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Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga
Cancer Letters 167 (2001) 145±150
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Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga Shingo Hiroishi a,*, Kazutomo Sugie a, Takashi Yoshida a, Junji Morimoto b, Yuzo Taniguchi c, Shunsuke Imai d, Junichi Kurebayashi e a
Faculty of Biotechnology, Fukui Prefectural University,1-1 Gakuen-cho, Obama, Fukui 917-0003, Japan b Animal Center, Osaka Medical College,2-7 Daigaku-cho, Takatsuki, Osaka 569-8686, Japan c Research Laboratory, Nippon Fine Chemical Co. Ltd., 1-1, 5-Chome, Umei, Takasago, Hyogo 676-0074, Japan d Nara Prefectural Institute of Public Health, 57-6 Omori-cho, Nara 630-8131, Japan e Department of Breast & Thyroid Surgery, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan Received 15 December 2000; received in revised form 13 February 2001; accepted 13 February 2001
Abstract Marginisporum crassissimum (Yendo) Ganesan, a marine red alga found in the ordinal coastal sea around Japan, revealed antitumor (antimetastatic) effects in vitro and in vivo. In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line), but also invasion of B16-BL6 cells in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially puri®ed by ®ltration and hydrophobic column chromatography, and was revealed to be sensitive to trypsindigestion and heat-treatment. The molecular weight of the substance was greater than 100 kDa. This is the ®rst study demonstrating antitumor (antimetastatic) effects of M. crassissimum. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Marginisporum crassissimum; Red alga; Antitumor; Antimetastasis
1. Introduction Marine bioresources are known to be attractive as they sometimes include new compounds showing several kinds of bioactivities. Among them, studies on antitumor effects have been published using algae such as Laminaria [1±4], Sargassum [2,5], and so on [2,6]. The active substances have been * Corresponding author. Tel.: 181-770-52-6300; fax: 181-77052-6003. E-mail address: [email protected] (S. Hiroishi).
demonstrated to be algal polysaccharides, and these antitumor effects were reported to arise from immunoactivation. Marginisporum crassissimum was reported to have both immuno-stimulating and -suppressive effects [7]. It showed stimulation of T cell blastogenesis and IgG production, whereas it showed a suppressive effect in murine bone marrow proliferation. As this alga includes immunologically interesting substance(s), we examined the antitumor (antimetastatic) effects of this alga.
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00460-8
146
S. Hiroishi et al. / Cancer Letters 167 (2001) 145±150
Here, we report the effects of the extracts of M. crassissimum in in vitro and in vivo experiments. 2. Materials and methods 2.1. Tumor cell culture We used three tumor cell lines as follows: B16BL6, a mouse melanoma cell line from Dr Fidler, M.D., Anderson Cancer Center, USA; JYG-B, a mouse mammary carcinoma cell line established by us [8,9]; and KPL-1, a human mammary carcinoma cell line established by Dr Kurebayashi [10]. They were cultivated at 378C in Dulbecco's modi®ed Eagle's medium (D-MEM, Life Technologies, Inc., Tokyo, Japan) supplemented with 10% fetal calf serum (Life Technologies, Inc.), penicillin (80 U/ml) and streptomycin (100 mg/ml) in CO2 incubator (5% CO2; Sanyo MCO-175, Osaka, Japan). 2.2. Preparation of crude extract from M. crassissimum Marginisporum crassissimum (100 g, wet weight), collected in Wakasa Bay (Japan) or from Dr Mizukoshi (Ishihara Sangyo Co., Osaka, Japan), was homogenized in 500 ml of water by ULTRA-TURRAX T25 (JANKE & KUNKEL IKA-Labotechnik, Germany) at
20 000 revs./min for 5 min, and stirred at 48C for 4 days. After it was centrifuged at 12 000 £ g for 30 min, the supernatant solution was ®ltrated through a 0.22 mm ®lter and used as crude extracts of this alga for further experiments (10 mg/ml, dry weight). The pH value of the extract was 7.2. 2.3. In vitro antitumor cell growth assay B16-BL6, JYG-B or KPL-1 cells (2.5 £ 10 5 cells/ml) were cultivated in 100 ml of D-MEM in wells of 96-well microculture plates (Nunc Co., NY), with 10 ml of extracts of the alga. The cell growth was assayed at 1, 2 and 3 days after the cultivation using a cell counting kit (Dojin Co., Japan) according to the manufacturer's instructions. 2.4. In vitro invasion culture assay An invasion culture assay was performed as described by Isoai et al. [11] with a little modi®cation. Brie¯y, ®lters of chambers (Transwell, pore size 8.0 mm; Corning Coster Co., Cambridge, MA) were coated by matrigel (80 mg/®lter) and set on 24-well plates. Seven hundred microliters of B16-BL6 cell suspension (2 £ 10 5 cells) with algal extract (15 ml) in D-MEM medium was added into the chamber, and ®bronectin (10 mg/ml) in the medium was added in the corresponding wells of 24-well plates. They were
Fig. 1. Growth inhibition of B16-BL6, JYG-B and KPL-1 cells by the extracts of Marginisporum crassissimum. (A) B16-BL6; (B), JYG-B; (C), KPL-1; (X), control; (O), addition of M. crassissimum extracts.
S. Hiroishi et al. / Cancer Letters 167 (2001) 145±150
147
Table 1 Effects of Marginisporum crassissimum extract on the invasion of B16-BL6 melanoma cells in vitro Sample
Number of invading cells (cells/mm 2 per day)
Inhibition (%)
Control (saline) Marginisporum crassissimum
195.1 ^ 4.2 125.2 ^ 9.1 a
0 37.0
a
Statistical signi®cance, P , 0:01.
incubated in the CO2 incubator for 24 h. The melanoma cells which invaded through the ®lter to the outside surface of the chamber ®lter were counted after ®xing with 70% methanol and staining with hematoxylin and eosin. 2.5. In vivo antimetastasis assay Into the tail vein of female C57BL/6J mice (5 weeks old, Clea Japan Co.), 50 000 B16-BL6 cells were inoculated. Among them, 0.5 ml of the extracts of this alga was injected intraperitoneally into ten mice, four times/week for 3 weeks. Similar volumes of saline were injected into another ten mice as a control group. Metastases of the lung were monitored by counting lung-surface colonies of the mice 3 weeks after the inoculation. 2.6. A mouse prolonged life assay One million B16-BL6 cells were suspended in phosphate-buffered saline (PBS) intraperitoneally inoculated into the mice as mentioned above. From 1 week before the inoculation of melanoma cells, 0.5 ml of the extracts of M. crassissimum was administered intraperitoneally into 12 mice, four times/week for 3 weeks. Similar volumes of saline were injected into another 12 mice as a control group. They were observed everyday until death. Prolonged life (days) was counted after the tumor cell inoculation.
2.7. A histological study Tissues of the mice used in the in vivo antimetastatic assay (lung, liver, kidney, heart, spleen and lymph node) were ®xed in 10% formaldehyde and embedded in paraf®n. The ®xed tissues were sliced by microtome and stained with hematoxylin and eosin, and histological alterations were observed under an optical microscope. 2.8. Partial puri®cation of M. crassissimum extract Ammonium sulfate was added to 10 ml of the crude extracts of the alga obtained above (30% saturation), and they were centrifuged at 12 000 £ g for 30 min. The supernatant fraction was fractionated using a Centriplus centrifugal ®lter YM-100 (Millipore Corporation, MA). A fraction remaining on the membrane was dissolved in 10 ml of distilled water, and applied to a Hi Trap Butyl Sepharose column (Amersham Pharmacia Biotech, Buckinghamshire, UK). An effective fraction was obtained by adding 20% saturated ammonium sulfate solution through the column. 2.9. Heat-treatment of partially puri®ed algal extracts The partially puri®ed algal extracts obtained above (500 ml) were incubated at 20, 40, 60 and 808C for 1 h. The inhibitory effects of the heated extracts on the
Table 2 Effect of administration of Marginisporum crassissimum extract on the lung metastasis of B16-BL6 cells in C57BL/6J mice Sample
Dose (mg/kg per day)
Number of metastasis foci/ mouse lung (mean ^ SD)
Inhibition (%)
Control (saline) Marginisporum crassissimum
0 260 80
265.9 ^ 42.3 146.5 ^ 27.9 a 183.3 ^ 62.2 a
0 44.9 31.1
a
Statistical signi®cance, P , 0:01.
148
S. Hiroishi et al. / Cancer Letters 167 (2001) 145±150
Fig. 2. Metastasis of B16-BL6 cells in the lung tissues of C57BL/6J mice ( £ 100).
growth of B16-BL6 cells were measured by the in vitro antitumor cell growth inhibition assay as described above. 2.10. Trypsin-digestion of partially puri®ed algal extracts The partially puri®ed algal extracts obtained above (1.6 ml) were added with 0.4 ml of 0.25% Trypsin 250 (W/V; Wako Jun-yaku Kogyo Co., Osaka, Japan), and they were incubated at 378C for 30 min. After incubation, trypsin was removed by ®ltration using ultra®lter units USY-5 (Advantec, Tokyo, Japan) at 5000 £ g for 5 min. The non-®ltrated fraction on the ®lter was dissolved in 2 ml of distilled water. The inhibitory effects of the trypsin-digested extracts on the growth of B16-BL6 cells were measured by the in vitro antitumor cell growth inhibition assay as described above. 2.11. Statistical analysis The numbers of growing cells, growth inhibition, metastatic foci, and prolonged days of mouse life
Fig. 3. A dose-dependent effect of the extract on the growth inhibition of B16-BL6 cells. Partially puri®ed active substances were diluted ®ve and ten times by distilled water. Inhibitory effects were measured by in vitro antitumor cell growth assay as described in Section 2. An asterisk represents statistical signi®cance (P , 0:01) from the growth inhibition by non-diluted algal extract.
were given as the means ^ SD. These data were analyzed for signi®cance using the Student's t-test. 3. Results and discussion In the in vitro experiments, the extract of M. crassissimum inhibited the growth of B16-BL6, JYG-B and KPL-1 cells completely (Fig. 1). In microscopic observations of the cells incubated with the extract, they were alive on the bottom of 96-well plates, and dead cells were not found, which suggests that the effect of the extract is cell-static. In addition, it also inhibited the invasion of B16-BL6 melanoma cells in
Table 3 Effect of administration of Marginisporum crassissimum on the life of C57BL/6J mice inoculated with B16-BL6 cells Sample
Number of mice used
Life after inoculation (mean ^ SD days)
Control (saline) Marginisporum crassissimum
12 12
12 ^ 1 20 ^ 2 a
a
Statistical signi®cance, P , 0:01.
S. Hiroishi et al. / Cancer Letters 167 (2001) 145±150
149
Fig. 4. Effect of Marginisporum crassissimum extracts fractionated using the Centriplus centrifugal ®lter (YM-100) on the growth of B16-BL6 melanoma cells. .100 kDa, a fraction remained on the ®lter (M.W. cut-off: 100 kDa); ,100 kDa, a fraction passed through the ®lter. Growth inhibitory effects of the fractions on the growth of B16-BL6 were measured by in vitro antitumor cell growth inhibition assay (n 6), after the volume of the fractions was adjusted to that before fractionation by distilled water. Growth inhibition (%) growth inhibition after fractionation/growth inhibition before fractionation £ 100.
tered with the extract (data not shown). The immunological effects of the extract might be small in vivo. More precise further experiments are needed. No toxic effects of the algal extract were observed in the mouse behavior or in the tissues of the lung, liver, kidney, heart, spleen and lymph nodes, except for the metastatic foci of the tumor cells in the lung tissue. A mouse prolonged life assay was also performed as described in Section 2. The lives of the mice were prolonged for about 8 days by the administration of the extract (Table 3). The properties of the active substance showing growth inhibition of B16-BL6 in vitro were examined after partial puri®cation. Five bands were observed in SDS-PAGE and Coomassie Brilliant Blue staining (data not shown). Harvesting much more algae from the sea for further puri®cation is needed. However, the algal materials are limited. Therefore, some characteristics of the extract were examined. At ®rst, dose-dependency of the effect was examined on the growth inhibition of B16-BL6 melanoma cells with diluted partially puri®ed substances (Fig. 3). Dose-dependent effects were observed among one, ®ve and ten times dilution. The molecular weight of the active substance was greater than 100 kDa (Fig. 4), and it was sensitive to heat-treatment and trypsin-digestion (Figs. 5 and
the in vitro culture assay (Table 1). In these experiments, the direct inhibitory effects of the algal extract on the growth and invasion of the tumor cells were demonstrated. In the in vivo antimetastasis assay, the extract inhibited the metastasis of B16-BL6 cells to the lungs of B57BL/6J mice. The number of foci produced by B16-BL6 cells was reduced by the administration of the algal extracts (Table 2). Although the alga was reported to have immunostimulating activity ([H 3]thymidine incorporation in T cell blastogenesis in vitro) [7], an in®ltration or proliferation of immunologically associated cells, such as lymphocytes, was not observed around the foci of the cancer cells (Fig. 2) or in the tissues of the spleen and lymph nodes, except for proliferation of macrophages in the spleens of two mice and in®ltration of lymphocytes in the livers of two mice out of 12 experimental mice with B16-BL6 cells adminis-
Fig. 5. Heat sensitivity of the B16-BL6 growth inhibitory effects of partially puri®ed Marginisporum crassissimum extracts. Growth inhibition (%) growth inhibition after heat-treatment at each temperature/growth inhibition without heat-treatment £ 100.
150
S. Hiroishi et al. / Cancer Letters 167 (2001) 145±150
References
Fig. 6. Trypsin sensitivity of the B16-BL6 growth inhibitory effects of partially puri®ed Marginisporum crassissimum extracts. Growth inhibition (%) growth inhibition after trypsin-digestion/growth inhibition without trypsin-digestion £ 100 Extract, partially puri®ed M. crassissimum extracts; Trypsin, 0.05% trypsin solution; (1), added; (2), not added.
6), which suggested that it might be a protein. This protein-like substance possibly affects the cancer cell directly (or indirectly) and inhibits its growth and metastasis. Further puri®cation of the substance and in vivo studies will be done in next experiment to clarify the mechanism of the antitumor (antimetastatic) effects of this alga.
[1] I. Yamamoto, M. Takahashi, E. Tamura, H. Maruyama, Antitumor activity of crude extracts from edible marine algae against L-1210 leukemia, Bot. Mar. 25 (1982) 455±457. [2] H. Noda, H. Amano, K. Arashima, S. Hashimoto, K. Nisizawa, Antitumor activity of polysaccharides and lipids from marine algae, Nippon Suisan Gakkaishi 55 (1989) 1265±1271. [3] Y. Suzuki, I. Yamamoto, I. Umezawa, Antitumor effect of seaweed in Japanese with English summary, Chemotherapy 28 (1980) 165±170. [4] B. Jolles, M. Remington, P.S. Andrews, Effects of sulphated degraded laminarin on experimental tumor growth, Br. J. Cancer 17 (1963) 109. [5] I. Yamamoto, T. Nagumo, K. Yagi, H. Tominaga, M. Aoki, Antitumor effect of seaweeds, Jpn. J. Exp. Med. 44 (1974) 543±546. [6] S. Mizukoshi, H. Matsuoka, K. Nakamura, H. Katou, H. Noda, Search for bioactive substances from marine algae, Bull. Faculty Biosources, Mie Univ. 8 (1992) 27±34. [7] S. Mizukoshi, H. Matsuoka, F. Kato, Effect of seaweed preparations on murine immunocytes, J. Appl. Phycol. 15 (1993) 629±637. [8] J. Morimoto, S. Imai, S. Haga, M. Iwai, S. Hiroishi, N. Miyashita, K. Moriwaki, H.L. Hosick, New murine mammary tumor cell lines, In Vitro Cell. Dev. Biol. 27A (1993) 349± 351. [9] S. Hiroishi, J. Morimoto, Y. Taniguchi, H. Mori, Multiple metastases of mammary carcinoma cell lines isolated from feral mouse, Cancer Lett. 92 (1995) 83±86. [10] J. Kurebayashi, M. Kurosumi, H. Sonoo, A new human breast cancer cell line, KPL-1 secretes tumor-associated antigens and grows rapidly in female athymic nude mice, Br. J. Cancer 71 (1995) 845±853. [11] A. Isoai, H. Goto-Tsukamoto, K. Murakami, H. Akedo, H. Kumagai, A potent anti-metastatic activity of tumor invasion-inhibiting factor-2 and albumin conjugate, Biochem. Biophys. Res. Commun. 192 (1993) 7±14.
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Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga Shingo Hiroishi a,*, Kazutomo Sugie a, Takashi Yoshida a, Junji Morimoto b, Yuzo Taniguchi c, Shunsuke Imai d, Junichi Kurebayashi e a
Faculty of Biotechnology, Fukui Prefectural University,1-1 Gakuen-cho, Obama, Fukui 917-0003, Japan b Animal Center, Osaka Medical College,2-7 Daigaku-cho, Takatsuki, Osaka 569-8686, Japan c Research Laboratory, Nippon Fine Chemical Co. Ltd., 1-1, 5-Chome, Umei, Takasago, Hyogo 676-0074, Japan d Nara Prefectural Institute of Public Health, 57-6 Omori-cho, Nara 630-8131, Japan e Department of Breast & Thyroid Surgery, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan Received 15 December 2000; received in revised form 13 February 2001; accepted 13 February 2001
Abstract Marginisporum crassissimum (Yendo) Ganesan, a marine red alga found in the ordinal coastal sea around Japan, revealed antitumor (antimetastatic) effects in vitro and in vivo. In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line), but also invasion of B16-BL6 cells in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially puri®ed by ®ltration and hydrophobic column chromatography, and was revealed to be sensitive to trypsindigestion and heat-treatment. The molecular weight of the substance was greater than 100 kDa. This is the ®rst study demonstrating antitumor (antimetastatic) effects of M. crassissimum. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Marginisporum crassissimum; Red alga; Antitumor; Antimetastasis
1. Introduction Marine bioresources are known to be attractive as they sometimes include new compounds showing several kinds of bioactivities. Among them, studies on antitumor effects have been published using algae such as Laminaria [1±4], Sargassum [2,5], and so on [2,6]. The active substances have been * Corresponding author. Tel.: 181-770-52-6300; fax: 181-77052-6003. E-mail address: [email protected] (S. Hiroishi).
demonstrated to be algal polysaccharides, and these antitumor effects were reported to arise from immunoactivation. Marginisporum crassissimum was reported to have both immuno-stimulating and -suppressive effects [7]. It showed stimulation of T cell blastogenesis and IgG production, whereas it showed a suppressive effect in murine bone marrow proliferation. As this alga includes immunologically interesting substance(s), we examined the antitumor (antimetastatic) effects of this alga.
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00460-8
146
S. Hiroishi et al. / Cancer Letters 167 (2001) 145±150
Here, we report the effects of the extracts of M. crassissimum in in vitro and in vivo experiments. 2. Materials and methods 2.1. Tumor cell culture We used three tumor cell lines as follows: B16BL6, a mouse melanoma cell line from Dr Fidler, M.D., Anderson Cancer Center, USA; JYG-B, a mouse mammary carcinoma cell line established by us [8,9]; and KPL-1, a human mammary carcinoma cell line established by Dr Kurebayashi [10]. They were cultivated at 378C in Dulbecco's modi®ed Eagle's medium (D-MEM, Life Technologies, Inc., Tokyo, Japan) supplemented with 10% fetal calf serum (Life Technologies, Inc.), penicillin (80 U/ml) and streptomycin (100 mg/ml) in CO2 incubator (5% CO2; Sanyo MCO-175, Osaka, Japan). 2.2. Preparation of crude extract from M. crassissimum Marginisporum crassissimum (100 g, wet weight), collected in Wakasa Bay (Japan) or from Dr Mizukoshi (Ishihara Sangyo Co., Osaka, Japan), was homogenized in 500 ml of water by ULTRA-TURRAX T25 (JANKE & KUNKEL IKA-Labotechnik, Germany) at
20 000 revs./min for 5 min, and stirred at 48C for 4 days. After it was centrifuged at 12 000 £ g for 30 min, the supernatant solution was ®ltrated through a 0.22 mm ®lter and used as crude extracts of this alga for further experiments (10 mg/ml, dry weight). The pH value of the extract was 7.2. 2.3. In vitro antitumor cell growth assay B16-BL6, JYG-B or KPL-1 cells (2.5 £ 10 5 cells/ml) were cultivated in 100 ml of D-MEM in wells of 96-well microculture plates (Nunc Co., NY), with 10 ml of extracts of the alga. The cell growth was assayed at 1, 2 and 3 days after the cultivation using a cell counting kit (Dojin Co., Japan) according to the manufacturer's instructions. 2.4. In vitro invasion culture assay An invasion culture assay was performed as described by Isoai et al. [11] with a little modi®cation. Brie¯y, ®lters of chambers (Transwell, pore size 8.0 mm; Corning Coster Co., Cambridge, MA) were coated by matrigel (80 mg/®lter) and set on 24-well plates. Seven hundred microliters of B16-BL6 cell suspension (2 £ 10 5 cells) with algal extract (15 ml) in D-MEM medium was added into the chamber, and ®bronectin (10 mg/ml) in the medium was added in the corresponding wells of 24-well plates. They were
Fig. 1. Growth inhibition of B16-BL6, JYG-B and KPL-1 cells by the extracts of Marginisporum crassissimum. (A) B16-BL6; (B), JYG-B; (C), KPL-1; (X), control; (O), addition of M. crassissimum extracts.
S. Hiroishi et al. / Cancer Letters 167 (2001) 145±150
147
Table 1 Effects of Marginisporum crassissimum extract on the invasion of B16-BL6 melanoma cells in vitro Sample
Number of invading cells (cells/mm 2 per day)
Inhibition (%)
Control (saline) Marginisporum crassissimum
195.1 ^ 4.2 125.2 ^ 9.1 a
0 37.0
a
Statistical signi®cance, P , 0:01.
incubated in the CO2 incubator for 24 h. The melanoma cells which invaded through the ®lter to the outside surface of the chamber ®lter were counted after ®xing with 70% methanol and staining with hematoxylin and eosin. 2.5. In vivo antimetastasis assay Into the tail vein of female C57BL/6J mice (5 weeks old, Clea Japan Co.), 50 000 B16-BL6 cells were inoculated. Among them, 0.5 ml of the extracts of this alga was injected intraperitoneally into ten mice, four times/week for 3 weeks. Similar volumes of saline were injected into another ten mice as a control group. Metastases of the lung were monitored by counting lung-surface colonies of the mice 3 weeks after the inoculation. 2.6. A mouse prolonged life assay One million B16-BL6 cells were suspended in phosphate-buffered saline (PBS) intraperitoneally inoculated into the mice as mentioned above. From 1 week before the inoculation of melanoma cells, 0.5 ml of the extracts of M. crassissimum was administered intraperitoneally into 12 mice, four times/week for 3 weeks. Similar volumes of saline were injected into another 12 mice as a control group. They were observed everyday until death. Prolonged life (days) was counted after the tumor cell inoculation.
2.7. A histological study Tissues of the mice used in the in vivo antimetastatic assay (lung, liver, kidney, heart, spleen and lymph node) were ®xed in 10% formaldehyde and embedded in paraf®n. The ®xed tissues were sliced by microtome and stained with hematoxylin and eosin, and histological alterations were observed under an optical microscope. 2.8. Partial puri®cation of M. crassissimum extract Ammonium sulfate was added to 10 ml of the crude extracts of the alga obtained above (30% saturation), and they were centrifuged at 12 000 £ g for 30 min. The supernatant fraction was fractionated using a Centriplus centrifugal ®lter YM-100 (Millipore Corporation, MA). A fraction remaining on the membrane was dissolved in 10 ml of distilled water, and applied to a Hi Trap Butyl Sepharose column (Amersham Pharmacia Biotech, Buckinghamshire, UK). An effective fraction was obtained by adding 20% saturated ammonium sulfate solution through the column. 2.9. Heat-treatment of partially puri®ed algal extracts The partially puri®ed algal extracts obtained above (500 ml) were incubated at 20, 40, 60 and 808C for 1 h. The inhibitory effects of the heated extracts on the
Table 2 Effect of administration of Marginisporum crassissimum extract on the lung metastasis of B16-BL6 cells in C57BL/6J mice Sample
Dose (mg/kg per day)
Number of metastasis foci/ mouse lung (mean ^ SD)
Inhibition (%)
Control (saline) Marginisporum crassissimum
0 260 80
265.9 ^ 42.3 146.5 ^ 27.9 a 183.3 ^ 62.2 a
0 44.9 31.1
a
Statistical signi®cance, P , 0:01.
148
S. Hiroishi et al. / Cancer Letters 167 (2001) 145±150
Fig. 2. Metastasis of B16-BL6 cells in the lung tissues of C57BL/6J mice ( £ 100).
growth of B16-BL6 cells were measured by the in vitro antitumor cell growth inhibition assay as described above. 2.10. Trypsin-digestion of partially puri®ed algal extracts The partially puri®ed algal extracts obtained above (1.6 ml) were added with 0.4 ml of 0.25% Trypsin 250 (W/V; Wako Jun-yaku Kogyo Co., Osaka, Japan), and they were incubated at 378C for 30 min. After incubation, trypsin was removed by ®ltration using ultra®lter units USY-5 (Advantec, Tokyo, Japan) at 5000 £ g for 5 min. The non-®ltrated fraction on the ®lter was dissolved in 2 ml of distilled water. The inhibitory effects of the trypsin-digested extracts on the growth of B16-BL6 cells were measured by the in vitro antitumor cell growth inhibition assay as described above. 2.11. Statistical analysis The numbers of growing cells, growth inhibition, metastatic foci, and prolonged days of mouse life
Fig. 3. A dose-dependent effect of the extract on the growth inhibition of B16-BL6 cells. Partially puri®ed active substances were diluted ®ve and ten times by distilled water. Inhibitory effects were measured by in vitro antitumor cell growth assay as described in Section 2. An asterisk represents statistical signi®cance (P , 0:01) from the growth inhibition by non-diluted algal extract.
were given as the means ^ SD. These data were analyzed for signi®cance using the Student's t-test. 3. Results and discussion In the in vitro experiments, the extract of M. crassissimum inhibited the growth of B16-BL6, JYG-B and KPL-1 cells completely (Fig. 1). In microscopic observations of the cells incubated with the extract, they were alive on the bottom of 96-well plates, and dead cells were not found, which suggests that the effect of the extract is cell-static. In addition, it also inhibited the invasion of B16-BL6 melanoma cells in
Table 3 Effect of administration of Marginisporum crassissimum on the life of C57BL/6J mice inoculated with B16-BL6 cells Sample
Number of mice used
Life after inoculation (mean ^ SD days)
Control (saline) Marginisporum crassissimum
12 12
12 ^ 1 20 ^ 2 a
a
Statistical signi®cance, P , 0:01.
S. Hiroishi et al. / Cancer Letters 167 (2001) 145±150
149
Fig. 4. Effect of Marginisporum crassissimum extracts fractionated using the Centriplus centrifugal ®lter (YM-100) on the growth of B16-BL6 melanoma cells. .100 kDa, a fraction remained on the ®lter (M.W. cut-off: 100 kDa); ,100 kDa, a fraction passed through the ®lter. Growth inhibitory effects of the fractions on the growth of B16-BL6 were measured by in vitro antitumor cell growth inhibition assay (n 6), after the volume of the fractions was adjusted to that before fractionation by distilled water. Growth inhibition (%) growth inhibition after fractionation/growth inhibition before fractionation £ 100.
tered with the extract (data not shown). The immunological effects of the extract might be small in vivo. More precise further experiments are needed. No toxic effects of the algal extract were observed in the mouse behavior or in the tissues of the lung, liver, kidney, heart, spleen and lymph nodes, except for the metastatic foci of the tumor cells in the lung tissue. A mouse prolonged life assay was also performed as described in Section 2. The lives of the mice were prolonged for about 8 days by the administration of the extract (Table 3). The properties of the active substance showing growth inhibition of B16-BL6 in vitro were examined after partial puri®cation. Five bands were observed in SDS-PAGE and Coomassie Brilliant Blue staining (data not shown). Harvesting much more algae from the sea for further puri®cation is needed. However, the algal materials are limited. Therefore, some characteristics of the extract were examined. At ®rst, dose-dependency of the effect was examined on the growth inhibition of B16-BL6 melanoma cells with diluted partially puri®ed substances (Fig. 3). Dose-dependent effects were observed among one, ®ve and ten times dilution. The molecular weight of the active substance was greater than 100 kDa (Fig. 4), and it was sensitive to heat-treatment and trypsin-digestion (Figs. 5 and
the in vitro culture assay (Table 1). In these experiments, the direct inhibitory effects of the algal extract on the growth and invasion of the tumor cells were demonstrated. In the in vivo antimetastasis assay, the extract inhibited the metastasis of B16-BL6 cells to the lungs of B57BL/6J mice. The number of foci produced by B16-BL6 cells was reduced by the administration of the algal extracts (Table 2). Although the alga was reported to have immunostimulating activity ([H 3]thymidine incorporation in T cell blastogenesis in vitro) [7], an in®ltration or proliferation of immunologically associated cells, such as lymphocytes, was not observed around the foci of the cancer cells (Fig. 2) or in the tissues of the spleen and lymph nodes, except for proliferation of macrophages in the spleens of two mice and in®ltration of lymphocytes in the livers of two mice out of 12 experimental mice with B16-BL6 cells adminis-
Fig. 5. Heat sensitivity of the B16-BL6 growth inhibitory effects of partially puri®ed Marginisporum crassissimum extracts. Growth inhibition (%) growth inhibition after heat-treatment at each temperature/growth inhibition without heat-treatment £ 100.
150
S. Hiroishi et al. / Cancer Letters 167 (2001) 145±150
References
Fig. 6. Trypsin sensitivity of the B16-BL6 growth inhibitory effects of partially puri®ed Marginisporum crassissimum extracts. Growth inhibition (%) growth inhibition after trypsin-digestion/growth inhibition without trypsin-digestion £ 100 Extract, partially puri®ed M. crassissimum extracts; Trypsin, 0.05% trypsin solution; (1), added; (2), not added.
6), which suggested that it might be a protein. This protein-like substance possibly affects the cancer cell directly (or indirectly) and inhibits its growth and metastasis. Further puri®cation of the substance and in vivo studies will be done in next experiment to clarify the mechanism of the antitumor (antimetastatic) effects of this alga.
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