Protective effect of Withaferin-A on micronucleus frequency and detoxication agents during experimental oral carcinogenesis

Our aim was to investigate the effect of Withaferin-A on bone marrow micronucleus frequency andbuccal mucosa detoxication agents during 7, 12–dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis. Oral squamous cell carcinoma was developed in hamsters’ buccal pouches by painting 0.5% DMBA in liquid paraffin, three times per week for 14 weeks. We observed 100% tumor formation in DMBA painted hamsters. Elevated frequency of bone marrow micronucleated polychromatic erythrocytes (MnPCEs) and decrease in buccal mucosa phase II detoxication agents were noticed in tumor bearing hamsters. Oral administration of Withaferin-A significantly reduced the micronucleus frequency and brought back the status of phase II detoxication agents in DMBA painted hamsters. Our study thus demonstrated the protective effect of Withaferin-A on DMBA-induced micronucleus frequency in the bone marrow of golden Syrian hamsters. Also, Withaferin-A maintained the status of buccal mucosa detoxication agents during DMBA-induced hamster buccal pouch carcinogenesis.Key words: Withaferin-A, DMBA, Oral cancer, Micronucleus, Detoxicatio

Protective role of Withaferin-a on red blood cell integrity during 7,12-dimethylbenz[A]anthracene induced oral carcinogenesis

The aim of the present study was to investigate the protective effect of Withaferin-A on red blood cell integrity during 7,12-dimethylbenz[a]anthracene (DMBA) induced oral carcinogenesis. The protective effect of Withaferin-A was assessed by measuring the status of glycoconjugates, membrane bound enzyme activity and red blood cell osmotic fragility. Oral squamous cell carcinoma was induced in the buccal pouch of Syrian golden hamsters by painting with 0.5% DMBA in liquid paraffin thrice a week for 14 weeks. The levels of glycoconjugates, membrane bound enzyme activity, osmotic fragility and thiobarbituric acid reactive substances (TBARS) were analyzed by using specific colorimetric methods. We observed 100% tumor formation in DMBA painted hamsters. Increase in plasma glycoconjugates at the expense of red blood cell membrane glycoconjugates levels were observed in DMBA painted hamsters as compared to control hamsters. Erythrocytes from DMBA painted hamsters were more fragile than those from control hamsters. The activity of membrane bound enzyme (Na+ K+ ATPase) decreased whereas TBARS level was increased in DMBA painted hamsters as compared to control hamsters. Oral administration of Withaferin-A at a dose of 20mg kg-1 bw significantly prevented the tumor formation as well as normalized the biochemical variables in DMBA painted hamsters. Our results thus demonstrate the protective effect of Withaferin-A on red blood cell integrity during DMBA induced oral carcinogenesis.Key words: DMBA, Withaferin-A, Oral cancer, glycoconjugates, osmotic fragility

Synthesis and antiproliferative activity of novel homopiperazine derivatives in leukemia cells

A series of novel homopiperazine derivatives were synthesized and characterized using 1H NMR, LC MS, IR and elemental analysis data. These novel molecules were evaluated for their antiproliferative activity against Reh, leukemia cells using trypan blue and MTT assays. All the molecules showed cytotoxicity with IC50 values between 50-100 μM as calculated by trypan blue assay and greater than 100 μM as calculated by MTT assay. Compound 6b with 3,5-dinitro substituents on phenyl ring of the aryl carboxamide moiety attached to homopiperazine ring showed good activity with IC50 value of 41 μM

Effect of venlafaxine on bone loss associated with ligature-induced periodontitis in Wistar rats

<p>Abstract</p> <p>Background</p> <p>The present study investigated the effects of venlafaxine, an antidepressant drug with immunoregulatory properties on the inflammatory response and bone loss associated with experimental periodontal disease (EPD).</p> <p>Materials and Methods</p> <p>Wistar rats were subjected to a ligature placement around the second upper left molar. The treated groups received orally venlafaxine (10 or 50 mg/kg) one hour before the experimental periodontal disease induction and daily for 10 days. Vehicle-treated experimental periodontal disease and a sham-operated (SO) controls were included. Bone loss was analyzed morphometrically and histopathological analysis was based on cell influx, alveolar bone, and cementum integrity. Lipid peroxidation quantification and immunohistochemistry to TNF-α and iNOS were performed.</p> <p>Results</p> <p>Experimental periodontal disease rats showed an intense bone loss compared to SO ones (SO = 1.61 ± 1.36; EPD = 4.47 ± 1.98 mm, p < 0.001) and evidenced increased cellular infiltration and immunoreactivity for TNF-α and iNOS. Venlafaxine treatment while at low dose (10 mg/kg) afforded no significant protection against bone loss (3.25 ± 1.26 mm), a high dose (50 mg/kg) caused significantly enhanced bone loss (6.81 ± 3.31 mm, p < 0.05). Venlafaxine effectively decreased the lipid peroxidation but showed no significant change in TNF-α or iNOS immunoreactivity.</p> <p>Conclusion</p> <p>The increased bone loss associated with high dose venlafaxine may possibly be a result of synaptic inhibition of serotonin uptake.</p

Withanolides and related steroids

Since the isolation of the first withanolides in the mid-1960s, over 600 new members of this group of compounds have been described, with most from genera of the plant family Solanaceae. The basic structure of withaferin A, a C28 ergostane with a modified side chain forming a δ-lactone between carbons 22 and 26, was considered for many years the basic template for the withanolides. Nowadays, a considerable number of related structures are also considered part of the withanolide class; among them are those containing γ-lactones in the side chain that have come to be at least as common as the δ-lactones. The reduced versions (γ and δ-lactols) are also known. Further structural variations include modified skeletons (including C27 compounds), aromatic rings and additional rings, which may coexist in a single plant species. Seasonal and geographical variations have also been described in the concentration levels and types of withanolides that may occur, especially in the Jaborosa and Salpichroa genera, and biogenetic relationships among those withanolides may be inferred from the structural variations detected. Withania is the parent genus of the withanolides and a special section is devoted to the new structures isolated from species in this genus. Following this, all other new structures are grouped by structural types. Many withanolides have shown a variety of interesting biological activities ranging from antitumor, cytotoxic and potential cancer chemopreventive effects, to feeding deterrence for several insects as well as selective phytotoxicity towards monocotyledoneous and dicotyledoneous species. Trypanocidal, leishmanicidal, antibacterial, and antifungal activities have also been reported. A comprehensive description of the different activities and their significance has been included in this chapter. The final section is devoted to chemotaxonomic implications of withanolide distribution within the Solanaceae. Overall, this chapter covers the advances in the chemistry and biology of withanolides over the last 16 years.Fil: Misico, Rosana Isabel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos Aplicados a la Química Orgánica (i); ArgentinaFil: Nicotra, V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; ArgentinaFil: Oberti, Juan Carlos María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; ArgentinaFil: Barboza, Gloria Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; ArgentinaFil: Gil, Roberto Ricardo. University Of Carnegie Mellon; Estados UnidosFil: Burton, Gerardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos Aplicados a la Química Orgánica (i); Argentin

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. © 2013 Dias et al

Abstract 3301: A three-pronged attack on cancer cells: Induction of non-protective autophagy, inhibition of lysosomal acidification and promotion of energetic impairment

Abstract Background and Aim: Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. At the functional level, autophagic process can be cytoprotective, cytotoxic, cytostatic or nonprotective. We investigated strategies to convert cellular autophagic response to non-protective autophagy which does not interfere with therapeutic regimens exploiting bioactive molecules. Methods: Utilizing in vivo xenograft models, we established that Withaferin A (WA), a bioactive molecule from Withania Somnifera inhibits breast tumorigenesis. Autophagy studies were conducted utilizing immunoblot, RT-PCR, and immunofluorescence analyses for autophagy markers, transmission electron microscopy and confocal imaging. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red and GFP-LC3/mCherryRAB7A. Protein degradation activity of lysosomes and ATP levels were examined by DQ-BSA assay, Cathepsin activity and quantitative ATP assay. Results: WA inhibited growth and induced apoptosis in breast cancer cells resulting in inhibition of breast carcinogenesis in vivo. Although WA increased tumor suppressor LKB1 which is known to be involved in autophagy, WA-mediated increased cleavage of Light Chain 3 type II (LC3-II) and punctated LC3-II staining was LKB1-independent. The redistribution of EGFP-LC3 from cytosol to autophagosome indicated increased formation of autophagosomes in WA-treated cells. However, WA-induced increased autophagosome-formation was not mediated by increased activation of autophagy by upstream processes but was due to blockade of lysosomal-degradation as evident by higher level of sequestosome 1 (SQSTM1/p62) and decreased turnover of LC3. WA was found to be a potent lysosomal deacidification agent capable of blocking autophagic flux. Accordingly, inhibiting autophagy by blocking formation of autophagosomes or elevating lysosomal pH did not interfere with WA-mediated growth-inhibition. WA blocked autophagic flux decreasing recycling of cellular fuels leading to reduced energy supply. Investigating this alternative mechanism, we discovered that indeed, WA reduced ATP levels and increased phosphorylation of AMP-activated protein kinase (AMPK). Modulating substrates for tricarboxylic acid (TCA) cycle with methyl pyruvate protected WA-treated cells while 2DG potentiated WA-induced cell death. Conclusion: Our results indicate that WA induces a non-protective autophagy and blocks energy fuels in cancer cells by reducing ATP levels and inhibiting lysosomal acidification hence offering a three-pronged approach to facilitate cancer cell death. WA might be a useful strategic addition to chemotherapy regimens to evade cytoprotective effects of autophagy. Citation Format: Nethaji Muniraj, Arumugam Nagalingam, Panjamurthy Kuppusamy, Neeraj K. Saxena, Dipali Sharma. A three-pronged attack on cancer cells: Induction of non-protective autophagy, inhibition of lysosomal acidification and promotion of energetic impairment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3301. doi:10.1158/1538-7445.AM2017-3301</jats:p