J Nutr Sci Vitaminol, 1997, 43, 455-461

Anti-Tumor Activity of Squid Ink

Jin-ichi SASAKI,1 Kunio ISHITA,1 Yoshiaki TAKAYA,2 Hidemitsu UCHISAWA2 and Hajime MATSUE2

1 Department of Bacteriology, Hirosaki University School of Medicine, Hirosaki 036, Japan

2Aomori Advanced Industrial Technology Center, Aomori 030-01, Japan

(Received December 19, 1996)

Summary The anti-tumor activity of a new type of peptidoglycan

isolated from squid ink was shown to have a cure rate of 64% for Meth A

tumor from BALB/c mice. The ink delipidated in acetone, which con

tained the peptidoglycan at 0.1% (w/w), was administered to tumor

transplanted mice so as to examine the anti-tumor activity. One-fifth of

the tumor-bearing mice was cured with 3 injections (1mg/head) of the

acetone delipidated squid ink or a prolongation of survival was observed

in the treated animals. Heat treatment at 100•Ž for 10 min did not affect

the anti-tumor activity of the delipidated ink, its potentiality being pre

served. The acetone-extractable fraction of the ink also brought about

a similar cure rate for Meth A tumor. The delipidated ink enhanced

the phagocytic activity of macrophages but no direct cytotoxicity was

observed for the Meth A tumor cells. Hence it may be said that the

anti-tumor activity of the delipidated ink was mainly due to the aug

mented cellular immunity in vivo.

Key Words squid ink, delipidated fraction, anti-tumor activity, macro

phage activation, lipid fraction

Squid ink has been traditionally used from the past to preserve raw squid in Japan. Japanese have already noticed, through experience, the anti-bacterial

potency of the ink and have used it as an additive to foodstuffs. However, most squid ink in the fish processing industry is discarded at present. It would be beneficial if we could find recycling methods for the ink.

On the other hand, extensive work has been conducted to find novel anti-tumor compounds from natural resources (1-6). Recently, we have succeeded in the isolation of a new type of peptidoglycan from squid ink and demonstrated its anti-tumor activity with a 60-70% cure rate against Meth A fibrosarcoma in BALB/c mice (7).

However, it remains questionable whether or not squid ink as a whole elicits anti-tumor activity, which has never been investigated. If anti-tumor or another

455

456 J SASAKI et al

biological activity can be demonstrated by intact ink, the ink that is discarded

becomes a new material for re-usage as an additive in the food industry.

In this paper, we describe anti-tumor activities of the delipidated squid ink as

well as its lipid fraction.

MATERIALS AND METHODS

Meth A fibrosarcoma was intraperitoneally (IP) maintained in BALB/c mice

at two-week intervals and provided for experiments.

The ink was separated at 4•Ž from frozen squid (Illex argentinus) transported

from Argentina, homogenized with 20 volumes of absolute acetone at -30•Ž and

stirred for about 24h at the same temperature before rapid filtration. The acetone

powder containing peptidoglycan and the filtrate were further evaporated to

dryness under reduced pressure. The yields of delipidated ink and acetone-soluble

material (lipids) were 20 and 3%, respectively, of the initial wet ink. The acetone

powder of the delipidated ink was dissolved in physiological saline and examined

for anti-tumor activity with the Meth A tumor model of BALB/c mice in a manner

previously described (7). In brief, tumor cells obtained from mice were rinsed in

Hanks balanced salt solution (HBSS, pH 7.2) and centrifuged at 1,500 rpm for 10

min, and the cell number was adjusted to 2•~106/mL in HBSS. After IP was

transplanted into the tumor cells (2•~106), mice (n=10) were IP treated with each

mL saline solution containing 200 dig or 1mg of the delipidated ink on days 2, 4 and

6. Control mice (n=10) were IP treated with non-containing saline. Mice were

observed until the death of the tumor. Tumor-free mice were fed for at least 2

months to confirm no recurrence.

The delipidated ink held in saline solution was heated at 100•Ž for 10 min to

examine its heat stability. The anti-tumor activity of the heat-treated delipidated

ink was measured by treatment of mice on days 2, 4 and 6 (n=5 each) after tumor

transplantation (2•~106).

A 1mg/mL solution of lipids in acetone was poured into a test tube and acetone

was evacuated under a reduced pressure condition to make a thin lipid film inside

the test tube. Then lipid film was thoroughly homogenized with 1,000 volumes of

saline to prepare liposomes. Five mice were treated with aliquots of the homo

genate (1mL/head) on days 2, 4 and 6 after tumor (2•~106 cells) transplantation.

Control mice (n=5) were similarly treated with saline only.

The macrophage activating property of the delipidated ink was tested as

follows. Each 1mg/mL solution of delipidated ink in saline was IP injected into 4

mice and peritoneal exudated cells (PEC) were collected 4 days later. The PEC

were incubated in 10% FBS-containing RPMI 1640 (Gibco, USA) for 2h at 37•Ž

under a 5% C02 air atmosphere, and non-adherent cells were washed out in

medium for separation of macrophages. The phagocytic activity of macrophages

was examined using yeast particles (8). Saline-induced macrophages were used as

a control to compare phagocytosis with the delipidated ink-induced macrophages.

J Nutr Sci Vitaminol

Anti-Tumor Activity of Squid Ink 457

A cytotoxicity test of the ink was performed against Meth A tumor cells

in vitro. Meth A tumor cells (1•~107/mL) were mixed with the ink in 10%

FBS containing RPMI 1640 medium (1mg/mL) and incubated at 37•Ž under a 5%

CO2- air atmosphere. The viability of tumor cells was counted, as usual, by the

trypan blue exclusion method.

Statistical significance between the test and control groups was evaluated by

Student's t-test.

RESULTS

The chemical analyses of a major peptidoglycan isolated from the delipidated

ink are quoted here from our previous data (7). They revealed the composition

of 7.8% peptide, 57% polysaccharide and 30% pigment (melanin). The poly

saccharide had a unique structure with equimolar ratios of glucuronic acid,

N-acetylgalactosamine and fucose. Neither mannuronic acid nor iduronic acid

was detected. From those analyses, the peptidoglycan was proposed to contain

unique polysaccharide chains consisting of three kinds of sugar bound to the

peptide-chain. The anti-tumor activity of the peptidoglycan against mouse tumor is

shown in Table 1.

The anti-tumor activity of the delipidated ink was first tested with the same

therapeutic dosage of the peptidoglycan (200ƒÊg/mL/head, three shots). However,

there was no apparent therapeutic efficiency obtained with this dosage (data not

shown). A dosage increase from 200ƒÊg to 1mg/mL/head of the delipidated ink

reduced tumor growth by 20% and the survival rate was greatly prolonged (Fig. 1).

The lack of cytotoxicity of the delipidated ink against the tumor cells is shown

in Table 2. It thus seems likely that the anti-tumor activity of the delipidated ink

was not due to direct cytotoxicity but to the enhancement of cellular immunity.

The delipidated ink was heated at 100•Ž for 10 min to examine its heat

stability from an anti-tumorigenic point of view. The results are illustrated in

Fig. 2. The anti-tumor activity of the delipidated ink was preserved despite heat

Table 1. Anti-tumor activity of peptidoglycan fractionated from squid ink.

This table was quoted from our previous report (7) to compare with results of the

present experiment. Meth A tumor cells (2•~106) were intraperitoneally (IP)

transplanted into mice. Mice were IP treated with 200ƒÊg/mL/head peptidoglycan,

three times on days 2, 4 and 6 after tumor transplantation. Molecular weight of Fr.

A: 100,000, Fr. B: 80,000, Fr. C: 40,000.

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458 J SAAI et al

Fig. 1. Anti4umor activity of delipidated squid ink. Meth A tumor cells (2•~106)

were intraperitoneally (IP) transplanted into mice. Mice were I treated with

1m/mL/head ink, three times on days 2, 4 and 6 after tumor transplantation.

Table 2. Cytotoxicity test of delipidated squid ink against Meth A tumor cells.

Meth A tumor cells (1•~107) were incubated with delipidated squid ink (1mg/mL) in

10% FBS RPMI 1640 medium at 37•Ž under 5% CO2-air. Cytokilling activity was

not observed in the delipidated ink.

treatment, and one of five mice was cured o tumor. Another animal had a

prolonged survival rate of 4 weeks.

The nti4uor activity of the lipid fraction from the ink is shown in Table 3.

Three injections of the fraction (1mg/head) cured 3 of 15 tumor®bearin mice,

which survived over 2 months without recurrence.

The haocytic activity of intraperitoneal macrophages was enhanced by

treatment with the delipidated ink. Such photographic examples are shown in

Fig. 3 Deli idated ink4nduced macrophages spread well as compared with those

of the control. The number of yeasts ingested by a macrophage was 8.2•}1.3 and

1.0•}007 per cell for the delipidated ink® and saline-induced groups, respectively;

the difference between both groups was significant at p<0.005.

J Nutr Sci Vitaminol

Anti-Tumor Activity of Squid Ink 459

Fig. 2. Anti-tumor activity of delipidated squid ink after heat treatment at 100•Ž

for 10 min, Meth A tumor cells (2•~106) were intraperitoneally (IP) trans

planted into mice. Mice were IP treated with 1m/mL/head of heat4reated

delipidated ink, three times on days 2, 4 and 6 after tumor transplantation.

Table 3. Anti4umor activity of lipid isolated from squid ink.

Meth A tumor cells (2•~106) were intraperitoneally (IP) transplanted into mice .

Mice were IP treated with 1mg/mL/head lipid, three times on days 2, 4 and 6 after

tumor transplantation.

* Survival day was described for nonmcured mice.

DISCUSSION

Extensive efforts have been made for the eradication of cancer from various

aspects using surgical, radio-, chemo- and iuno-therapeutical methods. The first

two are the main strategies of therapies and the latter two are considered to e

supplemental ones followed by surgical removal of cancer foci.

With respect to immuno®therapy for cancer, it is indispensable to employ

potential anti4umor agents without any serious sidepefects, because such immuno

potentiatin agents in therapy are usually used long®term after surgical operation, aiming at preventing the regro the of remaining or metastatic cancer cells.

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460 J SASAKI et al

Fig. 3. Augmentation of phagocytic activity of macrophages by delipidated squid ink. A: Saline-induced macrophages. B: Delipidated ink-induced macrophages.

Increasing phagocytic activity was observed in the ink-induced macrophages

(B).

We have been trying to find anti-tumor compounds in natural resources, and

have recently separated a novel type of peptidoglycan from squid ink which

demonstrates potential anti-tumor activity in mouse tumor models (7). The cure

rate for Meth A tumor was about 60% without showing any side-effects. However,

the content of peptidoglycan is less than 0.1% in the ink and it is not easy to

separate it. For this reason, our concern was directed toward anti-tumor activity of

intact squid ink.

Both the delipidated ink and the lipid fraction of the ink showed anti-tumor

activity at a dosage of 1mg/head (three times at intervals of two days) against the

Meth A tumor. Their cure rates were 20% each, and their survival rates were much

longer when compared to the control, as shown in Fig. 1. The mice cured of tumor

survived over 2 months without regrowth of the tumor. Anti-tumor activity of the

delipidated ink was stable against heat treatment at 100•Ž for 10 min.

Since the delipidated ink-induced macrophages exhibited increasing phago

cytic activity, we speculated that the anti-tumor action of the delipidated ink might

be mediated by the enhancement of cellular immunity in vivo. A similar explanation

may hold true for the effect of the lipid fraction. In general, phospholipids have the

potency to activate macrophages (8) and thereby may participate in tumor eradica

J Nutr Sci Vitaminol

Anti-Tumor Activity of Squid Ink 461

tion. Taking into consideration other reports on biological activities of the ink , such as regulation of gastric juice secretion (9) or anti-ulceration activity (10), it is very advantageous to utilize squid ink as a foodstuff or an additive for health improvement. Incidentally, no toxicity of the squid ink is supported by its use as a traditional food additive in Japan for over 100 years.

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