Comparative Studies on Antitumor Activity of Klebsiella O3 Lipopolysaccharide and Its Polysaccharide Fraction in Mice

Comparative

Studies

on Antitumor

Lipopolysaccharide Takaaki

and

HASEGAWA, Kenichi

Department *Department

of

of Klebsiella

Its Polysaccharide

Michio

OHTA,

MIYAMOTO* Bacteriology,

65 Tsurumai-cho, of Pharmacology Ho-3

Activity

and

Nagoya

University

School

Nagoya

, Hokuriku

University

Kanazawa April

15,

KIDO,

Nobuo

in Mice KATO,

KOSHIURA*

Showa-ku,

Kanagawa-machi, Accepted

Nobuo Ryozo

Fraction

03

466,

of Medicine, Japan

School

920-11,

of Pharmacy,

Japan

1985

Abstract-The antitumor activity of the polysaccharide fraction (OPS) obtained by the acid hydrolysis of Klebsiella O3 Lipopolysaccharide (KO3 LPS) isolated from the culture supernatant of the decapsulated mutant strain LEN-1 (03: K1-) against both allogeneic tumor and syngeneic tumor systems in mice was compared with that of KO3 LPS. OPS prolonged the life span of MM2-bearing C3H/He mice by intraperitoneal (i.p.) pre- and post-treatment at the doses of 100 and 1000 mg/kg. However, large amounts of OPS were needed to show the antitumor activity as compared with KO3 LPS. OPS showed no growth inhibitory activity against Meth-A sarcoma in BALB/c mice by i.p., intravenous (i.v.) or intratumoral (i.t.) administration. When 1000 mg/kg of OPS was i.p. administered once a day for 10 days, OPS significantly inhibited the tumor growth of Sarcoma-180 solid type tumor. On the other hand, KO3 LPS significantly suppressed the growth of Meth A tumor by Lt. administration at the doses of 0.3 and 1.0 mg/kg and showed com plete regression in 8 and 9 out of 10 mice, respectively. In MM2 tumor, KO3 LPS also showed complete regression in all mice post-treated by i.p. administration at the dose of 1 .0 mg/kg. These results suggest that OPS has antitumor activity on the tumors used in this study, but the activity was less than that of KO3 LPS. In our earlier studies, the Klebsiella 03 Lipopolysaccharide (K03 LPS) isolated from the culture supernatant of Klebsiella pneumoniae strain Kasuya (03 : K1) or its decapsulated mutant strain LEN-1 (03: K1-) has been shown to exhibit a much stronger adjuvant effect on antibody responses and delayed-type hypersensitivity to protein antigens in mice than other known adjuvants including LPSs from Escherichia co/i 055, 0111 and 0127 and Salmonella enteritidis (1-8). Further studies have indicated that K03 LPS shows antitumor activity on both allogeneic tumors and syngeneic tumors, and the antitumor activity is likely provided by host-mediated actions (9). It has been reported that LPSs from various gram-negative bacteria show anti tumor activity in animals, and the activity is

dependent on reactions of the host or the host-mediated immunostimulatory actions (10-12). On the other hand, polysaccharides isolated from diverse sources have been found to have antitumor activity against certain allogeneic tumors in mice (13-17). Their antitumor activities are assumed to depend on host-mediated actions (18, 19). In the present study, we compared the antitumor activity of the polysaccharide fraction (OPS) obtained by the acid hydrolysis of K03 LPS against allogeneic and syngeneic tumors in mice with that of K03 LPS. Materials and Methods Animals and tumor cells: Female 6-weeks old ddY, BALB/c and C3H/He mice were purchased from Shizuoka Laboratory Animal Center, Hamamatsu. Sarcoma-180 (S-180),

Meth-A fibrosarcoma and MM2 mammary carcinoma were maintained by weekly transplantation into the peritoneal cavity of ddY, BALB/c and C3H/He mice, respectively. Preparations: K03 LPS was prepared from the culture supernatant of decapsulated mutant strain LEN-1 (03: K1-) derived from Klebsiella pneumoniae strain Kasuya (03: K1) as described previously (20). OPS was obtained from K03 LPS by the hydrolysis with 1% acetic acid at 100°C for 1 hr as described previously (21). Evaluation of antitumor activity: To evaluate the antitumor activities against solid type tumor, mice were subcutaneously (s.c.) inoculated at the left inguinal region with tumor cells (3 x 106 S-180 cells/ddY mouse, 2 x 105 Meth-A cells/BALB/c mouse) on day 0 according to the treatment schedules indicated in the tables. Thirty-five days after the inoculation of S-180 cells and 24 days after the inoculation of Meth-A cells, the tumors were removed and weighed. For MM2 ascites type tumor, mice were intra peritoneally (i.p.) inoculated with 5x`105 cells on day 0 according to the treatment schedules indicated in the tables. Sixty days after the cell inoculation, survivors were

Table

1.

Antitumor

activity

of

OPS

and

killed and autopsied. Antitumor activities were evaluated by comparison of the treated group (T) with the control group (C), i.e. the percentage inhibition of tumor growth, [(C-T)/C] x 100 (%), and life span, [(T-C)/ C]x100 (% ILS). Single dosage was determined from the LD50 values in SMA mice supplied by the Inbred Animal Breeding Laboratory, Nagoya University School of Medicine. The LD50 value of OPS by the i.p. route was greater than 3 g/kg, and that of K03 LPS by the i.p. and i.v. route was 15.2 mg/kg and 7.9 mg/ kg, respectively. The results in the tables are expressed as the mean±standard error, and the statistical significance of differences between the groups was assessed by Student's t-test, with P<0.05 taken as the minimum level of significance. Results Antitumor activities of OPS and K03 LPS against S-180 solid type tumor: Mice were inoculated s.c. with 3x`106 cells on day 0 and were given i.p. or per os (p.o.) graded doses of OPS and K03 LPS once a day for 10 days from day 10 to day 20 after the cell inoculation

K03

LPS

against

S-180

solid

type

tumor

(Table 1). When the agents were administered i.p., 1000 mg/kg of OPS and 1 mg/kg of K03 LPS significantly inhibited the tumor growth. Administration by the p.o. route of K03 LPS also showed an inhibitory effect on the tumor growth, but about a thousand-fold dose of this agent by the p.o. route was necessary to obtain an effect similar in degree to that by the i.p. route. OPS did not show any significant effect by the p.o. route. Antitumor activities of OPS and K03 LPS against Meth-A solid type tumor: Mice were s.c. inoculated with 2 X 105 cells on day 0 and were given graded doses of OPS or K03 LPS once a day according to the indicated schedules and routes (Table 2). OPS sup pressed the tumor growth by i.v. or intra tumoral (i.t.) administration, but the effect of OPS was weaker than that of K03 LPS. K03 LPS significantly inhibited the tumor growth at doses of 0.1 to 1 .0 mg/kg by i.v. or i.t. administration. Antitumor activities of OPS and K03 LPS against MM2 ascites type tumor: C3H/He Table

2.

Antitumor

activity

mice were i.p. inoculated with 5 X105 cells on day 0 and were i.p. given graded doses of OPS or K03 LPS once a day on day -4 as the preadministration or on days +1 to +10 as the postadministration (Table 3). These agents markedly prolonged the life span of the mice bearing MM2 tumor by both pre and post-administrations. However, large amounts of OPS were needed to show the antitumor activity compared with that of K03 LPS. Discussion By hydrolysis in 1 % acetic acid at 100'C for 1 hr, K03 LPS is dissociated into 59-66% OPS and 25-27% lipid A moiety (22). Chemical properties of K03 LPS and the chemical structure of OPS have been investigated (21, 22). OPS consists of the mannan which has a-mannosyl-(1-3)-a mannosyl-(1-2)-a-mannosyl-(1-2)-a mannosyl-(1-2)-a-mannose units joined through a-mannosyl-(1-3)-linkages (22). The lipid A moiety of K03 LPS retains lethal

of OPS and K03 LPS against

Meth-A

solid type tumor

Table

3.

Antitumor

activity

of

OPS

and

toxicity for mice, whereas OPS is essentially non-toxic (23). Our recent studies have revealed that the polysaccharide moiety plays an important role as well as the lipid A moiety in the extraordinarily strong adjuvant activity of K03 LPS in augmenting antibody responses and delayed-type hypersensitivity to protein antigens in mice (7, 8, 24). We also found that the adjuvant activity of OPS was weak and about a fifty-fold greater amount of OPS was necessary to obtain activity similar in degree to that obtained by the lipid A moiety, the activity of which was also much weaker than that of K03 LPS (23). Recently, it has been reported that poly saccharides isolated from diverse sources have considerable antitumor activity against certain allogeneic tumors, particularly S-180 implanted in mice (13-17). Moreover, a protein-bound glucan has also been reported to show strong antitumor activity against S 180 and is used clinically as an antitumor drug (13). The glucans which were reported to have antitumor activity consist mainly of 1, 3 linkages, and their antitumor action may be host-mediated, although their exact mechanisms are still unknown. In the present study, we investigated whether OPS has antitumor activity against mice bearing syngeneic ascites tumor (MM2

K03

LPS

against

MM2

ascites

type

tumor

mammary carcinoma), syngeneic solid type tumor (Meth-A fibrosarcoma), or allogeneic solid type tumor (S-180). OPS could elicit a marked increase in the life span of mice bearing MM2 ascites tumor by i.p. adminis tration. However, OPS showed no growth inhibitory activity against Meth-A tumor by i.p., i.v. or i.t. administration. In the case of allogeneic tumor, large amounts of OPS showed growth-inhibitory activity by i.p. administration, but did not show any sig nificant activity by p.o. administration. On the other hand, it has been reported that mannans obtained from fungi exhibited significant antitumor activity against S-180 solid type tumor at low doses by i.p. adminis tration (25). The results of this study suggest that the differences of antitumor activity in the mannan and OPS seem to be based on the differences in their molecular structures. Moreover, in their screening test, treatment was started at 1 day after s.c. transplantation of the tumor cells. On the other hand, it was shown that K03 LPS exhibits a strong antitumor activity against both allogeneic and syngeneic tumors in mice. In particular, i.t. administration of K03 LPS at doses of 0.3 and 1.0 mg/kg significantly suppressed the growth of Meth A solid type tumor and showed complete

regression in 8 and 9 out of 10 mice, and in the ascites type tumor, K03 LPS also showed complete regression in all mice posttreated by i.p. administration at a dose of 1.0 mg/kg. Therefore, all the results in this study showed that the antitumor activity of OPS against allogeneic and syngeneic tumors is much weaker than that of K03 LPS.

References 1 Nakashima, I., Kobayashi, T. and Kato, N.: Alterations in the antibody response to bovine serum albumin by capsular polysaccharide of Klebsiella pneumoniae. J. Immunol. 107, 11121121 (1971) 2 Nakashima, I.: Adjuvant action of capsular polysaccharide of Klebsiella pneumoniae on antibody response. I. Intensity of its action. J. Immunol. 108, 1009-1016 (1972) 3 Nakashima, I. and Kato, N.: Nonspecific stimu lation of immunoglobulin synthesis in mice by capsular polysaccharide of Klebsiellapneumoniae. Immunology 27, 179-193 (1974) 4 Nakashima, I., Ohta, F., Kobayashi, T., Kato, O. and Kato, N.: Effect of antigen doses and time intervals between antigen injections on secondary, tertiary and quaternary antibody responses. Establishment of hyperimmunization with bovine serum albumin in mice treated with capsular polysaccharide of Klebsiellapneumoniae. Immunology 26, 443-454 (1974) 5 Nakashima, I., Nagase, F., Yokochi, T., Ohta, M. and Kato, N.: Adjuvant actions of polyclonal lymphocyte activators. I. Comparison and char acterization of their actions in antibody response to deaggregated bovine serum albumin. Cell. Immunol. 46, 69-76 (1979) 6 Nakashima, I., Nagase, F., Matsuura, A. and Kato, N.: Adjuvant actions of polyclonal lym phocyte activators. II. Comparison and character ization of their actions in initiation and poten tiation of immune responses to T-dependent and T-independent soluble antigens. Cell. Immunol. 49, 360-371 (1980)

7 Ohta, M., Nakashima, I. and Kato, N.: Adjuvant action of bacterial lipopolysaccharide in induction of delayed-type hypersensitivity to protein antigens. I. Action of the O3 antigen of Klebsiella from culture fluid. Cell. Immunol. 66, 111-120 (1982) 8 Ohta, M., Nakashima, I. and Kato, N.: Adjuvant action of bacterial lipopolysaccharide in in duction of delayed-type hypersensitivity to protein antigens. II. Relationships of intensity of the action to that of other immunological activities. Immunobiology 163, 460-469 (1982) 9 Miyamoto, K., Koshiura, R., Hasegawa, T. and Kato, N.: Antitumor activity of Klebsiella O3

19 Nakahara, W., Fukuoka, F., Tokuzen, R. and Aoki, T.: A transplantable mouse sarcoma associated with the Friend virus. Gann 56, 309 311 (1965). 20 Ohta, M., Mori, M., Hasegawa, T., Nagase, F., Nakashima, I., Naito, S. and Kato, N.: Further studies of the polysaccharide of Klebsiella pneumoniae possessing strong adjuvanticity. I. Production of the adjuvant polysaccharide by noncapsulated mutant. Microbiol. Immunol. 25, 939-948 (1981) 21 Hasegawa, T., Ohta, M., Mori, M., Nakashima, I. and Kato, N.: The Klebsiella O3 lipopolysac charide isolated from culture fluid: Structure of the polysaccharide moiety. Microbiol. Immunol. 27, 683-694 (1983) 22 Hasegawa, T., Ohta, M., Nakashima, I., Kato, N., Morikawa, K., Harada, T. and Okuyama, T.: Structure of the polysaccharide moiety of the Klebsiella O3 lipopolysaccharide isolated from culture supernatant of decapsulated mutant

(Klebsiella O3: K1-). Chem. Pharm. Bull. (Tokyo) 33, 333-339 (1985) 23 Kato, N., Kido, N., Ohta, M. and Naito, S.: Comparative studies on adjuvanticity of Klebsiella O3 lipopolysaccharide and its lipid A and polysaccharide fractions. Immunology (1985) 54, 317-324 (1985) 24 Kido, N., Ohta, M. and Kato, N.: Inhibition by succinyl concanavalin A of strong adjuvant activity of Iipopo!ysaccharides which possess mannans as the O-specific polysaccharide chains. Cell. Immunol. 92 (1985) (in press) 25 Ukai, S., Kiho, T., Hara, C., Morita, M., Goto, A., Imaizumi, N. and Hasegawa, Y.: Polysaccharides in fungi. XIII. Antitumor activity of various polysaccharides isolated from Dictyophora indusiata, Ganoderma japonicum, Cordyceps cicadae, Auricularia auricula-judae, and Auricularia Species. Chem. Pharm. Bull. (Tokyo) 31, 741-744 (1983)