Cancer Letters, 65 (1992) 51- 54

Elseviet Scientific Publishers Ireland Ltd.

51

Effect of ( - )-epigallocatechin gallate, the main constituent of green tea, on lung metastasis with mouse B16 melanoma cell lines

Shun’ichiro Taniguchia, Hirota Fujikib, Hiroaki Kobayashi”, Hiroki Go”, Kenji Miyado”, Hiroyuki Sadano’ and Rie Shimokawa”

“Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-I-1 Maidashi,

Higashi-ku, Fukuoka City 812, bCancer Preoention Division, National Cancer Center Research Institute, 5-I-1 Tsukiji, Chuo-ku, Tokyo 104 (Japan)

(Received 14 February 1992) (Revision received 13 April 1992)

(Accepted 12 May 1992)

Summary

(- )-Epigallocatechin gpllate (EGCG), the main polyphenolic constituent of green tea, in- hibits tumor promotion and chemical carcino- genesis in animal experimental systems. Here we report that the peroral administration of EGCG inhibited metastasis of B16 melanoma cell lines, such as B16-F10 and BL6, in both

experimental and spontaneous systems.

Keywords: ( - )-epigallocatechin gallate; green tea; anti-metastasis; B16 melanoma

Introduction

An important clinical problem is that most of the lethality of malignant tumors is attributable to metastasis of neoplastic cells. Complete attack of malignant cells is usually limited, due to side effects caused by treatment. Therefore, new devices and drugs to inhibit invasion and

Correspondence to: Shun’ichiro Taniguchi, Department of

Molecular and Cellular Biology, Medical Institute of Bioregula-

tion, Kyushu University, 3-l-l Maidashi, Higashi-ku, Fukuoka

City 812, Japan.

metastasis without severe side effects should be developed.

( -)-Epigallocatechin gallate (EGCG) is the main polyphenolic constituent of green tea in- fusion. It is reported that EGCG inhibits iumor promotion induced by teleocidin in a two-stage carcinogenesis experiment on mouse skin [l] and duodenal carcinogenesis with N-ethyl-N’- nitro-IV-nitrosoguanidine [Z] . An epidemio- logical study also indicated a decreased risk of gastric cancer among Japanese with high con- sumption of green tea [3]. In this paper, we report that peroral administration of EGCG at various concentrations inhibits metastasis of highly metastatic mouse B16 melanoma cell lines without severe side effects.

Materials and Methods

The preparation of EGCG used contained EGCG (85%), (-)-epicatechin (10%) and ( - )-epicatechin gallate (5%)) as described previously [ 11.

The inhibitory effects of EGCG on metastasis using highly metastatic B16-FlO cells as well as highly metastatic and invasive B16-BL6 cells, were studied in both artificial and spontaneous systems. Male C57BL/6

03043835/92/$05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland

52

mice at 6 - 7 weeks of age were obtained from the Animal Center of Kyushu University and used for the experiments.

In the experiment of artificial metastasis, 2 x lo5 or 5 x lo5 cells of B16-F10/0.5 ml of Hank’s balanced salt solution/mouse were injected into the tail vein. The animals in the experimental groups were ad Iibitum perorally given a solution of 0.05% EGCG or 0.1% EGCG. Animals in the control group were given deionized water. The administra- tion of EGCG solution was started 1 day before the intravenous injection of B16-FlO cells and continued until examination of the lung metastasis. All mice were sacrificed and dissected when a few control mice died with the lung metastasis, usually around 3 weeks after the i.v. injection.

In the experiment of spontaneous meta- stasis, a B16-BL6 cell line, which was more invasive than the B16-FlO cell line, was used to obtain a larger number of lung nodules. A 3 x lo5 quantity of B16-BL6 cells/30 ~1 per mouse was inoculated into the right foot pad of C57BL/6 mice. When tumor nodules reached 4.5-6 mm in diameter (around 9 days after the cell inoculation), the legs bear- ing tumors were amputated. The administra- tion of EGCG solution was started 1 day before the amputation and continued in the same way as in the experiment of artificial metastasis.

The lung of sacrificed mice was rinsed in water and infused and fixed with Bouin’s solu- tion; the appearance of the metastasis in the chest was determined and the total number of visible nodules on the lung surface per animal was counted.

We examined the effects of EGCG on growth of tumor cells inoculated into the foot pad. In the experiments, the peroral admin- istration of EGCG was started 1 day before inoculation of B16-BL6 cells.

Results

The volume of EGCG solution or water intaken by mice was almost the same among

Exp. 1 1 ~““,bL3

3ooF 1

Exp. 2

T

P

$ p 200 -

3 ii g loo-

0

8” 0

Fig. 1. Effect of EGCG peroral administration in the experiment of artificial metastasis with B16-FlO cells in mate C57BL/6 mice. Seven to ten animals were used for each group in the experiment. In experiment 1,2 x 10’ B16-FlO cells were i.v. injected. Control versus 0.1% EGCG: P c 0.05 (Wilcoxon t-test). In experiment 2, 5 x lo5 B16-FlO cells were i.v. injected. Control ver- sus 0.05% EGCG: P < 0.01; control versus 0.1% EGCG: P < 0.01; 0.05% EGCG versus 0.1% EGCG P < 0.01 (Wilcoxon t-test).

the groups (about 4 ml/mouse per day), although slightly less intake was observed in the animals given EGCG a few days after the start of its administration. There was no signifi- cant difference in body weights among the groups (data not shown), indicating that EGCG did not cause any severe toxicity.

The inhibitory effects of EGCG on the arti- ficial metastasis with B16-FlO cells were shown in Fig. 1. In both experiments 1 (inocul- ation of 2 x lo5 cells) and 2 (inoculation of 5 x lo5 cells), the number of lung nodules showed a tendency to decrease in the treated animals depending on the concentrations of EGCG. The average number of lung nodules of each group was > 150 (0% EGCG), 107 (0.05% EGCG) and 76 (0.1% EGCG) in experiment 1 and > 300 (0% EGCG), > 227 (0.05% EGCG), 136 (0.1% EGCG) in experi- ment 2, respectively. These results indicated that EGCG inhibits lung metastasis of B16-FlO cells. We also observed significant inhibition of lung metastasis, which was caused by i.v.

40

P

3

P

F 3 20

ij

z"

0

0

x

0 x

0

f% =I

0 0.05 0.1 % EGCG

Fig. 2. Effects of EGCG peroral administration on the spontaneous lung metastasis of B16-BL6 cells in male C57BL/6 mice. Each group contains 8 mice. When a few mice in the control group died (26 days after the amputation), all animals were sacrificed for examination of the lung metastasis. (a) No. of lung colonies. The average numbers of each group were 25 (0% EGCG), 7 (0.05% EGCG) and 10 (0.1% EGCG). Control versus 0.05% EGCG: P < 0.01; control versus 0.1% EGCG: P < 0.01; 0.05% EGCG versus 0.1% EGCG: P > 0.05 (Wilcoxon t-test). (b) Note the apparent growth of BL6 cells on the pleura in the mediastinum in the control group (top line) as compared with the treated groups with EGCG (middle line, 0.05% EGCG; bottom line, 0.1% ECCG).

f . 5 a- g 12 6-

6

$

4-

E 2-

Occ” I I

0 10 20

Days after cell inoculation

Fig. 3. Effect of EGCG peroral administration on in vivo growth of BL 6 cells. Each group contains 8 mice. Administration of EGCG was started 1 day before the cell inoculation (ifp, 5 x lo5 cells/30 ~1 per mouse). (0)

Control; (cdO.0546 EGCG; ( x ) 0.1% EGCG. Bars: SD. Control vs. 0.05% EGCG (P > 0.05%). Control vs. 0.1% EGCG (P < 0.05%) (Wilcoxon t-test).. _

injection of 1 X lo5 Lewis lung carcinoma cells per mouse, by the peroral administration of 0.1% EGCG. The average number of the lung nodules of control group (0% EGCG) was 24 (range: 11 - 39, 8 mice), while that of treated group (0.1% EGCG) was 17 (range: 6 -26, 8 mice) (P < 0.05).

In the experiment of spontaneous metastasis of B16-BL6 cells to the lung, the number of lung nodules was significantly reduced by administration of EGCG solution (Fig. 2a). Particularly, inhibition of growth of melanoma cells was clearly observed on the mediastinal pleura of the mice given EGCG (Fig. 2b).

We examined the effects of EGCG on growth of tumor cells inoculated into the foot pad. Tumor growth was slightly inhibited when the animals were administered with 0.1% EGCG (P < 0.05) (Fig.3).

54

Discussion

Information on the biological effects of EGCG has recently accumulated. EGCG is known to have anti-mutagenic activity [4] and inhibitory activity of protein kinase C by a tumor promoter, teleocidin [ 11. More recently, the antioxidative activity of a green tea fraction was reported [5]. It is also reported that super- oxide radicals enhance invasiveness of tumor cells [6]. Therefore, one of the inhibitory ac- tivities of EGCG on the metastasis can be understood by the scavenging activity of super- oxide radicals. In vitro invasiveness and motil- ity was partially inhibited at concentrations with little cytotoxicity (data not shown). However, it is still not known how EGCG affects tumor cells in vivo. It remains to be examined whether inhibition of lung metastasis and tumor growth by EGCG administration is due to the inhibition of the invasion or to tumor specific cytotoxic effects in vivo. Japanese people ingest green tea every day. It is also reported that orally administered green tea in- hibits carcinogenesis by ultraviolet B light and chemical carcinogens, such as tobacco-specific nitrosoamine [7]. Because the main consti- tuent of green tea possibly inhibits metastasis within doses with no apparent toxicity through simple peroral administration, EGCG seems to have potential for clinical use. Activity of EGCG should be further investigated in various human tumor cell lines applied in nude mouse systems. The presented results with EGCG indicate that there is great potential to

pursue this approach in combination with various other treatments.

Acknowledgments

This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Health and WeIfare and the Ministry of Educa- tion, Science and Culture and by the Princess Takamatsu Cancer Research Fund.

References

Yoshizawa, S., Horiuchi, T., Fujiki, H., Yoshida, T., Okuda, T. and Sugimura, T. (1987) Antitumor promoter activity of (- )-epigallocatechin gallate, the main consti- tuent of “tannin” in green tea. Phytother. Res., 1,44 - 47. Fujita, Y., Yamane, T., Tanaka, M., Kuwata, K., Okuzumi, J., Takahashi, T., Fujiki, H. and Okuda, T. (1989) Inhibitory effect of (-)-epigallocatechin gallate on carcinogenesis with IV-ethyl-IV’-nitro-N-nitrosoguanidine in

mouse duodenum. Jpn. J. Cancer Res., 80, 503-505. Kono, S., Ikeda, M., Tokudome, S. and Kuratsune, M. (1988) A case-control study of gastric cancer and diet in northern Kyushu, Japan. Jpn. J. Cancer Res., 79, 1067 - 1074. Okuda, T., Mori, K. and Hayatsu, H. (1984) Inhibitory effect of tannins on direct-acting mutagens. Chem. Pharm. Bull., 32, 3755 - 3758. Ruth, R.J., Cheng, S. and Klaunig, J.E. (1989) Preven- tion of cytotoxicity and inhibition of intercellular com- munication by anti-oxidant catechins isolated from Chinese green tea. Carcinogenesis, 10, 1003 - 1008. Shinkai, K., Mukai, M. and Akedo, H. (1986) Superoxide radical potentiates invasive capacity of rat ascites hapatoma cells in vitro. Cancer Lett., 32, 7- 13. Pennisi, E. (1991) Tea-totalling mice gain cancer protec- tion. Sci. News, 140, 133.