M. Tien Kuo

Expression of Multidrug Resistance Protein/GS-X Pump and γ-Glutamylcysteine Synthetase Genes Is Regulated by Oxidative Stress

Expression of the MRP1 gene encoding the GS-X pump and of the γ-GCSh gene encoding the heavy (catalytic) subunit of the γ-glutamylcysteine synthetase is frequently elevated in many drug-resistant cell lines and can be co-induced by many cytotoxic agents. However, mechanisms that regulate the expression of these genes remain to be elucidated. We report here that like γ-GCSh, the expression of MRP1 can be induced in cultured cells treated with pro-oxidants such astert-butylhydroquinone, 2,3-dimethoxy-1,4-naphthoquinone, and menadione. Intracellular reactive oxygen intermediate (ROI) levels were increased in hepatoma cells treated withtert-butylhydroquinone for 2 h as measured by flow cytometry using an ROI-specific probe, dihydrorhodamine 123. Elevated GSH levels in stably γ-GCSh-transfected cell lines down-regulated endogenous MRP1 and γ-GCShexpression. ROI levels in these transfected cells were lower than those in the untransfected control. In the cell lines in which depleting cellular GSH pools did not affect the expression of theMRP1 and γ-GCSh genes, only minor increased intracellular levels of ROIs were observed. These results suggest that intracellular ROI levels play an important role in the regulation ofMRP1 and γ-GCSh expression. Our data also suggest that elevated intracellular GSH levels not only facilitate substrate transport by the MRP1/GS-X pump as previously demonstrated, but also suppress MRP1 and γ-GCShexpression.

2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate NF-κB pathway

Overexpression of multidrug resistance genes and their encoded P-glycoproteins is a major mechanism for the development of multidrug resistance in cancer cells. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression. However, the underlying mechanisms are largely unknown. In this study, we demonstrated that a NF-κB site on the mdr1b promoter was required for this induction. Overexpression of antisense p65 and IκBα partially abolished the induction. We then delineated the pathway through which 2-AAF activates NF-κB. 2-AAF treatment led to the increase of intracellular reactive oxygen species (ROS) which causes activation of IKK kinases, degradation of IκBβ (but not IκBα), and increase in NF-κB DNA binding activity. Consistent with the idea that ROS may participate inmdr1b regulation, antioxidant N-acetylcysteine inhibited the induction of mdr1b by 2-AAF. Overproduction of a physiological antioxidant glutathione (GSH) blocked the activation of IKK kinase complex and NF-κB DNA binding. Based on these results, we conclude that 2-AAF up-regulates mdr1b through the generation of ROS, activation of IKK kinase, degradation of IκBβ, and subsequent activation of NF-κB. This is the first report that reveals the specific cis-elements and signaling pathway responsible for the induction of mdr1b by the chemical carcinogen 2-AAF.

Roles of reactive oxygen species in hepatocarcinogenesis and drug resistance gene expression in liver cancers

Hepatocellular carcinoma (HCC) has traditionally been an attractive system for cancer research because many animal HCC models are available. It is well known that liver tumors in animals can be induced by many different protocols, such as chronic hepatitis viral infections, carcinogens, toxins, steroid hormones, and dietary intervention. Although these different inducers have different cellular targets and modes of cytotoxic effects, their common denominator is the formation of reactive oxygen species (ROS). In this review, we present compelling evidence to support the hypothesis that ROS play important roles in hepatocarcinogenesis and the associated upregulation of drug resistance gene expression.

The human multidrug resistance-associated protein (MRP) gene family: from biological function to drug molecular design.

Abstract The ATP-binding cassette transmembrane proteins play an important role in transport of drugs as well as of biologically active endogenous substances. The human multidrug resistance-associated protein (MRP) subfamily consists of at least six members, exhibiting a wide spectrum of biological functions. MRP1 operates as an ATP-dependent primary active transporter for substrates conjugated with glucuronide, sulfate or glutathione. Leukotriene C4 is an important endogenous substrate for MRP1. Glutathione serves as a cofactor in MRP1-mediated drug transport as well. Genes encoding both MRP1 and the catalytic subunit of γ-glutamylcysteine synthetase (γ-GCS) are coordinately regulated in cultured cancer cell lines as well as colorectal cancer tissues from colon cancer patients. The induction of MRP1 and γ-GCS expression by oxidative stress varies among different cell lines, and p53 mutations are associated with elevated levels of induction. To modulate the transport function of MRP1, we have synthesized novel glutathione derivatives as photoreactive biochemical probes targeting the transporter protein. GIF-0019 restored the cellular sensitivity of MRP1-overexpressing drug-resistant cancer cells to anticancer prostaglandins in vitro, which was characterized by enhanced mRNA levels of the cyclin-dependent kinase inhibitor p21, suppressed c-myc expression and G1 arrest.

Differential sensitivities of the MRP gene family and γ-glutamylcysteine synthetase to prooxidants in human colorectal carcinoma cell lines with different p53 status

1Recent molecular cloning studies have identified six members in the multidrug-resistance protein (MRP) gene family. However, the regulation of expression of these genes is largely unknown. We previously reported that expression of MRP1, encoding multidrug-resistance associated protein, and gamma-GCSh, which encodes the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS), could be up-regulated by prooxidants [Yamane et al., J Biol Chem 1998;273:31075-85]. In the present study, we investigated whether different members of the MRP family exhibit different responses to induction by prooxidants, and whether p53 status influences the levels of induction. A panel of colorectal cancer cell lines with different p53 status, i.e. HCT116 containing wild-type p53, and HT29, SW480, and Caco2 containing mutant p53, was treated with tert-butylhydroquinone (t-BHQ) and pyrrolidinedithiocarbamate (PDTC). MRP1 and gamma-GCSh mRNA levels were determined by the RNase protection assay, using gene-specific probes. We report here that induction of MRP1 and gamma-GCSh expression by these prooxidants varied among the different cell lines, and p53 mutations were not always associated with elevated levels of induction. These results suggest that the effects of p53 on the induced expression of MRP1 and gamma-GCSh depend on the environment of the cell and/or nature of p53 mutations. In an isogenic HCT116 cell line containing p53(-/-) alleles, we demonstrated that, as for MRP1, expression of MRP2 and MRP3 was induced by the prooxidants, whereas expression of MRP4 and MRP5 was not. MRP6 mRNA was not detectable. Induction of MRP2 expression by prooxidants seemed to be independent of p53 status. Our results demonstrated the differential regulation of the MRP gene family by p53 mutation under oxidative stress.

Expression of heavy subunit of γ-glutamylcysteine synthetase (γ-GCSh) in human colorectal carcinoma

Gamma‐glutamylcysteine synthetase (γ‐GCS) is a heterodimer consisting of heavy (γ‐GCSh) and light (γ‐GCSl) subunits. γ‐GCS catalyzes the rate‐limiting de novo biosynthesis of glutathione (GSH), an abundant physiological antioxidant that plays important roles for regulating oxidative stress. Expression of γ‐GCSh and γ‐GCSl are sensitive to oxidative stress. To investigate whether expression of γ‐GCS is correlated with tumor progression, we used immunohistochemical approaches to examine 16 human colorectal adenomas and resected 57 carcinomas from untreated patients. In adjacent normal colorectal epithelium, levels of γ‐GCSh expression were low. Strong cytoplasmic staining for γ‐GCSh was detected in 3 (18.8%) adenoma and 48 (84.2%) carcinomas. The frequency of γ‐GCSh expression in carcinoma was significantly higher than in adenoma (p<0.0001). We used RNase protation assay and Western blot to determine levels of γ‐GCSh mRNA and protein from 10 pairs of matched carcinomas with adjacent normal controls. Elevated expression of both γ‐GCSh mRNA and protein were found in 6 cases, suggesting that transcriptional and/or posttranscriptional regulation play an important role in the upregulation of γ‐GCS during colorectal carcinogenesis. We also examined the expression of another redox‐regulated gene, multidrug resistance protein 1 (MRP1). Strong staining for MRP1 was detected in 1 (6.3%) adenoma and 40 (70.2%) carcinomas. The frequency of MRP1 expression in carcinoma was significantly higher than in adenoma ( p<0.0001). Nuclear p53 expression was detected in 30 (52.6%) of carcinomas. There is a significant correlation between γ‐GCSh and MRP1 expression (p=0.013) but not between γ‐GCSh and p53. Since γ‐GCS is a sensor of oxidative stress, these results are consistent with the notion that oxidative stress is associated with colorectal tumor progression.

The combination of ADI-PEG20 and TRAIL effectively increases cell death in melanoma cell lines

Current treatment for advanced, metastatic melanoma is not very effective, and new modalities are needed. ADI-PEG20 is a drug that specifically targets ASS-negative malignant melanomas while sparing the ASS-expressing normal cells. Although laboratory research and clinical trials showed promising results, there are some ASS-negative cell lines and patients that can develop resistance to this drug. In this report, we combined ADI-PEG20 with another antitumor drug TRAIL to increase the killing of malignant melanoma cells. This combination can greatly inhibit cell growth (to over 80%) and also enhanced cell death (to over 60%) in four melanoma cell lines tested compared with control. We found that ADI-PEG20 could increase the cell surface receptors DR4/5 for TRAIL and that caspase activity correlated with the increased cell death. These two drugs could also increase the level of Noxa while decrease that of survivin. We propose that these two drugs can complement each other by activating the intrinsic and extrinsic apoptosis pathways, thus enhance the killing of melanoma cells.

Redox regulation of matrix metalloproteinase gene family in small cell lung cancer cells

It has been implicated that reactive oxygen species (ROS) play important roles in modulating tumor progression. However, the mechanisms by which redox-regulated tumor progression are largely unknown. We previously demonstrated that reduced intracellular redox conditions could be achieved in stably transfected small cell lung cancer cells with γ-glutamylcysteine synthetase (γ-GCSh) cDNA which encodes a rate-limiting enzyme in the biosynthesis of glutathione (GSH), a major physiological redox regulator. In the present study, using DNA microarray analyses, we compared the expression profiles between the γ-GCSh-transfected cells and their nontransfected counterpart. We observed downregulation of several matrix metalloproteinases (MMPs), i.e., MMP1 and MMP3, and MMP10 in the transfected cells. Dot blot and Northern blot hybridizations confirmed that, among the 18 MMP gene family members and four tissue inhibitors of matrix metalloprotein family (TIMP) analyzed, the expression levels of these three MMPs were consistently reduced. Transiently increased γ-GCSh expression using tetracycline-inducible γ-GCSh adenoviral expression system also showed down-regulation of MMP3 and MMP10, but not MMP1. Our results demonstrated that redox regulation of MMP1, MMP3 and MMP10 expression depend upon different modes of redox manipulation. These results bear implication that antioxidant modulation of antitumor progression may be contributed at least in part by the downregulation of a subset of metrix metalloproteins.

MDR AND MRP GENE FAMILIES AS CELLULAR DETERMINANT FACTORS FOR RESISTANCE TO CLINICAL ANTICANCER AGENTS

The constant threat by a countless array of environmental poisons, natural products and synthetic agents, over evolutionary time has led living organisms to develop many elaborate mechanisms that combat the toxic effects of these insults. Among such mechanisms is one that decreases the intracellular accumulation of a toxic substance by directly pumping toxic molecules out of the cells, and another that modifies the metabolism of the toxic substances and effluxes the metabolized compounds. The former mechanism is typified by the mammalian multidrug resistance system mediated by P-glycoproteins (P-gp) that are encoded by the MDR gene family. The second mechanism is exemplified by the multidrug resistance protein (MRP). Both P-gp and MRP contain ATP-binding cassettes and therefore belong to the ABC superfamily of membrane transporters

MOLECULAR DESIGN OF GLUTATHIONE-DERIVED BIOCHEMICAL PROBES TARGETING THE GS-X PUMP

Abstract The syntheses of novel glutathione derivatives as photoaffinity and fluorescent biochemical probes for the GS-X pump are described. GIF-0017, an S -alkylated glutathione derivative with a benzophenone photophore, competitively inhibits the transport of leukotriene C 4 by the GS-X pump with an IC 50 value of 0.40 μM, indicating the existence of strong interaction between the probe molecule and the GS-X pump.