Stimuli-responsive colorimetric and NIR fluorescence combination probe for selective reporting of cellular hydrogen peroxide.

Hydrogen peroxide (H2O2) is a key reactive oxygen species and a messenger in cellular signal transduction apart from playing a vital role in many biological processes in living organisms. In this article, we present phenyl boronic acid-functionalized quinone-cyanine (QCy-BA) in combination with AT-rich DNA (exogenous or endogenous cellular DNA), i.e., QCy-BA⊂DNA as a stimuli-responsive NIR fluorescence probe for measuring in vitro levels of H2O2. In response to cellular H2O2 stimulus, QCy-BA converts into QCy-DT, a one-donor-two-acceptor (D2A) system that exhibits switch-on NIR fluorescence upon binding to the DNA minor groove. Fluorescence studies on the combination probe QCy-BA⊂DNA showed strong NIR fluorescence selectively in the presence of H2O2. Furthermore, glucose oxidase (GOx) assay confirmed the high efficiency of the combination probe QCy-BA⊂DNA for probing H2O2 generated in situ through GOx-mediated glucose oxidation. Quantitative analysis through fluorescence plate reader, flow cytometry and live imaging approaches showed that QCy-BA is a promising probe to detect the normal as well as elevated levels of H2O2 produced by EGF/Nox pathways and post-genotoxic stress in both primary and senescent cells. Overall, QCy-BA, in combination with exogenous or cellular DNA, is a versatile probe to quantify and image H2O2 in normal and disease-associated cells.

Epithelial atrophy in oral submucous fibrosis is mediated by copper (II) and arecoline of areca nut.

Exposure of oral cavity to areca nut is associated with several pathological conditions including oral submucous fibrosis (OSF). Histopathologically OSF is characterized by epithelial atrophy, chronic inflammation, juxtaepithelial hyalinization, leading to fibrosis of submucosal tissue and affects 0.5% of the population in the Indian subcontinent. As the molecular mechanisms leading to atrophied epithelium and fibrosis are poorly understood, we studied areca nut actions on human keratinocyte and gingival fibroblast cells. Areca nut water extract (ANW) was cytotoxic to epithelial cells and had a pro-proliferative effect on fibroblasts. This opposite effect of ANW on epithelial and fibroblast cells was intriguing but reflects the OSF histopathology such as epithelial atrophy and proliferation of fibroblasts. We demonstrate that the pro-proliferative effects of ANW on fibroblasts are dependent on insulin-like growth factor signalling while the cytotoxic effects on keratinocytes are dependent on the generation of reactive oxygen species. Treatment of keratinocytes with arecoline which is a component of ANW along with copper resulted in enhanced cytotoxicity which becomes comparable to IC(50) of ANW. Furthermore, studies using cyclic voltammetry, mass spectrometry and plasmid cleavage assay suggested that the presence of arecoline increases oxidation reduction potential of copper leading to enhanced cleavage of DNA which could generate an apoptotic response. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling assay and Ki-67 index of OSF tissue sections suggested epithelial apoptosis, which could be responsible for the atrophy of OSF epithelium.

Activation of TGF-ß pathway by areca nut constituents: a possible cause of oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as an important etiological factor for disease manifestation. Activation of transforming growth factor-ß signaling has been postulated as the main causative event for increased collagen production in OSF. Oral epithelium plays important roles in OSF, and arecoline has been shown to induce TGF-ß in epithelial cells. In an attempt to understand the role of areca nut constituents in the manifestation of OSF, we studied the global gene expression profile in epithelial cells (HaCaT) following treatment with areca nut water extract or TGF-ß. Interestingly, 64% of the differentially regulated genes by areca nut water extract matches with the TGF-ß induced gene expression profile. Out of these, expression of 57% of genes was compromised in the presence of ALK5 (TßRI) inhibitor and 7% were independently induced by areca nut, highlighting the importance of TGF-ß in areca nut actions. Areca nut water extract treatment induced p-SMAD2 and TGF-ß downstream targets in HaCaT cells but not in human gingival fibroblast cells (hGF), suggesting epithelial cells could be the source of TGF-ß in promoting OSF. Water extract of areca nut consists of polyphenols and alkaloids. Both polyphenol and alkaloid fractions of areca nut were able to induce TGF-ß signaling and its downstream targets. Also, SMAD-2 was phosphorylated following treatment of HaCaT cells by Catechin, Tannin and alkaloids namely Arecoline, Arecaidine and Guvacine. Moreover, both polyphenols and alkaloids induced TGF-ß2 and THBS1 (activator of latent TGF-ß) in HaCaT cells suggesting areca nut mediated activation of p-SMAD2 involves up-regulation and activation of TGF-ß. These data suggest a major causative role for TGF-ß that is induced by areca nut in OSF progression.

Caryophyllene-rich essential oil of Didymocarpus tomentosa: chemical composition and cytotoxic activity.

The essential oil from the leaves of Didymocarpus tomentosa was extracted by hydrodistillation and analyzed by GC/FID and GC/MS. Twenty five constituents amounting to 81.6% of the oil were identified. The leaf oil contained 78.7% sesquiterpenes and 2.9% monoterpenes. The leaf essential oil of D. tomentosa is a unique caryophyllene-rich natural source containing beta-caryophyllene, caryophyllene oxide, a-humulene and humulene oxide. The cytotoxic activity of the oil was determined by the BSLT using shrimp larva and the MTT assay using HeLa tumor cell line. The oil showed significant cytotoxic activity with LC50 and IC50 values of 12.26 and 11.4 microg/mL, respectively. This is the first report on the chemical composition and cytotoxic activity of the essential oil of D. tomentosa.