunction occur. EC dysfunction shifts the vascular system into a pro-coagulative, proinflammatory state, thereby increasing the risk of developing cardiovascular (CV) diseases and metabolic disorders. Studies have turned to the investigation of microRNA treatment for CV risk factors, as these post-transcription regulators are known to co-regulate ROS. In this review, we will discuss ROS pathways and generation, normal endothelial cell physiology and ROS-induced dysfunction, and the current knowledge of common metabolic disorders and their connection to oxidative stress. Therapeutic strategies based on microRNAs in response to oxidative stress and microRNA’s regulatory roles in controlling ROS will also be explored. It is important to gain an in-depth comprehension of the mechanisms generating ROS and how manipulating these enzymatic byproducts can protect endothelial cell function from oxidative stress and prevent the development of vascular disorders.

Glioblastoma multiforme (GBM) is a major aggressive primary brain tumor with dismal survival outcome and few therapeutic options. Although Temozolomide (TMZ) is a part of the standard therapy, over time, it can cause DNA damage leading to deleterious effects, necessitating the discovery of drugs with minimal side effects. To this end, we investigated the effect of cinnamaldehyde (CA), a highly purified, single ingredient from cinnamon, on the GBM cell lines U87 and U251 and the neuroglioma cell line H4. On observing similar impact on the viability in all the three cell lines, detailed studies were conducted with CA and its isomer/analog, trans-CA (TCA), and methoxy-CA (MCA) on U87 cells. The compounds exhibited equal potency when assessed at the cellular level in inhibiting U87 cells as well as at the molecular level, resulting in an increase in reactive oxygen species (ROS) and an increase in the apoptotic and multicaspase cell populations. To further characterize the key entities, protein profiling was performed with CA. The studies revealed differential regulation of entities that could be key to glioblastoma cell circuits such as downregulation of pyruvate kinase-PKM2, the key enzyme of the glycolytic pathway that is central to the Warburg effect. This allows for monitoring the levels of PKM2 after therapy using recently developed noninvasive technology employing PET [18F] DASA-23. Additionally, the observation of downregulation of phosphomevalonate kinase is significant as the brain tumor initiating cells (BTIC) are maintained by the metabolism occurring via the mevalonate pathway. Results from the current study, if translated in vivo, could provide additional efficacious treatment options for glioblastoma with minimal side effects.

Oxidative stress regulates many physiological and pathological processes. Indeed, a low increase in the basal level of reactive oxygen species (ROS) is essential for various cellular functions, including signal transduction, gene expression, cell survival or death, as well as antioxidant capacity. However, if the amount of generated ROS overcomes the antioxidant capacity, excessive ROS results in cellular dysfunctions as a consequence of damage to cellular components, including DNA, lipids and proteins, and may eventually lead to cell death or carcinogenesis. Both in vitro and in vivo investigations have shown that activation of the mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) pathway is frequently involved in oxidative stress-elicited effects. In particular, accumulating evidence identified a prominent role of this pathway in the anti-oxidative response. In this respect, activation of krüppel-like factor 2/4 and nuclear factor erythroid 2-related factor 2 emerged among the most frequent events in ERK5-mediated response to oxidative stress. This review summarizes what is known about the role of the MEK5/ERK5 pathway in the response to oxidative stress in pathophysiological contexts within the cardiovascular, respiratory, lymphohematopoietic, urinary and central nervous systems. The possible beneficial or detrimental effects exerted by the MEK5/ERK5 pathway in the above systems are also discussed.

Life on Earth has evolved in the presence of a gravity constraint. Any change in the value of such a constraint has important physiological effects. Gravity reduction (microgravity) alters the performance of muscle, bone and, immune systems among others. Therefore, countermeasures to limit such deleterious effects of microgravity are needed considering future Lunar and Martian missions. Our study aims to demonstrate that the activation of mitochondrial Sirtuin 3 (SIRT3) can be exploited to reduce muscle damage and to maintain muscle differentiation following microgravity exposure. To this effect, we used a RCCS machine to simulate microgravity on ground on a muscle and cardiac cell line. During microgravity, cells were treated with a newly synthesized SIRT3 activator, called MC2791 and vitality, differentiation, ROS and, autophagy/mitophagy were measured. Our results indicate that SIRT3 activation reduces microgravity-induced cell death while maintaining the expression of muscle cell differentiation markers. In conclusion, our study demonstrates that SIRT3 activation could represent a targeted molecular strategy to reduce muscle tissue damage caused by microgravity.

NEK6 is a central kinase in developing castration-resistant prostate cancer (CRPC). However, the pathways regulated by NEK6 in CRPC are still unclear. Cancer cells have high reactive oxygen species (ROS) levels and easily adapt to this circumstance and avoid cell death by increasing antioxidant defenses. We knocked out the NEK6 gene and evaluated the redox state and DNA damage response in DU-145 cells. The knockout of NEK6 decreases the clonogenic capacity, proliferation, cell viability, and mitochondrial activity. Targeting the NEK6 gene increases the level of intracellular ROS; decreases the expression of antioxidant defenses (SOD1, SOD2, and PRDX3); increases JNK phosphorylation, a stress-responsive kinase; and increases DNA damage markers (p-ATM and γH2AX). The exogenous overexpression of NEK6 also increases the expression of these same antioxidant defenses and decreases γH2AX. The depletion of NEK6 also induces cell death by apoptosis and reduces the antiapoptotic Bcl-2 protein. NEK6-lacking cells have more sensitivity to cisplatin. Additionally, NEK6 regulates the nuclear localization of NF-κB2, suggesting NEK6 may regulate NF-κB2 activity. Therefore, NEK6 alters the redox balance, regulates the expression of antioxidant proteins and DNA damage, and its absence induces the death of DU-145 cells. NEK6 inhibition may be a new strategy for CRPC therapy.

Insulin resistance onset in skeletal muscle is characterized by the impairment of insulin signaling, which reduces the internalization of glucose, known as glucose uptake, into the cell. Therefore, there is a deficit of intracellular glucose, which is the main source for energy production in the cell. This may compromise cellular viability and functions, leading to pathological dysfunction. Skeletal muscle fibers continuously generate reactive oxygen and nitrogen species (RONS). An excess of RONS produces oxidative distress, which may evoke cellular damage and dysfunction. However, a moderate level of RONS, which is called oxidative eustress, is critical to maintain, modulate and regulate cellular functions through reversible interactions between RONS and the components of cellular signaling pathways that control those functions, such as the facilitation of glucose uptake. The skeletal muscle releases peptides called myokines that may have endocrine and paracrine effects. Some myokines bind to specific receptors in skeletal muscle fibers and might interact with cellular signaling pathways, such as PI3K/Akt and AMPK, and facilitate glucose uptake. In addition, there are cytokines, which are peptides produced by non-skeletal muscle cells, that bind to receptors at the plasma membrane of skeletal muscle cells and interact with the cellular signaling pathways, facilitating glucose uptake. RONS, myokines and cytokines might be acting on the same signaling pathways that facilitate glucose uptake in skeletal muscle. However, the experimental studies are limited and scarce. The aim of this review is to highlight the current knowledge regarding the role of RONS, myokines and cytokines as potential signals that facilitate glucose uptake in skeletal muscle. In addition, we encourage researchers in the field to lead and undertake investigations to uncover the fundamentals of glucose uptake evoked by RONS, myokines, and cytokines.

Diabetes-associated bone complications lead to fragile bone mechanical strength and osteoporosis, aggravating the disease burden of patients. Advanced evidence shows that chronic hyperglycemia and metabolic intermediates, such as inflammatory factor, reactive oxygen species (ROS), and advanced glycation end products (AGEs), are regarded as dominant hazardous factors of bone complications, whereas the pathophysiological mechanisms are complex and controversial. By establishing a diabetic Sprague-Dawley (SD) rat model and diabetic bone loss cell model in vitro, we confirmed that diabetes impaired primary cilia and led to bone loss, while adding Icariin (ICA) could relieve the inhibitions. Mechanistically, ICA could scavenge ROS to maintain the mitochondrial and primary cilia homeostasis of osteoblasts. Intact primary cilia acted as anchoring and modifying sites of Gli2, thereby activating the primary cilia/Gli2/osteocalcin signaling pathway to promote osteoblast differentiation. All results suggest that ICA has potential as a therapeutic drug targeting bone loss induced by diabetes.

Cancer is one of the most important problems of modern societies. Recently, studies have reported the anticancer properties of rosiglitazone related to its ability to bind peroxisome proliferator receptor γ (PPARγ), which has various effects on cancer and can inhibit cell proliferation. In this study, we investigated the effect of new 4-thiazolidinone (4-TZD) hybrids Les-4369 and Les-3467 and their effect on reactive oxygen species (ROS) production, metabolic activity, lactate dehydrogenase (LDH) release, caspase-3 activity, and gene and protein expression in human foreskin fibroblast (BJ) cells and lung adenocarcinoma (A549) cells. The ROS production and caspase-3 activity were mainly increased in the micromolar concentrations of the studied compounds in both cell lines. Les-3467 and Les-4369 increased the mRNA expression of PPARG , P53 (tumor protein P53), and ATM (ATM serine/threonine kinase) in the BJ cells, while the mRNA expression of these genes (except PPARG ) was mainly decreased in the A549 cells treated with both of the tested compounds. Our results indicate a decrease in the protein expression of AhR, PPARγ, and PARP-1 in the BJ cells exposed to 1 µM Les-3467 and Les-4369. In the A549 cells, the protein expression of AhR, PPARγ, and PARP-1 increased in the treatment with 1 µM Les-3467 and Les-4369. We have also shown the PPARγ modulatory properties of Les-3467 and Les-4369. However, both compounds prove weak anticancer properties evidenced by their action at high concentrations and non-selective effects against BJ and A549 cells.

Irinotecan is the first line chemotherapy drug used for treatment of metastatic colorectal cancer worldwide. There is increasing evidence suggesting that liver damage, including steatosis and steatohepatitis, can be caused during the treatment involving irinotecan. However, molecular mechanisms by which irinotecan-induced liver injury remain elusive. In this study, we found that irinotecan treatment caused significant elevation of ALT, inflammation, and fat accumulation in the liver, which are associated with hepatic macrophage activation. Depletion of macrophages by clodronate liposome improved irinotecan induced liver injury and inflammatory response in mice. In vitro data indicated that irinotecan induced intracellular ROS production in primary hepatocyte and upregulating of toll-like receptor (TLRs) family expression in macrophages. Supernatant from irinotecan treated hepatocyte triggered macrophage activation and upregulation of TLRs in macrophage, and N-acetylcysteine (NAC) abolished these effects. By using co-culture system, we further revealed that irinotecan activated macrophage induced impairment of lipid metabolism and promoted apoptosis in hepatocyte and NAC prevented macrophage-induced cell death and partially revered impaired lipid metabolism in hepatocytes. By using the irinotecan liver injury model, we demonstrated that combining NAC with irinotecan prevented irinotecan-induced macrophage activation, TLR upregulation, liver injury, and partially prevented the accumulation of triglycerides in liver. Our results thus indicated that macrophages play a critical role in irinotecan-induced liver injury, and targeting ROS provides new options for development of hepatoprotective drugs in clinical practice.

Oocyte cryopreservation is widely used in assisted-reproductive technology and animal production. However, cryopreservation not only induces a massive accumulation of reactive oxygen species (ROS) in oocytes, but also leads to oxidative-stress-inflicted damage to mitochondria and the endoplasmic reticulum. These stresses lead to damage to the spindle, DNA, proteins, and lipids, ultimately reducing the developmental potential of oocytes both in vitro and in vivo. Although oocytes can mitigate oxidative stress via intrinsic antioxidant systems, the formation of ribonucleoprotein granules, mitophagy, and the cryopreservation-inflicted oxidative damage cannot be completely eliminated. Therefore, exogenous antioxidants such as melatonin and resveratrol are widely used in oocyte cryopreservation to reduce oxidative damage through direct or indirect scavenging of ROS. In this review, we discuss analysis of various oxidative stresses induced by oocyte cryopreservation, the impact of antioxidants against oxidative damage, and their underlying mechanisms. We hope that this literature review can provide a reference for improving the efficiency of oocyte cryopreservation.

Metabolic rewiring in glioblastoma (GBM) is linked to intra- and extracellular pH regulation. In this study, we sought to characterize the role of melatonin on intracellular pH modulation and metabolic consequences to identify the mechanisms of action underlying melatonin oncostatic effects on GBM tumor initiating cells. GBM tumor initiating cells were treated at different times with melatonin (1.5 and 3.0 mM). We analyzed melatonin’s functional effects on GBM proliferation, cell cycle, viability, stemness, and chemo-radiosensitivity. We then assessed the effects of melatonin on GBM metabolism by analyzing the mitochondrial and glycolytic parameters. We also measured the intracellular and extracellular pH. Finally, we tested the effects of melatonin on a mouse subcutaneous xenograft model. We found that melatonin downregulated LDHA and MCT4, decreasing lactate production and inducing a decrease in intracellular pH that was associated with an increase in ROS and ATP depletion. These changes blocked cell cycle progression and induced cellular death and we observed similar results in vivo. Melatonin’s cytotoxic effects on GBM were due, at least in part, to intracellular pH modulation, which has emerged as a newly identified mechanism, providing new insights into the oncostatic effect of melatonin on GBM.

The plasma membrane (sarcolemma) of skeletal muscle myofibers is susceptible to injury caused by physical and chemical stresses during normal daily movement and/or under disease conditions. These acute plasma membrane disruptions are normally compensated by an intrinsic membrane resealing process involving interactions of multiple intracellular proteins including dysferlin, annexin, caveolin, and Mitsugumin 53 (MG53)/TRIM72. There is new evidence for compromised muscle sarcolemma repair mechanisms in Amyotrophic Lateral Sclerosis (ALS). Mitochondrial dysfunction in proximity to neuromuscular junctions (NMJs) increases oxidative stress, triggering MG53 aggregation and loss of its function. Compromised membrane repair further worsens sarcolemma fragility and amplifies oxidative stress in a vicious cycle. This article is to review existing literature supporting the concept that ALS is a disease of oxidative-stress induced disruption of muscle membrane repair that compromise the integrity of the NMJs and hence augmenting muscle membrane repair mechanisms could represent a viable therapeutic strategy for ALS.

The capacity to induce tumour-cell specific apoptosis represents the most unique feature of the TNF receptor (TNFR) family member CD40. Recent studies on the signalling events triggered by its membrane-presented ligand CD40L (mCD40L) in normal and malignant epithelial cells have started to unravel an exquisite context and cell type specificity for the functional effects of CD40. Here, we demonstrate that, in comparison to other carcinomas, mCD40L triggered strikingly more rapid apoptosis in colorectal carcinoma (CRC) cells, underpinned by its ability to entrain two concurrently operating signalling axes. CD40 ligation initially activates TNFR-associated factor 3 (TRAF3) and subsequently NADPH oxidase (NOX)/Apoptosis signal-regulating kinase 1 (ASK1)-signalling and induction of reactive oxygen species (ROS) to mediate p38/JNK- and ROS-dependent cell death. At that point, p38/JNK signalling directly activates the mitochondrial pathway, and triggers rapid induction of intracellular TNF-related apoptosis-inducing ligand (TRAIL) that signals from internal compartments to initiate extrinsic caspase-10-asscociated apoptosis, leading to truncated Bid (tBid)-activated mitochondrial signalling. p38 and JNK are essential both for direct mitochondrial apoptosis induction and the TRAIL/caspase-10/tBid pathway, but their involvement follows functional hierarchy and temporally controlled interplay, as p38 function is required for JNK phosphorylation. By engaging both intrinsic and extrinsic pathways to activate apoptosis via two signals simultaneously, CD40 can accelerate CRC cell death. Our findings further unravel the multi-faceted properties of the CD40/mCD40L dyad, highlighted by the novel TNFR crosstalk that accelerates tumour cell-specific death, and may have implications for the use of CD40 as a therapeutic target.

Most conventional incubators used in cell culture do not regulate O2 levels, making the headspace O2 concentration ~18%. In contrast, most human tissues are exposed to 2–6% O2 (physioxia) in vivo. Accumulating evidence has shown that such hyperoxic conditions in standard cell culture practices affect a variety of biological processes. In this review, we discuss how supraphysiological O2 levels affect reactive oxygen species (ROS) metabolism and redox homeostasis, gene expression, replicative lifespan, cellular respiration, and mitochondrial dynamics. Furthermore, we present evidence demonstrating how hyperoxic cell culture conditions fail to recapitulate the physiological and pathological behavior of tissues in vivo, including cases of how O2 alters the cellular response to drugs, hormones, and toxicants. We conclude that maintaining physioxia in cell culture is imperative in order to better replicate in vivo-like tissue physiology and pathology, and to avoid artifacts in research involving cell culture.

Although the menstrual cycle driven by sex steroid hormones is an uncomplicated physiological process, it is important for female health, fertility and regenerative biology. However, our understanding of this unique type of tissue homeostasis remains unclear. Here, we examined the biological effects of mechanical force by evaluating the changing trend of extracellular matrix (ECM) stiffness, and the results suggested that ECM stiffness was reduced and that breaking of mechanotransduction delayed endometrium repair in a mouse model of simulated menses. We constructed an ECM stiffness interference model in vitro to explain the mechanical force conduction mechanism during endometrial regeneration. We discovered that ECM stiffness increased the expression and nuclear transfer of YAP, which improved the creation of a microenvironment, in a manner that induced proliferation and angiogenesis for endometrial repair by activating YAP. In addition, we observed that physiological endometrial hypoxia occurs during the menstrual cycle and that the expression of HIF-1α was increased. Mechanistically, in addition to the classical F-actin/YAP pathway, we also found that the ROS/HIF-1α/YAP axis was involved in the transmission of mechanical signals. This study provides novel insights into the essential menstrual cycle and presents an effective, nonhormonal treatment for menstrual disorders.

Although autophagy regulates the quality and quantity of cellular compartments, the regulatory mechanisms underlying peroxisomal autophagy (pexophagy) remain largely unknown. In this study, we identified several BRD4 inhibitors, including molibresib, a novel pexophagy inducer, via chemical library screening. Treatment with molibresib promotes loss of peroxisomes selectively, but not mitochondria, ER, or Golgi apparatus in HeLa cells. Consistently, depletion of BRD4 expression also induced pexophagy in RPE cells. In addition, the inhibition of BRD4 by molibresib increased autophagic degradation of peroxisome ATG7-dependency. We further found that molibresib produced reactive oxygen species (ROS), which potentiates ATM activation. Inhibition of ROS or ATM suppressed the loss of peroxisomes in molibresib-treated cells. Taken together, our data suggest that inhibition of BRD4 promotes pexophagy by increasing ROS and ATM activation.

Inflammation, oxidative stress, and copper (Cu) play an important role in cardiovascular disease, including atherosclerosis. We previously reported that cytosolic Cu chaperone antioxidant-1 (Atox1) translocates to the nucleus in response to inflammatory cytokines or exogenous Cu and that Atox1 is localized at the nucleus in the endothelium of inflamed atherosclerotic aorta. However, the roles of nuclear Atox1 and their function are poorly understood. Here we showed that Atox1 deficiency in ApoE−/− mice with a Western diet exhibited a significant reduction of atherosclerotic lesion formation. In vitro, adenovirus-mediated overexpression of nuclear-targeted Atox1 (Ad-Atox1-NLS) in cultured human endothelial cells (ECs) increased monocyte adhesion and reactive oxygen species (ROS) production compared to control cells (Ad-null). To address the underlying mechanisms, we performed genome-wide mapping of Atox1-regulated targets in ECs, using an unbiased systemic approach integrating sequencing data. Combination of ChIP-Seq and RNA-Seq analyses in ECs transfected with Ad-Atox1-NLS or Ad-null identified 1387 differentially expressed genes (DEG). Motif enrichment assay and KEGG pathway enrichment analysis revealed that 248 differentially expressed genes, including inflammatory and angiogenic genes, were regulated by Atox1-NLS, which was then confirmed by real-time qPCR. Among these genes, functional analysis of inflammatory responses identified CD137, CSF1, and IL5RA as new nuclear Atox1-targeted inflammatory genes, while CD137 is also a key regulator of Atox1-NLS-induced ROS production. These findings uncover new nuclear Atox1 downstream targets involved in inflammation and ROS production and provide insights into the nuclear Atox1 as a potential therapeutic target for the treatment of inflammatory diseases such as atherosclerosis.

The widespread environmental contaminant di-n-butyl phthalate (DBP) has been linked with reduced testosterone levels and adverse reproductive health outcomes in men. However, the underlying mechanisms of these anti-androgenic effects and the potential effects on other classes of steroid hormones remain to be elucidated. Here, we conducted mechanistic studies in human adrenocortical H295R cells exposed to 1–500 µM of DBP or its metabolite, mono-n-butyl phthalate (MBP), for 48 h. Quantification of steroid hormones in the cell medium by liquid chromatography-mass spectrometry revealed that both phthalates significantly decreased testosterone, androstenedione, corticosterone, and progesterone levels, in particular after dibutyryl-cyclic-AMP stimulation of steroidogenesis. Western blot analysis of key steroidogenic proteins showed that DBP induced a dose-dependent decrease of CYP11A1 and HSD3β2 levels, while MBP only significantly decreased CYP17A1 levels, indicating that the compounds affect early steps of the steroidogenesis differently. Both DBP and MBP exposure also lead to a dose-related decrease in HSD17β3, the enzyme which catalyzes the final step in the testosterone biosynthesis pathway, although these effects were not statistically significant. Interestingly, DBP increased the cortisol concentration, which may be due to the non-significant CYP11B1 increase in DBP-exposed cells. In contrast, MBP decreased cortisol concentration. Moreover, the analysis of superoxide generation and quantification of the protein oxidation marker nitrotyrosine demonstrated that DBP induced oxidative stress in H295R cells while MBP reduced protein nitrotyrosine levels. These findings confirm the anti-androgenic effects of DBP and MBP and reveal several differences in their toxicological mechanisms, with possible implications for future research on phthalate toxicity.

Mitochondrial disruption leads to the release of cytochrome c to activate caspase-9 and the downstream caspase cascade for the execution of apoptosis. However, cell death can proceed efficiently in the absence of caspase-9 following mitochondrial disruption, suggesting the existence of caspase-9-independent cell death mechanisms. Through a genome-wide siRNA library screening, we identified a network of genes that mediate caspase-9-independent cell death, through ROS production and Alox5-dependent membrane lipid peroxidation. Erk1-dependent phosphorylation of Alox5 is critical for targeting Alox5 to the nuclear membrane to mediate lipid peroxidation, resulting in nuclear translocation of cytolytic molecules to induce DNA damage and cell death. Consistently, double knockouts of caspase-9 and Alox5 in mice, but not deletion of either gene alone, led to significant T cell expansion with inhibited cell death, indicating that caspase-9- and Alox5-dependent pathways function in parallel to regulate T cell death in vivo. This unbiased whole-genome screening reveals an Erk1-Alox5-mediated pathway that promotes membrane lipid peroxidation and nuclear translocation of cytolytic molecules, leading to the execution of cell death in parallel to the caspase-9 signaling cascade.

Ferroptosis has recently been demonstrated to be a novel regulated non-apoptotic cell death characterized by iron-dependence and the accumulation of lipid peroxidation that results in membrane damage. Excessive iron induces ferroptosis by promoting the generation of both soluble and lipid ROS via an iron-dependent Fenton reaction and lipoxygenase (LOX) enzyme activity. Cytosolic glutathione peroxidase 4 (cGPX4) pairing with ferroptosis suppressor protein 1 (FSP1) and mitochondrial glutathione peroxidase 4 (mGPX4) pairing with dihydroorotate dehydrogenase (DHODH) serve as two separate defense systems to detoxify lipid peroxidation in the cytoplasmic as well as the mitochondrial membrane, thereby defending against ferroptosis in cells under normal conditions. However, disruption of these defense systems may cause ferroptosis. Emerging evidence has revealed that ferroptosis plays an essential role in the development of diverse cardiovascular diseases (CVDs), such as hemochromatosis-associated cardiomyopathy, doxorubicin-induced cardiotoxicity, ischemia/reperfusion (I/R) injury, heart failure (HF), atherosclerosis, and COVID-19–related arrhythmias. Iron chelators, antioxidants, ferroptosis inhibitors, and genetic manipulations may alleviate the aforementioned CVDs by blocking ferroptosis pathways. In conclusion, ferroptosis plays a critical role in the pathogenesis of various CVDs and suppression of cardiac ferroptosis is expected to become a potential therapeutic option. Here, we provide a comprehensive review on the molecular mechanisms involved in ferroptosis and its implications in cardiovascular disease.

The NRF2–KEAP1 system is a fundamental component of the cellular response that controls a great variety of transcriptional targets that are mainly involved in the regulation of redox homeostasis and multiple cytoprotective mechanisms that confer adaptation to the stress conditions. The pleiotropic response orchestrated by NRF2 is particularly relevant in the context of oncogenic activation, wherein this transcription factor acts as a key driver of tumor progression and cancer cells’ resistance to treatment. For this reason, NRF2 has emerged as a promising therapeutic target in cancer cells, stimulating extensive research aimed at the identification of natural, as well as chemical, NRF2 inhibitors. Excitingly, the influence of NRF2 on cancer cells’ biology extends far beyond its mere antioxidant function and rather encompasses a functional crosstalk with the mitochondrial network that can influence crucial aspects of mitochondrial homeostasis, including biogenesis, oxidative phosphorylation, metabolic reprogramming, and mitophagy. In the present review, we summarize the current knowledge of the reciprocal interrelation between NRF2 and mitochondria, with a focus on malignant tumors and cancer stem cells.

The loss-of-function conditions for an l(3)malignant brain tumour (l(3)mbt) in larvae reared at 29 °C results in malignant brain tumours and hyperplastic imaginal discs. Unlike the former that have been extensively characterised, little is known about the latter. Here we report the results of a study of the hyperplastic l(3)mbt mutant wing imaginal discs. We identify the l(3)mbt wing disc tumour transcriptome and find it to include genes involved in reactive oxygen species (ROS) metabolism. Furthermore, we show the presence of oxidative stress in l(3)mbt hyperplastic discs, even in apoptosis-blocked conditions, but not in l(3)mbt brain tumours. We also find that chemically blocking oxidative stress in l(3)mbt wing discs reduces the incidence of wing disc overgrowths. Our results reveal the involvement of oxidative stress in l(3)mbt wing discs hyperplastic growth.

Oxidative stress, caused by the accumulation of reactive oxygen species (ROS) during acute myocardial infarction (AMI), is one of the main factors leading to myocardial cell damage and programmed cell death. Phosphatidylinositol-3-kinase-AKT (PI3K-AKT) signaling is essential for regulating cell proliferation, differentiation, and apoptosis. Phosphoinositide-3-kinase (PI3K)-interacting protein 1 (PIK3IP1) is an intrinsic inhibitor of PI3K in various tissues, but its functional role during AMI remains unknown. In this study, the anti-ischemic role of PIK3IP1 in an in vitro AMI setting was evaluated using H9c2 cells. The MTT assay demonstrated that cell viability decreased significantly via treatment with H2O2 (200–500 μM). The TUNEL assay results revealed substantial cellular apoptosis following treatment with 200 μM H2O2. Under the same conditions, the expression levels of hypoxia-inducible factor (HIF-1α), endothelin-1 (ET-1), bcl-2-like protein 4 (BAX), and cleaved caspase-3 were elevated, whereas those of PIK3IP1, LC3II, p53, and Bcl-2 decreased significantly. PIK3IP1 overexpression inhibited H2O2-induced and PI3K-mediated apoptosis; however, PIK3IP1 knockdown reversed this effect, suggesting that PIK3IP1 functions as an anti-apoptotic molecule. To identify both the upstream and downstream molecules associated with PIK3IP1, ET-1 receptor type-specific antagonists (BQ-123 and BQ-788) and PI3K subtype-specific antagonists (LY294002 and IPI-549) were used to determine the participating isoforms. Co-immunoprecipitation was performed to identify the binding partners of PIK3IP1. Our results demonstrated that ROS-induced cardiac cell death may occur through the ETA-PI3Kγ-AKT axis, and that PIK3IP1 inhibits binding with both ETA and PI3Kγ. Taken together, these findings reveal that PIK3IP1 plays an anti-ischemic role by reducing the likelihood of programmed cell death via interaction with the ETA-PI3Kr-AKT axis.

Ischemia reperfusion injury (IRI) is a major obstacle in liver resection and liver transplantation. The initial step of IRI is mediated through ischemia which promotes the production of reactive oxygen species in Kupffer cells. This furthermore promotes the activation of pro-inflammatory signaling cascades, including tumor necrosis factor-alpha, IL-6, interferon, inducible nitric oxide synthase, TLR9/nuclear-factor kappa B pathway, and the production of damage-associated molecular patterns (DAMPs), such as ATP, histone, high mobility group box 1 (HMGB1), urate, mitochondrial formyl peptides and S100 proteins. With ongoing cell death of hepatocytes during the ischemic phase, DAMPs are built up and released into the circulation upon reperfusion. This promotes a cytokines/chemokine storm that attracts neutrophils and other immune cells to the site of tissue injury. The effect of IRI is further aggravated by the release of cytokines and chemokines, such as epithelial neutrophil activating protein (CXCL5), KC (CXCL1) and MIP-2 (CXCL2), the complement proteins C3a and C5a, mitochondrial-derived formyl peptides, leukotriene B4 and neutrophil extracellular traps (NETs) from migrating neutrophils. These NETs can also activate platelets and form Neutrophil-platelet microthrombi to further worsen ischemia in the liver. In this review we aim to summarize the current knowledge of mediators that promote liver IRI, and we will discuss the role of neutrophils and neutrophil extracellular traps in mediating IRI.

Cigarette smoke is a rich source of carcinogens and reactive oxygen species (ROS) that can damage macromolecules including DNA. Repair systems can restore DNA integrity. Depending on the duration or intensity of stress signals, cells may utilize various survival and adaptive mechanisms. ROS levels are kept in check through redundant detoxification processes controlled largely by antioxidant systems. This review covers and expands on the mechanisms available to cigarette smoke-exposed cancer cells for restoring the redox balance. These include multiple layers of transcriptional control, each of which is posited to be activated upon reaching a particular stress threshold, among them the NRF2 pathway, the AP-1 and NF-kB pathways, and, finally, TP53, which triggers apoptosis if extreme toxicity is reached. The review also discusses long noncoding RNAs, which have been implicated recently in regulating oxidative stress—with roles in ROS detoxification, the inflammatory response, oxidative stress-induced apoptosis, and mitochondrial oxidative phosphorylation. Lastly, the emerging roles of tunneling nanotubes in providing additional mechanisms for metabolic rescue and the regulation of redox imbalance are considered, further highlighting the expanded redox reset arsenal available to cells.

Reperfusion injury after extended ischemia accounts for approximately 50% of myocardial infarct size, and there is no standard therapy. HDAC inhibition reduces infarct size and enhances cardiomyocyte autophagy and PGC1α-mediated mitochondrial biogenesis when administered at the time of reperfusion. Furthermore, a specific autophagy-inducing peptide, Tat-Beclin 1 (TB), reduces infarct size when administered at the time of reperfusion. However, since SAHA affects multiple pathways in addition to inducing autophagy, whether autophagic flux induced by TB maintains mitochondrial homeostasis during ischemia/reperfusion (I/R) injury is unknown. We tested whether the augmentation of autophagic flux by TB has cardioprotection by preserving mitochondrial homeostasis both in vitro and in vivo. Wild-type mice were randomized into two groups: Tat-Scrambled (TS) peptide as the control and TB as the experimental group. Mice were subjected to I/R surgery (45 min coronary ligation, 24 h reperfusion). Autophagic flux, mitochondrial DNA (mtDNA), mitochondrial morphology, and mitochondrial dynamic genes were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with a simulated I/R injury to verify cardiomyocyte specificity. The essential autophagy gene, ATG7, conditional cardiomyocyte-specific knockout (ATG7 cKO) mice, and isolated adult mouse ventricular myocytes (AMVMs) were used to evaluate the dependency of autophagy in adult cardiomyocytes. In NRVMs subjected to I/R, TB increased autophagic flux, mtDNA content, mitochondrial function, reduced reactive oxygen species (ROS), and mtDNA damage. Similarly, in the infarct border zone of the mouse heart, TB induced autophagy, increased mitochondrial size and mtDNA content, and promoted the expression of PGC1α and mitochondrial dynamic genes. Conversely, loss of ATG7 in AMVMs and in the myocardium of ATG7 cKO mice abolished the beneficial effects of TB on mitochondrial homeostasis. Thus, autophagic flux is a sufficient and essential process to mitigate myocardial reperfusion injury by maintaining mitochondrial homeostasis and partly by inducing PGC1α-mediated mitochondrial biogenesis.

Retinopathy of prematurity (ROP) is a leading cause of vision impairment and blindness in premature infants. Oxidative stress is implicated in its pathophysiology. NADPH oxidase (NOX), a major enzyme responsible for reactive oxygen species (ROS) generation in endothelial cells, has been studied for its involvement in physiologic and pathologic angiogenesis. Erythropoietin (EPO) has gained interest recently due to its tissue protective and angiogenic effects, and it has been shown to act as an antioxidant. In this review, we summarize studies performed over the last five years regarding the role of various NOXs in physiologic and pathologic angiogenesis. We also discuss the effect of EPO in tissue and vasoprotection, and the intersection of EPO and NOX-mediated oxidative stress in angiogenesis and the pathophysiology of ROP.

p66Shc is a widely expressed protein that governs a variety of cardiovascular pathologies by generating, and exacerbating, pro-apoptotic ROS signals. Here, we review p66Shc’s connections to reactive oxygen species, expression, localization, and discuss p66Shc signaling and mitochondrial functions. Emphasis is placed on recent p66Shc mitochondrial function discoveries including structure/function relationships, ROS identity and regulation, mechanistic insights, and how p66Shc-cyt c interactions can influence p66Shc mitochondrial function. Based on recent findings, a new p66Shc mitochondrial function model is also put forth wherein p66Shc acts as a rheostat that can promote or antagonize apoptosis. A discussion of how the revised p66Shc model fits previous findings in p66Shc-mediated cardiovascular pathology follows.

Reducing the oxidative stress in neurons extends lifespan in Drosophila melanogaster, highlighting the crucial role of neuronal oxidative damage in lifespan determination. However, the source of the reactive oxygen species (ROS) that provoke oxidative stress in neurons is not clearly defined. Here, we identify dual oxidase (duox), a calcium-activated ROS-producing enzyme, as a lifespan determinant. Due to the lethality of duox homozygous mutants, we employed a duox heterozygote that exhibited normal appearance and movement. We found that duox heterozygous male flies, which were isogenized with control flies, demonstrated extended lifespan. Neuronal knockdown experiments further suggested that duox is crucial to oxidative stress in neurons. Our findings suggest duox to be a source of neuronal oxidative stress associated with animal lifespan.

Aging causes degenerative changes such as epigenetic changes and mitochondrial dysfunction in skeletal muscle. Exercise can upregulate muscle mitochondrial homeostasis and enhance antioxidant capacity and represents an effective treatment to prevent muscle aging. Epigenetic changes such as DNA methylation, histone posttranslational modifications, and microRNA expression are involved in the regulation of exercise-induced adaptive changes in muscle mitochondria. Reactive oxygen species (ROS) play an important role in signaling molecules in exercise-induced muscle mitochondrial health benefits, and strong evidence emphasizes that exercise-induced ROS can regulate gene expression via epigenetic mechanisms. The majority of mitochondrial proteins are imported into mitochondria from the cytosol, so mitochondrial homeostasis is regulated by nuclear epigenetic mechanisms. Exercise can reverse aging-induced changes in myokine expression by modulating epigenetic mechanisms. In this review, we provide an overview of the role of exercise-generated ROS in the regulation of mitochondrial homeostasis mediated by epigenetic mechanisms. In addition, the potential epigenetic mechanisms involved in exercise-induced myokine expression are reviewed.

Understanding early nervous system stress response mechanisms is crucial for studying developmental neurotoxicity and devising neuroprotective treatments. We used hiPSC-derived long-term self-renewing neuroepithelial stem (lt-NES) cells differentiated for up to 12 weeks as an in vitro model of human neural development. Following a transcriptome analysis to identify pathway alterations, we induced acute oxidative stress (OS) using tert-butyl hydroperoxide (TBHP) and assessed cell viability at different stages of neural differentiation. We studied NRF2 activation, autophagy, and proteasomal function to explore the contribution and interplay of these pathways in the acute stress response. With increasing differentiation, lt-NES cells showed changes in the expression of metabolic pathway-associated genes with engagement of the pentose phosphate pathway after 6 weeks, this was accompanied by a decreased susceptibility to TBHP-induced stress. Microarray analysis revealed upregulation of target genes of the antioxidant response KEAP1–NRF2–ARE pathway after 6 weeks of differentiation. Pharmacological inhibition of NRF2 confirmed its vital role in the increased resistance to stress. While autophagy was upregulated alongside differentiation, it was not further increased upon oxidative stress and had no effect on stress-induced cell loss and the activation of NRF2 downstream genes. In contrast, proteasome inhibition led to the aggravation of the stress response resulting in decreased cell viability, derangement of NRF2 and KEAP1 protein levels, and lacking NRF2-pathway activation. Our data provide detailed insight into the dynamic regulation and interaction of pathways involved in modulating stress responses across defined time points of neural differentiation.

Atherosclerosis is a chronic inflammatory arterial disease characterized by build-up of atheromatous plaque, which narrows the lumen of arteries. Hypercholesterolemia and excessive oxidative stress in arterial walls are among the main causative factors of atherosclerosis. Transient receptor potential channel M2 (TRPM2) is a Ca2+-permeable cation channel activated by oxidative stress. However, the role of TRPM2 in atherosclerosis in animal models is not well studied. In the present study, with the use of adeno-associated virus (AAV)-PCSK9 and TRPM2 knockout (TRPM2−/−) mice, we determined the role of TRPM2 in hypercholesterolemia-induced atherosclerosis. Our results demonstrated that TRPM2 knockout reduced atherosclerotic plaque area in analysis of En face Oil Red O staining of both whole aortas and aortic-root thin sections. Furthermore, TRPM2 knockout reduced the expression of CD68, α-SMA, and PCNA in the plaque region, suggesting a role of TRPM2 in promoting macrophage infiltration and smooth-muscle cell migration into the lesion area. Moreover, TRPM2 knockout reduced the expression of ICAM-1, MCP-1, and TNFα and decreased the ROS level in the plaque region, suggesting a role of TRPM2 in enhancing monocyte adhesion and promoting vascular inflammation. In bone-marrow-derived macrophages and primary cultured arterial endothelial cells, TRPM2 knockout reduced the production of inflammatory cytokines/factors and decreased ROS production. In addition, a TRPM2 antagonist N-(p-amylcinnamoyl) anthranilic acid (ACA) was able to inhibit atherosclerotic development in an ApoE−/− mouse model of atherosclerosis. Taken together, the findings of our study demonstrated that TRPM2 contributes to the progression of hypercholesterolemia-induced atherosclerosis. Mechanistically, TRPM2 channels may provide an essential link that can connect ROS to Ca2+ and inflammation, consequently promoting atherosclerotic progression.

Excessive exposure to solar radiation is associated with several deleterious effects on human skin. These effects vary from the occasional simple sunburn to conditions resulting from chronic exposure such as skin aging and cancers. Secondary metabolites from the plant kingdom, including phenolic compounds, show relevant photoprotective activities. In this study, we evaluated the potential photoprotective activity of a phytocomplex derived from three varieties of red orange (Citrus sinensis (L.) Osbeck). We used an in vitro model of skin photoaging on two human cell lines, evaluating the protective effects of the phytocomplex in the pathways involved in the response to damage induced by UVA-B. The antioxidant capacity of the extract was determined at the same time as evaluating its influence on the cellular redox state (ROS levels and total thiol groups). In addition, the potential protective action against DNA damage induced by UVA-B and the effects on mRNA and protein expression of collagen, elastin, MMP1, and MMP9 were investigated, including some inflammatory markers (TNF-α, IL-6, and total and phospho NFkB) by ELISA. The obtained results highlight the capacity of the extract to protect cells both from oxidative stress—preserving RSH (p < 0.05) content and reducing ROS (p < 0.01) levels—and from UVA-B-induced DNA damage. Furthermore, the phytocomplex is able to counteract harmful effects through the significant downregulation of proinflammatory markers (p < 0.05) and MMPs (p < 0.05) and by promoting the remodeling of the extracellular matrix through collagen and elastin expression. This allows the conclusion that red orange extract, with its strong antioxidant and photoprotective properties, represents a safe and effective option to prevent photoaging caused by UVA-B exposure.

This study addresses the eventual consequence of cytochrome c oxidase (CytOx) inhibition by ATP at high ATP/ADP ratio in isolated rat heart mitochondria. Earlier, it has been demonstrated that the mechanism of allosteric ATP inhibition of CytOx is one of the key regulations of mitochondrial functions. It is relevant that aiming to maintain a high ATP/ADP ratio for the measurement of CytOx activity effectuating the enzymatic inhibition as well as mitochondrial respiration, optimal concentration of mitochondria is critically important. Likewise, only at this concentration, were the differences in ΔΨm and ROS concentrations measured under various conditions significant. Moreover, when CytOx activity was inhibited in the presence of ATP, mitochondrial respiration and ΔΨm both remained static, while the ROS production was markedly decreased. Consubstantial results were found when the electron transport chain was inhibited by antimycin A, letting only CytOx remain functional to support the energy production. This seems to corroborate that the decrease in mitochondrial ROS production is solely the effect of ATP binding to CytOx which results in static respiration as well as membrane potential.

Cellular redox homeostasis is precisely balanced by generation and elimination of reactive oxygen species (ROS). ROS are not only capable of causing oxidation of proteins, lipids and DNA to damage cells but can also act as signaling molecules to modulate transcription factors and epigenetic pathways that determine cell survival and death. Hsp70 proteins are central hubs for proteostasis and are important factors to ameliorate damage from different kinds of stress including oxidative stress. Hsp70 members often participate in different cellular signaling pathways via their clients and cochaperones. ROS can directly cause oxidative cysteine modifications of Hsp70 members to alter their structure and chaperone activity, resulting in changes in the interactions between Hsp70 and their clients or cochaperones, which can then transfer redox signals to Hsp70-related signaling pathways. On the other hand, ROS also activate some redox-related signaling pathways to indirectly modulate Hsp70 activity and expression. Post-translational modifications including phosphorylation together with elevated Hsp70 expression can expand the capacity of Hsp70 to deal with ROS-damaged proteins and support antioxidant enzymes. Knowledge about the response and role of Hsp70 in redox homeostasis will facilitate our understanding of the cellular knock-on effects of inhibitors targeting Hsp70 and the mechanisms of redox-related diseases and aging.

In this study, we identified a novel pyrazole-based derivative (P3C) that displayed potent cytotoxicity against 27 human cancer cell lines derived from different tissue origins with 50% cytotoxic concentrations (CC50) in the low micromolar and nanomolar range, particularly in two triple-negative breast cancer (TNBC) cell lines (from 0.25 to 0.49 µM). In vitro assays revealed that P3C induces reactive oxygen species (ROS) accumulation leading to mitochondrial depolarization and caspase-3/7 and -8 activation, suggesting the participation of both the intrinsic and extrinsic apoptotic pathways. P3C caused microtubule disruption, phosphatidylserine externalization, PARP cleavage, DNA fragmentation, and cell cycle arrest on TNBC cells. In addition, P3C triggered dephosphorylation of CREB, p38, ERK, STAT3, and Fyn, and hyperphosphorylation of JNK and NF-kB in TNBC cells, indicating the inactivation of both p38MAPK/STAT3 and ERK1/2/CREB signaling pathways. In support of our in vitro assays, transcriptome analyses of two distinct TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells) treated with P3C revealed 28 genes similarly affected by the treatment implicated in apoptosis, oxidative stress, protein kinase modulation, and microtubule stability.

Chlorine (Cl 2 ) exposure poses a significant risk to ocular health, with the cornea being particularly susceptible to its corrosive effects. Antioxidants, known for their ability to neutralize reactive oxygen species (ROS) and alleviate oxidative stress, were explored as potential therapeutic agents to counteract chlorine-induced damage. In vitro experiments using human corneal epithelial cells showed decreased cell viability by chlorine-induced ROS production, which was reversed by antioxidant incubation. The mitochondrial membrane potential decreased due to both low and high doses of Cl 2 exposure; however, it was recovered through antioxidants. The wound scratch assay showed that antioxidants mitigated impaired wound healing after Cl 2 exposure. In vivo and ex vivo, after Cl 2 exposure, increased corneal fluorescein staining indicates damaged corneal epithelial and stromal layers of mice cornea. Likewise, Cl 2 exposure in human ex vivo corneas led to corneal injury characterized by epithelial fluorescein staining and epithelial erosion. However, antioxidants protected Cl 2 -induced damage. These results highlight the effects of Cl 2 on corneal cells using in vitro, ex vivo, and in vivo models while also underscoring the potential of antioxidants, such as vitamin A, vitamin C, resveratrol, and melatonin, as protective agents against acute chlorine toxicity-induced corneal injury. Further investigation is needed to confirm the antioxidants' capacity to alleviate oxidative stress and enhance the corneal healing process.

Mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodelling and wound healing, reducing tissue damage. Upon neutrophil activation, there is a release of myeloperoxidase (MPO), an oxidant enzyme. But little is known about the direct role of MSCs on MPO activity. The aim of this study was to investigate the effect of equine mesenchymal stem cells derived from muscle microinvasive biopsy (mdMSC) on the oxidant response of neutrophils and particularly on the activity of the myeloperoxidase released by stimulated equine neutrophils. After specific treatment (trypsin and washings in phosphate buffer saline), the mdMSCs were exposed to isolated neutrophils. The effect of the suspended mdMSCs was studied on the ROS production and the release of total and active MPO by stimulated neutrophils and specifically on the activity of MPO in a neutrophil-free model. Additionally, we developed a model combining adherent mdMSCs with neutrophils to study total and active MPO from the neutrophil extracellular trap (NET). Our results show that mdMSCs inhibited the ROS production, the activity of MPO released by stimulated neutrophils and the activity of MPO bound to the NET. Moreover, the co-incubation of mdMSCs directly with MPO results in a strong inhibition of the peroxidase activity of MPO, probably by affecting the active site of the enzyme. We confirm the strong potential of mdMSCs to lower the oxidant response of neutrophils. The novelty of our study is an evident inhibition of the activity of MPO by MSCs. The results indicated a new potential therapeutic approach of mdMSCs in the inhibition of MPO, which is considered as a pro-oxidant actor in numerous chronic and acute inflammatory pathologies.

Previous work examining the therapeutic efficacy of adjunct thymosin beta 4 (Tβ4) to ciprofloxacin for ocular infectious disease has revealed markedly reduced inflammation (inflammatory mediators and innate immune cells) with increased activation of wound healing pathways. Understanding the therapeutic mechanisms of action have further revealed a synergistic effect with ciprofloxacin to enhance bacterial killing along with a regulatory influence over macrophage effector cell function. As a natural extension of the aforementioned work, the current study uses an experimental model of P. aeruginosa-induced keratitis to examine the influence of Tβ4 regarding polymorphonuclear leukocyte (PMN/neutrophil) cellular function, contributing to improved disease response. Flow cytometry was utilized to phenotypically profile infiltrating PMNs after infection. The generation of reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and PMN apoptosis were investigated to assess the functional activities of PMNs in response to Tβ4 therapy. In vitro work using peritoneal-derived PMNs was similarly carried out to verify and extend our in vivo findings. The results indicate that the numbers of infiltrated PMNs into infected corneas were significantly reduced with adjunctive Tβ4 treatment. This was paired with the downregulated expression of proinflammatory markers on these cells, as well. Data generated from PMN functional studies suggested that the corneas of adjunctive Tβ4 treated B6 mice exhibit a well-regulated production of ROS, NETs, and limited PMN apoptosis. In addition to confirming the in vivo results, the in vitro findings also demonstrated that neutrophil elastase (NE) was unnecessary for NETosis. Collectively, these data provide additional evidence that adjunctive Tβ4 + ciprofloxacin treatment is a promising option for bacterial keratitis that addresses both the infectious pathogen and cellular-mediated immune response, as revealed by the current study.

Mitochondria are one of organelles that undergo significant changes associated with senescence. An increase in mitochondrial size is observed in senescent cells, and this increase is ascribed to the accumulation of dysfunctional mitochondria that generate excessive reactive oxygen species (ROS). Such dysfunctional mitochondria are prime targets for ROS-induced damage, which leads to the deterioration of oxidative phosphorylation and increased dependence on glycolysis as an energy source. Based on findings indicating that senescent cells exhibit mitochondrial metabolic alterations, a strategy to induce mitochondrial metabolic reprogramming has been proposed to treat aging and age-related diseases. In this review, we discuss senescence-related mitochondrial changes and consequent mitochondrial metabolic alterations. We assess the significance of mitochondrial metabolic reprogramming for senescence regulation and propose the appropriate control of mitochondrial metabolism to ameliorate senescence. Learning how to regulate mitochondrial metabolism will provide knowledge for the control of aging and age-related pathologies. Further research focusing on mitochondrial metabolic reprogramming will be an important guide for the development of anti-aging therapies, and will provide novel strategies for anti-aging interventions.

Porphyromonas gingivalis, a periodontal pathogen, has been proposed to cause blood vessel injury leading to cerebrovascular diseases such as stroke. Brain endothelial cells compose the blood-brain barrier that protects homeostasis of the central nervous system. However, whether P. gingivalis causes the death of endothelial cells and the underlying mechanisms remain unclear. This study aimed to investigate the impact and regulatory mechanisms of P. gingivalis infection in brain endothelial cells. We used bEnd.3 cells and primary mouse endothelial cells to assess the effects of P. gingivalis on endothelial cells. Our results showed that infection with live P. gingivalis, unlike heat-killed P. gingivalis, triggers brain endothelial cell death by inducing cell apoptosis. Moreover, P. gingivalis infection increased intracellular reactive oxygen species (ROS) production, activated NF-κB, and up-regulated the expression of IL-1β and TNF-α. Furthermore, N-acetyl-L-cysteine (NAC), a most frequently used antioxidant, treatment significantly reduced P. gingivalis-induced cell apoptosis and brain endothelial cell death. The enhancement of ROS production, NF-κB p65 activation, and proinflammatory cytokine expression was also attenuated by NAC treatment. The impact of P. gingivalis on brain endothelial cells was also confirmed using adult primary mouse brain endothelial cells (MBECs). In summary, our results showed that P. gingivalis up-regulates IL-1β and TNF-α protein expression, which consequently causes cell death of brain endothelial cells through the ROS/NF-κB pathway. Our results, together with the results of previous case-control studies and epidemiologic reports, strongly support the hypothesis that periodontal infection increases the risk of developing cerebrovascular disease.

Following efficient tumor therapy, some cancer cells may survive through a dormancy process, contributing to tumor recurrence and worse outcomes. Dormancy is considered a process where most cancer cells in a tumor cell population are quiescent with no, or only slow, proliferation. Recent advances indicate that redox mechanisms control the dormant cancer cell life cycle, including dormancy entrance, long-term dormancy, and metastatic relapse. This regulatory network is orchestrated mainly through redox modification on key regulators or global change of reactive oxygen species (ROS) levels in dormant cancer cells. Encouragingly, several strategies targeting redox signaling, including sleeping, awaking, or killing dormant cancer cells are currently under early clinical evaluation. However, the molecular mechanisms underlying redox control of the dormant cancer cell cycle are poorly understood and need further exploration. In this review, we discuss the underlying molecular basis of redox signaling in the cell life cycle of dormant cancer and the potential redox-based targeting strategies for eliminating dormant cancer cells.

The Transforming Growth Factor-beta (TGF-β) pathway plays essential roles in liver development and homeostasis and become a relevant factor involved in different liver pathologies, particularly fibrosis and cancer. The family of NADPH oxidases (NOXs) has emerged in recent years as targets of the TGF-β pathway mediating many of its effects on hepatocytes, stellate cells and macrophages. This review focuses on how the axis TGF-β/NOXs may regulate the biology of different liver cells and how this influences physiological situations, such as liver regeneration, and pathological circumstances, such as liver fibrosis and cancer. Finally, we discuss whether NOX inhibitors may be considered as potential therapeutic tools in liver diseases.

Von Hippel–Lindau disease (VHL) is a rare hereditary disease characterized by the predisposal to develop different types of highly vascularized tumors. VHL patients carry a VHL mutation that causes partial lack of functional VHL protein (pVHL) in all cells, and a total lack thereof in cells harboring a second hit mutation. Absence of pVHL generates a prolonged state of pseudo-hypoxia in the cell due to accumulation of hypoxia inducible factor, an important transcription factor regulating pro-tumorigenic genes. The work here presented focuses on characterizing the endothelium of VHL patients, by means of blood outgrowth endothelial cells (BOECs). Transcriptome analysis of VHL-derived BOECs, further supported by in vitro assays, shows that these cells are at a disadvantage, as evidenced by loss of cell adhesion capacity, angiogenesis defects, and immune response and oxidative metabolic gene downregulation, which induce oxidative stress. These results suggest that the endothelium of VHL patients is functionally compromised and more susceptible to tumor development. These findings contribute to shedding light on the vascular landscape of VHL patients preceding the second hit mutation in the VHL gene. This knowledge could be useful in searching for new therapies for these patients and other vascular diseases.