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Carcinogenesis Advance Access originally published online on January 3, 2008
Carcinogenesis 2008 29(2):440-447; doi:10.1093/carcin/bgm274
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Platelet-type 12-lipoxygenase accelerates tumor promotion of mouse epidermal cells through enhancement of cloning efficiency

Ying-Shi Piao1,2,3, Yu-Chen Du1,2, Hiroko Oshima1, Jing-Chun Jin4, Masaaki Nomura5, Tanihiro Yoshimoto2 and Masanobu Oshima1,*

1 Division of Genetics, Cancer Research Institute
2 Department of Pharmacology, Graduate School of Medicine, Kanazawa University, Kanazawa 920-0934, Japan
3 Department of Physiology and Pathophysiology
4 Department of Hematology, Medical College, Yanbian University, Yanji 133000, Jilin, China
5 Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan

* To whom correspondence should be addressed. Tel: +81 76 265 2721; Fax: +81 76 234 4519; Email: oshimam{at}kenroku.kanazawa-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Accumulating evidence suggests that platelet-type 12-lipoxygenase (p12-LOX) plays an important role in tumor development. However, how p12-LOX contributes to tumorigenesis is still not understood. The role of p12-LOX was therefore examined in tumor promotion using mouse epidermal JB6 P+ cells that are sensitive to 12-O-tetradecanoylphorbol-13-acetate-induced transformation. The expression of p12-LOX was significantly higher in JB6 P+ cells than in JB6 P– cells that were resistant to transformation, and its expression was further increased by tumor necrosis factor (TNF)-{alpha}. Importantly, the inhibition of p12-LOX in JB6 P+ cells by baicalein, a specific inhibitor or small interfering RNA significantly suppressed TPA-induced transformation. Moreover, treatment with 12(S)-hydroxyeicosatetraenoic acid (HETE), a metabolite of p12-LOX, enhanced TPA-induced neoplastic transformation either in the presence or absence of baicalein. These results indicate that p12-LOX is required for tumor promotion of epidermal cells and that 12(S)-HETE functions as a rate-limiting factor. Notably, treatment with baicalein significantly suppressed the proliferation of JB6 P+ cells when cells were seeded at a low density in a culture plate. Moreover, the cloning efficiency of JB6 P+ cells was dramatically decreased by inhibition of p12-LOX. In contrast, baicalein treatment did not affect the cloning efficiency of most malignant cancer cells. These results indicate that p12-LOX is induced by the inflammatory cytokine TNF-{alpha} in the early stage of tumorigenesis, and is required for tumor promotion through enhancing efficient proliferation of a small number of initiated cells. The present results suggest that the p12-LOX pathway may be an effective target of chemoprevention for skin carcinogenesis.

Abbreviations: COX, cyclooxygenase; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; HETE, hydroxyeicosatetraenoic acid; LOX, lipoxygenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide; NDGA, nordihydroguaiaretic acid; p12-LOX, platelet-type 12- lipoxygenase; PG, prostaglandin; RT–PCR, reverse transcription–polymerase chain reaction; siRNA, small interfering RNA; TNF-{alpha}, tumor necrosis factor-{alpha}; TPA, 12-O-tetradecanoylphorbol-13-acetate


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The cyclooxygenase (COX) and lipoxygenase (LOX) are two important enzyme classes that metabolize polyunsaturated fatty acids and play important roles in tumorigenesis (1). COX-2, one of the COX isoenzymes, plays a variety of pathological roles including cancer development (2). Previous studies have demonstrated that the induction of COX-2 is responsible for intestinal tumorigenesis (3) and that prostaglandin (PG)E2, one of downstream products of COX-2, is required for carcinogenesis in the stomach through cross-talk with activated Wnt signaling (4,5).

Among the LOX family members, it has been suggested that platelet-type 12-lipoxygenase (p12-LOX) plays an important role in tumorigenesis (6,7). The expression of p12-LOX is absent or at a basal level in the normal tissues (6,8). However, the induction of p12-LOX is widely found in human cancer tissues that develop in the colon (9), prostate (10,11), stomach (12) and skin (13). In animal models, the p12-LOX expression is also found in xenografts of melanoma (9) and in skin tumors developed by an initiation/promotion protocol (14). Moreover, it has been shown that p12-LOX expression is associated with the advanced stages of human prostate cancer and its levels increase in metastasized tumors in a mouse model (10,11). These analyses suggest that p12-LOX contributes to development and progression of various types of cancers.

The treatment of cancer cells with baicalein, a p12-LOX inhibitor, has also been shown to result in the induction of apoptosis and inhibition of tumor xenograft growth in immunodeficient mice (13,15–17). Consistently, the forced expression of the p12-LOX gene or stimulation with 12(S)-hydroxyeicosatetraenoic acid (HETE), a metabolite of p12-LOX, has been shown to induce cell proliferation and survival (16,18,19). Moreover, it has been reported that p12-LOX induces cell spreading, integrin-dependent cell survival (19,20) and angiogenesis through the induction of vascular endothelial growth factor expression (21,22). Accordingly, it is conceivable that the p12-LOX pathway also contributes to tumor progression through the acceleration of cell survival, migration and angiogenesis.

Genetic experiments have indicated that p12-LOX plays an important role in skin tumorigenesis. In the initiation/promotion skin carcinogenesis model, multiplicity and incidence of squamous cell carcinoma are suppressed significantly by the disruption of the p12-LOX gene (23). Accordingly, it is possible that p12-LOX is important for tumor promotion of the initiated cells during skin carcinogenesis. To assess this possibility, we have investigated the role of p12-LOX and 12(S)-HETE in promotion of epidermal cell transformation using mouse JB6 P+ cell line. The cells of this line are transformed by stimulation with tumor promoters such as TPA or tumor necrosis factor (TNF)-{alpha} and, therefore, are useful to examine the mechanisms involved in the promotion of epidermal cell transformation (24,25). Clonal variant JB6 P– cells are resistant to tumor promoter-induced transformation. As a result, we therefore used JB6 P– cells, in addition to P+ cells, to examine the role of p12-LOX in the transformation process.

We demonstrate here that expression of p12-LOX is increased significantly in JB6 P+ cells but not in JB6 P– cells. Moreover, inhibition of p12-LOX of JB6 P+ cells suppresses colony formation in soft agar, whereas treatment with 12(S)-HETE accelerates it. In addition, we show that the p12-LOX pathway is responsible for proliferation from a single cell or a small number of cells. These results suggest that p12-LOX contributes to tumor promotion through the acceleration of initiated cell proliferation and that lack of p12-LOX is thus considered to be one of the causes for the resistance of JB6 P– cells against tumor promoter-induced transformation.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Reverse transcription–polymerase chain reaction analysis
RNA samples were prepared from cultured cells or mouse tissue samples using the ISOGEN solution (Nippongene, Tokyo, Japan). Mouse gastric tumors and intestinal polyps were obtained from K19-Wnt1/C2mE mice (6) and Apc{Delta}716 mice (26), respectively. Control tissues were obtained from normal tissues of the same mice or wild-type mice. Extracted RNA was reverse transcribed and amplified by PCR with the following primer set: p12-LOX (F-5'-GCGGTCTTCGAATTGAACTT-3' and R-5'-CAGGAACAGTGTTGGAGCTG-3'); epidermal-type 12-LOX (F-5'-CAGGAGCTGGAGAACAGAAGG-3' and R-5'-GAACTGGTACCCAAAGAGAGCATC-3'); leukocyte-type 12-LOX (F-5'-GGCTCCAACAACGAGGTCTA-3' and R-5'-AGTTCCTCCTCCCTGTGGTT-3'); 5-LOX (F-5'-AATGGAGGTGGTGAGCATCTA-3' and R-5'-TCCTTCACTGGCTTCTCAATG-3'); 8-LOX (F-5'-CGAAATGCAGGGTGAGAGTA-3' and R-5'-TCCTGCAGTGTAGGGTGATG-3'). Animal experiments were carried out according to the protocol approved by the Committee on Animal Experimentation on Kanazawa University.

Cell culture and soft agar colony formation assay
JB6 P+ (Cl 41-5a) and JB6 P– (Cl 30-7b) cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in modified Eagle’s medium supplemented with 5% fetal bovine serum and incubated in a 5% CO2 at 37°C. For the soft agar colony formation assay, JB6 P+ and JB6 P– cells were exposed to TPA (Calbiochem, San Diego, CA) at 10 ng/ml in 1 ml of basal medium eagle (Invitrogen, Grand Island, NY) with 10% fetal bovine serum and 0.33% agar and then seeded in a six-well plate (104 cells per well). Soft agar cultures were maintained in a 5% CO2 incubator at 37°C for 14 days and stained with Giemsa stain solution (Wako, Osaka, Japan) and the number of colonies was scored using a stereomicroscope and Image J software program (National Institutes of Health). A non-selective LOX inhibitor nordihydroguaiaretic acid (NDGA), a selective 12-LOX inhibitor baicalein (Tocris Biosciences, Ellisville, MI) or a selective 5-LOX inhibitor AA-861 (Sigma, St Louis, MO) was added to the agar containing medium at 5 µM (NDGA and baicalein) or 10 µM (AA-861) to test their inhibitory effect on anchorage-independent transformation. To examine the effect of 12(S)-HETE, cells were pre-treated with 12(S)-HETE (Cayman Chemical, Ann Arbor, MI) at 0.1 µM or 1 µM for 1 day prior to stimulation with TPA and were plated as described above for the soft agar colony formation assay. All soft agar colony formation assays were repeated three times, and the mean colony numbers and standard deviation were calculated.

Subcutaneous transplantation of tumor cells to mice
JB6 P+ cells were stimulated with TPA at 10 ng/ml for 12 h and inoculated subcutaneously at 1.0 x 106 cells per site to three sites per mouse for four mice each for drug-treated and no-treated group (Balb/c-nu/nu: Charles River Laboratories Japan, Tokyo, Japan). At 5 days after inoculation, 50 µl of TPA (10 ng/ml in phosphate-buffered saline) was injected subcutaneously. into the inoculation site. After palpable tumors developed, baicalein was administered at 20 mg/kg/day in 0.5% methylcellulose (Wako) per os, and treatment was continued for 14 days. In another dosing experiment, baicalein treatment started from day 7 after cell inoculation and the treatment was continued until 1 week after palpable tumors developed in the control mice. The dosing protocol has been reported to inhibit p12-LOX activity in vivo (27). The tumor size was measured every 2 days during drug treatment using six developed tumors for each experiment and the mean tumor volume and standard error were calculated.

BrdU labeling analysis
Mice were injected intra-peritoneally with 100 µl of BrdU solution (BD Pharmingen, San Diego, CA) at 1 h before euthanasia. The tumor tissues were fixed in 4% paraformaldehyde, embedded and sectioned at 4-µm thickness. These sections were stained with anti-BrdU antibody (BD Pharmingen). The immunostaining analysis was repeated three times.

Small interfering RNA transfection
The target sequence of three small interfering RNAs (siRNAs) against p12-LOX were as follows: A, sense: GGGUGCAGGGAGAGGGAAUTT, antisense: AUUCCCUCUCCCUGCACCCTT; B, sense: GGAUGGAAUUCCAGCUAAUTT, antisense: AUUAGCUGGAAUUCCAUCCTT and C, sense: CAUCUCAGAUGGAGGAAUATT, antisense: UAUUCCUCCAUCUGAGAUGTT. We confirmed the siRNA sequences to be specific for mouse p12-LOX by GenBank database search. There is no transcript in the database that has >80% homology with siRNA sequences except for p12-LOX. These three siRNAs (A, B and C) were mixed and transfected into JB6 P+ cells at 50 nM using siFECTOR (B-Bridge, Sunnyvale, CA) prior to TPA stimulation. A decrease of p12-LOX mRNA by siRNA transfection was confirmed by RT–PCR and immunoblotting analysis.

Immunoblotting analysis
Cells were collected in buffer and sonicated. After centrifugation at 20 000g, 20 µg of the supernatant was separated in a 10% sodium dodecyl sulfate–polyacrylamide gel. Antibody for 12-LOX (Santa Cruz Biotechnology, Carlsbad, CA) was used as the primary antibody. The enhanced chemiluminescence detection system (Amersham Biosciences, Buckinghamshire, UK) was used to detect the specific signals.

NF-{kappa}B activation assay
JB6 P+ and JB6 P– cells were stimulated with TNF-{alpha} (Calbiochem) at 10 ng/ml in the presence or absence of baicalein (Tocris Biosciences) at 5 µM, and the nuclear extract of these cells was prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (PIERCE, Rockford, IL). Activity of NF-{kappa}B was measured using NF-{kappa}B p65 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA).

Cell proliferation assay
The cell proliferation rate was determined using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s protocol. Briefly, 102 or 103 cells were plated to each well of a 96-well microplate and cultured overnight, and the cells of the drug-treated group were treated with NDGA (5 µM) or baicalein (5 µM). Following the drug exposure period (4 or 6 days), 10 µl of MTT (5 mg/ml in phosphate-buffered saline) was added to each well and then the cells were cultured further for 4 h. The formazan crystals that were produced by these cultures were solubilized using 100 µl of isopropyl alcohol containing hydrochloric acid at 40 mM, and the absorbance at 570 nm was measured. All experiments were repeated three times and the mean relative MTT assay results to the no-drug control groups were then calculated and presented.

Cloning efficiency assay
JB6 P+ cells, two gastric cancer cells: AGS (American Type Culture Collection) and Kato-III (Cell Resource Center for Biomedical Research, Tohoku University, Japan) and three colon cancer cells: HCT-116 (American Type Culture Collection), DLD-1 and SW480 (Cell Resource Center for Biomedical Research) were used for the cloning efficiency experiments. The gastrointestinal cancer cells were maintained in Roswell Park Memorial Institute (AGS, Kato-III and DLD-1) or Dulbecco’s modified Eagle’s medium (SW480 and HCT-116) supplemented with 10% fetal bovine serum and cultured in a 5% CO2 incubator at 37°C. The cells were suspended in the medium at 10 cells/ml, and 100 µl was dispensed to each well of 96-well microplate to expect plating a single cell in each well. To examine the effect of p12-LOX on cloning efficiency, siRNA for p12-LOX was transfected prior to TPA stimulation or baicalein was added to the medium at 5 µM. After culture for 14 days, all wells were stained with Giemsa stain solution (Wako). All experiments were repeated three times and the cloning efficiency was calculated by dividing the number of wells containing proliferating cells with the total number of cell-plated wells.

Statistical analysis
The statistical significance of differences was determined by Student’s t-test. Differences with a value of P < 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Increased expression of p12-LOX in promotion-sensitive JB6 P+ cells
The expression level of p12-LOX was first examined by RT–PCR in the promotion-sensitive JB6 P+ cells as well as in promotion-resistant JB6 P– cells. The level of p12-LOX mRNA in the JB6 P+ cells was significantly higher than that in the JB6 P– cells (Figure 1A). Stimulation with TPA at 10 ng/ml increased the p12-LOX expression level in JB6 P– cells, although its highest level at 12 h was lower than that of the basal level of JB6 P+ cells. In contrast, p12-LOX in the TPA-stimulated JB6 P+ cells was increased only slightly possibly caused by constitutive elevation of the basal level. No significant differences were observed in either the expression of other LOX family members, such as epidermal-type 12-LOX, leukocyte-type 12-LOX, 5-LOX and 8-LOX between JB6 P+ cells and JB6 P– cells (Figure 1A).


Figure 1
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  		Fig. 1. The inhibition of p12-LOX in transformation-sensitive JB6 P+ cells suppresses colony formation in soft agar. (A) Representative RT–PCR results for the p12-LOX, epidermal type 12-LOX (e12-LOX), leukocyte type 12-LOX (l12-LOX), 5-LOX and 8-LOX are shown. The expression of p12-LOX is elevated in transformation-sensitive JB6 P+ cells, whereas it remains at the basal level in transformation-resistant JB6 P– cells. The expression of other LOX members is at the same level between JB6 P+ and JB6 P– cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. JB6 P+ and P– cells were stimulated with TPA (10 ng/ml) for 24 h, and the mRNA levels were examined by RT–PCR chronologically at the respective time points indicated. Relative band intensity of p12-LOX to the level at 0 h is indicated. (B) Representative photographs of soft agar cultures of JB6 P+ (top) and JB6 P– (bottom) cells are shown (Giemsa staining). TPA treatment (10 ng/ml) induced soft agar colony formation in JB6 P+ cells (top, second panel from left) but not in JB6 P– cells (bottom, second panel from left). Cells were treated with NDGA (5 µM), baicalein (5 µM) or AA-861 (10 µM) in the presence of TPA (right three panels). Note that treatment with a non-selective LOX inhibitor, NDGA, and a selective p12-LOX inhibitor, baicalein, resulted in significant suppression of soft agar colony formation, whereas the 5-LOX inhibitor, AA-861, treatment did not. (C) The mean number of colonies per field (mean ± SD) is shown. Treatments of the cells (drug treatment or siRNA transfection) are indicated below the histogram. *P < 0.05 versus no-drug control of TPA-stimulated JB6 P+ cells. (D) Representative RT–PCR for p12-LOX in the siRNA-transfected JB6 P+ cells is shown. GAPHD was used as an internal control. (E) Immunoblotting for p12-LOX in the siRNA-transfected JB6 P+ cells is shown. β-Actin was used as an internal control.

 
Suppression of anchorage-independent transformation of JB6 P+ cells by inhibition of p12-LOX
TPA treatment induced soft agar colony formation of JB6 P+ cells but not of JB6 P– cells that are a resistant line to tumor promoter-induced transformation (Figure 1B). When JB6 P+ cells were treated with a non-specific LOX inhibitor NDGA at 5 µM, the number of soft agar colonies decreased significantly by 88%. Moreover, treatment with baicalein, a p12-LOX specific inhibitor, at 5 µM also suppressed colony formation in soft agar of JB6 P+ cells by 81% (Figure 1B and C). It has been suggested that 5-LOX is also procarcinogenic lipoxygenase among the LOX family members (1,6). However, treatment with AA-861, a 5-LOX specific inhibitor, at 10 µM did not reduce the number of soft agar colonies (Figure 1B and C). These results indicate that p12-LOX but not 5-LOX plays an important role in the neoplastic transformation of JB6 P+ cells.

Baicalein has been reported to be a potent inhibitor of both reticulocyte 15-LOX and p12-LOX (28). Therefore, the effects of p12-LOX inhibition on soft agar colony formation were examined using a specific siRNA. Transient transfection of siRNA against the p12-LOX gene reduced the mRNA level in comparison with that of control cells (Figure 1D). We also confirmed the protein level of p12-LOX to have decreased in the siRNA-transfected JB6 cells (Figure 1E). Importantly, the inhibition of p12-LOX in JB6 P+ cells by siRNA transfection caused a significant suppression of TPA-induced neoplastic transformation by 61% compared with that in the control cells (Figure 1C). Therefore, the siRNA experiments confirmed the role of p12-LOX in promotion step of JB6 P+ cell transformation.

p12-LOX-independent growth of TPA-induced JB6 P+ tumors in nude mice
We examined the possibility that p12-LOX is required for the in vivo growth of tumor tissues consisting of JB6 P+ cells. TPA-stimulated JB6 P+ cells formed palpable tumors in nude mice at 22 days after subcutaneous inoculation (Figure 2A). In contrast to the anchorage-independent transformation assays, treatment of the mice with baicalein at 20 mg/kg/day from day 26 to 40 (14 days) did not suppress tumor growth (Figure 2A and B). There was no significant difference in the BrdU incorporation in tumor cells between baicalein-treated and non-treated mouse tumors (Figure 2C). Moreover, tumor development was not suppressed when the baicalein treatment started from day 7 after tumor cell inoculation (Figure 2D). These observations suggest that p12-LOX is less important for the JB6 tumor growth in vivo which develops from 106 inoculated cells. Rather, p12-LOX appeared to play an important role in the proliferation from a single JB6 cell (see below).


Figure 2
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  		Fig. 2. The inhibition of p12-LOX does not affect the tumor growth of JB6 P+ cells in vivo. (A) Photographs of TPA-stimulated JB6 P+ tumors developed in nude mice before (left) and after (right) baicalein treatment (20 mg/kg/day) at 26 and 40 days after the inoculation of tumor cells, respectively. (B) The tumor volume changes from day 25 to day 40 (mean ± SE) are shown. Solid lines indicate baicalein-treated mouse tumors, whereas dashed lines show no-drug control mouse tumors. (C) Representative photograph of BrdU incorporation (1 h) in the baicalein-treated tumor (right) and no-drug control tumor (left). Bars indicate 100 µm. Note that BrdU incorporation has increased in both baicalein-treated and baicalein-untreated mouse tumors. (D) Tumor volume at 1 week after palpable tumor developed in the baicalein-treated mice (from day 7) and no-treated mice (mean ± SE) is shown.

 
Acceleration TPA-induced transformation of JB6 P+ cells by 12(S)-HETE treatment
We next examined the role of 12(S)-HETE, a metabolite of p12-LOX, in tumor promotion. JB6 P+ cells were pre-treated with 12(S)-HETE and the TPA-induced soft agar colony formation in the presence or absence of baicalein was examined. Notably, the treatment of JB6 P+ cells with 12(S)-HETE reversed the inhibitory effects of baicalein on TPA-induced colony formation in a dose-dependent manner (Figure 3A and B), thus indicating that 12(S)-HETE is responsible for tumor promotion of JB6 P+ cells.


Figure 3
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  		Fig. 3. Treatment with 12(S)-HETE enhances TPA-induced neoplastic transformation of JB6 P+ cells. (A) Representative photographs of soft agar cultures are shown (Giemsa staining). TPA-negative control cells (left), TPA-stimulated and baicalein-treated cells (center) and TPA-stimulated but baicalein-untreated cells (right). The cells were pre-treated with 12(S)-HETE at 0.1 µM (middle panels) and 1 µM (bottom panels). Note that treatment with 12(S)-HETE restored soft agar colony formation of baicalein-treated JB6 P+ cells (center panels). The number of colonies increased by 12(S)-HETE even in the baicalein-untreated JB6 P+ cells (right panels). (B) The mean number of colonies per field is shown (mean ± SD). Treatments of the cells are indicated below the histogram. *P < 0.05 versus 12(S)-HETE-untreated control of each group.

 
Importantly, even in the absence of baicalein, the number of TPA-induced soft agar colonies of JB6 P+ cells was significantly increased by treatment with 12(S)-HETE in a dose-dependent manner (Figure 3A and B). In contrast, 12(S)-HETE treatment alone did not induce the soft agar colony formation of the TPA-untreated JB6 P+ cells. These results, taken together, indicate that 12(S)-HETE is a rate-limiting factor for the promotion of JB6 P+ cell transformation, although stimulation by a tumor promoter, such as TPA, is still necessary for such promotion.

Induction of p12-LOX by TNF-{alpha} stimulation
JB6 P+ cells are also transformed by TNF-{alpha} stimulation, and TNF-{alpha}-activated NF-{kappa}B is important for neoplastic transformation of JB6 P+ cells (29,30). Stimulation with TPA, acting through the mitogen-activated protein kinase pathway, activates AP-1 and subsequently NF-{kappa}B in JB6 P+ cells (31). Therefore, we examined the possibility that p12-LOX is involved in activation of NF-{kappa}B in the TNF-{alpha}-stimulated JB6 P+ cells. Consistent with the findings of a previous report, the treatment of JB6 P+ cells with TNF-{alpha} significantly increased the NF-{kappa}B activity (Figure 4A). Notably, the treatment of the cells with baicalein did not affect the NF-{kappa}B activity in the TNF-{alpha}-stimulated cells, indicating that the p12-LOX pathway is not required for the activation of NF-{kappa}B. Although NF-{kappa}B was activated by TNF-{alpha} in JB6 P– cells, the level was significantly lower than that in JP6 P+ cells (Figure 4A). We also confirmed that the TNF-{alpha}-induced NF-{kappa}B activation in JB6 P– cells was not suppressed by the baicalein treatment. In contrast, stimulation with TNF-{alpha} induced the p12-LOX expression both in JB6 P+ and JB6 P– cells (Figure 4B), thus suggesting that p12-LOX is induced by inflammatory responses through the TNF-{alpha}/NF-{kappa}B pathway.


Figure 4
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  		Fig. 4. p12-LOX pathway is not required for TNF-{alpha}-induced NF-{kappa}B activation, but is induced by TNF-{alpha} stimulation. (A) Relative NF-{kappa}B activity to the untreated JB6 P– level is shown (mean ± SD). Treatments of the cells are indicated below the histogram. *P < 0.05 versus TNF-{alpha}-untreated level of JB6 P– and JB6 P+ cells, respectively. {dagger}P < 0.05. (B) Representative RT–PCR results for p12-LOX in the TNF-{alpha}-stimulated JB6 cells are shown. The expression of p12-LOX is induced both in JB6 P+ and P– cells by TNF-{alpha} stimulation. GAPHD was used as internal control.

 
Suppression of cell proliferation by inhibition of p12-LOX
It has been shown that treatment with baicalein at 20–50 µM induces apoptosis and suppresses proliferation in several types of cancer cells (13,15–17). Therefore, the MTT assay was used to determine whether the proliferation of JB6 P+ cells is suppressed by baicalein at 5 µM, the same concentration used in this study. When 103 cells were plated in each well of a 96-well microplate, JB6 P+ cells showed approximately 20% decreased proliferation compared with that of the control cells both at day 4 and day 6 (Figure 5). Treatment with NDGA at 5 µM also yielded a similar suppression of proliferation.


Figure 5
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  		Fig. 5. Inhibition of p12-LOX significantly suppresses JB6 P+ cell proliferation when cells are plated at a low density. Relative cell densities to no-drug control at day 4 (left) and day 6 (right) after plating are shown (MTT assay, mean ± SD). Open bars indicate relative cell density in the wells plated with 103 cells, whereas black bars indicate 102 cells. Note that treatment with NDGA (5 µM) and baicalein (5 µM) suppressed proliferation significantly in the wells plated with 102 cells compared with that with 103 cells. *P < 0.05 versus no-drug control of the same cell number group; {dagger}P < 0.05 versus 103 cell-plated well of the same drug group.

 
Importantly, however, the proliferation of JB6 P+ cells was suppressed more significantly by baicalein or NDGA when 102 cells were plated in each well of a 96-well microplate (Figure 5). The number of drug-treated cells decreased by 90% of that of control cells at 6 days; the cells appeared to stop proliferation and they did not reach confluence after continuous culture for several days (data not shown). These results suggest that p12-LOX is therefore essential for the proliferation of JB6 P+ cells when cells were plated at a low density. This is consistent with the results of the in vivo experiments (Figure 2C) showing that baicalein treatment did not affect cell proliferation in the established tumors, where the cell density was very high.

Decrease of cloning efficiency of JB6 P+ cells by inhibition of p12-LOX
These results prompted us to examine the possibility that p12-LOX is required for the proliferation from a solitary single cell. Such ability of proliferation from a single cell is one of the important factors that is required for tumorigenesis. The cloning efficiency of TPA-untreated or TPA-treated JB6 P+ cells was 54% and 44%, respectively (Figure 6B), indicating that the cloning efficiency was independent from TPA treatment. Notably, the transfection of siRNA for p12-LOX suppressed the cloning efficiency significantly to 41% and 31% in the TPA-untreated and TPA-treated JB6 P+ cells, respectively (Figure 6A and B). More importantly, baicalein treatment dramatically reduced the cloning efficiency to 3% and 5% in the TPA-untreated and TPA-treated cells, respectively. These results indicate that p12-LOX is essential for the clonal proliferation from a solitary JB6 P+ cell, which should be important for the tumor promotion process. Although the suppression of cloning efficiency was more effective after baicalein treatment than that after siRNA transfection, this difference may be attributable to the transfection efficiency of siRNA.


Figure 6
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  		Fig. 6. Inhibition of p12-LOX suppresses the cloning efficiency of JB6 P+ cells. (A) Representative photographs of a microplate at 14 days after plating with no-drug control, siRNA-transfected and baicalein-treated cells (left to right) in which a single cell was seeded in each well (Giemsa staining). The wells containing proliferating cells are indicated with a circle. Note that treatment with baicalein (5 µM) dramatically suppressed the cloning efficiency. (B) The cloning efficiency of JB6 P+ cells is indicated as histogram (mean ± SD). Treatments of the cells are indicated below the histogram. *P < 0.05 versus baicalein-untreated control of each group. (C) Expression of p12-LOX is induced in gastric and intestinal tumors developed in K19-Wnt1/C2mE mice and Apc{Delta}716 mice, respectively. Representative RT–PCR results are shown. T indicates tumor, whereas N indicates adjacent normal tissue. GAPHD was used as internal control. (D) The cloning efficiencies of gastric and colon cancer cells are indicated as histogram (mean ± SD). Treatments of the cells are indicated below the histogram. *P < 0.05 versus baicalein-untreated control of each group.

 
p12-LOX-independent clonal proliferation of malignant cancer cells
To investigate whether p12-LOX is required for the tumor promotion step in other tissues, the expression of p12-LOX in gastric tumors of K19-Wnt1/C2mE mice (5) as well as intestinal polyps of Apc{Delta}716 mice was examined (26). Interestingly, the p12-LOX expression was significantly induced in both gastric and intestinal tumor tissues (Figure 6C), suggesting the role of p12-LOX in tumorigenesis also in the gastrointestinal tract. Therefore, the cloning efficiency of gastric cancer cells (AGS and Kato-III) and colon cancer cells (DLD-1, SW480 and HCT-116) was examined. The treatment with baicalein at 5 µM significantly decreased the cloning efficiency of SW480 cells, although the colonies still formed in 44% of the plate wells (Figure 6D). The cloning efficiencies in other cell lines did not show significant decrease by the inhibition of p12-LOX. Accordingly, it is possible that progressing cancer cells acquire the ability for p12-LOX-independent proliferation from a solitary cell, unlike JB6 P+ cells.


   Discussion
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 Materials and methods
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Funding
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Accumulating evidence indicates that expression of p12-LOX is induced in several types of cancer tissues, whereas its expression is absent or weak in the normal tissues (6). Moreover, p12-LOX expression increases in the advanced stage of cancer tissues, and the p12-LOX pathway is involved in the suppression of apoptosis, induction of cell survival and angiogenesis (10,15,19,21). These results suggest that the p12-LOX pathway plays a role in tumor development, progression and metastasis. In addition to these results, the present study demonstrated, by pharmacological and genetic experiments, that p12-LOX pathway is required for tumor promotion in mouse epidermal cell transformation. This is consistent with a previous report that disruption of the p12-LOX gene in mice suppresses the development of squamous cell carcinoma caused by an initiation/promotion protocol (23). Moreover, the present study has shown that treatment with 12(S)-HETE, a metabolite of p12-LOX, further increases the number of soft agar colonies in TPA-treated JB6 P+ cells. Accordingly, it is possible that formation of 12(S)-HETE functions as a rate-limiting factor for tumor promotion in epidermal cell transformation. This suggests that the induction level of p12-LOX may be positively correlated with the efficiency of tumor promotion.

It has been shown that baicalein, a p12-LOX inhibitor, induces apoptosis in several types of cancer cells (13,15–17). However, baicalein treatment at the concentration used in this study (5 µM) showed only mild suppressive effect on cell proliferation when 103 JB6 P+ cells were plated in the microplate well. When the number of plating cells was reduced to 102 cells, however, baicalein treatment at 5 µM significantly suppressed proliferation. Moreover, the cloning efficiency of JB6 P+ cells was suppressed dramatically by the same concentration of baicalein. These results collectively indicate that 12(S)-HETE is required for the proliferation of small number of cells or a single cell. In the promotion step of tumorigenesis, the original ‘initiated’ single cell or a small number of promoted cells need to proliferate to form cluster of tumor cells. Accordingly, it is possible that the p12-LOX/12(S)-HETE pathway plays a key role in clonal proliferation of initiated cells during the promotion step. In contrast, the cloning efficiency of malignant gastric and colon cancer cells was not affected by inhibition of p12-LOX. It is possible that tumor cells derived from progressed cancer tissues have already acquired an ability of 12(S)-HETE-independent proliferation from a single cell.

These experiments showed that the expression of p12-LOX is induced in epidermal cells by stimulation with TNF-{alpha}, suggesting that an inflammatory host response induces p12-LOX expression through TNF-{alpha}/NF-{kappa}B pathway. It has been shown that TPA stimulation also activates NF-{kappa}B in JB6 P+ cells (31), which is required for transformation (29,30). It has been established that inflammation plays an important role in cancer development (32). Genetic studies have demonstrated that the activation of the TNF-{alpha}/NF-{kappa}B pathway is required for tumor development in the mouse colon and liver through the suppression of apoptosis in tumor cells and the induction of growth factors from stromal cells (33,34). Importantly, the expression of p12-LOX is induced not only in epidermal tumor cells but also in gastrointestinal tumors where an inflammatory response is associated (4,5). Accordingly, it is possible that p12-LOX is one of the tumor-promoting factors induced by inflammatory responses through the TNF-{alpha}/NF-{kappa}B pathway in various types of tumors. It has been reported that AP-1 and NF-{kappa}B play a critical role in transformation of JB6 P+ cells, and activity of these factors is downregulated in JB6 P– cells (30,31). Our results indicate that the deficiency of p12-LOX in JB6 P– cells contributes to a resistant phenotype to tumor promoter-induced transformation. Reactive oxygen species also stimulate the neoplastic transformation of JB6 cells by activating AP-1 and NF-{kappa}B (31). Moreover, p12-LOX has also been reported to play a role in the reactive oxygen species generation in neuroblastoma cells (35). Accordingly, it is possible that the inhibition of p12-LOX suppresses neoplastic transformation of JB6 P+ cells which is caused, at least in part, by the decrease of TPA-induced reactive oxygen species generation.

These effects of the p12-LOX pathway on tumor promotion are similar to those of the COX-2/PGE2 pathway in the gastrointestinal tumorigenesis. The activation of Wnt/β-catenin signaling causes the development of intestinal microadenoma (26). The subsequent induction of the COX-2/PGE2 pathway in the microenvironment around the microadenoma is required for proliferation of small number of adenoma cells (3,36). Specifically, the inhibition of COX-2 or PGE2 signaling suppresses proliferation of tumor cells in microadenoma (36,37). Importantly, these gastrointestinal tumors in mouse models also show the induction of the p12-LOX pathway as indicated in the present study. Therefore, it is possible that p12-LOX and COX-2 are two important pathways for tumor cell proliferation in the early stages. This is consistent with the findings of recent reports in which the polymorphism of either COX-2 or p12-LOX was shown to be associated with an increased risk of colon cancer development (38). Moreover, both COX-2 expression and 12(S)-HETE synthesis are induced in mouse prostate tumors (39). On the other hand, baicalein treatment failed to induce a regression of the established tumors in the immunodeficient mice. Accordingly, it is conceivable that the p12-LOX/12(S)-HETE pathway plays an important role particularly in the early promotion step like COX-2/PGE2 pathway, rather than in the progression stage.

In conclusion, the present results demonstrated that p12-LOX is required for tumor promotion during epidermal cell transformation and 12(S)-HETE can accelerate tumorigenesis through enhancement of cloning efficiency. The expression of p12-LOX is induced also in the gastrointestinal tumors, suggesting that the p12-LOX pathway is an effective target for chemoprevention against skin carcinogenesis as well as gastrointestinal cancer.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Ministry of Education, Culture, Sports, Science and Technology of Japan, and Ministry of Health, Labor and Welfare of Japan.


   Acknowledgments
 
We thank Manami Watanabe for valuable technical assistance.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received July 10, 2007; revised October 29, 2007; accepted November 24, 2007.


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