| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 13, 2005 as doi:10.1096/fj.04-2991fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Division of Cardiovascular Medicine and Graduate Center for Nutritional Sciences, University of Kentucky; and
Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA
1 Correspondence: Division of Nephrology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900, USA. E-mail: asmis{at}uthscsa.edu
SPECIFIC AIMS
The aim of this study was to elucidate the mechanisms involved in adriamycin-induced cell injury in human macrophages.
PRINCIPAL FINDINGS
1. Prolonged exposure to low doses of adriamycin promotes caspase-independent cell death in mature, human monocyte-derived macrophages, a model of human tissue macrophages
Macrophages incubated with adriamycin for up to 24 h showed no significant decrease in cell viability at adriamycin concentration below 25 µM (Fig. 1
). At high concentrations of 25 to 100 µM, adriamycin decreased cell viability in a dose-dependent manner from 85.6% to 19.1% with an apparent LD50 of 65 µM (Fig. 1
, insert). We observed a significant left shift in the dose-response curve when the incubation time was increased to 48 h. The onset of macrophage injury now was observed at adriamycin concentrations as low as 1 µM. Cell viability did not decrease in a classic dose-dependent relationship but was triphasic. Although cell viability decreased to 42.6% with 2 µM adriamycin, cell viability was routinely higher in the presence of 5 µM (53.3%, P=0.011) and decreased only after adriamycin concentrations were increased to 25 µM, indicating that adriamycin induces both cell injury and cell survival pathways. Treatment of macrophages with caspase inhibitory peptides or calpain inhibitors prior to adriamycin exposure did not prevent cell death, suggesting adriamycin-induced cell injury does not involve classic apoptotic pathways.
|
2. Adriamycin-induced macrophage injury is independent of one-electron redox cycling and concomitant ROS formation
The membrane-permeable superoxide scavenger PEG-SOD and the potent peroxyl radical scavenger Trolox diminished basal DCFH oxidation and completely blocked the adriamycin-induced increase in DCFH oxidation attributable to ROS formation in macrophages. However, neither PEG-SOD nor Trolox prevented adriamycin-induced macrophage death, suggesting that ROS formation inhibitable by these scavengers is not required for adriamycin toxicity in human macrophages.
3. Adriamycin-induced changes in the cellular thiol redox system promote cell injury
The GSH/GSSG ratio is a principal determinant of the cellular redox environment, and alterations in the redox environment can lead to cellular dysfunction and cell death. Treatment of macrophages with adriamycin for 24 h stimulated significant changes in the glutathione (GSH)/glutathione disulfide (GSSG) ratio, which preceded and closely correlated with changes in macrophage viability observed 24 h later. Increased protein-S-glutathionylation and the formation of protein-bound glutathione (PSSG) paralleled GSH oxidation and accumulation of GSSG induced by adriamycin. The membrane-permeable thiol oxidant diamide mimicked the toxic effects of adriamycin in human macrophages. Diamide induced a dose-dependent decrease in the GSH/GSSG ratio, which correlated with the dose dependence of diamide-induced cell lysis. Diamide also showed similar increases in GSSG (2.4-fold) and PSSG (1.6-fold) at concentrations where it induced macrophage lysis as those observed for adriamycin (25 µM), supporting the interpretation that thiol oxidation contributes to adriamycin-induced macrophage injury.
4. Adriamycin has both inhibitory and stimulatory effects on glutathione reductase activity in human macrophages
Enhanced GSH oxidation may account at least in part for accumulation of GSSG in adriamycin-treated macrophages. However, inhibition of glutathione reductase, an enzyme susceptible to oxidative inactivation, would also promote GSSG accumulation, particularly under conditions of enhanced thiol oxidation. Adriamycin at 0.3 and 1 µM, inhibited glutathione reductase activity by 18.9% and 32.4%, respectively. Increasing adriamycin concentration to 2 and 3 µM led to restoration of glutathione reductase to control levels. Western blot analysis revealed that protein levels of glutathione reductase paralleled the changes in enzyme activity, indicating that the decrease in glutathione reductase activity induced by adriamycin appears to be due to decreased protein levels rather than changes in specific enzymatic activity. The subsequent increase in enzymatic activity coincided with increased protein levels, suggesting that at higher concentrations adriamycin stimulates glutathione reductase expression.
5. Inhibition of glutathione reductase and glutaredoxin potentiates adriamycin-induced macrophages injury
Glutathione reductase and glutaredoxin, the primary enzyme involved in the deglutathionylation of PSSG, are key regulators of the cellular thiol redox state. The presence of the glutathione reductase inhibitor 1,3-bis[2-chloroethyl]-1-nitrosourea (BCNU, 0.2 mM) during adriamycin stimulation dramatically enhanced macrophage death in response to adriamycin, suggesting that glutathione reductase protects human macrophages against adriamycin toxicity. Transfection of human macrophages with siRNA directed against human glutathione reductase alone did not affect macrophage viability, but potentiated adriamycin toxicity at both low (2 µM) and high (25 µM) drug concentrations confirming a protective role for glutathione reductase against adriamycin-induced macrophage injury. The siRNA-mediated inhibition of glutaredoxin also potentiated adriamycin toxicity in human macrophages indicating that protein-S-glutathionylation contributes to adriamycin-induced macrophage injury.
CONCLUSIONS AND SIGNIFICANCE
The use of adriamycin in patients has been limited primarily by its cardiotoxicity, but other side effects have also been reported, including immunosuppression and impaired wound healing. Little is known about the toxicity of adriamycin in other cells and tissues. Here we show that adriamycin is a potent inducer of thiol oxidation and cell injury in human macrophages.
Our data suggest that adriamycin affects the thiol redox system of macrophages in multiple ways (Fig. 2
). Adriamycin promotes the oxidation GSH to GSSG (Fig. 2
, 
), as indicated by the accumulation of GSSG in response to adriamycin. We found that adriamycin also decreases glutathione reductase (GR) activity, the enzyme responsible for the reduction of GSSG and regeneration of GSH (Fig. 2
, 
). Modification of glutathione reductase activity by adriamycin appears to correspond to changes in glutathione reductase protein content. Unless GSSG is efficiently reduced by glutathione reductase, it will accumulate and decrease the cellular GSH/GSSG ratio, altering the overall redox environment of macrophages. However, decreased glutathione reductase activity alone cannot explain the accumulation of GSSG because at low adriamycin concentrations the adriamycin-dependent changes in GSSG content did not coincide with the changes in glutathione reductase activity. Thus, adriamycin exposure leads to oxidation of GSH to GSSG, even under conditions where glutathione reductase activity is not changed, but inactivation of glutathione reductase potentiates the effect of adriamycin on GSSG accumulation.
|
Adriamycin inhibited glutathione reductase content and activity at concentrations as low as 0.3 µM. Like other sulfhydryl repair enzymes, glutathione reductase is sensitive to oxidative stress, being inactivated by oxidation or other modifications of its active site residue Cys-63. One might therefore expect that adriamycin could inactivate glutathione reductase by various mechanisms, including sulfhydryl oxidation. However, our data indicate that adriamycin decreases glutathione reductase activity by decreasing the protein levels of the enzyme, suggesting a process that involves increased degradation of the glutathione reductase protein, although the underlying mechanism is not clear.
Glutathione reductase activity appears to be restored if adriamycin concentrations are increased to 2–5 µM. One explanation is that adriamycin enhances the expression of glutathione reductase (Fig. 2
, ) and other phase 2 enzymes to a rate that exceeds the rate of adriamycin-mediated loss of the enzyme. Indeed, we observed an increase in glutathione reductase protein levels at these adriamycin concentrations. Adriamycin treatment was shown to induce phase 2 enzymes in cardiac tissues, including 
-glutamylcysteine synthase, the rate-limiting enzyme in GSH synthesis. We observed a dose-dependent increase in total glutathione levels in response to adriamycin, indicating that the drug induced -glutamylcysteine synthase, and subsequently GSH synthesis, in human macrophages. At high adriamycin concentrations (>5 µM), glutathione reductase expression seems substantially impaired.
The collective data suggest that glutathione reductase plays a key role in protecting macrophages from adriamycin-induced cell injury and that under conditions of enhanced (thiol) oxidative stress, even a partial inhibition of glutathione reductase synthesis would significantly potentiate cell injury. As predicted, diminishing glutathione reductase activity with BCNU or enzyme expression with siRNA dramatically increased adriamycin-induced macrophage death.
Thiol oxidation in response to adriamycin occurs to some extent even when glutathione reductase activity is diminished. Thiol oxidation induced by the cell-permeable thiol oxidant diamide promoted GSSG accumulation, enhanced PSSG formation, and was sufficient to promote macrophage death, further supporting the hypothesis that thiol oxidation is the primary mechanism of adriamycin-induced macrophage injury. Our studies show that one-electron redox cycling and concomitant ROS formation are not required for adriamycin-induced cell injury in macrophages. Rather, thiol oxidation is more proximally implicated in macrophage death.
Adriamycin also promoted S-glutathionylation of protein thiols in human macrophages. Protein-S-glutathionylation can occur in response to elevated GSSG levels (Fig. 2
, 
). However, protein thiols can also be oxidized to sulfenates (PSOH), which readily react with GSH or PSH to form protein-glutathione or protein-protein (mixed) disulfides (Fig. 2
, 
). It is currently unclear which of the two mechanisms leads to the formation of PSSG in adriamycin-treated macrophages. Many proteins have now been identified as targets for protein-S-glutathionylation and, in most instances, protein-S-glutathionylation results in the inactivation of these proteins. Protein-S-glutathionylation is reversible; however, once it reaches a critical level, macrophages are likely to become dysfunctional and die. When we treated macrophages with siRNA to decrease glutaredoxin and thereby inhibit protein deglutathionylation, macrophage injury was potentiated after adriamycin treatment, consistent with protein-S-glutathionylation contributing to adriamycin-induced macrophage death.
The mechanism by which adriamycin oxidizes thiols—in particular, GSH—is not clear. In cardiomyocytes, redox cycling is believed to occur by one-electron reduction involving either mitochondrial flavoproteins or cytosolic NADPH-cytochrome P450 reductase, NADH-cytochrome b reductase, and nitric oxide synthases. One-electron reduction of adriamycin yields a reactive semiquinone radical, which in the presence of O2 converts back to adriamycin generating superoxide. According to this model, thiol oxidation would occur downstream of such ROS formation. However, our data show that in macrophages cytotoxicity occurs independent of superoxide dismutase- and Trolox-inhibitable ROS formation, suggesting that mechanisms other than the reductive activation of adriamycin are involved.
Reszka and colleagues recently showed that in the presence of H2O2 and NO2-, adriamycin is oxidized at its hydroquinone moiety. Both monocytes and macrophages express peroxidase activity. Thus, oxidation of adriamycin by peroxidases could create a redox cycle in macrophages that continually oxidizes thiols and, once the antioxidant defenses are overwhelmed, would lead to cell injury.
We conclude that adriamycin promotes cell injury in human macrophages likely by promoting the persistent oxidation of GSH and protein thiols. The combination of thiol oxidation and loss of glutathione reductase activity (Fig. 2)
are likely to explain the sensitivity of human macrophages to adriamycin toxicity. Because macrophages are intimately involved in immune responses and tissue repair, our data suggest that adriamycin-induced macrophage dysfunction and cell death may contribute to the noncardiotoxic side effects of adriamycin therapy, especially impaired wound healing.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2991fje;
This article has been cited by other articles:
|
|
 |
|
  D. M. Townsend S-Glutathionylation: Indicator of Cell Stress and Regulator of the Unfolded Protein Response Mol. Interv., December 1, 2007; 7(6): 313 - 324. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Rutkute, R. H. Asmis, and M. N. Nikolova-Karakashian Regulation of neutral sphingomyelinase-2 by GSH: a new insight to the role of oxidative stress in aging-associated inflammation J. Lipid Res., November 1, 2007; 48(11): 2443 - 2452. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Qiao, M. Kisgati, J. M. Cholewa, W. Zhu, E. J. Smart, M. S. Sulistio, and R. Asmis Increased Expression of Glutathione Reductase in Macrophages Decreases Atherosclerotic Lesion Formation in Low-Density Lipoprotein Receptor-Deficient Mice Arterioscler. Thromb. Vasc. Biol., June 1, 2007; 27(6): 1375 - 1382. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
