Resveratrol and curcumin synergistically induces apoptosis in cigarette smoke condensate transformed breast epithelial cells through a p21Waf1/Cip1 mediated inhibition of Hh-Gli signaling

Combination therapy using two or more small molecule inhibitors of aberrant signaling cascade in aggressive breast cancers is a promising therapeutic strategy over traditional monotherapeutic approaches. Here, we have studied the synergistic mechanism of resveratrol and curcumin induced apoptosis using in vitro (cigarette smoke condensate mediated transformed breast epithelial cell, MCF-10A-Tr) and in vivo (tumor xenograft mice) model system. Resveratrol exposure increased the intracellular uptake of curcumin in a dose dependent manner and caused apoptosis in MCF-10A-Tr cells. Approximately, ten fold lower IC₅₀ value was noted in cells treated with the combination of resveratrol (3 μM) and curcumin (3 μM) in comparison to 30 μM of resveratrol or curcumin alone. Resveratrol + curcumin combination caused apoptosis by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and caspase 3 in cells. Interestingly, this combination unaltered the protein expressions of WNT-TCF and Notch signaling components, β-catenin and cleaved notch-1 val1744, respectively. Furthermore, the combination also significantly decreased the intermediates of Hedgehog-Gli cascade including SMO, SHH, Gli-1, c-MYC, Cyclin-D1, etc. and increased the level of p21^Waf/Cip1 in vitro and in vivo. A significant reduction of Gli- promoter activity was noted in combinational drug treated cells in comparison to individual drug treatment. Un-alteration of the expressions of the above proteins and Gli1 promoter activity in p21^Waf/Cip1 knockout cells suggests this combination caused apoptosis through p21^Waf/Cip1. Thus, our findings revealed resveratrol and curcumin synergistically caused apoptosis in cigarette smoke induced breast cancer cells through p2  ^Waf/Cip1 mediated inhibition of Hedgehog-Gli cascade.     

Eucalyptol protects lungs against bacterial invasion through attenuating ciliated cell damage and suppressing MUC5AC expression

This study was conducted to investigate whether eucalyptol plays a role in influencing bacterial growth in cigarette smoke-exposed lungs. Rats were exposed to air (control) and cigarette smoke (smoking) in the presence and absence of eucalyptol (260 mg/day). Morphological analysis of lung structures and status of airway mucous production were observed under microscope. Pathological changes of ciliated columnar epithelium in airways were examined using transmission electron microscopy. MUC5AC protein and messenger RNA (mRNA) expression in bronchoalveolar lavage fluid (BALF) and lungs were determined. Application of eucalyptol reduced pulmonary bullae formation and airway mucus overproduction in the smoke-exposed lungs. Treatment with eucalyptol attenuated ciliated cell damage in cigarette smoke-exposed lungs. Bacterial colonies of lungs were obviously lower in the eucalyptol-treated rats than that in the smoking rats (p < 0.01). Treatment with eucalyptol reduced the counts of bacterial colonization residing in the challenged lungs (p < 0.01). Application of eucalyptol not only decreased MUC5AC protein expression in BALF and tobacco-exposed lungs but also suppressed its mRNA expression in the lungs (all p < 0.05). Intervention of eucalyptol benefits elimination of bacterial organisms from tobacco-exposed lungs through attenuating ciliated cell damage and suppressing MUC5AC expression in the lungs.     

Tips for improving the quality and quantity of the extracted DNA from exhaled breath condensate samples

Abstract

There is a growing interest in the tracking of genetic and epigenetic alterations in exhaled breath condensate (EBC) samples. The effects of different procedures on the quality and quantity of DNA in EBC were studied. The results demonstrated that sodium acetate precipitation and oligo (dT) improved the quality of the extracted DNA significantly (p?<?0.01). Also, sodium acetate precipitation, using oligo (dT), incubation at 70?°C and SDS treatment increased the quantity of DNA significantly (p?<?0.01). These results showed the advantages of the chemical and physical manipulations for improving the quality and quantity of the extracted DNA from EBC samples.     

Social capital and cigarette smoking: New empirics featuring the Norwegian HUNT data

Using a rich Norwegian longitudinal data set, this study explores the effects of different social capital variables on the probability of cigarette smoking. There are four social capital variables available in two waves of our data set. Our results based on probit (and OLS) analyses (with municipality fixed-effects) show that the likelihood of smoking participation is negatively and significantly associated with social capital attributes, namely, community trust (–0.017), participation in organizational activities (–0.032), and cohabitation (–0.045). Significant negative associations were also observed in panel data, pooled OLS, and random effects models for community trust (–0.024; −0.010) and cohabitation (–0.040; −0.032). Fixed-effects models also showed significant negative effects for cohabitation (–0.018). Estimates of alternative instrumental variables (IV) based on recursive bivariate probit and IV-GMM models also confirmed negative and significant effects for three of its characteristics: cohabitation (–0.030; −0.046), community trust (–0.065; −0.075), and participation in organizational activities (–0.035; −0.046). The limitations of our conclusions are discussed, and the significance of our study for the field of social capital and health is described, along with suggested avenues for future research.     

Acute effects of smoking and high experimental exposure to environmental tobacco smoke (ETS) on the immune system

Controversial results have been published on the immune response to cigarette smoking while the effects of exposure to environmental tobacco smoke (ETS) have not yet been reported. In a controlled study, acute effects of smoking and of a high environmental exposure to ETS on immunological parameters have been investigated. The study consisted of four experimental days, two control and two exposure days. On control days, 1 and 3, smokers (n=5) and nonsmokers (n=5) sat in an unventilated 45 m³ room for 8 h. On the exposure days, 2 and 4, each of the smokers smoked 24 cigarettes in 8 h, while the nonsmokers were exposed to the ETS generated by the smoking volunteers. Blood was drawn before and after each exposure session on all four experimental days for dosimetry of tobacco smoke exposure and determination of the immune response. Flow cytometry using monoclonal antibodies was used to determine CD3⁺ cells (whole T cells), CD19⁺ cells (B lymphocytes), CD16⁺ and CD56⁺ cells (natural killer cells), CD4⁺ cells (T-helper cells), CD8⁺ cells (T-suppressor cells), the CD4⁺/CD8⁺ (helper/supressor ratio), and Fc receptors on granulocytes. Serum was analyzed for soluble CD14 receptors (scD14), interleukin 1, interleukin 6 and prostaglandin E₂ (PGE₂). Functional stimulation assays were performed to determine the basal and induced level of reactive oxygen intermediate (ROI) production by polymorphic neutrophils. Exposure to tobacco smoke in both groups was confirmed by dosimetry of carboxyhemoglobin, plasma nicotine, and cotinine levels. In comparison to nonsmokers, smokers had elevated granulocyte cell counts, increased CD16⁺ and CD56⁺ cell levels and decreased CD3⁺ and CD19⁺ levels. Acute smoking, but not exposure to ETS, resulted in a slight decrease in the number of CD19⁺ cells and an increase in the number of granulocytes; the latter was restricted to one subject. Acute smoking and exposure to high experimental concentrations of ETS resulted in a slight increase in CD16⁺ and CD56⁺ cells. None of the changes determined in immunological parameters after either acute smoking or exposure to ETS reached statistical significance. Serum sCD14, cytokine and PGE₂, functional stimulation of in vitro ROI production, and changes in Fc receptors were not affected by acute smoking or exposure to ETS. Although no clear guidelines exist to assess immunotoxicity in man, our data do not favor immunosuppression and the possibility of increased risk of infection in nonsmokers exposed to ETS under real-life conditions.Abbreviations AM alveolar macrophage - BALF bronchoalveolar lavage fluid - CO carbon monoxide - CO₂ carbon dioxide - COHb carboxyhemoglobin - ELISA enzyme linked immunoassay - ETS environmental tobacco smoke - FITC fluorescein isothiocyanate - IL interleukin - MHC major histocompatibility complex - NK natural killer cell - NO nitrogen oxide - NO₂ nitrogen dioxide - PBS phosphate-buffered saline - PE phycoerythrin - PGE₂ prostaglandin E₂ - PMA phorbol-12-myristate-13-acetate - PMN polymorphic neutrophils - RIA radioimmunoassay - ROI reactive oxygen intermediates - RSP respirable suspended particles - sCD14 soluble CD14 receptor     

Fuel smoke condensate induced DNA damage in human lymphocytes and protection by turmeric (Curcuma longa)

Summary Twigs-dry leaves smoke condensate (TDS) was investigated for its DNA damaging activity in human peripheral lymphocytes, by using a sensitive method, fluorescence analysis of DNA unwinding (FADU). An aqueous turmeric component (Aq.T) was studied as a protective agent. TDS at one to 100 folds dilution induced 55% DNA damage at 20 min, while 12-0-tetradecanoylphorbol-13-acetate (TPA) at 10 ng/ml induced only 25% damage. Aq.T at 300 ng/1 afforded 90% protection to DNA against TPA and 65% against TPA. The mechanism of Aq.T protection was investigated by using (i) inhibitors of arachidonate cascade, viz., indomethacin (28 M), NDGA (10 M), DBAP (36 M), (ii) antioxidant enzymes viz., CAT (0.2 U/l), SOD (0.6 U/1), (iii) antioxidants - BHA, curcumin (40 M), mixed gangliosides (20 nM) and protease inhibitor TLCK (100 M). These compounds offered the following extents of protection to DNA against TDS: indomethacin-40%, NDGA-83%, DBAP-70%, SOD-38%, CAT-40%, BHA-38%, curcumin-60%, mixed gangliosides-88%, TLCK-85%. Against TPA as clastogenic agent, the extents of protection were: indomethacin-73%, NDGA-32%, DBAP-72%, SOD-60%, CAT, BHA-negligible, curcumin-23%, mixed gangliosides - 60%, TLCK - 59%. These results indicate that (i) TDS and TPA induce DNA damage possibly by different mechanisms, (ii) Aq.T is a more effective protectant against TDS whereas it is on par with other inhibitors against TPA.Abbreviations FADU Fluoroscence Analysis of DNA Unwinding - Aq.T Aqueous component of turmeric - TDS Twigs-Dry leaves Smoke condensate - PBS Phosphate Buffered Saline, 20 mM, 150 mM NaCl, pH 7.4 - TPA 12-O-Tetradecanoyl Phorbol-13-Acetate - NDGA Nordihydroguaiaretic Acid - DBAP 2,4-Dibromo Acetophenone - CAT Catalase - SOD Superoxide Dismutase - BHA Butylated Hydroxyanisole - TLCK Tosyl Lysyl Chloromethyl Ketone - ROS Reactive Oxygen Species - PAH Polycyclic Aromatic Hydrocarbons - DMSO Dimethyl Sulfoxide - Buffer B 250 mM m-inositol, 10 mM sodium phosphate, 1 mM magnesium chloride, pH 7.3 - BSC Beedi Smoke Condensate - CSC Cigarette Smoke Condensate     

The role of after‐ripening in promoting germination of arid zone seeds: a study on six Australian species

The effects of after‐ripening (storage under warm, dry conditions) on seed germination was examined in six plant species from the arid zone of Western Australia with the aim of improving germination and germination rate for rehabilitation objectives. Study species (Acanthocarpus preissii, Anthocercis littorea, Dioscorea hastifolia, Eremophila oldfieldii, Thryptomene baeckeacea and Zygophyllum fruticulosum) were selected based on diverse plant habits, seed types and requirements for rehabilitation. After‐ripening was investigated by adjusting seed moisture content to 13 and 50 equilibrium relative humidity (eRH) at 23 °C and storing seeds at two temperatures (30 and 45 °C) from 1 to 18 months. Following storage, seeds were incubated in water, gibberellic acid (GA₃) or karrikinolide (KAR₁; the butenolide, 3‐methyl‐2H‐furo[2,3‐c]pyran‐2‐one). All after‐ripening conditions increased germination percentage and rate of A. littorea and D. hastifolia, with A. littorea only germinating when treated with GA₃ or KAR₁. The germination of Z. fruticulosum was dependent on after‐ripening temperature and seed moisture content. After‐ripening had a limited effect on the remaining three species. The restoration implications of the findings are discussed. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161 , 411–421.     

The effect of a plant-derived smoke extract on the germination of light-sensitive lettuce seed

A simple and rapid bioassay using seeds of Lactuca sativa L. Grand Rapids has been developed for the detection of germination-enhancing compound(s) in plant-derived smoke extracts. This light-sensitive species germinates within 24 h in the dark at 20 or 25°C and shows responsiveness to smoke-derived extracts over a wide concentration range. For some seed lots where the P _fr level is high and germination in the dark is unacceptably high, a brief (10 min) exposure to far-red light, one hour after the start of imbibition in the dark, is necessary to clearly demonstrate biological activity in the smoke extracts.