leupeptins and diethyl-maleate

Aldo-keto reductases (AKRs) play central roles in the reductive metabolism of endogenous signaling molecules and in the detoxification of xenobiotics. AKRC1-1C3, AKR1B1 and AKR1B10 have been shown to be regulated via nuclear factor-erythroid 2 related factor 2 (Nrf2), a transcription factor that is activated upon oxidative stress. Proteasome inhibitors bortezomib and MG-132 produce mild oxidative stress that activates Nrf2-mediated gene expression that in turn may have cytoprotective effects. Bortezomib is clinically approved to treat haematological malignancies and it has also proven activity in solid tumors such as colon cancer. The present study investigated the effect of bortezomib and MG-132 on the expression of AKR1C1-1C4, AKR1B1, and AKR1B10 in colon cancer cell lines HT-29 and SW-480. Human cancer cell lines derived from different organs (lung, colon, pancreas, skin, liver, ovary) were initially assayed for the expression of the AKRs, showing a very unequal distribution. Even among the colon cell lines HT-29, Caco-2, HCT116 and SW-480, the AKRs were expressed quite non-uniformly. HT-29 cells expressed all AKRs on the mRNA level including liver-specific AKR1C4, but AKR1B1 was almost undetectable. In SW-480 cells, treatment with bortezomib (50 nM, 48 h) dramatically increased mRNA levels of AKR1B10 (32-fold), AKR1B1 (5.5-fold), and, to a lesser extent, AKR1C1 and AKR1C3. Drug-efflux transporter MRP2 (ABCC2) and Cox-2 were induced as well. AKR1C2 mRNA was down-regulated in SW-480 but induced in HT-29 cells. MG-132 increased mRNA amounts of AKR1C1, 1C3, 1B1, and 1B10 in a concentration-dependent manner. AKR1B10 and AKR1B1 protein expression was inducible by bortezomib in HT-29 cells, but not detectable in SW-480 cells. In conclusion, treatment with proteasome inhibitors increased the expression of several AKRs as well as of MRP2. It remains to be investigated whether this enzyme induction may contribute to enhanced cell survival and thereby supporting the phenomenon of multidrug resistance upon cancer chemotherapy.

Topics: 20-Hydroxysteroid Dehydrogenases; 3-Hydroxysteroid Dehydrogenases; Alcohol Oxidoreductases; Aldehyde Reductase; Aldo-Keto Reductase Family 1 Member C3; Aldo-Keto Reductases; Boronic Acids; Bortezomib; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Induction; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Hydroxyprostaglandin Dehydrogenases; Isothiocyanates; Leupeptins; Maleates; Multidrug Resistance-Associated Protein 2; NF-E2-Related Factor 2; Protease Inhibitors; Proteasome Inhibitors; Pyrazines; RNA, Messenger; Sulfoxides; Thiocyanates; Time Factors

Carbonyl reduction is a central metabolic process that controls the level of key regulatory molecules as well as xenobiotics. Carbonyl reductase 3 (CBR3; SDR21C2), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, has been poorly characterized so far, and the regulation of its expression is a complete mystery. Here, we show that CBR3 expression is regulated via Nrf2, a key regulator in response to oxidative stress. In human cancer cell lines, CBR3 mRNA was expressed differentially, ranging from very high (A549, lung) to very low (HT-29, colon; HepG2, liver) levels. CBR3 protein was highly expressed in SW-480 (colon) cells but was absent in HCT116 (colon) and HepG2 cells. CBR3 mRNA could be induced in HT-29 cells by Nrf2 agonists [sulforaphane (SUL, 7-fold) and diethyl maleate (DEM, 4-fold)] or hormone receptor ligand Z-guggulsterone (5-fold). Aryl hydrocarbon receptor agonist B[k]F failed to induce CBR3 mRNA after incubation for 8 h but elevated CBR3 levels after 24 h, most likely mediated by B[k]F metabolites that can activate Nrf2 signaling. Inhibition of Nrf2-activating upstream kinase MEK/ERK by PD98059 weakened DEM-mediated induction of CBR3 mRNA. Proteasome inhibitors MG-132 (5 μM) and bortezomib (50 nM) dramatically increased the level of CBR3 mRNA, obviously because of the increase in the level of Nrf2 protein. While siRNA-mediated knockdown of Nrf2 led to a decrease in the level of CBR3 mRNA in A549 cells (30% of control), Keap1 knockdown increased the level of CBR3 mRNA expression in HepG2 (9.3-fold) and HT-29 (2.7-fold) cells. Here, we provide for the first time evidence that human CBR3 is a new member of the Nrf2 gene battery.

Topics: Alcohol Oxidoreductases; Animals; Cell Line; Cell Line, Tumor; Colonic Neoplasms; Cricetinae; Enzyme Inhibitors; Flavonoids; Gene Expression; Gene Expression Regulation; Humans; Hydroquinones; Leupeptins; Maleates; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Oxidative Stress; Proteasome Inhibitors; RNA, Messenger; Transfection