Functional Studies Of Human Carbonyl Reductase Gene Cbr1 And Dcxr Of Cloning And Its Encoding Protein

Carbonyl reductases (EC 1.1.1.184, CRs), belonging to the superfamily of oxidoreductases, are mostly monomeric and cytosolic enzymes with broad substrate specificity for many endogenous and xenobiotic carbonyl compounds. So far there are four human carbonyl reductases have been recognized, including CBR1, DCXR, CBR3 and CBR4. DCXR was cloned by homogenous screening strategy and submitted to Genebank with accession No. AF139841. In this thesis, we study the characteristics of CBR1 and DCXR and their association with Hepatic Cellular Carcinoma (HCC).Northern blot analysis of human CBR1 and DCXR was carried out on multiple tissue samples from 16 adult human tissues to confirm theirs natural existence and tissue distribution. CBR1 was found to be 1.65 kb in length and abundant in liver, kidney, brain, middle in heart, pancreas, placenta, lung, while little in other tissues. DCXR was 1.35 kb in length and abundant in liver, kidney, heart, pancreas, and middle in brain, placenta, lung, spleen, thymus, but little in peripheral blood lymphocyte and colon. Then the transcriptional expression pattern of DCXR in liver cancer was detected and found to be commonly down-regulated in liver cancer tissues. DCXR protein was expressed in E. coli and purified by chromatography. The purified protein was used to immunize rabbits and polyclonal antibody was obtained. With the immunohistochemistry method by the polyclonal antibody, DCXR was found obviously expressed lower in the HCC tissues than in the normal liver tissues. It figures out some clues of the relationship between DCXR and HCC and provides some useful indications that the decreased DCXR expression might be a marker of liver cancer.Recombinant CBR1 and DCXR were purified from E. coli to detect their enzyme activity. Both DCXR and CBR1 were found to show reductase activity on isatin and 9,10-phenanthrenedione with NADPH as cofactor at fittest pH value of about 6.2. Then we screened 17 anti-cancer drugs to identify the new substrates of CBR1 and DCXR. The results indicated that daunorubicin, vincristine, salvicine and (-)-epigallocatechin gallate (EGCG) were found to be the substrates of CBR1 and DCXR and the later three have not been reported so far. Based on the molecular structure comparing, we found that only EGCG was with epicatechin gallate that was confirmed to promote cell apoptosis. Therefore, we speculated the enzyme catalyzingposition located at the galloyl structure on the B ring of epigallocatechin gallate, which was important for cell apoptosis induction. The hepatic carcinoma cells (SK-HEP-1 cells) stably transfected with CBR1 showed dramatic insensitivity to EGCG induced apoptosis, suggesting that high expressed CBR1 might be used as molecular marker to exclude EGCG treatment for HCC.Likes CBR1, DCXR can catalyze daunorubicin, vincristine, salvicine and EGCG. These anti-cytotoxicity roles of DCXR, protecting cells from daunorubicin, vincristine and EGCG, have been assessed by forced expression of DCXR through stable transfection of cDNA encoding DCXR in FOCUS cells and Hela cells. Cells highly expressing DCXR showed increased resistance to daunorubicin and vincristine but EGCG compared to their parent cells when measured with MTS cyto-toxicity assays (p<0.05). This is the first report that DCXR can metabolize anti-cancer drugs and decrease the chemosensitivity of cancer cells to daunorubicin and vincristine. The cloning of human DCXR genes may initiate further investigation of the carbonyl reductase's role in tumor chemotherapy, and is important for study of pharmacological deactivation of drugs with carbonyl group.