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Originally published In Press as doi:10.1074/jbc.M414498200 on May 5, 2005

J. Biol. Chem., Vol. 280, Issue 27, 25369-25376, July 8, 2005
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{alpha}-Tocopheryl Succinate Inhibits Malignant Mesothelioma by Disrupting the Fibroblast Growth Factor Autocrine Loop

MECHANISM AND THE ROLE OF OXIDATIVE STRESS*

Michael Stapelberg{ddagger}§, Nina Gellert{ddagger}, Emma Swettenham{ddagger}, Marco Tomasetti¶, Paul K. Witting||, Antonio Procopio¶, and Jiri Neuzil{ddagger}**{ddagger}{ddagger}

From the {ddagger}Apoptosis Research Group, School of Medical Science, Griffith University, Southport, 4216 Queensland, Australia, the Department of Molecular Pathology, Polytechnic University of Marche, Ancona 60131, Italy,|| ANZAC Institute, Concorde Hospital, University of Sydney, Concord 2139, New South Wales, Australia, and** Laboratory of Apoptosis and Cell Signalling, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague 14220, Czech Republic

Received for publication, December 23, 2004 , and in revised form, April 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have studied the potential effect against human malignant mesotheliomas (MM) of {alpha}-tocopheryl succinate ({alpha}-TOS), a redox-silent vitamin E analog with strong pro-apoptotic and anti-cancer activity. {alpha}-TOS at sub-apoptotic levels inhibited proliferation of MM cell lines, while being nontoxic to nonmalignant mesothelial cells. Because MM cells are typified by a highly metastatic phenotype, we investigated the effect of {alpha}-TOS on genes playing a major role in MM progression. Of these, {alpha}-TOS down regulated fibroblast growth factor (FGF)-1 and, in particular, FGF-2 on the transcriptional level in MM cells, and this was not observed in their nonmalignant counterparts. FGF-2 short interfering RNA suppressed proliferation of MM cells. Down-regulation of FGF-2 was likely because of inhibition of the egr-1 transcription activity that was decreased in MM cells via oxidative stress induced by {alpha}-TOS, as evidenced by EPR spectroscopy, whereas nonmalignant cells did not show this response. Treatment of MM cells with egr-1 short interfering RNA suppressed proliferation, which was overridden by exogenously added recombinant FGF-1 and, in particular, FGF-2. An analog of coenzyme Q targeted to mitochondria and superoxide dismutase overrode inhibition of MM cell proliferation by {alpha}-TOS as well as {alpha}-TOS-induced inhibition of egr-1-dependent transactivation. Finally, {alpha}-TOS significantly suppressed experimental MM in immunocompromised mice. Our data suggest that {alpha}-TOS suppresses MM cell proliferation by disrupting the FGF-FGF receptor autocrine signaling loop by generating oxidative stress and point to the agent as a selective drug against thus far fatal mesotheliomas.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Malignant mesothelioma (MM)1 is a highly aggressive tumor that arises from mesothelial cells of the serosal surfaces of body cavities. It has a very grim prognosis, as patients frequently succumb within months after diagnosis. The incidence of MM is increasing, and a progressive rise has been predicted until at least the year 2020 (1, 2). The lack of effective treatment for MM makes studies aimed at understanding the molecular mechanism underlying proliferation and metastasis of MM of high relevance (3).

Of the cytokines that are known to elicit a potent mitogenic response, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-1 and FGF-2, and the transforming growth factor-{beta} (TGF-{beta}) are involved in the malignant phenotype of various cancers (4–6). These cytokines have been also suggested to control proliferation and migration of MM cells (7). Active VEGF is expressed in a variety of human malignancies including MM (8). The biological effects of VEGF are mediated via two distinct cell surface tyrosine kinase receptors, Flt and Flk. Upon VEGF binding, the receptors dimerize, and several intracellular signaling routes that induce proliferation are activated, including the mitogen-activated protein (MAP) kinase pathway (5, 7). FGF belongs to a family of 23 members, of which FGF-1 and FGF-2 have been proposed to play a role in MM cell (patho)physiology (9). Signaling mechanisms induced by FGFs are mediated by four high affinity tyrosine kinase receptors known as FGF receptors (FGFR), which are transmembrane proteins that, once bound to cognate FGFs, dimerize and signal via intracellular pathways, such as the MAP kinase route, that induce cell proliferation and migration (5). FGF receptors (FGFR) 1–3 are alternatively spliced into two specific exons denoted IIIb and IIIc. The expression of FGFR 1–3 isoforms is highly controlled and essential for initiating specific cellular responses (5). TGF-{beta} is a potent growth regulatory cytokine that exerts a diverse range of effects on many types of cells. TGF-{beta} has potent mitogenic effects on several types of MM cells, which produce it at relatively high levels (6, 10). All of these cytokines may control proliferation of MM cells via autocrine signaling (11, 12).

Because these inducers and mediators are essential for the process of formation and progression of MM, understanding the regulatory mechanisms of their expression is important, because they control pathways that may present a target for MM treatment (6, 13, 14). Conceivably, an ideal anti-cancer agent preventing progression of the metastatic disease would selectively down-regulate the expression of those cytokines that positively control tumor growth, while being nontoxic toward normal cells.

Recent data showed that analogs of vitamin E have potent anti-proliferative and pro-apoptotic effects on multiple cancer cell lines and inhibit cancer in pre-clinical models (15). These compounds are epitomized by {alpha}-tocopheryl succinate ({alpha}-TOS), a redox-silent compound that has been reported to suppress several types of neoplasia (16–19). The vitamin E analogs inhibit proliferation of cancer cells by several mechanisms, including inhibition of DNA synthesis, induction of apoptosis and cellular differentiation, and by affecting the protein kinase C and the MAP kinase pathways (19–24). More importantly, {alpha}-TOS exerts anti-proliferative/pro-apoptotic effects in malignant cell lines but is largely nontoxic toward normal cells and tissues (17, 23, 25). It has also been reported that {alpha}-TOS can modulate cytokine gene expression in cancer cells (26, 27). For example, {alpha}-TOS down-regulates FGFR-1 in MM cells, although the precise mechanism has not been resolved (28).

Because {alpha}-TOS is a potent inducer of apoptosis in the generally resistant MM cells (29) and inhibits MM in vivo (30), we investigated the effects of the vitamin E analog on expression of cytokines involved in control of cancer development and progression. We show here that {alpha}-TOS disrupts the FGF autocrine loop through suppression of egr-1 transcriptional activity in MM cells but not in their nonmalignant counterparts, further highlighting the potential of {alpha}-TOS as a therapeutic agent.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Human MM cell lines, MM-B1 (biphasic), Meso-2 (sarcomatose), Ist-Mes and Ist-Mes2 (both epithelioid) (31), and a nonmalignant mesothelial cell line, Met-5A (ATCC), were used in our studies. The cells were cultured in DMEM supplemented with 10% fetal calf serum (both from JRH Biosciences), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. All assays were performed when the cells reached 60–70% confluency.

Cell Proliferation Assay—Cell proliferation was determined by assessing DNA synthesis. An end point enzyme-linked immunosorbent assay (ELISA) colorimetric kit (Roche Applied Science) was used to determine cells in the S phase of the mitotic cycle, based on incorporation of 5-bromo-2-deoxyuridine (BrdUrd) into their DNA. Briefly, MM cells were seeded at 104 cells per well into a 96-well plate, treated as specified, and incubated with 10 µM BrdUrd for 2 h at 37 °C. The cells were then fixed and denatured by a 30-min incubation with Fixdenat (Roche Applied Science), incubated for 90 min with an anti-BrdUrd antibody, washed, and incubated further with a substrate solution (tetramethylbenzidine). After 30 min, 1 M H2SO4 was added to each well to stop the reaction, and the absorbance was read at 450 nm using a plate reader. In some cases, cells were assessed for the expression of a proliferation-specific gene, the proliferating cell nuclear antigen by Western blotting, using an anti-proliferating cell nuclear antigen IgG (Santa Cruz Biotechnology). Where indicated, cells were exogenously supplemented with human recombinant (hr) FGF-1 or hrFGF-2 (both from Sigma), at 10 ng/ml 24 h prior to treatment.

Cell Cycle Assay—MM and Met-5A cells were plated at 105 cells per well in 24-well plates. The cells were allowed to attach overnight and were then incubated for up to 3 days with {alpha}-TOS. The floating and attached cells were collected, washed with PBS, resuspended in buffer containing sodium citrate (1%), Triton X-100 (0.1%), RNase A (0.05 µg/ml), and propidium iodide (PI) at 5 µg/ml, and incubated in the dark for 30 min at 4 °C. The nuclear suspension was filtered through a 60-µm mesh and analyzed by flow cytometry.

Apoptosis Assessment—Apoptosis was quantified by the annexin V-fluorescein isothiocyanate (FITC) method, which detects phosphatidylserine externalized in the early phases of apoptosis (20). Briefly, cells were seeded at the density of 105 cells per well in 24-well plates and treated with {alpha}-TOS after overnight recuperation. Floating and attached cells were collected, washed with PBS, resuspended in 0.1 ml of binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM CaCl2, pH 7.4), incubated for 20 min at room temperature with 2 µl of annexin V-FITC supplemented with 10 µl of PI (10 µg/ml), and analyzed by flow cytometry (FACSCalibur; BD Biosciences) using channel 1 for annexin V-FITC binding and channel 2 for PI staining.

Detection of Reactive Oxygen Species (ROS)—Cellular ROS were detected indirectly by flow cytometry and directly by EPR spectroscopy, following treatment of cells with {alpha}-TOS as indicated in the figure legends. In some experiments, the cells were pretreated for 1 h with 2 µM mitochondrially targeted coenzyme Q (mito-Q) (32) or co-incubated with superoxide dismutase (SOD; EC 1.15.1.1 [EC] ; Sigma S4636) at 750 units/ml. For indirect evaluation, cells were treated with {alpha}-TOS and reacted with dihydrodichlorofluorescein diacetate (DCF; Molecular Probes) for 30 min, and scored by flow cytometry for cells with high fluorescence, which was evaluated on the basis of an increase in mean fluorescence intensity. EPR spectroscopy analysis of ROS generation was based on the use of the radical trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Sigma). In brief, cells were plated in T25 flasks and allowed to reach 60–70% confluency (~5 x 106 cells per flask). Cells were washed, overlaid with the PSS medium (33), and incubated with 50 µM {alpha}-TOS 5 min after addition of 10 mM DMPO. Analyses of DMPO adducts were performed with samples taken from the cell suspension as well as the cell-conditioned medium transferred into a quartz flat cell (Wilmad). The quartz cell was then placed into the cavity of the Bruker EMX bench-top spectrometer set at 293 K with the following spectrometer parameters: field sweep 10 millitesla, microwave power 20 milliwatts, microwave frequency 100 kHz, modulation amplitude, 0.1 millitesla, sweep time 83.9 s. The detection limit of the stable nitroxide (TEMPO) under identical conditions was ~50 nM.

Real Time mRNA Analysis—Relative quantification of mRNA expression was achieved using quantitative real time-PCR (Q-PCR). Briefly, this technique is based on the detection of a fluorescent signal produced by the incorporation of the fluorescent dye SYBR-green during PCR amplification (Prism 7700 sequence detection system; Applied Biosystems). The expression of all genes of interest was related to that of the 18 S RNA control. Total RNA was extracted from MM cell cultures using Trizol (Invitrogen). To minimize potential genomic contamination, RNA samples were treated with RQ1 RNase-free DNase (Promega) and purified using the RNeasy mini kit (Qiagen). Each assay was performed according to the manufacturer's protocol. First strand cDNA was synthesized using the Superscript III Reverse Transcriptase kit (Invitrogen) according to the manufacturer's protocol. PCR primers were specifically designed for real time PCR. Particular attention was given to maintaining a constant 60 °C annealing temperature of primer pairs, which is common for Q-PCR. Each set of primers was also designed across intron/exon boundaries to detect genomic DNA contamination.

The primers used were as follows: FGF-1, 5'-GGG CTT TTA TAC GGC TCA CA-3', and 5'-GGC CAA CAA ACC AAT TCT TC-3'; FGF-2, 5'-GAC CCT CAC ATC AAG CTA CAA CT-3', and 5'-AAA GAA ACA CTC ATC CGT AAC ACA-3'; VEGF,5'-AGG CCA GCA CAT AGG AGA GA-3', and 5'-TTT CTT GCG CTT TCG TTT TT-3'; TGF-{beta},5'-GAG CCT GAG GCC GAC TAC TA-3', and 5'-TCG GAG CTC TGA TGT GTT GA-3'.

RNA Interference (RNAi)—For RNAi, cells were seeded at a final density of 5 x 104 cells per well in 12-well plates, cultured until ~50% confluency, and then treated with egr-1, FGF-1, or FGF-2 siRNA (all designed and synthesized by Proligo) as follows: siRNA (0.5 µg/ml) was combined with 100 µl of serum-free DMEM supplemented with 20 µlof Oligofectamine (Invitrogen) and left for 15 min at room temperature. The transfection mixture was added to cells, which were then left in the incubator for 24 h, after which they were overlaid with complete DMEM. 24–48 h later, the cells were used in experiments. Typically, 90–95% of treated cells showed significant down-regulation of the targeted genes as estimated by flow cytometric analysis (data not shown). Nonsilencing RNA was used as a negative control and FITC-tagged nonspecific RNA as a control for transfection efficacy (both Qiagen).

Analysis of FGF-1 and FGF-2 Protein—The FGF-1 and FGF-2 protein levels were assessed using an ELISA kit (R & D Systems) according to the manufacturer's instructions. In brief, cells were seeded in 24-well plates and allowed to reach 60–70% confluency. Following treatment, 100 µl of cell-conditioned medium was transferred to the ELISA 96-well plate, mixed with 100 µl of the assay diluent, and incubated for 2 h at room temperature. After washing, each well was supplemented with 200 µl of FGF conjugate, which was followed by a 2-h incubation at room temperature and a 30-min incubation with 200 µl of the substrate solution. Absorbance at 450 nm was assessed using an ELISA plate reader. The system was calibrated using hrFGF-1 or hrFGF-2.



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  		FIG. 1.
{alpha}-TOS induces apoptosis in mesothelioma cells in a dose-dependent manner and is selective for malignant cells. MM and nonmalignant mesothelial cells (Met-5A) were seeded in 24-well plates, allowed to reach 50–70% confluency, and treated for 24 h with {alpha}-TOS at the concentrations shown, and the level of apoptosis was assessed by the annexin V-binding assay. Data shown represent mean values ± S.D. (n = 3).

 
Immunofluorescence Analysis of Protein Expression—MM and Met-5A cells were grown on coverslips and treated with {alpha}-TOS at 10 and 20 µM for 24 and 48 h. The cells were then fixed in 3.6% formalin in PBS and reacted on ice for 2 h with anti-egr-1, anti-FGF-1, or anti FGF-2 IgG (all from Santa Cruz Biotechnology) diluted 1:100 in PBS containing 2% fetal calf serum, followed by incubation with a secondary, FITC-, or Texas Red-conjugated IgG. Background fluorescence was accounted for in samples where primary antibody was omitted. The cells were then mounted in 4,6-diamidino-2-phenylindole-containing Vectashield and assessed for fluorescence, reflecting protein expression, using the Leica DMI ER2 fluorescence microscope. Images were taken, and the level of staining was estimated using the Leica image analysis/deconvolution software.

Assessment of egr-1 Transcription Activity—To assess whether {alpha}-TOS affects egr-1-dependent trans-activation, cells were transiently transfected with a plasmid comprising the egr-1-response element in the promoter followed by the lux gene (Stratagene) and treated as described in the figure legends. Luciferase activity was assessed using the Luciferase Reporter Gene Detection Kit (Sigma) according to the manufacturer's protocol, and the extent of luciferase activity was related to the activity in the untreated controls.

Animal Experiments—Immunocompromised (athymic) mice were injected subcutaneously with Ist-Met2 (2 x 106 cells per animal). After 15 days when tumors were established, the mice were injected into the peritoneum with 100 µl of 200 mM {alpha}-TOS (diluted per animal) per animal every 3 days or with the vehicle alone. Tumor volume was estimated with calipers, and the volume was calculated by using the equation height x length x width x 0.524 as described previously (34). The growth of tumors was expressed as an increase in their volume relative to the tumor volume at the onset of treatment.

Assessment of Intracellular Levels of {alpha}-TOS and {alpha}-TOH—To assess the levels of the two vitamin E analogs, a high pressure liquid chromatography method was applied as described elsewhere (35).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
{alpha}-TOS Is Selectively Toxic to MM Cells—{alpha}-TOS suppresses several types of cancer in pre-clinical models (16–19, 27), although the reasons for this effect are not completely understood. Here we studied the effect of {alpha}-TOS on several MM cell lines of different phenotypic origin and on their nonmalignant counterparts. {alpha}-TOS consistently induced apoptosis in all MM cell lines in a concentration-dependent manner, and the extent of apoptosis was comparable irrespective of the cell phenotype (Fig. 1). In all cases, doses of >30 µM were required for apoptosis induction, and such doses are achievable in vivo (17). In contrast, and important to the therapeutic potential of {alpha}-TOS, the vitamin E analog was nontoxic to Met-5A cells (Fig. 1).

We next tested the effect of {alpha}-TOS on cell proliferation. The cells were treated with the agent at 10–50 µM for up to 72 h. As shown in Fig. 2, significant inhibition of cell proliferation was observed in MM cells treated for more than 24 h with {alpha}-TOS at concentrations between 10 and 50 µM. It is noteworthy that the vitamin E analog inhibited proliferation at levels where it did not induce apoptosis, pointing to effects that are apoptosis-independent. Suppression of cellular proliferation by the vitamin E analog suggests that it may inhibit cell cycle progression (Fig. 3). Indeed, treatment of Meso-2 MM cells with a subapoptotic concentration of {alpha}-TOS resulted in accumulation of cells in G2 phase at the expense of cells in the S-phase and, to a lesser extent, cells in G1 phase (Fig. 3B). Cell cycle arrest was comparable in the other MM cell lines tested (data not shown). Cell cycle analysis also revealed a low number of cells accumulating in sub-G0 (data not shown), supporting the idea that {alpha}-TOS exhibits anti-proliferative activity in addition to induction of apoptosis. Again, little effect was observed in Met-5A cells (Fig. 3A).



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  		FIG. 2.
{alpha}-TOS inhibits proliferation in MM cells but not in nonmalignant mesothelial cells. Met-5A (A), MM-B1 (B), Meso-2 (C), and Ist-Mes2 cells (D) were seeded in 24-well plates and left overnight to recuperate. The cells were then at ~50% confluency and incubated with {alpha}-TOS at concentrations shown for 24, 48, or 72 h, and the level of proliferation was estimated by the BrdUrd assay. Cell proliferation is expressed relative to proliferation of control cells at the onset of the experiment. Data shown represent mean values ± S.D. (n = 3).

 
{alpha}-TOS Selectively Suppresses Expression of FGF-1 and FGF-2—Inhibition of cell cycle progression and proliferation by {alpha}-TOS is indicative of modulation by the agent of expression of genes that are involved in these processes. Therefore, we treated the cells with {alpha}-TOS at sub-apoptotic levels and assessed for the expression of several genes that have an important role in tumor growth, namely FGF-1 (acidic FGF), FGF-2 (basic FGF), TGF-{beta}, and VEGF. Overall, the MM and nonmalignant mesothelial cells express the individual mRNAs, with the exception of Ist-Mes2, where no VEGF mRNA was detected (Fig. 4). Although the relative levels of mRNA for TGF-{beta} and VEGF did not differ substantially, significant differences in the expression of FGFs were observed. FGF-2 mRNA was expressed ~2–3-times more than FGF-1 mRNA in all cell lines. Notably, Meso-2 cells expressed the highest levels of FGF-2 mRNA, whereas these cells expressed less FGF-1 mRNA. This pattern was similar for other MM cell lines. The nonmalignant Met-5A cells expressed relatively low levels of FGF-2 mRNA. When challenged with {alpha}-TOS at 10 or 20 µM, there was no significant effect on the level of expression of TGF-{beta} and VEGF mRNA. However, FGF-1 and FGF-2 mRNA was differentially regulated by the vitamin E analog (Fig. 5). In general, the pro-vitamin had an effect on both FGF-1 and FGF-2, particularly in Meso-2 and MM-B1 cells, whereas FGF-2 mRNA was consistently depressed to an increased extent. Finally, {alpha}-TOS exerted no effect on either FGF-1 or FGF-2 mRNAs in the nonmalignant Met-5A cells.



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  		FIG. 3.
{alpha}-TOS arrests MM cells in G1. Met-5A (A) and Meso-2 cells (B) were seeded in 24-well plates and left overnight to reach ~50% confluency. The cells were then treated with {alpha}-TOS for 24, 48, and 72 h at 10 and 20 µM, harvested, and assessed for cell cycle distribution as described under "Experimental Procedures." Data shown represent mean values ± S.D. (n = 3).

 



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  		FIG. 4.
Tumorigenic cytokines are expressed in MM cells. MM and nonmalignant mesothelial cells were assessed for relative expression of TGF-{beta} (A), VEGF (B), FGF-1 (C), and FGF-2 (mRNA) (D) by Q-PCR. The data are expressed relative to the level of FGF-2 mRNA in Meso-2 cells as detailed under "Experimental Procedures." All data are expressed relative to the corresponding level of FGF-2 mRNA in Meso-2 cells. Data shown represent mean values ± S.D. (n = 3).

 
To test whether suppression of the level of mRNA translates into a comparable change in protein expression, we studied FGF-1 and FGF-2 secretion from MM and Met-5A cells in the presence of {alpha}-TOS (Fig. 6). Inhibition of FGF-1 and FGF-2 secretion followed a similar trend to the corresponding mRNA, i.e. with no significant inhibition of FGF-1 and FGF-2 protein in Met-5A cells, whereas protein secretion was suppressed in MM-BI and, to an increased extent, in Meso-2 cells. Overall, the effect was more pronounced for FGF-2 as documented by immunostaining the cells for FGF-1 and FGF-2. We also observed less fluorescence in the pericellular regions of the MM cells treated with {alpha}-TOS compared with controls, whereas no difference in immunostaining was observed for Met-5A cells (data not shown).

{alpha}-TOS Suppresses FGF-1 and FGF-2 Expression by Inhibiting egr-1 Trans-activation—Because the inhibition of FGF-1 and FGF-2 occurred at a transcriptional level, we studied the effect of {alpha}-TOS on the transcription factor egr-1 that controls expression of FGF-2 (36, 37) and possibly FGF-1 expression (38). We first determined the effect of the pro-vitamin on the protein level of egr-1 in MM and Met-5A cells and found no difference (data not shown). Next, we studied the effect of {alpha}-TOS on egr-1 trans-activation. Thus, Met-5A and Meso-2 cells were transiently transfected with a plasmid harboring the egr-1 promoter, and luciferase activity was assessed as a surrogate for egr-1 trans-activation in the presence of {alpha}-TOS (Fig. 7). Both doses of {alpha}-TOS employed suppressed luciferase activity when compared with corresponding controls, whereas no effect was observed in Met-5A cells.

To obtain data that support a role for egr-1 trans-activation in FGF-1/FGF-2 expression and in proliferation, we used the RNAi approach. Thus, Meso-2 cells were challenged with egr-1, FGF-1, or FGF-2 siRNA, and proliferation was assessed. Treatment with the three different siRNAs suppressed proliferation of the cells, although the effect of egr-1 siRNA was most prominent, suggesting redundancy when only one of the two FGFs was knocked down. However, this effect appeared to be more pronounced for FGF-2. Inhibition of proliferation of siRNA-treated cells was overcome, at least partially, when the cells were supplemented with endogenous hrFGF-1 or, more significantly, with hrFGF-2. Next, we assessed the egr-1 siRNA-transfected cells for secretion of FGF-1 and FGF-2 (Fig. 8D). Overall, egr-1 knock-down also resulted in lower secretion of FGF-1 and FGF-2, again with a greater effect for FGF-2. These data strongly suggest that selectivity of inhibition of proliferation by {alpha}-TOS is linked to suppression of egr-1 trans-activation efficacy.

Selective Down-regulation of FGF-2 by {alpha}-TOS and ROS Generation—That inhibition of FGF-1 and FGF-2 mRNA expression is selective for MM cells strongly suggests a clinical potential of {alpha}-TOS. We were therefore interested in the mechanism underlying this selectivity. One possibility was that the nonmalignant cells hydrolyze the pro-vitamin to {alpha}-TOH, because this has been reported for several nonmalignant cell types, including hepatocytes and cardiomyocytes (25). Fig. 9 shows analysis of {alpha}-TOS and {alpha}-TOH in Met-5A and Meso-2 cells incubated with {alpha}-TOS. Although there was a 5–10-fold increase in {alpha}-TOH levels, this represents only a minor portion of the total intracellular {alpha}-TOS (50–100 ng/mg protein), indicating that hydrolysis of {alpha}-TOS is not responsible for the selectivity of the drug for MM cells.

We then investigated generation of radicals by the two cell lines in the presence of added {alpha}-TOS. Both the indirect flow cytometric assay, using the probe DCF, and the direct radical trapping consistently indicated ROS accumulation in {alpha}-TOS-treated Meso-2 but not Met-5A cells (Fig. 10). These data support the idea that Meso-2 cells respond to {alpha}-TOS by rapid accumulation of ROS. It is likely that superoxide is formed in Meso-2 cells exposed to {alpha}-TOS, because inclusion of exogenous SOD both abrogated the EPR detection of ROS and suppressed DCF fluorescence. Moreover, pre-loading Meso-2 cells with mito-Q suppressed significantly radical accumulation in Meso-2 cells. Mito-Q is a coenzyme Q analog specifically targeted to mitochondria because of the attachment of a trimethylphosphonium group (32). That mito-Q preferentially associates with mitochondria in mito-Q-exposed cells as well in animals fed mito-Q has been well documented (32, 39). It has also been shown that minutes upon uptake, the originally semiquinone form of mito-Q is reduced by the mitochondrial electron redox chain, acquiring a high activity to suppress ROS-dependent apoptosis (32, 40–42). Therefore, these data suggest that mitochondria are the source and/or the target for ROS generated by MM cells in response to added {alpha}-TOS.



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  		FIG. 5.
FGF-1 and FGF-2 are selectively down-regulated by {alpha}-TOS. MM and nonmalignant cells were seeded in 24-well plates, allowed to reach 50–70% confluency, and exposed to {alpha}-TOS at 10 and 20 µM for 24 h. Total RNA was then isolated, and the levels of mRNA for TGF-{beta} (A), VEGF (B), FGF-1 (C), and FGF-2 (D) were assessed by Q-PCR. Values for each cytokine are presented relative to its level in control Met-5A cells. Data shown represent mean values ± S.D. (n = 3). * indicates data significantly different from controls (p < 0.05).

 



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  		FIG. 6.
{alpha}-TOS suppresses secretion of FGF-1 and FGF-2 protein in MM cells. Met-5A (A and B), MM-B1 (C and D), and Meso-2 cells (E and F) were seeded in 24-well plates, allowed to reach ~50% confluency, and treated for 24, 48, and 72 h with {alpha}-TOS at 10 or 20 µM. The level of secreted FGF-1 (A, C, and E) and FGF-2 protein (B, D, and F) was assessed by ELISA and is expressed as total amount of protein in the medium per well. Data shown represent mean values ± S.D. (n = 3).

 
We next studied the effect of mito-Q on the inhibition of egr-1 trans-activation in MM cells exposed to {alpha}-TOS (Fig. 11). Addition of either mito-Q or SOD overcame {alpha}-TOS-inhibited egr-1-dependent trans-activation in cells transfected with egr-1-lux (Fig. 11A). In addition, mito-Q abrogated the down-regulation of FGF-2 by the added vitamin E analog in Meso-2 cells (Fig. 11B). Taken together with the data confirming generation of ROS upon {alpha}-TOS treatment, our findings support the notion that ROS are important mediators of the effect of {alpha}-TOS on the expression of FGF-2, which is regulated by egr-1. These data also provide evidence that resistance of nonmalignant cells to the toxic effects of {alpha}-TOS may be due to their lower capacity to generate ROS and/or their more efficient anti-oxidant system.



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  		FIG. 7.
{alpha}-TOS suppresses the egr-1 transcription activity in malignant but not nonmalignant mesothelioma cells. Met-5A and Meso-2 cells were seeded in 24-well plates and allowed to reach ~50% confluency, after which they were transiently transfected with a plasmid harboring the egr-1 promoter and the luciferase reporter. The cells were either left untreated or exposed for 24 h to 10 or 20 µM {alpha}-TOS. Luciferase activity was then assessed by a standard assay and expressed as relative luminescence compared with the relevant control cells. Data shown represent mean values ± S.D. (n = 3). * indicates data significantly different from controls (p < 0.05).

 
{alpha}-TOS Suppresses Mesotheliomas in Vivo—We have observed recently that {alpha}-TOS extended the life span of immunocompromised mice with experimental human peritoneal mesotheliomas (30). Here we investigated whether {alpha}-TOS retains this propensity also in the case of subcutaneously placed MM cell-derived xenografts in athymic mice. Fig. 12 shows that intraperitoneally administered {alpha}-TOS exerted a strong and highly significant effect, with ~90–95% inhibition of tumor growth over the 15-day period of treatment, further pointing to the potential of the vitamin E analog as an anti-mesothelioma agent.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this communication we present data pointing to a novel mode of action of {alpha}-TOS, a redox-silent vitamin E analog, relevant to its anti-tumor activity. Here we show that the pro-vitamin (i) selectively inhibits proliferation in MM cells; (ii) suppresses expression of FGF-1 and, in particular, FGF-2 in MM cells; (iii) causes high level of generation of ROS in MM cells, thereby inhibiting egr-1-dependent FGF-1/-2 trans-activation; and (iv) suppresses mesotheliomas in vivo. These major points of this communication epitomize an intriguing activity of {alpha}-TOS, by which it suppresses the FGF-FGFR autocrine signaling that results in decreased proliferation of MM cells, an effect that may translates into inhibition of mesothelioma progression. Our data with immunocompromised mice strongly support the idea that {alpha}-TOS is active against tumor progression and highlights its therapeutic potential of this vitamin E analog.



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  		FIG. 8.
Treatment of MM cells with egr-1 and FGF siRNA inhibits their proliferation. Meso-2 cells were seeded in 24-well plates, left to reach ~40% confluency, and transfected with egr-1 (A), FGF-1 (B), or FGF-2 siRNA (C) using specific oligonucleotide duplexes as detailed under "Experimental Procedures." Nonsilencing (NS) siRNA was used as a negative control. The cells were then assessed for proliferation using the BrdUrd method. D shows the levels of FGF-1 and FGF-2 secreted by the cells treated with egr-1 siRNA. Where indicated, cells were endogenously supplemented with hrFGF-1 or hrFGF-2 at 10 ng/ml 24 h prior to treatment. Data shown represent mean values ± S.D. (n = 3). * indicates data significantly different from controls (p < 0.05).

 



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  		FIG. 9.
Mesothelial cells do not hydrolyze {alpha}-TOS. Met-5A (A) or Meso-2 cells (B) were seeded in 24-well plates, allowed to reach ~50% confluency, and supplemented with 20 µM {alpha}-TOS. At the time points indicated, cells were extracted and assessed by high pressure liquid chromatography for the intracellular levels of {alpha}-TOH and {alpha}-TOS. Data shown represent mean values ± S.D. (n = 3).

 



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  		FIG. 10.
MM cells respond to {alpha}-TOS by generation of ROS. Met-5A (A and C) or Meso-2 cells (B and D) were seeded in 24-well plates, allowed to reach ~50% confluency, and exposed as indicated to 20 µM {alpha}-TOS following, as shown, pre-incubation (1 h prior to {alpha}-TOS treatment) with 2 µM mito-Q or 750 units/ml SOD. The cells were then washed and incubated for 30 min with DCF (A and B), after which the level of fluorescence was estimated by flow cytometry. C and D show the level of generation of ROS in the cells treated as above pre-loaded with the radical trap DMPO (final concentration 10 mM) and assessed for ROS by EPR spectroscopy. Data shown represent mean values ± S.D. (n = 3).

 



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  		FIG. 11.
ROS generated as a response to {alpha}-TOS inhibit egr-1 trans-activation. Meso-2 cells were seeded in 24-well plates, allowed to reach ~50% confluency, and exposed as indicated to 20 µM {alpha}-TOS for 24 h following, as shown, pre-incubation (1 h prior to {alpha}-TOS treatment) with 750 units/ml SOD or 2 µM mito-Q (mQ). The cells were then assessed for luciferase activity (A) or FGF-2 secretion (B). Data shown represent mean values ± S.D. (n = 3). * indicates data significantly different from controls (p < 0.05).

 
We and others (17, 21, 25, 26) have reported over the last few years anti-proliferative and pro-apoptotic activity of {alpha}-TOS and other analogs of vitamin E. The anti-proliferative/pro-apoptotic activity of these compounds is based on their structure; the molecule comprises three domains, the hydrophobic (phytyl chain), the signaling ({alpha}-tocopheryl moiety), and the functional domain (succinyl moiety in case of {alpha}-TOS) (24, 43). The major signaling pathway underlying the anti-proliferative/pro-apoptotic activity of {alpha}-TOS includes translocation of the pro-vitamin into the cell where it associates with subcellular structures, thereby causing their destabilization. This is followed/accompanied by generation of ROS, an event that can be suppressed by anti-oxidants (43–45). In line with this notion, scavengers of ROS also inhibit {alpha}-TOS-induced apoptosis (42, 46). Multiple additional pathways exist that modulate toxic effects of the vitamin E analog, either paralleling the major intrinsic signaling (proceeding via mitochondrial destabilization) and/or resulting from ROS-mediated downstream events. These include the TGF-{beta} pathway (26), activation of the nuclear factor-{kappa}B (41, 47), inhibition of the cell cycle transition (48, 49), and regulation of the c-Jun/c-Fos (50, 51) or the E2F1 transcriptional machinery (28, 48). Extracellular signal-regulated kinase/MAP kinase pathways have been suggested to link the upstream events and their ultimate downstream targets (22). Neither of these pathways explains the high toxicity of {alpha}-TOS toward malignant cells, while being largely nontoxic toward normal cells (17, 23, 29). Also, {alpha}-TOS has been shown to suppress several types of neoplasia in pre-clinical models, and it has been reported that the underlying cause is its ability to inhibit proliferation and induce apoptosis in tumor cells (17, 25). The potential of {alpha}-TOS as an anti-tumor agent and/or adjuvant has been further supported by findings that it agonized the tumor necrosis factor-related apoptosis-inducing ligand, inhibiting proliferation and killing MM cells while antagonizing the effects of the tumor necrosis factor-related apoptosis-inducing ligand in the case of normal mesothelial cells (29).



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  		FIG. 12.
{alpha}-TOS suppresses mesothelioma tumors in vivo. Nude mice were injected subcutaneously with Ist-Met2 cells (2 x 106 cells per mouse). The animals were left until solid tumors were established (~100 mm3), after which they were treated with intraperitoneal injections of 100 µl o f200 mM {alpha}-TOS in Me2SO or the vehicle every 2nd day. Tumor volume was estimated by micro-calipers and expressed as relative increase over the volume at the onset of treatment. The data shown represent mean values ± S.E. (n = 10). * indicates data significantly different from controls (p < 0.05).

 
We have observed recently (28) that {alpha}-TOS suppressed transcription of FGFR-1, likely via inhibition of its E2F1-dependent trans-activation. Of the possible ligands regulating proliferation, we found here that {alpha}-TOS suppressed expression of FGF-1 and FGF-2, ligands of FGFR-1, in all MM cell lines tested, whereas no effect was observed for the nonmalignant mesothelial cells. Because we observed no effect on VEGF or TGF-{beta}, the FGF-FGFR signaling loop is likely to be a major target for {alpha}-TOS in MM cells. Numerous reports document the importance of FGFs and their reporters for tumor growth and development, usually focusing on outcomes of paracrine signaling (53) such as angiogenesis (54). It has been also reported that the FGF-FGFR signaling may contribute to uncontrolled proliferation of cancer cells (55, 56) that may also be the case in the development of mesotheliomas.

Our major interest was to understand the selectivity with which {alpha}-TOS suppresses expression of FGF-1 and, in particular, FGF-2 in MM cells. The promoter of the FGF-2 gene contains an egr-1-binding site (57, 58). A link between egr-1 and FGF-1 is not well understood, although recent data suggest that there may be some degree of mutual regulation (38, 54). Both the FGF-2 mRNA and protein are depressed significantly more by {alpha}-TOS in MM cells than is FGF-1, which is consistent with the notion that FGF-2 plays an important role in MM progression and the clinical outcome of the pathology (6, 9). Several lines of evidence stipulate a role for egr-1 in {alpha}-TOS-induced FGF-2 and probably FGF-1 down-regulation. This follows from experiments in which {alpha}-TOS inhibited trans-activation of the lux gene under egr-1 control and from experiments where the pro-vitamin suppressed FGF-1 and FGF-2 secretion combined with the finding that egr-1 RNAi suppressed FGF-1 and FGF-2 secretion by MM cells. That these effects were more pronounced for FGF-2 than FGF-1 is consistent with the notion that FGF-2 has been associated with tumor promotion (59–61).

An intriguing finding is that these effects were observed in malignant cells, although their nonmalignant counterparts were not affected. The possibility that Met-5A cells were resistant to the toxic effects of {alpha}-TOS because they may hydrolyze it to its nontoxic vitamin form was ruled out because the concentration of intracellular {alpha}-TOS remained above toxicity levels in both cell lines. Such resistance to {alpha}-TOS was reported for cells like hepatocytes (62) or cardiomyocytes (63); moreover, it was stipulated that for such cells {alpha}-TOS is a rich source of vitamin E, rendering them more resistant to toxic insults (62, 64). We found that at least one major reason for resistance of nonmalignant mesothelial cells to {alpha}-TOS is because of their low level of accumulation of ROS (consistent with only marginally increased intracellular levels of {alpha}-TOH, cf. Fig. 9), whereas the malignant cells responded by generation of substantial ROS levels. This effect translated to inhibition of egr-1 trans-activation and secretion of FGF-2, because it was overridden in both cases by addition of SOD or mito-Q. The latter also suggests that mitochondria of the malignant cells are the source and/or target of ROS, although this needs more clarification. It is cannot be excluded at this stage that ROS are generated by other systems solely as a result of leakage from the mitochondrial electron transport chain, including the plasma membrane NADP(H) oxidase, the nonphagocytic gp91phox-like oxidase, xanthine oxidase, nitric-oxide synthase, phospholipase A2, or lipoxygenases (65–68). That ROS generation readily occurs following exposure of cancer cells to {alpha}-TOS has been reported before (42, 44, 69, 70). Moreover, it has been documented that a failure to respond to {alpha}-TOS by generation of ROS may render the cells resistant to the drug (69, 70).

The novel finding here is that ROS are probably responsible for the egr-1-mediated effect of {alpha}-TOS on FGF-1 and FGF-2 expression. Reports on the effect of oxidative stress on egr-1 are controversial. Although it has been suggested that hydrogen peroxide suppressed the transcriptional activity of egr-1 (71), others propose its role in cell survival following exposure to oxidative stress (72, 73). To complicate matters even further, a recent report (52) suggested up-regulation of egr-1 by hydrogen peroxide and showed inhibition of cell proliferation under identical conditions. It is thus possible that egr-1 is a component of a system that fine-tunes cellular responses to various levels of oxidative stress. Nevertheless, our data clearly suggest selective inhibition of egr-1 trans-activation by ROS generated by MM cells following exposure to {alpha}-TOS, which is based on its reversal and on reversal in expression of the egr-1-controlled FGF-2 by SOD and mito-Q.

Taken together, we propose that {alpha}-TOS, a redox-silent vitamin E analog with strong anti-cancer activity, selectively suppresses egr-1-dependent trans-activation of FGF-2, an important autocrine signaling molecule. By virtue of this, the pro-vitamin disrupts the FGF-FGFR autocrine signaling loop while efficiently suppressing proliferation of malignant mesothelioma cells. Because MM is currently a fatal type of neoplasia and because {alpha}-TOS, epitomizing a new group of anti-cancer agents (25), suppresses proliferation of MM cells, the vitamin E analog is a promising anti-mesothelioma agent.


   FOOTNOTES
 
* This work was supported in part by grants from the Dust Diseases Board of Australia, the Queensland Cancer Fund, the Australian Research Council Discovery grant, Project AV0Z5052014 from the Academy of Sciences of the Czech Republic (to J. N.), and by an Australian Research fellowship (to P. K. W.). 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. Back

§ Present address: Dept. of Dermatology, School of Medicine, University of Sydney, Sydney 2000, New South Wales, Australia. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Apoptosis Research Group, Heart Foundation Research Centre, School of Medical Science, Griffith University Gold Coast Campus, Southport, 4216 Queensland, Australia. Tel.: 61-7-555-29109; Fax: 61-7-555-28444; E-mail: j.neuzil{at}griffith.edu.au.

1 The abbreviations used are: MM, malignant mesothelioma; BrdUrd, 5-bromo-2-deoxyuridine; DCF, dihydrodichlorofluorescein diacetate; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; egr-1, early growth response factor-1; hr, human recombinant; FGF, fibroblast growth factor; FGFR, FGF receptor; FITC, fluorescein isothiocyanate; MAP, mitogen-activated protein; mito-Q, mitochondrially targeted coenzyme Q; PI, propidium iodide; Q-PCR, quantitative real time PCR; RNAi, RNA interference; ROS, reactive oxygen species; siRNA, short interfering RNA; SOD, superoxide dismutase; {alpha}-TOH, {alpha}-tocopherol; TGF-{beta}, transforming growth factor-{beta}; {alpha}-TOS, {alpha}-tocopheryl succinate; VEGF, vascular endothelial growth factor; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Drs. M. Murphy and R. Smith for providing mito-Q.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Steele, J. P. (2002) Semin. Oncol. 29,36 –40[Medline] [Order article via Infotrieve]
  2. Baas, P. (2003) Curr. Opin. Oncol. 15,127 –130[CrossRef][Medline] [Order article via Infotrieve]
  3. Tomek, S., Emri, S., Krejcy, K., and Manegold, C. (2003) Br. J. Cancer. 88,167 –174[CrossRef][Medline] [Order article via Infotrieve]
  4. Folkman, J. (1995) Nat. Med. 1,27 –31[CrossRef][Medline] [Order article via Infotrieve]
  5. Cross, M. J., and Claesson-Welsh, L. (2001) Trends Pharmacol. Sci. 22,201 –207[CrossRef][Medline] [Order article via Infotrieve]
  6. Kumar-Singh, S., Weyler, J., Martin, M. J., Vermeulen, P. B., and Van Marck, E. (1999) J. Pathol. 189,72 –78[CrossRef][Medline] [Order article via Infotrieve]
  7. Neufeld, G., Cohen, T., Gengrinovitch, S., and Poltorak, Z. (1999) FASEB J. 13,9 –22[Abstract/Free Full Text]
  8. Catalano, A., Romano, M., Martinotti, S., and Procopio, A. (2002) Oncogene 21,2896 –2900[CrossRef][Medline] [Order article via Infotrieve]
  9. Strizzi, L., Vianale, G., Catalano, A., Muraro, R., Mutti, L., and Procopio, A. (2001) Int. J. Oncol. 1,1093 –1098
  10. Marzo, A. L., Fitzpatrick, D. R., Robinson, B. W., and Scott, B. (1997) Cancer Res. 57,3200 –3207[Abstract/Free Full Text]
  11. Bermudez, E., Everitt, J., and Walker, C. (1990) Exp. Cell Res. 190,91 –98[CrossRef][Medline] [Order article via Infotrieve]
  12. Walker, C., Everitt, J., Ferriola, P. C., Stewart, W., Mangum, J., and Bermudez, E. (1995) Cancer Res. 55,530 –536[Abstract/Free Full Text]
  13. Ohta, Y., Shridhar, V., Bright, R. K., Kalemkerian, G. P., Du, W., Carbone, M., Watanabe, Y., and Pass, H. I. (1999) Br. J. Cancer 81,54 –61[CrossRef][Medline] [Order article via Infotrieve]
  14. Nowak, A. K., Lake, R. A., Kindler, H. L., and Robinson, B. W. (2002) Semin. Oncol. 29,82 –96[CrossRef][Medline] [Order article via Infotrieve]
  15. Neuzil, J. (2003) Br. J. Cancer 89,1822 –1826[CrossRef][Medline] [Order article via Infotrieve]
  16. Malafa, M. P., Fokum, F. D., Mowlavi, A., Abusief, M., and King, M. (2002) Surgery 131,85 –91[CrossRef][Medline] [Order article via Infotrieve]
  17. Weber, T., Lu, M., Andera, L., Lahm, H., Gellert, N., Fariss, M. W., Korinek, V., Sattler, W., Ucker, D. S., Terman, A., Schroder, A., Erl, W., Brunk, U. T., Coffey, R. J., Weber, C., and Neuzil, J. (2002) Clin. Cancer Res. 8,863 –869[Abstract/Free Full Text]
  18. Malafa, M. P., Fokum, F. D., Smith, L., and Louis, A. (2002) Ann. Surg. Oncol. 9,1023 –1032[Abstract/Free Full Text]
  19. Barnett, K. T., Fokum, F. D., and Malafa, M. P. (2002) J. Surg. Res. 106,292 –298[CrossRef][Medline] [Order article via Infotrieve]
  20. Neuzil, J., Weber, T., Schroder, A., Lu, M., Ostermann, G., Gellert, N., Mayne, G. C., Olejnicka, B., Negre-Salvayre, A., Sticha, M., Coffey, R. J., and Weber, C. (2001) FASEB J. 15,403 –415[Abstract/Free Full Text]
  21. You, H., Yu, W., Munoz-Medellin, D., Brown, P. H., Sanders, B. G., and Kline, K. (2002) Mol. Carcinog. 33,228 –236[CrossRef][Medline] [Order article via Infotrieve]
  22. Yu, W., Liao, Q. Y., Hantash, F. M., Sanders, B. G., and Kline, K. (2001) Cancer Res. 61,6569 –6576[Abstract/Free Full Text]
  23. Neuzil, J., Weber, T., Gellert, N., and Weber, C. (2001) Br. J. Cancer 84,87 –89[CrossRef][Medline] [Order article via Infotrieve]
  24. Birringer, M., EyTina, J. H., Salvatore, B. A., and Neuzil, J. (2003) Br. J. Cancer 88,1948 –1955[CrossRef][Medline] [Order article via Infotrieve]
  25. Neuzil, J., Tomasetti, M., Mellick, A. S., Alleva, R., Salvatore, B. A., Birringer, M., and Fariss, M. W. (2004) Curr. Cancer Drug Targets 4,267 –284[CrossRef][Medline] [Order article via Infotrieve]
  26. Turley, J. M., Funakoshi, S., Ruscetti, F. W., Kasper, J., Murphy, W. J., Longo, D. L., and Birchenall-Roberts, M. C. (1995) Cell Growth & Differ. 6,655 –663[Abstract]
  27. Malafa, M. P., and Neitzel, L. T. (2000) J. Surg. Res. 93,163 –170[CrossRef][Medline] [Order article via Infotrieve]
  28. Stapelberg, M., Tomasetti, M., Gellert, N., Procopio, A., and Neuzil, J. (2004) Biochem. Biophys. Res. Commun. 318,636 –641[CrossRef][Medline] [Order article via Infotrieve]
  29. Tomasetti, M., Rippo, M. R., Alleva, R., Moretti, S., Andera, L., Neuzil, J., and Procopio, A. (2004) Br. J. Cancer 90,1644 –1653[CrossRef][Medline] [Order article via Infotrieve]
  30. Tomasetti, M., Gellert, N., Procopio, A., and Neuzil, J. (2004) Int. J. Cancer 109,641 –642[CrossRef][Medline] [Order article via Infotrieve]
  31. Pass, H. I., Stevens, E. J., Oie, H., Tsokos, M. G., Abati, A. D., Fetsch, P. A., Mew, D. J., Pogrebniak, H. W., and Matthews, W. J. (1995) Ann. Thorac. Surg. 59,835 –844[Abstract/Free Full Text]
  32. Kelso, G. F., Porteous, C. M., Coulter, C. V., Hughes, G., Porteous, W. K., Ledgerwood, E. C., Smith, R. A., and Murphy, M. P. (2001) J. Biol. Chem. 276,4588 –4596[Abstract/Free Full Text]
  33. Thomas, S. R., Chen, K., and Keaney, J. F. (2002) J. Biol. Chem. 277,6017 –6024[Abstract/Free Full Text]
  34. Kogure, K., Manabe, S., Hama, S., Tokumura, A., and Fukuzawa, K. (2003) Cancer Lett. 192,19 –24[CrossRef][Medline] [Order article via Infotrieve]
  35. Fariss, M. W., Merson, M. H., and O'Hara, T. M. (1989) Toxicol. Lett. 47,61 –75[CrossRef][Medline] [Order article via Infotrieve]
  36. Biesiada, E., Razandi, M., and Levin, E. R. (1996) J. Biol. Chem. 271,18576 –18581[Abstract/Free Full Text]
  37. Wang, D., Mayo, M. W., and Baldwin, A. S. (1997) Oncogene 14,2291 –2299[CrossRef][Medline] [Order article via Infotrieve]
  38. Delbridge, G. J., and Khachigian, L. M. (1997) Circ. Res. 81,282 –288[Abstract/Free Full Text]
  39. Smith, R. A., Porteous, C. M., Gane, A. M., and Murphy, M. P. (2003) Proc. Natl. Acad. Sci. U. S. A. 100,5407 –5412[Abstract/Free Full Text]
  40. Dhanasekaran, A., Kotamraju, S., Kalivendi, S. V., Matsunaga, T., Shang, T., Keszler, A., Joseph, J., and Kalyanaraman, B. (2004) J. Biol. Chem. 279,37575 –37587[Abstract/Free Full Text]
  41. Alleva, R., Tomasetti, M., Andera, L., Gellert, N., Borghi, B., Weber, C., Murphy, M. P., and Neuzil, J. (2001) FEBS Lett. 503,46 –50[CrossRef][Medline] [Order article via Infotrieve]
  42. Wang, X. F., Witting, P. K., Salvatore, B. A., and Neuzil, J. (2005) Biochem. Biophys. Res. Commun. 326,282 –289[CrossRef][Medline] [Order article via Infotrieve]
  43. Neuzil, J. (2002) Biochem. Biophys. Res. Commun. 293,1309 –1313[CrossRef][Medline] [Order article via Infotrieve]
  44. Weber, T., Dalen, H., Andera, L., Negre-Salvayre, A., Auge, N., Sticha, M., Lloret, A., Terman, A., Witting, P. K., Higuchi, M., Plasilova, M., Zivny, J., Gellert, N., Weber, C., and Neuzil, J. (2003) Biochemistry 42,4277 –4291[CrossRef][Medline] [Order article via Infotrieve]
  45. Kogure, K., Morita, M., Nakashima, S., Hama, S., Tokumura, A., and Fukuzawa, K. (2001) Biochim. Biophys. Acta 1528,25 –30[Medline] [Order article via Infotrieve]
  46. Neuzil, J., Schroder, A., von Hundelshausen, P., Zernecke, A., Weber, T., Gellert, N., and Weber, C. (2001) Biochemistry 40,4686 –4692[CrossRef][Medline] [Order article via Infotrieve]
  47. Dalen, H., and Neuzil, J. (2003) Br. J. Cancer 88,153 –158[CrossRef][Medline] [Order article via Infotrieve]
  48. Turley, J. M., Fu, T., Ruscetti, F. W., Mikovits, J. A., Bertolette, D. C., and Birchenall-Roberts, M. C. (1997) Cancer Res. 57,2668 –2675[Abstract/Free Full Text]
  49. Ni, J., Chen, M., Zhang, Y., Li, R., Huang, J., and Yeh, S. (200) Biochem. Biophys. Res. Commun. 300,357 –363
  50. Qian, M., Kralova, J., Yu, W., Bose, H. R., Dvorak, M., Sanders, B. G., and Kline, K. (1997) Oncogene 15,223 –230[CrossRef][Medline] [Order article via Infotrieve]
  51. Zhao, B., Yu, W., Qian, M., Simmons-Menchaca, M., Brown, P., Birrer, M. J., Sanders, B. G., and Kline, K. (1997) Mol. Carcinog. 19,180 –190[CrossRef][Medline] [Order article via Infotrieve]
  52. Paron, I., D'Elia, A., D'Ambrosio, C., Scaloni, A., D'Aurizio, F., Prescott, A., Damante, G., and Tell, G. (2004) Biochem. J. 378,929 –937[CrossRef][Medline] [Order article via Infotrieve]
  53. Zhang, L., Kharbanda, S., Hanfelt, J., and Kern, F. G. (1998) Cancer Res. 58,352 –361[Abstract/Free Full Text]
  54. Santiago, F. S., Lowe, H. C., Day, F. L., Chesterman, C. N., and Khachigian, L. M. (1999) Am. J. Pathol. 154,937 –944[Abstract/Free Full Text]
  55. Fahmy, R. G., Dass, C. R., Sun, L. Q., Chesterman, C. N., and Khachigian, L. M. (2003) Nat. Med. 9,1026 –1032[CrossRef][Medline] [Order article via Infotrieve]
  56. Billottet, C., Elkhatib, N., Thiery, J. P., and Jouanneau, J. (2004) Mol. Biol. Cell 15,4725 –4734[Abstract/Free Full Text]
  57. Klint, P., and Claesson-Welsh, L. (1999) Front. Biosci. 4,D165 –D177[Medline] [Order article via Infotrieve]
  58. Polnaszek, N., Kwabi-Addo, B., Peterson, L. E., Ozen, M., Greenberg, N. M., Ortega, S., Basilico, C., and Ittmann, M. (2003) Cancer Res. 63,5754 –5760[Abstract/Free Full Text]
  59. Jin, Y., Sheikh, F., Dettillieux, K. A., and Cattini, P. A. (2000) Mol. Pharmacol. 57,984 –990[Abstract/Free Full Text]
  60. Straume, O., and Akslen, L. A. (2002) Am. J. Pathol. 160,1009 –1019[Abstract/Free Full Text]
  61. Lee, H. T., Lee, J. G., Na, M., and Kay, E. (2004) J. Biol. Chem. 279,32325 –32332[Abstract/Free Full Text]
  62. Tirmenstein, M. A., Ge, X., Elkins, C. R., and Fariss, M. W. (1999) Free Radic. Biol. Med. 26,825 –835[CrossRef][Medline] [Order article via Infotrieve]
  63. Roberg, K., and Ollinger, K. (1998) Am. J. Pathol. 152,1151 –1156[Abstract]
  64. Zhang, J. G., Nicholls-Grzemski, F. A., Tirmenstein, M. A., and Fariss, M. W. (2001) Chem. Biol. Interact. 138,267 –284[CrossRef][Medline] [Order article via Infotrieve]
  65. Thannickal, V. J., and Fanburg, B. L. (2000) Am. J. Physiol. 279,L1005 –L1028
  66. Babior, B. M., Lambeth, J. D., and Nauseef, W. (2002) Arch. Biochem. Biophys. 397,342 –344[CrossRef][Medline] [Order article via Infotrieve]
  67. Bokoch, G. M., and Knaus, U. G. (2003) Trends Biochem. Sci. 28,502 –508[CrossRef][Medline] [Order article via Infotrieve]
  68. Vaquero, E. C., Edderkaoui, M., Pandol, S. J., Gukovsky, I., and Gukovskaya, A. S. (2004) J. Biol. Chem. 279,34643 –34654[Abstract/Free Full Text]
  69. Kogure, K., Hama, S., Manabe, S., Tokumura, A., and Fukuzawa, K. (2002) Cancer Lett. 186,151 –156[CrossRef][Medline] [Order article via Infotrieve]
  70. Kang, Y. H., Lee, E., Choi, M. K., Ku, J. L., Kim, S. H., Park, Y. G., and Lim, S. J. (2004) Int. J. Cancer 112,385 –392[CrossRef][Medline] [Order article via Infotrieve]
  71. Nose, K., and Ohba, M. (1996) Biochem. J. 316,381 –383
  72. Esposito, F., Agosti, V., Morrane, G., Morra, F., Cuomo, C., Russo, T., Venutta, S., and Cimino, F. (1994) Biochem. J. 301,649 –653
  73. Hallahan, D. E., Dunphy, E., Virudachalam, S., Sukhatme, V. P., Kufe, D. W., and Weichselbaum, R. R. (1995) J. Biol. Chem. 270,30303 –30309[Abstract/Free Full Text]

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