Design, synthesis, in silico and biological evaluation of new indole based oxadiazole derivatives targeting estrogen receptor alpha.

A series of new indole-oxadiazole derivatives was designed and synthesized to develop potential anti-breast cancer agents. The compounds exhibited significant inhibitory activity with IC50 values ranging from 1.78 to 19.74 µM against ER-positive human breast cancer (BC) cell lines T-47D and MCF-7. Among them, compounds (5a, 5c, 5e-5h, 5j-5o) displayed superior activity against ER-α dominant (ratio of ER-α/ER-ß is 9/1) T-47D cells compared to the standard drug bazedoxifene (IC50 = 12.78 ± 0.92 µM). Compounds 5c and 5o exhibited remarkable anti-proliferative activity with IC50 values of 3.24 ± 0.46 and 1.72 ± 1.67 µM against T-47D cells, respectively. Further, compound 5o manifested 1589-fold higher ER-α binding affinity (213.4 pM) relative to bazedoxifene (339.2 nM) in a competitive ER-α binding assay, while compound 5c showed a binding affinity of 446.6 nM. The Western blot analysis proved that both compounds influenced the ER-α protein's expression, impeding its subsequent transactivation and signalling pathway within T-47D cells. Additionally, a molecular docking study suggests that compounds 5c and 5o bind in such a fashion that induces conformational changes in the protein, culminating in their antagonistic effect. Also, pharmacokinetic profiles showed that all compounds have drug-like properties. Further, molecular dynamic (MD) simulations and density functional theory (DFT) analysis confirmed the stability, conformational behaviour, reactivity, and biological feasibility of compounds 5c and 5o. In conclusion, based on our findings, compounds 5c and 5o, which exhibit significant ER-α antagonistic activity, can act as potential lead compounds for developing anti-breast cancer agents.

Design, synthesis, in vitro and in silico evaluation of indole-based tetrazole derivatives as putative anti-breast cancer agents.

A series of new indole-tetrazole derivatives were designed and synthesized to develop potential anti-breast cancer agents. The compounds exhibited in vitro anti-proliferative activity against ER-α positive T-47D (IC50 = 3.82-24.43 µM), MCF-7 (IC50 = 3.08-22.65 µM), and ER-α negative MDA-MB-231 (IC50 = 7.69-19.4 µM) human breast cancer cell lines. Compounds 5d and 5f displayed significant anti-proliferative activity compared to bazedoxifene (IC50 = 14.23 ± 0.68 µM), with IC50 values of 10.00 ± 0.59 and 3.83 ± 0.74 µM, respectively, against the ER-α dominant T-47D cell line. Also, both compounds showed non-significant cytotoxicity against normal cells HEK-293. Further, the ER-α binding affinity of 5d and 5f was assessed through a fluorescence polarization-based competitive binding assay, where 5d and 5f have shown significant binding with IC50 = 5.826 and 110.6 nM, respectively, as compared to the standard drug bazedoxifene (IC50 = 339.2 nM). Western blot analysis confirmed that compound 5d reduced ER-α protein expression in T-47D cells, hindering its transactivation and signalling pathways. Additionally, a molecular docking study suggests that compounds 5d and 5f bind in such a fashion that induces conformational changes in the protein, culminating in their antagonistic effect. Pharmacokinetic profiles showed that the compounds possessed drug-like properties. Furthermore, molecular dynamics simulation studies establish the dynamic stability and conformational behaviour of the ER-α protein and ligand complex of both compounds. Additionally, 5d and 5f ensure biological feasibility as per their DFT analysis through HOMO-LUMO energy gap analysis. In conclusion, compounds 5d and 5f, exhibiting significant ER-α antagonistic activity, can act as potential lead compounds for anti-breast cancer therapies.

Role of Cytoskeletal Elements in Regulation of Synaptic Functions: Implications Toward Alzheimer's Disease and Phytochemicals-Based Interventions.

Alzheimer's disease (AD), a multifactorial disease, is characterized by the accumulation of neurofibrillary tangles (NFTs) and amyloid beta (Aß) plaques. AD is triggered via several factors like alteration in cytoskeletal proteins, a mutation in presenilin 1 (PSEN1), presenilin 2 (PSEN2), amyloid precursor protein (APP), and post-translational modifications (PTMs) in the cytoskeletal elements. Owing to the major structural and functional role of cytoskeletal elements, like the organization of axon initial segmentation, dendritic spines, synaptic regulation, and delivery of cargo at the synapse; modulation of these elements plays an important role in AD pathogenesis; like Tau is a microtubule-associated protein that stabilizes the microtubules, and it also causes inhibition of nucleo-cytoplasmic transportation by disrupting the integrity of nuclear pore complex. One of the major cytoskeletal elements, actin and its dynamics, regulate the dendritic spine structure and functions; impairments have been documented towards learning and memory defects. The second major constituent of these cytoskeletal elements, microtubules, are necessary for the delivery of the cargo, like ion channels and receptors at the synaptic membranes, whereas actin-binding protein, i.e., Cofilin's activation form rod-like structures, is involved in the formation of paired helical filaments (PHFs) observed in AD. Also, the glial cells rely on their cytoskeleton to maintain synaptic functionality. Thus, making cytoskeletal elements and their regulation in synaptic structure and function as an important aspect to be focused for better management and targeting AD pathology. This review advocates exploring phytochemicals and Ayurvedic plant extracts against AD by elucidating their neuroprotective mechanisms involving cytoskeletal modulation and enhancing synaptic plasticity. However, challenges include their limited bioavailability due to the poor solubility and the limited potential to cross the blood-brain barrier (BBB), emphasizing the need for targeted strategies to improve therapeutic efficacy.

Helicobacter pylori secretary Proteins-Induced oxidative stress and its role in NLRP3 inflammasome activation.

Helicobacter pylori-associated stomach infection is a leading cause of gastric ulcer and related cancer. H. pylori modulates the functions of infiltrated immune cells to survive the killing by reactive oxygen and nitrogen species (ROS and RNS) produced by these cells. Uncontrolled immune responses further produce excess ROS and RNS which lead to mucosal damage. The persistent oxidative stress is a major cause of gastric cancer. H. pylori regulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), nitric oxide synthase 2 (NOS2), and polyamines to control ROS and RNS release through lesser-known mechanisms. ROS and RNS produced by these pathways differentiate macrophages and T cells from protective to inflammatory phenotype. Pathogens-associated molecular patterns (PAMPs) induced ROS activates nuclear oligomerization domain (NOD), leucine rich repeats (LRR) and pyrin domain-containing protein 3 (NLRP3) inflammasome for the release of pro-inflammatory cytokines. This study evaluates the role of H. pylori secreted concentrated proteins (HPSCP) related oxidative stress role in NLRP3 inflammasome activation and macrophage differentiation. To perceive the role of ROS/RNS, THP-1 and AGS cells were treated with 10 µM diphenyleneiodonium (DPI), 50 µM salicyl hydroxamic acid (SHX), 5 µM Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), which are specific inhibitors of NADPH oxidase (NOX), Myeloperoxidase (MPO), and mitochondrial oxidative phosphorylation respectively. Cells were also treated with 10 µM of NOS2 inhibitor l-NMMA and 10 µM of N-acetyl cysteine (NAC), a free radical scavenger·H2O2 (100 µM) treated and untreated cells were used as positive controls and negative control respectively. The expression of gp91phox (NOX2), NOS2, NLRP3, CD86 and CD163 was analyzed through fluorescent microscopy. THP-1 macrophages growth was unaffected whereas the gastric epithelial AGS cells proliferated in response to higher concentration of HPSCP. ROS and myeloperoxidase (MPO) level increased in THP-1 cells and nitric oxide (NO) and lipid peroxidation significantly decreased in AGS cells. gp91phox expression was unchanged, whereas NOS2 and NLRP3 downregulated in response to HPSCP, but increased after inhibition of NO, ROS and MPO in THP-1 cells. HPSCP upregulated the expression of M1 and M2 macrophage markers, CD86 and CD163 respectively, which was decreased after the inhibition of ROS. This study concludes that there are multiple pathways which are generating ROS during H. pylori infection which further regulates other cellular processes. NO is closely associated with MPO and inhibition of NLRP3 inflammasome. The low levels of NO and MPO regulates gastrointestinal tract homeostasis and overcomes the inflammatory response of NLRP3. The ROS also plays crucial role in macrophage polarization hence alter the immune responses duing H. pylori pathogenesis.

Implications of organophosphate pesticides on brain cells and their contribution toward progression of Alzheimer's disease.

The most widespread neurodegenerative disorder, Alzheimer's disease (AD) is marked by severe behavioral abnormalities, cognitive and functional impairments. It is inextricably linked with the deposition of amyloid ß (Aß) plaques and tau protein in the brain. Loss of white matter, neurons, synapses, and reactive microgliosis are also frequently observed in patients of AD. Although the causative mechanisms behind the neuropathological alterations in AD are not fully understood, they are likely influenced by hereditary and environmental factors. The etiology and pathogenesis of AD are significantly influenced by the cells of the central nervous system, namely, glial cells and neurons, which are directly engaged in the transmission of electrical signals and the processing of information. Emerging evidence suggests that exposure to organophosphate pesticides (OPPs) can trigger inflammatory responses in glial cells, leading to various cascades of events that contribute to neuroinflammation, neuronal damage, and ultimately, AD pathogenesis. Furthermore, there are striking similarities between the biomarkers associated with AD and OPPs, including neuroinflammation, oxidative stress, dysregulation of microRNA, and accumulation of toxic protein aggregates, such as amyloid ß. These shared markers suggest a potential mechanistic link between OPP exposure and AD pathology. In this review, we attempt to address the role of OPPs on altered cell physiology of the brain cells leading to neuroinflammation, mitochondrial dysfunction, and oxidative stress linked with AD pathogenesis.

Understanding the multifaceted role of miRNAs in Alzheimer's disease pathology.

Small non-coding RNAs (miRNAs) regulate gene expression by binding to mRNA and mediating its degradation or inhibiting translation. Since miRNAs can regulate the expression of several genes, they have multiple roles to play in biological processes and human diseases. The majority of miRNAs are known to be expressed in the brain and are involved in synaptic functions, thus marking their presence and role in major neurodegenerative disorders, including Alzheimer's disease (AD). In AD, amyloid beta (Aß) plaques and neurofibrillary tangles (NFTs) are known to be the major hallmarks. The clearance of Aß and tau is known to be associated with miRNA dysregulation. In addition, the ß-site APP cleaving enzyme (BACE 1), which cleaves APP to form Aß, is also found to be regulated by miRNAs, thus directly affecting Aß accumulation. Growing evidences suggest that neuroinflammation can be an initial event in AD pathology, and miRNAs have been linked with the regulation of neuroinflammation. Inflammatory disorders have also been associated with AD pathology, and exosomes associated with miRNAs are known to regulate brain inflammation, suggesting for the role of systemic miRNAs in AD pathology. Several miRNAs have been related in AD, years before the clinical symptoms appear, most of which are associated with regulating the cell cycle, immune system, stress responses, cellular senescence, nerve growth factor (NGF) signaling, and synaptic regulation. Phytochemicals, especially polyphenols, alter the expression of various miRNAs by binding to miRNAs or binding to the transcriptional activators of miRNAs, thus control/alter various metabolic pathways. Awing to the sundry biological processes being regulated by miRNAs in the brain and regulation of expression of miRNAs via phytochemicals, miRNAs and the regulatory bioactive phytochemicals can serve as therapeutic agents in the treatment and management of AD.

Understanding the neuronal synapse and challenges associated with the mitochondrial dysfunction in mild cognitive impairment and Alzheimer's disease.

Synaptic mitochondria are crucial for maintaining synaptic activity due to their high energy requirements, substantial calcium (Ca2+) fluctuation, and neurotransmitter release at the synapse. To provide a continuous energy supply, neurons use special mechanisms to transport and distribute healthy mitochondria to the synapse while eliminating the damaged mitochondria from the synapse. Along the neuron, mitochondrial membrane potential (ψ) gradient exists and is highest in the somal region. Lower ψ in the synaptic region renders mitochondria more vulnerable to oxidative stress-mediated damage. Secondly, mitochondria become susceptible to the release of cytochrome c, and mitochondrial DNA (mtDNA) is not shielded from the reactive oxygen species (ROS) by the histone proteins (unlike nuclear DNA), leading to activation of caspases and pronounced oxidative DNA base damage, which ultimately causes synaptic loss. Both synaptic mitochondrial dysfunction and synaptic failure are crucial factors responsible for Alzheimer's disease (AD). Furthermore, amyloid beta (Aß) and hyper-phosphorylated Tau, the two leading players of AD, exaggerate the disease-like pathological conditions by reducing the mitochondrial trafficking, blocking the bi-directional transport at the synapse, enhancing the mitochondrial fission via activating the mitochondrial fission proteins, enhancing the swelling of mitochondria by increasing the influx of water through mitochondrial permeability transition pore (mPTP) opening, as well as reduced ATP production by blocking the activity of complex I and complex IV. Mild cognitive impairment (MCI) is also associated with decline in cognitive ability caused by synaptic degradation. This review summarizes the challenges associated with the synaptic mitochondrial dysfunction linked to AD and MCI and the role of phytochemicals in restoring the synaptic activity and rendering neuroprotection in AD.

A cross-sectional study to correlate antioxidant enzymes, oxidative stress and inflammation with prevalence of hypertension.

AIMS: Hypertension a multifactorial consequence of environmental factors, life style and genetics is the well-recognized risk factor contributing to coronary heart diseases. The antioxidant imbalance, excessive reactive oxygen species (ROS) leads to oxidative stress which is pivotal in progression of hypertension. The present study aims to understand the complex interaction between oxidative stress, inflammation and antioxidant system which is crucial to maintain cellular homeostasis which further can exaggerate hypertension pathophysiology. MATERIALS AND METHODS: The metabolic profile of hypertensive and normotensive subjects from Malwa region, Punjab was compared by estimating lipid profile, cardiac, hepatic and renal markers. The oxidative stress markers (protein carbonyls and lipid peroxidation), inflammatory markers (Nitric oxide, Myeloperoxidase and advanced oxygen protein products), and antioxidant enzymes (Superoxide Dismutase, Catalase, and Total Antioxidant Capacity) were analyzed. KEY FINDINGS: It is observed that the metabolic markers are altered in hypertensive subjects which further these subjects showed increased oxidative, inflammatory profile and compromised antioxidant status when compared with normotensive subjects. Co-relation analysis validated the involvement of inflammation and oxidative stress in impaired endothelial function and vital organ damage. SIGNIFICANCE OF STUDY: These markers may act as early indicators of hypertension which usually do not show any physical symptoms, thus can be diagnosed and treated at the earliest. The current study suggests that disturbed homeostasis, a consequence of altered interaction between antioxidant system and inflammatory events raises the oxidative stress levels which eventually leads to hypertension and associated complications. These indicators can serve as early indicators of future chronic complications of hypertension.

Correlative study on heavy metal-induced oxidative stress and hypertension among the rural population of Malwa Region of Punjab, India.

Heavy metal-induced toxicity contributes to the progression of various metabolic disorders and possible mechanisms involved in disease progression are not well established. In this study, the correlation of  heavy metal exposure and  hypertension have been demonstrated. The results showed that in hypertensive subjects, the lipid profiles (triglycerides, LDL-C, HDL-C, and total cholesterol) and cardiac markers (CK-MB and LDH) were altered abruptly. As a consequence of heavy- induced oxidative stress, the oxidants (TBARS and protein carbonyls) and antioxidants (SOD, GSH, and TAC) were significantly increased and decreased, respectively in hypertension subjects. The concentrations of heavy metals (Pb, Cd, and As) exceeded the permissible limits in hypertensive subjects. The Nrf-2 genotyping indicated that heavy metals may induce mutations at molecular level. The results of correlation analysis revealed that the heavy metals interact with cellular components and interfere with metabolic processes which then results in disturbed lipid profile, enhanced oxidative stress, and reduced antioxidant status. The current study systematically estimated the association of hair and nail heavy metal concentrations with hypertension among the population residing in the Malwa region of Punjab. The proposed study highlighted that heavy metals act as a silent risk factor in the hypertension progression in the population of Malwa region. Future studies are required to confirm current findings and further scrutinize the effect of heavy metals exposure in early adulthood, early, and late mid-life to develop metabolic complications such as hypertension.

Gliadin induced oxidative stress and altered cellular responses in human intestinal cells: An in-vitro study to understand the cross-talk between the transcription factor Nrf-2 and multifunctional APE1 enzyme.

The present study examined the wheat protein gliadin-induced oxidative and nitrosative stress and its downstream responses in human intestinal HCT-116 and HT-29 cells. The beneficial role of dietary phytochemical curcumin and role of multifunctional enzyme Apurinic/aprymidinic endonuclease 1 (APE1) a major player involved in the base excision repair (BER)-pathway in gliadin intolerant intestinal HCT-116 and HT-29 cell lines were evaluated as an in vitro model study. The cultured cells were exposed to gliadin protein, H2 O2 , and curcumin followed by the assessment of oxidative stress and the consequences were measured using spectrophotometric, PCR, flow cytometer, Western blotting, confocal microscopy, and other methods. Results demonstrate that a 3 h pretreatment of curcumin, followed by the treatment of gliadin protein for 24 h time period protected both the HCT-116 and HT-29 cells via: (i) decreasing the ROS/RNS, restoring the mitochondrial transmembrane potential; (ii) re-establishing the cellular antioxidant defense system (superoxide dismutase, catalase, and GSH); (iii) enhancing the functions of APE1 viz. endonuclease activity and redox activation of transcription factor Nrf-2, the later binds with the antioxidant response elements (ARE) and activates downstream targets involved in cell survival. The cross-talk between APE1 and Nrf-2 was also established using immunofluorescence imaging and co-immunoprecipitation assays. In conclusion, gliadin protein induces oxidative/nitrosative stress, mitochondrial dysfunction and it damages cellular biomolecules in the intestinal cells. Hence it can be attributed to the tissue damage and disease pathogenesis in wheat intolerance-associated intestinal diseases. The gliadin-induced stress and its consequences are significantly reduced by the pretreatment of curcumin via BER-pathway and ARE-pathway; which is evident through the interaction between these two essential proteins. Hence suggesting for the intervention of curcumin and other natural dietary phytochemicals-based disease management and treatment of gliadin intolerance associated intestinal diseases like celiac disease.

Methods to Detect Nitric Oxide and Reactive Nitrogen Species in Biological Sample.

Oxidative stress has been implicated in various human diseases, including cancer, mainly through the generation of reactive nitrogen species (RNS), such as nitric oxide (NO), nitrite, nitroxyl, s-nitrosothiols, and reactive oxygen species (ROS) such as peroxides, superoxide, and hydroxyl radicals. NO being the main player among RNS induced altered cellular molecules and metabolisms, thus making it important to understand and detect the generation of NO in biological samples. There are many methods for direct and indirect detection of NO; out of these most commonly used are spectrophotometric-based Griess assay and fluorescence probe-based assays. In this chapter, we summarize these routinely used methods to detect NO and various challenges associated with these methods.

Methods to Assess Oxidative DNA Base Damage Repair of Apurinic/Apyrimidinic (AP) Sites Using Radioactive and Nonradioactive Oligonucleotide-Based Assays.

Reactive oxygen species (ROS) overproduction results in oxidative stress leading to genomic instability via the generation of small base lesions in the genome, and this unrepaired DNA base damage leads to various cellular consequences. The oxidative stress-mediated DNA base damage is involved in various human disorders like cancer, cardiovascular, ocular, and neurodegenerative diseases. Base excision repair (BER) pathway, one of the DNA repair pathways, is majorly involved in the repair of oxidative DNA base lesions, which utilizes a different set of enzymes, including endonuclease viz Apurinic/apyrimidinic endonuclease 1 (APE1). APE1 is a well-known multifunctional enzyme with DNA repair, REDOX regulatory, and protein-protein interaction/cross-talk functions associated with the cell survival mechanisms. APE1 acts as an important player in both normal and cancerous cell survival; thus, evaluating its endonuclease activity in the biological samples provide useful readout of the DNA repair capacity/ability, which can be used to tune for the development of therapeutic candidates via either stimulating or blocking its DNA repair function in normal vs. cancer cells, respectively. This chapter enlists two methods used for the determination of APE1's endonuclease activity by oligonucleotide-based radioactive P32-labeled and nonradioactive fluorescence dyes using the cell extracts and recombinant APE1 protein.

New pentacyclic triterpene from Potentilla atrosanguinea Lodd. as anticancer agent for breast cancer targeting estrogen receptor-α.

One new (compound 3) along with two previously known ursane type triterpenoids (compounds 1 and 2) were purified by chromatographic techniques from ethyl acetate extract of aerial parts of Potentilla atrosanguniea and characterized by HRMS, 1 D and 2 D-NMR. Compounds 1 (ursolic acid), 2 (euscaphic acid) and 3 (3α,20α-dihydroxy 2-oxo-urs-12-en-28-oic acid) were tested for their antiproliferative activity along with standard bazedoxifene. Compounds 1 and 3 were found to be of higher activity (3.71 and 6.05 µg/mL) as compared to compound 2 and bazedoxifene (IC50: 24.53 and 17.87 µg/mL). Anti-estrogenic activity of three compounds on breast cancer (BC) were studied in vitro by accessing their antiproliferative activity and binding with estrogen receptor alpha (ER-α). All three compounds have effective binding affinity towards ER-α and decreased cell growth by downregulating the expression of mRNA and its translational protein as tested by semi-qRT-PCR and western blotting. In terms of effectiveness compounds 1 and 3 were found more active due to their antiproliferative, and antiestrogenic activity as compared to standard bazedoxifene.

Brain Exosomes: Friend or Foe in Alzheimer's Disease?

Alzheimer's disease (AD) is the most common neurodegenerative disease. It is known to be a multifactorial disease and several causes are associated with its occurrence as well as progression. However, the accumulation of amyloid beta (Aß) is widely considered its major pathogenic hallmark. Additionally, neurofibrillary tangles (NFT), mitochondrial dysfunction, oxidative stress, and aging (cellular senescence) are considered as additional hits affecting the disease pathology. Several studies are now suggesting important role of inflammation in AD, which shifts our thought towards the brain's resident immune cells, microglia, and astrocytes; how they interact with neurons; and how these interactions are affected by intra and extracellular stressful factors. These interactions can be modulated by different mechanisms and pathways, in which exosomes could play an important role. Exosomes are multivesicular bodies secreted by nearly all types of cells. The exosomes secreted by glial cells or neurons affect the interactions and thus the physiology of these cells by transmitting miRNAs, proteins, and lipids. Exosomes can serve as a friend or foe to the neuron function, depending upon the carried signals. Exosomes, from the healthy microenvironment, may assist neuron function and health, whereas, from the stressed microenvironment, they carry oxidative and inflammatory signals to the neurons and thus prove detrimental to the neuronal function. Furthermore, exosomes can cross the blood-brain barrier (BBB), and from the blood plasma they can enter the brain cells and activate microglia and astrocytes. Exosomes can transport Aß or Tau, cytokines, miRNAs between the cells, and alter the physiology of recipient cells. They can also assist in Aß clearance and regulation of synaptic activity. The exosomes derived from different cells play different roles, and this field is still in its infancy stage. This review advocates exosomes' role as a friend or foe in neurodegenerative diseases, especially in the case of Alzheimer's disease.

A short review on cross-link between pyruvate kinase (PKM2) and Glioblastoma Multiforme.

Pyruvate kinase (PK) catalyzes the last irreversible reaction of glycolysis pathway, generating pyruvate and ATP, from Phosphoenol Pyruvate (PEP) and ADP precursors. In mammals, four different tissue-specific isoforms (M1, M2, L and R) of PK exist, which are translated from two genes (PKL and PKR). PKM2 is the highly expressed isoform of PK in cancers, which regulates the aerobic glycolysis via reprogramming cancer cell's metabolic pathways to provide an anabolic advantage to the tumor cells. In addition to the established role of PKM2 in aerobic glycolysis of multiple cancer types, various recent findings have highlighted the non-metabolic functions of PKM2 in brain tumor development. Nuclear PKM2 acts as a co-activator and directly regulates gene transcription. PKM2 dependent transactivation of various oncogenic genes is instrumental in the progression and aggressiveness of Glioblastoma Multiforme (GBM). Also, PKM2 acts as a protein kinase in histone modification which regulates gene expression and tumorigenesis. Ongoing research has explored novel regulatory mechanisms of PKM2 and its association in GBM progression. This review enlists and summarizes the metabolic and non-metabolic roles of PKM2 at the cellular level, and its regulatory function highlights the importance of the nuclear functions of PKM2 in GBM progression, and an emerging role of PKM2 as novel cancer therapeutics.

Mitigation of Gliadin-Induced Inflammation and Cellular Damage by Curcumin in Human Intestinal Cell Lines.

Wheat is a major diet from many years; apart from its nutritious value, the wheat protein gliadin is responsible for many inflammatory diseases like celiac disease (CD), and non-celiac gluten sensitivity (NCGS). In this study, the gliadin-induced inflammation and associated cellular damage along with the protective role of curcumin was evaluated using human intestinal cell lines (HCT-116 and HT-29) as a model. Cells were cultured and exposed to 160 µg/ml of gliadin, 100 µM H2O2, and 10 µM curcumin (3 h pretreatment) followed by the assessment of inflammation. Spectrophotometric methods, real-time-PCR, ELISA, Western blotting, and confocal microscopy techniques were used to assess inflammatory markers such as advanced oxidation protein products (AOPPs) level, activity of myeloperoxidase (MPO) and NADPH oxidase (NOX), cytokines, and cell damage markers. The results show that gliadin increases the AOPPs level and the activity of MPO and NOX expression. It enhances inflammation by increasing expression of pro-inflammatory cytokines, altered expression of anti-inflammatory, and regulatory cytokines. It exacerbates the cellular damage by increasing MMP-2 and 9 and decreasing integrin α and ß expression. Gliadin promotes disease pathogenesis by inducing the inflammation and cellular damage which further alter the cellular homeostasis. The pretreatment of curcumin counteracts the adverse effect of gliadin and protect the cells via diminishing the inflammation and help the cell to regain the cellular morphology suggesting phytochemical-based remedial interventions against wheat allergies.

A short review: Doxorubicin and its effect on cardiac proteins.

Doxorubicin (DOX) is a boon for cancer-suffering patients. However, the undesirable effect on health on vital organs, especially the heart, is a limiting factor, resulting in an increased number of patients with cardiac dysfunction. The present review focuses on the contractile machinery and associated factors, which get affected due to DOX toxicity in chemo-patients for which they are kept under life-long investigation for cardiac function. DOX-induced oxidative stress disrupts the integrity of cardiac contractile muscle proteins that alter the rhythmic mechanism and oxygen consumption rate of the heart. DOX is an oxidant and it is further discussed that oxidative stress prompts the damage of contractile components and associated factors, which include Ca2+ load through Ca2+ ATPase, SERCA, ryanodine receptor-2, phospholamban, and calsequestrin, which ultimately results in left ventricular ejection and dilation. Based on data and evidence, the associated proteins can be considered as clinical markers to develop medications for patients. Even with the advancement of various diagnosing tools and modified drugs to mitigate DOX-induced cardiotoxicity, the risk could not be surmounted with survivors of cancer.

Organophosphate-pesticides induced survival mechanisms and APE1-mediated Nrf2 regulation in non-small-cell lung cancer cells.

Epidemiological and molecular studies have indicated that environmental exposure to organophosphate pesticides (OPPs) is associated with increased cancer risk; however, the underlying molecular mechanisms still need to be explained. Increasing cancer incidence is linked to OPPs-induced oxidative stress (OS). Our study evaluates monocrotophos (MCP) and chlorpyrifos (CP)-induced OS responses and apurinic/apyrimidinic endonuclease 1 (APE1) role in human non-small-cell lung cancer (NSCLC) cells. Our prior study has implicated OPPs-induced base excision repair (BER)-pathway dysregulation and APE1-mediated regulation of transcription factor (TF) c-jun in A549 cells. We further investigated the effects of MCP and CP on apoptosis, proliferation, and APE1's redox-regulation of nuclear factor-like 2 (Nrf2). Data demonstrates that MCP and CP at subtoxic concentrations induced reactive oxygen species generation and oxidative DNA base damage 8-oxo-dG lesions in NCI-H1299 cells. CP moderately upregulated the apoptosis-inducing factor (AIF) in A549 cells, however, it did not trigger other pro-apoptotic factors viz. caspase-9 and caspase-3, suggesting early caspase-independent apoptosis. However, dose-dependent AIF-downregulation was observed for MCP treatment. Furthermore, CP and MCP treatments upregulated proliferating cell nuclear antigen levels. Immunofluorescent confocal imaging showed the colocalization of APE1 with Nrf2 in 10 µM CP- and MCP-treated NCI-H1299 cells. Immunoprecipitation confirmed that APE1 and Nrf2 physically interacted, indicating the role of APE1-mediated Nrf2 activation following OPPs treatment. This study suggests that low concentration MCP and CP exposure generates OS along with DNA damage, and modulates apoptosis, and APE1-mediated Nrf2 activation, which might be considered as the possible mechanism promoting lung cancer cell survival, suggesting that APE1 may have the potential to become a therapeutic target for the treatment of NSCLC.

Parthenium hysterophorus mediated inflammation and hyper-responsiveness via NF-κB pathway in human A549 lung cancer cell line.

Being one of the notorious weed P. hysterophorus has invaded almost every part India and is the lead cause of skin allergies and severe dermatitis among farmers and rural population. It is an invasive obnoxious weed capable of surviving extreme environmental conditions and various parts of this plant are reported to cause severe contact allergies in humans due to the presence of high concentrations of toxic sesquiterpene lactones viz. parthenin. It can stimulate numerous cellular and immune responses that may translate into Oxidative stress, allergies, and inflammation. The effect of P. hysterophorus flower extract was evaluated on cell viability, oxidative stress and inflammation in A549 lung cancer cell line by spectrophotometric and reverse transcriptase-polymerase chain reaction methods. Schrodinger software based docking was performed for possible interactions studies. The A549 cells treated with P. hysterophorus flower extract favors increase in cell viability, reactive oxygen species generation. The mRNA expression of proinflammatory cytokines such as IFN-γ, TNF-α, and IL-1ß was significantly increased whereas no change in IL-18 expression was observed. Significant increase in protein expression of NF-κB was observed, suggesting the role of NF-κB signalling in allergic responses. The docking studies demonstrated the potential interaction between Parthenin and NF-κB/IL-1ß/IL-18 suggesting their activation leading to inflammation. The current study emphasize that P. hysterophorus mediates oxidative stress, and inflammatory process via alterations in expression of proinflammatory cytokines such as IL-1ß, IFN-γ through NF-κB activation which was also confirmed in docking studies. Cellular and molecular mechanisms involved in pathogenesis of allergic/chronic inflammation and severe dermatitis need to be further investigated to identify specific binding partners responsible for severe inflammation which can provide some leads in developing effective targets against severe dermatitis and skin allergies.

Glycyrrhiza glabra (Licorice) root extract attenuates doxorubicin-induced cardiotoxicity via alleviating oxidative stress and stabilising the cardiac health in H9c2 cardiomyocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Doxorubicin (DOX) is an effective anti-neoplastic drug, however; it has downside effects on cardiac health and other vital organs. The herbal remedies used in day to day life may have a beneficial effect without disturbing the health of the vital organs. Glycyrrhiza glabra L. is a ligneous perennial shrub belonging to Leguminosae/Fabaceae/Papilionaceae family growing in Mediterranean region and Asia and widespread in Turkey, Italy, Spain, Russia, Syria, Iran, China, India and Israel. Commonly known as mulaithi in north India, G. glabra has glycyrrhizin, glycyrrhetic acid, isoliquiritin, isoflavones, etc., which have been reported for several pharmacological activities such as anti-demulcent, anti-ulcer, anti-cancer, anti-inflammatory and anti-diabetic. AIM OF THE STUDY: The objective of the present study is to investigate the interaction between the molecular factors like PPAR-α/γ and SIRT-1 during cardiac failure arbitrated by DOX under in vitro conditions and role of Glycyrrhiza glabra (Gg) root extract in alleviating these affects. MATERIALS AND METHODS: In the present study, we have examined the DOX induced responses in H9c2 cardiomyocytes and investigated the role of phytochemical Glycyrrhiza glabra in modulating these affects. MTT assay was done to evaluate the cell viability, Reactive Oxygen Species (ROS)/Reactive Nitrogen Species (RNS) levels, mitochondrial ROS, mitochondrial membrane potential was estimated using fluorescent probes. The oxidative stress in terms of protein carbonylation, lipid peroxidation and DNA damage was detected via spectrophotometric methods and immune-fluorescence imaging. The cardiac markers and interaction between SIRT-1 and PPAR-α/γ was measured using Real-Time PCR, Western blotting and Co-immunoprecipitation based studies. RESULTS: The Glycyrrhiza glabra (Gg) extracts maintained the membrane integrity and improved the lipid homeostasis and stabilized cytoskeletal element actin. Gg phytoextracts attenuated aggravated ROS level, repaired the antioxidant status and consequently, assisted in repairing the DNA damage and mitochondrial function. Further, the expression of hypertrophic markers in the DOX treated cardiomyocytes reconciled the expression factors both at the transcriptional and translational levels after Gg treatment. SIRT-1 mediated pathway and its downstream activator PPARs are significant in maintaining the cellular functions. It was observed that the Gg extract allows regaining the nuclear SIRT-1 and PPAR-γ level which was otherwise reduced with DOX treatment in H9c2 cardiomyocytes. The co-immunoprecipitation (Co-IP) documented that SIRT-1 interacts with PPAR-α in the untreated control H9c2 cardiomyocytes whereas DOX treatment interferes and diminishes this interaction however the Gg treatment maintains this interaction. Knocking down SIRT-1 also downregulated expression of PPAR-α and PPAR-γ in DOX treated cells and Gg treatment was able to enhance the expression of PPAR-α and PPAR-γ in SIRT-1 knocked down cardiomyocytes. CONCLUSIONS: The antioxidant property of Gg defend the cardiac cells against the DOX induced toxicity via; 1) reducing the oxidative stress, 2) maintaining the mitochondrial functions, 3) regulating lipid homeostasis and cardiac metabolism through SIRT-1 pathway, and 4) conserving the cardiac hypertrophy and hence preserving the cardiomyocytes health. Therefore, Gg can be recommended as a healthy supplement with DOX towards cancer therapeutics associated cardiotoxicity.