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Cancer Letters 160 (2000) 51±58
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Anti-transforming nature of ascorbic acid and its derivatives examined by two-stage cell transformation using BALB/c 3T3 cells Toshiyuki Tsuchiya a,*, Eiko Kato-Masatsuji a, Toshi Tsuzuki a, Makoto Umeda b a
Safety Evaluation Center, Central Research Laboratory, Showa Denko K.K., Ohnodai 1-1-1, Midori-ku, Chiba 267-0056, Japan b Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257-8523, Japan Received 29 June 2000; received in revised form 17 July 2000; accepted 17 July 2000
Abstract The anti-transforming effects of sodium ascorbate and its stable derivatives were examined in the two-stage transformation assay. When BALB/c 3T3 cells were treated with 0.2 mg/ml 20-methylcholanthrene as an initiator, and 100 ng/ml 12-Otetradecanoylphorbol-13-acetate as a promoter, the addition at the promotion stage of l-ascorbic acid-2-phosphate ester magnesium (APM) was most marked in the inhibition of transformation. The inhibitory effects of sodium ascorbate and ascorbic acid-2-glucoside (AG) were comparable, but weaker than those of APM; l (1)-ascorbic acid-2-sulfate ester disodium 2H2O showed little effect. When phorbol 12, 13-didecanoate or tumor necrosis factor a (TNF-a) were used as promoters, APM also effectively suppressed transformation. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ascorbic acid; Two-stage transformation; BALB/c 3T3 cells; Anti-transformation; Phorbol ester; Tumor necrosis factor a
1. Introduction Many epidemiological surveys, as well as experimental and clinical observations, have shown that ascorbic acid exerts an inhibitory effect on tumor promotion and tumor progression [1±8]. As vitamin C is unstable in light and heat, efforts have been made to obtain stable ascorbic acid derivatives for medical use. Several such compounds have been synthesized [9±11], and serum concentrations of these derivatives are maintained for longer periods. Two-stage cell transformation experiments using C3H10T1/2 or BALB/c 3T3 cells are considered to mimic the process of in vivo carcinogenesis, initiation and promotion, and have been used for the elucidation * Corresponding author. Tel.: 181-43-226-5216; fax: 181-43226-5222. E-mail address: [email protected] (T. Tsuchiya).
of the mechanisms of cellular events of cell transformation and for the detection of initiating and promoting compounds [12±17]. In the C3H10T1/2 cell transformation assay, ascorbic acid administered at the stage of cell maintenance has been shown to prevent focus formation induced by 20-methylcholanthrene (MCA) or X-rays [1,18,19]. In most of these studies, the vitamin was added daily to the medium because of its chemical instability. The inhibitory effect of ascorbic acid on transformation of JB6 P 1 cells treated with tumor-promoting agents has also been demonstrated [20]. Here, we examined the effects of ascorbic acid and its stable derivatives at the promotion stage in the twostage BALB/c 3T3 cell transformation assay. Tested compounds included ascorbic acid sodium salts (AscNa), l-ascorbic acid-2-phosphate ester magnesium (APM), ascorbic acid-2-glucoside (AG) and l (1)-
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00560-7
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T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
ascorbic acid-2-sulfate ester disodium 2H2O (AS; Fig. 1) [9±11]. We tested their inhibitory effects on transformation enhanced by 12-O-tetradecanoylphorbol13-acetate (TPA), as well as phorbol 12,13-didecanoate (PDD) and tumor necrosis factor a (TNF-a).
2. Materials and methods 2.1. Cells, media and culture conditions BALB/c 3T3 A31-1-1 clonal cells were originally supplied by the late Dr T. Kakunaga, and were grown in a medium consisting of minimum essential medium (MEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS). In the present transformation experiment, FBS from Moregate (lot no. 174014; Brisbane, Australia) was used. Other media were obtained as follows: DME/ F-12 from Gibco Laboratories (Grand Island, NY), and ITES (200 mg/ml bovine pancreas insulin, 200 mg/ml human transferrin, 12.2 mg/ml ethanolamine and 0.034 mg/ml sodium selenite) from Wako Pure Chemical Industries, Ltd. Osaka, Japan. The cells were passaged before con¯uence using a detaching solution containing 50 U/ml crystalline trypsin (Mochida Pharmaceutical Co., Tokyo, Japan). For the transformation assay, the cells were used at passage 11 after having been supplied to our laboratory. They were cultivated in a humidi®ed incubator under 5% CO2±95% air.
Fig. 1. Chemical structures of sodium ascorbate and its derivatives.
2.2. Chemicals Asc-Na was purchased from Tokyo Kasei K.K. (Tokyo, Japan). APM was synthesized by Showa Denko K.K. (Tokyo, Japan). AG was obtained from Hayashibara Biochemical Research Institute Co., Ltd. (Okayama, Japan); and AS, MCA, TPA and PDD were obtained from Wako Pure Chemical Industries, Ltd. Recombinant human TNF-a was obtained from Genzyme Techne Co. (Minneapolis, MN). Ascorbates and TNF-a were dissolved in distilled water and sterilized by ®ltration. MCA, TPA and PDD were dissolved in dimethyl sulfoxide and diluted in the medium. 2.3. Transformation assay The cell transformation assay procedure was described in detail in the previous report [21]. Brie¯y, exponentially growing BALB/c 3T3 A31-11 cells were plated at a density of 1 £ 10 4 cells/60mm dish (Sumilon MS-10600, Sumitomo Bakelite, Japan) in ten plates/condition (day 0). The medium was MEM 1 10% FBS. After a 24-h incubation, MCA was added in the medium to obtain 0.2 mg/ ml as the initiation treatment. The concentration of MCA was chosen to induce transformed foci slightly by the single treatment, but markedly by the posttreatment with TPA. On day 4, the dishes were replenished with DME/F-12 1 ITES 1 2% FBS (DFI2F) medium. Afterwards, the DFI2F medium was changed twice a week. TPA at 100 ng/ml was added on days 7, 11 and 14. When PDD or TNF-a was used, the cells were treated as in the case of TPA treatment. Ascorbic acid and its derivatives were added at the time of medium change. The cells were ®xed with methanol and stained with Giemsa solution on day 25. Scoring of transformed foci was performed according to the criteria described before [21], which identi®es transformed foci by four morphological characteristics: (1), random orientation of cells at the edge of foci; (2), formation of piled-up dense cell layers; (3), basophilic staining; and (4), foci of more than 2 mm in diameter. The data were statistically analyzed using the Mann±Whitney U-test.
T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
2.4. Cell growth assay The growth inhibition of MCA was determined in parallel with the transformation assay. Replicate dishes (four/group) were seeded and treated with MCA under the same conditions as the transformation assay. After a 4-day incubation, the dishes were ®xed with 10% formaldehyde for 15 min and stained with 1% crystal-violet (CV) solution for 15 min. After extraction of stained CV with 0.02 N HCl±50% ethanol, the OD570 value was measured [21]. To determine the effects of ascorbic acid and its derivatives on cells at the stationary phase, 5 £ 10 4 BALB/c 3T3 cells in 0.5 ml DFI2F medium were plated into each well of a 24-well plate and cultured for 48 h. The growth medium was removed and replaced with fresh medium containing Asc-Na or its derivatives. After a 3-day incubation the plates were ®xed and stained with CV solution. Stained CV was measured as in the MCA cytotoxicity test. 2.5. Stability of Asc-Na and its derivatives under culture conditions Asc-Na and its derivatives were added to DFI2F medium at a ®nal concentration of 100 mM in the absence or presence of BALB/c 3T3 cells at con¯uence, and incubated at 378C in an atmosphere of 5% CO2±95% air. At appropriate intervals, the remaining Asc-Na and its derivatives were measured by HPLC. In the case of Asc-Na, an aliquot of the medium was injected into a column (Shodex Asahipak NH2P50 of 250 £ 4.6 mm; Showa Denko K.K.) connected in a series circuit of Shimadzu HPLC system (LC10AD, Shimadzu Co. Ltd., Tokyo), and developed at 408C at a ¯ow rate of 0.8 ml/min. The mobile phase solution consisted of 0.1% H3PO4 and 80% acetonitrile. In the case of ascorbate, a Shodex NHpak J-411 column (150 £ 4.6 mm; Showa Denko K.K.) was used, and the mobile phase solution consisted of 0.05 M KH2PO4 and 50% acetonitrile. Asc-Na and its derivatives were detected with a UV detector (UVIDEC-100-III, Japan Spectroscopic Co. Ltd., Tokyo) at 257 nm. 3. Results The suppressive effect of in vitro cell transforma-
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tion with Asc-Na was compared with that of ascorbate derivatives. In the assay, BALB/c 3T3 cells were treated with 0.2 mg/ml MCA for 3 days as an initiation treatment, and continuously incubated for 25 days in total. On days 7, 11 and 14, 100 ng/ml TPA was added to the medium as a tumor promoter treatment. Tested ascorbate compounds were added in the medium at 30 or 100 mM on days 7, 11, 14, 18 and 21. Representative dishes are shown in Fig. 2, and the results are summarized in Table 1. Exposure to either TPA or MCA alone induced only two transformed foci/ten dishes, respectively, whereas treatment with MCA followed by TPA induced 45 transformed foci/ ten dishes (transformation control). When Asc-Na or its derivatives were included in the medium at the time of medium change, focus formation was clearly decreased, except with AS. The inhibition was most marked when APM was added in the medium; focus formation was reduced to 44 and 11% of that of the transformation control in the presence of 30 and 100 mM APM, respectively. The inhibitory effect of AG was weaker than that of APM, and was nearly comparable with that of Asc-Na. The inhibition by 100 mM Asc-Na (22%) and AG (31%) was statistically signi®cant compared with the transformation control. AS at 100 mM showed a minor inhibitory effect (84%) which was not statistically signi®cant.
Fig. 2. Photograph of representative Giemsa-stained dishes showing foci of transformed cells in the in vitro two-stage transformation assay of BALB/c 3T3 Cells. BALB/c 3T3 cells were treated with 0.2 mg/ml MCA, and then with or without 100 ng/ml TPA and 100 mM Asc-Na, APM, AG or AS as described in Section 2. Cultures were ®xed with methanol and stained with Giemsa solution.
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T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
Table 1 Inhibitory effects of ascorbates on TPA-enhanced transformation of MCA-treated BALB/c 3T3 cells Initiating treatment
Promoting treatment
Inhibitor treatment
Transformation
Treatment
No. of dishes with foci/no. of dishes examined
Total no. of foci
Foci/dish (mean ^ SD)
% of transformation
DMSO DMSO MCA b MCA MCA MCA MCA MCA MCA MCA MCA MCA
DMSO TPA a DMSO TPA TPA TPA TPA TPA TPA TPA TPA TPA
Medium Medium Medium Medium Asc-Na Asc-Na APM APM AG AG AS AS
0/10 2/10 1/10 10/10 10/10 7/10 9/10 4/10 9/10 8/10 10/10 10/10
0 2 2 45 31 10 d 20 d 5d 31 14 d 40 38
0.00 ^ 0.00 0.20 ^ 0.42 0.20 ^ 0.63 4.50 ^ 2.12 3.10 ^ 0.99 1.00 ^ 0.94 2.00 ^ 0.94 0.50 ^ 0.71 3.10 ^ 1.52 1.40 ^ 1.43 4.00 ^ 1.76 3.80 ^ 1.40
0 ± 4 100 c 69 22 44 11 69 31 89 84
a b c d
Concentration (mm)
30 100 30 100 30 100 30 100
The concentration of TPA was 100 ng/ml. The concentration of MCA was 0.2 mg/ml and the cell growth of the MCA-treated cells was 57% of the untreated control cells. Transformation control. P , 0:01 vs. transformation control by the Mann±Whitney U-test.
In order to further characterize the inhibitory effect of APM, various concentrations of APM were examined (Table 2). A dose-dependent suppression of transformation was observed. In this experiment, 100±300 mM APM inhibited the induction of trans-
formed foci to the level observed with the MCA-alone control exposure. Since Asc-Na and its derivatives have been reported to stimulate or inhibit the growth of various types of cells [22], it is possible that they affect in
Table 2 Inhibitory effects of APM on TPA-enhanced transformation of MCA-treated BALB/c 3T3 cells Initiating treatment
Promoting treatment
Inhibitor treatment
Transformation
Treatment
No. of dishes with foci/no. of dishes examined
Total no. of foci
Foci/dish (mean ^ SD)
% of transformation control
DMSO DMSO MCA b MCA MCA MCA MCA MCA
DMSO TPA a DMSO TPA TPA TPA TPA TPA
Medium Medium Medium Medium APM APM APM APM
0/10 0/10 3/10 10/10 10/10 6/10 2/10 2/10
0 0 3 35 23 10 d 2d 3d
0.00 ^ 0.00 0.00 ^ 0.00 0.30 ^ 0.48 3.50 ^ 1.72 2.30 ^ 1.06 1.00 ^ 1.05 0.20 ^ 0.42 0.30 ^ 0.67
0 0 9 100 c 66 29 6 9
a b c d
Concentration (mm)
10 30 100 300
The concentration of TPA was 100 ng/ml. The concentration of MCA was 0.2 mg/ml and the cell growth of the MCA-treated cells was 63% of the untreated cells. Transformation control. P , 0:01 vs. transformation control by the Mann±Whitney U-test.
T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
vitro cell transformation by modulating the cell growth. Therefore, the effects of Asc-Na and its derivatives on the BALB/c 3T3 clone A31-1-1 and its transformed clone cells at the stationary phase were examined. The results showed that these compounds at a concentration of 300 mM neither stimulated nor decreased the cell number (data not shown). This indicated that the inhibition of cell transformation by AscNa and its derivatives was not related to their effect on the modulation of the cells. In previous studies of Asc-Na inhibition of cell transformation in C3H10T1/2 cells, Asc-Na was added daily because of its instability. We therefore examined the stability of Asc-Na and its derivatives in DFI2F medium with or without BALB/c 3T3 cells. Similar results were obtained whether the cells were present or not. Fig. 3 illustrates the rate of disappearance of the compounds from the medium in the presence of the cells. Asc-Na rapidly disappeared, and less than 5% remained after a 6-h incubation. Its half-life was approximately 1.8 h. Thus, even with such rapid degradation and only a twice-weekly application to MCA/TPA-treated cells, Asc-Na showed a signi®cant inhibition of transformation (Table 1). APM degraded more slowly, and 13% remained after 48 h. The half-life of APM was 23 h. In contrast, AG degraded very slightly; after a 96-h
Fig. 3. The rates of disappearance of Asc-Na, APM, AG and AS in culture medium. Asc-Na, APM, AG or AS was added to DFI2F medium at a ®nal concentration of 100 mM in the presence of BALB/c 3T3 cells at con¯uence and then incubated at 378C for 96 h. Points are the means of measurements in duplicate cultures.
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incubation with cells, 93.1% of AG was detected. In the case of AS, little or no degradation was observed. We next examined whether Asc-Na and APM would be effective against PDD, a more potent phorbol ester tumor promoter than TPA in in vitro experiments [23]. As shown in Table 3, PDD at 0.3±1.0 ng/ml was about 2±3 times more effective than 100 ng/ml TPA in enhancing cell transformation after MCA initiation. This enhancement with PDD was markedly inhibited with 100 mM APM (84±92% inhibition), but less so with Asc-Na (9±48% inhibition). The inhibitory activity of APM was examined on the promotional effect of TNF-a, a promoter with a different mechanism of action to the phorbol esters. As shown in Table 4, a marked enhancement of transformation with TNF-a occurred at doses of 3 and 10 ng/ml, 65±70% of the cell transformation being induced by 100 ng/ml TPA. Under the assay conditions, 100 mM APM inhibited the TNF-a cell transformation by 70±75%. 4. Discussion Earlier work had shown that ascorbic acid prevented the expression of transformed foci of C3H10T1/2 cells treated with MCA. The fact that ascorbic acid was still effective for treatment begun as late as day 23 after MCA initiation suggested the anti-tumor promoter nature of ascorbic acid [1]. Subsequent work demonstrated that ascorbic acid also prevented the morphological transformation of X-ray-exposed C3H10T1/2 cells [19]. As for the mechanism of its action, Ibric et al. [18] suggested an association with regulation of the redox potential. In these studies, ascorbic acid was added in the medium daily because of its instability. Gol-Winkler et al. [24] showed in their short report that the twiceweekly addition of ascorbic acid also suppressed C3H10T1/2 cell transformation. Adding to the results in the C3H10T1/2 cell transformation system, Huang et al. showed that ascorbic acid could inhibit the transformation of JB6 P 1 cells treated with tumor-promoting agents [20]. Since AP-1 plays a major role in neoplastic progression in the JB6 cell system, these workers measured AP-1 and demonstrated the inhibition of AP-1 activity by ascorbic acid.
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T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
Table 3 Inhibitory effects of Asc-Na and APM on PDD-enhanced transformation of MCA-treated BALB/c 3T3 cells Initiating treatment
Promoting treatment
Inhibitor treatment
Treatment Concentration (ng/ml)
Treatment Concentration No. of dishes Total no. (mm) with foci/no. of of foci dishes examined
Foci/dish (mean ^ SD)
% of transformation control
DMSO DMSO MCA a MCA MCA MCA MCA MCA MCA MCA
DMSO TPA DMSO TPA PDD PDD PDD PDD PDD PDD
Medium Medium Medium Medium Medium Asc-Na APM Medium Asc-Na APM
0.00 ^ 0.00 0.00 ^ 0.00 0.30 ^ 0.48 3.50 ^ 1.72 6.50 ^ 3.03 3.40 ^ 1.43 0.50 ^ 0.71 9.10 ^ 2.38 8.30 ^ 1.95 1.50 ^ 1.18
± ± ± ± 100 b 52 8 100 b 91 16
a b c d
100.0 100.0 0.3 0.3 0.3 1.0 1.0 1.0
100 100 100 100
Transformation
0/10 0/10 3/10 10/10 10/10 10/10 4/10 10/10 10/10 7/10
0 0 3 35 65 34 c 5d 91 83 15 d
The concentration of MCA was 0.2 mg/ml and the cell growth of MCA-treated cells was 63% of the untreated control cells. Transformation control. P , 0:05 vs. transformation control by the Mann±Whitney U-test. P , 0:01 vs. transformation control by the Mann±Whitney U-test.
In the current set of experiments, we tried to examine the effect of ascorbic acid and stable ascorbic acid derivatives in the two-stage BALB/c 3T3 cell transformation. Our experimental system consisted of the two-stage transformation of BALB/c 3T3 cells. The cells were ®rst treated with a relatively low concentration of MCA (0.2 mg/ml in our case, and 1±5 mg/ml
in the previous papers using C3H10T1/2 cells without additional TPA treatment) as an initiation treatment, followed by TPA as a promotion treatment. Asc-Na and its derivatives were added in the medium twice/ week during cell maintenance. Our results showed that cell transformation was most markedly inhibited with APM and less strongly
Table 4 Inhibitory effects of APM on TNF-a-enhanced transformation of MCA-treated BALB/c 3T3 cells Initiating treatment
Promoting treatment
Treatment Concentration Treatment Concentration No. of dishes Total no. (ng/ml) (mm) with foci/no. of of foci dishes examined
Foci/dish (mean ^ SD)
% of transformation control
DMSO DMSO MCA a MCA MCA MCA MCA MCA MCA MCA
DMSO TPA DMSO TPA TNF-a TNF-a TNF-a TNF-a TNF-a TNF-a
0.00 ^ 0.00 0.00 ^ 0.00 0.30 ^ 0.48 3.50 ^ 1.72 1.00 ^ 1.15 0.30 ^ 0.67 2.40 ^ 2.41 0.60 ^ 0.70 2.30 ^ 1.77 0.70 ^ 0.67
± ± ± ± 100 b 30 100 b 25 100 b 30
a b c
100 100 1 1 3 3 10 10
Inhibitor treatment
Medium Medium Medium Medium Medium APM Medium APM Medium APM
100 100 100
Transformation
0/10 0/10 3/10 10/10 5/10 2/10 8/10 5/10 9/10 6/10
0 0 3 35 10 3 24 6c 23 7c
The concentration of MCA was 0.2 mg/ml and the cell growth of MCA-treated cells was 63% of the untreated control cells. Transformation control. P , 0:05 vs. transformation control by the Mann±Whitney U-test.
T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
with Asc-Na and AG. AS showed little effect (Table 1). The fact that a biweekly addition of unstable ascorbic acid was effective in TPA-induced transformation also attracted our attention. We then tried to elucidate these results from the viewpoint of the stability of the compounds and the use of PDD or TNF-a in place of TPA. In the stability studies, Asc-Na degraded rapidly in the culture medium during the incubation (half-life, 1.8 h). In contrast, the half-life of APM was 23 h. We considered that this marked difference may give rise to different activities against the more potent and more stable in vitro phorbol ester promoter, PDD (Table 3). As may be expected, the inhibitory effect of Asc-Na was markedly decreased, but the stable derivative, APM, was still very active in reducing the number of transformed foci. These results suggest that, in order to inhibit the promotional effect by phorbol esters, active ascorbic acid must be present in the medium for extended periods. APM is degraded to produce ascorbic acid by alkaline phosphatase which is present in serum. Our medium contains FBS, and therefore APM can be effectively reduced to ascorbic acid in the culture medium. In the case of AG, 93.1% remained even after a 96-h incubation. Glucosidase is responsible for degrading AG. We then examined the stability of APM and AG in sera from humans, mice and guinea pigs. APM degraded to produce ascorbic acid in every serum examined, but AG degradation was observed only in mouse serum (data not shown). Therefore, the reason why AG was active in the current cell transformation assay is suggested to be due either to the presence of glucosidase on the membrane of BALB/c 3T3 cells or to the presence of an active transport system for AG. In either explanation, only a small amount of AG was utilized and AG was less effective than APM and Asc-Na in inhibiting cell transformation. In contrast, AS was quite stable under the present culture conditions. AS can be hydrolyzed by sulfohydrase, which is detected in tissues of the rat, guinea pig and rabbit, but not in human tissues [25]. This enzyme is also considered to be absent from FBS and from the membranes of BALB/c 3T3 cells. Thus, in our transformation experiments little suppressive effect was observed. From this comparative study on the ascorbate deri-
57
vatives, it is concluded that APM was the most ef®cient `supplier' of ascorbic acid to effect the inhibition of in vitro cell transformation. In addition, the effect of ascorbates was examined on the transformation of MCA-treated BALB/c 3T3 cells enhanced by a tumor promoter other than phorbol esters. TNF-a, a pleiotropic cytokine, was reported to increase the transformation of initiatortreated BALB/c 3T3 cells when added at the promotion stage. The cytokine is produced by addition of okadaic acid, a potent tumor promoter, and is therefore considered to be an intrinsic promoter [26]. Our results showed that the in vitro promotion enhanced by TNF-a was indeed inhibited with APM (Table 4). The effect of TNF-a is related to free radical production [27]. Therefore, these results may support the notion that the mechanism of inhibition of cell transformation by ascorbic acid is related to the elimination of active oxygen as a radical scavenger. Further in vivo experiments are necessary to con®rm an anti-tumor-promoting effect of the ascorbate derivatives. References [1] W.F. Benedict, W.L. Wheatley, P.A. Jones, Inhibition of chemically induced morphological transformation and reversion of the transformed phenotype by ascorbic acid in C3H10T1/2 cells, Cancer Res. 40 (1980) 2796±2801. [2] M.P. Rosin, A.R. Peterson, H.F. Stich, The effect of ascorbate on 3-methylcholanthrene-induced cell transformation in C3H10T1/2 mouse-embryo ®broblast cell cultures, Mutat. Res. 72 (1980) 533±537. [3] W.F. Benedict, W.L. Wheatley, P.A. Jones, Differences in anchorage-dependent growth and tumorigenicities between transformed C3H/10T1/2 cells with morphologies that are or are not reverted to a normal phenotype by ascorbic acid, Cancer Res. 42 (1982) 1041±1045. [4] J. Bhattacharjee, A.S. Chakraborty, N.H. Sarkar, A. Basu, Study of ascorbic acid status in murine and human leukemias, Comp. Pathol. 95 (1985) 87±91. [5] J. Ghosh, S. Das, Evaluation of vitamin A and C status in normal and malignant conditions and their possible role in cancer prevention, Jpn. J. Cancer Res. 76 (1985) 1174±1178. [6] S. Bram, P. Frousard, M. Guichard, Vitamin C preferential toxicity of malignant melanoma, Nature 284 (1980) 629±631. [7] S.J. Harada, T. Masumoto, E. Ogata, Role of ascorbic acid in the regulation of proliferation in osteoblast-like MC3T3E1cells, J. Bone Miner. Res. 6 (1991) 903±907. [8] A. Campbell, T. Jack, E. Cameron, Reticulum cell sarcoma: two complete spontaneous regressions in response to high dose ascorbic acid therapy, Oncology 48 (1991) 495±497.
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[9] H. Nomura, H. Ishiguro, S. Morimoto, Studies on l-ascorbic acid derivatives. III. Bis (l-ascorbic acid-3, 3 0 ) phosphate and l-ascorbic acid 2-phosphate, Chem. Pharm. Bull. (Tokyo) 17 (1969) 387±393. [10] B.M. Tolbert, M. Downing, R.W. Carlson, K. Knight, E.M. Baker, Chemistry and metabolism of ascorbic acid and ascorbate sulfate, Ann. N. Y. Acad. Sci. 258 (1975) 48±69. [11] I. Yamamoto, N. Muto, K. Murakami, S. Suga, H. Yamaguchi, l-Ascorbic acid a-glucoside formed by regioselective transglucosylation with rat intestinal and rice seed a-glucosidases: its improved stability and structure determination, Chem. Pharm. Bull. 38 (1990) 3020±3023. [12] T. Kakunaga, A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3, Int. J. Cancer. 12 (1973) 463±473. [13] A.R. Kennedy, Promotion and other interactions between agents in the induction of transformation in vitro in ®broblasts, in: T.J. Slaga (Ed.), Mechanisms of Tumor Promotion, Vol. 3, CRC Press, Roca Raton FL, 1984, pp. 13±55. [14] C. Heidelberger, A.E. Freeman, R.J. Pienta, A. Sivak, J.S. Bertram, B.C. Casto, V.C. Dunkel, M.W. Francis, T. Kakunaga, J.B. Little, L.M. Schechtman, Cell transformation by chemical agents ± a review and analysis of the literature: a report of the United States Environmental Protection Agency Gene-Tox Program, Mutat. Res. 114 (1983) 283±385. [15] V.C. Dunkel, R.J. Pienta, A. Sivak, K.A. Traul, Comparative neoplastic transformation responses of Balb/3T3 cells, Syrian hamster embryo cells, and Rauscher murine leukemia virusinfected Fischer 344 rat embryo cells to chemical carcinogens, J. Natl. Cancer Inst. 67 (1981) 1303±1315. [16] A.L. Meyer, In vitro transformation assays for chemical carcinogens, Mutat. Res. 115 (1983) 323±338. [17] T. Tsuchiya, M. Umeda, Improvement in the ef®ciency of the in vitro transformation assay method using BALB/3T3 A31-11 cells, Carcinogenesis 16 (1995) 1887±1894. [18] L.L.V. Ibric, A.R. Peterson, A. Sevanian, Mechanisms of ascorbic acid-induced inhibition of chemical transformation in C3H/10T1/2 cells, Am. J. Clin. Nutr. 54 (1991) 1236S± 1240S. [19] M. Yasukawa, T. Terashima, M. Seki, Radiation-induced
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neoplastic transformation of C3H10T1/2 cells is suppressed by ascorbic acid, Radiat. Res. 120 (1989) 456±467. C. Huang, W. Ma, Z. Dong, Inhibitory effects of ascorbic acid on AP-1 activity and transformation of JB6 cells, Int. Natl. Oncol. 8 (1996) 389±393. T. Tsuchiya, M. Umeda, H. Nishiyama, I. Yoshimura, S. Ajimi, M. Asakura, H. Baba, Y. Dewa, Y. Ebe, Y. Fushiwaki, S. Hamada, T. Hamamura, M. Hayashi, Y. Iwase, Y. Kajiwara, Y. Kasahara, M. Kawabata, E. Kitada, K. Kubo, K. Mashiko, D. Miura, F. Mizuhashi, F. Mizuno, M. Nakajima, Y. Nakamura, N. Nobe, H. Oishi, E. Ota, A. Sakai, M. Sato, S. Shimada, T. Sugiyama, C. Takahashi, Y. Takeda, N. Tanaka, C. Toyoizumi, T. Tsutsui, S. Wakuri, S. Yajima, N. Yajima, 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-genotoxic Carcinogen Study Group, Altern. Lab. Anim. 27 (1999) 685±702. R. Hata, Vitamin C and cell growth: use of l-ascorbic acid 2phosphate in culture for construction of three-dimensional structure from isolated cell, Seikagaku 60 (1988) 201±206. T. Enomoto, N. Martel, Y. Kannno, H. Yamasaki, Inhibition of cell communication between Balb/c 3T3 cells by tumor promoters and protection by cAMP, J. Cell. Physiol. 121 (1984) 323±333. R. Gol-Winkler, Y. DeClerck, J. Gielen, Ascorbic acid effect on methylcholanthrene-induced cell transformation in C3H10T1/2 clone 8 cells, Toxicology 17 (1980) 237±239. T. Sakamoto, Development of the stabilized vitamin C derivative with prolonged effects, Fragrance J. 27 (1997) 62±70. A. Komori, J. Yatsunami, M. Suganuma, S. Okabe, S. Abe, A. Sakai, K. Sasaki, H. Fujiki, Tumor necrosis factor acts as a tumor promoter in BALB/3T3 cell transformation, Cancer Res. 53 (1993) 1982±1985. A. Mensink, L.H. de Haan, C.M. Lakemond, C.A. Koelman, J.H. Koeman, Inhibition of gap junctional intercellular communication between primary human smooth muscle cells by tumor necrosis factor alpha, Carcinogenesis 16 (1995) 2063±2067.
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Anti-transforming nature of ascorbic acid and its derivatives examined by two-stage cell transformation using BALB/c 3T3 cells Toshiyuki Tsuchiya a,*, Eiko Kato-Masatsuji a, Toshi Tsuzuki a, Makoto Umeda b a
Safety Evaluation Center, Central Research Laboratory, Showa Denko K.K., Ohnodai 1-1-1, Midori-ku, Chiba 267-0056, Japan b Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257-8523, Japan Received 29 June 2000; received in revised form 17 July 2000; accepted 17 July 2000
Abstract The anti-transforming effects of sodium ascorbate and its stable derivatives were examined in the two-stage transformation assay. When BALB/c 3T3 cells were treated with 0.2 mg/ml 20-methylcholanthrene as an initiator, and 100 ng/ml 12-Otetradecanoylphorbol-13-acetate as a promoter, the addition at the promotion stage of l-ascorbic acid-2-phosphate ester magnesium (APM) was most marked in the inhibition of transformation. The inhibitory effects of sodium ascorbate and ascorbic acid-2-glucoside (AG) were comparable, but weaker than those of APM; l (1)-ascorbic acid-2-sulfate ester disodium 2H2O showed little effect. When phorbol 12, 13-didecanoate or tumor necrosis factor a (TNF-a) were used as promoters, APM also effectively suppressed transformation. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ascorbic acid; Two-stage transformation; BALB/c 3T3 cells; Anti-transformation; Phorbol ester; Tumor necrosis factor a
1. Introduction Many epidemiological surveys, as well as experimental and clinical observations, have shown that ascorbic acid exerts an inhibitory effect on tumor promotion and tumor progression [1±8]. As vitamin C is unstable in light and heat, efforts have been made to obtain stable ascorbic acid derivatives for medical use. Several such compounds have been synthesized [9±11], and serum concentrations of these derivatives are maintained for longer periods. Two-stage cell transformation experiments using C3H10T1/2 or BALB/c 3T3 cells are considered to mimic the process of in vivo carcinogenesis, initiation and promotion, and have been used for the elucidation * Corresponding author. Tel.: 181-43-226-5216; fax: 181-43226-5222. E-mail address: [email protected] (T. Tsuchiya).
of the mechanisms of cellular events of cell transformation and for the detection of initiating and promoting compounds [12±17]. In the C3H10T1/2 cell transformation assay, ascorbic acid administered at the stage of cell maintenance has been shown to prevent focus formation induced by 20-methylcholanthrene (MCA) or X-rays [1,18,19]. In most of these studies, the vitamin was added daily to the medium because of its chemical instability. The inhibitory effect of ascorbic acid on transformation of JB6 P 1 cells treated with tumor-promoting agents has also been demonstrated [20]. Here, we examined the effects of ascorbic acid and its stable derivatives at the promotion stage in the twostage BALB/c 3T3 cell transformation assay. Tested compounds included ascorbic acid sodium salts (AscNa), l-ascorbic acid-2-phosphate ester magnesium (APM), ascorbic acid-2-glucoside (AG) and l (1)-
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00560-7
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T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
ascorbic acid-2-sulfate ester disodium 2H2O (AS; Fig. 1) [9±11]. We tested their inhibitory effects on transformation enhanced by 12-O-tetradecanoylphorbol13-acetate (TPA), as well as phorbol 12,13-didecanoate (PDD) and tumor necrosis factor a (TNF-a).
2. Materials and methods 2.1. Cells, media and culture conditions BALB/c 3T3 A31-1-1 clonal cells were originally supplied by the late Dr T. Kakunaga, and were grown in a medium consisting of minimum essential medium (MEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS). In the present transformation experiment, FBS from Moregate (lot no. 174014; Brisbane, Australia) was used. Other media were obtained as follows: DME/ F-12 from Gibco Laboratories (Grand Island, NY), and ITES (200 mg/ml bovine pancreas insulin, 200 mg/ml human transferrin, 12.2 mg/ml ethanolamine and 0.034 mg/ml sodium selenite) from Wako Pure Chemical Industries, Ltd. Osaka, Japan. The cells were passaged before con¯uence using a detaching solution containing 50 U/ml crystalline trypsin (Mochida Pharmaceutical Co., Tokyo, Japan). For the transformation assay, the cells were used at passage 11 after having been supplied to our laboratory. They were cultivated in a humidi®ed incubator under 5% CO2±95% air.
Fig. 1. Chemical structures of sodium ascorbate and its derivatives.
2.2. Chemicals Asc-Na was purchased from Tokyo Kasei K.K. (Tokyo, Japan). APM was synthesized by Showa Denko K.K. (Tokyo, Japan). AG was obtained from Hayashibara Biochemical Research Institute Co., Ltd. (Okayama, Japan); and AS, MCA, TPA and PDD were obtained from Wako Pure Chemical Industries, Ltd. Recombinant human TNF-a was obtained from Genzyme Techne Co. (Minneapolis, MN). Ascorbates and TNF-a were dissolved in distilled water and sterilized by ®ltration. MCA, TPA and PDD were dissolved in dimethyl sulfoxide and diluted in the medium. 2.3. Transformation assay The cell transformation assay procedure was described in detail in the previous report [21]. Brie¯y, exponentially growing BALB/c 3T3 A31-11 cells were plated at a density of 1 £ 10 4 cells/60mm dish (Sumilon MS-10600, Sumitomo Bakelite, Japan) in ten plates/condition (day 0). The medium was MEM 1 10% FBS. After a 24-h incubation, MCA was added in the medium to obtain 0.2 mg/ ml as the initiation treatment. The concentration of MCA was chosen to induce transformed foci slightly by the single treatment, but markedly by the posttreatment with TPA. On day 4, the dishes were replenished with DME/F-12 1 ITES 1 2% FBS (DFI2F) medium. Afterwards, the DFI2F medium was changed twice a week. TPA at 100 ng/ml was added on days 7, 11 and 14. When PDD or TNF-a was used, the cells were treated as in the case of TPA treatment. Ascorbic acid and its derivatives were added at the time of medium change. The cells were ®xed with methanol and stained with Giemsa solution on day 25. Scoring of transformed foci was performed according to the criteria described before [21], which identi®es transformed foci by four morphological characteristics: (1), random orientation of cells at the edge of foci; (2), formation of piled-up dense cell layers; (3), basophilic staining; and (4), foci of more than 2 mm in diameter. The data were statistically analyzed using the Mann±Whitney U-test.
T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
2.4. Cell growth assay The growth inhibition of MCA was determined in parallel with the transformation assay. Replicate dishes (four/group) were seeded and treated with MCA under the same conditions as the transformation assay. After a 4-day incubation, the dishes were ®xed with 10% formaldehyde for 15 min and stained with 1% crystal-violet (CV) solution for 15 min. After extraction of stained CV with 0.02 N HCl±50% ethanol, the OD570 value was measured [21]. To determine the effects of ascorbic acid and its derivatives on cells at the stationary phase, 5 £ 10 4 BALB/c 3T3 cells in 0.5 ml DFI2F medium were plated into each well of a 24-well plate and cultured for 48 h. The growth medium was removed and replaced with fresh medium containing Asc-Na or its derivatives. After a 3-day incubation the plates were ®xed and stained with CV solution. Stained CV was measured as in the MCA cytotoxicity test. 2.5. Stability of Asc-Na and its derivatives under culture conditions Asc-Na and its derivatives were added to DFI2F medium at a ®nal concentration of 100 mM in the absence or presence of BALB/c 3T3 cells at con¯uence, and incubated at 378C in an atmosphere of 5% CO2±95% air. At appropriate intervals, the remaining Asc-Na and its derivatives were measured by HPLC. In the case of Asc-Na, an aliquot of the medium was injected into a column (Shodex Asahipak NH2P50 of 250 £ 4.6 mm; Showa Denko K.K.) connected in a series circuit of Shimadzu HPLC system (LC10AD, Shimadzu Co. Ltd., Tokyo), and developed at 408C at a ¯ow rate of 0.8 ml/min. The mobile phase solution consisted of 0.1% H3PO4 and 80% acetonitrile. In the case of ascorbate, a Shodex NHpak J-411 column (150 £ 4.6 mm; Showa Denko K.K.) was used, and the mobile phase solution consisted of 0.05 M KH2PO4 and 50% acetonitrile. Asc-Na and its derivatives were detected with a UV detector (UVIDEC-100-III, Japan Spectroscopic Co. Ltd., Tokyo) at 257 nm. 3. Results The suppressive effect of in vitro cell transforma-
53
tion with Asc-Na was compared with that of ascorbate derivatives. In the assay, BALB/c 3T3 cells were treated with 0.2 mg/ml MCA for 3 days as an initiation treatment, and continuously incubated for 25 days in total. On days 7, 11 and 14, 100 ng/ml TPA was added to the medium as a tumor promoter treatment. Tested ascorbate compounds were added in the medium at 30 or 100 mM on days 7, 11, 14, 18 and 21. Representative dishes are shown in Fig. 2, and the results are summarized in Table 1. Exposure to either TPA or MCA alone induced only two transformed foci/ten dishes, respectively, whereas treatment with MCA followed by TPA induced 45 transformed foci/ ten dishes (transformation control). When Asc-Na or its derivatives were included in the medium at the time of medium change, focus formation was clearly decreased, except with AS. The inhibition was most marked when APM was added in the medium; focus formation was reduced to 44 and 11% of that of the transformation control in the presence of 30 and 100 mM APM, respectively. The inhibitory effect of AG was weaker than that of APM, and was nearly comparable with that of Asc-Na. The inhibition by 100 mM Asc-Na (22%) and AG (31%) was statistically signi®cant compared with the transformation control. AS at 100 mM showed a minor inhibitory effect (84%) which was not statistically signi®cant.
Fig. 2. Photograph of representative Giemsa-stained dishes showing foci of transformed cells in the in vitro two-stage transformation assay of BALB/c 3T3 Cells. BALB/c 3T3 cells were treated with 0.2 mg/ml MCA, and then with or without 100 ng/ml TPA and 100 mM Asc-Na, APM, AG or AS as described in Section 2. Cultures were ®xed with methanol and stained with Giemsa solution.
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T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
Table 1 Inhibitory effects of ascorbates on TPA-enhanced transformation of MCA-treated BALB/c 3T3 cells Initiating treatment
Promoting treatment
Inhibitor treatment
Transformation
Treatment
No. of dishes with foci/no. of dishes examined
Total no. of foci
Foci/dish (mean ^ SD)
% of transformation
DMSO DMSO MCA b MCA MCA MCA MCA MCA MCA MCA MCA MCA
DMSO TPA a DMSO TPA TPA TPA TPA TPA TPA TPA TPA TPA
Medium Medium Medium Medium Asc-Na Asc-Na APM APM AG AG AS AS
0/10 2/10 1/10 10/10 10/10 7/10 9/10 4/10 9/10 8/10 10/10 10/10
0 2 2 45 31 10 d 20 d 5d 31 14 d 40 38
0.00 ^ 0.00 0.20 ^ 0.42 0.20 ^ 0.63 4.50 ^ 2.12 3.10 ^ 0.99 1.00 ^ 0.94 2.00 ^ 0.94 0.50 ^ 0.71 3.10 ^ 1.52 1.40 ^ 1.43 4.00 ^ 1.76 3.80 ^ 1.40
0 ± 4 100 c 69 22 44 11 69 31 89 84
a b c d
Concentration (mm)
30 100 30 100 30 100 30 100
The concentration of TPA was 100 ng/ml. The concentration of MCA was 0.2 mg/ml and the cell growth of the MCA-treated cells was 57% of the untreated control cells. Transformation control. P , 0:01 vs. transformation control by the Mann±Whitney U-test.
In order to further characterize the inhibitory effect of APM, various concentrations of APM were examined (Table 2). A dose-dependent suppression of transformation was observed. In this experiment, 100±300 mM APM inhibited the induction of trans-
formed foci to the level observed with the MCA-alone control exposure. Since Asc-Na and its derivatives have been reported to stimulate or inhibit the growth of various types of cells [22], it is possible that they affect in
Table 2 Inhibitory effects of APM on TPA-enhanced transformation of MCA-treated BALB/c 3T3 cells Initiating treatment
Promoting treatment
Inhibitor treatment
Transformation
Treatment
No. of dishes with foci/no. of dishes examined
Total no. of foci
Foci/dish (mean ^ SD)
% of transformation control
DMSO DMSO MCA b MCA MCA MCA MCA MCA
DMSO TPA a DMSO TPA TPA TPA TPA TPA
Medium Medium Medium Medium APM APM APM APM
0/10 0/10 3/10 10/10 10/10 6/10 2/10 2/10
0 0 3 35 23 10 d 2d 3d
0.00 ^ 0.00 0.00 ^ 0.00 0.30 ^ 0.48 3.50 ^ 1.72 2.30 ^ 1.06 1.00 ^ 1.05 0.20 ^ 0.42 0.30 ^ 0.67
0 0 9 100 c 66 29 6 9
a b c d
Concentration (mm)
10 30 100 300
The concentration of TPA was 100 ng/ml. The concentration of MCA was 0.2 mg/ml and the cell growth of the MCA-treated cells was 63% of the untreated cells. Transformation control. P , 0:01 vs. transformation control by the Mann±Whitney U-test.
T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
vitro cell transformation by modulating the cell growth. Therefore, the effects of Asc-Na and its derivatives on the BALB/c 3T3 clone A31-1-1 and its transformed clone cells at the stationary phase were examined. The results showed that these compounds at a concentration of 300 mM neither stimulated nor decreased the cell number (data not shown). This indicated that the inhibition of cell transformation by AscNa and its derivatives was not related to their effect on the modulation of the cells. In previous studies of Asc-Na inhibition of cell transformation in C3H10T1/2 cells, Asc-Na was added daily because of its instability. We therefore examined the stability of Asc-Na and its derivatives in DFI2F medium with or without BALB/c 3T3 cells. Similar results were obtained whether the cells were present or not. Fig. 3 illustrates the rate of disappearance of the compounds from the medium in the presence of the cells. Asc-Na rapidly disappeared, and less than 5% remained after a 6-h incubation. Its half-life was approximately 1.8 h. Thus, even with such rapid degradation and only a twice-weekly application to MCA/TPA-treated cells, Asc-Na showed a signi®cant inhibition of transformation (Table 1). APM degraded more slowly, and 13% remained after 48 h. The half-life of APM was 23 h. In contrast, AG degraded very slightly; after a 96-h
Fig. 3. The rates of disappearance of Asc-Na, APM, AG and AS in culture medium. Asc-Na, APM, AG or AS was added to DFI2F medium at a ®nal concentration of 100 mM in the presence of BALB/c 3T3 cells at con¯uence and then incubated at 378C for 96 h. Points are the means of measurements in duplicate cultures.
55
incubation with cells, 93.1% of AG was detected. In the case of AS, little or no degradation was observed. We next examined whether Asc-Na and APM would be effective against PDD, a more potent phorbol ester tumor promoter than TPA in in vitro experiments [23]. As shown in Table 3, PDD at 0.3±1.0 ng/ml was about 2±3 times more effective than 100 ng/ml TPA in enhancing cell transformation after MCA initiation. This enhancement with PDD was markedly inhibited with 100 mM APM (84±92% inhibition), but less so with Asc-Na (9±48% inhibition). The inhibitory activity of APM was examined on the promotional effect of TNF-a, a promoter with a different mechanism of action to the phorbol esters. As shown in Table 4, a marked enhancement of transformation with TNF-a occurred at doses of 3 and 10 ng/ml, 65±70% of the cell transformation being induced by 100 ng/ml TPA. Under the assay conditions, 100 mM APM inhibited the TNF-a cell transformation by 70±75%. 4. Discussion Earlier work had shown that ascorbic acid prevented the expression of transformed foci of C3H10T1/2 cells treated with MCA. The fact that ascorbic acid was still effective for treatment begun as late as day 23 after MCA initiation suggested the anti-tumor promoter nature of ascorbic acid [1]. Subsequent work demonstrated that ascorbic acid also prevented the morphological transformation of X-ray-exposed C3H10T1/2 cells [19]. As for the mechanism of its action, Ibric et al. [18] suggested an association with regulation of the redox potential. In these studies, ascorbic acid was added in the medium daily because of its instability. Gol-Winkler et al. [24] showed in their short report that the twiceweekly addition of ascorbic acid also suppressed C3H10T1/2 cell transformation. Adding to the results in the C3H10T1/2 cell transformation system, Huang et al. showed that ascorbic acid could inhibit the transformation of JB6 P 1 cells treated with tumor-promoting agents [20]. Since AP-1 plays a major role in neoplastic progression in the JB6 cell system, these workers measured AP-1 and demonstrated the inhibition of AP-1 activity by ascorbic acid.
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T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
Table 3 Inhibitory effects of Asc-Na and APM on PDD-enhanced transformation of MCA-treated BALB/c 3T3 cells Initiating treatment
Promoting treatment
Inhibitor treatment
Treatment Concentration (ng/ml)
Treatment Concentration No. of dishes Total no. (mm) with foci/no. of of foci dishes examined
Foci/dish (mean ^ SD)
% of transformation control
DMSO DMSO MCA a MCA MCA MCA MCA MCA MCA MCA
DMSO TPA DMSO TPA PDD PDD PDD PDD PDD PDD
Medium Medium Medium Medium Medium Asc-Na APM Medium Asc-Na APM
0.00 ^ 0.00 0.00 ^ 0.00 0.30 ^ 0.48 3.50 ^ 1.72 6.50 ^ 3.03 3.40 ^ 1.43 0.50 ^ 0.71 9.10 ^ 2.38 8.30 ^ 1.95 1.50 ^ 1.18
± ± ± ± 100 b 52 8 100 b 91 16
a b c d
100.0 100.0 0.3 0.3 0.3 1.0 1.0 1.0
100 100 100 100
Transformation
0/10 0/10 3/10 10/10 10/10 10/10 4/10 10/10 10/10 7/10
0 0 3 35 65 34 c 5d 91 83 15 d
The concentration of MCA was 0.2 mg/ml and the cell growth of MCA-treated cells was 63% of the untreated control cells. Transformation control. P , 0:05 vs. transformation control by the Mann±Whitney U-test. P , 0:01 vs. transformation control by the Mann±Whitney U-test.
In the current set of experiments, we tried to examine the effect of ascorbic acid and stable ascorbic acid derivatives in the two-stage BALB/c 3T3 cell transformation. Our experimental system consisted of the two-stage transformation of BALB/c 3T3 cells. The cells were ®rst treated with a relatively low concentration of MCA (0.2 mg/ml in our case, and 1±5 mg/ml
in the previous papers using C3H10T1/2 cells without additional TPA treatment) as an initiation treatment, followed by TPA as a promotion treatment. Asc-Na and its derivatives were added in the medium twice/ week during cell maintenance. Our results showed that cell transformation was most markedly inhibited with APM and less strongly
Table 4 Inhibitory effects of APM on TNF-a-enhanced transformation of MCA-treated BALB/c 3T3 cells Initiating treatment
Promoting treatment
Treatment Concentration Treatment Concentration No. of dishes Total no. (ng/ml) (mm) with foci/no. of of foci dishes examined
Foci/dish (mean ^ SD)
% of transformation control
DMSO DMSO MCA a MCA MCA MCA MCA MCA MCA MCA
DMSO TPA DMSO TPA TNF-a TNF-a TNF-a TNF-a TNF-a TNF-a
0.00 ^ 0.00 0.00 ^ 0.00 0.30 ^ 0.48 3.50 ^ 1.72 1.00 ^ 1.15 0.30 ^ 0.67 2.40 ^ 2.41 0.60 ^ 0.70 2.30 ^ 1.77 0.70 ^ 0.67
± ± ± ± 100 b 30 100 b 25 100 b 30
a b c
100 100 1 1 3 3 10 10
Inhibitor treatment
Medium Medium Medium Medium Medium APM Medium APM Medium APM
100 100 100
Transformation
0/10 0/10 3/10 10/10 5/10 2/10 8/10 5/10 9/10 6/10
0 0 3 35 10 3 24 6c 23 7c
The concentration of MCA was 0.2 mg/ml and the cell growth of MCA-treated cells was 63% of the untreated control cells. Transformation control. P , 0:05 vs. transformation control by the Mann±Whitney U-test.
T. Tsuchiya et al. / Cancer Letters 160 (2000) 51±58
with Asc-Na and AG. AS showed little effect (Table 1). The fact that a biweekly addition of unstable ascorbic acid was effective in TPA-induced transformation also attracted our attention. We then tried to elucidate these results from the viewpoint of the stability of the compounds and the use of PDD or TNF-a in place of TPA. In the stability studies, Asc-Na degraded rapidly in the culture medium during the incubation (half-life, 1.8 h). In contrast, the half-life of APM was 23 h. We considered that this marked difference may give rise to different activities against the more potent and more stable in vitro phorbol ester promoter, PDD (Table 3). As may be expected, the inhibitory effect of Asc-Na was markedly decreased, but the stable derivative, APM, was still very active in reducing the number of transformed foci. These results suggest that, in order to inhibit the promotional effect by phorbol esters, active ascorbic acid must be present in the medium for extended periods. APM is degraded to produce ascorbic acid by alkaline phosphatase which is present in serum. Our medium contains FBS, and therefore APM can be effectively reduced to ascorbic acid in the culture medium. In the case of AG, 93.1% remained even after a 96-h incubation. Glucosidase is responsible for degrading AG. We then examined the stability of APM and AG in sera from humans, mice and guinea pigs. APM degraded to produce ascorbic acid in every serum examined, but AG degradation was observed only in mouse serum (data not shown). Therefore, the reason why AG was active in the current cell transformation assay is suggested to be due either to the presence of glucosidase on the membrane of BALB/c 3T3 cells or to the presence of an active transport system for AG. In either explanation, only a small amount of AG was utilized and AG was less effective than APM and Asc-Na in inhibiting cell transformation. In contrast, AS was quite stable under the present culture conditions. AS can be hydrolyzed by sulfohydrase, which is detected in tissues of the rat, guinea pig and rabbit, but not in human tissues [25]. This enzyme is also considered to be absent from FBS and from the membranes of BALB/c 3T3 cells. Thus, in our transformation experiments little suppressive effect was observed. From this comparative study on the ascorbate deri-
57
vatives, it is concluded that APM was the most ef®cient `supplier' of ascorbic acid to effect the inhibition of in vitro cell transformation. In addition, the effect of ascorbates was examined on the transformation of MCA-treated BALB/c 3T3 cells enhanced by a tumor promoter other than phorbol esters. TNF-a, a pleiotropic cytokine, was reported to increase the transformation of initiatortreated BALB/c 3T3 cells when added at the promotion stage. The cytokine is produced by addition of okadaic acid, a potent tumor promoter, and is therefore considered to be an intrinsic promoter [26]. Our results showed that the in vitro promotion enhanced by TNF-a was indeed inhibited with APM (Table 4). The effect of TNF-a is related to free radical production [27]. Therefore, these results may support the notion that the mechanism of inhibition of cell transformation by ascorbic acid is related to the elimination of active oxygen as a radical scavenger. Further in vivo experiments are necessary to con®rm an anti-tumor-promoting effect of the ascorbate derivatives. References [1] W.F. Benedict, W.L. Wheatley, P.A. Jones, Inhibition of chemically induced morphological transformation and reversion of the transformed phenotype by ascorbic acid in C3H10T1/2 cells, Cancer Res. 40 (1980) 2796±2801. [2] M.P. Rosin, A.R. Peterson, H.F. Stich, The effect of ascorbate on 3-methylcholanthrene-induced cell transformation in C3H10T1/2 mouse-embryo ®broblast cell cultures, Mutat. Res. 72 (1980) 533±537. [3] W.F. Benedict, W.L. Wheatley, P.A. Jones, Differences in anchorage-dependent growth and tumorigenicities between transformed C3H/10T1/2 cells with morphologies that are or are not reverted to a normal phenotype by ascorbic acid, Cancer Res. 42 (1982) 1041±1045. [4] J. Bhattacharjee, A.S. Chakraborty, N.H. Sarkar, A. Basu, Study of ascorbic acid status in murine and human leukemias, Comp. Pathol. 95 (1985) 87±91. [5] J. Ghosh, S. Das, Evaluation of vitamin A and C status in normal and malignant conditions and their possible role in cancer prevention, Jpn. J. Cancer Res. 76 (1985) 1174±1178. [6] S. Bram, P. Frousard, M. Guichard, Vitamin C preferential toxicity of malignant melanoma, Nature 284 (1980) 629±631. [7] S.J. Harada, T. Masumoto, E. Ogata, Role of ascorbic acid in the regulation of proliferation in osteoblast-like MC3T3E1cells, J. Bone Miner. Res. 6 (1991) 903±907. [8] A. Campbell, T. Jack, E. Cameron, Reticulum cell sarcoma: two complete spontaneous regressions in response to high dose ascorbic acid therapy, Oncology 48 (1991) 495±497.
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