TCDD as a biological response modifier for Mitomycin C: Oxygen tension affects enzyme activation, reactive oxygen species and cell death☆
Introduction
The anticancer compound Mitomycin C (MMC) is a DNA alkylating agent that is administered as a pro-drug and activated intracellularly by the enzymes NAD(P)H quinone oxido-reductase (NQO1), xanthine dehydrogenase (XDH), xanthine oxidase (XO), Cytochrome P450 reductase (CYPR) and Cytochrome b5 reductase (Pan et al., 1984, Gustafson and Pritsos, 1992, Siegel et al., 1992, Hodnick and Sartorelli, 1993). MMC is more toxic under hypoxic than normoxic conditions and this is especially important for its clinical use in chemotherapy of solid tumors that possess hypoxic interior regions and often over-express activation enzymes such as NQO1 (Rauth et al., 1984).
2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is a s-, multi-ringed organic compound that is highly lipophilic and rapidly and completely bioavailable and is the prototypical agonist of the aryl-hydrocarbon receptor (AhR) (Rowlands and Gustafsson, 1997). This receptor exists in the cytoplasm and, upon activation, translocates to the nucleus and binds to response elements in the enhancers of target genes (Rowlands and Gustafsson, 1997). Activation of the AhR is associated with transcriptional induction of a number of metabolizing enzymes including CYP1A, CYPR, XDH, XO, NQO1, UGT1A6, GST and ALHD3 (Rowlands and Gustafsson, 1997). As many of these are activators of MMC, activation of the AhR may be expected to increase the toxicity of MMC due to enzyme induction although activation of genes through AhR commonly involves balance, wherein detoxicative as well as activating enzymes are upregulated.
Promising recent investigations have demonstrated that partial AhR agonists such as dimethyl fumarate or sulforaphane, can enhance the cytotoxicity of bioreductive drugs and modulate disease states through intracellular signaling (Sebok et al., 1994, Munday et al., 1999, Wang et al., 1999, Begleiter et al., 2003). Compounds used to study dietary chemoprevention may also involve AhR activation and this is also an area where AhR agonists are receiving attention (McPherson et al., 2001). Although the precise mechanisms are yet to be fully elucidated, it has been speculated that several of these compounds may induce Phase I activation enzymes without a concurrent increase in Phase II clearance enzymes and also exhibit intrinsic apoptotic, oxidative and cell cycle effects (Sebok et al., 1994, Munday et al., 1999, Wang et al., 1999, McPherson et al., 2001, Begleiter et al., 2003).
Although the AhR is known to up-regulate several Phase I enzymes that are responsible for MMC activation, the influences of hypoxia and oxidative stress on AhR signaling are not well characterized. Additionally, the mechanisms of TCDD toxicity remain undefined but effects on cell cycle control, apoptosis and mitochondrial function have been demonstrated (Nebert et al., 2000, Senft et al., 2002, Jin et al., 2004). There has recently been a move toward combining active drugs with chemicals that have little or no demonstrated tumor suppression of their own yet which act synergistically to increase the effect of the active drug (so-called biological response modifiers). The emergence of Thalidomide, which has small anti-angiogenic and cell cycle effects, as an enhancer of chemotherapy in the treatment multiple myeloma is a good example of this (Joglekar and Levin, 2004). The use of biological response modifiers may bypass some of the technical difficulties of laboratory-based methods such as antibody-directed or gene-directed approaches to enhance pro-drug therapy and may increase bystander effects in surrounding cells. Here we have investigated TCDD as a biological response modifier for increasing MMC toxicity using human MCF-7 cells under both aerobic and hypoxic (radio-opaque) conditions. It was hypothesized that concurrent administration of TCDD would increase the toxicity of MMC through AhR-mediated induction of enzymes that activate MMC. We further hypothesized that hypoxic and normoxic conditions would have differential effects on the ability of AhR agonists to modify MMC toxicity because of alterations in intracellular oxidative stress and subsequent effects on AhR signaling.
Section snippets
Materials and methods
3-[4,4-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reagent, 4-MU, 4-MUG, ethoxyresorufin, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), 2′,7′-dichlorofluorescein (DCF), penicillin/streptomycin, 1-chloro,2-4-dinitrobenzene (CDNB), glutathione, 3,3′-methylenebis[4-hydroxycoumarin] (dicumarol), dichloroindophenol, UDPGA, NAD(P)H, NADH, NAD+, TCDD and xanthine were obtained from the Sigma Chemical Company (St Louis Mo); cell culture media and plastic ware, agarose, BSA, EDTA and
Enzyme expression and activity assays
Biochemical activity assays were performed using S9 protein from MCF-7 cells treated as described above. S9 protein was prepared by harvesting cells and resuspending them in 0.01 M Tris–HCl with 2 mM PMSF pH 7.5, sonication for 30 s with a probe sonicator then centrifugation at 10,000 ×g for 20 min whereupon the supernatant was removed and used for biochemical determination of enzyme activities. Biochemical assays for enzyme activity were carried out according to previously described methods
Statistical analysis and data transformations
All statistics were performed using Graph Pad Prism 3.0 (GraphPad Software Inc. San Diego, California). Non-linear regression fits for IC50 were performed using a four-parameter logistic regression with no weighting. Convergence was reached when two consecutive iterations changed the sum of squares by less than 0.01%. To generate the curves, zero dose controls (100% survival) were modeled as a theoretical concentration of MMC which does not cause cell death (10− 12 M). Comparisons of combined
Cell Viability
The fit of the four parameter logistic regression showed small standard errors for the LogLD50, Upper Plateaus and Hill Slopes, typically < 3%. Larger variation was observed in the standard error of the fit for the bottom plateaus. All fits were considered appropriate and acceptable although, under hypoxia, the fit of the four-parameter logistic regression for cells treated with both MMC and TCDD (Fig. 1A and B, dashed line) was not excellent.
Using a clonogenic assay, Mitomycin C alone was more
Discussion
In recent years, focus has moved away from redox and DNA-binding interactions of MMC and towards its enzymology in the hope of increasing the efficacy and application of the drug. Thus, by choosing TCDD as a biological response modifier, the present series of experiments employed an AhR/enzyme-mediated approach in an attempt to increase MMC cytotoxicity (efficacy). In the present results, activation of the AhR and increased enzyme activity was clearly demonstrated, particularly through the
Conclusion
Increased potency is often considered redundant for clinically used drugs, the rationale being that so long as one remains within the therapeutic index, one can simply increase the amount of drug administered to obtain greater effect. In the case of anti-cancer drugs, however, side-effects are often dose-limiting with maximum tolerated doses in patients commonly being below the strictly toxic thresholds for the drug. Therefore, eliciting greater drug effect at equivalent concentrations with a
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Funding acknowledgement: Funding for this project was provided in part by the Nevada Agricultural Experiment Station.