Pathophysiological relationship between hypoxia associated oxidative stress,Epithelial-mesenchymal transition,stemness acquisition and alteration of Shh/ Gli-1 axis during oral sub-mucous fibrosis and oral squamous cell carcinoma

Oral sub-mucous fibrosis (OSF) is a pathophysiological state of oral cavity or oropharynx having a high chance of conversion to oral squamous cell carcinoma (OSCC). It involves fibrotic transformation of sub-epithelial matrix along with epithelial abnormalities. The present work aims to unveil the mechanistic domain regarding OSF to OSCC conversion exploring the scenario of hypoxia associated oxidative stress, epithelial-mesenchymal transition (EMT), metastasis and stemness acquisition. The study involves histopathological analysis of the diseased condition along with the exploration of oxidative stress status, assessment of mitochondrial condition, immunohistochemical analysis of HIF-1α, E-cadherin, vimentin, ERK, ALDH-1, CD133, Shh, Gli-1 and survivin expressions in the oral epithelial region together with the quantitative approach towards collagen deposition in the sub-epithelial matrix. Oxidative stress was found to be associated with type-II EMT in case of OSF attributing the development of sub-epithelial fibrosis and type-III EMT in case of OSCC favoring malignancy associated metastasis. Moreover, the acquisition of stemness during OSCC can also be correlated with EMT. Alteration of Shh and Gli-1 expression pattern revealed the mechanistic association of hypoxia with the phenotypic plasticity and disease manifestation in case of OSF as well as OSCC. Shh/ Gli-1 signaling can also be correlated with survivin mediated cytoprotective phenomenon under oxidative stress. Overall, the study established the correlative network of hypoxia associated oxidative stress, EMT and manifestation of oral pre-cancerous and cancerous condition in a holistic approach that may throw rays of hope in the therapeutic domain of the concerned diseases.     

Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis

BACKGROUND: Plant-derived flavonoids, which occur abundantly in our daily dietary intake, possess antitumor, antibacterial, and free radical scavenging properties. They form active constituents of a number of herbal and traditional medicines. Several flavonoids have been shown to exert their action by interacting with DNA topoisomerases and promoting site-specific DNA cleavage. Therefore, flavonoids are potential candidates in drug design. We report here that, although the flavonoids luteolin and quercetin are potent antileishmanial agents, luteolin has great promise for acting as a lead compound in the chemotherapy of leishmaniasis, a major concern in developing countries. MATERIALS AND METHODS: Kinetoplast DNA (kDNA) minicircle cleavage in drug-treated parasites was measured by electrophoresis of the total cellular DNA, followed by Southern hybridization using 32P labeled kDNA as a probe. Cell cycle progression and apoptosis were measured by flow cytometry using propidium iodide and fluorescein isothiocyanate (FITC)-labeled Annexin V. RESULTS: Luteolin and quercetin inhibited the growth of Leishmania donovani promastigotes and amastigotes in vitro, inhibited DNA synthesis in promastigotes, and promoted topoisomerase-II-mediated linearization of kDNA minicircles. The IC50 values of luteolin and quercetin were 12.5 microM and 45.5 microM, respectively. These compounds arrest cell cycle progression in L. donovani promastigotes, leading to apoptosis. Luteolin has no effect on normal human T-cell blasts. Both luteolin and quercetin reduced splenic parasite burden in animal models. CONCLUSION: Luteolin and quercetin are effective antileishmanial agents. Quercetin has nonspecific effects on normal human T cells, but luteolin appears nontoxic. So, luteolin can be a strong candidate for antileishmanial drug design.     

MsrA Efflux Pump Inhibitory Activity of Piper cubeba L.f. and its Phytoconstituents against Staphylococcus aureus RN4220

MsrA, an efflux pump belonging to ATP‐binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strains. Herein, we report the isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds, viz. pellitorine, sesamin, piperic acid and tetrahydropiperine studied in combination with erythromycin in S. aureus RN4220, exhibited 2–8‐fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8‐fold. The real‐time fluorometry‐based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post‐antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment.     

Role of caffeine in DNA recognition of a potential food‐carcinogen benzo[a]pyrene and UVA induced DNA damage

Electron transfer (ET) reactions are important for their implications in both oxidative and reductive DNA damages. The current contribution investigates the efficacy of caffeine, a xanthine alkaloid in preventing UVA radiation induced ET from a carcinogen, benzo[a]pyrene (BP) to DNA by forming stable caffeine–BP complexes. While steady‐state emission and absorption results emphasize the role of caffeine in hosting BP in aqueous medium, the molecular modeling studies propose the energetically favorable structure of caffeine–BP complex. The picosecond‐resolved emission spectroscopic studies precisely explore the caffeine‐mediated inhibition of ET from BP to DNA under UVA radiation. The potential therapeutic activity of caffeine in preventing DNA damage has been ensured by agarose gel electrophoresis. Furthermore, time‐gated fluorescence microscopy has been used to monitor caffeine‐mediated exclusion of BP from various cell lines including squamous epithelial cells, WI‐38 (fibroblast), MCF‐7 (breast cancer) and HeLa (cervical cancer) cells. Our in vitro and ex vivo experimental results provide imperative evidences about the role of caffeine in modified biomolecular recognition of a model carcinogen BP by DNA resulting dissociation of the carcinogen from various cell lines, implicating its potential medicinal applications in the prevention of other toxic organic molecule induced cellular damages. Copyright © 2014 John Wiley & Sons, Ltd.     

Atomistic details of effect of disulfide bond reduction on active site of Phytase B from Aspergillus niger: A MD Study

The molecular integrity of the active site of phytases from fungi is critical for maintaining phytase function as efficient catalytic machines. In this study, the molecular dynamics (MD) of two monomers of phytase B from Aspergillus niger, the disulfide intact monomer (NAP) and a monomer with broken disulfide bonds (RAP), were simulated to explore the conformational basis of the loss of catalytic activity when disulfide bonds are broken. The simulations indicated that the overall secondary and tertiary structures of the two monomers were nearly identical but differed in some crucial secondary–structural elements in the vicinity of the disulfide bonds and catalytic site. Disulfide bonds stabilize the β-sheet that contains residue Arg66 of the active site and destabilize the α-helix that contains the catalytic residue Asp319. This stabilization and destabilization lead to changes in the shape of the active–site pocket. Functionally important hydrogen bonds and atomic fluctuations in the catalytic pocket change during the RAP simulation. None of the disulfide bonds are in or near the catalytic pocket but are most likely essential for maintaining the native conformation of the catalytic site.

Abbreviations

PhyB - 2.5 pH acid phophatese from Aspergillus niger, NAP - disulphide intact monomer of Phytase B, RAP - disulphide reduced monomer of Phytase B, Rg - radius of gyration, RMSD - root mean square deviation, MD - molecular dynamics.     

Effect of nimesulide on acetic acid- and leukotriene-induced inflammatory bowel disease in rats

Inflammatory bowel disease (IBD) is a relapsing inflammation of intestine, which is mediated by release of inflammatory mediators. Both cyclo-oxygenase product prostaglandin (PGE2) and lipo-oxygenase product leukotriene (LTB4), may contribute to the pathogenesis of the inflammatory response. Nimesulide, a preferential COX-2 inhibitor was evaluated for its efficacy against experimental colitis in two different models (acetic acid- and LTB4-induced IBD) in rats. Inflammatory response was induced by intrarectal single administration of acetic acid or LTB4. Nimesulide (9 and 18 mg/kg, p.o.) significantly prevented development of inflammatory changes, decreased myeloperoxidase (MPO) activity, and also restored the altered contractility response of the isolated colon segment to KCl. The results suggested the involvement of both cyclo-oxygenase (COX) and lipo-oxygenase-mediated proinflammatory agents in colonic inflammatory process associated with IBD. Further, this study suggests that such therapeutic interventions may be of value in the treatment of IBD.     

Antileishmanial phenylpropanoids from Alpinia galanga (Linn.) Willd

Hexane, chloroform and ethyl acetate extracts (100 microg/ml) of Alpinia galanga rhizomes exhibited significant activity in vitro against promastigotes of L. donovani. Twelve compounds namely, methyleugenol (1), p-coumaryl diacetate (2), 1'-acetoxychavicol acetate (3), 1'-acetoxyeugenol acetate (4), trans-p-acetoxycinnamyl alcohol (5), trans-3,4-dimethoxycinnamyl alcohol (6), p-hydroxybenzaldehyde (7), p-hydroxycinnamaldehyde (8), trans-p-coumaryl alcohol (9), galangin (10), trans-p-coumaric acid (11) and galanganol B (12) were isolated from these extracts. Of these, compounds 2, 3, 4 and 5 were found most active in vitro against promastigotes of L. donovani with IC50 values of 39.3, 32.9, 18.9 and 79.9 microM respectively. This is the first report of antileishmanial activity of the extracts and isolated constituents of A. galanga.     

Probing kinetic drug binding mechanism in voltage-gated sodium ion channel: open state versus inactive state blockers

The kinetics and nonequilibrium thermodynamics of open state and inactive state drug binding mechanisms have been studied here using different voltage protocols in sodium ion channel. We have found that for constant voltage protocol, open state block is more efficient in blocking ionic current than inactive state block. Kinetic effect comes through peak current for mexiletine as an open state blocker and in the tail part for lidocaine as an inactive state blocker. Although the inactivation of sodium channel is a free energy driven process, however, the two different kinds of drug affect the inactivation process in a different way as seen from thermodynamic analysis. In presence of open state drug block, the process initially for a long time remains entropy driven and then becomes free energy driven. However in presence of inactive state block, the process remains entirely entropy driven until the equilibrium is attained. For oscillating voltage protocol, the inactive state blocking is more efficient in damping the oscillation of ionic current. From the pulse train analysis it is found that inactive state blocking is less effective in restoring normal repolarisation and blocks peak ionic current. Pulse train protocol also shows that all the inactive states behave differently as one inactive state responds instantly to the test pulse in an opposite manner from the other two states.