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Tamoxifen Induces Oxidative Stress and Mitochondrial Apoptosis via Stimulating Mitochondrial Nitric Oxide Synthase

¹ Vascular Surgery, Davis Heart and Lung Research Institute, and Institute of Mitochondrial Biology, Ohio State University Medical Center, Columbus, Ohio and ² Department of Community Health and Family Medicine, University of Florida School of Medicine, Gainesville, Florida

Requests for reprints: Pedram Ghafourifar, 510 Davis Heart and Lung Research Institute, Ohio State University Medical Center, 473 West 12th Avenue, Columbus, OH 43210. Phone: 614-247-7373; E-mail: Pedram.Ghafourifar{at}osumc.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Tamoxifen is an anticancer drug that induces oxidative stress and apoptosis via mitochondria-dependent and nitric oxide (NO)–dependent pathways. The present report shows that tamoxifen increases intramitochondrial ionized Ca²⁺ concentration and stimulates mitochondrial NO synthase (mtNOS) activity in the mitochondria from rat liver and human breast cancer MCF-7 cells. By stimulating mtNOS, tamoxifen hampers mitochondrial respiration, releases cytochrome c, elevates mitochondrial lipid peroxidation, increases protein tyrosine nitration of certain mitochondrial proteins, decreases the catalytic activity of succinyl-CoA:3-oxoacid CoA-transferase, and induces aggregation of mitochondria. The present report suggests a critical role for mtNOS in apoptosis induced by tamoxifen. [Cancer Res 2007;67(3):1282–90]

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Tamoxifen is a cancer chemotherapeutic agent of a drug family known as estrogen receptor (ER) modulators. Tamoxifen exerts ER antagonistic and agonistic properties in various cells and tissues (1–3). Many cancer chemotherapeutic agents (4), including tamoxifen (5), exert their anticancer properties by inducing apoptosis through mechanisms that involve mitochondria. Therapeutic doses of tamoxifen are 150 to 300 µg d^–1 kg^–1 body weight with an average therapeutic blood concentration of 120 ng/mL that is equal to 0.3 µmol/L (6). Treatment of isolated rat liver mitochondria with 20 to 100 µmol/L tamoxifen, concentrations 60 to 300 times greater than the therapeutic, inhibited mitochondrial permeability transition pore opening (7). Likewise, these high concentrations of tamoxifen increased the mitochondrial respiration and proton permeability and decreased mitochondrial transmembrane potential () and oxidative phosphorylation (8, 9), typifying uncoupling mitochondria. The present study tested the effect of submicromolar concentrations of tamoxifen on mitochondria.

Tamoxifen induces apoptosis in both ER-positive and ER-negative cells via nitric oxide (NO)–dependent pathways (10). Tamoxifen increased the NO synthase (NOS) activity in 10T1/2 murine fibroblasts (11) and in human erythroleukemia K562 cells undergoing apoptosis induced by tamoxifen (10). Moreover, exogenously added NO-potentiated tamoxifen-induced apoptosis and inhibition of NOS activity prevented the apoptosis induced by tamoxifen (10). We have shown that mitochondria possess a NOS [mitochondrial NOS (mtNOS)] that generates NO in response to elevation of intramitochondrial ionized Ca²⁺ concentration ([Ca²⁺]_m; refs. 12, 13). NO generated by mtNOS regulates mitochondrial bioenergetics via reversible regulation of cytochrome oxidase activity (12, 13). mtNOS-derived NO also generates peroxynitrite that induces oxidative stress and apoptosis (13). The present study investigated the function of mtNOS for oxidative stress and apoptosis induced by tamoxifen. It was found that tamoxifen increases [Ca²⁺]_m that stimulates mtNOS activity of isolated rat liver mitochondria and human breast cancer MCF-7 cells. It was found that tamoxifen also induces oxidative stress and release of cytochrome c from mitochondria by mechanisms resembling mtNOS-derived peroxynitrite formation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mitochondria and mitochondrial subfraction preparations. Liver mitochondria were purified from female Sprague-Dawley rats as described (14). The purity of the mitochondria was verified by measuring the cytochrome a content at 605 to 630 nm using the extinction coefficient of 12 mmol/L^–1 cm^–1 (14). Only mitochondria with <5% impurity were used in this study. Broken mitochondria were prepared from freshly isolated intact mitochondria by freeze-thawing followed by hypoosmolar shock as described (12, 14).

Mitochondria and cytoplasm of MCF-7 cells were isolated as described (15). Briefly, after washing cells twice in PBS, protease inhibitor cocktail [aprotinin, pepstatin A, phenylmethylsulfonyl fluoride (PMSF), and leupeptin (10 µmol/L each); refs. 13, 15] was added and cells were homogenized with 20 to 30 strokes with a glass homogenizer while visualizing microscopically to avoid overhomogenizing that damages the mitochondria. The homogenate was centrifuged at 1,000 x g and the pellet was discarded. The supernatant was centrifuged at 10,000 x g for 30 min and the crude cytoplasmic (supernatant) and mitochondrial (pellet) fractions were separated. The crude cytoplasmic fraction was centrifuged at 100,000 x g and the cytoplasm (supernatant) was collected. Mitochondria were purified from crude mitochondria fraction by 20% Percoll purification at 100,000 x g followed by rinsing twice in cold buffer.

Incubation procedure for liver mitochondria. Unless mentioned otherwise, intact or broken mitochondria [1 mg protein in 100 µL HEPES buffer (100 mmol/L; pH 7.10)] were incubated with 100 µmol/L N-monomethyl-L-arginine (L-NMMA; to inhibit mtNOS; ref. 14) or 100 µmol/L glutathione monoethyl ester (GME; to supplement mitochondria with glutathione; ref. 16) for 30 min on ice. After this incubation time, tamoxifen (0.1 or 0.5 µmol/L) or equal volume of its solvent (ethanol) was added and mitochondria were incubated for 20 min at room temperature with occasional gentle shaking. When used, horse heart cytochrome c (680 pmol; ref. 17) was added immediately before tamoxifen.

Incubation procedure for MCF-7 cells. Cells were cultured in phenol red–free DMEM. At 80% confluence, cells were passaged using 5% trypsin and 0.03% EDTA and seeded at 1 x 10⁶ cells/mL. At 80% confluence, cells were treated with tamoxifen (0.1–10 µmol/L; ref. 18) in the absence or presence of L-nitroarginine methyl ester (L-NAME; 100 µmol/L; NOS inhibitor) for 12 to 72 h.

Mitochondrial oxygen consumption. Liver mitochondria samples were suspended in a final volume of 1 mL HEPES buffer, respiration was supported by K⁺-succinate (0.8 mmol/L), and the oxygen consumption was determined using a Clark-type oxygen electrode (Harvard Apparatus, Holliston, MA) as described (14).

NOS activity. NOS activity was determined by citrulline assay, NO-sensitive electrode (World Precision Instrument, Sarasota, FL; Apollo 4000), and NO chemiluminescence analyzer (Sievers 280i, General Electric, Boulder, CO) as described (14). For citrulline assay, liver mitochondria were incubated as under incubation procedure for liver mitochondria, whereas buffer was supplemented with 30,000 to 50,000 cpm L-[³H]arginine. At the end of the incubation, mtNOS activity was terminated by adding ice-chilled stop solution [2 mmol/L EDTA and 1 mmol/L unlabeled L-citrulline in 20 mmol/L Na⁺-acetate buffer (pH 5.00)]. Samples were run through Dowex columns prepared as described (14), and mtNOS activity was determined by measuring the radioactivity of the effluent. To measure mtNOS activity with the NO electrode, 1 mg broken mitochondria incubated as under incubation procedure for liver mitochondria were added to the HEPES buffer (1,000 µL final volume) and NO formation was detected with a NO-sensitive electrode (amiNO 100, Innovative Instruments, Tampa, FL) using a 7-µm tip. Tamoxifen, L-NMMA, or EGTA at the concentrations used in this study did not interfere with the signal detected by the NO electrode. To measure NOS activity in MCF-7 cells, their cytoplasm or mitochondria, samples were injected into the purge vessel containing vanadium chloride (0.8% in 1 mol/L HCl) thermostated at 95°C and the chemiluminescence of the NO released was measured using the NO analyzer (Sievers 280i). The vessel was depleted of oxygen by purging with N₂ at least 20 min before injecting the samples and during the entire measurement. NaNO₂ was used to prepare the standard curve.

[Ca²⁺]_m determination. [Ca²⁺]_m was done at 675 to 685 nm in the presence of Arsenazo III (5 µmol/L) by using an Aminco DW-2000 spectrophotometer (17, 19), by a Ca²⁺-sensitive electrode (Accumet-Fisher Scientific, Hanover Park, IL; ref. 20) and by fluorometery using the Ca²⁺-sensitive fluorescent probe, fura-2 (21, 22). In the former assay, carbonyl cyanide m-chlorophenylhydrazone (cccp; 1 µmol/L) was added to the intact mitochondria samples to collapse the and allow the [Ca²⁺]_m to equilibrate with extra mitochondrial buffer. The same approach was taken with the Ca²⁺ electrode, except 50 nmol/L antimycin A (AA) was added to collapse the because cccp interfered with the signal detected by the electrode. The [Ca²⁺]_m was also measured in broken mitochondria. No cccp or AA was added to the broken mitochondria preparation. Standard curves were obtained by using known concentrations of Ca²⁺ added to the broken mitochondria suspensions. Tamoxifen or L-NMMA at the concentrations used in this study did not interfere with the Ca²⁺ signal detected by either method.

Both [Ca²⁺]_m detection assays are very sensitive; however, they require larger amounts of mitochondria than can be obtained from cultured cells. Therefore, the Ca²⁺ fluorescent probe fura-2 was used and a series of experiments was conducted to establish a sensitive assay to measure [Ca²⁺]_m in very small amounts of mitochondria. Fura-2 (the active form) in the absence or presence of Ca²⁺ was excited from 320 to 400 nm and emission was collected at 510 nm using an Aminco DW-2000 dual-beam dual-wavelength spectrophotometer equipped with original Aminco fluorescent unit. A clear peak and a sharp isosbestic point were detected at 352 and 362 nm, respectively. Next, isolated mitochondria were loaded with the membrane-permeable fura-2/acetoxymethylester (fura-2/AM; 15 min followed by twice wash in cold buffer), excited from 320 to 400 nm, and emission was collected at 510 nm. Identical peak and isosbestic point of 352 and 362 nm were detected. To do a highly sensitive [Ca²⁺]_m measurement, fura-2-loaded mitochondria were excited at dual wavelengths of peak minus isosbestic point (352–362 nm) and emission was detected at 510 nm. A [Ca²⁺]_m-dependent fluorescence was successfully obtained. This sensitive dual-wavelength excitation fluorometric assay was used to test the [Ca²⁺]_m of mitochondria of MCF-7 cells.

Mitochondrial . The of intact liver mitochondria was supported by K⁺-succinate (800 µmol/L) and measured at 511 to 533 nm in the presence of 10 µmol/L safranin as described (13, 17).

Cytochrome c release and lipid peroxidation. Cytochrome c was determined by Western blot using monoclonal anti–cytochrome c antibody (eBioscience, San Diego, CA) as described (13, 17). Lipid peroxidation (LPO) was determined by measuring thiobarbituric acid-reactive substances (TBARS) at 535 nm as described (13).

Protein tyrosine nitration. Samples were lysed in 100 µL lyses buffer containing 133 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.00), 0.1% SDS, 0.5% sodium deoxycholate, 0.1% NP40, and protease inhibitors (10 µmol/L each): PMSF, pepstatin A, leupeptin, and aprotinin. Proteins (20 µg) of each sample were separated by SDS-PAGE on a 10% gel and blotted onto nitrocellulose membrane, probed with monoclonal anti-nitrotyrosine antibody (Alexis Biochemicals, San Diego, CA) or monoclonal anti–cytochrome oxidase subunit VIc antibody (Molecular Probes, Eugene, OR) as loading control, and visualized with alkaline phosphatase.

Activity of succinyl-CoA:3-oxoacid CoA-transferase. Liver mitochondria samples were thrice frozen-thawed in liquid nitrogen followed by addition of ice-cold H₂O (four times the volume of mitochondria suspension) containing protease inhibitors (leupeptin, aprotinin, pepstatin-A, and PMSF; 10 µmol/L each) to fully rupture mitochondrial membranes (14). The suspension was centrifuged at 100,000 x g at 4°C for 30 min and the supernatant [soluble fraction (SF)] was collected. One hundred microgram SF was added to the incubation mixture containing 50 mmol/L Tris-HCl (pH 8.50), 0.1 mmol/L succinyl-CoA, 5 mmol/L acetoacetate, 5 mmol/L MgCl₂, and 4 mmol/L iodoacetamide, and the succinyl-CoA:3-oxoacid CoA-transferase (SCOT) catalytic activity was measured by following the formation of acetoacetyl-CoA at 313 nm as described (23).

Mitochondria aggregation. Aggregation of liver mitochondria was studied as described (24) by using a light microscope (BalPlan-Bausch&Lomb, Rochester, NY) connected to a digital camera (Nikon Coolpix 4500, Nikon, Koga Ku, Japan). Mitochondria samples (3 µg mitochondrial protein/µL) were placed on a Petroff-Hausser counting chamber with 20 µm cell depth (Hausser Scientific, Horsham, PA). Tamoxifen (0.1 and 0.5 µmol/L) or its solvent (control) was added and microphotographs were taken in triplicate every 5 min.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Apoptosis is a fundamental mechanism that regulates cell and tissue homeostasis and eliminates cancer cells (25). It is generally believed that insufficient apoptosis causes cancer (26). Many cancer chemotherapeutic agents (4), including tamoxifen (5, 27), induce oxidative stress and apoptosis via mechanisms that involve mitochondria and NO (10, 11). We (12) and several other laboratories (refs. 27–31; reviewed in ref. 32) have shown that mitochondria generate NO through the Ca²⁺-sensitive mtNOS. Stimulation of mtNOS leads to formation of peroxynitrite that induces oxidative stress (33) and mitochondrial apoptosis (13). The present study investigated the function of mtNOS for tamoxifen-induced oxidative stress and apoptosis.

Tamoxifen increases mtNOS activity, decreases mitochondrial oxygen consumption, and increases [Ca²⁺]_m. Figure 1A shows that submicromolar concentrations of tamoxifen increased mtNOS activity of intact isolated rat liver mitochondria and that this effect of tamoxifen was prevented with L-NMMA. Figure 1B shows that NO formation by broken mitochondria was increased by tamoxifen and that this effect of tamoxifen was prevented when mtNOS was inhibited with L-NMMA, or [Ca²⁺]_m was removed by EGTA. An L-NMMA– and EGTA-sensitive increase in NO formation of broken mitochondria was also observed for 0.1 µmol/L tamoxifen (data not shown). These results indicate that tamoxifen stimulates mtNOS activity of rat liver mitochondria. NO is the pharmacologic competitive antagonist of O₂ (i.e., NO reversibly decreases mitochondrial respiration by competing with O₂ for the O₂ binding site of cytochrome oxidase; ref. 34). Several reports have shown that NO produced by mtNOS decreases mitochondrial O₂ consumption (12, 13). Thus, the present study tested the effect of tamoxifen on mitochondrial oxygen consumption. Figure 1C shows that tamoxifen decreased the oxygen consumption of intact liver mitochondria. Some studies suggested that tamoxifen increased the oxygen consumption of mitochondria (7–9). This can be explained by considering that those studies used 20 to 100 µmol/L tamoxifen, which is 60 to 300 times >0.3 µmol/L, the submicromolar concentration of tamoxifen (6). Tamoxifen is highly lipophilic and high concentrations of lipophilic compounds readily increase mitochondrial respiration by perturbing the mitochondrial membrane integrity and uncoupling the mitochondria. Figure 1C shows that the decrease of mitochondrial oxygen consumption was prevented when mtNOS was inhibited by L-NMMA. This finding indicates that tamoxifen decreased the mitochondrial oxygen consumption by stimulating mtNOS activity. Figure 1C shows that tamoxifen also decreased the respiration of broken mitochondria and that this effect of tamoxifen was prevented when [Ca²⁺]_m was chelated by EGTA. This finding together with those presented in Fig. 1B strongly suggest that tamoxifen increases mtNOS activity by increasing [Ca²⁺]_m.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Tamoxifen increases mtNOS activity. A, effect of tamoxifen [0.1 µmol/L (Tam 0.1) or 0.5 µmol/L (Tam 0.5)] or its solvent [control (Ctrl)] on mtNOS activity of rat liver mitochondria (black columns) and the inhibitory effect of L-NMMA (white columns). mtNOS activity was determined by citrulline assay and expressed as counts per minute L-[3H]citrulline per milligram mitochondrial protein per minute. B, effect of 0.5 µmol/L tamoxifen (Tam 0.5) or its solvent [control (Cont)] on mtNOS activity of broken liver mitochondria and the effect of L-NMMA (+ L-NMMA) or EGTA (+ EGTA). mtNOS activity was measured with a NO-sensitive electrode and expressed as picoampere. Arrowhead, addition of broken mitochondria to NO electrode chamber. C, effect of tamoxifen on succinate-supported (0.8 mmol/L K+-succinate; arrowhead) oxygen consumption of intact and broken liver mitochondria. D, NOS activity of MCF-7 cells (whole cell) and the mitochondria of MCF-7 cells (mitochondria) treated with tamoxifen (3 µmol/L, 48 h) in the absence (black columns) or presence (white columns) of L-NAME. NOS activity was measured by using chemiluminescence assay and expressed as nmol NO (Nox)/106 cells or pmol NO/µg mitochondrial protein. Traces are representative of three to four experiments. Columns, mean of four to six experiments; bars, SE. *, P < 0.05; **, P < 0.01, significantly different from control.

Mitochondria contain sufficient substrates and cofactors that mtNOS requires, and elevation of [Ca²⁺]_m per se stimulates the mtNOS activity (12, 13, 30, 31). Because tamoxifen increased the mtNOS activity and this effect of tamoxifen was inhibited when [Ca²⁺]_m was removed, the present study tested whether tamoxifen increases [Ca²⁺]_m. Figure 2A shows that tamoxifen increased the [Ca²⁺]_m stored in intact liver mitochondria. Figure 2A also shows that L-NMMA did not alter the effect of tamoxifen on [Ca²⁺]_m, indicating that the increased [Ca²⁺]_m efflux was not due to decreasing the by mtNOS-derived NO (12, 19). To rule out possible direct effect of tamoxifen on Ca²⁺ transport system of intact mitochondria, the effect of tamoxifen on broken mitochondria was tested. Figure 2B shows that tamoxifen increased the Ca²⁺ concentration in broken mitochondria and that L-NMMA did not alter this effect of tamoxifen. Elevation of [Ca²⁺]_m neutralizes the negative charges of the inner mitochondrial membrane and decreases the . Thus, the present study tested the effect of tamoxifen on . Figure 2C shows that tamoxifen decreased the and that this effect of tamoxifen was prevented when [Ca²⁺]_m was chelated by EGTA. This finding further supports the findings presented in Fig 2A and B and indicates that tamoxifen increases the [Ca²⁺]_m in liver mitochondria.

View larger version (10K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Tamoxifen, [Ca2+]m, and of liver mitochondria. A, effect of tamoxifen [0.1 µmol/L (Tam 0.1) or 0.5 µmol/L (Tam 0.5)] or its solvent (Cont) on [Ca2+]m of intact mitochondria in the absence or presence of L-NMMA. The was collapsed by AA to allow [Ca2+]m efflux. Where indicated, EGTA (10 mmol/L) was added. B, effect of tamoxifen (0.1 and 0.5 µmol/L) on the [Ca2+]m of broken mitochondria in the absence (black columns) or presence (white columns) of L-NMMA. C, the of mitochondria (0.6 mg/mL) treated with 0.1 µmol/L tamoxifen (Tam 0.1) or solvent (Cont) was supported by K+-succinate (Succ; 0.8 mmol/L) and measured at 511 to 533 nm. At the end of the test, was collapsed by uncoupling mitochondria with cccp (1 µmol/L). Where indicated, 5 mmol/L EGTA (Tam 0.1 + EGTA) was present in the buffer. Traces are representative of three to four experiments. Columns, mean of four to six experiments; bars, SE. **, P < 0.01, significantly different from control.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Tamoxifen and [Ca2+]m of MCF-7 cells. A, fura-2 (active form; 10 µmol/L) in the absence (no Ca2+) or presence of Ca2+ (1–50 µmol/L) was excited from 320 to 400 nm and emission was recorded at 510 nm using Aminco DW-2000 original fluorescent unit equipped with precision 510 nm bandpass filter. Peak and isosbestic points were identified 352 and 362 nm, respectively. B, liver mitochondria (1 mg/mL) loaded with fura-2/AM were excited from 320 to 400 and emission was collected at 510 nm. Peak and isosbestic points were identified 352 and 362 nm, respectively. C, fura-2-loaded liver mitochondria (1 mg/mL) were excited at dual wavelengths of 352 – 362 nm and emission was recorded at 510 nm. Arrowheads, the [Ca2+]m was elevated by loading mitochondria with shown concentrations of Ca2+. Where indicated, the was collapsed with cccp to deplete the mitochondria of [Ca2+]m. D, mitochondria (10 µg) of MCF-7 cells treated with tamoxifen (3 µmol/L, 48 h) in the presence or absence of L-NAME were loaded with fura-2/AM as in (B), and [Ca2+]m was measured as in (C) (exciting at 352–362 nm and emission at 510 nm). Where indicated, mitochondria from untreated cells were loaded with 0.1 µmol/L Ca2+. Traces are representative of four to six experiments. Columns, mean (n = 4–6); bars, SE. *, P < 0.05; **, P < 0.01, significantly different from the control.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Scheme. Mitochondria, mtNOS, and tamoxifen. Mitochondria consist of the inner (IM) and the outer membrane (OM), matrix, and the intermembrane space (IMS). The respiratory chain consists of four complexes (I–IV), coenzyme Q (Q; ubiquinone), and ATP synthase also called complex V, which are embedded in the IM. Electrons (e–) flow down the chain to complex IV and reduce O2 to H2O. Coupled to the electron flow, protons (H+) are pumped from the matrix into the IMS. The proton extrusion establishes a transmembrane potential (; negative inside) and an electrochemical gradient (pH; alkaline inside) across the coupling membrane. The is the driving force for mitochondria to take up Ca2+. Whereas mitochondria take up relatively large quantities of Ca2+, intramitochondrial ionized Ca2+ concentration ([Ca2+]m) is maintained very low by mechanisms, including precipitation of the [Ca2+]m to nonionized calcium pools, the matrix electron-dense granules. Mitochondria generate NO through the mtNOS. mtNOS is Ca2+ sensitive and elevation of [Ca2+]m stimulates the mtNOS activity. mtNOS-derived NO reversibly inhibits mitochondrial respiration by competing with O2 for the O2 binding site on complex IV. mtNOS-derived NO also reacts with superoxide anion (O2–) to produce peroxynitrite (ONOO–). Peroxynitrite induces cytochrome c release, elevates LPO, and nitrates tyrosine residues of susceptible proteins, including SCOT. Tamoxifen affects intramitochondrial calcium homeostasis by shifting the mitochondrial nonionized/ionized calcium equilibrium in the favor of the ionized form. Elevated [Ca2+]m stimulates mtNOS that increases intramitochondrial ONOO– that induces oxidative stress and mitochondrial apoptosis.

Tamoxifen releases cytochrome c and elevates LPO. Release of cytochrome c from the mitochondria is one of the key events during most forms of apoptosis, including that induced by tamoxifen (43). Stimulation of mtNOS increases mitochondrial peroxynitrite that releases cytochrome c from the mitochondria (13). mtNOS stimulation also increases LPO that is a widely used peroxynitrite biomarker (13, 44). Figure 4A and B shows that tamoxifen released cytochrome c from liver mitochondria and increased LPO and that those effects of tamoxifen were prevented when mtNOS was inhibited. These findings suggest that tamoxifen released the cytochrome c and elevated LPO by stimulating mtNOS. To test whether these effects of tamoxifen were mediated by peroxynitrite, mitochondria were supplemented with glutathione. Supplementing mitochondria with glutathione prevented the apoptosis induced by tamoxifen (45) and decreased the oxidative damage induced by peroxynitrite (46–48). Elegant work of Nakamura et al. (46) has shown that glutathione reverses the peroxynitrite-induced oxidative damage by converting peroxynitrite to S-nitrosating species (47). Figure 4A and B shows that supplementing mitochondria with glutathione prevented tamoxifen-induced release of cytochrome c and increase of LPO. Supplementing cells with cytochrome c prevents apoptosis induced by tamoxifen (45) and supplementing mitochondria with cytochrome c prevents elevated LPO induced by mtNOS-derived peroxynitrite (13). Figure 4B shows that supplementing mitochondria with cytochrome c prevented tamoxifen-induced LPO elevation. These findings strongly suggest that tamoxifen increases peroxynitrite in liver mitochondria.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Tamoxifen, cytochrome c release, and LPO. Liver mitochondria were treated with ethanol (Cont) and tamoxifen [0.1 µmol/L (Tam 0.1) or 0.5 µmol/L (Tam 0.5)], whereas mtNOS was inhibited with L-NMMA (Tam + L-NMMA), or mitochondria were supplemented with glutathione (Tam + GME) or cytochrome c (Tam + cyto c). Cytochrome c released from the mitochondria into the supernatant or remained in the mitochondria pellet (A) was determined by Western blot, and LPO (B) was measured by measuring TBARS. LPO is expressed as nanomol TBARS/milligram mitochondrial protein. Horse heart cytochrome c (60 ng) was used as positive control in (A). C, MCF-7 cells were treated with tamoxifen (3 µmol/L 48 h) in the absence or presence of L-NAME as in Fig. 1E. LPO was measured in the cytoplasm (white columns) or mitochondria (black columns) and expressed as picomolar TBARS per milligram protein. Columns, mean of four experiments; bars, SE. *, P < 0.05; **, P < 0.01, significantly different from control.

Tamoxifen increases protein tyrosine nitration and decreases SCOT activity. Peroxynitrite readily reacts with tyrosine residues to produce nitrotyrosine. Determination of protein tyrosine nitration has been widely used as a reliable peroxynitrite biomarker (49). Figure 5A shows that tamoxifen increased the tyrosine nitration of certain liver mitochondrial proteins and that this effect of tamoxifen was prevented when mtNOS was inhibited. This finding further suggest that tamoxifen increases mitochondrial peroxynitrite via stimulating mtNOS. Figure 5A shows substantial increase in the tyrosine nitration of a protein with molecular weight of 50 kDa. SCOT is a 52-kDa mitochondrial matrix protein that is involved in energy production in malignant cells (50) and undergoes peroxynitrite-induced tyrosine nitration (23). Figure 5B shows that tamoxifen decreased the activity of SCOT and that this effect of tamoxifen was prevented when mtNOS was inhibited. This finding suggests that the 50-kDa tyrosine-nitrated protein shown in Fig. 5A is SCOT. Figure 5A and B also shows that supplementing mitochondria with glutathione or cytochrome c prevented tamoxifen-induced increase in protein tyrosine nitration and decrease in the SCOT activity, which confirms the results presented in Fig. 4A and B and further indicate tamoxifen increases mitochondrial peroxynitrite.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Tamoxifen and mitochondrial tyrosine nitration and SCOT activity. Liver mitochondria were treated with tamoxifen [0.1 µmol/L (Tam 0.1) or 0.5 µmol/L (Tam 0.5)] or its solvent (Cont), whereas mtNOS was inhibited with L-NMMA (Tam + L-NMMA), or mitochondria were supplemented with glutathione (Tam + GME) or cytochrome c (Tam + cyto c). A, tyrosine nitration of mitochondrial proteins was determined by Western blot. Tyrosine-nitrated bovine serum albumin (BSA-TyrNO; 60 ng) was used as positive control. Cytochrome oxidase (CytOx) was determined to serve as loading control. Numbers indicate the molecular weight in kDa. B, the catalytic activity of SCOT was measured as described in Materials and Methods. Columns, mean of four experiments; bars, SE. *, P < 0.05, significantly different from control; #, P < 0.05, significantly different from 0.1 µmol/L tamoxifen (Tam 0.1).

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Tamoxifen and mitochondrial aggregation. Microphotographs were taken immediately (T0) and after 20 min (T20) after addition of ethanol (Cont), or 0.1 µmol/L tamoxifen (Tam 0.1) or 0.5 µmol/L tamoxifen (Tam 0.5). Inhibition of mtNOS (+ L-NMMA) or supplementing mitochondria with glutathione (+ GME) or cytochrome c (+ cyto c; 125 pg/mg mitochondrial protein; ref. 35) were done as described in Materials and Methods. No aggregation was observed at T0 in any experimental group (only the T0 photograph for the control experiment has been shown). Arrow, typical aggregation. Microphotographs are representative of four to six experiments.

These findings suggest a pivotal role for mtNOS in oxidative stress and apoptosis induced by tamoxifen and propose a novel mechanism for the action of a widely used cancer chemotherapeutic agent.

	Acknowledgments

 
Grant support: National Institute on Aging (award AG023264-02), American Heart Association (award 0565221B), Marshall University Center of Biomedical Research Excellence (award 1 P20 RR020180 NCRR), and Gdynia Maritime University, Poland (R.R. Nazarewicz).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/21/06. Revised 10/12/06. Accepted 11/20/06.

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