Predicting response to platinum and non-platinum drugs through bioluminescence resonance energy transfer (BRET) based bio-molecular interactions in platinum resistant epithelial ovarian cancer

Therapy induced rewiring of signalling networks often lead to acquirement of platinum-resistance, thereby necessitating the use of non-platinum agents as second-line treatment particularly for epithelial ovarian cancer (EOC). A prior subject-specific assessment can guide the choice of optimal non-platinum agent/s and possible targeted therapeutic/s. Assessment of protein-protein interactions are superior to simple cytotoxicity assays to determine therapeutic efficacy and associated molecular responses. Utilizing improved PIP3-AKT and ERK1/2 activation Bioluminescence Resonance Energy Transfer (BRET) sensors, we report chemotherapy-induced ERK1/2 activation predominantly in cisplatin-paclitaxel resistant EOC cells and increased activation of both ERK1/2 and AKT in malignant ascites derived cancer cells from platinum-resistant patients but not from treatment-naive or platinum-sensitive relapse patients. Further, majority of the non-platinum drugs except irinotecan increased ERK1/2 activation in platinum-taxol resistant cells as observed by live-cell BRET assessment which were associated with p90RSK1/2 and BAD activation along with upregulation of multidrug transporter gene ABCC1 and cell survival genes like cyclin D1 and Bcl2. Interestingly, only irinotecan was able to sensitize these resistant cells. Altogether, this first report of BRET based sensing of molecular pathway activations in platinum resistant cell lines and patient's derived cancer cells highlight the clinical potential of BRET sensors in management of therapy resistant cancer.     

Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/invasive phenotype

Transforming growth factor-beta (TGF-beta) induces epithelial-mesenchymal transition (EMT) of epithelial cells in both normal embryonic development and certain pathological contexts. Here, we show that TGF-beta induced-EMT in human lung cancer cells (A549; adenocarcinoma cells) mediates tumor cell migration and invasion phenotypes. To gain insights into molecular events during EMT, we employed a global stable isotope labeled profiling strategy using iTRAQ reagents, followed by 2DLC-MS/MS, which identified a total of 51 differentially expressed proteins during EMT; 29 proteins were up-regulated and 22 proteins were down-regulated. Down-regulated proteins were predominantly enzymes involved in regulating nutrient or drug metabolism. The majority of the TGF-beta-induced proteins (such as tropomyosins, filamin A, B, & C, integrin-beta1, heat shock protein27, transglutaminase2, cofilin, 14-3-3 zeta, ezrin-radixin-moesin) are involved in the regulation of cell migration, adhesion and invasion, suggesting the acquisition of a invasive phenotype.     

Low degree metabolites explain essential reactions and enhance modularity in biological networks

Background  

Recently there has been a lot of interest in identifying modules at the level of genetic and metabolic networks of organisms, as well as in identifying single genes and reactions that are essential for the organism. A goal of computational and systems biology is to go beyond identification towards an explanation of specific modules and essential genes and reactions in terms of specific structural or evolutionary constraints.     

Oral administration of orthovanadate and Trigonella foenum graecum seed powder restore the activities of mitochondrial enzymes in tissues of alloxan-induced diabetic rats

The effect of oral administration of sodium orthovanadate (SOV) and Trigonella foenum graecum seed powder (TSP), a medicinal plant used extensively in Asia, on the mitochondrial metabolism in the alloxan diabetic rats has been investigated. Rats were injected with alloxan monohydrate (20 mg/100 g body wt) or vehicle (Na-acetate buffer), the former were treated with either 2 IU insulin i.p., 0.6 mg/ml SOV ad libitum, 5% TSP ad libitum, and a combination of 0.2% SOV and 5% TSP ad libitum for 21 days. Selected rate-limiting enzymes of the tricarboxylic acid cycle, hydrogen shuttle system, ketone body metabolism, amino acid metabolism and urea cycle were measured in the mitochondrial and cytosolic fractions of liver, kidney and brain tissues of the experimental rats. Majority of the mitochondrial enzymes in the tissues of the diabetic rats had significantly higher activities compared to the control rats. Similarly, the activities of mitochondrial and cytosolic aminotransferases and arginase were significantly higher in liver and kidney tissues of the diabetic rats. The separate administrations of SOV and TSP to diabetic rats were able to restore the activities of these enzymes to control values. The lower dose of SOV (0.2%) administered in combination with TSP to diabetic rats lowered the enzyme activities more significantly than when given in a higher dose (0.6%) separately. This is the first report of the effective combined action of oral SOV and TSP in ameliorating the altered mitochondrial enzyme activities during experimental type-1 diabetes. Our novel combined oral administration of SOV and TSP to diabetic rats thus conclusively proves as a possible method to minimize potential vanadate toxicity without compromising its positive effects in the therapy of experimental type-1 diabetes.     

Subject Index

Index

Subject Index     

Activation of caspase-9 is required for UV-induced apoptosis of human keratinocytes

UV radiation from the sun activates both the membrane death receptor and the intrinsic or mitochondrial apoptotic signaling pathways in epidermal keratinocytes, triggering apoptosis and affording protection against skin cancer formation. We have investigated the involvement of caspase-9 in the UV death effector pathway in human keratinocytes, since this is the initiating caspase in the mitochondrial pathway required for UV-induced apoptosis in some, but not all, cell types. UV radiation triggered activation of caspase-3, caspase-9, and caspase-8 with similar kinetics, although the rank order of activation was caspase-3 > caspase-9 > caspase-8. Inhibition of caspase-9 with either the peptide inhibitor benzyloxycarbonyl-Leu-Glu(OCH(3))-His-Asp(OCH(3))-fluoromethyl ketone, or expression of a catalytically inactive caspase-9 by retroviral transduction, protected normal keratinocytes from UV-induced apoptosis. HaCaT keratinocytes harboring mutant p53 alleles were also protected from UV-induced apoptosis by the dominant negative caspase-9. The dominant negative caspase-9 blocked UV-induced activation of caspase-3, caspase-9, and caspase-8, and also protected cells from the loss of mitochondrial membrane potential. In contrast, the dominant negative caspase-9 did not protect from anti-Fas-induced apoptosis or caspase activation. These results identify caspase-9 as the critical upstream caspase initiating apoptosis by UV radiation in human keratinocytes, the relevant cell type for this important environmental carcinogen.