anti-tumor activity of squid ink
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Anti-Tumor Activity of Squid InkTRANSCRIPT
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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
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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.
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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.
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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
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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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