Inhibition of oxidative modification of proteins by RU486*†

FERTILITY AND STERILITY® Copyright

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Vol. 66, No.1, July 1996

1996 American Society for Reproductive Medicine

Printed on acid-free paper in U. S. A.

Inhibition of oxidative modification of proteins by RU486*t

Sue Ellen K. Carpenter, M.D. Nalini Santanam, Ph.D. Ana A. Murphy, M.D.

John A. Rock, M.D. Sampath Parthasarathy, Ph.D.:!:

Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia

Objectives: To elucidate further the antioxidant properties of RU486. We determined whether it can protect biologic molecules such as proteins (albumin, low-density lipoprotein [LDL] and oxidized LDL) from damage by pre-existing lipid peroxides. Design: In vitro study. Interventions: We tested the effects of RU486 on the formation of fluorescent oxidatively modified proteins by pre-existing lipid peroxides. We used two model systems, the incubation of oxidized linoleic acid with serum albumin and the incubation of human LDL with copper. Main Outcome Measures: The formation of modified protein was established by determining fluorescence at excitation wavelength of 330 nm and emission wavelength between 390 and 500 nm. Modified protein has a characteristic emission between 425 and 430 nm. Results: The addition of increasing amounts of RU486 inhibited the formation of fluorescent oxidatively modified protein products in both model systems. Conclusion: These results provide evidence that RU486 not only can prevent the formation oflipid peroxide, but also can block the formation of fluorescent protein adducts in the presence of pre-existing lipid peroxides. Fertil Steril® 1996; 66:90-4 Key Words: Oxidation, LDL, OX-LDL, RU486, mifepristone, oxidatively modified protein, fluorescent oxidatively modified protein, lipid peroxide

The anti-P and antiglucocorticoid RU486 or mifepristone has attracted worldwide attention because of its efficacy in the termination of pregnancy (1,2). However, its other potential clinical uses have not been examined adequately. RU486 has been suggested to be effective in providing relief from pain associated with endometriosis (3). It also induces a marked reduction in leiomyoma volume and appears to be effective in retarding the progression of breast cancer and meningiomas (4). Its efficacy as an antiglucocorticoid has received attention in the treatment of patients with Cushing syndrome. The mechanism(s) of actions of RU486 have been explained

Received December 28, 1995; revised and accepted January 25, 1996. * Roussel-Uclaf, Romainville, France. t Supported by the Department of Gynecology and Obstetrics at Emory University. :j: Reprint requests: Sampath Parthasarathy, Ph.D., Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia 30322 (FAX: 404-727-8615; E-mail: [email protected]).

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Carpenter et al. RU486 inhibits oxidation of protein

based on its ability to bind to intracellular P or glucocorticoid receptors and block specific transcriptional activation of gene action (1, 2, 5-7). Very few studies have looked at the actions of RU486 on cells that do not express specific hormone receptors. We recently have reported that RU486 is a potent antioxidant and that it prevents the oxidation of low-density lipoprotein (LDL) in vitro (8). This inhibition is evident at micromolar concentrations that are physiologically relevant. The inhibitory activity can be attributed to the presence of the dimethyl amino phenyl side chain of the molecule. Analogues that do not have this moiety are devoid of antioxidant activity. The potential of RU486 to act as an antioxidant in vivo is suggested by the resistance ofLDL isolated from subjects treated with RU486 to in vitro oxidation. Antioxidants not only decrease the rate of initiation of oxidation by reacting with oxygen radicals, but they also function as chain terminators and prevent propagation of oxidation. The potential of antioxidants to inhibit the damage to biologic molecules, such as proteins and nucleic acids, from pre-existing Fertility and Sterility®

lipid peroxides has not been studied. We recently described a novel reaction in which preformed lipid peroxides reacted with E-amino groups oflysine residues of proteins to generate oxidatively modified proteins (9). The proposed mechanism of such a reaction involves the oxidation of a second double bond in the same peroxidized fatty acid molecule by an aminecatalyzed release of reactive oxygen species from the peroxy group and the covalent modification of the amino groups oflysine residues. These modified proteins are characterized by fluorescent epitopes that are antigenic. The antioxidant enzyme, superoxide dismutase (SOD), and its mimic, copper diisopropyl salicylate, can block the generation of the fluorescent polypeptides. Recent evidence suggests that oxidatively modified proteins can have potent biologic properties. The protein component of oxidatively modified LDL recently was shown to induce interleukin-1 (IL-l) synthesis from macrophages (10). Oxidized LDL and similarly modified serum albumin is chemotactic to monocyte-macrophages and can induce vascular cell adhesion molecule-1 expression from endothelial cells (11). In these studies, the effect appears to be due to a transcriptional activation of specific genes. In the current study we demonstrate that RU486 is a potent inhibitor of such reactions and the generation of an oxidatively modified protein. Such proteins can be generated both extracellularly and intracellularly in biologic systems. Because of its lipophilic nature and the ability of RU486 to be associated with lipoproteins (8), we suggest that RU486 may have profound effects on the generation of biologically active oxidized lipid-protein complexes and thus may have additional cellular effects that are independent of its ability to bind to intracellular hormonal receptors.

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Figure 1 Effect of RU486 and levonorgestrel on the formation of fluorescent oxidized linoleic acid-protein adducts. Two hundred nanomoles ofhydroperoxylinoleic acid was incubated with 200 j.tg of rabbit serum albumin in the presence of RU486 or levonorgestrel for 24 hours. Samples were analyzed for fluorescence as described in the Materials and Methods section. Sample 1, no addition; sample 2, 2.5 mM RU486; sample 3, 5 j.tM RU486; sample 4, 10 j.tM RU486; sample 5, 10 j.tM levonorgestrel. Results presented are from a representative experiment from four separate experiments.

oxidatively modified protein had a characteristic emission between 425 and 430 nm. Low-density lipoprotein was isolated from plasma of normal human volunteers and subjected to oxidation using 5 mM copper (12). The rate of oxidation was measured by the determination of the formation of conjugated dienes and thiobarbituric acid reactive substances as described earlier (13). Sodium dodecylsulfate-polyacrylamide gel electropheresis was performed on 7.5% cross-linked acrylamide gels. Proteins were transferred to nitrocellulose membranes and Western blot analysis was performed using a rabbit polydonal antibody to oxidatively modified rabbit serum albumin at the dilution of 1 to 250 (14). Protein was detected by peroxidase-conjugated goat anti-rabbit serum immunoglobulin (goat IgG).

MATERIALS AND METHODS RESULTS

Linoleic acid, soybean lipoxygenase, levonorgestrel, and bovine serum albumin were obtained from Sigma Chemical Company (St. Louis, MO). Linoleic acid was oxidized by incubation of 1 JLM linoleic acid in 1 mL of phosphate-buffered saline (PBS) with 100 units of soybean lipoxygenase (9). The formation of conjugated diene was monitored by measuring the increase in absorption at 234 nm in a spectrophotometer (12). Modification of protein was initiated by the incubation of 200 JLM oxidized linoleic acid (13hydroperoxy octadecadienoic acid [13-HPODE]) with 200 JLg of serum albumin in 1 mL of PBS at 37°C for 24 hours as described earlier (9). The formation of modified protein was established by determining the fluorescence at excitation wavelength of 330 nm and emission wavelength between 390 and 500 nm. The Vol. 66, No.1, July 1996

We tested the effects of RU486 on the formation of fluorescence during the incubation of oxidized linoleic acid (13-HPODE). As seen in Figure 1, incubation of oxidized linoleic acid with serum albumin resulted in the formation of fluorescent products. The addition of increasing amounts of RU486 inhibited the formation of fluorescent products. The inhibition could be seen at as little as 1 JLM RU486, and, at 10 JLM RU486, complete inhibition could be noted. Levonorgestrel is structurally similar to RU486 but lacks the presence of the dimethyl amino phenyl group, an essential component of the antioxidant property of RU486. When levonorgestrel was included in the incubation system, even at 10 JLM concentrations, it failed to show inhibition of the formaCarpenter et at. RU486 inhibits oxidation of protein

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Figure 2

(Al, Oxidation of LDL in the presence of RU486. Human LDL (100 J.!g/mLl was incubated in spectrophotometric cuvettes in the presence or absence of 10 J.!M RU486. Oxidation was initiated by the addition of 5 J.!M copper (final concentration). The formation of conjugated dienes (lipid peroxidation products) was monitored continuously by measuring the increase in absorption at 234 nm. Sample 1, unoxidized LDL; samples 2 and 4, oxidized LDL; sample 3, LDL oxidized in the presence of 10 J.!M RU486. Results presented are from a representative experiment from a set of four experiments. (B), The formation offiuorescent oxidized lipid-protein adducts from LDL subjected to oxidation. Low-density lipoprotein was oxidized as described in the previous experiment. After 3 hours (when lipid peroxidation was complete as determined by lack of further increase in OD 234 nm), 10 J.!M RU486 was added to sample 2 and the incubation was continued for an additional 21 hours. Fluorescence was determined as described in the text. Results presented are from a representative experiments from a set of four experiments.

tion of fluorescent product. In fact, a small increase in the formation of fluorescent products could be seen. These results suggested that RU486 can prevent the formation of fluorescent products even from pre-existing lipid peroxide. To establish that RU486 has an additional antioxidant capability inhibiting the formation of fluorescent products, we performed the following experiment. Low-density lipoprotein was subjected to oxidation in the presence of 5 ",M copper for 180 minutes. As seen in Figure 2A, the LDL that was incubated with copper was oxidized readily and the maximum level of conjugated dienes were formed at approximately 180 minutes (lines 2 and 4). The presence of RU486 completely inhibited the oxidation (line 3) and, at 180 minutes, <5% of the LDL lipids were oxidized. We added RU486 to LDL that 92

Carpenter et al. RU486 inhibits oxidation of protein

was oxidized with copper for 3 hours and continued the incubation for an additional 21 hours. The results are presented in Figure 2B. The LDL that was subjected to oxidation for 3 hours and then was incubated for additional 21 hours generated fluorescent products as shown in line 1. As expected, LDL that was incubated in the presence of RU486 during the oxidation step generated few fluorescent products (line 2). In contrast, the addition of RU486 to LDL that was subject to 3 hours oxidation failed to show an increase in the formation of fluorescence (line 3) despite complete oxidation as determined by the formation of conjugated dienes. RU486 added after 3 hours, however, did not inhibit the decomposition of lipid peroxides into aldehydes as seen in Figure 3 . In fact, a marginal but insignificant increase could be observed, indicating that RU 486 or products derived from RU486 also might have contributed to the formation of thiobarbituric acid reactive substances. These results provide additional evidence that RU486 not only can prevent the formation of lipid peroxide, but also can block the formation offluorescent protein adducts in the presence of pre-existing lipid peroxides. It is possible that the presence of RU486 may have interfered with the measurement of fluorescence. However, the addition of RU486 to an already modified protein had no effect on the measurement of fluorescence. Furthermore, as seen in Figure 4, on Western blot analysis the antibody to modified proteins readily recognized serum albumin that was modified by lipid peroxides in the absence of RU486. However, the presence of RU486 during the modification process failed to generate a fluorescent epitope as determined by very weak presence of the antigenic epitope.

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Figure 3 Formation of thiobarbituric acid reactive substances during the oxidation of LDL in the presence and absence of RU486. The experimental setup was the same as in Figure 2A. 1, LDL only incubated for 24 hours, 2, LDL incubated with copper, 3, LDL incubated with copper in the presence of 10 J.!M RU486, 4, LDL incubated with copper and RU486 added after 3 hours of incubation. Results presented are averages of duplicate sets of experiments. P < 0.05 for 2 and 3; P < 0.001 for 3 and 4.

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Figure 4 Western blot analysis of serum albumin subjected to modification in the presence and absence ofRU486. Proteins were separated by electrophoresis, transferred on to nitrocellulose membranes, and probed with rabbit polyclonal antibody to oxidatively modified protein (rabbit serum albumin). Lane 1, albumin incubated with lipid peroxides; lane 2, albumin incubated with lipid peroxides in the presence of 10 /LM RU486; lane 3, albumin incubated with lipid peroxides in the presence of 2.5 /LM RU486.

DISCUSSION

Currently, it is believed that the physiological and pharmacologic effects of RU486 are dependent on the presence of P or glucocorticoid receptors (1, 2, 5-7). We recently have reported that RU486 is a potent antioxidant and prevents the oxidation of LDL in vitro (8). Its antioxidant actions, however, do not depend on the presence of hormone receptors. The inhibitory activities of RU486 can be attributed to the presence of dimethyl amino phenyl side chain of the molecule. Significantly, this inhibition was noted at micromolar concentrations that are physiologically observed in subjects treated with RU486 (2, 8). In fact, patients treated with 5 mg RU486 daily had LDL that was resistant to in vitro oxidation (8). Other antiprogestin analogues that do not have this moiety and levonorgestrel, a progestin, do not have antioxidant activity. This paper furthers our studies on the antioxidant properties ofRU486 by exploring its ability to inhibit the production of oxidatively modified protein resulting from the reaction of pre-existing lipid peroxides with E-amino groups of lysine residues of proteins. We recently have described this novel reaction and proposed a mechanism for it (9). These modified proteins are characterized by fluorescent epitopes that are antigenic and against which we have developed an antibody (14). Its characteristic fluorescence and our ability to detect these oxidatively modified proteins forms the basis for this study. This paper provides evidence that RU 486 is a potent inhibitor of such reactions and thus not only can prevent the formation of lipid peroxide, but also can block the Vol. 66, No.1, July 1996

generation of an oxidatively modified protein in the presence of pre-existing lipid peroxides. Concentrations of RU486 used in this study (2.5 to 10 J.lM) correlate well with amounts of RU486 that have been noted in the plasma of subjects who have been given daily doses of RU486 (2,8). Recent evidence suggests that RU486 may have effects independent of the presence of the receptor (15). These studies suggest that some of the agonistlike transcriptional effects may be seen without binding to P response elements. And we have shown that RU486 has antioxidant activity in cells that do not possess Preceptors (8). The ability of RU486 to be lipophilic and at the same time cross cell membranes may provide an important tool to regulate the redox cycle of cells. Redox regulation has been suggested to affect a number of genes, including those coding for vascular cell adhesion molecule and monocyte chemotactic protein. Such regulation may playa major role in programmed cell death (1620). The transportation of RU486 across the nuclear membrane by specific receptors also may provide an important tool for preventing lipid peroxide-mediated DNA damage, thus providing vital control of cell functions. The intracellular effects ofRU486 currently are under investigation. For example, RU486, by acting as an antioxidant, should decrease the expression of genes that are regulated by oxidative stress. The gene for monocyte chemotactic protein1, which determines the chemotactic recruitment of monocytes into the uterine tissue, may be one of the relevant genes that may be subject to regulation by RU486. Acknowledgments. We thank Roussel-Uelaf (Romainville, France) for the generous supply of RU486. We thank Ms. Toni Edwards for typing the manuscript.

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