Black Tea Extract, via Modulation of TGF-ß Pathway, Prevents Inorganic Arsenic-induced Development of Squamous Cell Carcinoma of the Skin in Swiss Albino Mice.

Chronic exposure to inorganic arsenic (iAs) elevates reactive oxygen species (ROS) generation and up-regulates TGF-ß signalling. This promotes induction of epithelial to mesenchymal transition (EMT) and causes the development of squamous cell carcinoma (SCC) of skin. Black tea is a popular beverage worldwide and an effective antioxidant. Chemopreventive potential of black tea extract (BTE) against iAs induced carcinogenicity has been explored here. The study aims to investigate the role of BTE in prevention of iAs-induced SCC of skin in Swiss albino mice via the modulation of TGF-ß signalling and EMT. Mice were divided into (1) control, (2) iAs, (3) iAs+BTE, and (4) BTE groups and were administered iAs and BTE alone, or in combination for 330 days. Histological studies were performed to assess development of SCC. ROS generation was estimated by flowcytometry. Expression of TGF-ß and downstream proteins belonging to suppressor of mothers against decapentaplegic (Smad), phosphoinositide-3-kinase (PI3K)-protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) pathways was assessed by immunoblotting. Expression of EMT markers was evaluated by immunoblotting, immunohistochemistry and semi-quantitative reverse transcriptase-PCR. After 330 days of iAs treatment, development of invasive SCC of skin probably due to excess ROS generation, elevation of TGF-ß, downregulation of the Smad pathway, upregulation of PI3K-AKT and MAPK signalling molecules and induction of EMT was observed. All these modulations were found to be reversed by BTE, which inhibits iAs induced SCC of skin by quenching excess ROS, promoting Smad mediated TGF-ß signalling, downregulating signalling intermediates of PI3K-AKT and MAPK pathways and inhibiting EMT.

Black tea extract prevents inorganic arsenic induced uncontrolled proliferation, epithelial to mesenchymal transition and induction of metastatic properties in HaCaT keratinocytes - an in vitro study.

A major global problem is chronic exposure to inorganic arsenic (iAs) which causes various health hazards including cancer. Escalation of reactive oxygen species (ROS) generation by chronic iAs exposure promotes Epithelial to Mesenchymal transition (EMT) which is followed by metastatic progression. In the present study, skin keratinocyte cells (HaCaT) were divided into three groups: (i) untreated, (ii) chronically iAs exposed, (iii) black tea extract (BTE) along with iAs treated. ROS was estimated by flowcytometry, expression of EMT markers were assessed by flowcytometry, western-blotting and Immunofluorescence. For metastatic studies, wound-healing assay, gelatin zymography, western-blot, transwell migration/invasion assay had been performed. Long term exposure of HaCaT cells to iAs causes excess generation of ROS. Morphological transformation and EMT were apparent at 210 days of exposure. Development of metastatic characteristics were observed at 240 days. Alterations in the parameters induced by iAs were found to be ameliorated by BTE. BTE was found to quench excess generation of ROS by iAs, subsequently inhibiting the chain of events like EMT and metastasis. Therefore, BTE may be considered as a potential phytochemical to prevent the deleterious effect of iAs. Skin carcinogenesis induced by iAs may thus be prevented by BTE via inhibition of EMT.

Prevention of inorganic arsenic induced squamous cell carcinoma of the skin in Swiss albino mice by black tea through epigenetic modulation.

Consumption of inorganic Arsenic (iAs) above the safe level may lead to many diseases including cancers of skin. It is known that carcinogenicity of iAs is mediated through generation of excessive reactive oxygen species and polyphenols present in black tea extract (BTE) ameliorate the deleterious effect. Epigenetics also plays vital roles in carcinogenesis. The aim of this paper is to study the influence of iAs on epigenetics and the modulatory effect of BTE. Male Swiss albino mice were divided into three groups, (i) control, (ii) iAs-administered and (iii) iAs + BTE administered. Group (ii) developed invasive squamous cell carcinoma (SCC) of the skin after 330 days, while only hyperplasic and dysplastic changes were observed in group (iii). Expression levels of histone methylation, acetylation marks and several histone methylases, demethylases and acetylases due to iAs were studied; most aberrant expression levels due to iAs were modulated by BTE. JARID1B, a histone demethylase implicated as one of the markers in SCC and a therapeutic target gets upregulated by iAs, but is not influenced by BTE. However, SCC is prevented by BTE. Upregulation of JARID1B by iAs represses H3K4me3; BTE upregulates H3K4me3 without influencing JARID1B expression level. It is known that theaflavin compounds in BTE are transported to the nucleus and interact with histone proteins. in-silico findings in this paper hint that theaflavin compounds present in BTE are very good inhibitors of JARID1B and BTE inhibits its demethylating activity. BTE reverses the epigenetic alterations caused by iAs, thus aids in prevention of SCC.

Chemopreventive Role of Black Tea Extract in Swiss Albino Mice Exposed to Inorganic Arsenic.

OBJECTIVE: Chronic exposure to inorganic arsenic (iAs) may cause a number of health problems including skin cancer. Present study is aimed to look into the potential of black tea extract (BTE) to prevent the development of skin carcinoma in Swiss albino mice. METHODS: The study was done on Swiss albino mice, chronically exposed to inorganic arsenic. 150 mice were housed in different cages, 5 in each cage. The control mice did not receive any treatment. Mice were sacrificed at 30, 90, 180, 270 and 330 days. Development of carcinogenesis was assessed by histological studies. Generation of Reactive Oxygen Species (ROS) and Reactive Oxygen Species (RNS) were estimated using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Greiss reagent respectively, and their consequences on DNA (by Micronuclei and Comet assay), protein (by protein carbonyl assay kit) and lipid (by lipid peroxidation) were estimated. Activity of antioxidant enzymes, along with total antioxidant capacity were measured by respective kits. Repair percentage was obtained by Comet assay. Western blotting was employed to study the expression of repair enzymes and expression of cytokines. Sandwich Enzyme-linked Immunosorbent Assay (ELISA) technique was employed to study the activity of various cytokines. RESULTS: At 330 days, invasive squamous cell carcinoma of the skin developed. With increasing time generation of ROS and RNS increased, causing damage to DNA, protein and lipid. Antioxidant defence system gets repressed with time. Capacity to repair the DNA damage is inhibited by iAs, due to alteration in repair enzymes - XRCC I, DNA Ligase I, PARP I, ERCC1, ERCC2, XPA, DNA Ligase IV, DNA PKc and Ku-70. Another consequence of iAs exposure is chronic inflammation due to disrupted cytokine level. Intervention with BTE reverses these deleterious effects, preventing development of skin carcinogenesis.

Curcumin Rescues Doxorubicin Responsiveness via Regulating Aurora a Signaling Network in Breast Cancer Cells.

BACKGROUND: Insensitivity towards anthracycline drugs like doxorubicin poses a significant challenge in the treatment of breast cancer. Among several factors, Aurora A (a mitotic serine threonine kinase) plays crucial roles in acquiring non-responsiveness towards doxorubicin. However, the mechanisms underlying need to be elucidated. The present study was therefore designed to evaluate the underlying mechanisms of Aurora A mediated doxorubicin insensitivity in MCF-7Dox/R, an isolated resistant-subline of MCF-7 (breast adenocarcinoma cell line). Effect of curcumin, a natural phytochemical in restoring doxorubicin sensitivity by targeting Aurora A was assessed furthermore. METHODS: A doxorubicin resistant subline (MCF-7Dox/R) was isolated from the parental MCF-7 cells by treating the cell with gradual step-wise increasing concentration of the drug. Expressions of Aurora A and its target proteins (Akt, IκBα and NFκB) were assessed in both parental and MCF-7Dox/R cells. Both the cell lines were pretreated with curcumin prior to doxorubicin treatment. Cellular proliferation rate was measured using BrdU (5-bromo-2'-deoxyuridine) assay kit. Intracellular doxorubicin accumulation was estimated spectrofluorimetrically. Cellular uptake of curcumin (spectrophotometric and spectrofluorimetric method) and its nuclear localization was confirmed by confocal microscopic study. Protein expressions were determined by western blot analysis. Localization of Aurora A was ascertained by immunofluorescence assay. To explore the possible outcome of impact of curcumin on Aurora A, cell-cycle distribution and apoptosis were performed subsequently. RESULTS: Higher expressions of Aurora A in MCF-7Dox/R cells led to phosphorylation of Akt as well as IκBα. Phosphorylated IκBα preceded release of NFκB. Phospho-Akt, NFκB consequentially decreased doxorubicin accumulation by enhancing the expressions of ABCG2 and Pgp1 respectively. Curcumin by regulating Aurora A and its target molecules sensitized resistant subline towards doxorubicin mediated G2/M-arrest and apoptosis. CONCLUSION: Molecular targeting of Aurora A by curcumin restores chemosensitivity by increasing the efficacy of doxorubicin in breast cancer.
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