Enhancement of natural killer cytotoxicity delayed murine carcinogenesis by a probiotic microorganism

Akimitsu Takagi,1, Takeshi Matsuzaki, Mikiko Sato, Koji Nomoto, Masami Morotomi and Teruo Yokokura

Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Regulation of innate immunity may be an effective means of cancer control. Delaying cancer onset is regarded as an important mode of action in cancer prevention. We have been investigating the chemopreventive mechanisms of Lactobacillus casei Shirota (LcS), a probiotic strain. In this study, we evaluated the effect of LcS on tumor onset and the involvement of natural killer (NK) cells using a 3-methylcholanthrene-induced carcinogenesis model. C3H/HeN mice were divided into three groups, according to treatment: vehicle-treated, treated with vehicle only; control, 3-methylcholanthrene treated; LcS, 3-methylcholanthrene and LcS treated. 3-Methylcholanthrene was injected intradermally at 7 weeks of age. LcS was mixed into the diet (0.05%, w/w), which the mice were fed from the day of 3-methylcholanthrene injection onward. Tumor incidence was observed weekly. Profiles of splenic NK cells, in vitro cytotoxicity and the proportion, in the early stage of carcinogenesis were analyzed at 5 weeks after the injection. The tumor suppressive effect of LcS was also evaluated in a beige mouse model that is genetically deficient in NK cells. LcS delayed tumor onset and reduced tumor incidence in the results with C3H/HeN mice (P < 0.05). More specifically, tumor incidence in the control group was 33% at 6 weeks after the injection and 83% at 11 weeks as opposed to 0 and 42%, respectively, in the LcS group. NK cell cytotoxicity was significantly higher than in the control group, and the number of NK cells also increased in the LcS group of C3H/HeN mice. However, LcS failed to suppress tumorigenesis in the beige mouse. These findings suggest that enhancement of the cytotoxicity of NK cells by LcS delays tumor onset.

Abbreviations: Con A, concanavalin A; ELISA, enzyme linked immunosorbent assay; LcS, Lactobacillus casei Shirota; IL, interleukin; mAb; monoclonal antibody; MC, 3-methylcholanthrene; NK, natural killer.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cancer prevention can be defined as the inhibition, delay or reversal of carcinogenesis. Delayed cancer onset has been proposed as an important mode of cancer control (1). This is because delaying onset into later life in man results in an increase life expectancy (2). This may occur as a result of reduced exposure to carcinogenic factors in the diet, life style changes, or using a chemopreventive agent that can inhibit carcinogen uptake, maintain tumor suppressor function or modulate immune responses. An increase in immune response has been shown in patients with cancer (36), and patients with relapses had significantly lower preoperative NK cytotoxicity than relapse-free patients (7). These observations indicate that NK cells are among the candidates for cells producing direct tumor cell destruction and may be the first line of host defense against tumorigenesis in humans.

Probiotics can be defined as a microbial food supplement that affects the host animal beneficially. Such microorganisms are regarded as safe because they have been used for centuries in the production of daily foods. In terms of cancer prevention, most reports on probiotics have focused on gut ecological modification (812). Few reports have discussed host- immune responses (13,14). Moreover, the effects of probiotics on the early stage of tumor onset are as yet unknown. We have been studying the mechanism of cancer prevention using probiotic microorganisms which evokes host-mediated immunity. One of these probiotic strains, Lactobacillus casei Shirota (LcS), was isolated from the healthy human intestine. An important feature of LcS, a Gram-positive bacterium, is that it has no pathogenicity. It has been reported that systemically administrated heat-killed LcS exerts anti-tumor activity in transplantable tumor-bearing mice (1517). A clinical study revealed that oral administration of LcS to patients with superficial bladder cancer after transurethral resection of the tumor lowered the risk of recurrence (18). We previously reported that oral administration of LcS lowered the 3-methylcholanthrene (MC), polycyclic aromatic hydrocarbon, induced tumor incidence in mice and this effect in the late stage of tumor development was mediated by host immune responses (19).

The MC-induced carcinogenesis model has been used in studies of host-mediated cancer control strategies (20,21). Various MC tumor models were also utilized such as colon, liver, lung, uterine cervix and mammary gland cancers (2226). A unique advantage of the MC-induced tumor is its high frequency of p53 tumor suppressor gene mutations (2729), and it has one of the highest mutation frequencies among the various chemically induced tumors (3035). Moreover, mutant p53 product in MC-induced murine tumor cells was recognized by host lymphoid cell (36).

In the present study, we investigated the effect of LcS on the onset of MC-induced tumors, and evaluated the involvement of natural killer cells, focusing on the early stage of tumor development.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental animal and diet
C3H/HeN male mice (CLEA Japan, Tokyo, Japan) and C57BL/6J and C57BL/6LystbgJ (beige) female mice (Charles River Japan, Kanagawa, Japan) were housed in plastic cages (six mice/cage) with wood chips under controlled conditions: 12 h light/12 h dark cycle, 55 ± 10% humidity, 24 ± 1°C temperature. They were fed a commercially available diet (Oriental MF diet, Oriental Yeast, Tokyo, Japan). MF diet was supplied as the control diet. Food and water were provided ad libitum and body weight and dietary intake were measured weekly during the experiments.

Tumor induction
3-Methylcholanthrene (MC; Sigma Chemical, St Louis, MO) was dissolved in olive oil as a vehicle at a concentration of 10 mg/ml. Seven-week-old mice were treated once with MC (1.0 mg/0.1 ml/mouse) by intradermal injection in the flank. Tumorigenesis was determined by manipulation of the tumor nodule. A nodule with a mean diameter of >3 mm was judged to be putative tumor and this tumor was monitored at weekly intervals until the end of the experiment. Two observers scored each tumor blindly and independently.

Preparation of the probiotic microorganism
Viable L.casei Shirota (LcS) was used in this study. LcS was cultured for 24 h at 37°C in MRS medium (Difco Laboratories, Detroit, MI). After cultivation, cells were collected by centrifugation, washed three times with distilled ion-exchanged water and then lyophilized. The number of viable LcS organisms was estimated from the number of colonies formed on LcS selective medium. These colonies were identified by enzyme linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) against LcS. Preparation of the selective medium and the ELISA procedure were described previously (43).

Experimental design
Mice were fed either the control diet or the LcS supplemented MF diet. LcS was mixed into the diet (0.05%, w/w), which was fed to the mice from the day of MC injection until the end of the experimental period. For tumor monitoring, 36 seven-week-old mice were randomly divided into three groups (12 mice per group). Treatments were as follows: vehicle-treated, treated with vehicle only; control, treated with MC; LcS, treated with MC and given LcS. The total experimental period was 15 weeks. For analysis of immune parameters, mice were dissected at 5 weeks after MC treatment. During this week, MC-treated mice did not have detectable tumors. Two or three independent experiments were done per analysis (n = 3/group/experiment).

Cell preparation
Spleens were removed from mice under anesthesia and a single cell suspension was prepared by pressing the tissue gently. After debris removal, erythrocytes were depleted by hypotonic lysis (Tris-buffered NH4Cl solution). The cells were washed three times with cold Hanks' solution and then resuspended in RPMI 1640 medium (Gibco BRL, Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS; Boehringer Mannheim GmbH, Germany). To purify NK cells from spleen cells, a Magnetic Antibody Cell Sorting (MACS®; Miltenyi Biotec GmbH, Bergish Gladbach, Germany) system was used according to the manufacturer's instructions. In brief, DX5 positive selection was performed using anti-NK cell (DX5) Micro Beads (Miltenyi Biotec) against spleen cells. DX5 positive cells were separated by passage over a MACS® column (Miltenyi Biotec). The purity of the NK cells was confirmed by flow cytometry after staining of the column-separated cells with streptoavidin–phycoerythrin conjugate (Pharmingen, San Diego, CA).

Antibodies and flow cytometry
The mAb used for phenotypic analysis of the splenic NK cells were directed against the DX5 antigen. Cells at a density of 5x105 cells/ml in wash buffer (PBS/0.2% FCS) were incubated at 4°C for 30min with 1µg biotin-conjugated primary mAb. Cells were washed three times in wash buffer at 4°C. Streptoavidin–phycoerythrin conjugate, a secondary reagent designed to detect binding to biotinylated mAb, was added and incubated at 4°C for 30 min in the dark. Expression of cell surface markers was analyzed by standard flow cytometry using an EPICS® Elite Flow Cytometer (Coulter, Miami, FL). All antibodies and secondary reagents used for these analyses were purchased from PharMingen.

Analysis of NK cell function
A standard chromium release assay was used to assess the cytolytic function of splenocytes. YAC-1 target cells (5x106) were labeled with 100 µCi sodium [51Cr]chromate (DuPont-NEN Research Products, Boston, MA) for 1 h, washed three times with Hanks' solution and resuspended in RPMI 1640/FCS. The 51Cr-labeled target cell suspension was aliquoted into 96-well round-bottomed plates (Nunc, Roskilde, Denmark) at a concentration of 2x104 cells/well. The effector cells, single cell suspensions of splenocytes or purified NK cells, were incubated in the presence of the target cells in a total volume of 200 µl/well to produce the E:T ratios shown in the Results section. Plates were incubated for 4 h at 37°C in 95% air–5% CO2, and 51Cr-release from lysed target cells was measured using a {gamma} counter. The percentage of specific chromium release at each well was calculated using the following formula,

where test c.p.m. represents the counts in experimental cultures of target cells and effector cells; spontaneous c.p.m., counts in cultures containing only target cells and medium; and total c.p.m., counts obtained by adding 100 µl 1 N HCl to target cells to lyse all cells.

Statistical analyses
Tumor incidences were expressed as the percentage of tumor-bearing mice out of all mice tested. The cumulative tumor incidence was determined by the Cox–Mantel test, the incidences at each week by Fisher's exact test. Differences in other results were analyzed by the Bonferroni multiple comparison test. Probability values of <5% were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor suppressive effect of LcS
Figure 1aGo shows the tumor onset delay and suppressive effects of LcS in C3H/HeN mice. No tumors were ever detected in mice injected with the olive oil vehicle alone (vehicle-treated group). In the control group, tumors began to appear at 6 weeks after MC treatment and the tumor incidence at this time point was 33%. Then, the incidence increased until 11 weeks after MC treatment, reaching an incidence of 83%. In the LcS group, tumor incidences at 6 and 11 weeks after MC treatment were 0 and 42%, respectively. There was a significant difference at 6 weeks after MC treatment (P < 0.05), and the cumulative incidences at 11 weeks were also significantly different (P < 0.05). The tumor nodules formed at the site of MC injection were identified as rhabdomyosarcoma by histological examination and no histological differences were found between the experimental groups (data not shown). There were no significant differences in body weight gain changes among the groups during the experimental period (Figure 1bGo). These results indicated that LcS has the effect of delaying tumor onset in mice injected with MC. We decided that the observational period needed for the mechanisms of action of LcS to manifest was 5 weeks after MC injection, because mice had no detectable tumors during the fifth week which was just before the appearance of tumors.



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Fig. 1. (a) Inhibition of 3-methylcholanthrene (MC)-induced carcinogenesis and (b) effects of MC treatment or LcS administration on body weight gain. C3H/HeN mice were injected intradermally with 1.0 mg of MC. Lactobacillus casei Shirota (LcS) containing diet (0.05%, w/w) was provided from the day of 3-methylcholanthrene injection until 15 weeks after the injection. Tumor incidences and body weights were observed weekly. Symbols: open circles, vehicle-treated; closed circles, control; closed squares, LcS. Each group consisted of 12 mice. Each value represents the mean (bars, SD). (a) Statistically significant differences were found between the two groups as follows: {dagger}, Fisher's exact test for the incidences at 6 weeks after MC treatment (P < 0.05); , Cox–Mantel test for the cumulative incidence up to 11 weeks after the MC treatment (P < 0.05).

 
Growth parameters and fecal recovery of LcS
There were no differences in body, liver and spleen weights, or dietary intake, among the groups at 5 weeks after MC treatment (Table IGo). The actual dose of LcS per day, calculated from dietary intake, was ~1.6 mg/mouse/day for the LcS group, which corresponded with ~1.6x109 cells/mouse/day. During this week, viable LcS cells were detected in feces obtained from the LcS group at a level of 5.4 ± 0.8 log/g feces. Viable LcS cells were not detected in fecal samples from other groups.


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Table I. Body, liver and spleen weights, and dietary intake, at death
 
Profiles of NK cells
To evaluate the association of NK cells with the LcS-mediated delay of tumor onset, profiles of NK cells were analyzed just before the tumor appearance period. Cytotoxicities of NK cells in spleen cells, from each treated mouse, against YAC-1 target cells were analyzed at 5 weeks after MC treatment. As shown in Figure 2Go, NK cell cytotoxicity was significantly enhanced in splenocytes from the LcS group as compared with the control group (P < 0.05) when we used whole spleen cells as the effector cells. In contrast, there were no differences in NK cytotoxicity among the groups when we used purified splenic NK cells as the effector cells (Figure 3aGo). The activity of purified NK cells obtained from the control and the LcS groups were at the same level as the vehicle-treated group. However, the proportion of NK cells in splenocytes from the LcS group was significantly higher than in the control group (Figure 3bGo; P < 0.05). We, therefore, conclude that the observed enhancement of in vitro NK cytotoxicity in splenocytes from the LcS group depended mainly on the increased proportion of NK cells.



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Fig. 2. Enhancement of cytotoxicity of natural killer (NK) cells by LcS. Spleen cells derived from each group at 5 weeks after MC treatment were used as effector cells against YAC-1 target cells (2 x 104 cells/well). Results are represented as the means (bars, SD) of triplicate wells on the y-axis, with the effector cell to target cell ratio (E:T) on the x-axis. Symbols: open circles, vehicle-treated; closed circles, control; closed squares, LcS. Each experiment and group consisted of 3 mice. Similar results were obtained in three independent experiments conducted in the same manner. , P < 0.05 versus control group.

 


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Fig. 3. (a) Cytotoxicity at the cellular level in splenic NK cells and (b) proportions of NK cells in splenocytes. Spleen cells derived from each group at 5 weeks after MC treatment were analyzed. (a) Anti-DX5 mAb positive (NK) cells were separated using a MACS® column, as described in Materials and methods, and cytotoxicity of the separated cells against YAC-1 cells (2x104 cells/well) was then measured in vitro. Results are presented as the means (bars, SD) of triplicate wells on the y-axis, with the effector cell to target cell ratio (E:T) on the x-axis. Symbols: open circles, vehicle-treated; closed circles, control; closed squares, LcS. (b) DX5 antigen expressing cells in splenocytes were detected by flow cytometry. Results are presented as means (bars, SD). Each experiment and group consisted of three mice. Similar results were obtained in two (a) or three (b) independent experiments conducted in the same manner. , P < 0.05 versus control group.

 
Tumor suppressive effect of LcS on NK cell deficient mice
To determine whether the tumor onset delay produced by LcS depended on NK cells in vivo, we used the beige mouse, an NK cell deficient mutant. LcS was administered to MC-treated beige mice and C57BL/6 mice (the background strain of the beige mouse) under the same experimental conditions as those used for C3H/HeN mice and tumor incidences were monitored. Figure 4Go shows that LcS failed to suppress MC-carcinogenesis in the beige mouse, whereas LcS suppressed tumor onset in C57BL/6 mice (P = 0.054). We also reconfirmed the significantly enhanced natural cytotoxicity of spleen cells from LcS-treated C57BL/6 mice (Figure 5Go). Thus, it was demonstrated that the delayed tumor onset mediated by LcS depended on NK cells in vivo.



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Fig. 4. Effect of LcS on MC-carcinogenesis in NK cell deficient (beige) mice and C57BL/6 mice. Dietary LcS (0.05%, w/w) was administered to MC-injected (1.0 mg/mouse, i.d.) beige mice and background C57BL/6 mice. Tumor incidence was monitored weekly. Symbols: closed circles, control; closed squares, LcS. Each group consisted of 12 mice.

 


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Fig. 5. Cytotoxicity of NK cells in beige mice and C57BL/6 mice. Spleen cells derived from each strain and group at 5 weeks after MC treatment were used as effector cells against YAC-1 target cells (2x104 cells/well). Results represent means (bars, SD) of triplicate wells on the y-axis, with the effector cell to target cell ratio (E:T) on the x-axis. Symbols: open circles, vehicle-treated; closed circles, control; closed squares, LcS. Each experiment and group consisted of three mice.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effectiveness of enhanced NK cytotoxicity in cancer prevention has been reported. Treatment of breast cancer patients with retinoid augmented peripheral NK activity as compared with that of patients given a placebo (38). In an animal study, bovine lactoferrin enhanced NK activity in azoxymethane-treated rats (39). The importance of NK cells was clearly demonstrated in the early period of carcinogenesis. For example, tumor incidence was increased when anti-asialo GM1 was administrated to depress NK cells in the early stage of chemical carcinogenesis before tumor development, though the incidence was not increased when anti-asialo GM1 was administrated at a later stage (40). This suggested that NK cells play an important role in the early stage of carcinogenesis. Therefore, we focused on the involvement in NK cells in the cancer preventive effect of LcS in the early stage of carcinogenesis.

We found the tumor latency period in the beige mouse, which is deficient in NK cells (41), to be shorter than that of the background C57BL/6 mouse. It was reported that beige mice (bg/bg) injected with benzo[a]pyrene had a shorter latency of tumor development than heterozygous littermate controls (+/bg) (42). This observation is consistent with our results, because +/bg mice show the same level of NK cell cytotoxicity as C57BL/6 mice (41). It is possible, therefore, that tumor onset was inhibited in part by the NK cytotoxicity of the host. One important aspect of cancer control is the delay of tumor onset, as mentioned in the Introduction (1,2). In the present study, LcS delayed MC-induced tumor onset and suppressed the incidence in C3H/HeN and C57BL/6 mice. However, LcS failed to delay tumor onset in the beige mouse. Therefore, NK cells were indicated to play a key functional role in the delay of tumor onset, i.e. in the cancer preventive effect of LcS. We thus examined the status of NK cells as a mechanism for analyzing delayed tumor onset, due to administering LcS, at the early stage of MC-carcinogenesis.

Mice that had been injected with MC and had not yet developed tumors were examined in this study to elucidate the mechanism of tumorigenesis, and C3H/HeN and C57BL/6 mice were used as control strains. These strains are susceptible to MC-induced carcinogenesis (60). The relative susceptibility of MC-sensitive strains to MC-carcinogenesis depended on the dose of MC, because the rank order of strain-susceptibility was reversed with dosage of administrated MC (61). The strains used in the present study exhibited the reversibility phenomenon to an extreme (62), indicating that the response of these two strains to the same dose of MC was different. In our study, the cytotoxicity of the NK cells obtained from the control group of C3H/HeN mice was slightly higher than in the vehicle-treated group, whereas among the C57BL/6 mice the cytotoxicity in the control group was lower than in the vehicle-treated group before tumor development. It has been reported that the function of NK and T cells gradually diminishes as tumors grow (43). Before tumor development, however, NK cell cytotoxicity was suppressed especially in the initial period after MC injection (63). The depressed cytotoxicity recovered and rebounded to above the normal level immediately before tumor development, and then returned to the normal level in the early period of tumor development (63). It was noteworthy that the timing of recovery from depressed cytotoxicity varied with the dose of MC (40). Therefore it was concluded that the difference in the control level of cytotoxicity between C3H/HeN and C57BL/6 mice in the early stage of MC-carcinogenesis under our experimental conditions reflected a strain difference in susceptibility to MC-carcinogenesis.

The present study demonstrated that splenic NK cytotoxicity is enhanced by LcS before tumor onset. Moreover, the proportion of splenic NK cells was increased but functional enhancement of splenic NK cells was not detected at a cellular level by our experimental method. Therefore, the enhanced NK cytotoxicity in spleen cells may simply reflect a higher number of cells in the NK subset able to express lytic activity. However, assessment of NK cell activity as measured against the YAC-1 cell line may not reflect in vivo events completely. It was reported that cytotoxic action was initiated after contact between tumor and NK cells (44). Recently, cognitive mechanisms operating between tumor and NK cells were reported. A receptor for the cytotoxic response of NK cells was identified, and ligands for the receptor were expressed on the surfaces of various human carcinoma specimens (4548). These reports suggest that a killing activation trigger was pulled by physical contact and, specifically, by the interaction between cancer and NK cells. Based on the above, we speculate that enhancement of NK cell functions by LcS is involved in detection and removal of transformed cells at the site of carcinogenesis, and these processes would result in retardation of tumor onset. However, further study is needed to define this functional enhancement at the cellular level in NK cells under systemic conditions.

Although chemopreventive mechanisms mediated by LcS were demonstrated, it is still unclear, in terms of the results obtained in this study, how the administered LcS increases the number of NK cells. IL-15 treatment increased the activity and the proportion of splenic NK cells in tumor-bearing mice, and treatment of mice with IL-15 enhances anti-tumor responses when administered in combination with a therapeutic agent such as cyclophosphamide (49). Meanwhile, NK cell development depends critically on IL-15 (50), which can be substituted by IL-2 in vitro (51). In addition, IL-2 allows Pax5–/– cells to differentiate into phenotypic and functional NK cells in vitro, although it was not possible for the cells to progress further (52). Our previous reports demonstrated the production of splenic IL-2 in MC-injected mice to be significantly enhanced by LcS (19), and the expression of the murine IL-15 gene to be induced by treatment with LcS (16). In the current study, the expanded NK cell population might depend mainly on soluble mediators induced by LcS. Further experiments to elucidate the relationship between NK cells and oral administrated LcS in carcinogenesis are in progress.

The detailed nature in which probiotics work is not known. It has been suggested that stimulation of the host immune system by probiotics was exerted via translocation of these probiotics (53), and some Lactobacillus species have been isolated from patients (5456). However, the safety of LcS has been demonstrated in a clinical study (18). Possible mechanisms of recognition between ingested LcS cells and the host have been partially elucidated. In the Peyer's patch, LcS phagocytosed by macrophages was observed by histological examination in an animal study (57). Furthermore, murine macrophage stimulated with LcS produced IL-12 in vitro (58,59). These observations suggest that LcS may be degraded in gut-associated lymphoid tissue, and their signal from immunocompetent cells leads to a systemic effect. Therefore, in order for LcS to exert its systemic effects it may not be necessary for viable LcS cells to migrate from the gut to the peripheral circulation. Delineating the precise mechanism of action will provide insights into the development of rational approaches to cancer control. We believe that regulation of innate immunity by probiotics may be among the possible ways to achieve safe cancer prevention.


    Notes
 
1 To whom correspondence should be addressed Back


    Acknowledgments
 
We are grateful to Dr Yoshinori Umesaki and Satoshi Matsumoto for valuable suggestions and technical advice. We also thank Dr Kazumi Uchida for excellent technical assistance in the histological preparation of the tumors and their diagnosis.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 17, 2000; revised November 20, 2000; accepted December 6, 2000.