Overexpressed HDAC8 in cervical cancer cells shows functional redundancy of tubulin deacetylation with HDAC6.

BACKGROUND: Histone deacetylases (HDACs) are involved in epigenetic gene regulation via deacetylation of acetylated lysine residues of both histone and non-histone proteins. Among the 18 HDACs identified in humans, HDAC8, a class I HDAC, is best understood structurally and enzymatically. However, its precise subcellular location, function in normal cellular physiology, its protein partners and substrates still remain elusive. METHODS: The subcellular localization of HDAC8 was studied using immunofluorescence and confocal imaging. The binding parterns were identified employing immunoprecipitation (IP) followed by MALDI-TOF analysis and confirmed using in-silico protein-protein interaction studies, HPLC-based in vitro deacetylation assay, intrinsic fluorescence spectrophotometric analysis, Circular dichroism (CD) and Surface Plasmon Resonance (SPR). Functional characterization of the binding was carried out using immunoblot and knockdown by siRNA. Using one way ANOVA statistical significance (n = 3) was determined. RESULTS: Here, we show that HDAC8 and its phosphorylated form (pHDAC8) localized predominantly in the cytoplasm in cancerous, HeLa, and non-cancerous (normal), HEK293T, cells, although nucleolar localization was observed in HeLa cells. The study identified Alpha tubulin as a novel interacting partner of HDAC8. Further, the results indicated binding and deacetylation of tubulin at ac-lys40 by HDAC8. Knockdown of HDAC8 by siRNA, inhibition of HDAC8 and/or HDAC6 by PCI-34051 and tubastatin respectively, cell-migration, cell morphology and cell cycle analysis clearly explained HDAC8 as tubulin deacetylase in HeLa cells and HDAC6 in HEK 293 T cells. CONCLUSIONS: HDAC8 shows functional redundancy with HDAC6 when overexpressed in cervical cancer cells, HeLa, and deacetylaes ac-lys40 of alpha tubulin leading to cervical cancer proliferation and progression.

Anti-inflammatory profile of Aegle marmelos (L) Correa (Bilva) with special reference to young roots grown in different parts of India.

BACKGROUND: Aegle marmelos (Bilva) is being used in Ayurveda for the treatment of several inflammatory disorders. The plant is a member of a fixed dose combination of Dashamoola in Ayurveda. However, the usage of roots/root bark or stems is associated with sustainability concerns. OBJECTIVES: The present study is aimed to compare the anti-inflammatory properties of different extracts of young roots (year wise) and mature parts of Bilva plants collected from different geographical locations in India, so as to identify a sustainable source for Ayurvedic formulation. MATERIALS AND METHODS: A total of 191 extracts (petroleum ether, ethyl acetate, ethanol and aqueous) of roots, stems and leaves of A. marmelos (collected from Gujarat, Maharashtra, Odisha, Chhattisgarh, Karnataka and Andhra Pradesh region) were tested for anti-inflammatory effects in vitro on isolated target enzymes cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), lymphocyte proliferation assay (LPA), cytokine profiling in LPS induced mouse macrophage (RAW 264.7) cell line and in vivo carrageenan induced paw edema in mice. RESULTS: Of 191 extracts, 44 extracts showed COX-2 inhibition and 38 extracts showed COX-1 inhibition, while none showed 5-LOX inhibition. Cytokine analysis of the 44 extracts showing inhibition of COX-2 suggested that only 17 extracts modulated the cytokines by increasing the anti-inflammatory cytokine IL-2 and reducing the pro-inflammatory cytokines like IL-1ß, MIP1-α and IL-6. The young (2 and 3 years) roots of Bilva plants from Gujarat and young (1 yr) roots from Odisha showed the most potent anti-inflammatory activity by suppressing the pro-inflammatory cytokines and inducing anti-inflammatory cytokines. These three extracts have also shown in vivo anti-inflammatory activity comparable to that in adult stem and root barks. CONCLUSION: The present study reveals that young roots of Bilva plants from Gujarat and Odisha region could form a sustainable source for use in Ayurvedic formulations with anti-inflammatory activities. The present study also indicates that the region in which the plants are grown and the age of the plants play an important role in exhibiting the anti-inflammatory effect.

Design, synthesis, in silico and in vitro evaluation of thiophene derivatives: A potent tyrosine phosphatase 1B inhibitor and anticancer activity.

A series of novel methyl 4-(4-amidoaryl)-3-methoxythiophene-2-carboxylate derivatives were designed against the active site of protein tyrosine phosphatise 1B (PTP1B) enzyme using MOE.2008.10. These molecules are also subjected for in silico toxicity prediction studies and considering their corresponding drug scores, it implied that, the molecules are promising as anticancer agents. The designed compounds were synthesized by using suitable methods and characterized. They were subjected to inhibitory activity against PTP1B and in vitro anticancer activity by MTT assay. Most of the tested compounds showed potent inhibitory activity against PTP1B, among the compounds tested, compound 5b exhibited the highest activity (IC50=5.25µM) and remarkable cytotoxic activity at 0.09µM of IC50 against the MCF-7 cell line. In addition to this, compound 5c also showed potential anticancer activity at 2.22µM of IC50 against MCF-7 and 0.72µM against HepG2 cell lines as well as PTP1B inhibitory activity at IC50 of 6.37µM.

Thieno[3,2-c]pyran-4-one based novel small molecules: their synthesis, crystal structure analysis and in vitro evaluation as potential anticancer agents.

Novel thieno[3,2-c]pyran-4-one based small molecules were designed as potential anticancer agents. Expeditious synthesis of these compounds was carried out via a multi-step sequence consisting of few steps such as Gewald reaction, Sandmeyer type iodination, Sonogashira type coupling followed by iodocyclization and then Pd-mediated various C-C bond forming reactions. The overall strategy involved the construction of thiophene ring followed by the fused pyranone moiety and then functionalization at C-7 position of the resultant thieno[3,2-c]pyran-4-one framework. Some of the compounds synthesized showed selective growth inhibition of cancer cells in vitro among which two compounds for example, 5d and 6c showed IC(50) values in the range of 2.0-2.5 µM. The crystal structure analysis of an active compound along with hydrogen bonding patterns and molecular arrangement present within the molecule is described.

A new approach to construct a fused 2-ylidene chromene ring: highly regioselective synthesis of novel chromeno quinoxalines.

Regioselective construction of a fused 2-ylidene chromene ring was achieved for the first time by using AlCl(3)-induced C-C bond formation followed by Pd/C-Cu mediate coupling-cyclization strategy. A number of chromeno[4,3-b]quinoxaline derivatives were prepared by using this strategy. Single crystal X-ray diffraction study of a representative compound e.g. 6-(2,2-dimethylpropylidene)-4-methyl-6H-chromeno[4,3-b]quinoxalin-3-ol confirmed the presence of an exocyclic C-C double bond with Z-geometry. The crystal structure analysis and hydrogen bonding patterns of the same compound along with its structure elaboration via propargylation followed by Sonogashira coupling of the resulting terminal alkyne is presented. A probable mechanism for the formation of 2-ylidene chromene ring is discussed. Some of the compounds synthesized showed anticancer properties when tested in vitro.

Solvent effect on copper-catalyzed azide-alkyne cycloaddition (CuAAC): synthesis of novel triazolyl substituted quinolines as potential anticancer agents.

A regioselective route to novel mono triazolyl substituted quinolines has been developed via copper-catalyzed azide-alkyne cycloaddition (CuAAC) of 2,4-diazidoquinoline with terminal alkynes in DMF. The reaction provided bis triazolyl substituted quinolines when performed in water in the presence of Et(3)N. A number of the compounds synthesized showed promising anti-proliferative properties when tested in vitro especially against breast cancer cells.

Synthesis of novel 2-mercapto benzothiazole and 1,2,3-triazole based bis-heterocycles: their anti-inflammatory and anti-nociceptive activities.

A focused library of novel bis-heterocycles encompassing 2-mercapto benzothiazole and 1,2,3-triazoles were synthesized using click chemistry approach. The synthesized compounds have been tested for their anti-inflammatory activity by using biochemical cyclooxygenase (COX) activity assays and carrageenan-induced hind paw edema. Among the tested compounds, compound 4d demonstrated a potent selective COX-2 inhibition with COX-2/COX-1 ratio of 0.44. Results from carrageenan-induced hind paw edema showed that compounds 4a, 4d, 4e and 4f posses significant anti-inflammatory activity as compared to the standard drug Ibuprofen. The compounds showing significant activity were further subjected to anti-nociceptive activity by writhing test. These four compounds have shown comparable activity with the standard Ibuprofen. Further ulcerogenic studies shows that none of these compounds causing gastric ulceration.

Formation of volatiles and fattyacids of therapeutic importance in the probiotic Lactobacillus plantarum LPcfr adapted to resist GIT conditions.

Probiotics are the microorganisms that impart therapeutic effect and promote health by preventing various diseases. In the present work, the volatile compounds were studied in the native isolate Lactobacillus plantarum (LP) and after adaptation to resist gastro intestinal tract (GIT) conditions, which was coded as LPcfr. A number of therapeutically important compounds were present in LPcfr like butanediol (2.5%) and propionic acid (2.8%), which were not found in LP. Hexadecane (3%), butanoic acid (2%), dodecanal (2%), hexanal (7.5%), hexadecanoic acid (4%) and heptanal (6%) were found in higher concentrations in LPcfr as compared to the parent strain LP. Production of oleic acid (LP-19.2%; LPcfr -33.5%), known for reducing blood cholesterol and linoleic acid (LPcfr 2.3%), and a conjugated fatty acid known as a novel beneficial functional lipid was noticed. Linoleic acid was absent in LP. These important fatty acids were found in larger quantities in the probiotic adapted culture strain LPcfr as compared to the parent strain LP.

Cytotoxic studies of anti-neoplastic drugs on human lymphocytes--in vitro studies.

All the anti-cancer drugs proved to be highly cytotoxic agents to normal cells like lymphocyte cells used in our study which do not come under rapidly dividing cells like bone marrow cells, fetal cells, germ cells, hair follicle cells, intestinal cells, etc. but will proliferate when maintained in growth media with proliferation agents. In this investigation, we determined the effect of anti-cancer drugs on lymphocyte cultures from peripheral blood of healthy non-smoking donors under in vitro conditions by employing MTT assay. All the experiments were carried out with seven anti-cancer drugs; Carboplatin, Avastin, Vinorelbine, Tamoxifen Citrate, Methotrexate, Gemcitabine and Paclitaxel which are broad-spectrum cancer drugs and being used in six different types of chemotherapy in cancers. We have documented the results that were evaluated by statistical analysis. The MTT assay is now widely adopted by researchers and industry, as it is a rapid spectrophotometric method for determining cell viability in cell lines and in vitro. The initial or dose-finding studies determine the drug toxicity relative to dose and subsequent studies define the spectrum of activity of the drug. And moreover, no anti-neoplastic drug is devoid of side effects. Hence, this investigation determines the lethal concentrations and thereby the dosages at which individual anti-neoplastic drugs and also combinations can bring about certain degree of cytotoxicity in human lymphocytes which with comparative study on cancer cell lines would be useful in defining drug dosages.

Isolation, PCR based identification, and sensitivity pattern of environmental mycobacteria from leprosy and tuberculosis patients.

We have isolated and identified the biotype of environmental mycobacteria from the expectorate of leprosy patients, their contacts, their drinking water supply and also from the sputa samples of tuberculosis patients. 78% of the isolates from lepromatous leprosy patients and their contacts wereMycobacterium fortuitum- chelonae complex (MFC), 9%Mycobacterium avium complex (MAC), 9%Mycobacterium scrofulaceum and 4% wereMycobacterium smegmatis. Among the isolates from tuberculosis patients 63% belonged toM. fortuitum- chelonae complex, 19% toM. avium complex, 12% toMycobacterium Kansasii and 6% toM. smegmatis. All the isolates were multi-drug resistant when tested for sensitivity total of 21 drugs. TheMycobacterium fortuitum-chelonae complex organisms from leprosy contacts were more sensitive to rifampicin than those isolated from lepromatous leprosy and tuberculosis patients. Among 23 isolates from leprosy patients one isolate was resistant to 20 drugs, one isolate to 17 drugs and another isolate was resistant to 13 drugs. Among the 18 isolates from drinking water supply six showed resistance to more than 12 drugs. Polymerase Chain Reaction (PCR) and subsequent hybridisation with specific probes confirmed all the isolated strains as nontuberculous mycobacteria (Using genus primers and probe sensitivity 100%) and none asM. tuberculosis, suggesting that PCR could be used to rapidly identify mycobacteria at the genus level and to rule out tuberculosis in leprosy patients at an early stage to decide on appropriate course of therapy.

Some observations on skin smear examination.

40 slit and scrape smears in a planned study and 35 routine smears picked up from the laboratory were examined. An end-to-end examination of the smears detected additional positives and gave a higher bacterial index than what was reported in the routine. Acid-fast bacilli were found to be distributed in only 1 to 3 per cent of the fields in the smears. The bacilli were found mostly in the centre and in narrow bands between centre and periphery of the smears. Some of the high BI smears were found to contain areas completely free from bacilli.

Bacterial flora of ulcers treated by three types of dressings.

Effect of three types of dressings on bacterial flora in ulcers is presented. Debrisan seemed to be more effective than Zinc tape and collagen sheet in reducing the number of bacterial pathogens.