Orthologs of NOX5 and EC-SOD/SOD3: dNox and dSod3 Impact Egg Hardening Process and Egg Laying in Reproductive Function of Drosophila melanogaster.

The occurrence of ovarian dysfunction is often due to the imbalance between the formation of reactive oxygen species (ROS) and the ineffectiveness of the antioxidative defense mechanisms. Primary sources of ROS are respiratory electron transfer and the activity of NADPH oxidases (NOX) while superoxide dismutases (SOD) are the main key regulators that control the levels of ROS and reactive nitrogen species intra- and extracellularly. Because of their central role SODs are the subject of research on human ovarian dysfunction but sample acquisition is low. The high degree of cellular and molecular similarity between Drosophila melanogaster ovaries and human ovaries provides this model organism with the best conditions for analyzing the role of ROS during ovarian function. In this study we clarify the localization of the ROS-producing enzyme dNox within the ovaries of Drosophila melanogaster and by a tissue-specific knockdown we show that dNox-derived ROS are involved in the chorion hardening process. Furthermore, we analyze the dSod3 localization and show that reduced activity of dSod3 impacts egg-laying behavior but not the chorion hardening process.

Structural basis of human NOX5 activation.

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

Proteasome inhibitors induce apoptosis by superoxide anion generation via NADPH oxidase 5 in human neuroblastoma SH-SY5Y cells.

The ubiquitin-proteasome system (UPS) is a major proteolytic system that plays an important role in the regulation of various cell processes, such as cell cycle, stress response, and transcriptional regulation, especially in neurons, and dysfunction of UPS is considered to be a cause of neuronal cell death in neurodegenerative diseases. However, the mechanism of neuronal cell death caused by UPS dysfunction has not yet been fully elucidated. In this study, we investigated the mechanism of neuronal cell death induced by proteasome inhibitors using human neuroblastoma SH-SY5Y cells. Z-Leu-D-Leu-Leu-al (MG132), a proteasome inhibitor, induced apoptosis in SH-SY5Y cells in a concentration- and time-dependent manner. Antioxidants N-acetylcysteine and EUK-8 attenuated MG132-induced apoptosis. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase (NOX), an enzyme that produces superoxide anions, also attenuated MG132-induced apoptosis. It was also found that MG132 treatment increased the expression of NOX5, a NOX family member, and that siRNA-mediated silencing of NOX5 and BAPTA-AM, which inhibits NOX5 by chelating calcium, suppressed MG132-induced apoptosis and production of reactive oxygen species in SH-SY5Y cells. These results suggest that MG132 induces apoptosis in SH-SY5Y cells through the production of superoxide anion by NOX5.

Endothelial NOX5 overexpression induces changes in the cardiac gene profile: potential impact in myocardial infarction?

Cardiovascular diseases and the ischemic heart disease specifically constitute the main cause of death worldwide. The ischemic heart disease may lead to myocardial infarction, which in turn triggers numerous mechanisms and pathways involved in cardiac repair and remodeling. Our goal in the present study was to characterize the effect of the NADPH oxidase 5 (NOX5) endothelial expression in healthy and infarcted knock-in mice on diverse signaling pathways. The mechanisms studied in the heart of mice were the redox pathway, metalloproteinases and collagen pathway, signaling factors such as NFκB, AKT or Bcl-2, and adhesion molecules among others. Recent studies support that NOX5 expression in animal models can modify the environment and predisposes organ response to harmful stimuli prior to pathological processes. We found many alterations in the mRNA expression of components involved in cardiac fibrosis as collagen type I or TGF-β and in key players of cardiac apoptosis such as AKT, Bcl-2, or p53. In the heart of NOX5-expressing mice after chronic myocardial infarction, gene alterations were predominant in the redox pathway (NOX2, NOX4, p22phox, or SOD1), but we also found alterations in VCAM-1 and β-MHC expression. Our results suggest that NOX5 endothelial expression in mice preconditions the heart, and we propose that NOX5 has a cardioprotective role. The correlation studies performed between echocardiographic parameters and cardiac mRNA expression supported NOX5 protective action. (AU)

Endothelial NOX5 overexpression induces changes in the cardiac gene profile: potential impact in myocardial infarction?

Cardiovascular diseases and the ischemic heart disease specifically constitute the main cause of death worldwide. The ischemic heart disease may lead to myocardial infarction, which in turn triggers numerous mechanisms and pathways involved in cardiac repair and remodeling. Our goal in the present study was to characterize the effect of the NADPH oxidase 5 (NOX5) endothelial expression in healthy and infarcted knock-in mice on diverse signaling pathways. The mechanisms studied in the heart of mice were the redox pathway, metalloproteinases and collagen pathway, signaling factors such as NFκB, AKT or Bcl-2, and adhesion molecules among others. Recent studies support that NOX5 expression in animal models can modify the environment and predisposes organ response to harmful stimuli prior to pathological processes. We found many alterations in the mRNA expression of components involved in cardiac fibrosis as collagen type I or TGF-ß and in key players of cardiac apoptosis such as AKT, Bcl-2, or p53. In the heart of NOX5-expressing mice after chronic myocardial infarction, gene alterations were predominant in the redox pathway (NOX2, NOX4, p22phox, or SOD1), but we also found alterations in VCAM-1 and ß-MHC expression. Our results suggest that NOX5 endothelial expression in mice preconditions the heart, and we propose that NOX5 has a cardioprotective role. The correlation studies performed between echocardiographic parameters and cardiac mRNA expression supported NOX5 protective action.

Structure, regulation, and physiological functions of NADPH oxidase 5 (NOX5)

NOX5 is the last member of the NADPH oxidase (NOXs) family to be identified and presents some specific characteristics differing from the rest of the NOXs. It contains four Ca2+ binding domains at the N-terminus and its activity is regulated by the intracellular concentration of Ca2+. NOX5 generates superoxide (O2•−) using NADPH as a substrate, and it modulates functions related to processes in which reactive oxygen species (ROS) are involved. Those functions appear to be detrimental or beneficial depending on the level of ROS produced. For example, the increase in NOX5 activity is related to the development of various oxidative stress-related pathologies such as cancer, cardiovascular, and renal diseases. In this context, pancreatic expression of NOX5 can negatively alter insulin action in high-fat diet-fed transgenic mice. This is consistent with the idea that the expression of NOX5 tends to increase in response to a stimulus or a stressful situation, generally causing a worsening of the pathology. On the other hand, it has also been suggested that it might have a positive role in preparing the body for metabolic stress, for example, by inducing a protective adipose tissue adaptation to the excess of nutrients supplied by a high-fat diet. In this line, its endothelial overexpression can delay lipid accumulation and insulin resistance development in obese transgenic mice by inducing the secretion of IL-6 followed by the expression of thermogenic and lipolytic genes. However, as NOX5 gene is not present in rodents and human NOX5 protein has not been crystallized, its function is still poorly characterized and further extensive research is required. (AU)

Structure, regulation, and physiological functions of NADPH oxidase 5 (NOX5).

NOX5 is the last member of the NADPH oxidase (NOXs) family to be identified and presents some specific characteristics differing from the rest of the NOXs. It contains four Ca2+ binding domains at the N-terminus and its activity is regulated by the intracellular concentration of Ca2+. NOX5 generates superoxide (O2•-) using NADPH as a substrate, and it modulates functions related to processes in which reactive oxygen species (ROS) are involved. Those functions appear to be detrimental or beneficial depending on the level of ROS produced. For example, the increase in NOX5 activity is related to the development of various oxidative stress-related pathologies such as cancer, cardiovascular, and renal diseases. In this context, pancreatic expression of NOX5 can negatively alter insulin action in high-fat diet-fed transgenic mice. This is consistent with the idea that the expression of NOX5 tends to increase in response to a stimulus or a stressful situation, generally causing a worsening of the pathology. On the other hand, it has also been suggested that it might have a positive role in preparing the body for metabolic stress, for example, by inducing a protective adipose tissue adaptation to the excess of nutrients supplied by a high-fat diet. In this line, its endothelial overexpression can delay lipid accumulation and insulin resistance development in obese transgenic mice by inducing the secretion of IL-6 followed by the expression of thermogenic and lipolytic genes. However, as NOX5 gene is not present in rodents and human NOX5 protein has not been crystallized, its function is still poorly characterized and further extensive research is required.

Ca2+ -binding-region-dependent cell surface localization of NADPH oxidase Nox5.

Six gene splice variants of superoxide-generating NADPH oxidase 5 (Nox5) have been identified in humans, and they differ in the sequence of their N-terminal cytoplasmic domains, which comprise four EF-hand motifs. Here, we demonstrated that the Ca2+ -dependent association and dissociation between the N- and C-terminal cytoplasmic domains of the Nox5ß variant are affected by the alanine substitution of the conserved Ile-113 or Leu-115 at the connecting loop between the third and fourth EF-hand motifs. These substitutions impair the cell surface localization of Nox5ß. In addition, the Nox5ε/S variant, lacking all EF-hand motifs, does not localize to the plasma membrane. Thus, the Ca2+ -sensitive intramolecular interaction determines the Nox5 subcellular localization, that is, whether Nox5 variants generate superoxide in the extracellular or intracellular space.

Impact of calcium and reactive oxygen species on human sperm function: Role of NOX5.

NOX5 is introduced as a new therapeutic target for infertility treatment. This study aimed to compare the basal and stimulated reactive oxygen species (ROS) production and sperm function in human teratozoospermic (n = 15) and normozoospermic (n = 17) semen samples following calcium overload and NOX5 activation. Washed spermatozoa incubated for 1 h under five various conditions: control group, adding a calcium ionophore A23187, phorbol myristate acetate (PMA), A23187 + PMA, and diphenylene iodonium (DPI) + A23187 + PMA. ROS generation was measured immediately after treatment for 30 min. Motility, viability, acrosome reaction, and apoptosis were evaluated after 1-h incubation. ROS production significantly increased when A23187 or PMA was added to the sperm medium. DPI had suppressive effects on ROS generation. Progressive and total motility significantly decreased following calcium elevation and NOX5 activation, which was somewhat returned by DPI. Necrotic and live cells in teratozoospermia was, respectively, higher and lower than normozoospermia samples. Incubation with A23187 significantly increased the percentage of early and late apoptosis. Teratozoosperm are more vulnerable than normal spermatozoa, and produce more basal and stimulated ROS. It seems that calcium overload induces apoptosis in spermatozoa and loss of viability through MPT pore opening and increased intracellular ROS.

Independent of Renox, NOX5 Promotes Renal Inflammation and Fibrosis in Diabetes by Activating ROS-Sensitive Pathways.

Excessive production of renal reactive oxygen species (ROS) plays a major role in diabetic kidney disease (DKD). Here, we provide key findings demonstrating the predominant pathological role of the pro-oxidant enzyme NADPH oxidase 5 (NOX5) in DKD, independent of the previously characterized NOX4 pathway. In patients with diabetes, we found increased expression of renal NOX5 in association with enhanced ROS formation and upregulation of ROS-sensitive factors early growth response 1 (EGR-1), protein kinase C-α (PKC-α), and a key metabolic gene involved in redox balance, thioredoxin-interacting protein (TXNIP). In preclinical models of DKD, overexpression of NOX5 in Nox4-deficient mice enhances kidney damage by increasing albuminuria and augmenting renal fibrosis and inflammation via enhanced ROS formation and the modulation of EGR1, TXNIP, ERK1/2, PKC-α, and PKC-ε. In addition, the only first-in-class NOX inhibitor, GKT137831, appears to be ineffective in the presence of NOX5 expression in diabetes. In vitro, silencing of NOX5 in human mesangial cells attenuated upregulation of EGR1, PKC-α, and TXNIP induced by high glucose levels, as well as markers of inflammation (TLR4 and MCP-1) and fibrosis (CTGF and collagens I and III) via reduction in ROS formation. Collectively, these findings identify NOX5 as a superior target in human DKD compared with other NOX isoforms such as NOX4, which may have been overinterpreted in previous rodent studies.

Central role of c-Src in NOX5- mediated redox signalling in vascular smooth muscle cells in human hypertension.

AIMS: NOX-derived reactive oxygen species (ROS) are mediators of signalling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here, we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction. METHODS AND RESULTS: VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signalling molecules, and actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were down-regulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs, NOX5 was up-regulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1), and hyperphosphorylation (c-Src, PKC, ERK1/2, MLC20) of signalling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2. CONCLUSION: We define NOX5/ROS/c-Src as a novel feedforward signalling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury.

The evolutionary origins of peroxynitrite signalling.

Peroxynitrite is a reactive intermediate formed in vivo through uncatalysed reaction of superoxide and nitric oxide radicals. Despite significant interest in detecting peroxynitrite in vivo and understanding its production, little attention has been given to the evolutionary origins of peroxynitrite signalling. Herein we focus on two enzymes that are key to the biosynthesis of superoxide and nitric oxide, NADPH oxidase 5 (NOX5) and endothelial nitric oxide synthase (eNOS), respectively. Multiple sequence alignments of both enzymes including homologues from all domains of life, coupled with a phylogenetic analysis of NOX5, suggest eNOS and NOX5 are present in animals as the result of horizontal gene transfer from ancestral cyanobacteria to ancestral eukaryotes. Therefore, biochemical studies from other laboratories on a NOX5 homologue in Cylindrospermum stagnale and an eNOS homologue in Synechococcus sp. PCC 7335 are likely to be of relevance to human NOX5 and eNOS and to the production of superoxide, nitric oxide and peroxynitrite in humans.

NOX5 mediates the crosstalk between tumor cells and cancer-associated fibroblasts via regulating cytokine network.

Activation of cancer-associated fibroblasts (CAFs) is a crucial feature for tumor malignancy. The reciprocal interplay between tumor cells and CAFs not only facilitates tumor progression and metastasis but also sustains the tumor-promoting function of CAFs. Nevertheless, how tumor cells readily adapt to these functional CAFs is still unclear. NADPH oxidase 5 (NOX5) is a strong reactive oxygen species producer overexpressed in esophageal squamous cell carcinoma (ESCC) cells. In this study, we showed that NOX5-positive ESCC cells induced normal fibroblasts (NFs) or adipose-derived mesenchymal stem cells (MSCs) to express the marker of CAFs-α smooth muscle actin. Moreover, these tumor cells reprogrammed the cytokine profile of the activated CAFs, which further stimulated NFs or MSCs to CAFs and induced lymphangiogenesis to facilitate ESCC malignancy. NOX5 activated intratumoral Src/nuclear factor-κB signaling to stimulate secretion of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and lactate from tumor cells. Subsequently, TNF-α, IL-1ß, and lactate activated CAFs, and facilitated the secretion of IL-6, IL-7, IL-8, CCL5, and transforming growth factor-ß1 from CAFs. These CAFs-derived cytokines reciprocally induced the progression of NOX5-positive ESCC cells. Our findings together indicate that NOX5 serves as the driving oncoprotein to provide a niche that is beneficial for tumor malignant progression.

NADPH oxidase 5 has a crucial role in cellular motility of colon cancer cells.

NADPH oxidases (NOXs) are a family of transmembrane proteins that generate reactive oxygen species. It was previously reported that patients with colon cancer who had high NOX5 expression had poor prognosis. However, no studies have investigated the cellular functions of NOX5 in colon cancer. The present study aimed to clarify the relationship between NOX5 and cancer development using an in vitro model. Reverse transcription­quantitative PCR was performed to determine the NOX5 expression levels of colon cancer cell lines. NOX5­knockdown experiments were conducted, and the effect on cell proliferation, migration, and invasion were analyzed. In addition, mRNA microarray was conducted to assess changes in gene profile. NOX5 mRNA expression was high in HCT116 cells and moderate in SW48 cells. NOX5 knockdown significantly inhibited cell migration and invasion in both HCT116 and SW48 cells; however, NOX5 knockdown reduced cell proliferation in only HCT116 cells. mRNA microarrays revealed a strong relationship between NOX5 expression levels and integrin­linked kinase signaling pathways. The NOX5 expression in colon cancer cells affected cancer progression, especially cell motility. NOX5 may be a novel therapeutic target for the future development of treatments for colon cancer.

Lysophosphatidylcholine induces oxidative stress in human endothelial cells via NOX5 activation - implications in atherosclerosis.

OBJECTIVE: The mechanisms involved in NOX5 activation in atherosclerotic processes are not completely understood. The present study tested the hypothesis that lysophosphatidylcholine (LPC), a proatherogenic component of oxLDL, induces endothelial calcium influx, which drives NOX5-dependent reactive oxygen species (ROS) production, oxidative stress, and endothelial cell dysfunction. APPROACH: Human aortic endothelial cells (HAEC) were stimulated with LPC (10-5 M, for different time points). Pharmacological inhibition of NOX5 (Melittin, 10-7 M) and NOX5 gene silencing (siRNA) was used to determine the role of NOX5-dependent ROS production in endothelial oxidative stress induced by LPC. ROS production was determined by lucigenin assay and electron paramagnetic spectroscopy (EPR), calcium transients by Fluo4 fluorimetry, and NOX5 activity and protein expression by pharmacological assays and immunoblotting, respectively. RESULTS: LPC increased ROS generation in endothelial cells at short (15 min) and long (4 h) stimulation times. LPC-induced ROS was abolished by a selective NOX5 inhibitor and by NOX5 siRNA. NOX1/4 dual inhibition and selective NOX1 inhibition only decreased ROS generation at 4 h. LPC increased HAEC intracellular calcium, important for NOX5 activation, and this was blocked by nifedipine and thapsigargin. Bapta-AM, selective Ca2+ chelator, prevented LPC-induced ROS production. NOX5 knockdown decreased LPC-induced ICAM-1 mRNA expression and monocyte adhesion to endothelial cells. CONCLUSION: These results suggest that NOX5, by mechanisms linked to increased intracellular calcium, is key to early LPC-induced endothelial oxidative stress and pro-inflammatory processes. Since these are essential events in the formation and progression of atherosclerotic lesions, the present study highlights an important role for NOX5 in atherosclerosis.

Endothelial Nox5 Expression Modulates Glucose Uptake and Lipid Accumulation in Mice Fed a High-Fat Diet and 3T3-L1 Adipocytes Treated with Glucose and Palmitic Acid.

Obesity is a global health issue associated with insulin resistance and altered lipid homeostasis. It has been described that reactive oxygen species (ROS) derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity are involved in the development of these pathologies. The present study describes the role of endothelial NOX5 expression over adipose tissue by using two experimental systems: NOX5 conditional knock-in mice fed with a high-fat diet and 3T3-L1 adipocytes cultured with conditioned media of NOX5-expressing endothelial cells previously treated with glucose and palmitic acid. Animals expressing NOX5 presented lower body weight gain and less mesenteric and epididymal adipose mass compared to control mice fed with the same diet. NOX5-expressing mice also showed significantly lower glycaemia and improved insulin-induced glucose uptake. In addition, Glut4 and Caveolin 1 (Cav1) expression were significantly increased in the adipose tissue of these animals. Likewise, 3T3-L1 adipocytes treated with conditioned media from NOX5-expressing endothelial cells, incubated with high glucose and palmitic acid, presented a reduction in lipid accumulation and an increase in glucose uptake. Moreover, a significant increase in the expression of Glut4 and Cav1 was also detected in these cells. Taken together, all these data support that, in response to a highly caloric diet, NOX5 endothelial activity may regulate glucose sensitivity and lipid homeostasis in the adipose tissue.

NOX5-induced uncoupling of endothelial NO synthase is a causal mechanism and theragnostic target of an age-related hypertension endotype.

Hypertension is the most important cause of death and disability in the elderly. In 9 out of 10 cases, the molecular cause, however, is unknown. One mechanistic hypothesis involves impaired endothelium-dependent vasodilation through reactive oxygen species (ROS) formation. Indeed, ROS forming NADPH oxidase (Nox) genes associate with hypertension, yet target validation has been negative. We re-investigate this association by molecular network analysis and identify NOX5, not present in rodents, as a sole neighbor to human vasodilatory endothelial nitric oxide (NO) signaling. In hypertensive patients, endothelial microparticles indeed contained higher levels of NOX5-but not NOX1, NOX2, or NOX4-with a bimodal distribution correlating with disease severity. Mechanistically, mice expressing human Nox5 in endothelial cells developed-upon aging-severe systolic hypertension and impaired endothelium-dependent vasodilation due to uncoupled NO synthase (NOS). We conclude that NOX5-induced uncoupling of endothelial NOS is a causal mechanism and theragnostic target of an age-related hypertension endotype. Nox5 knock-in (KI) mice represent the first mechanism-based animal model of hypertension.

An Integrative Gene Expression and Mathematical Flux Balance Analysis Identifies Targetable Redox Vulnerabilities in Melanoma Cells.

Melanomas harboring BRAF mutations can be treated with BRAF inhibitors (BRAFi), but responses are varied and tumor recurrence is inevitable. Here we used an integrative approach of experimentation and mathematical flux balance analyses in BRAF-mutated melanoma cells to discover that elevated antioxidant capacity is linked to BRAFi sensitivity in melanoma cells. High levels of antioxidant metabolites in cells with reduced BRAFi sensitivity confirmed this conclusion. By extending our analyses to other melanoma subtypes in The Cancer Genome Atlas, we predict that elevated redox capacity is a general feature of melanomas, not previously observed. We propose that redox vulnerabilities could be exploited for therapeutic benefits and identify unsuspected combination targets to enhance the effects of BRAFi in any melanoma, regardless of mutational status. SIGNIFICANCE: An integrative bioinformatics, flux balance analysis, and experimental approach identify targetable redox vulnerabilities and show the potential for modulation of cancer antioxidant defense to augment the benefits of existing therapies in melanoma.

Epoxyazadiradione exhibit activities in head and neck squamous cell carcinoma by targeting multiple pathways.

The head and neck squamous cell carcinoma (HNSCC) constitute about 90% of all head and neck cancers. HNSCC falls in the top 10 cancers in men globally. Epoxyazadiradione (EPA) and Azadiradione (AZA) are the limonoids derived from the medicinal plant Azadirachta indica (popularly known as Neem). Whether or not the limonoids exhibit activities against HNSCC and the associated mechanism remains elusive. Herein, we demonstrate that EPA exhibits stronger activity in HNSCC in comparison to AZA. The limonoids obeyed the Lipinski's rule of 5. EPA exhibited activities in a variety of HNSCC lines like suppression of the proliferation and the induction of apoptosis. The limonoid suppressed the level of proteins associated with anti-apoptosis (survivin, Bcl-2, Bcl-xL), proliferation (cyclin D1), and invasion (MMP-9). Further, the expression of proapoptotic Bax and caspase-9 cleavage was induced by the limonoid. Exposure of EPA induced reactive oxygen species (ROS) generation in the FaDu cells. N-acetyl-L-cysteine (ROS scavenger) abrogated the down-regulation of tumorigenic proteins caused by EPA exposure. EPA induced NOX-5 while suppressing the expression of programmed death-ligand 1 (PD-L1). Further, hydrogen peroxide induced NF-κB-p65 nuclear translocation and EPA inhibited the translocation. Finally, EPA modulated the expression of lncRNAs in HNSCC lines. Overall, these results have shown that EPA exhibit activities against HNSCC by targeting multiple cancer related signalling molecules. Currently, we are evaluating the efficacy of this molecule in mice models.