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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Human U251 Glioma Cell Proliferation Is Suppressed by HET0016 [N-Hydroxy-N'-(4-butyl-2-methylphenyl)formamidine], a Selective Inhibitor of CYP4A

Eye Care Services, Henry Ford Hospital, Detroit, Michigan (M.G., P.A.E.); Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin (R.J.R.); Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.); and Eye Care Services, Henry Ford Hospital, and Department of Anesthesiology, Wayne State University, Detroit, Michigan (A.G.S.)

Received May 6, 2005; accepted August 2, 2005.

	Abstract

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We have previously reported that HET0016 [N-hydroxy-N'-(4-butyl-2 methylphenyl)formamidine], a selective inhibitor of CYP4A and thus 20-HETE (20-hydroxyeicosatetraenoic acid) synthesis, inhibits endothelial cell proliferation and decreases angiogenesis induced by human glioma cell U251. A stable 20-HETE agonist, WIT003 [20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (1 µM)], increased U251 cell proliferation from 3.9- to 4.8-folds from T₀ (time of the treatment). We examined the effects of HET0016 on the growth of U251. HET0016 inhibited U251 basal cell proliferation in a dose-dependent manner. 10 µM HET0016 suppressed 56% of U251 proliferation and significantly increased the proportions of the cells arrested in the G₀/G₁ phase of the cell cycle. Exposure to HET0016 (as early as 4 h) reduced protein tyrosine and p42/p44 MAPK (mitogen-activated protein kinase) phosphorylation. Furthermore, HET0016 significantly inhibited the U251 proliferation and phosphorylation of both the epidermal growth factor (EGF) receptor and p42/p44 MAPK induced by EGF. CYP4A mRNA and proteins were both present in U251. This suggests that HET0016 inhibited U251 proliferation by inhibiting 20-HETE synthesis. However, U251 did not synthesize 20-HETE in the presence of arachidonic acid. This implies that HET0016 suppresses U251 proliferation by mechanisms that are not yet clear but may involve activities other than inhibition of 20-HETE synthesis. We concluded that HET0016 may be the prototype of novel compounds that suppress human glioma cell proliferation.

Cancer cells are known for their ability to produce autocrine growth factors that contribute to their abnormal growth rate. We asked whether HET0016 could also modulate growth factor signal transduction in some cancer cells and thereby lower their basal growth rate. Brain tumors, particularly malignant glioma, are highly aggressive tumors of the central nervous system. They are resistant to conventional therapies, including radiation and chemotherapy (Rich and Bigner, 2004). In this study, U251, a human glioma cell line, was chosen as our in vitro model because we already had evidence that HET0016 inhibits the effects of angiogenic growth factors produced by these cells (Chen et al., 2005). We investigated the effects of HET0016 on U251 cell growth. Our studies showed that HET0016 inhibits the growth of U251 cells and markedly reduces their mitogenic response to EGF. Furthermore, we found that these effects of HET0016 may be independent of 20-HETE synthesis inhibition.

	Materials and Methods

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Cell Lines and Reagents. U251 human glioma cells were obtained from Dr. Stephen Brown (Department of Radiation Oncology, Henry Ford Health System, Detroit, MI). Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex Bio Science Baltimore, Inc. (Baltimore, MD). Primary human keratinocytes were obtained from Dr. George Murakawa (Department of Dermatology, Wayne State University, Detroit, MI). HET0016 [N-hydroxy-N'-(4-butyl-2 methylphenyl)formamidine] was a gift from Taisho Pharmaceuticals Co., Ltd. (Saitama, Japan). Dibromododecenyl methylsulfonimide (DDMS), palmitic acid, and EGF were purchased from Sigma-Aldrich (St. Louis, MO). WIT003 was synthesized by John R. Falck. Cell culture reagents for HUVECs were purchased from Cambrex. All other cell culture reagents were purchased from Invitrogen (Carlsbad, CA). Phosphotyrosine (Tyr102), phospho-Ser/Thr-Pro-MPM2, phospho-p42/p44 MAPK (Thr202/Tyr204) (20G11), phospho-SAPK/JNK (Thr183/Tyr185) (98F2) monoclonal antibodies, and phospho-EGFR (Tyr992) antibodies were all purchased from Upstate Biotechnology (Lake Placid, NY). In addition, CYP4A11 polyclonal antibody was purchased from Research Diagnostics (Flanders, NJ).

Culture Conditions. The U251 cells were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (10 IU/ml), streptomycin (10 µg/ml), and 10% nonessential amino acids (all purchased from Invitrogen). Cells were maintained at 37°C in a humidified incubator containing 5% CO₂.

Cell Proliferation Assays. Proliferation studies were performed with cultures plated at a density that ensured exponential growth for at least 5 days. Cells were then serum-starved overnight before treating with various concentrations of a given compound for 24 or 48 h. All treatments and subsequent experiments were performed in serum-free medium. U251 grows exponentially in serum-free conditions. HET0016, DDMS, and WIT003 were all dissolved in ethanol (EtOH), which was used as vehicle control in all experiments. Organic solvent never exceeded 0.1% of total culture volume. Cells were harvested by exposure to 0.25% trypsin/EDTA and counted using a hemocytometer.

[³H]Thymidine Incorporation Studies. Thymidine incorporation studies were performed with cells grown in 35-mm culture dishes. Cultures were pulsed with [methyl-³H]thymidine (1 µCi/ml culture medium) for 1 h at various times after treatment with HET0016. Palmitic acid and EtOH served as fatty acid and vehicle controls, respectively. At the end of the pulse, the medium was aspirated, and the cells were rinsed twice with ice-cold phosphate-buffered saline (PBS). The rinsed cultures were fixed by exposure to 5% ice-cold trichloracetic acid overnight at 4°C, after which these cells were processed as described previously (Scholler et al., 1994). A second set of nonfixed dishes was treated with 0.05% trypsin/EDTA to estimate cell numbers. Cellular incorporation of [³H]thymidine was detected by scintillation counting and expressed as disintegrations per minute/10³ cells.

Flow Cytometry. Cells were cultured in 100-mm dishes at densities that ensured exponential growth at the time of harvest. The harvesting and processing protocols used to detect DNA by flow cytometry with propidium iodide were done as described previously (Reiners et al., 1999). Cells were analyzed with a BD Biosciences FACScan (San Jose, CA) at Wayne State University Flow Cytometry Core Facility (Detroit, MI). Percentages of cells in the G₀/G₁, S, and G₂/M stages of the cell cycle were determined with a DNA histogram-fitting program (MODFIT; Verity Software, Topsham, ME). A minimum of 10⁴ events/sample was collected.

DNA Fragmentation and TUNEL Assays. HET0016-treated cultures were washed twice with PBS and incubated with lysis buffer (20 mM Tris-HCl, 10 mM EDTA, and 0.3% Triton X-100). Genomic DNA was extracted and separated on a 2% agarose gel. Separated DNA was visualized by staining gels with ethidium bromide. U251 cultures were also seeded onto coverslips and treated with HET0016 to perform TUNEL assays. Coverslips were washed three times with PBS and air-dried. Samples were then fixed with a freshly prepared fixation solution (4% paraformaldehyde in PBS, pH 7.4) for 1 h at room temperature followed by incubation in fresh permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. Finally, samples were processed using in situ Cell Death Detection Kit AP (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's recommendations.

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction. U251 cultures were harvested at various times after overnight serum starvation. In brief, total RNA was isolated with TRIzol reagent and 1 to 2 µg of RNA were used to synthesize cDNA using a first strand synthesis kit. We used polymerase chain reaction (PCR) primers that specifically recognize CYP4A11 and -actin-specific primers. All PCR reagents were purchased from Invitrogen. The PCR conditions used to amplify CYP4A11 and -actin consisted of a precycle of 95°C for 3 min followed by 35 cycles of 95°C for 45 s, 52°C for 1 min, and 72°C for 2 min, with final extension at 72°C for 10 min. The primers used were as follows: -actin forward primer, 5'-TGC GTG ACA TTA AGG AGA AG-3'; -actin reverse primer, 5'-GCT CGT AGC TCT TCT CCA-3'; CYP4A11 forward primer, 5'-CCA CCT GGA CCA GAG GCC CTA CAC CAC C-3'; and CYP4A11 reverse primer, 5'-AGG ATA TGG GCA GAC AGG AA-3'. The PCR products were subjected to electrophoresis on 2% Nusieve 3:1-agarose gels (FMC Bioproducts, Rockland, MA) and visualized by ethidium bromide.

Western Blotting. Cells were treated with 10 µM HET0016 and washed twice with ice-cold PBS. Cells were lysed by adding radioimmune precipitation 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 Inhibitor Set 1 (Calbiochem, La Jolla, CA)]. Plates were then scraped, and cells were collected in 1.5-ml centrifuge tubes followed by incubation at 4°C for 30 min. Homogenates of the cell suspension were centrifuged for 10 min at 14,000g and 4°C. The pellets were discarded, and protein concentrations in the supernatant were determined by the BCA protein assay.

Typically, 20 µg of protein was separated on a 14% Tris-glycine gel (Invitrogen) and electroblotted onto a PVDF membrane (Biotrace, Bothell, WA). Membranes were blocked for 1 h at room temperature with blocking buffer [0.2% I-Block reagent (Tropix, Bedford, MA) and 0.1% Tween 20 in 1x PBS] before incubating with primary antibodies in blocking buffer (overnight at 4°C). Phosphotyrosine (Tyr102) and phospho-Ser/Thr-Pro-MPM2 were detected using a 1:2000 dilution of the corresponding antibodies, whereas phospho-p42/p44 MAPK, phospho-SAPK/JNK, and phospho-EGFR (Tyr992) were detected using 1:1000 antibody dilutions. The CYP4A11 antibodies were used at 1:500 dilutions. After washing three times in washing buffer (1x TBS and 0.1% Tween 20), membranes were incubated for 1 h at room temperature with a peroxidase-conjugated goat anti-mouse or anti-rabbit antibody (Upstate Biotechnology) (diluted 1:4000 in blocking buffer). Membranes were then washed three times in washing buffer, and chemiluminescence detection was performed using an enhanced chemiluminescence kit (Upstate) according to the manufacturer's protocol. Actin was used as a loading control.

HPLC Detection of Arachidonic Acid Metabolites. U251 cultures were plated and grown overnight followed by serum starvation. Cells were harvested, pelleted, and snap-frozen in liquid N₂. Protein extracts were prepared and incubated in a 0.1 M KPO₄ buffer containing 1 mM NADPH for 30 min at 37°C in the presence and absence of 10 µM HET0016. Incubations were stopped by acidification with formic acid and homogenized, and the homogenate was extracted with chloroform:methanol (2:1) after the addition of 10 ng of internal standard, EEZE. Samples were reconstituted in 50% acetonitrile, cleaned using an online reverse-phase high-performance liquid chromatography (HPLC) trapping column, 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 (150 x 213 µm; BetaBasic18; Thermo Hypersil-Keystone, Bellefonte, PA) 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. Samples were ionized using negative ion electrospray, 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 mass spectroscopy mode using an Agilent LSD ion trap mass spectrometer 1100 (Agilent Technologies, Palo Alto, CA). The ratio of ion abundances 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 were analyzed by the Tukey Honestly Significantly Difference test using the Statistica 5.0 software package (StaSoft, Tulsa, OK). Differences were considered statistically significant at p < 0.05.

	Results

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Proliferative Effects of WIT003, a Stable 20-HETE Analog. Serum-starved U251 cultures were treated with 0.1 and 1.0 µM WIT003 for 48 h, and cell counts were obtained. For this experiment, EGF was used as a positive control. It was observed that 1.0 µM WIT003 stimulates U251 growth from 3.9- to 4.8-folds from T₀, comparable with the stimulation induced by 200 ng/ml EGF (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1. WIT003, a stable 20-HETE analog, stimulates U251 proliferation. U251 cultures were serum-starved overnight before the addition of 0.1 and 1 µM WIT003. EGF was used as positive control. Cell numbers were counted 48 h later, and EtOH treated cultures were used as control. Data are expressed in fold of increase in cell numbers from T0. 1 µM WIT003 increases U251 growth as much as 200 ng/ml EGF. Data are mean ± S.D. of three separate experiments, each by triplicate. *, p < 0.05.

View larger version (39K): [in this window] [in a new window]   Fig. 2. CYP4A is expressed in U251 at both mRNA and protein levels. A, U251 cells were plated and allowed to grow overnight. U251 cells were then serum-starved overnight. They were harvested at 0, 24, and 48 h at the end of overnight serum starvation. Reverse transcriptase-PCR was performed using CYP4A11-specific primers. PCR products were visualized by ethidium bromide. B, Western blots analyses were performed using polyclonal CYP4A11 antibodies. Images are the representative of three separate experiments.

HET0016 Decreases U251 Cell Proliferation. U251 cultures were treated with various concentrations of HET0016 for 2 days, and then the numbers of viable cells per culture were counted. Basal proliferation of U251 was suppressed by HET0016 in a concentration-dependent manner (Fig. 3A). This was considered a cytostatic effect, because trypan blue exclusion showed that culture viability was not affected by HET0016. These studies showed that 10 µM HET0016 inhibited proliferation by 56%. This concentration was employed for all subsequent studies. Analysis of [³H]thymidine incorporation data indicated 50 and 65% inhibition of DNA synthesis at 24 and 48 h post-HET0016 treatment (10 µM), respectively (Fig. 3B). Flow cytometry of cellular DNA content was performed to determine whether the antiproliferative effects of HET0016 reflected arrest at a specific point in the cell cycle (Fig. 3C). The numbers of cells containing G₀/G₁ DNA increased at 24 and 48 h after HET0016, whereas S and G₂/M phase cells decreased. This indicates that HET0016 leads to a cell-cycle arrest in G₀/G₁ phase.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Growth pattern and cell-cycle profile of U251-treated with HET0016. A, equal numbers of U251 cells (0.75 x 104) were plated and serum-starved for 1 day before exposure to four different concentrations of HET0016. Cell counting was performed at 24 and 48 h. B, [3H]thymidine incorporation into DNA was assessed in cultures treated with 10 µM HET0016. [3H]Thymidine incorporation data were calculated as disintegrations per minute/103 cells and then normalized to the EtOH controls. HET0016 was added to cultures at T0. C, U251 cells were plated and treated with 10 µM HET0016. Cells were stained with propidium iodide, and their cell-cycle distribution was determined by analyzing total DNA content of the stained cells by fluorescence-activated cell sorter. Percentage of cells in various stages of the cell cycle is indicated in each figure. Data in A through B are mean ± S.D. of three to four separate experiments, each by triplicate. *, p < 0.05; **, p < 0.01.

DNA fragmentation and TUNEL assays were performed to examine whether HET0016 induces apoptosis in U251 cells. Because both were negative (data not shown), we concluded that HET0016 does not induce apoptosis in these cells.

DDMS Results in a Modest Inhibition of U251 Proliferation. Another CYP4A and 20-HETE synthesis inhibitor, DDMS, that is structurally and mechanistically unrelated to HET0016 was also used to study U251 proliferation. At 10 µM, DDMS only inhibit U251 growth by 20% (Fig. 4).

View larger version (14K): [in this window] [in a new window]   Fig. 4. DDMS partially inhibits U251 cell proliferation. Partial inhibitions were observed with 10 µM DDMS. Data are mean ± S.D. of three separate experiments, each by triplicate. *, p < 0.05.

View larger version (22K): [in this window] [in a new window]   Fig. 5. HET0016 inhibits EGF-stimulated U251 proliferation. U251 cultures were serum-starved and then treated with 200 ng/ml EGF, 10 µM HET0016, or both together. Cells were counted 24 and 48 h later. Data are mean ± S.D. of four separate experiments, each by triplicates, and are expressed as fold of increase in cell number from T0. *, p < 0.05; **, p < 0.01 versus controls; #, p < 0.05; ##, p < 0.01 versus EGF-stimulated cultures. There is no statistical significance between 10 µM HET0016 and EGF + HET0016 at both 24 and 48 h.

View larger version (18K): [in this window] [in a new window]   Fig. 6. WIT003 alters the antiproliferative effects of HET0016. Cultures were serum-starved and then treated with either 10 µM HET0016 alone or in combination with 1 µM WIT003. Cell proliferation was assessed by cell counting 24 and 48 h after treatment. Data are mean ± S.D. of three separate experiments, each by triplicate, and are expressed as fold of increase in cell number from T0. *, p < 0.05; **, p < 0.01 versus controls; #, p < 0.05; ##, p < 0.01 versus HET0016-treated cultures.

U251 Cells Do Not Synthesize 20-HETE. For HET0016 to be antiproliferative through inhibition of 20-HETE synthesis, U251 cells should produce 20-HETE. To examine this possibility, we performed HPLC and mass spectrometry analysis of U251 cell extracts. We were surprised to find that U251 cells do not synthesize 20-HETE (Fig. 7). Furthermore, although U251 cells do produce other HETEs and EETs, HET0016 failed to inhibit any of them (Fig. 7).

View larger version (24K): [in this window] [in a new window]   Fig. 7. U251 cells do not synthesize 20-HETE. U251 cells were plated on 100-mm dishes. Cells were scraped and pelleted. Cell pellet was then resuspended in serum-free medium and quickly frozen in liquid N2. Fluorescent HPLC analysis was performed. 20-HETE (0.1 ng), 1.3 ng of HETEs/EETs, and 10 ng of EEZE were used as positive controls (in red). U251 controls are in purple, and some samples treated with 1 µM HET0016 are shown in blue.

HET0016 Alters Several Signal Transduction Pathways. To clarify downstream signaling effects of HET0016, protein extracts were isolated from U251 cultures treated with HET0016 at different times. To examine the changes induced by HET0016 in the tyrosine and serine/threonine protein phosphorylation status of U251, Western blotting was performed using a phospho-Tyr102 antibody and a phospho-Ser/Thr-Pro MPM2 antibody. Tyrosine phosphorylation in U251 was altered by HET0016, leading to a noticeable decrease as early as 4 h and a marked decrease at 24 and 48 h after the addition of HET0016 (Fig. 8A); however, no significant changes in total serine/threonine phosphorylation were observed (data not shown). Two additional antibodies were used to examine how blocking CYP4A with HET0016 would affect phosphorylation of p42/p44 MAPK and SAPK/JNK. As early as 4 h after HET0016 addition, we observed a modest inhibition of p42/p44 MAPK and SAPK/JNK phosphorylation. By 24 and 48 h, HET0016 markedly inhibited their phosphorylation (Fig. 8B).

View larger version (33K): [in this window] [in a new window]   Fig. 8. HET0016 inhibits the phosphorylation status of several signal transduction pathways. For phosphotyrosine and basal phospho-p42/p44 MAPK and SAPK/JNK experiments, U251 cultures were treated with 10 µM HET0016 for 0, 4, 24, and 48 h. Total lysates were analyzed by Western blot using specific antibodies. Actin was used as the loading control. A, HET0016 induces a decrease in tyrosine phosphorylation. B, inhibition in tyrosine phosphorylation was accompanied by a similar decrease in the phosphorylation of both p42/44 MAPK and SAPK/JNK. To examine the effects of HET0016 on EGF-induced EGF receptor and p42/p44 MAPK phosphorylation, cultures were either pretreated with 10 µM HET0016 for 30 min followed by 200 ng/ml EGF for 5 and 30 min or treated with EGF alone for 5 and 30 min. Western blots were performed using phospho-EGFR and phospho-p42/p44 MAPK antibody. C, HET0016 markedly decreased the phosphorylation of both EGFR and p42/p44 MAPK induced by EGF at 5 and 30 min. Images are representative of three to four separate experiments.

HET0016 Is Not Cytostatic to Normal Cells. To determine whether HET0016 has cytotoxic effects on normal cells, HUVECs and human primary keratinocytes were treated with 10 µM HET0016 and cells were counted after 48 h of treatment. There were no effects on their proliferation rates (Fig. 9).

View larger version (9K): [in this window] [in a new window]   Fig. 9. HET0016 is not cytostatic to normal cells. Human keratinocytes, HUVECs, and U251 cells were plated and treated with 10 µM HET0016, and cell number was counted after 48 h. Solid symbol, untreated; open symbols, HET0016-treated. Data represent the mean ± S.D. of three experiments. **, p < 0.01 versus U251 controls.

	Discussion

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EGF was reported to play a role in the growth of glioblastoma (Rich and Bigner, 2004). We have shown that HET0016 is an effective inhibitor of the angiogenic response to EGF and the angiogenesis induced by U251. We then hypothesized that HET0016 would alter the growth rate of glioblastoma-derived cells.

We determined whether U251 responded to 20-HETE with a growth response. For this, we used WIT003, an analog of 20-HETE that has the advantage over the natural compound of being stable (Yu et al., 2004). At the maximum effective dose used (1 µM), WIT003 was as mitogenic as 200 ng/ml EGF. The higher dose resulted in cell detachment. We found that U251 contained mRNA for CYP4A and immunoreactive proteins of 55 kDa, the molecular mass predicted for CYP4A11 (Capdevila and Falck, 2002). These results were consistent with 20-HETE possibly playing a role in regulation of U251 growth. We then assessed the effects of HET0016 on U251 growth (Sato et al., 2001; Yu et al., 2004). We found that HET0016 caused dose-dependent inhibition of the basal growth rate of U251. At 10 µM HET0016, growth inhibition was 56% and we used this dose for all subsequent studies.

At the time of these experiments, we assumed that any action of HET0016 was due to the inhibition of CYP4A, thus 20-HETE synthesis. To confirm this association, we used another highly selective CYP4A inhibitor, DDMS, which is structurally and mechanistically different from HET0016 (Gebremedhin et al., 2000; Yu et al., 2004). DDMS at the same concentration only resulted in 20% inhibition of U251 cell growth. When used at this or even lower concentrations, DDMS is thought to be a selective and potent inhibitor of CYP4A and CYP4F enzymes (Roman, 2002). This finding was not consistent with both compounds acting via the inhibition of 20-HETE synthesis. Because HET0016 seemed far more potent as an inhibitor of cancer cell growth than DDMS, we focused on HET0016.

To ascertain whether the inhibitory effects of HET0016 could be ascribed at least in part to inhibition of the metabolism of arachidonic acid to 20-HETE, we added arachidonic acid to microsomes obtained from U251 and tested whether 20-HETE was synthesized. U251 synthesized a number of arachidonic acid derivatives, including epoxides and 5- and 12-HETE, but no evidence of 20-HETE synthesis was found. Thus, this clearly suggests that the effects of HET0016 on U251 cells could not really be due to the inhibition of 20-HETE synthesis because no such synthesis seemed to occur. Because CYP4A11 mRNA and protein are both present in U251, this indicates that there is no enzymatically active CYP4A11 or that there is negligible enzymatically active CYP4A11, at least against AA. The reasons for this absence of catalytic activity are not clear (Ito and Roman, 1999; Wang et al., 2003).

There are few reports (Jiang et al., 2004; Parmentier et al., 2005) in which others have used HET0016 at the concentrations used for this study. However, the concentration of HET0016 needed to act effectively as antiproliferative agent is higher than the concentration reportedly needed to selectively inhibit CYP4A (Wang et al., 1998; Miyata et al., 2001). It is possible that, at these concentrations, HET0016 is acting on pathways other than 20-HETE generation from AA by CYP4A. It is also possible that it acts by mechanisms unrelated to interference with synthesis of arachidonic acid derivatives. It is interesting that 10 µM DDMS also induced a modest inhibition of U251 proliferation. Again, this suggests that, at the concentration used, DDMS acted on a mechanism other than inhibition of 20-HETE formation.

A common approach to ensure that the effects observed with an inhibitor are indeed related to inhibition of a product is to add the supposedly absent compound and determine whether it "rescues" the cells from inhibition. Inhibition of U251 proliferation by HET0016 was reduced by the presence of WIT003. Because the cells do not produce 20-HETE, this suggests that, at least for these experiments, there could not be rescue since there is no synthesis of 20-HETE. These data just show that HET0016 only weakly, if at all, alters growth responses induced by WIT003. Based on our data, we cannot exclude that HET0016 acts by inhibiting the biological activity of an unidentified mediator derived from the catalytic activity of CYP4A.

Treatment with HET0016 did not alter basal proliferation of growth-arrested HUVECs and keratinocytes. These data suggest that, at the concentration of HET0016 that inhibits cancer cell proliferation, HET0016 is neither cytostatic nor cytotoxic to normal cells. This apparent cancer-specific effect may simply be due to the properties of highly proliferating cells. Such cells differ from their quiescent counterparts in their differentiation state, in degree of cell adhesion and migration, and in basic morphological features such as nuclear/cytoplasmic ratio (Green and Evan, 2002). The important point is that HET0016 inhibits growth of a malignant glioma cell line, without apparent apoptotic effects.

Cell-cycle analysis showed that HET0016 increased the number of cells in G₀/G₁ arrest and decreased both S and G₂/M phases. Thus, HET0016 seems to control progression through the cell cycle by arresting some of the U251 cells in the G₀/G₁ phase, an antiproliferative effect (Blagosklonny and Pardee, 2002). Antiproliferative agents are often associated with their ability to alter progression of the cell cycle by regulating the phosphorylation status of its components. We studied how exposure to HET0016 might alter general serine/threonine and tyrosine protein phosphorylation. A significant progressive decrease in tyrosine phosphorylation was detected between 4 and 48 h after HET0016 addition. Mitogen-activated protein kinases (MAPK) are important mediators of signal transduction and play a key role in the regulation of many cellular processes, such as cell growth and differentiation. It is well documented that p42/p44 MAPK (extracellular signal-regulated kinase) is typically stimulated by growth-related signals (Blagosklonny, 2003; Kohno and Pouyssegur, 2003). We studied whether HET0016 alters tyrosine phosphorylation of p42/p44 MAPK. Under basal conditions (no growth agonist present), a single pulse of HET0016 decreased tyrosine phosphorylation of p42/p44 MAPK. Reduced phosphorylation of MAPK was already observed 4 h after HET0016 treatment. Thus, HET0016 may interfere with phosphorylation of some signal transduction components that regulate the cell cycle. This may explain why HET0016 caused increased number of U251 cells to be arrested in the G₀/G₁ phase of the cell cycle. We have no direct evidence that HET0016 acts by inhibiting MAPK phosphorylation. Changes in MAPK phosphorylation may be secondary to the much decreased growth rate of the HET0016-treated cells.

To address this concern, we studied whether HET0016 would affect agonist-stimulated growth. EGF has been implicated as a factor in the abnormal growth of glioma cancer cells (Nieder et al., 2003). We studied whether HET0016 would affect EGF-induced growth responses in the U251 cells. HET0016 suppressed growth responses to EGF. The fact that the presence of HET0016 inhibits both basal growth and EGF-stimulated growth suggests that its presence causes the cells to become refractory to endogenous and exogenous growth factors; clear inhibition of the phosphorylation of the EGFR after EGF addition was observed. This was accompanied by decreased phosphorylation of p42/p44 MAPK. Inhibition of EGF phosphorylation and its associated intracellular signaling explains the inhibition of EGF growth stimulation by HET0016. This indicates that HET0016 may be acting early at the level of interaction of the growth factor with its membrane receptor. The precise manner by which HET0016 interferes with the stimulation of EGF receptors by EGF is not clear at this time. Although HET0016 seems to be effective in inhibiting EGF-stimulated responses, it only weakly inhibited WIT003-induced U251 growth. The reason for this difference remains to be elucidated.

HET0016 seems to be a novel tool to interfere with the growth of the highly malignant U251 glioma cells. Further studies would be needed to elucidate the precise mechanisms by which exposure to HET0016 inhibits basal and EGF-stimulated growth responses and EGFR phosphorylation in U251 as well as to study the effects of HET0016 on experimentally induced human gliomas in vivo.

Our laboratory has published that HET0016 affects angiogenesis (Chen et al., 2005). We interpreted that HET0016 acted by inhibiting 20-HETE synthesis. In light of the present results, this interpretation may need to be reassessed. In that study, we did not determine whether endothelial cells or the cornea actually produced 20-HETE. In conclusion, the results shown here suggest that HET0016 may be the prototype of compounds with antitumor activity in glioma.

	Acknowledgements

 
We thank Dr. Stephen Brown and Dr. George Murakawa for providing us U251 and human primary keratinocytes and Gloria Scicli for assistance.

	Footnotes

 
This work was supported by National Institutes of Health Grants EY014385 (to A.G.S.), GM31278 (to J.R.F.), and HL 036279 (to R.J.R.) and a grant from the Robert A. Welch Foundation (to J.R.F.). This study has been presented in part at the 2004 American Association for Cancer Research Annual Meeting (Mar 21–31), Orlando, FL.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.088567.

ABBREVIATIONS: AA, arachidonic acid; P450, cytochrome P450; 20-HETE, 20-hydroxyeicosatetraenoic acid; HET0016, N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine; WIT003, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid; DDMS, dibromododecenyl methylsulfonimide; EEZE, 14,15-epoxyeicosa-5(Z)-enoic-methyl sulfonylimide; EET, epoxyeicosatrienoic acid; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; HUVEC, human umbilical vein endothelial cell(s); SAPK, stress-activated protein kinase; JNK, c-Jun NH₂-terminal kinase; EtOH, ethanol; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; GBM, glioblastoma multiforme.

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

	References

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