Inactivation of uPA and its receptor uPAR by 3,3′‐diindolylmethane (DIM) leads to the inhibition of prostate cancer cell growth and migration

3,3′‐Diindolylmethane (DIM) has been studied for its putative anti‐cancer properties, especially against prostate cancer; however, its exact mechanism of action remains unclear. We recently provided preliminary data suggesting down‐regulation of uPA during B‐DIM (a clinically active DIM)‐induced inhibition of invasion and angiogenesis in prostate cancer cells. Since the expression and activation of uPA plays important role in tumorigenicity, and high endogenous levels of uPA and uPAR are found in advanced metastatic cancers, we investigated their role in B‐DIM‐mediated inhibition of prostate cancer cell growth and motility. Using PC3 cells, we found that B‐DIM treatment as well as the silencing of uPA and uPAR by siRNAs led to the inhibition of cell growth and motility. Conversely, over‐expression of uPA/uPAR in LNCaP and C4‐2B cells resulted in increased cell growth and motility, which was effectively inhibited by B‐DIM. Moreover, we found that uPA as well as uPAR induced the production of VEGF and MMP‐9, and that the down‐regulation of uPA/uPAR by siRNAs or B‐DIM treatment resulted in the inhibition of VEGF and MMP‐9 secretion which could be responsible for the observed inhibition of cell migration. Interestingly, silencing of uPA/uPAR led to decreased sensitivity to B‐DIM indicating important role of uPA/uPAR in B‐DIM‐mediated regulation of prostate cancer cell growth and migration. Our data suggest that chemopreventive and/or therapeutic activity of B‐DIM is in part due to down‐regulation of uPA–uPAR leading to reduced production of VEGF/MMP‐9 which ultimately leads to the inhibition of cell growth and migration of aggressive prostate cancer cells. J. Cell. Biochem. 107: 516–527, 2009. © 2009 Wiley‐Liss, Inc.     

Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing.

BACKGROUND: Clonogenic assays are used frequently to measure the cell killing and mutagenic effects of radiation and other agents. Clonogenic assays carried out manually are tedious and time-consuming and involve a significant element of subjectivity. However, several commercial automatic colony counters are available. Based on CCD video imaging and image analysis they are relatively expensive and can analyze only one petri dish at a time. METHOD: We have developed a cheaper and more efficient device, which employs a flat bed scanner to image 12 60-mm petri dishes at a time. Two major problems in automated colony counting are the clustering of colonies and edge effects. By using standard image analysis and implementing an inflection point algorithm, these problems were greatly diminished. The resulting system was compared with two manual colony counts, as well as with automated counts with the Oxford Optronix ColCount colony counter for cell lines V79 and HaCaT. RESULTS: Comparisons assuming the manual counts to be correct showed that our automatic counter was slightly more accurate than the commercial unit. CONCLUSIONS: As a whole, our automated colony counter performed significantly better than the commercial unit with regard to processing time, cost and accuracy.     

Acidic preconditioning protects endothelial cells against apoptosis through p38- and Akt-dependent Bcl-xL overexpression

To analyze the underlying cellular mechanisms of adaptation to ischemia-induced apoptosis through short acidic pretreatment, i.e. acidic preconditioning (APC), Wistar rat coronary endothelial cells (EC) were exposed for 40 min to acidosis (pH 6.4) followed by a 14 h recovery period (pH 7.4) and finally treated for 2 h with simulated in vitro ischemia (glucose-free anoxia at pH 6.4). APC led to a transient activation of p38 and Akt kinases, but not of JNK and ERK1/2 kinases, which was accompanied by significant reduction of the apoptotic cell number, caspase-12/-3 cleavage and Bcl-xL overexpression. These effects of APC were completely abolished by prevention of Akt- or p38-phosphorylation during APC. Furthermore, knock-down of Bcl-xL by siRNA-transfection also abolished the anti-apoptotic effect of APC. Therefore, APC leads to protection of EC against ischemic apoptosis by activation of Akt and p38 followed by overexpression of Bcl-xL, which is a key anti-apoptotic mechanism of APC.     

Optimum DNA Curvature Using a Hybrid Approach Involving an Artificial Neural Network and Genetic Algorithm

Abstract

In the present paper, a hybrid technique involving artificial neural network (ANN) and genetic algorithm (GA) has been proposed for performing modeling and optimization of complex biological systems. In this approach, first an ANN approximates (models) the nonlinear relationship(s) existing between its input and output example data sets. Next, the GA, which is a stochastic optimization technique, searches the input space of the ANN with a view to optimize the ANN output. The efficacy of this formalism has been tested by conducting a case study involving optimization of DNA curvature characterized in terms of the R_L value. Using the ANN-GA methodology, a number of sequences possessing high R_L values have been obtained and analyzed to verify the existence of features known to be responsible for the occurrence of curvature. A couple of sequences have also been tested experimentally. The experimental results validate qualitatively and also near-quantitatively, the solutions obtained using the hybrid formalism. The ANN-GA technique is a useful tool to obtain, ahead of experimentation, sequences that yield high R_L values. The methodology is a general one and can be suitably employed for optimizing any other biological feature.     

Identification of Phosphoribosyl-AMP cyclohydrolase,as drug target and its inhibitors in Brucella melitensis bv. 1 16M using metabolic pathway analysis

Brucella melitensis is a pathogenic Gram-negative bacterium which is known for causing zoonotic diseases (Brucellosis). The organism is highly contagious and has been reported to be used as bioterrorism agent against humans. Several antibiotics and vaccines have been developed but these antibiotics have exhibited the sign of antibiotic resistance or ineffective at lower concentrations, which imposes an urgent need to identify the novel drugs/drug targets against this organism. In this work, metabolic pathways analysis has been performed with different filters such as non-homology with humans, essentially of genes and choke point analysis, leading to identification of novel drug targets. A total of 18 potential drug target proteins were filtered out and used to develop the high confidence protein–protein interaction network The Phosphoribosyl-AMP cyclohydrolase (HisI) protein has been identified as potential drug target on the basis of topological parameters. Further, a homology model of (HisI) protein has been developed using Modeller with multiple template (1W6Q (48%), 1ZPS (55%), and 2ZKN (48%)) approach and validated using PROCHECK and Verify3D. The virtual high throughput screening (vHTS) using DockBlaster tool has been performed against 16,11,889 clean fragments from ZINC database. Top 500 molecules from DockBlaster were docked using Vina. The docking analysis resulted in ZINC04880153 showing the lowest binding energy (?9.1 kcal/mol) with the drug target. The molecular dynamics study of the complex HisI-ZINC04880153 was conducted to analyze the stability and fluctuation of ligand within the binding pocket of HisI. The identified ligand could be analyzed in the wet-lab based experiments for future drug discovery.