INTERNATIONAL JOURNAL OF ONCOLOGY 41: 1495-1503, 2012
Abstract. The arachidonic acid pathway is important in the development and progression of numerous malignant dias, including prostate cancer. To more fully evaluate the role of individual cyclooxygenas (COXs), lipoxygenas (LOXs) and their metabolites in prostate cancer, we measured mRNA and protein levels of COXs and LOXs and their arachidonate metabolites in androgen-dependent (LNCaP) and androgen-independent (PC-3 and DU145) prostate cancer cell lines, bone metastasis-derived MDA PCa 2a and MDA PCa 2b cell lines and their corresponding xenograft models, as well as core biopsy specimens of primary prostate cancer and nonneo-plastic prostate tissue taken ex vivo after prostatectomy. Relatively high levels of COX-2 mRNA and its product PGE2 were obrved only in PC-3 cells and their xenografts. By contrast, levels of the exogenous 12-LOX product 12-HETE were consistently higher in MDA PCa 2b and PC-3 cells and their corresponding xenograft tissues than were tho in LNCaP cells. More strikingly, the mean endogenous level of 12-HETE was significantly higher in the primary pros-tate cancers than in the nonneoplastic prostate tissue (0.094 vs. 0.010 ng/mg protein, respectively; p=0.019). Our results suggest that LOX metabolites such as 12-HETE are critical in prostate cancer progression and that the LOX pathway may be a target for treating and preventing prostate cancer.Introduction
Rates of prostate cancer incidence vary dramatically in different geographic locations (1,2). Dietary fat intake, which is sometimes ud to help explain this variance, is among the most widely studied dietary risk factors for prostate cancer, yet its role in influencing cancer risk remains controversial (3). We do know that dietary fat composition may greatly influence the risk of prostate cancer (4) as it has been obrved in prostate cancer cell lines that n-6 fatty acids, such as linoleic acid and arachidonic acid (AA), promote cell proliferation, whereas long-chain polyunsaturated n-3 fatty acids, which are abun-dant, for example, in fish oil, inhibit cell proliferation (5-8). The promotional and inhibitory effects of n-6 and n-3 fatty acids, respectively, have also been demonstrated in prostate carcinogenesis and progression in vivo (9). Evidence is also available from specimens obtained during radical prosta-tectomy that AA turnover is 10 times greater in tumor tissue than it is in normal prostate tissue (7).
omaniOf the possible mechanisms responsible for AA-induced cell proliferation in prostate cancer, one of the most likely is the generation of certain eicosanoids, key mediators of the inflammatory respon (10). They are produced by both tissue cells and tumor-infiltrating leukocytes (10), and they may act as autocrine and/or paracrine factors. Synthesized from polyunsaturated fatty acids, eicosanoids play important biologic roles in cell proliferation and tissue repair, blood clot-ting, blood vesl perme
ability, inflammation, and immune cell behavior (10). They fall into three general groups: pros-taglandins (PGs), including PGE2; leukotrienes (LTs); and thromboxanes. They also are associated with the enzymatic pathways of the cyclooxygenas (COX-1 and COX-2), lipoxy-genas (LOXs), and cytochrome P450.
The PGs are synthesized by the action of COX enzymes: the constitutively expresd COX-1 and the inducible COX-2. COX-2 can be stimulated by inflammatory mediators, cytokines, growth factors, and tumor promoters and can be inhibited by steroids and certain nonsteroidal anti-inflamma-tory drugs (11). Higher than usual COX-2 and PGE2 levels have been obrved in numerous malignancies, including tho of the colon, lung, head and neck, breast, pancreas, bladder, and prostate (11). Both AA and PGE2 stimulate cell proliferation and tumor growth in vitro in PC-3 human prostate cancer cells (12). In PC-3, LNCaP, and DU145 prostate cancer cell
Arachidonic acid metabolism in human prostate cancer
PEIYING YANG1,2, CARRIE A. CARTWRIGHT2, JIN LI3, SIJIN WEN4, INA N. PROKHOROV A5, IMAD SHUREIQI6, PATRICIA TRONCOSO5, NORA M. NA VONE7, ROBERT A. NEWMAN8 and JERI KIM7 Departments of 1General Oncology, 2Cancer Biology, 3Systems Biology, 4Biostatistics, 5Pathology,
学府考研
6Clinical Cancer Prevention, 7Genitourinary Medical Oncology and 8Experimental Therapeutics,
The University of Texas MD Anderson Cancer Center, Houston, TX, USA
Received April 11, 2012; Accepted June 15, 2012
DOI: 10.3892/ijo.2012.1588
Correspondence to: Dr Peiying Yang, Department of General
Oncology, Unit 462, The University of Texas MD Anderson Cancer
Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA
E-mail: pyang@mdanderson
Abbreviations: AA, arachidonic acid; BHT, butylated hydro-
xytoluene; COX, cyclooxygena; FBS, fetal bovine rum; HETE,
hydroxyeicosatetraenoic acid; 13-HODE, 13-hydroxyoctadecadienoic
acid; HPLC, high-pressure liquid chromatography; LC/MS/MS,
liquid chromatography/tandem mass spectrometry; LT, leukotriene;
LOX, lipoxygena; PCR, polymera chain reaction; PG, prosta-
glandin; PIN, prostatic intraepithelial neoplasia; PPARγ, peroxisome
proliferator-activated receptor γ
Key words: arachidonic acid, cyclooxygena, lipoxygena, prostate
cancer, xenograft, eicosanoid
YANG et al: ARACHIDONATE METABOLISM AND PROSTATE CANCER 1496
lines, upregulation of COX-2 and PGE2 has been inverly correlated with apoptosis (13).
Relative to the effects of the PGs, tho of the LOX products, such as LTs and hydroxyeicosatetraenoic acids (HETEs), are more diver and may be cell-type specific. An AA metabolite derived from 12-LOX, 12-HETE, promotes the proliferation of human colon, pancreatic, a
minimum
nd breast cancer cell lines and plays an important role in cell adhesion and promotion of metastasis (14). Expression of 12-LOX also appears to stimu-late angiogenesis in human prostate carcinoma cells (14). More recently, study results have suggested that 12-LOX functions as a potential biomarker and therapeutic target for prostate cancer stem cells (15). Further, the 5-LOX product 5-HETE has been suggested as playing a role as a potent pro-growth survival factor for human prostate cancer cells (16).
By contrast, we and others have reported that 15-LOX-1, 15-LOX-2, and their related products 13-hydroxyoctadeca-dienoic acid (13-HODE) and 15-HETE actually function as tumor suppressors in, respectively, colorectal and prostate cancer cells (17,18). In addition, 15-deoxy-∆12,14-PGJ2, the metabolite of PGJ2 and a peroxisome proliferator-activated receptor γ (PPARγ) ligand, has been reported to induce cell death in three human prostate cancer cell lines, PC-3, LNCaP, and DU145 (19).
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Thromboxanes, additional metabolites of COX enzymes, have also been studied in cancer. For example, higher thromboxane syntha mRNA levels have been obrved in prostate and uterine cancers relative to levels in matched normal tissues (20). In prostate cancer, higher expression of thromboxane syntha was associated with advanced-stage and high-grade dia (21). Thrombox
ane syntha expresd in prostate cancer cells was enzymatically active and might play a contributory role in tumor progression, especially in tumor cell motility (22,23).
In vitro studies have demonstrated that eicosanoids appear to have key roles in the biology of human prostate cancer, but the role of endogenous eicosanoids in vivo in prostate cancer development and progression remains unclear. We and others have investigated the alteration of AA metabolites in the TRAMP mou model and found that 12-HETE and COX-2 levels were significantly higher in prostate tissues of the mice than they were in tho of control or wild-type mice (24,25). However, endogenous PGE2 was almost five times lower in the prostate of TRAMP mice than it was in wild-type mice (24). Shappell et al (26) found in human samples from radical prostatectomy specimens that expression of COX-2 was elevated only in high-grade tumors. By comparison, the reduction of 15-LOX-2 and 15-HETE formation is the most characteristic alteration of AA metabolism in prostate cancer (26). We also reported that 15-LOX-2 expression was lost in all the prostate cancer cell lines we tested (including PC-3, LNCaP, and DU145 cells), yet easily detectable levels of 15-LOX-2 were expresd in normal human prostate cells (17,27). Not surprisingly, eicosanoid profiles differ between cancer and normal prostate cells and between mou and human prostate tissues.
Changes in eicosanoid content appear to be important for a more complete understanding of prostate cancer progression. We therefore undertook this study in androgen-dependent and androgen-independent cell lines from bone, brain, and lymph node metastas; in rodent xenografts of human tumors with bone microenvironment influence; and in human tumor tissues. Our findings from the studies show that eicosanoids may play an important role in prostate cancer progression in a cell type- and microenvironment-dependent manner. Materials and methods
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Eicosanoids and antibodies. The eicosanoids PGE2 , 5-HETE, 12-HETE, and 15-HETE, and the corresponding deuterated eicosanoid standards were purchad from Cayman Chemical. AA, butylated hydroxytoluene (BHT), citric acid, and EDTA were obtained from Sigma Chemical. All high-pressure liquid chromatography (HPLC) solvents ud for analys of eico-sanoids were purchad from Fisher Scientific. Anti-COX-2 and anti-15-LOX antibodies were obtained from Cayman Chemical. Anti-5-LOX antibody was purchad from Rearch Diagnostics and anti-β-actin antibody was purchad from Sigma Chemical. Anti-platelet-type 12-LOX antibody was obtained from Oxford Biomedical Rearch.
Cell lines. Human prostate cancer cell lines LNCaP, PC-3, and DU145 were obtained from the American Type Culture Collection and maintained in a humidified atmosphere containing 5% CO2at
37˚C. LNCaP and PC3 cells were routinely cultured in RPMI-1640 medium (Invitrogen), supplemented with 10% heat-inactivated fetal bovine rum (FBS) (Hyclone Laboratories) containing 50 IU/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine [all from Gibco (Invitrogen)]. DU145 cells were cultured under similar conditions except that cells were grown in DMEM medium (Mediatech).
The human prostate cancer cell lines MDA PCa 2a and MDA PCa 2b were gifts from Dr Nora Navone (28). The cell lines were grown in BRFF-HPC1 medium (AthenaES) supplemented with 20% FBS in tissue culture flasks coated with FNC coating mix (AthenaES). All cell lines were authen-ticated via microscopic morphology check and DNA analysis.
Human prostate xenografts. All animal experiments were approved by the Institutional Animal Care and U Committee at MD Anderson. Xenograft models of PC-3, LNCaP, DU145, and MDA PCa 2b cells were developed as previously described (28). In brief, PC-3 and DU145 (1x106), LNCaP (5x106), and MDA PCa 2b (1x107) cells were suspended in 50% Matrigel (BD Biosciences) and subcutaneously implanted into the flanks of 8-week-old male BALB/c athymic (Nu/Nu) mice. Mice were euthanized with CO2 approximately 1 month later, when the tumor volume reached 1 cm3. A portion of each tumor was snap frozen and stored at -80˚C until measurements of eicosanoid profile
s and protein expression of eicosanoid enzymes were obtained.
Human core biopsy specimens from prostatectomy specimens. Ex vivo biopsy specimens were obtained from eight patients undergoing prostatectomy who connted to the u of their tissues in rearch, according to the Institutional Tissue Banking protocol at MD Anderson. Whenever possible, multiple core biopsy specimens were obtained from each prostatectomy specimen. The prence of tumor in each core
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was determined by making a touch prep of the cores before the specimens were snap frozen in liquid nitrogen, placed in cryovials, and archived at -80˚C in the Prostate Tissue Bank. Technicians ensured conformance with the prostatectomy specimen map delineating individual tumor foci. Seven core biopsy specimens containing tumor and three without tumor were ud to test for the expression of COXs and LOXs; ven core biopsy specimens without tumor and 19 with tumor were ud for evaluating eicosanoid profiles. In this proof-of-principle analysis, the limited amount of tissue prevented the testing of matching specimens across all two-sample types and evaluating intrapatient as well as interpatient variabilities.
RNA extraction and real-time polymera chain reaction. Total RNA was extracted from the five prostate cancer cell lines using TRI reagent (Molecular Rearch Center). The RNA from each sample was rever transcribed and then measured quantitatively using real-time polymera chain reaction (PCR) and a comparative threshold cycle method, as previously described (29,30).
Western blot analysis. Cells growing in log pha were washed with cold PBS and scraped free in the prence of a lysis buffer (Invitrogen) with protea inhibitor cocktail (Sigma). Cell lysates were then sonicated on ice for 3 min, incubated for 10 min on ice, and centrifuged at 14,000 x g for 10 min at 4˚C. Protein levels were quantified via the detergent-compatible protein assay (Bio-Rad). Equal levels of protein (60 µg) were fractionated on precast gels (Bio-Rad) and then transferred onto polyvinylidene diflouride membranes, according to standard methods. After a 1- to 2-h incubation in 5% nonfat dry milk blocking buffer prepared in Tris-buffered saline with 0.1% Tween 20, the membranes were probed with primary anti-bodies diluted 1:2,000 in blocking buffer. Protein bands were visualized via chemiluminescence, using the ECL+ detec-tion kit (Amersham Biosciences) and hyper-film (Amersham Biosciences). Equal loading of samples was illustrated by Western blotting for the prence of β-actin.
Eicosanoid analys. The levels of eicosanoids in the prostate cancer cells, xenograft tissues, and h
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uman biopsy samples were determined according to the methods of Kempen et al and Yang et al (31-33). In brief, the human prostate cancer cells (5x106) were harvested by trypsinization, washed with PBS, and then resuspended in 0.5 ml of PBS containing 1 mM CaCl2. For exogenous eicosanoid analysis, samples were incubated with 2.5 µl of calcium ionophore A23187 (1 mM; Sigma) for 2 min at 37˚C, followed by addition of an aliquot of 2.5 µl of 10 mM AA. Samples were then incubated for a further 10 min under similar conditions. The reaction was terminated by the addition of aliquots of 40 µl of 1 N citric acid and 5 µl of 10% BHT. An aliquot of 10 µl of the relevant deuterated eicosanoids (PGE2-d4, 15-HETE-d8, 12-HETE-d8, and 5-HETE-d8) per 100 ng/ml) was added to the reaction mixtures as internal standards. The eicosanoids were extracted three times with 2 ml of hexane-ethyl acetate (1:1, v/v). The upper organic phas were then pooled and evaporated to dryness under a stream of nitrogen at room temperature. All extraction procedures were performed under conditions of minimal light. Samples were then reconstituted in 100 µl of methanol with 10 mM ammonium acetate buffer (70:30, v/v; pH, 8.5) before analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). For the measurement of endogenous eicosanoids, the resuspended cells were acidified by the addition of an aliquot of 40 µl of 1 N citric acid followed by the addition of the deuterated internal standards and extrac-tion of eicosanoids as previously described (33).
For analysis of eicosanoids in the xenograft tissues, each frozen tissue specimen (25-50 mg) was ground to a fine powder in a liquid nitrogen-cooled mortar (Fisher). Samples were then transferred to microcentrifuge tubes. Ice-cold PBS buffer, triple each sample's volume and containing 0.1% BHT and 1 mM EDTA, was added, and the tubes were aled. The mixtures were then homogenized using an ultrasonic processor (Misonix) at 0˚C for 3 min. A 100-µl aliquot of each homog-enate was transferred to a glass tube (13x100 mm), and the eicosanoids were extracted using the method of Yang et al (33). We next measured the AA metabolites in the human pros-tate core biopsy samples using a modification of the method of Yang et al (33). In brief, the frozen specimens were mixed with 150 µl of homogenization buffer [500 mM Tris-HCl (pH 7.2), 0.5 M sucro, 200 M EDTA, 100 mM EGTA, 0.4 M NaF, 10% Triton X-100, and 10 mM sodium orthovanadate (Sigma)] and incubated at 0˚C for 30 min. The samples were then homogenized and procesd for eicosanoid extraction as described above.
涉及到Rever-pha HPLC electrospray ionization MS was ud to measure eicosanoid (PGE2, 5-HETE, 12-HETE) levels in cells using our previously published method (31,34).
A Micromass Quattro Ultima tandem mass spectrometer (Waters) was equipped with an Agilent 1100-HP binary pump HPLC inlet for u in the studies. Eicosanoids were parated by a Luna 3-
µ phenyl-hexyl (2x150 mm) LC column (Phenomenex). The mobile pha consisted of 10 mM ammonium acetate (pH 8.5) and methanol; the flow rate was 250 µl/min, the column temperature was 50˚C, and the sample injection volume was 25 µl. Samples were kept at 4˚C in an autosampler before their injection into the analytical column. The mass spectrometer was operated in the elec-trospray negative-ion mode with a cone voltage of 100 V. Fragmentation of all compounds was performed using argon as the inert collision gas at a cell pressure of 2.1x10-3 torr. The collision energy was 19 V. All eicosanoids were detected using negative ionization and multiple-reaction monitoring of the transition ions for eicosanoid products and their internal standards (33).
Statistical analys. Descriptive statistics were calculated and exploratory data analys were performed. Categorical data were summarized by frequency counts, proportions, or percentages. Continuously scaled measures were summarized as means and standard deviations or as medians with ranges. Gene expression image plots, bar graphs, and scatter plots were ud to graphically prent the data. Student's t-test was ud for analyzing continuous variables. A mixed-effects model was ud in the analysis of repeated measurements from the same patients. All statistical tests were two sided, and p-values of <0.05 were considered to indicate statistically significant differences. Statistical analys were performed with S-PLUS software (TIBCO Software).
YANG et al : ARACHIDONATE METABOLISM AND PROSTATE CANCER
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Results
AA metabolism enzyme expression in human prostate cancer cell lines. The data from the quantitative analysis of mRNA of eicosanoid enzymes in the five different human prostate cancer cell lines are shown in Fig. 1. COX-2 mRNA was detectable only in PC-3 and DU145 cells, with PC-3 cells containing 10 times more than DU145 cells (Fig. 1A). By contrast, levels of 12-LOX mRNA were obrved only in the MDA PCa 2b metastatic prostate cancer cells (Fig. 1B). Notably, the level of 5-LOX mRNA in LNCaP cells was almost five times higher than that in PC-3 cells, whereas very limited expression of 5-LOX was detected in MDA PCa 2a and MDA PCa 2b cells (Fig. 1C). The mR
NA level of 15-LOX-2 was also highest in the LNCaP cells, relative to that in the other cells tested (Fig. 1D).
Expression of COX-2 and the LOXs in prostate cancer cell lines. The protein expression of COX-2 and LOX enzymes was examined in the five different prostate cancer cell lines by Western blotting to confirm that the RNA expression of the enzymes had resulted in protein expression. Although the protein expression of COX-2 in PC-3 cells was similar to that of mRNA in this cell line, the protein expression levels of 5-LOX, 12-LOX, and 15-LOX-2 were somewhat different from that of mRNA (Fig. 2). This could be reflective of either assay differences or, more importantly, differences in mRNA translation efficiency between the various cancer cell lines.Endogenous and exogenous AA metabolism in the human prostate cancer cell lines. The mRNA and protein levels of each eicosanoid enzyme were differently regulated in the
five prostate cancer cell lines we tested, which suggested that the relevant metabolites of the eicosanoids may also differ. Therefore, we measured both endogenous and exogenous eico-sanoid levels in tho cell lines. As shown in Fig. 3A, levels of both endogenous and exogenous PGE 2, a COX-2 metabolite, were highest in the PC-3 cells, suggesting a high capacity to produce this pro-inflammatory eicosanoid. The level of exog -enous PGE 2 in PC-3 cells was almost 15 times higher t
han that produced endogenously.
By comparison, the levels of endogenous 12-HETE in the metastatic prostate cancer cells MDA PCa 2a (0.48±0.10 ng/million cells) and MDA PCa 2b (0.41±0.19 ng/million cells) were almost twice tho in the PC-3, DU145, and LNCaP cells (0.16-0.25 ng/million cells) (Fig. 3B). When AA was
ud as a supplement for the cells, the formation of 12-HETE
Figure 1. Quantitative analysis of mRNA of eicosanoid enzymes in five human prostate cancer cell lines. Cells (1x106) were plated in 100-mm dishes and allowed to attach overnight. They were then collected and subjected to RNA extraction as described in Materials and methods. The expression levels were
calculated as the values relative to that of a calibrator sample (PC-3). Data are prented as the means ± SDs of three parate experiments.
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Figure 2. Protein expression of the eicosanoid enzymes in prostate cancer cells. Cells that reached approximately 80% confluency were collected and lyd in the lysis buffer as described in Materials and methods. The expres-sion of protein was determined by Western blot analysis using their relevant antibodies. Data are prented as reprentative blots of replicate experiments.
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was markedly incread in the MDA PCa 2b and PC-3 cells. The level of exogenous 12-HETE was ranked from highest to lowest in the MDA PCa 2b, PC-3, MDA PCa 2a, DU145, and LNCaP cells.实现伟大中国梦
The level of endogenous 5-HETE was highest in the LNCaP cells, but the exogenous level of 5-HETE was notice-ably higher in the PC-3 cells, in fact, more than 1.5 times the levels in the other cell lines (Fig. 3C). This suggests that the capacity to produce 5-LOX is greater in PC-3 cells than in the other cancer cell lines tested, and it correlated with the highest expression of the 5-LOX protein in this particular cell line (Fig. 2). The level of endogenous 15-HETE was also highest in LNCaP cells, but after exposure of the cell lines to AA, the MDA PCa 2a cells showed the highest level of 15-HETE formation (Fig. 3D).
Expression of COX-2 and the LOXs in human prostate cancer xenograft tissues. To explore the role of eicosanoids in prostate cancer progression, we further investigated the expression of COX-2 and various LOX proteins in human prostate xenograft tissues (Fig. 4, top panel). As the tumorigenicity of the MDA PCa 2a cells was extremely low, we evaluated the protein expression of eicosanoid enzymes in only the other four human prostate cancer xenograft tissues. The protein expres-sion of the COX-2 and 5-LOX enzymes was highest in the PC-3 xenografts, whereas the protein levels of 12-LOX were higher in both PC-3 and PCa 2b cells. In line with the protein expression of 15-LOX-2 i
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n the cells in vitro , its expression was consistently highest in the two tumor tissues derived from MDA PCa 2b xenografts (Fig. 4, top panel).
Endogenous AA metabolism in human prostate cancer xenograft tissues. AA metabolites were further examined in xenografts of the DU145, LNCaP, PC-3, and MDA PCa 2b human prostate cancer cells (Fig. 4, bottom panel). In a pattern similar to that obrved in the cultured prostate cancer cell lines, the concentration of endogenous PGE 2 was significantly higher in PC-3 xenograft tissues (10.5±2.17 ng/mg protein) than it was in the other three xenograft tissues (range, 0.07-1.1 ng/mg of protein) (p<0.001) (Fig. 4A, bottom panel). The levels of 12-HETE in both PC-3 and MDA PCa 2b xenograft tissues were also much higher (13.9 and 10.9 ng/mg protein, respec-tively) than tho in the LNCaP xenograft tissues (3.9 ng/mg protein) (p<0.01) (Fig. 4B, bottom panel). The highest levels of both 5- and 15-HETE in PC3 and MDA PCa 2b tumor tissues were only about one third or one fourth (2.07±0.46 for 5-HETE and 3.24±0.45 ng/mg protein) the levels of PGE 2 and 12-HETE (Fig. 4C and D, bottom panel). The results appear to correspond with the endogenous levels of the AA metabolites in the same prostate cancer cells in vitro . Expression of COXs and LOXs in prostate core biopsy speci-mens containing tumor. To test the role of AA metabolism in prostate cancer, the expression of COX and LOX proteins in human prostate core biopsy specimens
containing tumor was compared with that in normal, nontumorous prostate tissue specimens. As shown in Fig. 5, the expression of COX-1 and COX-2 enzymes did not differ between the tumorous and normal tissues. In almost all of the tumorous specimens, the expression of 12-LOX was moderately greater than it
was in the specimens of normal tissue. The protein levels of
Figure 3. Endogenous (clear bars) and exogenous (striped bars) eicosanoid metabolism in the five human prostate cancer cell lines. For the endogenous eicosanoid analysis, cells (3x106) were plated and allowed to attach overnight. They were then harvested by trypsinization and subjected to eicosanoid analysis using LC/MS/MS, as described in Materials and methods. For the exogenous eicosanoid analysis, cells (5x106) were treated with 50 µM arachidonic acid for 10 min and then analyzed for eicosanoid content. Data are prented as the means ± SDs of three parate experiments.
