C. Channa Reddy, PhD

Research

Regulation of prostaglandin and leukotriene biosynthesis

We have a long-standing interest in understanding the molecular mechanism or mechanisms underlying the regulation of biosynthesis of prostaglandins (PGs) and leukotrienes (LTs). Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of PGs. It exists in two isoforms, COX-1 and COX-2. An expanding body of evidence indicates that down regulation of COX-1 and COX-2 will be an important strategy for preventing cancer because PGs have multiple effects that favor tumorigenesis. Thus, non-steroidal anti-inflammatory drugs (NSAIDs) that target COX-2 have great potential as chemopreventive agents against colon cancer. Accordingly, research in our laboratory has focused on the strategies to selectively inhibit and down regulate the COX-2 isoform. Through understanding the biochemistry of these enzymes and the regulation of COX-1 and COX-2 gene expression employing transgenic animals, we hope to understand how COX-2 can be regulated selectively as a target for chemopreventive therapy. A second aspect our research is concerned with LTs, a class of potent biological mediators of inflamma-tion and anaphylaxis. Their biosynthesis derives from 5-LOX-catalyzed oxygenation of arachidonic acid in granulocytes, macrophages and mast cells. Recently we have isolated a full-length 5-LOX cDNA clone from potato tubers and have over expressed the cDNA in E. coli. The mechanistic details underlying stereospecificity and regiospecificity of the recombinant enzyme are being investigated. Through molecular modeling and combinatorial chemistry, we are designing the synthesis of mechanism-based selective inhibitors for this enzyme.

Another major interest in our laboratory is concerned with glutathione S-transferases (GSTs). We have provided convincing evidence that GSTs are directly involved in the biosynthesis of PGs and LTs, particularly PGF_2a , PGD2, PGE2, LTC4, and hepoxillins. We have also demonstrated that specific GST isozymes are responsible for the formation of these eicosanoids. Most of these enzymes have been purified to homogeneity and their primary amino acid sequence deduced from the respective cDNA sequences. We have focused our attention on the alterations in expression of these enzymes caused by changes in amounts of dietary antioxidants, particularly by the altered concentrations of Se in the diet. Expression of genes for certain GSTs is increased during Se deficiency; however, in very few cases have the genetic changes that alter the patterns of expression been characterized. However, it is becoming clear from investigations in mammals that regulation of these genes occurs to a great extent at the transcriptional level, and that the factors involved include binding sites for regulatory proteins upstream from the start sites of transcription. We are investigating the effects of oxidative stress on the expression of GSTs at transcriptional level. Recently we have isolated a unique cationic GST with a subunit Mr of 25 kDa from sheep liver microsomes and have cloned a full-length cDNA. We have also overexpressed this isozyme in E. coli. Currently, we are focusing on the elucidation of the crystal structure of the recombinant protein as well as the role of this GST isozyme in the protection against membrane peroxidative damage.