diethyl-maleate and Colonic-Neoplasms

Uncontrolled accumulation of reactive oxygen species (ROS) causes oxidative stress and induces harmful effects. Both high ROS levels and p53 mutations are frequent in human cancer. Mutant p53 forms are known to actively promote malignant growth. However, no mechanistic details are known about the contribution of mutant p53 to excessive ROS accumulation in cancer cells. Herein, we examine the effect of p53(R273H), a commonly occurring mutated p53 form, on the expression of phase 2 ROS-detoxifying enzymes and on the ability of cells to readopt a reducing environment after exposure to oxidative stress. Our data suggest that p53(R273H) mutant interferes with the normal response of human cells to oxidative stress. We show here that, upon oxidative stress, mutant p53(R273H) attenuates the activation and function of NF-E2-related factor 2 (NRF2), a transcription factor that induces the antioxidant response. This effect of mutant p53 is manifested by decreased expression of phase 2 detoxifying enzymes NQO1 and HO-1 and high ROS levels. These findings were observed in several human cancer cell lines, highlighting the general nature of this phenomenon. The failure of p53(R273H) mutant-expressing cells to restore a reducing oxidative environment was accompanied by increased survival, a known consequence of mutant p53 expression. These activities are attributable to mutant p53(R273H) gain of function and might underlie its well-documented oncogenic nature in human cancer.

Topics: Amino Acid Substitution; Apoptosis; Cell Proliferation; Cell Survival; Colonic Neoplasms; Gene Knockdown Techniques; HCT116 Cells; Heme Oxygenase-1; Humans; Maleates; Metabolic Detoxication, Phase II; Mutant Proteins; Mutation; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species; RNA, Small Interfering; Superoxides; Tumor Suppressor Protein p53

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

The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S-transferases. Higher concentrations of monochlorobimane achieved more complete labelling of the human cellular GSH pool but gave increased background fluorescence due to non-GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with approximately 20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, o-phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labeling.

Topics: Animals; Breast Neoplasms; Bridged Bicyclo Compounds; Buthionine Sulfoximine; Carcinoma; Carcinosarcoma; CHO Cells; Colonic Neoplasms; Cricetinae; Ethylmaleimide; Eukaryotic Cells; Evaluation Studies as Topic; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Glutathione; Humans; Maleates; Mammary Neoplasms, Experimental; Methionine Sulfoximine; Mice; o-Phthalaldehyde; Phenylmercury Compounds; Pyrazoles; Species Specificity; Tumor Cells, Cultured; Urinary Bladder Neoplasms