Pharmacology and Safety Research Department, Pharmaceutical Development Research Laboratories, TEIJIN Pharma Limited, 4-3-2, Asahigaoka, Hino, Tokyo 191-8512, Japan

    ABSTRACT

    Cell transforming activity of palytoxin, a non-TPA type tumor-promoter, was investigated with the two-stage transformation assay using Balb/c 3T3 cells. Palytoxin showed potent promoting activity; treatment at 1.9 pM or more increased the number of transformed foci after initiation by 3-methylcholanthrene (MCA). Determination of prostaglandin (PG) E2 and PGF2 concentrations in the culture medium revealed that palytoxin (1.9–3.7 pM for 24 h) stimulated the production of PG in Balb/c 3T3 cells (the concentration reached 3–4 μM), and treatment with PGE2 or PGF2 itself increased the number of transformed foci of Balb/c 3T3 cells after initiation by MCA. Neither palytoxin nor PGs showed initiating activity. Indomethacin suppressed the promoting activity of palytoxin, but not that of PGE2 and PGF2. Interestingly, concomitant treatment with PGE2 or PGF2 in addition to indomethacin markedly reversed the suppressive effect of indomethacin. These findings indicated that the in vitro transformation model could reproduce experiments that have been performed in animal models regarding the inhibitory effect of indomethacin on carcinogenic responses and reversal of indomethacin's effect by exogenous prostaglandin and, therefore, may provide insight into molecular modes of action of palytoxin. In the present study, palytoxin also induced prostaglandin synthesis, and therefore, the Balb/c 3T3 cell model should provide insight into the molecular mechanism by which palytoxin regulates prostaglandin biosynthesis.

    Key Words: palytoxin; prostaglandin; cell transformation assay; tumor-promoter.

    INTRODUCTION

    It is widely accepted that multiple mechanisms underlie the two main stages of carcinogenesis, initiation and promotion. The cell transformation assay in vitro is regarded as a valid model of carcinogenesis in vivo (Smets, 1980). Among mammalian cloned cell lines, Balb/c 3T3 (Cortesi et al., 1983; Kakunaga et al., 1973) and C3H 10T1/2 (Reznikoff et al., 1973a,b) have been widely used in cell transformation assays; these cells are very sensitive to post-confluence inhibition of cell division and to the induction of transformed foci by chemical carcinogens. As in the mouse skin papilloma model, there are two stages, initiation and promotion, in the transformation assay (Hirakawa et al., 1982; Mondal et al., 1976), and two-stage transformation assays have been used for testing tumor-promoters and studies on the two-stage process of carcinogenesis (Frixten and Yamazaki, 1987; Mondal et al., 1978). Many tumor-promoters have been reported to enhance the number of transformed foci, including 12-O-tetradecanoylphorbol-13-acetate (TPA), dihydroteleocidin B, aplysiatoxin, phorbol-12,13-dedecanoate, 1-oleoyl-2-acethyl glycerol, okadaic acid, insulin, catechol, lithocholic acid, 3-hydroanthranilic acid, and transforming growth factor  (Atchison et al., 1982; Frixten and Yamazaki, 1987; Hamel et al., 1988; Hirakawa et al., 1982; Sakai and Fujiki, 1991; Shimomura et al., 1983; Umeda et al., 1983, 1989). Cell transformation assays have been also used in mechanistic studies on carcinogenesis and to elucidate the possible mechanisms of action of carcinogens (Combes et al., 1999).

    Many reports have revealed that prostaglandin (PG) enhances cancer development, acting as a tumor-promoter, and has profound effects on carcinogenesis (Lupulescu, 1996). Regarding tumor-promoting activity in vivo, the development of skin tumors (squamous cell carcinomas and papillomas) was markedly enhanced by the administration of PGE2 and PGF2 in mice treated with 3-methylcholanthrene (MCA) (Lupulescu, 1978). In addition, typical tumor-promoters, including both TPA type and non-TPA type promoters, were reported to stimulate the release of PG from mouse epidermal cells and human keratinocytes (Aizu et al., 1992; Goldyne and Evans, 1994). Inhibitors of PG synthesis were reported to suppress the expression of PG synthase and prevent TPA-induced tumor-promotion in vivo and cell transformation in vitro (Müller-Decker et al., 1995; Wlfle et al., 2000). In the clinical setting, an inhibitor of PG synthesis, indomethacin, was reported to affect human rectal adenomatosis and the regression of polyps (Hirota et al., 1996).

    Palytoxin, which was found in a coelenterate of the genus Palythoa, is a highly poisonous agent of low molecular weight. The receptor for palytoxin is the plasma membrane Na+,K+-ATPase, which stimulates ion flux by transforming the pump into an ion channel (Habermann,1989). Palytoxin is also known as a non-TPA type tumor-promoter (Fujiki et al., 1986). The stimulation of ion flux may be a critical component of palytoxin-stimulated signaling (Li and Wattenberg, 1998); however, the biochemical mechanisms by which palytoxin-stimulated signaling contributes to tumor promotion have not been elucidated.

    Meanwhile, Fujiki et al. (1986) showed tumor-promoting activity of palytoxin in an animal model, but its effect on transformed foci in vitro has not been examined. Lazzaro et al. (1987) reported stimulation of PG production by palytoxin in mouse calvariae cells in vitro. Interestingly, the relative effect of palytoxin to stimulate the metabolism of arachidonic acid was very potent (1000–3000 times that of the TPA type promoters). Considering the many reports of the profound effects of PG on carcinogenesis as mentioned above, regulation of PG biosynthesis by palytoxin would play an important role; however, the relationship between PG production and the tumor-promoting activity of palytoxin has not been elucidated. Investigating the biochemical mechanism of palytoxin action may help to define fundamental events in tumor promotion, and the in vitro model, which can reproduce the results of experiments performed in animal models, should provide insight into molecular modes of action of palytoxin.

    In this study, we investigated the cell transforming activity of palytoxin by conducting two-stage transformation assays using Balb/c 3T3 cells in vitro. Palytoxin showed strong promoting activity in this system. Determination of PGE2 and PGF2 concentrations in the culture medium revealed that palytoxin stimulated the production of PG in Balb/c 3T3 cells, and treatment with PGE2 or PGF2 itself increased the number of transformed foci of Balb/c 3T3 cells after initiation by MCA. Indomethacin suppressed the promoting activity of palytoxin, but not that of PGE2 and PGF2. Interestingly, concomitant treatment with PGE2 or PGF2 in addition to indomethacin markedly reversed the suppressive effect of indomethacin. The present study indicated that this in vitro transformation model may provide insight into the molecular modes of action by which palytoxin regulates prostaglandin biosynthesis and tumor-promoting activity.

    MATERIALS and METHODS

    Chemicals.

    The chemicals used were obtained from the following sources: MCA, TPA, palytoxin, and indomethacin from Wako Pure Chemical Industries (Osaka, Japan), and PGE2 and PGF2 from Funakoshi (Tokyo, Japan).

    Antibodies.

    The antibodies were obtained from the following sources: polyclonal rabbit antibodies against phospho-extracellular signal-related kinases (pERK) 1/2 (respective molecular weights of 44 and 42 kDa; phosphorylated on Thr202/Tyr-204) and ERK 1/2 from Cell Signaling (Beverly, MA) and horseradish peroxidase-conjugated goat anti-rabbit IgG from Pierce (Rockford, IL).

    Cells and culture conditions.

    Balb/c 3T3 clone A31-1-1 cells (JCRB 0601) were provided by the Japanese Cancer Research Resource Bank, National Institute of Hygienic Sciences (Tokyo, Japan). The cells were multiplied, frozen with medium containing 10% dimethyl sulfoxide (DMSO) and 10% fetal bovine serum (FBS) in a deep freezer (–80°C), and stored in liquid nitrogen. Transformation assays were conducted with the frozen stock.

    Eagle's minimal essential medium (MEM) with Earle's salt, supplemented with 10% heat-inactivated FBS, was used as a culture medium throughout the assay. MEM was purchased from Invitrogen (Carlsbad, CA). The FBS used was a single lot, which was prescreened to confirm a failure to produce spontaneously transformed foci in Balb/c 3T3 cells and to ensure an ability to induce appreciable numbers of foci treated with 5 μg/ml MCA for 72 h (Kakunaga and Yamazaki, 1985). Cultures were maintained in a humidified incubator with an atmosphere of 5% CO2 in air at 37°C.

    Transformation assay.

    The two-stage transformation assay was carried out as shown in Figure 1, basically according to the protocol described by Sakai and Sato (1989). The frozen stock of Balb/c 3T3 clone A31-1-1 cells was rapidly thawed and cultured for 3 days until seeding. Actively growing cells were seeded at a density of 104 cells/60-mm culture dish (10 dishes were used per concentration) in 5 ml of the culture medium. Two days after seeding, cells were treated with MCA or DMSO added to the medium for 72 h (initiating treatment). The medium was replaced with fresh normal medium, and the cells were grown for 3 days. The cultures were then treated with medium containing the test chemical (palytoxin, PG, etc.) for 2 weeks (promoting treatment). The cells were subsequently cultured in normal medium for 2 or 3 weeks. The medium was changed twice a week during the first 4 weeks and once a week thereafter. The cells were fixed with methanol and stained with a 10% Giemsa solution.

    The test chemicals (palytoxin, PG, and indomethacin), MCA, and TPA were dissolved in DMSO. The final concentration of the vehicle in the culture medium was 0.5%, which did not affect the generation of transformed foci. The scoring of transformed foci was performed according to the criteria described by the IARC/NCI/EPA Working Group (1985); foci that showed a basophilic, dense multi-layering of cells, random cell orientation at the focus edge, invasion into the surrounding contact-inhibited monolayer, and domination of spindle-shaped cells were scored as positively transformed. Each transformation assay included negative and positive controls; as for negative controls, cultures were treated with 0.5% DMSO (initiating treatment) followed by 0.5% DMSO (promoting treatment), 0.5% DMSO (initiating treatment) followed by 324.3 nM (200 ng/ml) of TPA (promoting treatment), 0.5 μg/ml of MCA (initiating treatment) followed by 0.5% DMSO (promoting treatment); as for positive controls, 0.5μg/ml of MCA (initiating treatment) followed by 324.3 nM of TPA (promoting treatment), and 5 μg/ml of MCA (initiating treatment) followed by 0.5% DMSO (promoting treatment).

    Relative cell growth.

    Cytotoxic effects of chemicals were determined in parallel with the transformation assay as shown in Figure 1. Replicate dishes (four dishes per concentration) were seeded and treated with palytoxin or vehicle in the initiating treatment, and TPA, vehicle, or the test chemical in the promoting treatment, as in the transformation assay. At the end of promoting treatment, the cultures were fixed with 10% formaldehyde for 15 min and stained with a 1% crystal-violet solution for 15 min. The optical density at 570 nm (OD570) of stained-dishes was measured using a Monocellator (Olympus, Tokyo, Japan) (Tsuchiya et al., 1999). Relative cell growth was determined by standardizing the control OD570 as 100%.

    Measurement of PG concentration in medium.

    Triplicate dishes per concentration, at a density of 104 cells/60-mm culture dish, were seeded and treated with the test chemicals. After treatment, the culture medium was collected, and the concentration of PGE2 or PGF2 in the medium was determined by EIA or ELISA. For PGE2, an EIA kit was obtained from Invitrogen, and measurements were made according to the manufacturer's instructions. For PGF2, an ELISA kit was obtained from Neogen (Lansing, MI), and measurements were made according to the manufacturer's instructions.

    Preparation of whole cell extracts.

    Actively growing cells were seeded at a density of 104 cells/60-mm culture dish in 5 ml of the culture medium. The cells were cultured for 1 week. The medium was changed twice a week. The cultures at subconfluent state (approximate 80–90% confluency) were switched to serum-free medium overnight and then treated with the test chemicals. After the treatments, the cells were washed with ice-cold phosphate buffer saline (PBS), and lysed by addition of lysis buffer (250 mM NaCl, 0.1% Nonidet P-40, 50 mM HEPES at pH 7.0, 25 μM sodium fluoride, 250 μM sodium vanadate, 0.12 IU aprotinin). Cell lysate was centrifuged at 10,000 x g for10 min at 4°C. The protein concentration of the supernatant was determined using the Bradford assay (Bio-Rad, Hercules, CA).

    Western blot analysis of pERK 1/2 or ERK 1/2.

    Proteins from whole-cell lysate (40 μg) were incubated for 3 min at 95°C in Laemmli buffer, separated on a 8.5% SDS–polyacrylamide gel, and then transferred to Immobilon-P-PVDF (Millipore, Bedford, MA). The membrane was blocked with PBS containing 10% skimmed milk for 1 h at room temperature, then incubated for 1 h with rabbit polyclonal antibodies against pERK 1/2 or ERK 1/2, and followed by goat anti-rabbit IgG conjugated with horseradish peroxidase for another 1 h. The signal for immunoblots was detected using an enhanced chemiluminescence detection system purchased from Amersham Pharmacia Biotech (Amersham, UK) according to the manufacture's manual. pERK 1/2 levels were quantified using the ImageGauge program Ver. 3.46 (Fuji Photo Film, Tokyo, Japan).

    Statistical analysis.

    Statistical analyses were performed as follows: Dunnett's test for comparison among multiple groups and Student t-test for comparison between two groups for the data of relative cell growth, PG concentration in medium, and pERK level; Chi-square test for the data of number of dishes with foci/total number of dishes examined; Kruskal-Wallis test for comparison among multiple groups; and Fisher's exact test (one tailed) for comparison between two groups for the data of number of foci. When significant differences were observed in the Kruskal-Wallis test, Steel test was performed as posthoc test for the data of number of foci. Significant levels were p < 0.05, p < 0.01 and p < 0.001. Analysis was performed using the EXSAS software (Ver.7.10).

    RESULTS

    Promoting Activity of Palytoxin

    Table 1 shows the results of the two-stage transformation assay using Balb/c 3T3 cells to evaluate the promoting activity of palytoxin. In the assay, 0.5 μg/ml of MCA produced very few transformed foci without subsequent treatment with a promoter, while 5 μg/ml caused a marked increase in the number of transformed foci in the absence of a promoter. Palytoxin showed potent promoting activity; treatment at 1.9 pM markedly enhanced the generation of transformed foci initiated by 0.5 μg/ml of MCA. In cultures pretreated with vehicle alone, treatment with palytoxin did not increase the number of transformed foci. We tested the cytotoxicity of the chemicals using the monocellator methods. This method using crystal-violet staining has been employed in a chromosomal aberration test (Sugiki et al., 1994). Usually, the cytotoxic effect in a transformation assay is determined by the clonal colony-formation method; however, Tsuchiya et al. (1999) reported that the monocellator method was applicable and provided reproducible results. As shown by relative cell growth, palytoxin added during the treatment-phase did not cause a decrease in relative cell growth; therefore, the above transformation assay was carried out with a nontoxic dose. Rather than a decrease, palytoxin caused an increase in cell growth.

    Effect of Palytoxin on Production and Promoting Activity of PG

    Figure 2 shows the concentrations of PGE2 (A) and PGF2 (B) measured in the culture medium after treatment with palytoxin for 6 or 24 h. Palytoxin at 3.7 pM (10 pg/ml) increased both concentrations after 6 h. A dose-dependent increase was observed after 24 h in the range of 0.4 pM (1 pg/ml) to 3.7 pM.

    The results indicated that the production of PG may be involved in the promoting activity of palytoxin. Thus we investigated the effects of PGE2 and PGF2 using the two-stage transformation assay. As shown in Tables 2 and 3, both PGE2 and PGF2 markedly enhanced the production of transformed foci initiated by 0.5 μg/ml of MCA. In cultures pretreated with vehicle alone, treatment with PGE2 did not affect the transformed foci (Table 2). As shown in Table 3, while a statistical significance was observed in the DMSO/PGF2 (initiating/promoting) treatment groups, maximum number of foci/dish was 0.4 (at 13 μM of PGF2). This value was within the negative control criteria (not exceed 0.5 foci/dish) described by the IARC/NCI/EPA Working Group (1985). As for relative cell growth, neither PGE2 nor PGF2 added during the treatment-phase caused a decrease; rather, an increase in cell growth was observed, the same as after palytoxin treatment.

    Effects of Indomethacin on Promoting Activity of Palytoxin or PG

    We next examined the effect of a cyclooxygenase inhibitor, indomethacin, on the concentration and activity of PG. Figure 3 shows the effect of indomethacin on the concentration in medium. For both PGE2 and PGF2, the concentration drastically decreased when 55.9 μM (20 μg/ml) of indomethacin was added. Table 4 shows the effect of indomethacin on the promoting activity of palytoxin or PG. The enhancement of transformed foci after treatment with 0.5 μg/ml of MCA and palytoxin was completely suppressed by indomethacin. In contrast to palytoxin, the enhancement of transformed foci after treatment with 0.5 μg/ml of MCA and PG was not suppressed by indomethacin. Interestingly, when PGE2 or PGF2 was added to cultures along with indomethacin, this suppression was markedly reversed (Table 5). As shown in Figure 4A, relative cell growth decreased in the palytoxin and indomethacin-treated group as compared with the palytoxin-treated group. In contrast to palytoxin, decrease by the indomethacin treatment was not observed in the PGE2- or PGF2-treated groups. Interestingly, the decrease was reversed by concomitant treatment with PG (Fig. 4B).

    Effects of Palytoxin or PG on pERK 1/2 Level

    Mitogen-activated protein (MAP) kinases are important signal-transducing enzymes, and palytoxin or PGs had been reported to stimulate MAP kinase signaling cascade (Wang et al., 2005; Warmka et al., 2002). Therefore, we examined the ability of palytoxin or PGs to activate ERK in Balb/c 3T3 cells. The ERK activation was determined with Western blotting using anti-pERK 1/2. As shown in Figure 5A, palytoxin treatment for 24 h resulted in a dose-dependent increase in ERK phosphorylation. Figures 5B and 5C show the quantified data obtained by the densitometoric analyses using the ImageGauge program; increase of pERK level was dose- and time-dependent. PGE2 or PGF2 also increased ERK phosphorylation level in a dose- and time-dependent manner in Balb/c 3T3 cells (Figs. 5D and 5E). As shown in the right column in Figure 5A, the palytoxin-induced pERK level was inhibited in the presence of indomethacin. Figure 6A shows the quantified pERK levels in the presence or absence of indomethacin. In contrast to palytoxin, the PG-induced pERK level was not inhibited. Interestingly, when PGE2 or PGF2 was added to cultures along with indomethacin, the inhibitory effect of indomethacin on palytoxin-induced pERK level was reversed (Fig. 6B).

    DISCUSSION

    In this study, we investigated the cell transforming activity of palytoxin, especially the tumor-promoting activity, by conducting two-stage cell transformation assays using Balb/c 3T3 cells in vitro. The results as shown in Table 1 clearly indicated that palytoxin increased the number of transformed foci of Balb/c 3T3 cells after initiation by MCA. The in vitro activity of palytoxin revealed in the present study is considered to be consistent with the in vivo tumor-promoting activity reported by Fujiki et al. (1986). Malignant cell transformation in vitro is regarded as a good model system, and there are a number of similarities between carcinogenesis in vivo and transformation in vitro (Sakai and Sato, 1989; Smets, 1980). Adding to such studies, the results obtained from the present study support the use of transformation assays to detect tumor-promoters, including non-TPA type promoters such as palytoxin, in vitro.

    As shown in Figure 2, palytoxin stimulated PG production in Balb/c 3T3 cells. Regarding other tumor-promoters, many reports have suggested a critical role for PG in cancer development and a relationship between the modulation of PG synthesis and tumor-promoter activity. Lupulescu (1978) reported skin tumor (squamous cell carcinoma and papillomas) was markedly enhanced by PGE2 or PGF2 administration to mice treated with MCA. The enhanced release of PGE2 from mouse epidermis (Aizu et al., 1992) or human keratinocytes (Goldyne and Evans, 1994) after TPA treatment in vitro was reported, and Müller-Decker et al. (1995) suggested a critical role for the increase in PG synthase expression during the multistage carcinogenesis of mouse epidermis in vivo. Wlfle et al. (2000) indicated a critical role for the enhancement of COX-1 and COX-2 expression and stimulation of arachidonic acid metabolism in the promoting effect on transformed foci of C3H cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment, and later, Wlfle (2003) reported activity for cell transformation by PGF2 in C3H cells. Inhibitors of PG synthesis (aspirin, ibuprofen, indomethacin, piroxicam, and sulinduc), commonly called NSAIDS (nonsteroidal anti-inflammatory drugs), have also been reported to significantly inhibit cancer development and recently have been used in epidemiological studies for cancer prevention and treatment (Lupulescu, 1996). In the present study, we revealed an increase in the concentration of PG in the culture medium after palytoxin treatment (Fig. 2), enhancement of transformed foci by PG treatment (Tables 2 and 3), and suppressive effect of indomethacin on palytoxin-activity and reversibility of indomethacin's effect by concomitant treatment with PG (Tables 4 and 5) in Balb/c 3T3 cells. Considering these results, it was strongly suggested that the promoting activity of palytoxin on cell transformation was mediated by stimulating PG production. While Lazzaro et al. (1987) reported stimulation of PG production by palytoxin in mouse calvariae cells in vitro, they did not mention the relationship between such a biological response and tumor-promoting activity. Our data clarified the critical role of stimulated PG production in the tumor-promoting activity of palytoxin.

    Kimura et al. (2000) reported that PGE2 and PGF2 stimulated DNA synthesis and cell proliferation in primary cultures of rat hepatocytes. Thompson et al. (2001) showed that production of PGE2 was implicated in skin carcinogenesis, and indomethacin decreased cell growth; the effect of indomethacin was reversed by addition of PGE2 or EP1 agonists. In the present study, treatment with palytoxin or PG increased cell growth as shown in Tables 1– 3. In addition, while the increase in cell growth caused by PG was not suppressed by indomethacin, palytoxin-activity was suppressed by indomethacin, and reversibility of indomethacin's effect by concomitant treatment with PG was also observed (Fig. 4). Considering the previous reports and results of the present study, the increase in cell growth caused by palytoxin would have resulted from the stimulation of PG biosynthesis.

    As for the biochemical mechanism by which PG regulates cell growth, Wang et al. (2005) indicated that PGE2 promoted cell proliferation in intestinal adenomas, and the effect was mediated by activation of the MAP kinase signaling cascade. Interestingly, palytoxin had been also reported to stimulate MAP kinases (Li and Wattenberg, 1998, 1999) and the activation of ERK and c-Jun N-terminal kinase (Warmka et al., 2002; Zeliadt et al., 2003). ERK activity is regulated through the phosphorylation of threonine and tyrosine residues within the catalytic domain of ERK (Cobb and Goldsmith, 1995). As shown in Figure 5, palytoxin or PGs increased ERK phosphorylation in a dose- and time-dependent manner. The palytoxin-induced pERK level was inhibited by indomethacin (Fig. 6A), and interestingly, when PGE2 or PGF2 was added to cultures along with indomethacin, the inhibition on palytoxin-induced pERK levels was reversed (Fig. 6B). In addition, in our preliminary examination, the effect on transformed foci after treatment with 0.5 μg/ml of MCA (initiating treatment) and palytoxin, PGE2, or PGF2 (promoting treatment) was suppressed by adding PD 98059, which inhibits the activation of MAP kinase (data not shown). Based on these results, it was suggested that palytoxin regulates intracellular kinase activity and protein phosphorylation by stimulating PG synthesis, and such biochemical changes may be behind palytoxin's tumor-promoting activity.

    In the present study, we have established an in vitro model that reproduces experiments performed in animal models regarding the inhibitory effect of indomethacin on carcinogenesis, using transformed foci as an index, and reversal of indomethacin effect by exogenous PGs. Also, PG biosynthesis induced by palytoxin was observed; therefore, the transformation assay using Balb/c 3T3 cells should provide insight into the molecular modes of action and mechanism by which palytoxin regulates PG biosynthesis and tumor-promotion. In Figure 2, palytoxin increased PG levels to approximately 3–4 μM in the culture medium, while the promotion of transformed foci by exogenous PG required at least 10-fold this concentration, as shown in Tables 2 and 3. A previous report (Lupulescu, 1996) revealed large amounts of PGs in tumor tissue and in circulating blood in several experimental tumors and human cancers. However, levels of exogenous PGs in the present study were obviously much higher. Several interpretations, such as a lower sensitivity due to low expression levels of the PG receptors in Balb/c 3T3 cells or stability of the PGs added in the present experimental conditions (the medium containing PGs was changed twice a week during the treatment period), may be possible. However, the exact reason for differences between the concentration of PGs in the present in vitro study and the previous in vivo data remains unclear. In addition, the effect on the cognizant PG receptors has not been examined. Further study to solve the issues pertaining to PG concentrations and effects of palytoxin (i.e., activation of the cognizant PG receptor) is considered to be important for understanding the molecular mechanism of tumor-promotion by palytoxin.

    In the present study, we indicated cell transforming activity of palytoxin in vitro, especially tumor-promoting activity, consistent with the data obtained from in vivo carcinogenicity experiments. In addition, the present study suggested the involvement of production of PG. The cell transformation assay is considered a useful tool, not only for examining the tumor-promoting activity of a wide range of tumor promoters including palytoxin, but also for providing insight into the molecular modes of action by which palytoxin regulates prostaglandin biosynthesis and tumor-promoting activity.

    REFERENCES

    Aizu, E., Yamamoto, S., and Kato, R. (1992). Anthralin, a non-TPA type tumor promoter, synergistically enhances phorbol ester-caused prostaglandin E2 release from primary cultured mouse epidermal cells. Jpn. J. Pharmacol. 60, 9–17.

    Atchison, M., Chu, C., Kakunaga, T., and Van Duuren, B. L. (1982). Chemical cocarcinogenesis with the use of a subclone derived from Balb/c 3T3 cells with catechol as cocarcinogen. J. Natl. Cancer Inst. 69, 503–508.

    Cobb, M. H., and Goldsmith, E. J. (1995). How MAP kinases are regulated. J. Biol. Chem. 270, 14843–14846.

    Combes, R., Balls, M., Curren, R., Fischbach, M., Fusenig, N., Kirkland, D., Lasne, C., Landolph, J., LeBoeuf, R., Marquardt, H., et al. (1999). Cell transformation assays as predictor of human carcinogenicity. ATLA 27, 745–767.

    Cortesi, E., Saffiotti, U., Donovan, P. J., Rice, J. M., and Kakunaga, T. (1983). Dose-response studies on neoplastic transformation of BALB/3T3 clone A31-1-1 cells by aflatoxin B1, benzidine, benzo[a]pyrene, 3-methylcholanthrene, and N-methyl-N'-nitro-N-nitrosoguanidine. Teratog. Carcinog. Mutagen. 3, 101–110.

    Frixten, U., and Yamasaki, H. (1987). Enhancement of transformation and continuous inhibition of intracellular communication by 1-oleoyl-2-acetyl glycerol in BALB/c 3T3 cells. Carcinogenesis 8, 1101–1104.

    Fujiki, H., Suganuma, M., Nakayasu, M., Hakki, H., Horiuchi, T., Takayama, S., and Sugimura, T. (1986). Palytoxin is a non-12-O-tetradecanoylphorbol-13-acetate type tumor promoter in two-stage mouse skin carcinogenesis. Carcinogenesis 7, 707–710.

    Goldyne, M. E., and Evans, C. B. (1994). 12-O-Tetradecanoylphorbol-13-acetate and the induction of prostaglandin E2 generation by human keratinocytes: A re-evaluation. Carcinogenesis 15, 141–143.

    Habermann, E. (1989). Palytoxin acts through Na+,K+-ATPase. Toxicon 27, 1171–1187.

    Hamel, E., Katoh, F., Mueller, G., Birchmeier, W., and Yamasaki, H. (1988). Transforming growth factor  as a potent promoter in two-stage BALB/c 3T3 cell transformation. Cancer Res. 48, 2832–2836.

    Hirakawa, T., Kakunaga, T., Fujiki, H., and Sugimura, T. (1982). A new tumor-promoting agent, dihydroteleocidin B, markedly enhances chemically induced malignant cell transformation. Science 216, 527–529.

    Hirota, C., Ida, M., Aoyagi, K., Matsumoto, T., Tada, S., Yao, T., and Fujishima, M. (1996). Effect of indomethacin suppositories on rectal polyposis in patients with familial adenomatous polyposis. Cancer 78, 1660–1665.

    IARC/NCI/EPA Working Group (1985). Cellular and molecular mechanism of cell transformation and standardization of transformation assays of established cell lines for the prediction of carcinogenic chemicals: Overview and recommended protocols. Cancer Res. 45, 2395–2399.

    Kakunaga, T. (1973). A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3. Int. J. Cancer 12, 463–473.

    Kakunaga, T., and Yamasaki, H. (1985). Recommendations for experimental protocols and for scoring transformed foci in BALB/c 3T3 and C3H 10T1/2 cell transformation. In Transformation Assay of Established Cell Lines: Mechanism and Application (T. Kakunaga, and H. Yamasaki, Eds.), IARC Sci. Publ. No. 67, pp. 205–219. IARC, Lyon.

    Kimura, M., Osumi, S., and Ogihara, M. (2000). Stimulation of DNA synthesis and proliferation by prostaglandins in primary cultures of adult rat hepatocytes. Eur. J. Pharmacol. 404, 259–271.

    Lazzaro, M., Tashjian, A. H., Fujiki, H., and Levine, L. (1987). Palytoxin: An extraordinarily potent stimulation of prostaglandin production and bone resorption in cultured mouse calvariae. Endocrinology 120, 1338–1345.

    Li, S., and Wattenberg, E. V. (1998). Differential activation of mitogen-activated protein kinases by palytoxin and ouabain, two-ligands for the Na+,K+-ATPase. Toxicol. Appl. Pharmacol. 151, 377–384.

    Li, S., and Wattenberg, E. V. (1999). Cell-type-specific activation of p38 protein kinase cascades by the novel tumor promoter palytoxin. Toxicol. Appl. Pharmacol. 160, 109–119.

    Lupulescu, A. (1978). Enhancement of carcinogenesis by prostaglandins in male albino Swiss mice. J. Natl. Cancer Inst. 61, 97–101.

    Lupulescu, A. (1996). Prostaglandins, their inhibitors and cancer. Prostaglandins Leukot. Essent. Fatty Acids 54, 83–94.

    Mondal, S., Brankow, D. W., and Heidelberger, C. (1976). Two-stage chemical oncogenesis in cultures of C3H/10T1/2 cells. Cancer Res. 36, 2254–2260.

    Mondal, S., Brankow, D. W., and Heidelberger, C. (1978). Enhancement of oncogenesis in C3H/10T1/2 mouse embryo cell cultures by saccharin. Science 201, 1141–1142.

    Müller-Decker, K., Scholz, K., Marks, F., and Fürstenberger, G. (1995). Differential expression of prostaglandin H synthase isozymes during multistage carcinogesis in mouse epidermis. Mol. Carcinog. 12, 31–41.

    Reznikoff, C. A., Bertram, J. S., Brankow, D. W., and Heidelberger, C. (1973a). Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to post-confluent inhibition of cell division. Cancer Res. 33, 3239–3249.

    Reznikoff, C. A., Brankow, D. W., and Heidelberger, C. (1973b). Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to post confluent inhibition of division. Cancer Res. 33, 3231–3238.

    Sakai, Y., and Fujiki, H. (1991). Promotion of BALA/3T3 cell transformation by okadaic acid class tumor promoters, okadaic acid and dinophysistoxin-1. Jpn. J. Cancer Res. 82, 518–523.

    Sakai, Y., and Sato, M. (1989). Improvement of carcinogen identification in BALB/3T3 cell transformation by application of a 2-stage method. Mutat. Res. 214, 285–296.

    Shimomura, K., Mullinix, M. G., Kakunaga, T., Fujiki, H., and Sugimura, T. (1983). Bromine residue at hydrophilic region influences biological activity of aplysiatoxin, a tumor promoter. Science 222, 1242–1244.

    Smets, L. A. (1980). Cell transformation as a model for tumor induction and neoplastic growth. Biochim. Biophys. Acta. 605, 93–111.

    Sugiki, Y., Yamazaki, N., Matsuoka, A., Suzuki, T., Hayashi, M., and Sofuni, T. (1994). Comparison of six cytotoxicity tests to find optimal dose range for the in vitro chromosomal aberration test. Environ. Mut. Res. Commun. 16, 37–43.

    Thompson, E. J., Gupta, A., Vielhauer, G. A., Regan, J. W., and Bowden, G. T. (2001). The growth of malignant keratinocytes depends on signaling through the PGE2 receptor EP1. Neoplasia 3, 402–410.

    Tsuchiya, T., Umeda, M., Nishiyama, H., Yoshimura, I., Ajimi, S., Asakura, M., Baba, H., Dewa, Y., Ebe, Y., Fushiwaki, Y., et al. (1999). An interlaboratory validation study of the improved transformation assay employing Balb/c 3T3 cells: Results of a collaborative study on the two-stage cell transformation assay by the non-genomic carcinogen study group. ATLA 27, 685–702.

    Umeda, M., Tanaka, K., and Ono, T. (1983). Effect of insulin on the transformation of BALB/3T3 cells by X-ray irradiation. Gann 74, 864–869.

    Umeda, M., Tanaka, K., and Ono, T. (1989). Promotional effect of lithocholic acid and 3-hydroxyanthranilic acid on transformation of X-ray-initiated BALB/3T3 cells. Carcinogenesis 10, 1665–1668.

    Wang, D., Buchanan, F. G., Wang, H., Dey, S. K., and DuBois, R. N. (2005). Prostaglandin E2 enhances intestinal adenoma growth via activation of the ras-mitogen-activated protein kinase cascade. Cancer Res. 65, 1822–1829.

    Warmka, J. K., Winston, S. E., Zeliadt, N. A., and Wattenberg, E. V. (2002). Extracellular signal-regulated kinase transmits palytoxin-stimulated signals leading to altered gene expression in mouse keratinocytes. Toxicol. Appl. Pharmacol. 185, 8–17.

    Wlfle, D. (2003). Enhancement of carcinogen-induced malignant cell transformation by prostaglandin F2. Toxicology 188, 139–147.

    Wlfle, D., Marotzki, S., Dartsch, D., Schfer, W., and Marquardt, H. (2000). Induction of cyclooxygenase expression and enhancement of malignant cell transformation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Carcinogenesis 21, 15–21.

    Zeliadt, N. A., Warmka, J. K., and Wattenberg, E. V. (2003). Mitogen activated protein kinases selectively regulate palytoxin-stimulated gene expression in mouse keratinocytes. Toxicol. Appl. Pharmacol. 192, 212–221.

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《毒物学科学杂志》2006年1月第89卷第1期