A global multicenter study on reference values: 2. Exploration of sources of variation across the countries.

This global multicenter study on reference values (RVs) allowed us to explore biological sources of variation (SVs) of RVs across the world.As described in the first part of this paper, RVs of 50 major analytes from 13,396 healthy individuals living in 12 countries were obtained. Analyzed in this study were 23 clinical chemistry analytes and 8 analytes measured by immunoturbidimetry. Multiple regression analysis was performed for each gender, country by country, analyte by analyte, by setting four major SVs (age, BMI, and levels of drinking and smoking) as a fixed set of explanatory variables. For analytes with skewed distributions, log-transformation was applied. The association of each source of variation with RVs was expressed as partial correlation coefficient (rp).Obvious gender and age-related changes in the RVs were observed in many analytes, almost consistently between countries. Compilation of age-related change profiles of RVs after adjusting for between-country differences revealed peculiar patterns specific to each analyte. Judged fromrp, BMI related changes were observed in many nutritional and inflammatory markers in almost all countries. However, the slope of linear BMI vs. RV relationship differed greatly among countries for some analytes. Alcohol and smoking-related changes were observed less conspicuously in a limited number of analytes.Features of sex, age, alcohol, and smoking-related changes in RVs of major analytes were almost comparable worldwide. The finding of differences in BMI-related changes among countries in some analytes is quite relevant to understanding ethnic differences in susceptibility to nutritionally related diseases

Role And Regulation Of Hausp In Cancer

Cancer is a disease of uncontrolled cell growth resulting from deregulated cellular signaling. Signal transduction is subject to several modes of regulation.This study is focussed on post-translational modifications, especially deubiquitination. The thesis work is divided into four sections, the first three emphasize on the role of HAUSP and the fourth one is on the regulation of HAUSP.In the first part of the work, we have found a large number of putative substrates of HAUSP by MALDI-TOF/TOF MS/MS analysis. We have shown the effects of exogenously expressed HAUSP on the overall proteome. To further investigate the interacting partners of HAUSP, the pool of interacting proteins was enriched by immunoprecipitation. Gene ontology based analysis of the proteins identified, indicate a probable involvement of HAUSP in tumor suppression, metabolism, cytoskeletal organization and transport, gene expression, chaperone system, apoptosis, etc. A few uncharacterized/ novel proteins have also been identified. From the analysis, three putative bona fide substrates – TRRAP, Keratin and FBF-1 have been identified which were also found to colocalize with HAUSP. In this part of the work we have shown that HAUSP plays an important role in cancer by regulating cMyc via TRRAP.In the second part, we have identified Rb as a novel substrate for HAUSP mediated deubiquitination. This has added an edge to the MDM2 – p53 mediated functions of HAUSP. We have shown the role of HAUSP in preferential stabilization of MDM2 over Rb in cancer/ GBM cell lines. In delineating the mechanism of the phenomena, we show how HAUSP stabilizes Rb in a contextPreface dependent manner, using a p53-independent switching mechanism in normal and cancer cells, where MDM2 is the critical regulator. In the third part, we have identified the role of HAUSP specifically in glioma. In clinical specimens of glioma we have shown that HAUSP protein levels increase during progression from lower to higher grad(immunohistochemistry). This is very well corroborated by the in vitro data showing knockdown of HAUSP leads to huge decrease in the cellular proliferation as seen in U87MG cells while upregulation of HAUSP led to larger sized and more number of colonies in a colony formation assay.In the fourth and last part of the work on regulation of HAUSP, first we found that in U87MG (GBM) cells HAUSP is regulated by EGF stimulation, wherein HAUSP transcript levels show a huge increase upon EGF treatment and at the protein level we see a clear nuclear exclusion within a short period of induction. The most appropriate explanation here at this point of time would be that in U87MG cells which harbor wild type p53, HAUSP nuclear exclusion would lead to destabilization of p53 in the nucleus, thus preventing its function as a tumor suppressive transcription factor. Upon in silico analysis of the HAUSP promoter sequence we identified a large number of p53 binding sites giving rise to the possible existence of a feedback regulation of HAUSP by p53. Additionally the presence of TCF4/LEF sites is also suggestive of a role of Wnt signaling in regulating HAUSP

Cell Death and Deubiquitinases: Perspectives in Cancer

The process of cell death has important physiological implications. At the organism level it is mostly involved in maintenance of tissue homeostasis. At the cellular level, the strategies of cell death may be categorized as either suicide or sabotage. The mere fact that many of these processes are programmed and that these are often deregulated in pathological conditions is seed to thought. The various players that are involved in these pathways are highly regulated. One of the modes of regulation is via post-translational modifications such as ubiquitination and deubiquitination. In this review, we have first dealt with the different modes and pathways involved in cell death and then we have focused on the regulation of several proteins in these signaling cascades by the different deubiquitinating enzymes, in the perspective of cancer. The study of deubiquitinases is currently in a rather nascent stage with limited knowledge both in vitro and in vivo, but the emerging roles of the deubiquitinases in various processes and their specificity have implicated them as potential targets from the therapeutic point of view. This review throws light on another aspect of cancer therapeutics by targeting the deubiquitinating enzymes

2,2′-Diphenyl-3,3′-Diindolylmethane: A Potent Compound Induces Apoptosis in Breast Cancer Cells by Inhibiting EGFR Pathway

<div><p>Despite recent advances in medicine, 30–40% of patients with breast cancer show recurrence underscoring the need for improved effective therapy. In this study, by <i>in vitro</i> screening we have selected a novel synthetic indole derivative 2,2'-diphenyl-3,3'-diindolylmethane (DPDIM) as a potential anti- breast cancer agent. DPDIM induces apoptosis both <i>in vitro</i> in breast cancer cells MCF7, MDA-MB 231 and MDA-MB 468 and <i>in vivo</i> in 7,12-dimethylbenz[α]anthracene (DMBA) induced Sprague-Dawley (SD) rat mammary tumor. Our <i>in vitro</i> studies show that DPDIM exerts apoptotic effect by negatively regulating the activity of EGFR and its downstream molecules like STAT3, AKT and ERK1/2 which are involved in the proliferation and survival of these cancer cells. <i>In silico</i> predictions also suggest that DPDIM may bind to EGFR at its ATP binding site. DPDIM furthermore inhibits EGF induced increased cell viability. We have also shown decreased expression of pro-survival factor Bcl-XL as well as increase in the level of pro-apoptotic proteins like Bax, Bad, Bim in DPDIM treated cells <i>in vitro</i> and <i>in vivo</i>. Our results further indicate that the DPDIM induced apoptosis is mediated through mitochondrial apoptotic pathway involving the caspase-cascade. To the best of our knowledge this is the first report of DPDIM for its anticancer activity. Altogether this report suggests that DPDIM could be an effective therapeutic agent for breast cancer.</p> </div

Reduction of breast tumor growth by DPDIM in animal model.

<p>Six tumor bearing Sprague Dawley rats were taken for each of the DPDIM treated and untreated group and six normal rats were also taken as control. (A), Inhibition of DMBA-induced breast tumor growth after DPDIM (5 mg/kg) treatment for every alternative days upto 21 days in rats was shown. (<i>n = </i>6 in each group). Data is represented as mean ± SEM and *indicates <i>P<0.001.</i> (B), Tumor growth curve of rats after oral administration of DPDIM for 21 days was represented in graph. Growth pattern of tumors for another 21 days after discontinuation of DPDIM treatment was also represented in graph. Representation of growth pattern of untreated tumors for 42 days is there in the graph. For each time point a group of 6 rats were taken. Bars represent standard deviation of the volume of tumors at each time point. (C), Time-course of plasma concentration over 48 hr following oral administration of 5 mg/kg DPDIM to the tumor bearing rats was shown in the figure. Mean compound concentration in rat (n = 6) plasma at each time point (at 0.5, 1, 2, 3, 4, 5, 8, 16, 24 and 48 hrs) was studied by HPLC analysis on a Shimadzu Model SPD-M10Avp equipped with LC-10ATvp HPLC pump, Hamamatsu Deuterium Lamp type L6585 photodiode array detector. Main parameters studied for this compound in rat was given in the inserted table. C_max = Maximum plasma concentration of a drug after oral administration; t_max = Time to reach C_max; AUC = Area under the curve; Kel = elimination rate constant and T1/2 = Biological half life.</p

Activation of mitochondrial caspases and induction of apoptosis in DPDIM treated breast cancer cells.

<p>(A), Activation of mitochondrial caspases-9, 7 and 3 in 24 hr DPDIM treated cells were shown by IB. (B), Analysis of PARP cleavage was done by IB in MCF7 and MDA-MB 231 cells after 24 hr treatment with DPDIM<b>.</b> (C), Apoptotic cell population was evaluated after 24 hr of treatment by FACS analysis using double staining with Annexin V and PI. (D), <i>In situ</i> TUNEL assay showing inter-nucleosomal degradation of genomic DNA in 48 hr treated MCF7 cells. Cells were stained with DAB and counterstained with methylgreen. The % TUNEL positive cells were calculated and the quantitative evaluation represented in the bar diagram with SD. *indicates <i>P</i><0.0001. All results are representative of three independent experiments.</p

<i>In vivo</i> determination of DPDIM induced EGFR pathway regulation directed to apoptosis.

<p>(A), Expression and activation of EGFR, AKT, STAT3, ERK1/2, Bcl-XL, Bax and Caspases-3, 7 and 9 in tumor tissue lysates isolated from untreated and treated groups were checked by IB. (B and C), Tissue sections were prepared from tumors excised at day 21. (B), IHC analysis of phospho-EGFR, Bcl-XL and cleaved caspase-3 was shown in figure. Percentage of positive cells per microscopic field of 5 different fields for each antibody were calculated and represented as a bar diagram with SD. * indicates <i>P<0.0001.</i> (C), H&E staining were done for the histological analysis. TUNEL assay of normal breast, untreated and treated tumor tissue sections. All images were captured under bright field microscope with 20X magnification.</p

Evaluation of the inhibitory effect of DPDIM on EGFR pathway and induction of mitochondrial cytochrome c release.

<p>(A), The change of Phospho-EGFR levels were examined in MCF7, MDA-MB 231 and MDA-MB 468 cells following DPDIM treatment for 24 hr by IB. (B), Alteration in normal and activated level of EGFR, HER2 and HER3 in 24 hr DPDIM treated ZR-75-1 cells were examined by IB. (C), Whole cell lysates (WCL) were prepared from 24 hr DPDIM treated cells and immunoblotted for normal and activated forms of AKT, ERK1/2 and STAT3. (D), IB analyses of Bcl-XL, Bax, Bad and Bim in MCF7, MDA-MB 231 and MDA-MB 468 cells treated with DPDIM for 24 hr. (E), Fluorescence micrographs of vehicle control (Leftmost panel) and treated (Right panels) MCF7 cells showing Cyt c release after 24 hr DPDIM treatment. All results are representative of three independent experiments.</p

Cytotoxicity study of DPDIM.

<p>(A, B and D), Figures show percent (%) chromosomal aberration and micronucleus (MN) frequency (Mean ± SEM) due to 48 hr DPDIM treatment in human lymphocytes. All the analyzed data for quantification are inserted in the respective tables. (C), Graphical representation of percentage (%) of reverse mutation on <i>Salmonella typhimurium</i> (TA100) in untreated (-ve control), Sodium azide treated (+ve control) and DPDIM (1 µM, 10 µM and 50 µM) treated wells in 96 well plate. (PC = positive control and *indicates p<0.001). Data are representative of three independent experiments.</p

Binding analysis of DPDIM with EGFR.

<p>(A), Ribbon representation of human EGFR kinase domain docked with DPDIM into its ATP binding site. Arrow shows the binding of DPDIM to the receptor. (B), Two-dimensional representation of DPDIM-EGFR binding interactions. Green solid lines represents hydrophobic interactions, π-cation interaction is shown in green dotted line and the hydrogen bonding with black dotted line. Residues were numbered according to the PDB ID 1M17. (C) and (D) display the thermodynamic analysis of statistical ensemble of the ligand/receptor complex. (C), Energy spectrum distribution of system states (observed conformations) as determined by molecular docking computation and (D), concordant cluster distribution of the ensemble structures over the energy axis. Conformations within 2 Å root mean square deviation (irrespective of their binding energy) were clustered together. The graphs were plotted with OriginPro 8.</p