Antitumor Activity of Klebsiella O3 Lipopolysaccharide in Mice

Antitumor

Activity Kenichi

of Klebsiella

M IYAMOTO,

Ryozo and

Department

Kanagawa-machi, of Bacteriology

65

Tsurumai-cho,

*Department

Lipopolysaccharide

KOSH I U RA, Takaaki Nobuo

of Pharmacology, Ho-3

03

Hokuriku

University

School

920-11,

, Nagoya

University

Showa-ku,

Nagoya

June

HASEGAWA*

KATO*

Kanazawa

Accepted

in Mice

School 466,

of Pharmacy,

Japan of Medicine,

Japan

5, 1984

Abstract-The antitumor activity of Klebsiella 03 lipopolysaccharide (K03 LPS) isolated from the culture supernatant against S180 sarcoma, Ehrlich carcinoma, MM2 mammary carcinoma and Meth A fibrosarcoma in mice was investigated. K03 LPS significantly prolonged the lifespan of S180-bearing ddY mice and MM2 bearing C3H/He mice by intraperitoneal pre or postmedication at doses ranging from 0.1 to 1.0 mg/kg. The LPS also inhibited the growth of subcutaneously inoculated Ehrlich carcinoma in ddY mice and Meth A sarcoma in BALB/c mice by intraperitoneal, intravenous or intratumoral administration. The intratumoral injection of K03 LPS was most effective and results by the intravenous and the intraperitoneal administrations followed in effectiveness, but the administration through the subcutaneous route was hardly effective. Thus, K03 LPS was shown to have antitumor activity on both allogeneic tumors and syngeneic tumors. It was also indicated in this study that the lifeprolonging effect of K03 LPS on S180 ascites type tumor-bearing mice was significantly minimized by pretreatment of cyclophosphamide and that the LPS did not influence the cell viability of HeLa cells, Ehrlich cells and MM2 cells in vitro. These results suggest that the antitumor activity of K03 LPS is provided by host-mediated actions. It has been indicated that lipopolysac charides (LPSs) from various gram-negative bacteria show antitumor activity in animals, and the activity is attributable to their host mediated immunostimularoty action (1-3). Our previous studies have shown that Klebsiella 03 LPS (K03 LPS) isolated from the culture supernatant of Klebsiella pneumoniae strain Kasuya (03:K1) or its decapsulated mutant strain LEN-1 (03:K1-) exhibits a much stronger adjuvant effect on both antibody response and delayed type hypersensitivity to protein antigens in mice than other known adjuvants, including the LPSs of Escherichia coli and Salmonella (4-7). In the present study, we investigated the antitumor activity of K03 LPS against al logeneic and syngeneic tumors in mice and discussed the relationship between the anti

tumor LPS.

activity

and

other

activities

of

K03

Materials and Methods Animals and tumors: Female 6-weeks-old ddY, C3H/He and BALB/c mice were purchased from Shizuoka Agricultural Co operative for Laboratory Animals, Hama matsu. Ehrlich carcinoma and S180 were maintained in the peritoneal cavity of ddY mice by weekly transplantation. MM2 mammary carcinoma and Meth A fibrosar coma were maintained by intraperitoneal passage at weekly intervals in C3H/He mice and BALB/c mice, respectively. HeLa S3 cells were maintained in Engle's minimum essential medium supplemented with 5% calf serum in gum-stoppered square bottles at 37 °C. Agents: K03-LPS was prepared from the

Fig.

Table 1.

1.

Chemical

structure

Chemical composition

culture supernatant of decapsulated mutant strain LEN-1 (03:K1-) derived from K. pneumoniae strain Kasuya as described previously (8). Its chemical composition and structure are in Table 1 and Fig. 1, respec tively (9). Other agents used were LPS from Escherichia coli 055:B5 (E055 LPS, Difco) and cyclophosphamide (CPA, Shionogi). Antitumor activities in mice: To determine the antitumor activities against ascites type tumors, mice were inoculated with tumor cells (allogeneic tumor system: 1 X106 S180 cells/ddY mouse, syngeneic tumor system: 5 X105 MM2 cells/C3H/He mouse) on day 0 and were medicated intraperitoneally (i.p.) according to the treatment schedule indicated in the tables. Sixty days after the cell inocu lation, survivors were killed and autopsied. For solid type tumors, mice were inoculated subcutaneously (s.c.) at the left inguinal region with tumor cells (allogeneic tumor system: 5x106 Ehrlich cells/ddY mouse, syngeneic tumor system: 2x105 Meth A cells/BALB/c mouse) on day 0 and were medicated i.p., intravenously (i.v.) at the tail, s.c. at the right inguinal region or intra tumorally (i.t.) according to the treatment schedule indicated in the tables. Thirty-eight days after inoculation of Ehrlich cells or twenty-four days after inoculation of Meth A cells, the tumors were removed and weighed. Single dosage was determined from the LD50 values (15.2 mg/kg i.p., 7.9 mg/kg i.v.) in SMA mice which are a sensitive strain to the LPS.

of

K03

LPS

(%) of K03 LPS

Assay of direct cytotoxic activity against tumor cells: HeLa cells, Ehrlich cells and MM2 cells were used in this study. The activity of K03 LPS against HeLa cells was evaluated using the colony-forming method. The cells (1 X104/2 ml) plated on 35 mm plastic petri dishes were incubated with graded concen trations of the agent at 37°C in a humidified 5% C02 incubator for 48 hr. The treated cells were washed with Hanks' solution and were further incubated for 48 hr. The colonies formed with 8-10 cells were fixed, stained with Crystal Violet, and then counted micro scopically. For Ehrlich cells and MM2 cells, a 2 ml aliquot of tumor cells (5x105/ml) prepared in RPMI 1640 medium supple mented with 10% fetal calf serum and 10 etM mercaptoethanol was placed in a 35 mm plastic dish and was added with graded concentrations of K03 LPS. The mixture was incubated at 37'C in a C02 incubator for 48 hr. The viable cells were assayed by a dye exclusion method with 0.2% Trypan Blue. Results Antitumor activity of K03 LPS against S180 ascites type tumor: Mice were inocu lated i.p. with 1 X106 cells on day 0 and received intraperitoneal medication with several treatment schedules (Table 2). K03 LPS significantly prolonged the lifespan of the tumorbearing mice by either pre or postmedication. Even when the LPS was given by a single intraperitoneal injection 7 days before tumor inoculation, a marked suppression of tumor growth was observed.

The effect of CPA on the antitumor activity of K03 LPS was also examined. CPA (40 mg/ kg) was injected i.p. once a day from day 6 to day 2 before the tumor inoculation. The lifeprolonging effect of K03 LPS in both medication schedules was cancelled or minimized by the pretreatment with CPA, while CPA alone hardly influenced the survival time of the mice bearing S180 ascites tumor (Table 2). Table activity

2.

Antitumor

activity

of K03

LPS against

Antitumor activity of K03 LPS against MM2 ascites type tumor: C3H/He mice were inoculated i.p. with 5X105 cells on day 0, and K03 LPS was administered i.p. once a day on days -7 to -2 as the premedication or on days +1 to +10 as the postmedication. The agent showed a growth-inhibitory activity against MM2 ascites type tumor (Table 3). The postmedication was more effective than the premedication, and all mice treated with S180

ascites

type

tumor

and

effect

of K03 LPS

Table

3.

Antitumor

activity

of

K03

LPS

against

MM2

ascites

type

tumor

of CPA on the

1.0 mg/kg of K03 LPS on days +1 to +10 survived for 60 days after the tumor in oculation. Antitumor activity of K03 LPS against Ehrlich solid type tumor: Mice were inocu lated s.c. with 5 x 106 cells and were given K03 LPS in the peritoneal cavity once a day for 10 days from 10 days after the cell inoculation or were given the agent i.v. by a single injection on day +10. As shown in Table 4, the tumor growth in mice given K03 LPS i.p. or i.v. was significantly suppressed compared with that of the non-treated control. Particularly, three-tenths of the mice were cured by the treatment of 1.0 mg/kg of K03 LPS through each administration route, and the tumor growth in other mice was inhibited below 30% of the control. Antitumor activity of K03 LPS against Meth A solid type tumor: Mice were inocu lated s.c. with 2 x 105 cells on day 0 and were given graded doses of K03 LPS through various routes according to the indicated schedules. The antitumor activity of K03 LPS against Meth A solid type tumor was compared with that of E055 LPS (Table 5). K03 LPS showed growth-inhibitory activity against the tumor to almost the same extent as that of E055 LPS. When K03 LPS was administered i.v. or i.t., the tumor growth was inhibited significantly, and the intratumoral administration of K03 LPS was especially effective, causing almost complete tumor regression in mice, even if given a single administration of 10 mg/kg on day +10. When administered i.p. or s.c., the tumor growth was mildly suppressed, but the Table

4.

Antitumor

activity

of

K03

tumor weight was not significantly different from that of the control. However, in every case of administration, the tumors weighing below about 30% of the control weight became necrotic with hemorrhage. Direct cytotoxic activity of K03 LPS against tumor cells in vitro: Table 6 shows the results of experiments for the direct cytotoxic activity of K03 LPS against three target cell lines in the culture system. K03 LPS concen trations ranging from 10 to 100 ,cg/ml did not influence the colony-forming ability of HeLa cells treated with the agent for 48 hr. Moreover, significant decreases in the surviving fraction of Ehrlich cells and MM2 cells were not observed upon addition of 100 tag/ml of K03 LPS to the culture medium. These results indicate that K03 LPS has no direct cytotoxic activity against tumor cells i n vitro. Discussion Many investigators showed the antitumor activity of LPSs from several microorganisms such as Serratia marcescens (1), E. coli (2), Salmonella entiritidis (10) and Proteus vulgaris (3). However, in general, LPSs do not show antitumor activity against the ascites type tumors and by treatment before tumor inoculation. In this paper, we show that the LPS from Klebsiella pneumoniae (K03 LPS) has an antitumor activity against both allogeneic and syngeneic ascites type tumors and solid type tumors. K03 LPS significantly prolonged the lifespan of mice bearing ascites tumors such as S180 and MM2, even if it was given by intraperitoneal LPS

against

Ehrlich

solid

type

tumor

ment of munogenic Moreover,

premedication. It was also indicated in this study that the lifeprolonging effect of K03 LPS was significantly minimized by pretreat

Table

5.

Antitumor

Table

6.

activity

Direct

of K03

cytotoxic

LPS

activity

and

E055

of K03

LPS

LPS

CPA, which decreases the im potency of the host animals. the viability of tumor cells was not against

against

Meth

tumor

A solid

cells

type

in vitro

tumor

influenced by K03 LPS in vitro. Consequently, it is suggested that the antitumor activity of K03 LPS is provided by host-mediated actions. Earlier, it was thought that the tumor regressing effect of LPSs depends on the intensity of the stimulation of the reticulo endothelial system, which provokes sub sequent antibody formation (3). LPSs generally consist of an 0-specific polysac charide moiety, a core moiety and a lipid A moiety. Our previous studies have revealed that LPSs including K03 LPS whose 0 specific polysaccharides consist of mannans showed a much stronger adjuvant activity than other LPSs such as E055 LPS and LPSs from Salmonella which don't possess mannans in their polysaccharide moiety (4-7). However, in this study, the antitumor activity of K03 LPS against Meth A fibrosar coma was indicated to be similar to that of E055 LPS by each administration route. Therefore, it may be difficult to attribute the antitumor activity of K03 LPS to its own adjuvant activities, and it seems that the antitumor activity of this LPS is based on the action of its lipid A moiety. We have reported that K03 LPS stimulates macrophages in vitro (11) and in vivo (12) and produces a large amount of interleukin 1 in macrophage cultures (13), interferon in mice (14, 15) and a cytotoxic factor (cytotoxin) in sera of BCG-infected mice (15). The properties of the cytotoxin were similar but not identical to those of the tumor necrosis factor (TNF) (15). TNF is produced in the sera of tumor bearing or BCG-infected mice by LPSs, and it is considered as a kind of monokine (16). In this study, tumors which were suppressed from growing by K03 LPS almost became necrotic with hemorrhage as in the cases of other LPSs (2, 17, 18). This seems to suggest that TNF may also contribute to the antitumor effect of K03 LPS. From these results, it is thought that the antitumor effect of K03 LPS is provided by cell-mediated cytotoxicity, including the stimulation of macrophages.

bacterial polysaccharide. 4, 461-476 (1944) 2 Ikawa,

tumor. Cancer

tumor

to injection

of a hemorrhage-producing

J.B.,

Mudd,

S.G.

and

I. Isolation and properties. Inst. 13, 157-166 (1952)

J.

Natl.

3 Mizuno, D., Yoshioka, 0., Akamatu, M. and Kataoka, T.: Antitumor effect of intracutaneous injection of bacterial lipopolysaccharide. Cancer Res. 28, 1531 -1 537 (1968) 4 Nakashima, I., Nagase, F., Matsuura, A. and Kato, N.: Adjuvant actions of polyclonal lym phocyte activators. 11.Comparison and character

5 Nakashima, I., Nagase, F., Matsuura, A., Yokochi, T. and Kato, N.: Adjuvant actions of polyclonal lymphocyte activators. III. Two distinct types of T-initiating adjuvant action demonstrated under different experimental con ditions. Cell. Immunol. 52, 429-437 (1980) 6 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 03 antigen of K/ebsie//a from culture fluid. Cell. Immunol. 66, 111-120 (1982) 7 Ohta, M., Nakashima, I. and Kato, N.: Adjuvant action of bacterial lipopolysaccharide in induction of delayed-type hypersensitivity to protein anti gens. II. Relationships of intensity of the action to that of other immunological activities. Immunobiology 163, 460-469 (1982) 8 Ohta, M., Mori, M., Hasegawa, T., Nagase, F., Nakashima, I., Naito, S. and Kato, N.: Further studies of the polysaccharide of K/ebsie//a possessing strong adjuvanticity. I. Production of the adjuvant polysaccharide by noncapsulated mutant. Microbiol. Immunol. 25, 939-948 (1981 ) 9 Hasegawa, T., Ohta, M., Mori, M., Nakashima. I. and Kato, N.: The Klebsiella 03 lipopolysac charide isolated from culture fluid: Structure of the polysaccharide moiety. Microbiol. Immunol. 27, 683-694 (1983) 10

Strausser,

H.R. and

Bober,

L.A.: Inhibition

tumor growth and survival inoculated with Moloney tumor treated with endotoxin. Cancer 2159

of tumors. IX. subcutaneous

Koepfli,

Inst.

Nieman, C.: An agent from E. coli causing hemorrhage and regression of an experimental

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