An acidic polysaccharide Chlon A, from Chlorella
Pyrenoidosa

Kanki Komiyama, Yumiko Hirokawa, Takashi Morota and lwao Umezawa
The Kitasato Institute

An acidic polysaccharide, chlon A, from Chlorella PYrenOid0sa

2. Antitumor activity and immunological response
Chlon A, an acidic polysaccharide purified from the hot water extract of Chlorella pyrenoidosa, possessed antitumor activity against transplantable murine tumors in vivo. In allogeneic systems, Chlon A showed remarkable life prolongation effects in mice bearing Sarcoma 180 with a broad optimal dose range. Chlon A was also active in syngeneic systems such as IMC carcinoma, Meth A fibrosarcoma, B16 melanoma and Lewis lung carcinoma growing in the intraperitoneal cavity. When Meth A cells were admixed with Chlon A and inoculated sc, remarkable growth inhibition of tumor cells was observed. Chlon A enhanced cytotocicity of mice macrophages for EL-4 tumor cells in vivo, lymphoproliferative effects in vitro, and carbon clearance activity (reticuloendothelial system) in vivo. The electrophoretic pattern showed that LA (ceruloplasmin) and/or LB (hemopexin)like components rapidly appeared in serum proteins of mice receiving Chlon A. These findings indicate that Chlon A is a potent modifier of some biological responses.

Table 2. Antitumor activity of Chlon A on Sarcoma 180 and Meth-A fibrosarcoma.
Mice were given with Chlon A from days 1 to 5 and 7 to 1 1.

a) Numbers in parentheses indicate the number of cured mice/seven treated mice. Cured mice were excluded from the calculation of I LS (%).
b) Zymosan was purchased from SIGMA Chemical Company (ST. Louis, USA).

Table 3. Antitumor activity of Chlon A on IMC carcinoma, P388 leukemia Lewis lung carcinoma and B-16 melanoma.
Mice were given with Chlon A from days 1 to 5 and 7 to 1 1.

Table 4. In vivo growth of Meth A cells mixed with Chlon A.
Meth A cells suspended in minimum essential medium were admixed with Chlon A in vitro, and incubated for 30 minutes at 37^oC. After incubation, tumor cells were inoculated subcutaneously into BALB/c mice, and tumor growth was measured periodically by calipers.

Table 5. Lymphoproliferative effect of Chlon A.
Spleen cells from normal mice were mixed with Chlon A, incubated for 72 hours at 37^oC, and the uptake of ³ H-TdR into spleen cells was measured.

Table 6. Carbon clearance in mice treated with Chlon A.
Normal ddY mice received Chlon A (once a day for 2 days, iv). On day 3, mice were injected iv with India ink (Pelican Fount India) and blood was withdrawn periodically from the retro-orbital plexus. Phagocytic activity was measured by the method of Halpern³).

Fig. 1. Antitumor activity of Chlon A on Sarcoma 180 solid tumors.
Mice were given Chlon A iv on days 1, 3, 5, 7 and 9.

Fig. 2. Effect of Chlon A on the cytolytic activity of peritoneal exudate cells.
Adherent peritoneal exudate cells (PEC) of ddY mice were collected on day 1 or 4 after injection of Chlon A and were mixed with EL-4 cells previously labeled with ³ H-uridine. After incubation for 16 hours, the radioactivity of E L-4 cells was measured. (1-T/C) x 100 (%). The mean radio-activity of the control group was 5,931 cpm.

Fig. 3. Effect of Chlon A on the cytostatic activity of peritoneal exdate cells.
Adhearent peritoneal exudate cells (PEC) of ddY mice were collected on day 1 or 4 after injection of Chlon A. Then ³ H-TdR (1 ug/ml) was added to the cell mixture, and incubation was further continued for 5 hours. The uptake of ³ H-TdR into EL-4 cells was measured. (1 -T/C) x 1 00 (%). The mean radioactivity of the control group was 6,332 cpm.

Fig. 4. Effect of Chlon A on the electrophoretic pattern of mouse serum.
Chlon A was injected iv into normal ddY mice on day 0, and sera were obtained on days 3, 5, 7, and 9. Sera were subjected to polyacrylamide gel electrophoresis on gradient gel. After electrophoresis, the gel was stained with 15 Amidoblack 10B solution.

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