12-Lipoxygenase inhibition induced apoptosis in human gastric cancer cells
Benjamin Chun Yu Wong3,
Wei Ping Wang,
Chi Hin Cho1,
Xiao Ming Fan,
Marie Chia Mi Lin2,
Hsiang Fu Kung2 and
Shiu Kum Lam
Department of Medicine,
1 Department of Pharmacology and
2 Institute of Molecular Biology, University of Hong Kong, Hong Kong, China
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Abstract
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Arachidonic acid release from membrane phospholipids is essential for tumour cell proliferation. Lipoxygenases constitute a pathway for arachidonate metabolism. The present study investigated the expression of 12-lipoxygenase and its effect on cell proliferation as well as survival in two human gastric cancer cell lines (AGS and MKN-28). RTPCR and western blots, respectively, showed 12-LOX mRNA and protein expression in both AGS and MKN-28 cell lines. Treatment with a 12-LOX inhibitor, baicalein, significantly inhibited cancer cell proliferation, but a metabolite of 12-LOX activity, 12 hydroxyeicosatetraenoic acid (12-HETE) reversed baicalein-induced growth inhibition. Furthermore, the blockade of the 12-LOX pathway through a 12-LOX inhibitor and antisense induced apoptosis of gastric cancer cell lines. The biochemical characteristics of apoptosis were p53-independent combined with a decrease in bcl-2 expression. Caspase-7 was proteolytically activated and responsible for the apoptosis execution.
Abbreviations: AA, arachidonic acid; baicalein, 5,6,7-trihydroxyflavone; cdks, cyclin dependent kinases; COX, cyclooxygenase; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; 12-HETE, 12 hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; JNK, c-Jun NH2-terminal kinase; LOX, lipoxygenase; MAPK, mitogen activated protein kinase; TCA, trichloroacetic acid.
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Introduction
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Arachidonic acid (AA) is cleaved from membrane phospholipids by phospholipases. The metabolism of AA can be catalyzed by either one of two enzyme families, cyclooxygenase (COX) or lipoxygenase (LOX). Cyclooxygenase converts AA to prostaglandins and LOX converts AA into hydroperoxyeicosatetraenoic acid (HPETE) and eventually to hydroxyeicosatetraenoic acid (HETE) or to leukotrienes. It has been shown that the metabolites from COX or LOX are involved in modulating cell proliferation and apoptosis (1,2). Mammalian LOXs constitute a heterogeneous family of lipid-peroxidizing enzymes that are categorized with respect to their positional specificity of AA oxygenation into various subfamilies: 5-LOX, 8-LOX, 12-LOX and 15-LOX (3,4). The 5-LOX pathway leads to the formation of 5-HETE and leukotrienes, while 8-, 12- and 15-LOX can form 8-, 12- and 15-HETEs.
Studies show two major isoforms of 12-LOX. A platelet-type that was cloned from human platelet cells (5) and a leukocyte-type from porcine leukocytes, which shares 65% homology to platelet-type cDNA (6). Platelet-type 12-LOX occurs across mammalian species, whereas the leukocyte-type 12-LOX is found in rat, mouse, pig and cow, but not in humans or rabbits which have a structurally related reticulocyte-type of 15-LOX. Platelet 12-LOX differs from leukocyte 12-LOX in substrate specificity. The former is much less active with 18 fatty acids such as linoleic acid in comparison with AA, while leukocyte 12-LOX has broader substrate specificity reacting with C18 and C22 unsaturated fatty acids as efficiently as with AA (7). 12-LOX is overexpressed in tumour tissues including prostate cancer, breast cancer, colorectal cancer and lung cancer (811). Various cancer cell lines express 12-LOX and produce 12-HETE (1,1114). Moreover, 12-HETE facilitated the invasion and metastasis of tumours in a multi-faceted way, such as enhancing tumour cell motility, proteinase secretion and angiogenesis (1417).
Gastric cancer is the second most common cancer type in the world and the second leading cause of cancer death in Asia. The survival rate for gastric cancer is poor. Two important factors that influence survival in resectable gastric cancer are the depth of invasion through the gastric wall and presence or absence of regional lymph node metastasis (18). Although 12-LOX expression has been studied in various epithelial cancer cell lines such as colon, prostate, breast and pancreas, no information is available as to whether 12-LOX and its metabolite are involved in the regulation of gastric cancer cell survival. In the present study, we investigate the effect of 12-LOX inhibition on growth and apoptosis of gastric cancer cell lines.
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Materials and methods
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Cell culture
Human AGS cell line was purchased from American Type Culture Collection (Rockville, MD). Human MKN-28 cell line, which contains a mutation in p53 (codon 251, isoleucine to leucine) was from RIKEN (The Institute of Physical and Chemical Research) Cell Bank (Japan). Both cells were passaged into a RPMI-1640 medium with 10% fetal bovine serum (FBS), 100 U/ml penicillin G and 100 µg/ml streptomycin in an incubator at 37°C, with 100% humidity and 5% CO2.
Chemicals and drug treatment
Baicalein (5,6,7-trihydroxyflavone), a specific 12-LOX inhibitor, was purchased from Calbiochem (La Jolla, CA). Stock solutions of baicalein were made at 1000x concentration in dimethyl sulfoxide (DMSO) and added to the culture medium. 12-HETE was from Cayman Chemical (Ann Arbor, MI).
RTPCR analysis
Total RNA was isolated form AGS and MKN-28 cells using TRIzol Reagent (Gibco-BRL, Gaithersburg, MD). A 4 µg aliquot of RNA was reverse transcribed to cDNA by Thermoscript RTPCR System reagent (Gibco-BRL). Primers were designed according to the published sequences (11): sense, 5'-CTTCCCGTGCTACCGCTG-3' and antisense, 5'-TGGGGTTGGCACCATTGAG-3' primers for human platelet-type 12-LOX (337 bp fragment). PCR amplification consisted of 35 cycles: 15 s at 94°C for denaturing, 30 s at 58°C for annealing and 45 s (7 min in the final cycle) at 72°C for elongation.
[3H]Thymidine incorporation
Cells were seeded into 24-well plates at 1x104 cells/well and grown for 24 h. Media was replaced with fresh media, with different concentrations of baicalein for 48 h. Following treatment, cells were exposed to 0.5 µCi/ml [3H]thymidine (Amersham, Arlington Heights, IL) for 5 h at 37°C to allow incorporation of [3H]thymidine into cellular DNA. Incubation solutions were aspirated and cells washed with 0.5 ml ice-cold 0.15 M NaCl to remove non-incorporated [3H]thymidine. Then trichloroacetic acid (TCA) was added to the cells to terminate the uptake of [3H]thymidine. After washing with distilled water, the cells were exposed to 1% SDS at 37°C for 15 min to disrupt the cell membranes. Cell lysates were then transferred to separate scintillation vials and another 0.5 ml 1% SDS was added to each well and transferred to the appropriate vial. [3H]Thymidine incorporation into cellular DNA was then quantified by adding liquid scintillation and counting the counts per minute (c.p.m.) on a beta-counter (LS-6500; Beckman Instruments, CA, USA).
12-LOX antisense oligonucleotide transfection
Oligonucleotides were synthesized by Genset (Singapore) with HPLC purification. Sequences of the 12-LOX antisense and sense oligonucleotides were as follows: antisense, 5'-GTGACTATGCGGTCAGCA-3' and sense, 5'-TGTTTACACCCTCCTGAG-3' (2,19). Cells (1x105) cultured in 6-well plates for 24 h were transfected with oligonucleotides in 10 µg/ml lipofectin in serum-free RPMI 1640 medium for 4 h after which oligonucleotides were washed off and cells were cultured in 10% RPMI 1640 medium for up to 24 h. Treatment was then repeated for another 24 h. 12-LOX expression was determined by western blot 48 h after the transfection of 12-LOX antisense or sense oligonucleotides.
Acridine orange stain
Single-cell suspensions were fixed in 1% formalin/PBS and stained with acridine orange at a final concentration of 1 µg/ml. One drop (about 15 µl) of the stained cell suspension was placed on a microscope slide. Cells were visualized under a UV fluorescence microscope with a blue-green filter. Apoptotic cells were defined as cells showing cytoplasmic and nuclear shrinkage and chromatin condensation or fragmentation morphologically. At least 300 cells were counted and the percentage of apoptotic cells was determined.
DNA fragmentation analysis
After drug treatment or oligonucleotide transfection, cells were harvested and rinsed in ice-cold PBS. The final pellet was lysed in 0.1 ml 10 mM TrisHCl (pH 7.4) buffer containing 25 mM EDTA, 0.5% SDS on ice for 10 min, and then treated with 0.1 mg proteinase K or 0.1 mg RNase A for 1 h for drug treatment or oligonucleotide transfection, respectively. Following DNA isopropanol precipitation, DNA was dissolved in distilled water and electrophoresed on a 1.8% agarose gel. DNA fragments were visualized by UV illumination.
Western blot analysis
Total cell lysates (30 µg) were fractionated by 10% SDSPAGE, then transferred to nitrocellulose membrane. After incubating in blocking buffer containing 5% skimmed milk dissolved in 10 mM TrisHCl pH 7.5, 100 mM NaCl and 0.1% Tween-20, blots were respectively probed with human platelet-type 12-LOX polyclonal antiserum (Cayman Chemical), p53, p21waf1, bcl-2, bax, caspase-3 and caspase-7 (Santa Cruz Biotechnology, Santa Cruz, CA) as primary antibodies for 1 h at room temperature. Antigenantibody complexes were visualized using horseradish peroxidase-conjugated goat antimouse and antirabbit or rabbit antigoat antibodies. Band intensity on Fuji Medical X-ray film (Tokyo, Japan) was estimated using gel image analyses on a Bio-Rad Gel Doc 1000 system.
Statistical analysis
Results were expressed as means ± SEM. Statistical analysis was performed with analysis of variance (ANOVA) followed with unpaired Student's t-test. Values of P < 0.05 were considered statistically significant.
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Results
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Expression of platelet-type 12-LOX in gastric cancer cells
Both human gastric cancer cell lines AGS and MKN-28 expressed platelet-type 12-LOX mRNA (Figure 1a
). The presence of platelet-type 12-LOX in AGS and MKN-28 was also confirmed by western blot. The antigen of human platelet-type 12-LOX, which gives a band of 73 kDa, was used as a positive control (Figure 1b
). The upper band (75 kDa) is thought to represent a phosphorylated form of platelet-type 12-LOX (20).

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Fig. 1. (a) RTPCR analysis of 12-LOX mRNA in human gastric cancer cell lines. Total RNA from human platelet cells (as positive control; lane 1), AGS (lane 2) and MKN-28 (lane 3) were amplified by human platelet-type specific 12-LOX primers. (b) Western blot analysis of cellular proteins from AGS (lane 2) and MKN-28 (lane 3). Human platelet-type antigen acted as a positive control (lane 1).
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Baicalein-induced growth inhibition in gastric cancer cells
At concentrations from 5 to 20 µM, the specific 12-LOX inhibitor baicalein caused a concentration-dependent decrease of thymidine incorporation in AGS and MKN-28 cell lines following 48 h treatment. There was also a time-dependent inhibition of cell growth in both cell lines from 12 to 48 h (Figure 2
). The degree of inhibition at each concentration and time point was similar in AGS and MKN-28 cells.

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Fig. 2. Concentration and time effect of baicalein on growth of AGS and MKN28 cells. (a) AGS and MKN-28 cells were incubated with various concentrations of baicalein for 48 h. (b) AGS and MKN-28 cells were incubated with 20 µM baicalein at 12, 24 and 24 h. The value is represented as mean ± SEM (n = 4). *P < 0.05 and **P < 0.005 versus corresponding control group.
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12-HETE reversed baicalein-induced growth inhibition of gastric cancer cells
12-HETE, a metabolite of 12-LOX, directly stimulated cell proliferation concentration of 100 nM and significantly reversed 20 µM baicalein-induced growth inhibition of AGS and MKN-28 cells (Figure 3
).

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Fig. 3. Effect of 12-HETE on baicalein-induced growth inhibition in AGS and MKN-28 cells. AGS and MKN-28 cells were incubated with either 100 nM 12-HETE or 20 µM baicalein or both. Values are represented as mean ± SEM (n = 4). *P < 0.05, **P < 0.005 versus corresponding control group and #P < 0.05 compared with baicalein-treated group.
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12-LOX inhibition induced-apoptosis in gastric cancer cells
To clarify the mechanism of growth inhibition induced by baicalein, apoptosis of cells was evaluated. Acridine orange staining showed that both AGS and MKN-28 underwent typical apoptotic morphological changes upon treatment with 20 µM baicalein at 48 h. Cytoplasmic shrinkage and chromatin condensation were evident 48 h after baicalein treatment (Figure 4
). Baicalein induction of apoptosis was also studied by DNA fragmentation assay. Baicalein caused the generation of a nucleosomal-sized ladder of DNA fragments in treated cells (Figure 5
). Moreover, the transfection of 12-LOX antisense oligonucleotides into AGS and MKN-28 also resulted in DNA fragmentation in a concentration-dependent manner and increased the numbers of apoptotic cell (Figure 6a and b
). Antisense oligonuleotides to 12-LOX inhibit the accumulation of 12-LOX protein in AGS and MKN-28, whereas sense oligonucleotides had no effect (Figure 6c
).

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Fig. 4. Fluorescent photography of AGS and MKN-28 cell lines. Cell were stained with acridine orange and examined under a fluorescent microscope. AGS and MKN-28 treated with 20 µM baicalein for 48 h.
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Fig. 5. DNA fragmentation induced by baicalein in cell lines AGS and MKN-28. Lanes 14, 0, 10, 20 and 30 µM baicalein, respectively.
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Fig. 6. (a) DNA fragmentation induced by 12-LOX antisense oligonucleotides in cell lines AGS and MKN-28. Lane 1, control; lane 2, sense oligonucleotide; lane 3, 5 µM antisense oligonucleotide and lane 4, 10 µM antisense oligonucleotides. (b) The percentage of apoptotic cells caused by 12-LOX antisense oligonucleotides in AGS and MKN-28. The number of apoptotic cells was quantified by acridine orange staining over 300 cells. Values are represented as mean ± SEM (n = 3). *P < 0.05 versus corresponding control group. (c) The effect of antisense or sense oligonucleotide transfection on the accumulation of 12-LOX protein in AGS and MKN-28.
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Apoptosisrelative gene expression in baicalein-treated gastric cancer cells
Time course experiments were carried out to detect the protein level of apoptosis-related genes in 20 µM baicalein-treated AGS and MKN-28 cells from 0 to 48 h. p53 and p21waf1/cip1 protein levels in AGS and MKN-28 cells remained unchanged. Bcl-2 protein level was reduced from 12 to 48 h, while bax protein level remained constant. The Bcl-2/Bax protein ratio decreased with increased time of baicalein treatment (Figure 7
).

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Fig. 7. Protein levels of p53, p21waf1, bcl-2 and bax in baicalein-treated AGS and MKN-28 cells. AGS cells were treated with 20 µM baicalein for different time intervals (0, 4, 8, 12, 24, 36 and 48 h). Protein levels of p53, p21waf1, bcl-2 and bax were determined by western blot. Band intensity of bcl-2 and bax proteins was quantified by gel image analyses. The bcl-2/bax ratio is an average calculated on the results from two separate experiments.
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Caspase-3 and caspase-7 western analysis
During apoptosis, active caspase-3 and -7 are derived from the processing of two procaspase zymogens. Caspases are activated by cleavage into two subunits. Western blot was performed on the lysates of 20 µM baicalein-treated AGS and MKN-28 cells from 0 to 48 h. Caspase-3 remained in its inactive form for the entire period of treatment and no change in intensity of its signal was shown (Figure 8
). Caspase-7 cleavage was reflected by the progressive disappearance of its zymogen (Figure 8
).

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Fig. 8. Baicalein-induced proteolytic activation of caspase-7 but not caspase-3. Immunoblot analysis of caspase-3 or caspase-7 follows the different time points after baicalein treatment. Inactive caspase-3 zymogen (32 kDa) or inactive caspase-7 zymogen (35 kDa) reacted with the specific peptide-realizing antibodies. No proteolytic activation of caspase-3 was evident.
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Discussion
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Arachidonic acid metabolite of 12-LOX, 12-HETE, is one of the most important lipid metabolites to influence tumour progression. This eicosanoid stimulates tumour cell proliferation and motility, protects tumour cell from apoptosis and promotes angiogenesis. Studies have shown 12-LOX to be expressed in a variety of tumour cells and 12-HETE to be involved in modulating cell growth. However, the exact role of 12-LOX in human gastric cancer cells is still far from clear. In this study, expression of platelet-type 12-LOX was shown in two human gastric cancer cell lines. 12-LOX inhibition decreased gastric cancer cell proliferation and induced apoptosis.
The expression of platelet-type 12-LOX mRNA in two human gastric cancer cell lines, AGS and MKN-28, was shown by RTPCR (Figure 1a
). Furthermore, Western blotting with human platelet-type 12-LOX antibody in both cancer cell lines revealed the presence of 12-LOX. In order to evaluate the potential functional significance of altered 12-LOX in gastric cancer cells, we examined the effect of the specific 12-LOX inhibitor baicalein. Baicalein led a marked inhibition of serum-induced growth of AGS and MKN-28 cell lines. The results indicate 12-LOX metabolites of AA influence the growth of human gastric cancer cells. Arachidonic acid and its metabolites as intermediates of growth factor signaling pathway appear to play an important role in the modulation of cell growth. Evidence suggests that 12-LOX can be activated by growth factors and cytokines. In epidermoid carcinoma cells, epidermal growth factor (EGF) and transform growth factor-
could induce platelet-type 12-LOX mRNA expression (21,22) and leukocyte-type 12-LOX in vascular smooth muscle cells was also markedly up-regulated by cytokines such as interleukine-1, -4 and -8 (23). Thus an increase in 12-LOX activity and expression may promote a growth effect through these factors. In this study, 12-HETE not only stimulated gastric cancer cell proliferation, as shown in colon and lung cancer cells in vitro (10,24), but also significantly reversed the growth inhibition induced by 20 µM baicalein. However, in the case of the non-selective LOX inhibitor NDGA, NDGA-induced growth inhibition was partially reversed by 12-HETE (data not shown). Thus the involvement of 5- and 15-LOX in the regulation of gastric cancer cell proliferation cannot be ruled out. The mechanism by which 12-HETE mediates cellular effects was reported through the activation of growth and stress-related kinases, which are signalling components of gene transcription for cell proliferation. Exogenous 12-HETE could activate mitogen activated protein kinase (MAPK) ERK1/2 via the Ras or PKC pathway in epidermoid carcinoma cells (25,26) and directly induce the activation of c-Jun NH2-terminal kinase (JNK), another member of the MAPK family (27). In addition, 12-HETE was also reported to induce the expression of c-fos and c-jun protein and activating protein (AP-1) activity (28,29). However, the precise role of 12-HETE in signal transduction in gastric cancer cells remains to be investigated.
We noticed a massive apoptosis of gastric cancer cells when baicalein was employed to inhibit 12-LOX. Therefore apoptosis, an intrinsic suicide program of the cell, may be another mechanism responsible for the growth inhibition of gastric cancer cells by blocking LOX pathway. Several lines of evidence showed that the LOX pathway could be critical for the survival of certain cancer cells, such as prostate, lung and pancreas (2,30,31). Apoptosis induced by baicalein in human gastric cancer cell lines was evaluated by morphology and DNA fragment assay. Acridine orange staining revealed apoptosis-like morphologic changes after baicalein treatment. Fragmented chromosome DNA of apoptosis was confirmed by DNA ladder assay. Apoptosis in both AGS and MKN-28 cells was in a concentration-dependent manner (Figures 5 and 6
). Next we explored the potential molecular mechanism associated with baicalein (12-LOX inhibition) induced apoptosis. Three well-known important mediators of apoptosis, p53, bcl-2 and bax, were examined in AGS and MKN-28 cells treated with baicalein. Tumour suppressor gene p53 initiates cell cycle arrest, especially G to S phase transition, allowing the repair of damaged DNA after DNA damage (32). Another function of p53 is the removal of damaged cells through the triggering of apoptosis via transcript induction of genes that encode pro-apoptotic factors, such as bax (33). p53 protein level did not change following baicalein treatment. This result implies that apoptosis induced by baicalein in AGS cells may be p53 independent. p53 independent apoptosis induced by baicalein in AGS was also reflected by apoptosis triggered in p53 mutant-type MKN-28 cell line. It has been understood that the ability of p53 to induce arrest within the G1 phase of the cell cycle is through p53 stimulating transcription of the gene for cyclin dependent kinases (cdks) inhibitory protein p21, preventing cdk from promoting cell cycle progression (34). No alteration of p21 protein levels was observed in AGS and MKN-28 apoptotic cells, strengthening the hypothesis of p53-independent apoptosis induced by baicalein.
Members of the bcl-2 gene family are major regulators of apoptosis. In this family, bcl-2 and bcl-XL are anti-apoptotic, whereas bax, bcl-XS, bad, bak and bik are pro-apoptotic (35). The ratio of anti-apoptotic factor to pro-apoptotic factor in the cell is assumed to be a critical mechanism in maintaining normal homeostasis. The cell continues to survive if bcl-2 predominates over bax. Nevertheless, a higher concentration of bax compared with bcl-2 enhances susceptibility to apoptosis. In this study, baicalein treatment led to a dramatic decrease in bcl-2. In contrast to bcl-2, bax protein remained unaltered after baicalein treatment. The net result following baicalein treatment was a time-dependent decrease in the ratio bcl-2/bax, thus shifting the homeostasis toward a preference for apoptosis. Bcl-2-regulated apoptosis is linked to the activation of caspases, a family of proteases catalyzing the cellular proteins and initiating processes associated with apoptosis. Members of bcl-2 family anchored to the mitochondrial membrane exert their apoptosis-regulatory effects partially by influencing membrane permeability, which consequently controls the release of cytochrome c from mitochondria into the cytosol (3638). Cytochrome c released from the mitochondria combined with other factors can activate executioner caspases. Caspase-3 and caspase-7 are the two major executioner caspases at the final stage of apoptosis. As baicalein triggered the apoptotic process in AGS and MKN-28 cell lines, we found that only caspase-7, and not caspase-3, was activated. Cytochrome c dependent activation of caspase-7 has been documented in prostatic carcinoma cell lines during Fas ligation-induced apoptosis (39). However, the present data are unable to rule out the possibility of other proteases conferring nuclear apoptotic activity.
In summary, this study provides evidence that cell growth and apoptosis of human gastric cancer cells are regulated by the 12-LOX pathway of AA metabolism. Growth inhibition of gastric cancer cells by blocking of the 12-LOX pathway was associated with induction of apoptosis. It is proposed that the decrease of bcl-2 and consequent activation of caspase-7 is one of pathways responsible for the apoptotic process in gastric cancer cell lines. Furthermore down-regulation of the AA metabolizing enzyme 12-LOX is a new strategic approach to provide new anticancer therapies.
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Notes
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Benjamin Chun Yu Wong and Wei Ping Wang contributed equally to this work
3 To whom correspondence should be addressedEmail: bcywong{at}hku.hk 
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Acknowledgments
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This study is supported in part by the Research Grant Council of Hong Kong, RGC 7010/99M to B.C.Y.W. and the Gastroenterological Research Fund, University of Hong Kong, Hong Kong.
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Received January 17, 2001;
revised April 17, 2001;
accepted April 27, 2001.