Antitumor activity of Eubacterium lentum (TYH - 11) on various tumor cell lines

Vol 5, No 3, July - September 1996 _{Antitumor Activity of E. Ientun 135}

Antitumor Activity

of

Ewbacterium

Eentum

(TYH

-

11)

on

Various Tumor

Cell Lines

Mochammad Hatta

Abstrak

Proliferasifibrosarkoma Meth-A dan AH6\C yang diinokulasi secara intraperitoneal pada nencit, terhattbat dengan penùerian Eubacteriuntlentun(WH-11) intraperitoneal.ProliferasihepatomaMl34danleukenialittrfoidLl2tTjugaterhanùalpadàsebagian ntencit dengan pemberian strain bal<teri _{tersebut. Diantara benîuk tutnor padat, pentberian strain baktiri}

ini

_{ntenghasitkan}

prry"r,

buhan pada beberapa nencit yang mengandung

Mlj4

hepatorna. waktu hidup nencit yang diinol<atasi dengàn ur"bnàrri 816 secara subkutan memanjang setelah pentberian _{Eubacteriun lentwn. Penberian strain ini sebelum iiokutasiiel hepatonn M134,} leuketnia lintfuid L1210 dan leuknùa P388 _{ntenanpaklcan aktivitas antitunor yang lebih baik daripada petnberian sesudah inokulasi.} Strain ini tidak menunjukknn _{aktivitas antitu,nor terhadap lintfonn EIA dan kariinonn paru}

Leiis-Abstract

The proliferation of Meth-A and AH6oCfibrosarcoma inoculated intraperitoneally was suppressed in all ntice given Eubacteriun lentu,tt (TYH-11) intraperitoneally. The proliferation of MH 134 hepatona and Ll2Io _{lytnphoitl ieukenria was also suppressed in sonte} of the _{nice receivingthis strain. Artrongforns of solidtutnors, this bacterial strain curedsone micewith MHl34 hepanira The sunival} tines of nice inoculated with 016 melanona were prolonged by injection of Eubacteriurn lentutn. The antitutttor activity induced by pre-adninistration of this sttain against MHli4 hepator,ra, L12l ) Iyntphoid leukenûa and p388 leukctnia cells was superior to that produced by _{post-administration. This strain, however, had no antitunror activity against EL4 lyntphonrc and lzwis lung carcinotna.}

Keywords : _{Antitumor, Eubacteriunr lentun, Tuutor Cell lines}

In a previous study, _{we have reported that I I bacterial} strains, which are indigenous in the intestinal tract

of

humans, guinea pigs and mice,.have antitumor activity

against Ehrlich ascites

tumor,l

At present, antitumor

cytotoxicity against tumor cells. Furthermore, these

bacteria, except Lactobacillus cassei, are not normal microorganisms in humans intestinal tract.

The mechanisms of antitumor activity of Eubacterium lentum include the improvement of cytotoxic effect

of

Natural

killer

_{and cytotoxic}

T

lymphocyte

_{ially indicated}

that no

other

_{antitumor ac_}

livily

of Eubacterium lentum.

In the present study, the antitumor activity of

Eubac-terium lentum

(TYH-ll),

an indigenous bacterium in

human intestinal flora, was examined against various experimental tumor cell lines.

METIIODS

Animals and tumor cell lines : Six to 8 week-old male

mice and

4 to 5

week-old Donryu male rats were obtained from Sankyo Labo. Service Co., Ltd., Japan. They were housed under standard laboratory condi-tions and given a commercial pallet diet and water ad

libitum. Tumor cell lines used

for

experiments were

maintained as

follows

:

Meth-A

fibrosarcoma in

BALB/c mice,

MH134 hepatoma

in

C3H/He mice.

Ll2lO

lymphoid

leukemia and

p388

leukemia in DBAI2 mice and EL4 lymphoma in C57BL/6 mice by successive ascites passages. AH60C was maitained by successive ascites passage in 4 week-old Donryu rats. 816 melanoma and Lewis lung carcinoma were main-tained by successive solid-form passage subcutaneous-ly in C57BL/6 mice.

r36 Hatta

Bacterial

strain

:

Eubacterium

lentum (TYH-11)

isolated from a human fecal sample was cultured in

GAM

broth (Nissui, Japan) under anaerobic condi-tions. After 24 hours of incubation,

it was killed with

0.3% formalin and washed with sterilized salino.

Antitumor activity

: Each tumor cell line suspended

inRPMI-I

FCS was neally, or terium len

peritoneally or intravenously for 7 days prior to or after tumor inoculation.

All

mice inoculated

with

ascites

form

of

tumor,

received intraperitoneal

injection of

Eubacterium

lentum

and those inoculated

with

solid

form

of

tumor, received intravenous injection of Eubacterium

lentum.

The assessment of survival was made lor 42 days for

the ascites form, and

for

80

to 100 days for

the solid form. Tumor weight was determined using the

follow-ing formula^: tumor weight

(mg)

=

{major axis x (minor axis)'112.

Statistical analysis :

All

data were recorded on special

forms and were managed with a data base and a

statis-tical software

package (Version

6, Epi Info)

in

ap-l5 20

Suwivel dry

a Mice were inoculatedwith Meth-Afibrosarcona cells

inna-peritoneally

[image:2.595.307.544.451.693.2] [image:2.595.52.286.456.716.2]

b. Killed Eubacteilun, lentum given intraperitoneallyfor 7 days

Figure

l.

Effect of Eubacteriu,n lentun on Meth-A _{fibrosarcoilta} (ascitesfonn) in BALB/C nice

MedJ Indones

propriate hardware. The comparison of tumor weight

and survival

time between the treated and control

group was determined by applying Student's t test. P value of 0.05 was taken as the limit of significance.

RESULTS

Meth-A fibrosarcoma tumor :

All mice in

the control group

(20

animals) died within 20 days

after

in-traperitoneal inoculation

of

tumor cells (17.2

+ 1.5

days).

All

2O mice given Eubacterium lentum, both before and after inoculation, did not develop ascites and survived for more than 42 days after inoculation (Figure 1).

When solid

form

of

tumor was inoculated

sub-cutaneously, all mice in the control group (11 animals)

died between day 35 and day 63

t38.7

+ 8.1 days). Only one

of

12 mice receiving intravenous Eubac-terium lentum after tumor inoculation were cured

com-pletely, with survival time of more than 80 days. The

other l1 treated mice died within same time period as

the controls.

Tumor

growth

in

the treated

group was also

sig-nificantly

suppressed

in

comparison to the control group (0.32 + 0.02 vs

3.83

0.08 gr) measured at day 28 after tumor inoculation (p < 0.01) (Figure 2).

a. Mice were inoculated with Meth-A _fibrosarconn cells subcutaneowly

b. Killed Eubacteriun lentun given intravenouslyfor 7 days

Figure 2. Effect of Eubacteriun lentwn on Meth-À _{fibrosarcoilta} .

(solidforn) in BALB/C nice

a

d

.t È

ta a

C

È

o

{

8

X

N a

o

F

\

\'

Vol 5, No 3, July - Septenber 1996

MII134

hepatoma

:

Intraperitoneal inoculation

of

MH 134 hepatoma lead to the death of all control mice

(n : 20) by day 17. No difference of survival time was observed between the post-treated and control group

(16.7

+

2.0 vs

16.2

+

1.4 days),

but g

of l0

in-traperitoneally pre-treated

mice

survived without tumor growth, and the other

2

mice died on days 2g and 31, respectively (Figure 3).

Among the mice with tumor inoculated subcutaneous-ly, all control _{mice (13 animals) died between day 22}

and

day

54

(

26.5

+

9.7 days).

In

contrast, 2

of

12

int

red

complete-lY

than 80 days

aft

between days

34 _{and,60. Tumor weight in} post-treated mice showed significant _{suppression compared to the control group}

( 0.39 + 0.03 vs 3.08 + _0.07 gr) at day

2!

aftq tumor

inoculation (p < 0.01) (Figure 4).

Ll210

lymphoid

leukemia

: All of

the conrrol

(l

l

animals) and intraperitoneally post-treated mice (lO

animals) died by day 9 and day lO, respectively (g.5 + 0.6 vs 8.5 + 9.7 days). On the other hand, 9

of

10 intraperitoneally _{pre-treated mice died between day 9} and day 11. The remaining one survived for more than 16 days after inoculation _{and showed no tumor growth} (Figure 5).

lated x107 d

in-f

the control mice (10 animals) died between day l1 and day 46

(2I.5 +

12.9 days), but 8

of

lO post-treared and all 10 pre-treated mice showed no tumor growth. Two rats were died

in at

46 and 50 after tumor inoculation. (Figure 7).

816

melanoma

:

When

mice

were inoculated in_

of

mice

(lO

animals)

15

(16.3 +

2.4

_days).

e

i

aly

pre_treated and post-treated mice (n

=

l0

each) died between day 22 and 30 (28.6 + 2.4 days) and between day

lg

and27

Antituntor Activiry of E. kntu,n 137

(25.3 + 2.7 days), respectively. These data confirmed

the prolongation

of

survival time

in

both

in-traperitoneally treated groups (Figure 8).

When mice were inoculated subcutaneously, no dif-ferences in tumor weight (2.71 _{+ 0.28 vs 2.85 + O.72} gr) and survival time (21.4 + 3.6 vs 21.6 + 3.2 days) were observed between the intravenously post-treated and control group. (Figure 9).

EL4 lymphoma

and Lewis lung carcinoma

:

C57BL|6 mice

were inoculated

with

104

ro

105 cells/animal

of

subcutaneously, venously

with I

days

piror

to

or

_{after inoculation. No differences in} tumor _{growth and survival time were observed}

be-tween the control

and

the

treated

group.

Other C57BL|6 mice were inoculated

with

5x104

to

106

cells/anim

rcinoma into the foot pad, and 107

ce

_{lentumwas administered}

intravenously for 7 days before or after inoculation. In this experiment, no differences were found between the control and treated groups both

in

tumor growth and survival time (data not shown).

DISCUSSION

Eubacterium lentum

is an

anaerobic gram_positive bacterium, short rod form, non-spore-bearing and non_ an enzyme

of

the its growth is vastly

ne both in vitro and

bile

salt

into their

3

and

7-ket.

i::iiiiJ"'r'.'fr:::

derivatives are transformed to an unsaturated form by Clostridium paraputrificurn, _{which has been shown to} be present

in

llre

feces

of

bowel cancer patient. l8 Acevedo et al.tg found that Eubacteriunr lentum

iso-In our prev

of bacilli,

terium acn

Ba

L Èa

È

û

Èa

G

È

û

s

G

.È

t

6

138 Hatta

17 2D 25

Sunival day

a. Mice were inoculatedwith MH134 hepatonra cells intraperi-toneally

[image:4.595.56.288.75.356.2] [image:4.595.314.550.91.355.2] [image:4.595.308.548.103.647.2] [image:4.595.54.288.421.647.2]

b. Killed Eubacteriuut lentunt give,t intraperitoneallyfor 7 days

Figure 3. Effect of Eubacteriun lentum on MH134 hepatotna in (ascites _fonn) C3H/He nice.

2.tEr0lr

Sunivel day

a- Mice were inoculated with L1210 lynphoid leukenùa cells intraperitoneally

b. Killed Eubacteriun lentuil given intraperitoneallyfor 7 days

Figure 5- Effect of Eubacteriw,t lentum on L1210 lynphoid

leukenia in DB,4,f2 nice

Med J hdones

a. Mice were inoculated with MH134 hepatona cells sub-cutaneously

b. Killed Eubacteriw,t lentun given intravenousll,for 7 dayg

Figure 4. Effect of Eubacteriutn lentun on MIt 134 hepatonru

(solid _forn) in C3H/He nice

a. Mice were inoculatedwith P388 leukenia cells

intaperito-neally

b. KiUed Eubacteriuna lentun given intraperitoneally _for 7 days

Figure 6. Effect of Eubacteriun lentun on P388 Leukenia in

DBAQ, ntice

m

I

N

o

E

F

l6

\\,

\\

....

\

\\

Vol 5, No 3, JuIy - September 1996 _{Antitunor Activiry, of E.}

k,ltu,,,

139

Suruival day

É

':

È

û 6a

o

È

6

a. Rats _{were inoculated with AH6OC intraperitonealll,}

[image:5.595.29.262.91.339.2] [image:5.595.288.523.96.343.2]

b. Klled _{Eubacteriurtr lentun given intraperitoneallT, for 7 dal,s}

Figure 7. _{Effect of Eubacteriuu lentuttt} on AH6OC itr Donryu rats

a. Rats _{were inoculated with B16 nrelanotna cells i,ltruperito,leallI} b. Killed Eubacteriutn lentutn given intraperitoneally, _for 7 tla1,s

Figu.re 8. Effect of _{Eubacteriun lentum on B16 tnelanotna}

(ascites _{form) in C578Ir/6 tuice}

èo

Ê

o

X

O N

çt,

tr o Ê

H

\a 6

È (t)

--

Post-adnr I

nlslratl

on

+-

_Control

+-

_Posl-adm

I nl

stratlon

*

Control

15

20

Surrlval

a. Mice were inoculated u'ith 816 ntelanona cells

sub

usl1,

b. Killed Eubecteriun lenrum given intravenousll, _for

140 Hatta

species, the antitumor activity of Eubacterium lentum

had never been reported previously. In the past, many

researchers had carried out studies

of

antitumor

ac-tivity of

bacteria

terium

rorur^.2

Bcé,3'4

Nocar

432,8,ç,ro

Lac-tobacillus

cassei

onocytogenes,l4

all of which have host-mediated effects. Inctobacillus

cassei inhabits the human intestinal microflora. Mat-suzaki et.

al.rr

have

found

that Lactobacillus casei

shows antitumor activity, especially an

immunomod-ulating effect against Lewis lung carcinoma. However,

we were unable to find any antitumor activity of this

species, while Lactobacillus acidopftilzs showed mild activity agairst Ehrlich ascites tumor.

Eubacterium lentum showed noticable antitumor

ac-tivity

against 3 tumor lines,

mild

antitumor activity

against 3 tumor

cell

lines and no antitumor activity

against

EL4

lymphoma and

Lewis

lung carcinoma

(table 1),

When Eubacterium lentum was injected

before inoculation of tumor cells, it exhibited

remark-able antitumor activity. It may be due to host-mediated

effects such as those shown

with

Corynebacterium

paryum, etc. In particular, the growth of leukemia cells

such as P388 and

LI2IO,

and MH134 hepatoma was

suppressed as

the

result

of

pre-administration

of

Eubacterium

lentum-Table l. Inhibitory effectof Eubacterium lentunt on the growth of tumor cells in mice and rats

Highly effective Effective Ineffective

Med J ltrdottes

show any antitumor activity against EL4 at all. These

result may indicate the existence

of

a

relationship between the virulence of a tumor and the capability

of

effector cells. When the tumor cell line was inoculated

intraperitoneally and Eubacterium lentunt was injected

in the same way, the proliferation

of

tumor cells was

completely suppressed

in

some

tumor

cell

lines.

Eubacterium lentum was particularly effective against

the ascites form

of

meth-A fibrosarcoma, but not the ascites form

of

Ll2lO

lymphoid leukemia.

In

other-words, the activity can vary according to the tumor cell line used, and that susceptibility depends on the kind

of

effector

cell

(for example, activated macrophage,

NK cell,

LAK

cell, etc) present in the host.

As yet, we have no evidence to suggest whether

Eubac-terium lentum can produce cytokines and other sub-stances, and what type of effector cells

it

activates in the host. The mechanisms

of

antitumor

activity of

Eubacterium lentum remain

to

be clarified

in

sub-sequent studies.

ACKNOWLEDGEMENTS

I am most grateful to Prof. Dr. A. Muraguchi and Dr.

K. Kawai for their expert help in the preparation of the

manuscript and to Dr. T. Hayashi for financial support

through

a

grant

from

Japan Indonesia Medical

As-sociation (JIMA).

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