Identification of human Thioredoxin as a potent redox protein in carcinogenesis

Background: Thioredoxins ("TRX") are ubiquitous 12-kDa oxidoreductase enzyme containing a dithiol-disulfide active site facilitating the reduction of other proteins by cysteine thiol-disulfide exchange. When thioredoxin levels are elevated there is increased cell growth and resistance to the normal mechanism of programmed cell death. An increase in thioredoxin levels seen in many human primary cancers compared to normal tissue appears to contribute to increased cancer cell growth and resistance to chemotherapy. Mechanisms by which thioredoxin increases cell growth include an increased supply of reducing equivalents for DNA synthesis, activation of transcription factors that regulate cell growth, and an increase in the sensitivity of cells to other cytokines and growth factors thereby thioredoxin offers a target for the development of drugs to treat and prevent cancer. Aim: To characterize molecular cloning, expression, purification and bioassay of the most potent redox protein in carcinogenesis. Research methodology: The expression of thioredoxin (Trx) in human lung adenocarcinoma epithelial cells (A549) was studied by cDNA isolation using RT PCR and it was cloned into pRSET A vector at Nde1 and BamH1 site (restriction sites) and then transformed in E.coli GJ1158/BL21 DE3. The cells were cultured in mass and lysed so as to generate recombinant thioredoxin, then purified to obtain a single band of appropriate molecular weight in SDS PAGE analysis. The gene sequence was confirmed by DNA sequencing and the protein by western blot using anti-thioredoxin antibody. The biological activity of recombinant thioredoxin was tested by insulin reduction assay. Results: The biological activity of recombinant thioredoxin was studied by monitoring the dithiol-disulphide oxidoreductase activity that catalyzes reduction of insulin disulphides by dithiothreitol spectrophotometrically at 650 nm at 25°C as turbidity formation from the precipitation of the free insulin β chain.