Prevention of aspirin-mediated secondary toxicity by combined treatment of carotenoids in macrophages.

Upon understanding the boosting role of carotenoids on the endogenous anti-inflammatory system, it is vital to explore their role in reducing the use of high doses of non-steroidal anti-inflammatory drug (NSAIDs), and their mediated secondary toxicity during the treatment of chronic diseases. The current study investigates the carotenoids potential on inhibition of secondary complications induced by NSAIDs, aspirin (ASA) against lipopolysaccharide (LPS) stimulated inflammation. Initially, this study evaluated a minimal cytotoxic dose of ASA and carotenoids (ß-carotene, BC/lutein, LUT/astaxanthin, AST/fucoxanthin FUCO) in Raw 264.7, U937, and peripheral blood mononuclear cells (PBMCs). In all three cells, carotenoids + ASA treatment reduced the LDH release, NO, and PGE2 efficiently than an equivalent dose of carotenoid or ASA treated alone. Based on cytotoxicity and sensitivity results, RAW 264.7 cells were selected for further cell-based assay. Among carotenoids, FUCO + ASA exhibited an efficient reduction of LDH release, NO, and PGE2 than the other carotenoids (BC + ASA, LUT + ASA, and AST + ASA) treatment. FUCO + ASA combination decreased LPS/ASA induced oxidative stress, pro-inflammatory mediators (iNOS, COX-2, and NF-κB), and cytokines (IL-6, TNF-α, and IL-1ß) efficiently. Further, apoptosis was inhibited by 69.2% in FUCO + ASA, and 46.7% in ASA than LPS treated cells. A drastic decrease in intracellular ROS generation with the increase in GSH was observed in FUCO + ASA compared to LPS/ASA groups. The results documented on the low dose of ASA with a relative physiological concentration of FUCO suggested greater importance for alleviating secondary complications and optimize prolonged chronic disease treatments with NSAID's associated side effects. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03632-w.

A systems biology investigation of curcumin potency against TGF-ß-induced EMT signaling in lung cancer.

Curcumin (diferuloylmethane) is bioactive phenolic compound which exerts diverse antimetastatic effect. Several studies have reported the antimetastatic effect of curcumin by its ability to modulate the epithelial-to-mesenchymal transition (EMT) process in different cancers, but underlying molecular mechanism is poorly understood. EMT is a highly conserved biological process in which epithelial cells acquire mesenchymal-like characteristics by losing their cell-cell junctions and polarity. As a consequence, deviation in cellular mechanism leads to cancer metastasis and thereby death. In this perspective, we explored the antimetastatic potential and mechanism of curcumin on the EMT process by establishing in vitro EMT model in lungs cancer (A549) cells induced by TGF-ß1. Our results showed that curcumin mitigates EMT by regulating the expression of crucial mesenchymal markers such as MMP2, vimentin and N-cadherin. Besides, the transcriptional analysis revealed that the curcumin treatment differentially regulated the expression of 75 genes in NanoString nCounter platform. Further protein-protein interaction network and clusters analysis of differentially expressed genes revealed their involvement in essential biological processes that plays a key role during EMT transition. Altogether, the study provides a comprehensive overview of the antimetastatic potential of curcumin in TGF-ß1-induced EMT in lung cancer cells. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03360-7.

Turmeric, red pepper, and black pepper affect carotenoids solubilized micelles properties and bioaccessibility: Capsaicin/piperine improves and curcumin inhibits carotenoids uptake and transport in Caco-2 cells.

This study aimed to evaluate the role of spices/spice active principles on physical, biochemical, and molecular targets of bioaccessibility/bioavailability. Carotenoids-rich micellar fraction obtained through simulated digestion of green leafy vegetables (GLV) with individual or two/three combinations were correlated to their influence on bioaccessibility, cellular uptake, and basolateral secretion of carotenoids in Caco-2 cells. Results suggest that carotenoids' bioaccessibility depends on micelles physicochemical properties, which is affected due to the presence of co-treated dietary spices and their composition. Increased bioaccessibility of ß-carotene (BC) and lutein (LUT) is found in GLV (spinach) digested with turmeric (TM) than red pepper (RP) and black pepper (BP). In contrast, enhanced cellular uptake and secretion of BC and LUT-rich triglyceride-rich lipoprotein is observed in the presence of RP and BP compared to the control group. In contrast, TM inhibited absorption, while retinol levels significantly reduced in the presence of TM and RP than BP. Control cells have indicated higher cleavage of ß-carotene to retinol than the spice-treated group. Besides, spice active principles modulate facilitated transport of carotenoids by scavenger receptor class B type 1 (SR-B1) protein. The effect of spices on carotenoids' bioavailability is validated with active spice principles. Overall, carotenoids' bioavailability (cellular uptake and basolateral secretion) was found in the following order of treatments; piperine > capsaicin > piperine + capsaicin > curcumin + capsaicin + piperine > control > turmeric. These findings suggested that the interaction of specific dietary factors, including spice ingredients at the enterocyte level, could provide greater insight into carotenoid absorption. PRACTICAL APPLICATION: Spices/spice active principles play a role in the digestion process by stimulating digestive enzymes and bile acids secretion. Since carotenoids are lipid soluble and have low bioavailability, spice ingredients' influence on intestinal absorption of carotenoids is considered crucial. Hence, understanding the interaction of co-consumed spices on the absorption process of carotenoids may help to develop functional foods/formulation of nutraceuticals to improve their health benefits.

Computational insights into the binding mode of curcumin analogues against EP300 HAT domain as potent acetyltransferase inhibitors.

Acetylation plays a key role in maintaining and balancing cellular regulation and homeostasis. Acetyltransferases are an important class of enzymes which mediate this acetylation process. EP300 is a type 3 major lysine (K) acetyl transferase, and its aberrant activity is implicated in many human diseases. Hence, targeting EP300 mediated acetylation is a necessary step to control the associated diseases. Currently, a few EP300 inhibitors are known, among which curcumin is the most widely investigated molecule. However, due to its instability, chemical aggregation and reactivity, its inhibitory activity against the EP300 acetyltransferase domain is disputable. To address this curcumin problem, different curcumin analogues have been synthesized. These molecules were selected for screening against the EP300 acetyltransferase domain using in silico docking and MD analysis. We have successfully elucidated that the curcumin analogue CNB001 is a potential EP300 inhibitor with good drug-like characteristics.

Exploring the Potential of Capsaicin Against Cancer Metastasis Based on TGF-ß Signaling Modulation Through Module-based Network Pharmacology Approachx.

BACKGROUND: Capsaicin is an active alkaloid /principal component of red pepper responsible for the pungency of chili pepper. Capsaicin by changing the intracellular redox homeostasis regulate a variety of signaling pathways ultimately producing a divergent cellular outcome. Several reports showed the potential of capsaicin against cancer metastasis, however unexplored molecular mechanism is still an active part of the research. Several growth factors have a critical role during cancer metastasis among them TGF- ß signaling play a vital role. METHODS: The present study aimed at analyzing capsaicin modulation of TGF-ß signaling using network pharmacology approach. The chemical and protein interaction data of capsaicin was curated and abstracted using STITCH4.0, PubChem and ChEMBL database. Further, the compiled data set was subjected to the pathway and functional enrichment analysis using Protein Analysis THrough Evolutionary Relationship (PANTHER) and, Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. Meanwhile, the pattern of amino acid composition across the capsaicin targets was analyzed using the EMBOSS Pepstat tool. Capsaicin targets involved in TGF- ß were identified and their Protein-Protein Interaction (PPI) network constructed using STRING v10 and Cytoscape (v 3.2.1). From the above-constructed network, the clusters were mined using the MCODE clustering algorithm and finally binding affinity of capsaicin with its targets involved in TGF-ß signaling pathway was analyzed using Autodock Vina. RESULTS: The analysis explored capsaicin targets and, their associated functional and pathway annotations. Besides, the analysis also provides a detailed distinct pattern of amino acid composition across the capsaicin targets. The capsaicin targets described as MAPK14, JUN, SMAD3, MAPK3, MAPK1 and MYC involved in TGF-ß signaling pathway through pathway enrichment analysis. The binding mode analysis of capsaicin with its targets has shown high affinity with MAPK3, MAPK1, JUN and MYC. CONCLUSION: The study explores the potential of capsaicin as a potent modulator of TGF-ß signaling pathway during cancer metastasis and proposes new methodology and mechanism of action of capsaicin against TGF- ß signaling pathway.

A systems biology approach to identify the key targets of curcumin and capsaicin that downregulate pro-inflammatory pathways in human monocytes.

VEGFR1 (Flt-1), is a high-affinity tyrosine kinase receptor of VEGF found primarily on vascular endothelial cells. Recently, Flt-1 has shown to be expressed in human monocytes. However, the key intracellular signaling pathway mediated by Flt-1 receptor has been yet to be identified in monocytes. In this regard, using a robust systems biology approach, the key druggable target(s) involved in inflammatory angiogenesis mediated through VEGFR1 signaling was identified. Furthermore, experimental validation of key drug targets is conducted using PMA- and VEGF- stimulated human monocyte THP-1 cell lines. The key network pathways and corresponding disease modules were analyzed to identify the important biological processes perturbed in diseases. Using topological analysis, ICAM1 was identified as putative regulator of monocytes migration into tumor-micro environment. And these targets were examined by treating with curcumin and capsaicin molecules. Our results showed that these two molecules inhibited the over expression of targets such as ICAM1, Flt-1, and NF-κB in the VEGFR1 signalling pathway by reducing THP-1 chemotaxis. Besides, Curcumin and Capsaicin down-regulated expression of pro-inflammatory cytokines TNF-α, IL-6, and CXCL8/IL-8 and up regulated the expression of IL-10, a sign of lowered M1/M2 ratio relating to abrogation of inflammation.

Curcumin and capsaicin modulates LPS induced expression of COX-2, IL-6 and TGF-ß in human peripheral blood mononuclear cells.

The mechanism of action of treatment of either curcumin or capsaicin or in combination on LPS (Lipopolysaccharide) induced inflammatory gene expression in peripheral blood mononuclear cells (PBMCs) was investigated using RT-PCR and in silico docking methods. RT-PCR analysis has shown that the curcumin and capsaicin significantly reduced LPS induced over expression of COX-2, IL-6 and TGF-ß in PBMCs. Whereas combined molecules demonstrated synergistic response on the reduction of COX-2, IL-6 and TGF-ß over expression in LPS induced PBMCs as compared to individual molecules. Further, The docking of curcumin and capsaicin at the active pockets of COX-2, IL-6 and TGF-ß has shown - 3.90, - 4.49 and - 5.61 kcal/mol binding energy for curcumin and - 3.80, - 4.78 and - 5.76 kcal/mol binding energy for capsaicin, while multiple ligand simultaneous docking (MLSD) of both molecules has shown higher binding energy of - 4.24, - 5.35 and - 5.83 kcal/mol respectively. This has demonstrated the efficacy of combined curcumin and capsaicin against the LPS induced expression of pro-inflammatory cytokines in PBMCs. These results attributed the coordinated positive modulation on biochemical and molecular cellular process by combined curcumin and capsaicin as compared to individual molecules.

Expression, purification, and characterization of pyruvate kinase from Mycobacterium tuberculosis: A key allosteric regulatory enzyme.

Background: The current research aims to isolate pyruvate kinase (Pyk) gene from Mycobacterium tuberculosis and expression of the gene (Rv1617) to obtain a purified enzyme. The enzyme activity and secondary structural features were assessed through biochemical assays and circular dichroism (CD) spectroscopy, respectively. Methods: The Pyk-encoding gene from the complete genome of M. tuberculosis was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). The enzyme was purified by nickel-nitrilotriacetic acid affinity chromatography and enzyme activity was determined by a lactose dehydrogenase-coupled assay system. Further, far ultraviolet CD spectra of the enzyme and the substrate bound enzyme were analyzed using a Jasco J712 spectrophotometer. Results: A single protein with an approximate molecular mass of 54 kDa was purified and a specific activity of 5.31 units/mg was determined from purified M. tuberculosis Pyk. The activity of the enzyme indicating a protein is defined by separate domain for each catalytic function. The secondary structure analysis of CD spectra of the recombinant Pyk has revealed a content of 17% α-helix, 34% ß-sheet, and 49% turns in the enzyme. Conclusion: The growing evidence has impacted M. tuberculosis central carbon metabolism as a key determinant of the survival and pathogenicity in the host. The purified Pyk was observed to have increased enzyme activity in all steps of purification. Retention of Pyk activity indicates a possible catalytic role for the lower part of the glycolytic pathway. The overall results of the spectra obtained from the CD suggest that the substrate phosphoenolpyruvate and adenosine diphosphate binding to the enzyme can cause conformational changes resulting in the exposure or shielding of residues susceptible to modification.

A new signal sequence for recombinant protein secretion in Pichia pastoris.

Pichia pastoris is one of the most widely used expression systems for the secretory expression of recombinant proteins. The secretory expression in P. pastoris usually makes use of the prepro MATα sequence from Saccharomyces cerevisiae, which has a dibasic amino acid cleavage site at the end of the signal sequence. This is efficiently processed by Kex2 protease, resulting in the secretion of high levels of proteins to the medium. However, the proteins that are having the internal accessible dibasic amino acids such as KR and RR in the coding region cannot be expressed using this signal sequence, as the protein will be fragmented. We have identified a new signal sequence of 18 amino acids from a P. pastoris protein that can secrete proteins to the medium efficiently. The PMT1-gene-inactivated P. pastoris strain secretes a ~30 kDa protein into the extracellular medium. We have identified this protein by determining its N-terminal amino acid sequence. The protein secreted has four DDDK concatameric internal repeats. This protein was not secreted in the wild-type P. pastoris under normal culture conditions. We show that the 18-amino-acid signal peptide at the N-terminal of this protein is useful for secretion of heterologous proteins in Pichia.

In vitro antioxidant and H(+), K(+)-ATPase inhibition activities of Acalypha wilkesiana foliage extract.

AIMS: The aim of this study was to evaluate the antioxidant activty and anti-acid property of Acalypha wilkesiana foliage extract. MATERIALS AND METHODS: Hot and cold aqueous extracts were prepared from healthy leaves of A. wilkesiana. Free radical scavenging activity and H(+), K(+)-ATPase inhibition activities of aqueous foliage extracts was screened by in vitro models. STATISTICAL ANALYSIS USED: All experiments were performed in triplicate and results are expressed as mean ± SEM. RESULTS: A. wilkesiana hot aqueous extract (AWHE) showed significant antioxidants and free radical scavenging activity. Further, AWHE has shown a potent H(+), K(+)-ATPase inhibitory activity (IC50: 51.5 ± 0.28 µg/ml) when compare to standard proton pump inhibitor omeprazole (56.2 ± 0.64 µg/ml); however, latter activity is equal to A. wilkesiana cold aqueous extract (AWCE). Quantitative analysis of AWHE has revealed more content of phenols and flavonoids; this is found to be the reason for good antioxidant activity over AWCE. Molecular docking was carried out against H(+), K(+)-ATPase enzyme crystal structure to validate the anti-acid activity of A. wilkesiana major phytochemicals. CONCLUSIONS: The present study indicates that the constituents of AWHE and AWCE have good antacid and free radical scavenging activity.

PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris.

Protein mannosyltransferases (PMTs) catalyze the O-mannosylation of serine and threonine residues of proteins in the endoplasmic reticulum. The five PMT genes coding for protein mannosyltransferases, designated as PMT1, 2, 4, 5 and 6, were identified from Pichia pastoris genome based on the homology to PMT genes in Saccharomyces cerevisiae genome, which has seven PMT genes. The homologues of S. cerevisiae PMT 3 &7 genes are absent in P. pastoris genome. Approximately 5% of the recombinant insulin precursor expressed in P. pastoris is O-mannosylated. In this study, we attempted to prevent O-mannosylation of insulin precursor in vivo, through inactivation of the Pichia PMT genes. Since multiple PMTs are found to be expressed, it was important to understand which of these are involved in O-mannosylation of the insulin precursor. The genes encoding PMT1, 4, 5 and 6 were knocked out by insertional inactivation method. Inactivation of PMT genes 4, 5 and 6 showed ∼16-28% reductions in the O-mannosylation of insulin precursor. The PMT1 gene disrupted Pichia clone showed ∼60% decrease in O-mannosylated insulin precursor, establishing its role as an important enzyme for insulin precursor O-mannosylation.