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Tumor Biology |
Intervention Section, Cell and Cancer Biology Department, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, Maryland 20892-1906
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using a panel of selective biochemical AA metabolizing enzyme inhibitors, we then evaluated the effect of these agents on cell lines with known expression status for the AA metabolizing enzymes. For the enzymes that were not universally expressed, growth inhibition by selective biochemical inhibitors did not closely correlate with the expression status of specific enzymes (P > 0.05). For the universally expressed enzymes, the LOX inhibitors were more potent growth inhibitors than the COX inhibitors. The frequent expression of the AA metabolizing enzymes suggests that AA metabolism pathway may be modulated in response to xenobiotic exposure during carcinogenesis. Although establishing a priori AA metabolizing enzyme status was not consistently informative about what AA metabolizing enzyme inhibition would be most growth inhibitory, the frequent inhibition of many epithelial cancers by these biochemical inhibitors opens a new avenue for cancer therapy and intervention in carcinogenesis.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The LOX pathway converts AA to a hydroxyeicosanoid by different LOXs, which catalyze the transfer of single O2 molecules into unsaturated fatty acids to yield hydroperoxy acids containing a conjugated diene. These enzymes show not only marked stereospecificity but also specificity to the position in the fatty acid chain at which they incorporate the oxygen molecule. The COX pathway processes AA to prostaglandins by the action of COXs, which catalyze the insertion of two molecules of oxygen into AA to yield endoperoxidase derivatives, and then, the same enzyme reduces the newly formed peroxide at position 15 to a hydroxy derivative. These families of enzymes produce a wide range of oxidation products. All of these enzymes share free radical and iron dependency.
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Inhibitors of AA metabolism can reverse the production of these metabolites, resulting in growth inhibition and recruitment of apoptotic cell clearance. Although the exact mechanism is not clear, we reported that blocking the 5-LOX pathway of AA metabolism reestablished apoptotic clearance in lung cancer (6) and breast cancer cells (13) . A similar effect has been reported for the COX pathway in colon cancer cell lines (9) . These findings suggest the importance of AA metabolizing enzymes in tumorigenesis, but they also highlight the complexity of the potential contribution of eicosanoid biochemistry to cancer.
Because all of the AA metabolizing enzymes are so closely related in mechanisms of action and substrate preference, it is difficult to determine what the exclusive biochemical effect of a single enzyme is. Consequently, it is not clear what the exact contribution of the particular enzymes to growth stimulation is. To approach the issue of their relative contribution to cancer, we first asked the question about the relative frequency of expression of the individual AA metabolizing enzymes as well as FLAP in 20 different human colon, lung, prostate, and breast cancer cell lines. Then, to analyze the relationship of enzyme expression to cell growth, we tested a series of epithelial cancer cell lines with known AA metabolizing enzyme expression status using a panel of selective AA metabolizing enzyme inhibitors. Our hypothesis was that cells expressing particular AA metabolizing enzymes would be most vulnerable to growth inhibition when exposed to the corresponding selective enzyme inhibitors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
General LOX inhibitor, NDGA, general LOX/COX inhibitor, ETYA, 5-LOX inhibitor, 2(12-hydroxydodeca-5–10-dinyl)-3,5,6-trimethyl-1–4-benzoquinone (AA861), FLAP inhibitors MK 886 (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol]-2,2-dimethylpropanoic acid), 12-LOX inhibitors 5,6,7-trihydroxyflavone (baicalein) and CDC, COX inhibitor ASA, and COX-2 inhibitor NS-398 were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). The reported IC50 of AA metabolizing enzyme inhibitors used for evaluating growth effect is shown in Table 1
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Hot-start PCR reactions were performed for 35 cycles with 94°C denaturation for 15 s, 60°C annealing for 15 s, and 72°C extension for 1 min. RT-PCR products were fractionated on 1.2% agarose gels and Southern blotted onto nitrocellulose. Southern blots were hybridized to radiolabeled internal primer probes for each gene. Amplification products for 5-LOX, FLAP, 12-LOX, 15-LOX, COX-1, and COX-2 were of the expected sizes (416, 224, 465, 459, 574, and 690 bp, respectively), as determined by ethidium bromide staining and Southern blot analysis.
Growth Inhibition Studies.
We used a modification of the CellTiter 96 (Promega, Madison, WI) semiautomated 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay (23)
, which quantitates cell numbers based on the reduction of a tetrazolium compound by tumor cells to a colored formazan end product, which is quantitated by measuring the change in absorbance (570 nm) compared with a control. Cells were fed with media containing serum 48 h prior to adding inhibitors. After trypsin treatment and washing, cells were maintained in serum-free HITES medium (Life Technologies). Stock solutions of inhibitors were made with ethanol, DMSO, or water (pH 7.0) based on solubility. Appropriate dilution was made for each inhibitor as shown in "Results." Seeding densities were 1–2 x 104 cells/well, and cells either with inhibitors or without inhibitors were grown for 4 days in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Experiments were repeated at least three times.
Statistics.
Significance of difference between samples was determined using Student’s paired t test. P < 0.05 was regarded as significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the transcripts of 5-LOX, FLAP, and COX-1 enzymes were expressed in all cell lines tested. In contrast to the uniform expression of 5-LOX, FLAP, and COX-1, the expression of 12-LOX mRNA, 15-LOX mRNA, and COX-2 mRNA was variable. The expression of 12-LOX mRNA (30% overall) was relatively low compared with that of other AA metabolizing enzymes. All colon and lung cancer cell lines expressed 15-LOX mRNA, whereas 6 of 7 breast and 2 of 3 prostate cancer cells expressed 15-LOX. The expression of inducible COX-2 mRNA was variable such that 4 of 7 colon cancer cell lines, 2 of 3 lung cancer cell lines, 5 of 7 breast cancer cell lines, and 1 of 3 prostate cancer cell lines showed enzyme expression. To verify correlation between mRNA expression and the corresponding protein, we studied the immunostaining of COX-1 and COX-2 using immunological reagents that have been previously reported to be isotype specific. We used control cell lines that have a previously published COX phenotype (24)
. The expression of protein status accessed by immunohistochemistry is consistent with the mRNA expression pattern revealed by RT-PCR (data not shown).
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. There was no obvious relation with the expression of particular AA metabolizing enzymes and specific histological cancer types.
Correlation of Expression Status with Growth Inhibition for AA Inhibitors.
To evaluate the relationship between enzyme expression status and growth inhibition, we used several representative cell lines with defined expression status for 12-LOX, 15-LOX, and COX-2. These cell lines are: SKBR3, ZR75, T47D, and COLO205. We asked what the effect of biochemical inhibitors on cell line growth as a function of their expression status of specific enzymes was.
We first analyzed the general LOX/COX inhibitors using either cell lines that expressed all of the AA metabolizing enzymes or other cell lines that lacked expression of specific AA metabolizing enzymes as shown in Fig. 2
. ETYA, the LOX/COX dual inhibitor, showed very strong growth inhibition (IC50 
6.44 µM) for the four cancer cell lines tested. The general LOX inhibitor and free radical scavenger NDGA also showed strong growth inhibition of the four cancer cells tested (IC50 3.7 µM; Fig. 2
), whereas the general COX inhibitor ASA showed little growth inhibition (IC50 > 50 µM) in all cell lines tested (Fig. 3)
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summarizes the growth inhibition for each compound evaluated. MK886, a 5-LOX selective inhibitor that functions by blocking FLAP activity showed strong growth inhibition of the four cancer cell lines tested at the concentration of 10 µM (IC50 2.73 µM), whereas another 5-LOX selective inhibitor, AA861, showed little growth inhibition at 10 µM (IC50 > 50 µM). At 10 µM, baicalein, a selective inhibitor of 12-LOX, showed strong inhibition not only on cell lines that express 12-LOX mRNA but also in cell lines that did not express 12-LOX (Fig. 3)
. Another 12-LOX selective inhibitor, CDC, showed moderate growth inhibition by 36% at 10 µM; however, CDC did not show the difference of growth inhibition between 12-LOX-expressing cell lines and nonexpressing cell lines at 10 µM. The COX-2 selective inhibitor NS398 did not show growth inhibition at 10 µM (Fig. 3)
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. The mRNA expression status for AA metabolizing enzymes did not reliably predict the level of growth inhibition using defined AA metabolism inhibitors. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our first question was to investigate the frequency of mRNA expression of AA metabolizing enzymes for a range of epithelial tumors. In this study, the mRNAs for the 5-LOX, FLAP, and COX-1 transcripts were expressed in all of the cancer cell lines evaluated (n = 20). Conversely, mRNAs of 12-LOX, 15-LOX, and COX-2 were expressed in 35, 90, and 60% of cell lines, respectively. We had reported previously that 5-LOX mRNA was widely expressed in normal and malignant lung (6)
as well as breast tissue (13)
. The data showed that all of the AA metabolizing enzymes tested were expressed in all epithelial cancer types, but the frequency of expression for individual AA metabolizing enzymes varied among the histological cancer type. The conserved expression of 5-LOX, FLAP, and COX-1 in all cancer cell lines suggest that these molecules may have a fundamental importance. The expression of 12-LOX, 15-LOX, and COX-2 is variable among the cell lines and histological cancer types. These enzymes are known to be induced by particular intracellular and extracellular stimuli (27)
. Induction of 12-LOX mRNA was demonstrated in human erythroleukemia cells by phorbol ester (28)
and in a human epidermoid carcinoma cell, A4341, by epidermal growth factor (29)
. Also, 15-LOX mRNA and protein in human monocytes were induced by interleukin 4 (30)
. In contrast to COX-1, COX-2 proteins are not detected in tissue under normal physiological conditions. Expression of COX-2 mRNA is induced by various stimuli including, lipopolysaccharides, retinoic acid, endothelin, phorbol esters, nuclear factor-
B, and inflammatory cytokines (31)
. One question was to determine whether a specific enzyme phenotype occurred in specific organ sites. However, we did not find predictable AA metabolizing enzyme phenotypes for particular histologies tested in our study.
To define the relative contribution of the individual AA metabolizing enzymes to growth regulation, we attempted to specifically inhibit each enzyme to determine the relative importance of those enzymes in tumor cell growth. Because various signaling compounds are produced through AA metabolism, the inhibitors of AA metabolizing enzymes can directly or indirectly induce biochemical and morphological changes in cultured cell lines. In particular, we evaluated the effects of inhibitors on cancer cell growth.
Using a proliferation assay, we tested a panel of biochemical AA metabolizing enzyme inhibitors to elucidate the most significant AA metabolizing enzyme relative to growth regulation. The selectivity of the biochemical inhibitors for the various AA metabolizing enzymes is shown in Table 1
. Table 3
shows that most AA metabolizing enzyme inhibitors reduce growth.
The dual LOX/COX inhibitor ETYA is an AA analogue and a competitive inhibitor for the catalytic sites of both lipoxygenase and cyclooxygenase. In our study, ETYA was a potent inhibitor of cancer cell growth. The general LOX inhibitor NDGA was also a potent growth inhibitor in this study. Establishing the specificity of NDGA is difficult because its mechanism of action is as a free radical scavenger. For example, NDGA is an antioxidant and has been shown to suppress the DNA synthesis of glioma cells (32) and to inhibit the progression of rat mammary tumors (33) . NDGA has a more significant effect on LOX activity compared to COX. Our previous results show that bcl-2 expression is down-regulated by treatment of NDGA (13) . In rat W256 cells, free radical quenching is shown to be involved in NDGA-mediated apoptosis (10) . In this study, we cannot conclusively determine whether the known inhibition by NDGA of 5-LOX activity or some other biochemical effect is actually responsible for the growth effect of this drug. For this reason, we used a panel of AA metabolizing enzyme drugs to attempt to investigate the problem of relative mechanistic selection of the antagonists.
To evaluate the contribution of 5-LOX to growth, we used other biochemical inhibitors that interfere with 5-LOX activity through a distinct mechanism. A FLAP inhibitor, MK886, was also a potent inhibitor of cancer cell growth in this study. MK886 is a strong inhibitor of leukotriene biosynthesis, inhibiting translocation of 5-LOX by altering the active site of FLAP (34) . The 5-LOX-specific inhibitor AA861 inhibited the growth of some breast cell lines by acting as an alternative substrate of 5-LOX by virtue of its redox potential (34) . This quinone analogue is not so potent as other LOX inhibitors like NDGA or MK886.
A selective 12-LOX inhibitor, baicalein shows a strong inhibition of cancer cell growth. Also another selective 12-LOX inhibitor, CDC shows growth inhibition in our study. However, the inhibition occurs in the cell lines lacking 12-LOX expression, suggesting that growth inhibition is independent of 12-LOX enzyme inhibition by these inhibitors.
In this study, regardless of COX-2 expression status, COX inhibitors do not appear to reduce cell growth in vitro as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Neither the cyclooxygenase inhibitor ASA nor a COX-2-specific inhibitor NS-398 was an effective growth inhibitor at 10 µM. In conclusion, our biochemical inhibition studies suggest that there is not a reliable predictable value in determining AA metabolizing enzyme expression status relative to in vitro growth inhibition with the biochemical inhibitor used in this study. We have described both examples where a specific enzyme is expressed, but the cell line is not inhibited by the relevant antagonist as well as when the enzyme is absent, but the relevant enzyme antagonist still results in growth inhibition. This finding may relate to the complexity of the target, such as with collateral interactions with other AA metabolic pathways or perhaps to the lack of biochemical specificity for the agents selected. The data still support the central conclusion that LOX inhibitors are more potent than the COX inhibitors (5-LOX > 12-LOX > COX-2). Even with the known limitation of specificity of the inhibitors used in the study, the magnitude and consistency of difference in the effect of LOX-selective inhibitors compared with COX-selected inhibitors suggest that there are fundamental differences in how these specific enzymes effect cell growth. Recent experimental work with plasmacytogenesis (35 , 36) provides a framework for understanding how COX biology may effect growth, which is distinct from the present understanding of 5-LOX mechanism of action (33 , 34) .
For this study, the expression frequency of AA metabolizing enzymes as determined by RT-PCR is more frequent than we had expected. A possible explanation is that individuals who develop cancer activate a wide range of new biochemical pathways in the face of xenobiotic challenge. AA metabolism not only produces a variety of important growth-related signaling metabolites but also is a major source of reactive oxygen species. Reactive oxygen species, such as free radicals and oxidized intermediates of lipids, are important mediators of cellular processes, including apoptosis. AA metabolizing enzymes are either directly or indirectly involved in cell proliferation or programmed cell death. Therefore, AA metabolizing enzyme inhibitors are important in regulating both cell survival and apoptosis by controlling intracellular redox homeostasis and regulating apoptosis-related molecules.
Success in targeting AA metabolizing enzymes for chemoprevention and therapeutic in cancer will depend on clarifying the relationship between AA metabolizing enzyme expression and growth inhibition. The significant growth reduction shown in this study supports the evidence that the AA pathway is a very promising target. Additional studies to dissect the independent contribution of the various enzymes to carcinogenesis will be useful in establishing the contribution of specific AA metabolizing enzymes to the process of carcinogenesis.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Intervention Section, Cell and Cancer Biology Department, Medicine Branch, National Cancer Institute, Building 10/12N226, 9000 Rockville Pike, Bethesda, MD 20892-1906. Phone: (301) 402-3721; Fax: (301) 402-3767; E-mail: mulshinej{at}bprb.nci.nih.gov 
2 The abbreviations used are: AA, arachidonic acid; LOX, lipoxygenase; COX, cyclooxygenase; FLAP, 5-lipoxygenase activating protein; NDGA, nordihydroguaiaretic acid; ETYA, 5,8,11,14-eicosatetraynoic acid; CDC, cinamyl-3,4-dihydroxy-a-cyanocinnamate; ASA, acetylsalicylic acid; RT-PCR, reverse transcription-PCR.
Received 9/16/98. Accepted 3/ 3/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), ZR75, breast cancer cell line (
), T47D, breast cancer cell line (•), and COLO205, colon cancer cell line (
). The experimental conditions were as outlined in "Materials and Methods," and the results are expressed as percentage of growth, with no drug added serving as control and expressed as 100% growth; bars, SD. Dose range concentrations used with drugs were a range of 1–20 µM.