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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

9L Gliosarcoma Cell Proliferation and Tumor Growth in Rats Are Suppressed by N-Hydroxy-N'-(4-butyl-2-methylphenol) Formamidine (HET0016), a Selective Inhibitor of CYP4A

Henry Ford Hospital, Detroit, Michigan (M.G., J.D.F., S.L.B., A.S.A., P.A.E.); Medical College of Wisconsin, Milwaukee, Wisconsin (R.J.R.); University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.); and Henry Ford Hospital and Wayne State University, Detroit, Michigan (A.G.S.)

Received October 28, 2005; accepted December 8, 2005.

	Abstract

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The present study examined the effects of N-hydroxy-N'-(4-butyl-2 methylphenyl) formamidine (HET0016), a selective inhibitor of the formation of 20-hydroxyeicosatrienoic acid (20-HETE) on the growth of 9L rat gliosarcoma cells in vitro and in vivo. After 48 h of incubation, HET0016 reduced the proliferation of 9L in vitro by 55%, and this was associated with a fall in p42/p44 mitogen-activated protein kinase and stress-activated protein kinase/c-Jun NH₂-terminal kinase phosphorylation and increased apoptosis. HET0016 inhibited epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-induced proliferation and diminished phosphorylation of PDGF receptors. A stable 20-HETE analog increased 9L cell proliferation. In vivo, chronic administration of HET0016 (10 mg/kg/day i.p.) for 2 weeks reduced the volume of 9L tumors by 80%. This was accompanied by a 4-fold reduction in the mitotic index, a 3- to 4-fold increase in the apoptotic index, and a 50% decrease in vascularization in the tumor. HET0016 treatment increased mean survival time of the animals from 17 to 22 days. Liquid chromatography/mass spectrometry experiments indicated that neither 9L cells grown in vitro nor 9L tumors removed produce 20-HETE when incubated with arachidonic acid. The normal surrounding brain tissue, however, avidly makes 20-HETE, and this activity is selectively inhibited by HET0016. These results suggest that HET0016 may be the prototype of a class of antigrowth compounds that may be efficacious for treating malignant brain tumors. In vivo, it may act in part by inhibiting the formation of 20-HETE by the surrounding tissue. However, the antiproliferative effects of HET0016 on 9L cells in vitro seem unrelated to its ability to inhibit the formation of 20-HETE.

20-HETE has been implicated as playing a role in promoting the growth of several cell types (Roman et al., 2002). Blockade of 20-HETE formations attenuated the growth responses to serum, norepinephrine, and EGF (Lin et al., 1995). Muthalif et al. (1998) reported that activation of the Ras/MAPK by norepinephrine, angiotensin II, and EGF is dependent on the formation of 20-HETE, which is generated after activation of phospholipase A₂ by calcium/calmodulin protein kinase II. This suggests that 20-HETE plays a central role in the regulation of other cellular signaling molecules involved in cell proliferation and growth.

N-Hydroxy-N'-(4-butyl-2 methylphenyl) formamidine (HET0016) has recently been reported to be a highly selective inhibitor of the synthesis of 20-HETE by enzymes of the CYP4A and CYP4F families (Miyata et al., 2001; Seki et al., 2005). In subsequent studies, HET0016 was found to inhibit the angiogenic responses to EGF, VEGF, and FGF and electrical stimulation in rats (Amaral et al., 2001; Chen et al., 2005). HET0016 also blocked angiogenesis in the cornea stimulated by the implantation of human U251 glioblastoma cells (Chen et al., 2005) as well as the proliferation of U251 cells grown in vitro (Guo et al., 2005). In the latter study, we also found that HET0016 inhibited the phosphorylation of the EGF receptor (EGFR) and decreased growth responses of U251 cells to EGF.

These studies suggested that HET0016 might have therapeutic potential for the treatment of glioma. The 9L gliosarcoma, which was chemically induced in an inbred Fischer rat, has been one of the most widely used of all rat brain tumor models and has provided much useful information relating to brain tumor biology and therapy. There is no information on the role of CYP4A products in the growth of 9L gliosarcoma cells. Thus, the present study examined the effects of HET0016 on the proliferation of a rat 9L gliosarcoma cell in vitro and the growth of 9L tumors after implantation of these cells in the brains of rats (Schmidek et al., 1971; Barth, 1998).

View larger version (19K): [in this window] [in a new window]   Fig. 1. HET0016 inhibits the proliferation of 9L gliosarcoma cells. A, equal numbers of 9L cells (0.75 x 104) were plated and serum starved for 1 day before exposure to various concentrations of HET0016. Cell counting was performed at 24 and 48 h thereafter. B, [3H]thymidine incorporation into DNA was assessed in cultures treated with 10 µM HET0016. Incorporation data were calculated as disintegrations per minute per 103 cells and normalized to the EtOH controls. Data presented are means ± S.D. from four separate experiments, each performed in triplicate. *, P < 0.05.

	Materials and Methods

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Cell Proliferation Assays. Proliferation studies were performed with cultures plated at a density that ensured exponential growth for at least 5 days. Unless indicated otherwise, the growth medium was replaced with serum-free medium 24 h after the cells were plated, because HET0016 is avidly bound by serum proteins. Cell growth was followed for 24 or 48 h after stimulation with the addition of EGF or PDGF (200 ng/ml) to the medium in the presence or absence of 10 µM HET0016 (Taisho Pharmaceuticals Co. Ltd., Satiama, Japan). Other cultures were treated with 20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid (WIT003), a stable 20-HETE agonist. All of the test agents were dissolved in EtOH and an equal volume of EtOH was added to the cultures as vehicle control. The concentration of EtOH in the media never exceeded 0.1%. After 24 or 48 h of exposure to the test agents, the cells were harvested by exposure to a solution of 0.25% trypsin-EDTA and counted using a hemocytometer.

View larger version (41K): [in this window] [in a new window]   Fig. 2. HET0016 induces apoptosis in 9L cells. Cells were plated and treated with 10 µM HET0016 for 48 h as described before. Attached and floating cells were harvested, pooled, and pelleted. Cell pellets were resuspended into a 1 x 106 cell/ml suspension and labeled with annexin V and PI as recommended by the manufacturer. Flow cytometry was performed to examine the effects of HET0016 on inducing apoptosis in 9L. Representative charts are shown. Lower left (LL), annexin V- and PI-negative—viable cells; lower right (LR), annexin V-positive—early apoptotic cells; upper left (UL), PI-positive—necrotic cells; upper right (UR), annexin V- and PI-double positive—mix of necrotic and late apoptotic cells. HET0016 significantly increased early apoptotic (LR) and dead cells (UR). Data represent means ± S.D. of two different experiments, each performed in triplicate. *, P < 0.05 in HET0016-treated versus control cells.

Assessment of Apoptosis. The effects of HET0016 to induced apoptosis in 9L cells were examined by fluorescence-activated cell sorting analysis after the cells were labeled with an annexin V-fluorescein isothiocyanate (FITC) antibody (Sigma Chemical, St. Louis, MO). After incubation of 9L cells with either vehicle or 10 µM HET0016 for 48 h, the cells were trypsinized, washed, and pelleted. Nonattached floating cells were also collected by centrifugation. The cells were resuspended in a binding buffer, treated with 5 µl of annexin V-FITC conjugate and 10 µl of propidium iodide (PI), and incubated in the dark at room temperature for 20 min before performance of a fluorescence-activated cell sorting analysis using a FAC-Scan (Becton Dickinson, Franklin Lakes, NJ) cell sorter. A minimum of 10⁴ events/sample was collected. Data were analyzed using CellQuest Pro software (BD Biosciences, San Jose, CA).

Western Blotting. 9L cells were treated with vehicle or 10 µM HET0016 for various times and washed twice with ice-cold PBS. Cells were then lysed using a radioimmunoprecipitation assay buffer [20 mM HEPES (pH 7.4), 100 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% deoxycholic acid, 10% glycerol, 1 mM EDTA, 1 mM NaVO₃, 50 mM NaF, and protease inhibitors Set 1 (Calbiochem, La Jolla, CA)]. The plates were scraped, and the cells were collected into a centrifuge tube, followed by incubation on ice for 30 min. The cell homogenates were centrifuged for 10 min at 14,000g and 4°C, and the pellets were discarded. The protein concentration of the supernatant was determined using the BCA protein assay (Pierce Biotechnology Inc., Rockford, IL). Typically, 10 µg of homogenate protein was separated on a 10 to 14% Tris-glycine gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Biotrace, Bothell, WA). The membranes were treated for 1 h at room temperature with blocking buffer [0.2% I-Block reagent (Tropix, Bedford, MA), 0.1% Tween 20 in 1x PBS] and then incubated with primary antibodies in blocking buffer overnight at 4°C. The primary antibodies used were phospho-p42/p44 MAPK (T202/Y204) (20G11) and phospho-SAPK/JNK (T183/Y185) (98F2) monoclonal antibodies, phospho-EGF receptor, and phospho-PDGF- (Y76) antibodies (Upstate, Waltham, MA). The phospho-p44/p42 MAPK and phospho-SAPK/JNK antibodies were used at a dilution of 1:1000, whereas the EGFR and PDGF receptor antibodies were used at a 1:500 dilution. After washing, the membranes were incubated for 1 h at room temperature with a 1:4000 dilution of peroxidase-conjugated secondary anti-goat, -mouse, or -rabbit antibodies (Upstate). The membranes were then washed three times and developed using an enhanced chemiluminescence kit (Upstate). Stripped membranes were reprobed with actin antibody that served as a loading control.

Tumor Implantation. Brain tumors were seeded by injecting 9L cell suspensions into the cerebral cortex of male Fisher 344 rats (Charles River Laboratories, Inc., Wilmington, MA). The rats were anesthetized with ketamine (80 mg/kg i.m.) and xylazine (10 mg/kg i.m.). The head was immobilized in a stereotactic frame (David Kopf Instruments, Tujunga, CA). After a skin incision to expose the skull, a small hole was drilled 2 mm lateral and 2.5 mm anterior of the bregma. Immediately before each implantation, cells were trypsinized and resuspended in serum-free DMEM, containing no additional supplements. A 10-µl Hamilton syringe attached to a 26-gauge needle was inserted 3.5 mm into the cerebral cortex and 1 x 10⁴ cells in 5 µl of DMEM was injected over a 5-min period. A 5-min waiting period was observed before slow withdrawal of the needle. The hole in the skull was sealed with bone wax, and the incision was closed (Kim et al., 1999, 2003). The tumor was allowed to grow for 2 days before the rats received twice-daily i.p. injections of HET0016 (10 mg/kg/day in 10% lecithin in water) or vehicle (10% lecithin in water). Others have used this dose (Hoagland et al., 2003b; Caron et al., 2004). Under these conditions the average HET0016 values in plasma are close to the 10 µM dose used in vitro (R. J. Roman, personal communication).

After 2 weeks, the rats were anesthetized, a catheter was inserted into the left ventricle of the heart, and blood was flushed from the vasculature by perfusion with 250 ml of 0.9% saline solution. This was followed by perfusion fixation with 200 ml of a 10% solution of buffered formalin in PBS. The brains were then collected and stored in 10% formalin.

Histology and Immunohistochemistry. The formalin-fixed brains were sliced into 3-mm-thick blocks. These blocks were embedded in paraffin and cut into 6-µm-thick sections that were mounted on positively charged frosted slides. Alternated serial sections were either stained with hematoxylin and eosin (H&E) to assess the size of the tumor or processed for immunohistochemical identification of markers of proliferation (Ki-67 antigen), apoptosis [terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay] or vascular density by staining with fluorescein Lycopersicon esculentum lectin or von Willebrand factor (VWF) immunoreactivity. Images of H&E-stained serial sections containing tumor were captured using a SONY charge-coupled device camera using a 2x objective. Because the sizes of tumor between vehicle- and HET0016-treated animals are substantially different, the distance between the first and the last serial sections that were identified as positive for tumor staining by H&E was used to define the sagittal distance of the tumor. The area of each section was measured using AIS Image Analysis System software (Imaging Research, St. Catherines, ON, Canada). Tumor volume was determined from the area of tumor in each section multiplied by the thickness of the sections and then summed to obtain the total tumor volume for each brain.

For Ki-67 immunohistochemical analysis, the sections prepared were deparaffinized and boiled in a citric acid buffer (pH 6.0) for 20 min. The sections were allowed to cool for 20 min, placed in a blocking buffer (2% normal goat serum, 1% bovine serum albumin in PBS) for 1 h, rinsed twice in a wash buffer (0.5% Tween 20 in PBS), blocked with H₂O₂ for 10 min, and then rinsed in wash buffer. The sections were incubated for 30 min with a 1:200 dilution rabbit polyclonal anti-Ki-67 antibody in 1.0% bovine serum albumin in PBS (Abcam Inc., Cambridge, MA). The sections were rinsed in a washing buffer, incubated with biotinylated goat anti-rabbit IgG diluted 1:500 in PBS (Vector Laboratories, Burlingame, CA) for 30 min, and rewashed. The sections were then incubated with a 1:500 dilution of horseradish peroxidase-streptavidin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min, washed, and incubated with 3,3'-diamino-benzidine substrate (Vector Laboratories) for 8 min. The sections were washed, counterstained with Meyer's hematoxylin for 5 s and exposed to ammonia in water, and then they were dehydrated, cleared, and mounted.

Tumor apoptosis was assessed with an ApopTag Plus Peroxidase detection kit (Chemicon International, Temecula, CA) according to the manufacturer's recommendations. Briefly, rehydrated sections were digested with proteinase K (20 µg/ml) for 15 min at room temperature. This was followed by quenching in 3.0% solution of H₂O₂ for 5 min and washing. The sections were then labeled with a terminal deoxynucleotidyl transferase enzyme in a humidified chamber at 37°C for 1 h, and anti-digoxigenin conjugate was applied for 30 min at room temperature. Sections were washed, developed in peroxidase substrate, counterstained in 0.5% solution of methyl green for 10 min, dehydrated, and mounted for light microscopy.

Proliferation and Apoptosis Indices. Five animals per group were used to assess the effects of HET0016 on the proliferative and apoptotic indices in the 9L tumors. Images of Ki-67 staining and TUNEL assay were captured using a 10x, 20x, or 40x objective. The images were overlaid with a grid, and the total number of Ki-67 positively stained and TUNEL-positive cells were counted. The proliferation and apoptosis indices were calculated as the number of positively stained cells divided by the total number of cells in the grid multiplied by 100. The proliferation and apoptosis indices of at least six fields per section were averaged, and this value was used to calculate mean values ± S.E.M.

Tumor Vascularization. Tumor vascularization was determined by immunostaining the sections with von Willebrand factor or fluorescein L. esculentum (tomato) lectin. The sections were stained for von Willebrand factor as follows. The brain sections were deparaffinized and boiled in citric acid buffer (pH 6.0) for 30 min. After samples were cooled for 20 min, the tissue sections were digested with proteinase K (20 µg/ml) for 15 min at 37°C. The sections were incubated with a 1:500 dilution of an anti-VWF antibody (Chemicon International) followed by a 1:500 dilution of secondary antibody as described for Ki-67. The images were taken from two random fields per section using a 20x objective. The sections were obtained from the midtumor region of five animals per group. Vessel densities were assessed by counting VWF-positive structures and expressed as numbers of these structures per field. In another experiment, vessels were also visualized in five animals per group after the tissue sections were stained with a tomato lectin. Briefly, tumor sections were deparaffinized and rehydrated in distilled water. The sections were labeled with a 1:40 dilution of a tomato lectin (Vector Laboratories) at 4°C overnight. The sections were then rinsed with water followed by application of Vectashield mounting media with DAPI (Vector Laboratories). Finally, the slides were covered and mounted for fluorescent microscopy. Section representative images were captured using a 10x and 50x objectives.

Survival Studies. Rats were implanted with a 9L tumor. After 2 days, they begin to receive twice-daily i.p. injections of vehicle or HET0016 (10 mg/kg/day). Rats were monitored daily for loss of weight, signs of toxicity, and behavioral changes. They were sacrificed if a loss of mobility, lethargy, hemorrhaging around the eyes, or a hunched posture was observed.

Cytochrome P450 Metabolism of Arachidonic Acid. 9L cultures were plated and grown overnight followed by serum starvation. The cells were harvested, pelleted, and snap-frozen in liquid N₂. The metabolism of AA was also studied in the homogenates of brain tumors and normal tissue from the control hemisphere. The edge of each tumor was trimmed to remove surrounding normal tissue. The tissues and cells were homogenized in a 10 mM KPO₄ buffer containing 300 mM sucrose and centrifuged at 3000g followed by 11,000g for 20 min. In preliminary studies we performed kinetic studies using 1 mg of protein from brain microsomes incubated with 10 to 100 µM arachidonic acid for 30 min. We found that V_max was obtained at 42 µM substrate. Under these conditions the amount of 20-HETE produced was such that it was easily detected and measured by mass spectrometry. Based on these data, aliquots of the supernatant (1 mg of protein) were incubated with a saturating concentration of arachidonic acid (40 µM) in a 0.1 M KPO₄ buffer containing 1 mM NADPH for 30 min at 37°C in the presence or absence of 10 µM HET0016. The incubations were stopped by acidification with formic acid and extracted with chloroform-methanol (2:1) after addition of 1 ng of an internal standard, 14,15-epoxyeicosa-5(Z)-enoic-methyl sulfonylimide (EEZE). The samples were reconstituted in 50% acetonitrile, and then the HETEs and epoxyeicosatrienoic acids (EETs) in the samples were separated using an isocratic step gradient on an 18C-RP 2 x 250-mm microbore HPLC column (150 x 21 3 µm; BetaBasic18; Thermo Hypersil-Keystone) using a mobile phase consisting of acetonitrile/water/acetic acid (57:43:0.1) for 20 min to resolve the HETEs followed by acetonitrile-water-acetic acid (63:37:0.1) for 15 min to resolve the EETs. The samples were ionized using an electrospray source, and the peaks eluting with a mass/charge ratio (m/z) of 319 (HETEs and EETs) or 323 (internal standard) were isolated and monitored in the selective ion mode using an Agilent LSD ion trap mass spectrometer 1100 (Agilent Technologies, Palo Alto, CA). The ratio of ion abundance in the peaks of interest (HETEs and EETs; m/z 319) versus that corresponding to the closely eluting internal standard (EEZE; m/z 323) was determined and compared with a standard curve generated over a range from 0.1 to 2 ng of 20-HETES and EETs with each batch of samples.

Statistical Analysis. Data from the in vitro experiments were analyzed using analysis of variance followed by a Tukey's test. A t test was used to evaluate the significance of differences in tumor volume, mitotic index, apoptotic index, flow cytometric analysis, and density of VWF-positive structures in vehicle- and HET0016-treated rats. A Wilcoxon rank-sum test was used to analyze the survival data. P < 0.05 was considered to be significant.

	Results

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Study of 9L Cells in Vitro
HET0016 Inhibits 9L Cell Proliferation. HET0016 produced a dose-dependent inhibition of the growth of 9L cells in culture (Fig. 1A). HET0016 at concentration of 1 and 10 µM significantly reduced cell number at 48 h by 30 and 55%, respectively. This led to the selection of 10 µM HET0016 for the subsequent in vitro experiments. As is presented in Fig. 1B, HET0016 (10 µM) reduced thymidine incorporation in 9L cells by 60%.

To determine whether 20-HETE stimulates proliferation of 9L cells, we examined the effects of WIT003, a 20-HETE stable analog, on their growth. At a concentration of 1 µM WIT003 increased the proliferation of 9L cells by 32% (P < 0.05, n = 3). Concentrations higher than 5 µM led to cell detachment and death.

Effect of HET0016 on Apoptosis in 9L Cells. 9L cells grown in culture for 48 h were labeled with annexin-V and PI, and the number of apoptotic cells was analyzed by flow cytometry. We found that HET0016 induced a significant increase in the percentage of cells labeled by annexin V, an indicator of cells in the early stages of apoptosis, as well as in cells labeled by annexin V and PI, an indicator of mixed necrotic and late apoptotic cell populations (Fig. 2).

Effect of HET0016 on PDGF-Stimulated Proliferation and Phosphorylation of the PDGF Receptor. The results of these experiments are presented in Fig. 3. We studied whether HET0016 would affect growth responses to exogenously added growth factors. We first performed a dose-response curve with EGF and found that 9L cells are not very responsive to this growth factor, because their proliferation was barely affected by concentrations of EGF up to 100 ng/ml. At a concentration of 200 ng/ml, EGF did increase their growth by 28 ± 7.5% (P < 0.05, n = 3). Treatment of the cells with HET0016 not only completely inhibited the effects of EGF on the growth of these cells, but decreased the cell count to 70 ± 3.2% of the number of cells seen in cultures treated with vehicle alone (P < 0.05, n = 4). 9L cells show considerable basal phosphorylation of the EGFR, and we were unable to document increases in EGFR phosphorylation by EGF stimulation or any effect of HET0016. We also tested the effects of hepatocyte growth factor, VEGF, bovine FGF-2, and PDGF on the proliferation of 9L cells grown in culture. Of these, only PDGF (200 ng/ml), increased proliferation rate 30% on average. The proliferative effects of PDGF were significantly (P < 0.05) inhibited by 10 µM HET0016 (Fig. 3A). Subsequently, we tested the effects of HET0016 on the phosphorylation of PDGF receptor in 9L cultures treated with 200 ng/ml PDGF. We found that phosphorylation of PDGF receptor increased by 3.5-fold in 9L cells treated with PDGF, and 10 µM HET0016 inhibited this effect by 50% (Fig. 3, B and C).

View larger version (23K): [in this window] [in a new window]   Fig. 3. HET0016 inhibits PDGF-stimulated 9L proliferation and PDGF receptor phosphorylation. A, serum-starved 9L cultures were treated with either 200 ng/ml PDGF alone, 10 µM HET0016, or both together. Cell proliferation was evaluated 48 h later. Data are presented as folds of increase in cell numbers from T0. *, P < 0.05 versus controls; #, P < 0.05 versus PDGF-stimulated cultures. B, HET0016 decreased the phosphorylation of PDGF- receptor (PDGF R) induced by PDGF measured 30 min after PDGF addition. Images are representative of three separate experiments. C, densitometry analysis of the effects of HET0016 on PDGF- receptor phosphorylation.

Effects of HET0016 on the Activation of the MAPK and SAPK/JNK Pathways in 9L Cells. The phosphorylation and activation of MAPKs are known to play a key role in the regulation of cell growth and proliferation. Treatment of 9L cells with HET0016 (10 µM) for 4 h significantly reduced the phosphorylation of p42/p44 MAPK (Fig. 4A). The reduction in the phosphorylation of p42/p44 MAPK was greater after 24 h of exposure to HET0016. A similar trend was observed for the phosphorylation of SAPK/JNK (Fig. 4B). No changes were detected in the levels of total p42/p44 MAPK or SAPK/JNK in these cells.

View larger version (53K): [in this window] [in a new window]   Fig. 4. HET0016 inhibits p42/p44 MAPK and SAPK/JNK phosphorylation. Cultures were treated with 10 µM HET0016 for 4, 24, and 48 h. Total and phosphorylated p42/p44 MAPK and SAPK/JNK were assessed by Western blotting. Actin was used to control for uniformed loading. A, HET0016 decreases the phosphorylation of p42/p44 MAPK as early as 4 h. B, HET0016 markedly decreases the phosphorylation of SAPK/JNK. Data shown are representative blots of three separate experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 5. 20-HETE synthesis was not detected after incubation of 9L cell extracts with arachidonic acid. 9L cells were plated, harvested, pelleted, and resuspended in serum-free medium and quickly frozen in liquid N2. LC/MS analysis was performed as described in Materials and Methods; 0.1 ng of 20-HETE, 1.3 ng of HETEs/EETs, and 10 ng of EEZE were used as positive controls. 9L controls are in blue, and samples treated with 10 µM HET0016 are shown in green.

View larger version (82K): [in this window] [in a new window]   Fig. 6. HET0016 decreases 9L tumor growth in vivo. 9L cells (1 x 104) were injected into brains of rats. After 2 days, the rats were treated with lecithin (vehicle) or HET0016 (10 mg/kg/day) for 2 weeks. A, appearance of brain tumors from vehicle- and HET0016-treated rats. B, H&E sections through the midpoint of the tumors in rats treated with either vehicle or HET0016. C, the tumor volumes were measured as described in Materials and Methods. Data shown are presented as means ± S.D. of six animals per group. *, P < 0.05.

View larger version (112K): [in this window] [in a new window]   Fig. 7. HET0016 decreases the proliferative index. A, rabbit polyclonal anti-Ki-67 antibody was used to label mitotic cells in tumor sections. Representative images are shown at 10x and 40x magnifications. B, the proliferation index was calculated as described in Materials and Methods. Results shown are means ± S.D. of five animals per group. *, P < 0.05.

The effects of HET0016 on the percentage of apoptotic cells in the 9L tumors are presented in Fig. 8A. A considerable number of TUNEL-positive cells were present in the tumors of the vehicle-treated rats. The number of apoptotic cells was 3- to 4-fold greater in 9L tumors obtained from rats treated with HET0016 than that seen in the control rats (Fig. 8B).

View larger version (113K): [in this window] [in a new window]   Fig. 8. HET0016 increases the apoptotic index. A, TUNEL assay was performed using an ApopTag Plus Peroxidase in situ apoptosis detection kit according to the manufacturer's instructions. Representative images are shown at 20x and 40x magnifications. B, the apoptosis index was calculated as described in Materials and Methods. Results shown are means ± S.D. of five animals per group. *, P < 0.05.

View larger version (144K): [in this window] [in a new window]   Fig. 9. HET0016 alters vascularization in 9L tumors. Tomato lectin was used to stain 9L tumor serial sections for blood vessel structures. Images were captured at 10x and 50x objectives. DAPI was used to visualize nuclei. Images shown are representative of merged pictures of lectin positive (green fluorescence) and DAPI (blue) of three animals per group.

To further confirm the effects of HET0016 to inhibit vessel recruitment into the tumors, sections obtained from the middle of the tumors were also stained using a von Willebrand factor antibody that stains endothelial cells. We found that HET0016 significantly decreased the number of VWF-positive structures by almost 50% compared with the number of structures in vehicle-treated rats (30 ± 6 in control versus 16 ± 4 in the treated animals; P < 0.05) (Fig. 10).

View larger version (115K): [in this window] [in a new window]   Fig. 10. HET0016 decreases the number of von Willebrand factor-positive structures in 9L tumors. A, immunohistochemical analysis using anti-VWF antibodies was performed in 9L tumor sections. Representative images were captured using 20x objectives. B, VWF-positive structures were counted in random 20x fields and expressed as the number of positive structures per field. Results shown are presented as means ± S.D. of five animals per group. *, P < 0.05.

View larger version (12K): [in this window] [in a new window]   Fig. 11. HET0016 prolongs the survival of 9L tumor-bearing rats. 9L-implanted rats were injected every 12 h with either vehicle or 5 mg/kg HET0016. Animals were either found dead or sacrificed when they showed signs of overt neurological stress as described in Materials and Methods. Results are presented as means ± S.D. of five animals per group. *, P < 0.05.

View larger version (25K): [in this window] [in a new window]   Fig. 12. 9L-induced gliosarcomas produce negligible amounts of 20-HETE in vivo. Normal brain (Normal) and tumor core tissues were excised and snap-frozen in liquid N2. Care was taken to avoid including peritumoral tissues in the brain tumor samples. Fractions were incubated with arachidonic acids, and fluorescent HPLC analysis was performed to detect 20-HETE synthesis. A, representative HPLC and chromatogram of arachidonic metabolites of both normal brain tissues and 9L tumors. B, 20-HETE production in normal and tumor tissue is presented as means ± S.E.M. of five samples per group. *, P < 0.05.

	Discussion

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HET0016 was synthesized as a highly selective inhibitor of CYP4A (Miyata et al., 2001; Sato et al., 2001; Yu et al., 2004). We recently found that HET0016, markedly decreased angiogenic responses induced by a glioma cell line and by VEGF, EGF, and FGF-2 (Chen et al., 2005). In addition, HET0016 inhibited growth of U251 human glioma cells in vitro (Guo et al., 2005). These studies indicated that HET0016 may have therapeutic potential to retard the growth of glial tumors. Many investigators have used 9L rat gliosarcoma cells as a model system to study the effects of different therapies on the growth of glioblastoma tumors (Barth, 1998; Straubinger et al., 2004). We examined the effects of HET0016 on the proliferation of 9L cells in culture and the growth of 9L gliosarcoma in vivo.

We found that addition of 10 µM HET0016 to 9L cells in culture markedly decreases their rate of proliferation. These inhibitory effects were associated with a significant reduction in the phosphorylation of p42/p44 MAPK and SAPK/JNK. HET0016 suppressed growth responses of 9L cells to EGF, as it did in U251 cells (Guo et al., 2005). Another growth factor, PDGF, triggered a significant growth response that was also inhibited by HET0016. This inhibition was associated with a reduction in the phosphorylation of the PDGF receptor. HET0016 also inhibited phosphorylation of the EGF receptor by EGF in U251 (Guo et al., 2005). These results suggest that HET0016 interferes with signals that drive some glioma cell proliferation in vitro, appearing to act upstream of growth factor receptor activation.

We examined the effects of HET0016 on the apoptosis of the 9L cells by annexin V staining followed by flow cytometry. For this analysis, we used both attached and detached cells. Annexin V binds to externalized phosphatidylserine in all stages of the cell death program, including the early stages (Maiese and Vincent, 2000). Exposure to HET0016 for 48 h markedly increased the number of annexin V-positive cells, thus suggesting that HET0016 triggers the programmed cell death. Cells that are labeled by both PI and annexin V are a mix of necrotic and late apoptotic cell populations and constituted a small fraction of the cell population. HET0016 doubled the number of cells labeled with both markers. In some tumor cells, treatment with compounds that inhibit cell proliferation can lower the apoptotic threshold (Milella et al., 2005), and HET0016 may act by such a general mechanism. The precise pathways affected by HET0016 that lead to apoptosis will require further study. There may be cell specificity in the proapoptotic effect of HET0016 because we did not observe apoptosis by TUNEL assay in cultured U251 cells treated with HET0016 (Guo et al., 2005). In that work we did not look at detached cells, only at attached cells. In addition, the TUNEL assay may not detect cells in the early stages of apoptosis as flow cytometry for annexin V binding does.

Together with the studies on U251 cells (Guo et al., 2005), these data suggest that HET0016 has antitumor activity against gliomas. We tested whether HET0016 would affect the growth of experimentally induced 9L brain tumors in rats. We used a Winn-type preventive approach, starting treatments 2 days after 9L cell implantation. The animals were sacrificed after 2 weeks of treatment. At this time, vehicle-treated rats have large tumors. Visually, the HET0016-treated rats had a healthier appearance than the untreated animals. A well defined tumor was still present in the HET0016-treated rats, but the volume of the tumor was, on average, 80% less than that seen in the vehicle-treated rats. Most of the sections of the rats treated with HET0016 showed tumor edges with less infiltrating tumor satellites compared with the edge of the untreated control rats, which show many satellites, infiltrating tumor cells, or clusters at the periphery of the tumor. Untreated 9L tumors also show zones of necrosis. There was little necrosis in the tumors of the HET0016-treated rats, perhaps because the tumors are smaller than those in the control rats.

HET0016-treated rats survived 5 days longer than control animals. Thus, HET0016 slows down the growth of this tumor. 9L brain tumors are very resistant to therapy. The 5-day growth delay observed for this very aggressive tumor (Barth, 1998) is exciting and highly significant in light of the dismal prognosis of patients with brain tumors, being equivalent to a 3- to 4-month extension in life for a patient with a 1-year life expectancy.

HET0016 markedly decreased the number of cells undergoing mitosis while increasing the number of cells that are apoptotic in 9L brain tumors. These results parallel those we found in the in vitro experiments with annexin V binding. Thus, the tumor-reducing effects of HET0016 may be explained by decreases in growth rate in combination with increases in the rate of cell death.

We have previously reported that HET0016 was antiangiogenic (Chen et al., 2005). Angiogenesis controls the new blood supply routes into the tumor via the host's endothelial cells and the supportive pericytes, and gliomas are well-vascularized tumors (Plate and Risau, 1995). We used FITC-labeled tomato lectin to stain the endothelial cells within the lumen of the blood vessel, and in some sections we also stained endothelial cells with anti-von Willebrand factor antibodies. Tomato lectin has been used previously to evaluate the vascularization of 9L brain tumors (Mazzetti et al., 2004). The lectin-positive structures in the untreated tumors appear short and highly branched and with the appearance of being connected as a complex network. There were abundant vascular structures on the growing edge of the tumor. Conversely, in the HET0016-treated rats, there were fewer vessels, and the appearance of the lectin-positive structures was indistinguishable from those in the normal contralateral brain, as if HET0016 treatment leads to normalization of the vasculature. In addition, the numbers of von Willebrand factor-positive structures were reduced by almost 50% in HET0016-treated tumors. These data suggest that part of the antitumor effects of HET0016 may be secondary to reduced tumor vascularization. Different from pure antiangiogenic drugs, HET0016 may also directly interfere with the growth of the tumors, obviously an advantage. In general, the antitumor effects of HET0016 appear to be more visible at the margins of the tumor edge, which is the site of both tumor progression and new vessel formation.

The results of the LC/MS experiments indicated that although normal brain tissue avidly produces 20-HETE, cultured 9L cells or 9L tumors removed from rats did not produce 20-HETE. This result is similar to the one we previously reported in U251 cells (Guo et al., 2005). We also found that normal brain tissue produced substantial amounts of 20-HETE, which was completely inhibited by HET0016. These results indicate that the antiproliferative effects of HET0016 on cultured 9L cells is not due to direct inhibition of the formation of 20-HETE, but it remains to be determined whether it is due to inhibition of the -hydroxylation of other fatty acid substrates in these cells. This latter possibility however, seems remote since in further studies we found that 9L cells in culture do not even metabolize lauric acid (data not shown). In our previous experiments, we demonstrated that U251 cells do express the message and protein for the CYP4A enzyme that metabolizes AA. We have also found that the 9L cells also express CYP4A enzyme (data not shown). These results indicate that the tumor must produce some factors that inhibit the activity of CYP4A enzymes (Alonso-Galicia et al., 1997; Hoagland et al., 2003b) or that 20-HETE is rapidly metabolized in 9L cells (Collins et al., 2005). On the other hand, inhibition of the synthesis of 20-HETE by normal brain tissue may play a role in the ability of HET0016 to reduce 9L tumor growth in vivo. One can envision that the 9L tumor may secrete growth factors that stimulate the formation of 20-HETE in surrounding normal brain tissue. 20-HETE then might act as a paracrine factor to stimulate the growth of the 9L cells as well as promote neovascularization of the tumor. There is support for this hypothesis, because addition of WIT003, a 20-HETE stable analog, increases the growth of 9L cells in vitro, as it did in U251 cells (Guo et al., 2005). Furthermore, WIT003 stimulates proliferation of endothelial cells and induces angiogenesis in the rat cornea (Chen et al., 2005). However, one cannot exclude the possibility that the effects of HET0016 to inhibit the growth of 9L gliosarcoma in vivo are completely unrelated to CYP4A inhibition. It will be important to elucidate the mechanisms by which HET0016 inhibits tumor growth and angiogenesis.

In summary, the results of this study indicate that HET0016, a compound initially synthesized as a selective inhibitor of CYP4A and CYP4F enzymes, inhibits proliferation of 9L gliosarcoma cells in vitro and induced a marked decrease in the volume of 9L-induced tumors. The precise cellular mechanisms explaining the antitumor activity of HET0016 in vivo appears to be complex and may involve mechanisms beyond mere inhibition of CYP4A activity. HET0016 may be the prototype of a new class of compounds with antitumor and antiangiogenic activity.

	Acknowledgements

 
We thank Gloria Scicli, Dana Wygle, and Dr. Ping Chen for assistance with the in vivo and in vitro experiments and Lisa Henderson and Averia Steinman for help with the LC/MS analysis.

	Footnotes

 
This work was supported by National Institutes of Health Grants EY014385 (A.G.S.), GM31278 (J.R.F.), and HL36279, HL059996, and HL29587 (R.J.R.), and from the Robert A. Welch Foundation to J.R.F.

This work was presented in part at 2005 American Association for Cancer Research annual meeting in Anaheim, CA, on April 16–20, 2005.

doi:10.1124/jpet.105.097782.

ABBREVIATIONS: EGF, epidermal growth factor; FGF, fibroblast growth factor; VEGF, vascular endothelial growth factor; 20-HETE, 20-hydroxyeicosatetraenoic acid; MAPK, mitogen-activated protein kinase; HET0016, N-hydroxy-N'-(4-butyl-2-methylphenol) formamidine; EGFR, EGF receptor; DMEM, Dulbecco's minimal essential medium; PDGF, platelet-derived growth factor; WIT003, 20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid; EtOH, ethanol; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PI, propidium iodide; SAPK/JNK, stress-activated protein kinase/c-Jun NH₂-terminal kinase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; VWF, von Willebrand factor; DAPI, 4,6-diamidino-2-phenylindole; AA, arachidonic acid; EEZE, 14,15-epoxyeicosa-5(Z)-enoic-methyl sulfonylimide; HPLC, high-performance liquid chromatography; EET, epoxyeicosatrienoic acid; LC/MS, liquid chromatography/mass spectrometry.

Address correspondence to: Dr. Meng Guo, Eye Care Services, Henry Ford Hospital, One Ford Place, 4 D, Detroit, MI 48202-3450. E-mail: mguo1{at}hfhs.org

	References

Top Abstract Materials and Methods Results Discussion References

 

Alonso-Galicia M, Drummond HA, Reddy KK, Falck JR, and Roman RJ (1997) Inhibition of 20-HETE production contributes to the vascular responses to nitric oxide. Hypertension 29: 320–325.[Abstract/Free Full Text]

Amaral SL, Papanek PE, and Greene AS (2001) Angiotensin II and VEGF are involved in angiogenesis induced by short-term exercise training. Am J Physiol 281: H1163–H1169.

Barth RF (1998) Rat brain tumor models in experimental neuro-oncology: the 9L, C6, T9, F98, RG2 (D74), RT-2 and CNS-1 gliomas. J Neurooncol 36: 91–102.[CrossRef][Medline]

Caron N, El Hajjam A, Decleves AE, Joly E, Falck JR, and Kramp R (2004) Changes in renal haemodynamics induced by indomethacin in the rat involve cytochrome P450 arachidonic acid-dependent epoxygenases. Clin Exp Pharmacol Physiol 31: 683–690.[CrossRef][Medline]

Chen P, Guo M, Wygle D, Edwards PA, Falck JR, Roman RJ, and Scicli AG (2005) Inhibitors of cytochrome P450 4A suppress angiogenic responses. Am J Pathol 166: 615–624.[Abstract/Free Full Text]

Collins XH, Harmon SD, Kaduce TL, Berst KB, Fang X, Moore SA, Raju TV, Falck JR, Weintraub NL, Duester G, et al. (2005) -Oxidation of 20-hydroxyeicosatetraenoic acid (20-HETE) in cerebral microvascular smooth muscle and endothelium by alcohol dehydrogenase 4. J Biol Chem 280: 33157–33164.[Abstract/Free Full Text]

Dunn IF, Heese O, and Black PM (2000) Growth factors in glioma angiogenesis: FGFs, PDGF, EGF and TGFs. J Neurooncol 50: 121–137.[CrossRef][Medline]

Guo M and Reiners JJ Jr (2000) Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha-ras-transformed human breast cell lines. Carcinogenesis 21: 1303–1312.[Abstract/Free Full Text]

Guo M, Roman RJ, Falck JR, Edwards PA, and Scicli AG (2005) Human U251 glioma cell proliferation is suppressed by HET0016, a selective inhibitor of CYP4A. J Pharmacol Exp Ther 315: 526–533.[Abstract/Free Full Text]

Hoagland KM, Flasch AK, and Roman RJ (2003a) Inhibitors of 20-HETE formation promote salt-sensitive hypertension in rats. Hypertension 42: 669–673.[Abstract/Free Full Text]

Hoagland KM, Maier KG, and Roman RJ (2003b) Contributions of 20-HETE to the anti-hypertensive effects of Tempol in Dahl salt-sensitive rats. Hypertension 41: 697–702.[Abstract/Free Full Text]

Kim JH, Khil MS, Kolozsvary A, Gutierrez JA, and Brown SL (1999) Fractionated radiosurgery for 9L gliosarcoma in the rat brain. Int J Radiat Oncol Biol Phys 45: 1035–1040.[Medline]

Kim JH, Lew YS, Kolozsvary A, Ryu S, and Brown SL (2003) Arsenic trioxide enhances radiation response of 9L glioma in the rat brain. Radiat Res 160: 662–666.[CrossRef][Medline]

Lin F, Rios A, Falck JR, Belosludtsev Y, and Schwartzman ML (1995) 20-Hydroxyeicosatetraenoic acid is formed in response to EGF and is a mitogen in rat proximal tubule. Am J Physiol 269: F806–F816.[Medline]

Maiese K and Vincent AM (2000) Membrane asymmetry and DNA degradation: functionally distinct determinants of neuronal programmed cell death. J Neurosci Res 15: 568–580.

Mazzetti S, Frigerio S, Gelati M, Salmaggi A, and Vitellaro-Zuccarello L (2004) Lycopersicon esculentum lectin: an effective and versatile endothelial marker of normal and tumoral blood vessels in the central nervous system. Eur J Histochem 48: 423–428.[Medline]

Milella M, Precupanu CM, Gregorj C, Ricciardi MR, Petrucci MT, Kornblau SM, Tafuri A, and Andreeff M (2005) Beyond single pathway inhibition: MEK inhibitors as a platform for the development of pharmacological combinations with synergistic anti-leukemic effects. Curr Pharm Des 11: 2779–2795.[CrossRef][Medline]

Miyata N, Taniguchi K, Seki T, Ishimoto T, Sato-Watanabe M, Yasuda Y, Doi M, Kametani S, Tomishima Y, Ueki T, et al. (2001) HET0016, a potent and selective inhibitor of 20-HETE synthesizing enzyme. Br J Pharmacol 133: 325–329.[CrossRef][Medline]

Muthalif MM, Benter IF, Karzoun N, Fatima S, Harper J, Uddin MR, and Malik KU (1998) 20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. Proc Natl Acad Sci USA 95: 12701–12706.[Abstract/Free Full Text]

Plate KH and Risau W (1995) Angiogenesis in malignant gliomas. Glia 15: 339–347.[CrossRef][Medline]

Roman RJ (2002) P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev 82: 131–185.[Abstract/Free Full Text]

Sato M, Ishii T, Kobayashi-Matsunaga Y, Amada H, Taniguchi K, Miyata N, and Kameo K (2001) Discovery of a N'-hydroxyphenylformamidine derivative HET0016 as a potent and selective 20-HETE synthase inhibitor. Bioorg Med Chem Lett 11: 2993–2995.[CrossRef][Medline]

Schmidek HH, Nielsen SL, Schiller AL, and Messer J (1971) Morphological studies of rat brain tumors induced by N-nitrosomethylurea. J Neurosurg 34: 335–340.[Medline]

Seki T, Wang MH, Miyata N, and Laniado-Schwartzman M (2005) Cytochrome P450 4A isoform inhibitory profile of N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016), a selective inhibitor of 20-HETE synthesis. Biol Pharm Bull 28: 1651–1654.[CrossRef][Medline]

Straubinger RM, Arnold RD, Zhou R, Mazurchuk R, and Slack JE (2004) Antivascular and antitumor activities of liposome-associated drugs. Anticancer Res 24: 397–404.[Abstract/Free Full Text]

Wheeler KT and Kaufman K (1981) Efficacy of continuous treatment with radiation in a rat brain-tumor model. J Neurosurg 55: 52–54.[Medline]

Yu M, Cambj-Sapunar L, Kehl F, Maier KG, Takeuchi K, Miyata N, Ishimoto T, Reddy LM, Falck JR, Gebremedhin D, et al. (2004) Effects of a 20-HETE antagonist and agonists on cerebral vascular tone. Eur J Pharmacol 486: 297–306.[CrossRef][Medline]

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