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Originally published In Press as doi:10.1074/jbc.M203895200 on July 16, 2002

J. Biol. Chem., Vol. 277, Issue 39, 36204-36215, September 27, 2002
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Tyrphostins Protect Neuronal Cells from Oxidative Stress*

Yutaka SagaraDagger §, Kumiko Ishige, Cindy TsaiDagger , and Pamela Maher||

From the Dagger  Department of Neurosciences, University of California, San Diego, La Jolla, California 92093-0624,  Department of Pharmacology, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba-ken 274-8555, Japan, and || Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037-1000

Received for publication, April 22, 2002, and in revised form, July 10, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tyrphostins are a family of tyrosine kinase inhibitors originally synthesized as potential anticarcinogenic compounds. Because tyrphostins have chemical structures similar to those of the phenolic antioxidants, we decided to test the protective efficacy of tyrphostins against oxidative stress-induced nerve cell death (oxytosis). Many commercially available tyrphostins, at concentrations ranging from 0.5 to 200 µM, protect both HT-22 hippocampal cells and rat primary neurons from oxytosis brought about by treatment with glutamate, as well as by treatment with homocysteic acid and buthionine sulfoximine. The tyrphostins protect nerve cells by three distinct mechanisms. Some tyrphostins, such as A25, act as antioxidants and eliminate the reactive oxygen species that accumulate as a result of glutamate treatment. These tyrphostins also protect cells from hydrogen peroxide and act as antioxidants in an in vitro assay. In contrast, tyrphostins A9 and AG126 act as mitochondrial uncouplers, collapsing the mitochondrial membrane potential and thereby reducing the generation of reactive oxygen species from mitochondria during glutamate toxicity. Finally, the third group of tyrphostins does not appear to be effective as antioxidants but rather protects cells by increasing the basal level of cellular glutathione. Therefore, the effects of tyrphostins on cells are not limited to their ability to inhibit tyrosine kinases.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxygen may induce many human diseases despite its importance in survival (1). For example, atherosclerosis (2), liver diseases (3), inflammatory-immune injuries (4, 5), and some eye diseases (6) may be initiated or exacerbated by oxygen and its derivatives. Neuronal death observed in ischemia and stroke (7), Alzheimer's disease (8, 9), Parkinson's disease (10, 11), and amyotrophic lateral sclerosis (12) may also result from oxidative stress. Oxidative stress arises from an excessive production or decreased clearance of reactive oxygen species (ROS),1 resulting in accumulation of ROS, cellular damage, and eventual cell death. Sources of ROS in cells include oxygenases, oxidases, and nitric-oxide synthetases (13). Mitochondria also produce a significant level of ROS during normal respiration as well as during cell death (13, 14). Molecules in the cell that can eliminate ROS include catalase and glutathione peroxidases and the nonenzymic, small antioxidants glutathione (GSH), vitamin C, and vitamin E (13).

Molecular and cellular events underlying neuronal death induced by oxidative stress have been characterized in the hippocampal cell line HT-22 and cortical neurons (15-18). This pathway of programmed cell death, termed oxytosis (19), is initiated by the addition of glutamate to the extracellular medium. Glutamate then inhibits the uptake of cystine, which is required for GSH synthesis (20), resulting in the depletion of GSH in neurons (15, 21-23). Subsequently, this decrease in cellular GSH results in the production of ROS by mitochondria. The production and accumulation of ROS proceed in two phases: an initial slow increase up to 5- to 10-fold of the control level, followed by an explosive generation up to 200 times greater than the control level. The higher rate of ROS increase begins after the cellular GSH levels fall below 20% of the control (18). The importance of mitochondria in ROS production is supported by the observation that the mitochondrial uncoupler cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and other mitochondrial inhibitors protect neuronal cells from glutamate toxicity (18). The ROS accumulation then causes Ca2+ influx from the extracellular medium, which leads to cell death. The central role of ROS in this cell death cascade is evidenced by the observation that exogenously added antioxidants such as vitamin E and propyl gallate (PG) (Fig. 1) protect cells from glutamate toxicity (15, 18, 21, 24). In addition to the elucidation of the steps involved in oxytosis, HT-22 cells have been used to screen potentially therapeutic drugs for the treatment of clinical conditions involving oxidative stress (24-28).

The tyrphostins are a family of structurally related phenolic compounds that inhibit protein tyrosine kinases (PTKs) (29, 30). Because the elevated activities of specific PTKs may cause cancers and other proliferative disorders, Levitzki and his co-workers (29, 31) synthesized a variety of tyrphostins from tyrosine and erbstatin. Thus, some tyrphostins inhibit specific PTKs such as the epidermal growth factor receptor without affecting other PTKs such as the insulin receptor (32). Because of their selective inhibition and low toxicity (33), tyrphostins may have therapeutic value for the treatment of pathological conditions caused by the excessive activity of specific PTKs (34). However, some tyrphostins appear to affect not only PTKs but also other enzymes and biological functions independent of their effects on PTKs. For example, tyrphostins can affect phosphodiesterases (35, 36), calcium entry in Jurkat T cells (37), and mitochondrial membrane potential in HL-60 cells (38) independently of their effects on PTKs.

Tyrphostins generally have a phenolic structure that closely resembles that of antioxidants such as vitamin E and PG (Fig. 1). Because these latter compounds protect HT-22 cells from oxidative glutamate toxicity, we decided to test the protective efficacy of tyrphostins in this system. We report here that many tyrphostins protect the nerve cell line HT-22, as well as rat primary neurons from cell death induced by oxidative glutamate toxicity. Additionally, tyrphostins protect nerve cells from oxidative injury caused by homocysteic acid and buthionine sulfoximine. The protection by tyrphostins is independent of their ability to inhibit tyrosine kinases. Instead, three distinct mechanisms appear to be involved in the protection of the nerve cells from oxidative stress-induced cell death. First, some tyrphostins (A23 and A25, for example) protect the nerve cells by acting as antioxidants. This conclusion is derived from the ability of these tyrphostins to quench free radicals both in a cell-free system and directly in nerve cells that have accumulated high levels of ROS. Second, many tyrphostins induce gamma -glutamylcysteine synthetase, the rate-limiting enzyme in GSH biosynthesis, resulting in an increased basal GSH level and protection of the cells. Third, tyrphostins A9 and AG126 protect cells from glutamate toxicity by their ability to decrease the mitochondrial membrane potential, which results in a decrease in ROS production after glutamate treatment. Thus, many tyrphostins possess additional, important biological activities that are independent of their ability to inhibit PTKs.


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  		Fig. 1.   Chemical structures of tyrphostins and other phenolic compounds used in this study. The position of hydroxyl groups and alternative designations are listed in Table I.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- Tyrphostins were purchased from Alexis (San Diego, CA) or Calbiochem. Because of stability issues (39, 40), stock solutions of tyrphostins were prepared in 100% Me2SO and kept in the dark at -20 °C. 2',7'-Dichlorodihydrofluorescein (H2DCF)-diacetate, indo-1, Pluronic 127, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolcarbocyanine iodide (JC-1), 4',6-diamidino-2-phenylindole, and rhodamine-123 were purchased from Molecular Probes (Eugene, OR). All other chemicals were from Sigma.

Cell Culture-- Fetal bovine serum (FBS) and dialyzed FBS were from Hyclone (Irvine, CA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Invitrogen. HT-22 cells (15, 16) were derived from the immortalized mouse hippocampal cell line HT-4 (41) and grown on tissue culture dishes (Falcon, Indianapolis, IN) in DMEM supplemented with 10% FBS. Pancreatin (Invitrogen) was used to dissociate the cells from the culture dishes. Short-term cultures of primary cortical neurons from 17-day-old rat embryos were prepared according to Abe and Kimura (42). The primary cells were used for experiments within 3 days after plating and do not express functional ionotropic glutamate receptors.

Cytotoxicity Assay-- Cell viability was determined by a modified version of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (43, 44) based on the standard procedure (45). Briefly, cells were seeded onto 96-well microtiter plates at a density of 2.0 × 103 cells/well in 100 µl of 10% dialyzed FBS. The next day, cells were treated with various protective reagents for 30 min before the addition of a toxic agent (glutamate, HCA, and BSO). 24 h after the addition of a toxic agent, the cell culture medium in each well was aspirated and replaced with fresh 10% dialyzed FBS containing 2.5 µg/ml MTT. After 4 h of incubation at 37 °C, cells were solubilized with 100 µl of a solution containing 50% dimethylformamide and 20% SDS (pH 4.7). The absorbance at 560 nm was measured on the following day with a microplate reader (ICN Flow Titertec Multiskan PLUS-Mk 11). Results obtained from the MTT assay correlated directly with the extent of cell death as confirmed visually (see, for example, Fig. 2) as well as the lactate dehydrogenase release assay (data not shown).

For hydrogen peroxide (H2O2) toxicity, cells were preincubated with a tyrphostin for 30 min before the addition of H2O2. 2 h later, the cell culture medium was aspirated and replaced with fresh medium. The next day, cell viability was assessed by the MTT assay as described above.

Microscopy-- A light microscope (Inverted Microscope Diaphot-TMD; Nikon) equipped with a phase-contrast condenser (Phase contrast-2 ELWD 0.3; Nikon), 10× objective lens, and a digital camera (Coolpix 990; Nikon) was used to capture the images with the manual setting.

Total Intracellular GSH-- Cells were washed twice with ice-cold phosphate-buffered saline, collected by scraping, and lysed with 3% sulfosalicylic acid. Lysates were incubated on ice for 10 min, and supernatants were collected after centrifugation in an Eppendorf microfuge. Upon neutralization of the supernatant with triethanolamine, the concentration of total glutathione (reduced and oxidized) was determined by the method of Tietze (46) with modifications (24). Briefly, a neutralized supernatant from above (25 µl) was mixed with 175 µl of a reaction mixture containing 143 mM sodium phosphate (pH 7.5), 6.3 mM Na4EDTA, 6 mM 5,5'-dithiobis(2-nitrobenzoic acid), and 0.25 mg/ml NADPH. The reaction was started by adding 1 unit/ml glutathione reductase. Color development was monitored at 405 nm in a kinetic mode with a microplate reader (ICN Flow Titertec Multiskan PLUS-Mk 11). Pure GSH was used to obtain a standard curve. The protein content of each sample was determined using the BCA protein assay kit from Pierce with bovine serum albumin as a standard.

ROS Level-- The intracellular accumulation of ROS in HT-22 cells was determined with H2DCF-diacetate (44). This nonfluorescent compound accumulates within cells upon deacetylation. H2DCF then reacts with ROS to form fluorescent dichlorofluorescein (DCF) (47). HT-22 cells were dissociated from tissue culture dishes with pancreatin in DMEM in the presence of 10 µM H2DCF-diacetate for 10 min at 37 °C, washed once with room temperature DMEM (without phenol red) supplemented with 2% dialyzed FBS, and resuspended in 750 µl of the same solution containing 2 µg/ml propidium iodide. The use of pancreatin did not affect the outcome of the flow cytometric experiments as confirmed by fluorescence microscopy. Flow cytometric analysis was performed using a FACScan instrument (BD PharMingen) with an excitation wavelength (lambda ex) of 475 nm and an emission wavelength (lambda em) of 525 nm. Data were collected in list mode on 10,000 cells after gating only for characteristic forward versus orthogonal light scatter and low propidium iodide fluorescence to exclude dead cells. Median fluorescence intensities of control and test samples were determined with CellQuestTM software (BD PharMingen).

Determination of Mitochondrial Membrane Potential-- JC-1 was used to determine the mitochondrial membrane potential (44, 48). This lipophilic cation dye aggregates reversibly in mitochondria due to the membrane potential, resulting in a fluorescence shift from green (527 nm of the monomer) to orange (590 nm of the aggregate) (49). Cells were washed once with DMEM supplemented with 10% FCS to remove test compounds before loading with 5 µg/ml JC-1 for 20 min at 37 °C. The cells were then dissociated with 1× trypsin/EDTA, centrifuged, and washed once with DMEM lacking phenol red. Cell pellets were resuspended in DMEM without phenol red, and 4',6-diamidino-2-phenylindole (2 µg/ml) was added to the samples to allow the gating out of dead cells. Flow cytometric analysis was performed using a LSR three-laser six-color analytic flow cytometer (BD PharMingen). lambda ex = 475 nm, and lambda em = 530 nm (the monomer JC-1), and lambda em = 585 nm (the aggregate JC-1); lambda ex = 345 nm, and lambda em = 455 nm for 4',6-diamidino-2-phenylindole.

Determination of the Trolox Equivalent Activity Concentration (TEAC)-- Values of TEAC for tyrphostins and other compounds were determined according to Rice-Evans and Miller (24, 50). Briefly, 150 µM 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonate), 2.5 µM metomyoglobin, and 75 µM H2O2 were mixed in Dulbecco's phosphate-buffered saline solution. Then, the change in absorbance due to the formation of the 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) free radical was measured at 734 nm for 7.5 min. The inhibition of this free radical formation by the inclusion of 1.0 mM Trolox in the assay solution was used as a basis for the determination of TEAC values for tyrphostins and other compounds.

Tyrosine Kinase Assay-- Protein tyrosine kinase activity was measured on extracts from untreated and tyrphostin-treated HT-22 cells exactly as described previously (51). Briefly, cells on 60-mm dishes were scraped into 500 µl of 50 mM Tris-HCl (pH 7.4) containing 2 mM MgCl2, 1 mM EDTA, 1 mM Na2VO4, and 1 mM phenylmethylsulfonyl fluoride and lysed by sonication. After centrifugation for 10 min at 16,000 × g at 4 °C, 20 µl of each supernatant were assayed for protein tyrosine kinase activity in a total volume of 50 µl containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 10 mM MnCl2, 50 µM Na2VO4, 50 µM ATP (Sigma), 2 µCi of [gamma -32P]ATP (10 mCi/ml; ICN), and 1 mg/ml poly(Glu:Tyr; 4:1) (MWviscosity 45,700; Sigma). The assay was carried out for 20 min at 30 °C and stopped by the addition of 15 µl of 5× SDS sample buffer and boiling for 5 min. 30 µl of each reaction were separated on a 10% SDS-polyacrylamide gel. The gels were stained with 1% Coomassie Blue, destained, dried, and autoradiographed overnight. To quantify the amount of 32P incorporated into the poly amino acid substrate, the section of each lane containing the radiophosphorylated substrate was cut out and counted in a scintillation counter (Cerenkov counts). Control reactions containing the cell extracts but no substrate were run in parallel, and the counts from these lanes were subtracted from those of lanes containing the substrate. The results were normalized to the amount of protein in each extract as determined using the BCA protein assay. Similar results were obtained when overall protein tyrosine phosphorylation was measured using immunoblotting with anti-phosphotyrosine antibodies (data not shown).

Immunoblot-- For Western blotting, cells from subconfluent cultures were washed twice in cold phosphate-buffered saline and then scraped into lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 50 mM NaF, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 15 µg/ml aprotinin, 1 µg/ml pepstatin, and 5 µg/ml leupeptin. Lysates were incubated at 4 °C for 30 min and then cleared by centrifugation at 14,000 rpm for 10 min. Protein concentrations were determined using the BCA protein assay (Pierce). Equal amounts of protein were solubilized in 2.5× SDS sample buffer, separated on 10% SDS-polyacrylamide gels, and transferred to nitrocellulose. Transfers were blocked for 2 h at room temperature with 5% nonfat milk in Tris-buffered saline/0.1% Tween 20 and then incubated overnight at 4 °C in the primary antibody diluted in 5% bovine serum albumin in Tris-buffered saline/0.05% Tween 20. The primary antibodies used were anti-catalytic subunit of gamma -GCS (from Dr. H. J. Forman) and anti-actin. The transfers were rinsed with Tris-buffered saline/0.05% Tween 20 and incubated for 1 h at room temperature in horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibody (Bio-Rad) diluted 1:5000 in 5% nonfat milk in Tris-buffered saline/0.1% Tween 20. The immunoblots were developed with the Super Signal reagent (Pierce).

Statistical Analysis-- Experiments presented were repeated at least three times with triplicate samples. The data are presented as means ± S.E.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protection from Glutamate Toxicity-- Glutamate kills the hippocampal cell line HT-22 by a mechanism that involves the accumulation of ROS (15, 18). This system is useful for screening chemical compounds that are protective against glutamate toxicity and other oxidative insults (15, 24, 26). As shown in Fig. 2, A and D, HT-22 cells are killed by glutamate within 24 h. To determine whether tyrphostins can protect the cells from glutamate toxicity, HT-22 cells were exposed to 5 mM glutamate for 24 h in the presence of 10 µM tyrphostin A25 or 1 µM tyrphostin A9, and cell viability was examined by light microscopy. The addition of tyrphostin A25 (Fig. 2, B and E) or tyrphostin A9 (Fig. 2, C and F) strongly protects the HT-22 cells from glutamate toxicity. Protection by these tyrphostins is not transient; the nerve cells are viable in toxic doses of glutamate for at least 3 days if a protective tyrphostin is continuously present (data not shown).


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  		Fig. 2.   The cytotoxic response of HT-22 cells to glutamate and protection by tyrphostins. HT-22 cells were plated (2.0 × 105 cells/60-mm dish) in 4 ml of DMEM containing 10% FBS. 16 h later, the cells were exposed to either no glutamate (A-C) or 5 mM glutamate (D-F) in the presence of no tyrphostins (A and D), 10 µM tyrphostin A25 (B and E), or 1 µM tyrphostin A9 (C and F). 24 h later, the cells were examined and photographed by phase-contrast microscopy.

To quantify the protection provided by the different tyrphostins, HT-22 cells were exposed to various concentrations of glutamate in the presence of each tyrphostin for 24 h, and then cell viability was assessed by the MTT assay (43). The half-maximal concentration for glutamate toxicity is 2.0 mM, whereas 5 mM glutamate causes 90-100% of the cells to die (Fig. 3A). The inclusion of 10 µM A25 or 1 µM A9 in the assay completely protects the HT-22 cells from death, even in the presence of 10 mM glutamate. Similar results were obtained when the lactate dehydrogenase release assay was used to determine cell viability (data not shown). Tyrphostins A25 and A9, as well as A23, are also effective in protecting rat primary cortical neurons from oxidative glutamate toxicity (Fig. 3B).


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  		Fig. 3.   The cytotoxic response of nerve cells to glutamate and protection by tyrphostins. A, exponentially dividing HT-22 cells were dissociated with pancreatin and plated into 96-well microtiter plates at 2000 cells/well in 100 µl of DMEM with 10% FBS. 12 h later, cells were exposed to the indicated concentrations of glutamate in the presence of no tyrphostin (open circle ), 10 µM tyrphostin A25 (), 1 µM tyrphostin A9 (black-triangle), or 10 µM AG10 (black-down-triangle ) for 24 h, and cell viability was assessed by the MTT assay as described under "Experimental Procedures." B, rat primary cortical neurons were exposed to the indicated concentrations of glutamate in the presence of no tyrphostin (open circle ), 10 µM tyrphostin A25 (), 0.5 µM tyrphostin A9 (black-triangle), or 10 µM tyrphostin A23 (black-square) for 24 h, and cell viability was determined as described above. C, the viability of HT-22 cells was measured as described above in the presence of 5 mM glutamate and the indicated concentrations of tyrphostin A25 (), tyrphostin A9 (black-triangle), or AG10 (black-down-triangle ).

Whereas some tyrphostins (A9, A23, and A25) were very effective in protecting the HT-22 cells from oxidative glutamate toxicity, other tyrphostins were either less effective or completely ineffective. To directly compare the protective efficacy of the different tyrphostins, HT-22 cells were exposed to 5 mM glutamate in the presence of various concentrations of each tyrphostin, and after 24 h, cell viability was determined by the MTT assay (Fig. 3C). Tyrphostin A25 has an effective half-maximal concentration for protection (EC50) of 6 µM, and maximal protection is observed with concentrations higher than 10 µM (Fig. 3C). Tyrphostin A9 is more effective than A25; the EC50 for A9 is 0.2 µM, and maximal protection is achieved at 1 µM (Fig. 3C). Among the less effective tyrphostins are A1, which is commonly used as a negative control for the tyrosine kinase inhibitory activity of other tyrphostins (29), and AG10 (Fig. 3, A and C), both of which have effective half-maximal concentrations for protection 8- to 15-fold higher than that for A25. Other tyrphostins, such as RG13022 and RG14620, are completely ineffective at protecting cells from oxidative stress (Table I). These results suggest certain structural requirements for protection from oxidative stress and will be addressed further in the "Discussion."

                              
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Table I
Protective efficacy of various tyrphostins against pro-oxidants
HT-22 cells were exposed to various concentrations of a tyrphostin to determine the half-maximal lethal dose LD50 (µM) using the MTT assay. Half-maximal effective concentrations (EC50) were determined by exposing HT-22 cells to 5 mM glutamate, 2 mM HCA, 50 µM BSO, or 50 µM H2O2. See Figs. 3, 4, and 8. TEAC values were determined as described under "Experimental Procedures" in two triplicate experiments and values are the mean ± SE. Values for half-maximal inhibitory concentration (IC50) for platelet-derived growth factor kinase (PDGFR-K) and epidermal growth factor receptor kinase (EGFR-K) were obtained from Refs. 29, 30, and 34.

Protection from HCA and BSO-- Glutamate kills HT-22 and other nerve cells by depleting cellular GSH (15). To determine whether the protection by tyrphostins extends to other agents that deplete cellular GSH, the protective efficacy of tyrphostins was determined in the presence of HCA or BSO. HCA blocks the system X<UP><SUB>c</SUB><SUP>−</SUP></UP> similarly to glutamate, resulting in a decreased cellular uptake of cystine and a reduction in GSH biosynthesis (20). HT-22 cells were incubated with HCA in the presence of various concentrations of a tyrphostin, and cell viability was determined as described before. In the presence of 2 mM HCA, cell viability decreases to 10% (Fig. 4A). Tyrphostins A9, A23, and A25 protect HT-22 cells from HCA, even though higher concentrations are required for total protection from HCA than from glutamate (Fig. 4A; Table I). This trend is particularly pronounced in tyrphostins that are only moderately efficacious against glutamate (Table I). For example, tyrphostin AG10 is only weakly protective against HCA (Fig. 4A), but it is not possible to achieve greater levels of protection due to the toxicity of the compound at higher concentrations. Therefore, the EC50 values for tyrphostins A1 and AG10 against HCA toxicity were not determined (Table I).


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  		Fig. 4.   Protection from HCA and BSO. A, HT-22 cells were plated into 96-well microtiter plates as described in the Fig. 3 legend and exposed 12 h later to 2 mM HCA in the presence of the indicated concentrations of tyrphostins. B, HT-22 cells prepared as described in A were exposed to either 50 or 5 µM BSO in the presence of the indicated tyrphostins. Cell viability was determined 24 h later by the MTT assay.

Unlike HCA, BSO decreases cellular GSH by inhibiting the GSH synthetic enzyme gamma -GCS (52). As shown in Fig. 4B, viability decreases to 7% when cells are treated with 50 µM BSO. The protection profile of many tyrphostins against BSO is similar to that against HCA. For instance, tyrphostins A23 and A25 protect HT-22 cells from BSO at concentrations that are higher than those required for protection against glutamate toxicity (Fig. 4B; Table I). In addition, tyrphostins A1, AG10, and AG1295 are not effective against BSO toxicity (Fig. 4B; Table I). However, tyrphostins A9, AG126, and AG1288 behave very differently in BSO toxicity as compared with HCA toxicity. In the presence of 5 µM BSO, cell viability is 80%, which is reduced to 3% if 0.5 µM tyrphostin A9 is added (Fig. 4B). AG126 also potentiates BSO toxicity when added at concentrations of 80 µM or higher (data not shown). AG1288 neither protects nor potentiates BSO toxicity at concentrations up to 200 µM (data not shown). In summary, some tyrphostins protect HT-22 cells to varying degrees from reagents that decrease cellular GSH. Tyrphostins exemplified by A25 protect HT-22 cells from glutamate, HCA, and BSO, all of which decrease cellular GSH. Another class of tyrphostins (tyrphostins A1, AG10, and AG1295) has a low efficacy against glutamate and HCA and is ineffective against BSO. Finally, tyrphostins A9, AG126, and AG1288 protect cells from glutamate and HCA in the low micromolar range, but both tyrphostins A9 and AG126 potentiate BSO toxicity, whereas tyrphostin AG1288 is not protective against BSO.

Inhibition of Tyrosine Kinase Activity-- To characterize further the mechanism of protection from oxytosis by tyrphostins, we initially focused on the fact that tyrphostins were originally designed to inhibit tyrosine kinases (30). Thus, the mechanism of cellular protection by tyrphostins could be attributable to their ability to inhibit a tyrosine kinase that is required for the progression of the cell death cascade initiated by glutamate. Inhibition of such a tyrosine kinase would stop the cell death cascade, resulting in protection. Inhibition of known tyrosine kinases such as the platelet-derived growth factor receptor kinase or the epidermal growth factor receptor kinase is not involved in the protection by the tyrphostins. This conclusion is based on a comparison between the protective efficacy of specific tyrphostins against oxidative stress in the HT-22 cells and the reported efficacy of inhibition of the tyrosine kinase activity of these growth factor receptors by the same tyrphostins. For example, the platelet-derived growth factor receptor tyrosine kinase is inhibited equally by tyrphostins A9 and AG1295 with an IC50 of 0.5 µM (30, 33), but AG1295 does not protect the HT-22 cells from oxidative stress, whereas tyrphostin A9 has an EC50 of 0.5 µM (Table I). As the first step to determine whether tyrosine kinase inhibition is important in the protection of cells from glutamate toxicity by the tyrphostins, inhibition of total tyrosine kinase activity was measured in cells treated with the tyrphostins (Fig. 5). There is no correlation between the efficacy of protection against glutamate toxicity and the inhibition of total tyrosine kinase activity. For example, total tyrosine kinase activity is inhibited to a similar level by 10 µM A25, 0.5 µM A9, 10 µM RG13022, and 100 µM AG1295. Whereas the former two tyrphostins protect HT-22 cells from glutamate toxicity at these concentrations, AG1295 is protective only at high concentrations, and RG13022 is not protective at any concentration tested (Table I). Furthermore, genistein, a general tyrosine kinase inhibitor (53), decreases the total protein tyrosine kinase activity to 34.1% and 24% of the control level when present at 100 and 300 µM, respectively. Genistein at 100 µM does not protect against glutamate toxicity, but in the presence of 300 µM genistein, 67% of the cells survive. These data do not eliminate unequivocally the possibility that tyrphostins protect HT-22 cells by inhibiting a tyrosine kinase whose activity may be a minor component of the total tyrosine kinase activity present in the cell-free system used here. However, the fact that many tyrphostins with various specificities to PTKs protect neuronal cells from pro-oxidants argues against a protective mechanism involving the inhibition of a specific tyrosine kinase. Also, when considered along with the results described below, protection by tyrphostins appears unlikely to involve the inhibition of a tyrosine kinase.


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  		Fig. 5.   Effects of tyrphostins on total protein tyrosine kinase activity. HT-22 cells were untreated (Control) or treated with the indicated tyrphostins for 1 h, and then cell lysates were prepared. The lysates were assayed for tyrosine kinase activity as described under "Experimental Procedures" using poly(Glu:Tyr; 4:1) as a substrate. The results presented are from a single experiment. Similar results were obtained in three independent experiments.

GSH Metabolism-- Glutamate and HCA inhibit cystine uptake in HT-22 cells, resulting in the total loss of the cellular GSH within 8 h (15, 18). Some compounds that protect HT-22 cells from these toxic agents do so by increasing the basal level of cellular GSH (24, 26). To determine whether any of the protective tyrphostins alter the basal metabolism of GSH, HT-22 cells were treated with each tyrphostin for 17 h and harvested, and total GSH was determined. As shown in Fig. 6A, tyrphostins A1 and A25 increase the basal levels of cellular GSH, whereas tyrphostin A9 decreases cellular GSH to 50-70% of the level in the untreated cells. The mitochondrial uncoupler FCCP also decreases cellular GSH similarly to tyrphostin A9 (Fig. 6A). To determine whether the increase in the basal GSH levels is attributable to an increased level of the GSH synthetic enzyme gamma -GCS (54), HT-22 cells were treated for 17 h with tyrphostins and then harvested for immunoblotting. When HT-22 cells are treated with the flavonoid quercetin, the basal GSH level is increased along with the increased level of gamma -GCS protein (Fig. 6A, inset, lane 2) (24). A similar increase in gamma -GCS protein is observed in cells treated with tyrphostins A25 and A1 (Fig. 6A, inset, lanes 3-5), whereas there was no significant change in gamma -GCS protein level in cells treated with tyrphostin A9 (Fig. 6A, inset, lane 6). Other protective tyrphostins also affect the basal levels of cellular GSH (Table II). Higher concentrations of tyrphostins A1, AG10, and A25 progressively increase the cellular GSH levels, whereas tyrphostin A9 further decreases the cellular GSH levels at higher concentrations (Fig. 6B). The changes in GSH levels are observed within 8 h of tyrphostin addition (Fig. 6C).


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  		Fig. 6.   Effects of tyrphostins on cellular levels of GSH. Exponentially dividing HT-22 cells were dissociated with pancreatin and plated into 60-mm dishes at the density of 3 × 105 cells/ml in 4 ml of DMEM with 10% FBS. 14 h later, the following experiments were performed. A, HT-22 cells were treated with the indicated tyrphostins, and cellular levels of total GSH were measured as described under "Experimental Procedures." The GSH level of the control sample (36.5 ± 2 nmol/mg protein) was taken as 100%. Inset, HT-22 cells (4 × 105) in 60-mm dishes were treated with 10 µM quercetin (lane 2), 10 µM tyrphostin A25 (lane 3), 25 µM A25 (lane 4), 50 µM A1 (lane 5), or 0.5 µM A9 (lane 6) for 17 h. Cells were lysed in the SDS sample buffer and probed with antibodies to the catalytic subunit of gamma -GCS (G) or actin (A) by using immunoblotting as described under "Experimental Procedures." Lane 1, lysates from untreated cells. B, HT-22 cells were treated with tyrphostin A25 (), A9 (black-triangle), AG10 (black-down-triangle ), or A1 (black-square), and total GSH was determined 17 h later. C, HT-22 cells were treated with tyrphostin A25 (), A9 (black-triangle), or A1 (black-square), and samples were collected for the GSH assay every 2 h after the addition. Control samples in the absence of tyrphostins (open circle ) were also collected at the indicated time points. D, HT-22 cells were treated with 2 mM glutamate alone (open circle ), 2 mM glutamate and tyrphostin A25 (), A9 (black-triangle), or A1 (black-square), and samples were collected for the GSH assay every 2 h after the addition of 2 mM glutamate. All GSH results are the means of triplicate determinations ± S.E. Similar results were obtained from at least two independent experiments.

                              
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Table II
Effects of tyrphostin on cellular GSH
HT-22 cells (3 × 105 cells) in 60-mm dishes were treated with the indicated concentrations of tyrphostins for 17 h, and cells were processed for GSH determination in triplicate samples as described under "Experimental Procedures." Similar results were obtained in at least two independent experiments.

To determine whether the up-regulation of basal GSH induced by tyrphostins is also observed during glutamate toxicity, cells were treated with a toxic dose of glutamate in the presence of protective tyrphostins for 24 h, and total GSH was determined. The cells treated with glutamate for 24 h lose all of their GSH (Fig. 6A) (15). In contrast, the cells maintain 30-60% of the GSH level of untreated cells if protective tyrphostins are included at the beginning of the incubation with glutamate (Fig. 6A). Even tyrphostin A9 and FCCP, which decrease the basal level of GSH, maintain the cellular GSH level during glutamate treatment (Fig. 6A). To see whether the rate of GSH decrease is also affected by the protective tyrphostins during glutamate toxicity, HT-22 cells were harvested for the determination of total cellular GSH every 2 h after the addition of glutamate and tyrphostins (Fig. 6D). The cells lose 90% of their GSH within 10 h of glutamate addition (15, 18). In contrast, cells treated with protective tyrphostins have slower rates of GSH loss. Even cells treated with tyrphostin A9 and FCCP, both of which decrease the basal level of GSH (Fig. 6A), show a decrease in the rate of GSH loss caused by glutamate (Fig. 6D). Therefore, protective tyrphostins increase basal GSH and/or maintain cellular GSH levels in the presence of glutamate, which likely contributes to cellular protection.

ROS Levels-- When more than 80% of the cellular GSH is depleted after glutamate treatment, mitochondria produce ROS, which accumulate to levels up to 100-fold above control values (18). The critical role of ROS accumulation in the cell death cascade is shown by the fact that antioxidants such as vitamin E and PG protect HT-22 cells from glutamate despite the loss of cellular GSH (15, 24). Because tyrphostins contain a phenolic functional group similar to other antioxidants (Fig. 1), tyrphostins could block ROS production during oxytosis. HT-22 cells were treated with a toxic dose of glutamate in the presence of a tyrphostin for 12 h. Then, the cells were loaded with the fluorescent probe DCF, and the level of ROS accumulation was measured by flow cytometry (15, 18). The mean DCF fluorescence increases dramatically in the HT-22 cells treated with glutamate (Fig. 7A, compare Control with Glu). If the antioxidant PG is added to the cells along with glutamate, the DCF fluorescence level after 12 h is greatly decreased (Fig. 7A, PG + Glu) (15, 24). Tyrphostin A25 prevents the increase in the DCF fluorescence caused by glutamate treatment similarly to PG (Fig. 7B, compare A25 + Glu to Glu). All other protective tyrphostins such as A1 (Fig. 7C), A9 (Fig. 7D), and A23 (data not shown) also prevented the increase in DCF fluorescence caused by glutamate treatment if these tyrphostins were present at the start of the treatment.


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  		Fig. 7.   Effects of tyrphostins on ROS levels. HT-22 cells were treated with 5 mM glutamate in the presence or absence of the indicated tyrphostins for 11 h. Then, the cells were loaded with H2DCF-diacetate and processed as described under "Experimental Procedures." The levels of ROS were measured with DCF fluorescence, and the intensity of DCF fluorescence was plotted with respect to the counts in each channel. A, HT-22 cells were untreated (Control) or treated either with 10 µM PG alone (PG) or with 5 mM glutamate for 12 h in the absence (Glu) or presence of 10 µM PG (PG + Glu). In another set of samples, HT-22 cells were treated with 5 mM glutamate for 11 h, and then 10 µM PG was added for 1 h (12hr Glu + PG). B, HT-22 cells were treated either with 10 µM tyrphostin A25 alone (Tyr A25) or with 5 mM glutamate for 12 h in the absence (Glu) or presence of 10 µM tyrphostin A25 (A25 + Glu). In another set of samples, HT-22 cells were treated with 5 mM glutamate for 11 h, and then 10 µM tyrphostin A25 was added for 1 h (12hr Glu + A25). C, HT-22 cells were treated either with 50 µM tyrphostin A1 alone (Tyr A1) or with 5 mM glutamate for 12 h in the absence (Glu) or presence of 50 µM tyrphostin A1 (A1 + Glu). In another set of samples, HT-22 cells were treated with 5 mM glutamate for 11 h, and then 50 µM tyrphostin A1 was added for 1 h (12hr Glu + A1). D, HT-22 cells were treated either with 1 µM tyrphostin A9 alone (Tyr A9) or with 5 mM glutamate for 12 h in the absence (Glu) or presence of 1 µM tyrphostin A9 (A9 + Glu). In another set of samples, HT-22 cells were treated with 5 mM glutamate for 11 h, and then 1 µM tyrphostin A9 was added for 1 h (12hr Glu + A9). E, HT-22 cells were treated either with 1 µM FCCP alone (FCCP) or with 5 mM glutamate for 12 h in the absence (Glu) or presence of 1 µM FCCP (FCCP + Glu). In another set of samples, HT-22 cells were treated with 5 mM glutamate for 11 h, and then 1 µM FCCP was added for 1 h (12hr Glu + FCCP).

Antioxidant Efficacy-- Because the above experiment does not distinguish between direct antioxidant activity and inhibition of a step required for ROS production, we asked whether tyrphostins can directly quench endogenously produced ROS that have accumulated within the cell. HT-22 cells were treated with glutamate for 11 h, a time point at which the cells are still alive but have accumulated high levels of ROS (Fig. 7A) (18). Then, tyrphostins were added to these cells in the presence of glutamate for an additional 1 h before the determination of ROS levels by DCF fluorescence (24) using flow cytometry. As shown in the previous section, HT-22 cells accumulate high levels of ROS after a 12-h incubation with 5 mM glutamate (Fig. 7A, Glu) compared with the control sample treated only with the antioxidant PG (Fig. 7A, PG). HT-22 cells treated with PG and 5 mM glutamate accumulate lower levels of ROS after 12 h (Fig. 7A, PG + Glu). PG can also act directly as an antioxidant, as shown by its ability to decrease the level of ROS already accumulated inside the cells (Fig. 7A, 12hr Glu + PG). Similarly, tyrphostin A25 quenches ROS, indicating that it has antioxidant properties (Fig. 7B, 12hr Glu + A25). Other protective tyrphostins (A23, AG10, and AG213) also acted as direct antioxidants in this system (data not shown). Although tyrphostin A1 decreases the level of DCF fluorescence if added at the start of glutamate treatment (Fig. 7C, A1 + Glu), it does not quench ROS already accumulated in the cell (Fig. 7C, 12hr Glu + A1), indicating that, in contrast to some of the other tyrphostins, it does not have antioxidant properties. Similarly, tyrphostin A9 is unable to reduce the levels of accumulated ROS (Fig. 7D, 12hr Glu + A9). Indeed, the ROS level in cells treated for 1 h with tyrphostin A9 after an 11-h treatment with glutamate was similar to that in the cells treated with glutamate alone (Fig. 7D, compare Glu with 12hr Glu + A9). Similar results were obtained when tyrphostin AG126 was used (data not shown). The mitochondrial uncoupler FCCP decreases the levels of ROS and protects HT-22 cells from glutamate toxicity if added before the accumulation of ROS (Fig. 7E, FCCP + Glu) (18). FCCP, however, cannot scavenge ROS if added after the accumulation of ROS (Fig. 7E, 12hr Glu + FCCP), consistent with the role of mitochondria as the major source of ROS in this system.

To examine further the possible antioxidant capacity of tyrphostins, the TEAC (50) of tyrphostins was determined to quantify their efficacy as antioxidants in a cell-free system. In this well-established procedure, a substance is compared with Trolox, a water-soluble vitamin E analog, in its ability to suppress the radical cation of 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) in an aqueous solution (55). Many tyrphostins have TEAC values higher than that of Trolox, indicating that they can act as antioxidants (Table I). For example, tyrphostins A23 and A25 have TEAC values of 3.42 ± 0.60 and 2.30 ± 0.28, respectively. Indeed, there was an inverse relationship between EC50 values for protection against glutamate and the TEAC values of the tyrphostins (Table I). Most tyrphostins, therefore, can function as antioxidants and thereby have the potential to directly protect cells from oxidative stress.

Protection from H2O2-- If tyrphostins act as antioxidants, then they should protect cells from peroxidizing agents such as H2O2. To test the protective efficacy of the tyrphostins against H2O2, HT-22 cells were exposed to H2O2 for 2 h in the presence of a tyrphostin, and cell viability was determined by the MTT assay. A number of the tyrphostins that are protective against glutamate toxicity also protect the HT-22 cells from H2O2 (Fig. 8A; Table I). The protective efficacy for glutamate and for H2O2 correlate well (Table I; Fig. 8B). This observation, in addition to the results presented in the previous section (Fig. 7; Table I), supports the hypothesis that certain tyrphostins can function as antioxidants in preventing oxytosis.


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  		Fig. 8.   Protection from H2O2 toxicity and correlation to glutamate toxicity. A, exponentially dividing HT-22 cells were dissociated with pancreatin and plated into 96-well microtiter plates in 100 µl of DMEM with 10% FBS. 12 h later, cells were exposed to 200 µM H2O2 in the presence of the indicated concentrations of tyrphostin A25 (), A23 (black-diamond ), AG10 (black-down-triangle ), A1 (black-square), A9 (black-triangle), or FCCP (X) for 2 h, and cell viability was assessed by the MTT assay as described under "Experimental Procedures." B, values for the EC50 for protection against glutamate toxicity were plotted against the values for the EC50 for protection against H2O2 toxicity taken from Table I. Statistical analysis using Pearson product-moment correlation gave r = 0.801 and two-tailed p = 0.017. C, HT-22 cells were plated (2.0 × 105 cells/60-mm dish) in 4 ml of DMEM containing 10% FBS. 16 h later, the cells were treated for 30 min with 10 µM tyrphostin A25, 0.5 µM tyrphostin A9, or 1 µM FCCP before the addition of 200 µM H2O2. 10 min later, the cells were examined and photographed by phase-contrast microscopy.

In contrast, tyrphostins A9 and AG126 potentiate H2O2 toxicity (Fig. 8A). The potentiation is obvious by light microscopic observation within 10 min of H2O2 addition (Fig. 8C). We speculated that this could be due to an effect of these tyrphostins on the mitochondrial membrane potential (38) similar to FCCP. To provide further evidence for this idea, mitochondrial inhibitors were tested in H2O2 toxicity. FCCP, which depolarizes mitochondria and protects HT-22 cells from glutamate toxicity (18), strongly potentiates H2O2 toxicity in a manner similar to tyrphostin A9 (Fig. 8C). Other mitochondrial inhibitors such as rotenone and antimycin A also potentiate H2O2 toxicity (Table I).

Protection by Collapsing Mitochondrial Membrane Potential-- To directly determine whether tyrphostins A9 and AG126 can alter the mitochondrial membrane potential, HT-22 cells were treated with these tyrphostins, and the mitochondrial membrane potential was determined fluorometrically using JC-1. JC-1 accumulates in mitochondria as the result of the membrane potential and forms aggregates, resulting in a shift of the fluorescence emission wavelength from 527 nm (monomer fluorescence) to 590 nm (aggregate fluorescence) (56). As shown in Fig. 9, neither tyrphostin A1 nor A25 alters the ratio between the monomer and aggregate fluorescence of JC-1. In contrast, tyrphostins A9 (Fig. 9) and AG126 (data not shown) increase the monomer fluorescence similarly to the mitochondrial membrane uncoupler FCCP, indicating that these tyrphostins decrease the mitochondrial membrane potential. From the above results, it can be concluded that tyrphostins A9 and AG126 protect the HT-22 cells from glutamate toxicity not by acting as antioxidants but rather by acting as mitochondrial uncouplers.


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  		Fig. 9.   The effects of tyrphostins on the mitochondrial membrane potential. HT-22 cells were incubated with the indicated compounds, and the membrane potential of the mitochondria was determined with 4 µg/ml JC-1 as described under "Experimental Procedures." The vertical axis indicates the ratio between the monomer fluorescence and the aggregate fluorescence (56).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The above results show that many tyrphostins protect nerve cells from oxidative stress induced by glutamate as well as by HCA, BSO, and H2O2. Using glutamate toxicity as a well-characterized model of oxidative injury, we determined the structural requirements of tyrphostins necessary to protect nerve cells from death. We found that tyrphostins protect nerve cells from oxidative stress by three distinct mechanisms: altering GSH metabolism, acting as antioxidants, and decreasing the mitochondrial membrane potential, thereby reducing the production of ROS after glutamate treatment. In some cases, the same tyrphostin protects by more than one mechanism. For example, A25 not only acts as an antioxidant but also increases the basal level of cellular GSH.

Structural Requirements for Protection

Because tyrphostins protect nerve cells from glutamate toxicity by at least three distinct mechanisms, it is difficult to arrive at a single structure-activity relationship for their protective efficacy. However, two main structural determinants appear to control the efficacy of protection: the hydroxyl groups and hydrophobicity.

The Hydroxyl Groups-- The hydroxyl group on the benzene ring of the tyrphostins is critical for the protection of HT-22 cells from oxidative stress. Indeed, all tyrphostins that are protective against glutamate, HCA, and BSO contain at least one hydroxyl group. Thus, tyrphostins without a hydroxyl group (A1, RG13022, RG14620, and AG1295) are either nonprotective or require very high concentrations to achieve even partial protection. Furthermore, the efficacy of protection improves with an increased number of hydroxyl groups. For example, as the number of hydroxyl groups increases from tyrphostin AG10 to A23 to A25, so does the protective efficacy against glutamate and other pro-oxidants (Table I). These hydroxyl groups may be essential for the antioxidant activity of the tyrphostins. The antioxidant efficacy of tyrphostins, as determined by TEAC values, generally correlates well with the protective efficacy against glutamate, in agreement with the observation that antioxidants protect nerve cells from oxytosis (15, 21).

However, not all tyrphostins with the hydroxyl group act primarily as antioxidants. Tyrphostins A1, A9, and AG126 protect cells from glutamate, but they have low TEAC values and do not protect cells from H2O2 (Table I). These observations indicate that different protective mechanisms are used by these tyrphostins. Tyrphostins A9 and AG126 protect cells by inhibiting ROS production by mitochondria (see below). Tyrphostin A1, in contrast, appears to protect cells by up-regulating GSH synthesis. The protection by tyrphostin A1 may also be attributable to demethoxylation, which converts tyrphostin A1 into the more protective tyrphostin AG10.

Hydrophobicity-- In general, the more hydrophobic a tyrphostin, the better its protective efficacy against oxidative stress. For example, the protective potency of tyrphostin A23 is increased if the substituent group on the R4 position is more hydrophobic than the nitrile group on A23. Thus, AG114, AG213, AG494, and AG556 have EC50 values lower than that of A23, with AG556 being the most hydrophobic and the most efficacious (EC50 = 1) of the group (Table I). This dependence of protective efficacy on hydrophobicity can be seen more clearly in HCA and BSO toxicity. Tyrphostins AG114, AG213, AG494, and AG556 are all more protective than A23 against HCA and BSO toxicity (Table I). Likewise, the protective efficacy against oxidative stress decreases as the hydrophilicity of the substituent group on the R4 position increases. For example, the substitution of the two nitrile groups in tyrphostin A23 with one hydrogen and the hydrophilic carboxylic acid group results in caffeic acid (3,4-dihydroxycinnamic acid). Caffeic acid does not protect HT-22 cells from glutamate, HCA, or BSO unless the carboxylic acid is esterified to reduce hydrophilicity (24). In general, hydrophobic polyphenolic antioxidants are more protective against oxidative stress (24) and amyloid beta  toxicity (25) than their more water-soluble counterparts. This dependence on hydrophobicity is probably due to the enhanced ability of hydrophobic compounds to penetrate lipid bilayers in order to reach the sites of ROS generation (mitochondria) and accumulation (the cytoplasm) (24).

Other Functional Groups-- Two other functional groups that alter the properties of tyrphostins are the nitro group and the t-butyl group on the phenolic ring. The presence of the nitro group decreases the efficacy of protection against all of the pro-oxidants tested. For instance, AG1288 is identical to A23 in structure except for the nitro group at the R1 position (Table I). Tyrphostin AG1288 is half as efficacious as A23 against glutamate, HCA, and H2O2. This difference may be attributable to the nitro group destabilizing either the reaction intermediate or the product after reaction with ROS. The presence of the t-butyl group, as seen in tyrphostin A9, has a much more dramatic impact on both the protection profile and the mechanism of protection (see below).

In contrast, the structural determinants of the tyrphostins that are important for protection against oxidative stress do not appear to be as critical for tyrosine kinase inhibition. For example, tyrphostins A23 and A24, both of which have two hydroxyl groups, protect similarly against glutamate, HCA, and BSO but show a 6-fold difference in the IC50 for inhibition of epidermal growth factor receptor kinase activity (Table I). Similarly, there is no direct correlation between the hydrophobicity of a tyrphostin and its IC50 for inhibition of tyrosine kinase activity (compare, for example, AG18 with AG1288).

Mechanisms of Protection

Tyrphostins protect HT-22 cells from oxytosis induced by glutamate at three distinct steps in the cell death cascade: GSH metabolism, the generation of ROS from mitochondria, and the accumulation of ROS in the cell. Because the initial insult in glutamate toxicity is a decrease in the level of the endogenous antioxidant GSH (21), the up-regulation of GSH metabolism may provide protection from glutamate toxicity. Indeed, many tyrphostins increase the basal GSH level, which correlates quite well with their protective efficacies (Fig. 6; Table II). A similar mechanism of protection due to an increase in GSH levels is observed with some flavonoids and gallic acid esters (24) and with the metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (26). Such an increase in the basal level of GSH is often caused by the up-regulation of the rate-limiting enzyme for GSH metabolism, gamma -GCS (Fig. 6A, inset) (24, 54, 58). It has been shown previously that the upstream region of the gamma -GCS catalytic gene contains an antioxidant response element (59), and some tyrphostins may be able to activate this promoter. The up-regulation of enzymes involved in GSH metabolism is an important mechanism of protection against oxidative stress and other toxic insults. For example, cells that have acquired resistance to pro-oxidants such as glutamate have increased levels of the GSH metabolic enzymes gamma -GCS, glutathione reductase, and glutathione peroxidase (54, 60). These cells also have an increased tolerance for H2O2.

The control of GSH metabolism alone, however, is unlikely to be sufficient for the protection of cells from oxidative stress by tyrphostins. This conclusion is based on two observations. First, tyrphostin A1 at low concentrations does not protect the HT-22 cells from glutamate toxicity, even though the basal GSH level is increased by these same concentrations of A1 (Fig. 6B). Second, many tyrphostins are protective against both glutamate and BSO. Because BSO inhibits gamma -GCS, the induction of this enzyme cannot be protective against BSO. However, tyrphostins may induce a subset of genes that contain the antioxidant response element. For example, the induction of quinone reductase, whose gene contains the antioxidant response element, is protective against glutamate toxicity (61).

In addition to the effect on GSH metabolism, many tyrphostins protect cells by acting as antioxidants and scavenging accumulated ROS. This conclusion is supported by four pieces of evidence. First, analogous to the antioxidants propyl gallate (Fig. 7) (24) and vitamin E (15), the antioxidant tyrphostins maintain low levels of ROS in the cells in the presence of glutamate (Fig. 7). Second, these compounds are also able to remove ROS that have already accumulated in cells (Fig. 7). Third, the TEAC values of these compounds indicate the potential to act as antioxidants (Table I). Fourth, these tyrphostins also protect HT-22 cells from H2O2 (Fig. 8; Table I).

The third mechanism of protection was observed with tyrphostins A9 and AG126 (Table II). As noted before, these tyrphostins do not act as antioxidants (Table I; Figs. 7 and 8), nor do they increase the basal level of GSH as seen with tyrphostins A1 and A25 (Fig. 6A). Instead, these tyrphostins act on mitochondria, resulting in a lower accumulation of ROS during glutamate toxicity. This conclusion is based on the observation that these tyrphostins decrease the mitochondrial membrane potential similarly to the mitochondrial uncoupler FCCP (Fig. 9). Additionally, these tyrphostins protect cells from glutamate and HCA in the low micromolar range, whereas they potentiate BSO and H2O2 toxicity (Figs. 4B and 8A; Table I). FCCP and other mitochondrial inhibitors show similar protection and potentiation profiles (Table I). Burger et al. (38) also found tyrphostin A9 to decrease the membrane potential of mitochondria.

The protection by tyrphostin A9 and FCCP, both of which act on mitochondria, points to the crucial role of mitochondria in the cell death cascade induced by glutamate. According to the time course experiments, when the cellular GSH level falls below 20% of the original value, the generation and accumulation of ROS increase at an exponential rate (18). We also see a concomitant rise in the membrane potential of mitochondria as the cellular GSH level falls below 20% of the basal level (data not shown). The addition of tyrphostin A9 or FCCP to cells not only results in the decreased generation and accumulation of ROS (Fig. 7) but also prevents the GSH loss caused by glutamate (Fig. 6D). These data suggest that mitochondrial activity consumes the majority of cellular GSH and that, in the absence of GSH, mitochondria actively produce a large amount of ROS, ultimately resulting in cell death.

In addition to the characterization of three distinct mechanisms of protection by tyrphostins, this study highlights subtle differences in pro-oxidants. For example, HCA kills the cells more effectively than glutamate, even though these compounds inhibit cystine uptake equally (62). Consequently, higher concentrations of tyrphostins are required to protect cells from HCA than from glutamate (Table I). This difference may be attributable to the presence of glutamate-specific transporters on the cell surface that reduce the glutamate concentration in the medium. Without such a transporter, HCA may remain in the medium and inhibit cystine uptake more effectively than glutamate.

More striking differences are shown between glutamate and BSO. Both glutamate and BSO decrease cellular GSH at a similar rate (18). However, mitochondrial inhibitors such as FCCP and tyrphostin A9 potentiate BSO toxicity while they protect cells from glutamate (Figs. 3 and 4B; Table I). This difference may stem from the different roles of mitochondria in the death caused by these pro-oxidants. As noted above, glutamate induces the hyperpolarization of mitochondria, resulting in ROS generation. Therefore, inhibition of mitochondria (or depolarization) decreases the generation and accumulation of ROS (18). Also, cytochrome c is not released during glutamate toxicity.2 In contrast, depolarization of mitochondria is observed in BSO toxicity under some conditions (63), along with the release of cytochrome c (64, 65). As a consequence, cells do not produce an appreciable amount of ROS (18, 66). Likewise, cell death caused by H2O2 appears to be similar to that caused by BSO in this respect: the mitochondrial membrane potential decreases, and cytochrome c is released upon H2O2 treatment (67, 68).

In summary, we have shown that tyrphostins protect nerve cells against several types of oxidative stress. We also demonstrate that certain tyrphostins act as antioxidants. Because tumor promotion and progression are controlled by the generation of ROS (57, 69), some tyrphostins may be particularly effective as anticancer drugs because they can not only inhibit the activity of specific PTKs but can also reduce the levels of ROS in cancer cells.

    ACKNOWLEDGEMENTS

We thank Drs. David Schubert and Toni Paladino for comments on the manuscript.

    FOOTNOTES

* This work was supported by a grant from Nihon University, Japan (to K. I.) and National Institutes of Health Grants NS28212 (to P. M.) and AG01029 (to Y. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Neurosciences, School of Medicine, MTF345, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0624. Tel.: 858-534-4975; Fax: 858-534-6232; E-mail: ysagara@ucsd.edu.

Published, JBC Papers in Press, July 16, 2002, DOI 10.1074/jbc.M203895200

2 P. Maher, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; GSH, glutathione; BSO, buthionine sulfoximine; HCA, homocysteic acid; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; DCF, dichlorofluorescein; H2DCF, 2',7'-dichlorodihydrofluorescein; TEAC, Trolox equivalent activity concentration; gamma -GCS, gamma -glutamylcysteine synthetase; PG, propyl gallate; FCCP, cyanide p-trifluoromethoxyphenylhydrazone; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolcarbocyanine iodide; PTK, protein tyrosine kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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