Tohoku
J. exp.
Med.,
1978,
Antitumor
Activity
Delipidated
BCG
HIROSI
125, 247-252
of
Hot-Water
Extract
SATO, ATSUNOBU YOKOSAWA, HIDEO
NAGAI,
NOBUKO
KIYOSHI
KONNO
KUMANO,
MASAKICHI
from
ARAI,
HIROMI
MOTOMIYA
and
The Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai 980
SATO, H., YOKOSAWA,A., ARAI, H., NAGAI, H., KUMANO, N., MOTOMIYA, M. and KONNO, K. Antitumor Activity of Hot Water Extract from. Delipidated BCG. Tohoku J. exp. Med., 1978, 125 (3), 247-252The antitumor activity of hot water extract of delipidated BCG was investigated in mice inoculated with Sarcoma-180 cells and Ehrlich carcinoma cells, respectively. The hot-water extract was found to be effective when administered after and ineffective when administered before the inoculation of tumor cells. When this extract was given with anticancer drugs, such as Mitomycin C and cyclophosphamide, a combined effect was obtained in the treatment of Sarcoma-180 and of Ehrlich carcinoma. water-soluble adjuvant; BCG
The mycobacterial preparations such as whole bacilli of BCG, cell wall skeleton (Meyer et al. 1974), methanol extraction residue (Shlomo et al. 1973), and cord factor (trehalose dimycolate) (Bekierkunst 1976), which are all adjuvant-active, have been employed in the treatment of neoplasms in recent years. Also the adjuvant activity of these preparations is known to be essential for their antitumor activity. Since 1972, several water-soluble substances from delipidated mycobacteria (Adam et al. 1973; Migliore-Samour and Jolles 1972, 1973) and cell walls (Adam et al. 1972) have been reported to possess adjuvant activity. The hot-water extract obtained from defatted Mycobacterium bovis strain BCG was found to have adjuvant activity and was designated as HSA (Hot-water Soluble Adjuvant) in our previous paper (Sato et al. 1976). In the inoculum
present
study
of Sarcoma-180
the
antitumor
and
Ehrlich
effect
of the
carcinoma
HSA
in mice
on the
intraperitoneal
was investigated.
MATERIALS AND METHODS Throughout
the
experiments,
4-week-old
ddI
mice
of both
sexes
weighing
22-24
g
were employed. Sarcoma-180 and Ehrlich carcinoma in ascitic form have been maintained in ddI mice by weekly inoculations. In each experiment the suspension of the tumor cells was diluted with saline immediately inoculated intraperitoneally. The test. Received
for publication,
before viability
December
use and the desired number of the cells were of cells was examined by the dye exclusion
7, 1977. 247
248
H. Sato et al.
The hot-water extract was prepared as previously reported taken up in 0.25 ml of saline and injected intraperitoneally.
(Sato et al. 1976) and was
Antitumor effect on Sarcoma-180 Experiment received of
3•~108
HSA as
I.
Mice
were
intraperitoneal tumor
for a
cells.
The
7 consecutive
control
(Fig.
divided
injections
The
after
3
equal
HSA
for
group
was
inoculation
number
of
the
of
mice.
was
The
days
with
tumor
survivors
20
consecutive
treated
of
the
groups 7
first
before
intraperitoneal
cells.
The
recorded
in
group
inoculation injections
third the
group
above
of
was
three
used groups
1). Experiment ‡U.
Eighty
mice
four
groups
Group
1
The
fourth
before
(control, group
and
(Group
after
3 in
inoculation
with
3•~108
saline
tumor
at
1,
received The
7
cells.
3,
5
group groups.
days
after
used
as
above
cells
Sixty
of
7
size
consecutive
of
for
control
5
4 mg
inoculation the
days
from
compared
a
the
day
for
5
of
20
of
Mitomycin
tumor
after
in
of
the
Experiment
I.
consecutive
days
the
mice
were
cells.
The
inoculation same
inoculated
C (1
of
the
as
in
manner
mg/kg)
second
in group
tumor the
cells. first
and
control.
the
manner
to
days
among
after
The in
groups
HSA
every
same
dose
third
time
were
group
terms
of day of
days
20
mice.
before HSA
received
of
the
2
and
experimental
were
other two of
saline
received
saline
The former
the after
of
mice
for
7
saline
was
Group
A
inoculation
of
consecutive
alone
compared
and
was
among
the
two
groups
tumor and
the
of
cells.
20
mice
Group
each. 1
inoculation
of
tumor
inoculation
of
the
inoculation to
groups and
the
groups
In
this
received cells.
tumor
HSA Group
3
cells.
The
alone.
an given
groups
4
1.0•~10,
after
before
injections
in
into to
Group
hundred
the
divided reduced
days
inoculation
four
equal
of the
cells.
and
cells.
as in
3
mg
received
was
received
tumor
into
0.25
survival
cyclophosphamide
2
1
HSA
divided
B
mice
One
of same
in
1).
prior
group
of
mice. Three as
HSA
injection
of
as
of
tumor
conseuctive
Experiment ‡V. and
of
consisting an
from and
served
inoculum
days
HSA
fourth
HSA
were
the
Eighty
the
received
of 20
treated
cells. each
received
C
groups
were
injection
inoculation
of
mean
(Table
Experiment ‡U.
for
2)
an
mice
after
Group
The
groups
experiment
group
mice
(1.2•~107).
control.
three
first
4 equal
Fig.
tumor of
injections
inoculation a
in
effect on Ehrlich carcinoma
intraperitoneal
tumor
3•~108
group
into
2
received
Mitomycin
fourth
I.
7
2)
injections
The
Experiment received
Fig.
7 days
received
Antitumor
the
and
divided
Group
groups The
consecutive
third
second
Four
were
and
of
Experiment ‡V.
in
the
second
days
group.
into
of
two
of groups
were
2•~106 of
injected
were
treated
cells.
The
control
with with
at
cells 2
.
days
2
after
cyclophosphamide
HSA
numbers
(Table
tumor
mice
for of
7 consecutive survivors
were
2).
RESULTS
Antitumor effect on Sarcoma-180 Fig. 1 shows the results of the experiment in which HSA was injected for 7 consecutive days each before or after inoculation of Sarcoma-180 cells. The survival time of the mice received HSA before inoculation of the tumor cells was prolonged only slightly. On the other hand an increase of survival time was evident in the mice treated with HSA after inoculation of the sarcoma, hells The results summarized mice
that
of another
in Fig. 2. had
received
experiment
In this HSA
in which
experiment before
survival
inoculation
HSA
alone
was administered
time was not prolonged of
the
group which received HSA for 5 consecutive days before the tumor cells only a very slight effect was observed .
tumor and
are in the
cells . In after inoculation
the of
Antitumor
Fig.
1.
Antitumor
Group
1,
activity
injected
Fig.
inoculation
0.25
106
tumor
2.
Antitumor
Group
mg
of
Fig.
3.
for
activity
1,
received
0.3
7
from
Sarcoma-180
3•~106
0.25
tumor
BCG
249
. mg
of
cells;
consecutive
of
HSA
mg
of
inoculation
cells;
Group and
Effect
of
1,
7
given
intraperitoneal
3,
after
HSA
for
Group
days
7 consecutive
2
, injected intraperitoneal
after
days
before
intraperitoneally inoculation of
3•~
mg
tumor
cells;
Group
I
and
Group Group
2.
tumor
0.3
of
of
consecutive
3,
given
of
HSA
both
C and
3•~
days Mitomycin
cells;
after
for
106
tumor
on at
and
HSA
mg
cells,
2,
3,
5
and
given
0.25
inoculation in
before of
of 5
HSA 3•~106
consecutive respectively.
Sarcoma-180. 1,
intraperitoneal C
0.3
inoculation
HSA
Group
days
received
intraperitoneally of
C intraperitoneally tumor
consecutive
2,
intraperitoneal
Mitomycin
3•~106
7
after
7
Group
inoculation
Mitomycin of
mg
for
cells;
days
received
inoculation for
intraperitoneally
consecutive
regimen
0.02
Sarcoma-180.
3•~106
intraperitoneal
combined
intraperitoneally
on HSA
of
for
before
Group
on
with
of
HSA
intraperitoneally
days
HSA
of Extract
cells.
intraperitoneal
tumor
of
intraperitoneally
intraperitoneal with
Activity
the
same
7
days mg of
manner
after of
HSA
3•~106 as
in
250
H. Sato
The combined experiment
III
C alone. Mitomycin
effect of HSA
the survival
et al.
plus Mitomycin
time became
longer
C is demonstrated in the mice treated
In addition it was found that in the group treated C the survival time became markedly prolonged.
in Fig. 3.
In
with Mitomycin
with
both
HSA
and
TABLE 1.
Antitumor activity of HSA on Ehrlich carcinoma
Group
A:
Injected for
intraperitoneally
7
inoculation Group
B:
4.
Antitumor
Group
1,
activity injected
7
intraperitoneal t oneally with of
of
HSA
on
0.3
mg
tumor
of
HSA
days
for
7
Group
before
and
after
mg
of
HSA
cells.
of
tumor
injected
HSA
for
cells;
consecutive
3,
mg
intraperitoneal
carcinoma 0.3
1.0•~105
0.25
after tumor
Ehrlieh with
of
cells;
1.2•~107
of HSA
cells.
with days
mg
intraperitonal
tumor
consecutive
intraperitoneally inoculation
1.0•~105
consecutive
of
1.2•~107
0.25
before
intraperitoneally
inoculation
Fig.
days
of
Injected for
with
consecutive
7
days
days
. injected intraperitoneal
after
before
2
intraperitoneally
intraperitoneal
consecutive
Group
inoculation
with
0
of
1
.3 mg .0•~105
intraperi inoculation of
HSA
for
tumor
5
cells
respectively.
Antitumor effect on Ehrlich carcinoma Table n
this
1
obtained which size longer ever tumor
either was
of
the
in
the
treated
with was
HSA
mice
which
was
observed
received in
had
after to
group
effect
of
inoculated received
as after treated
of in
HSA .
HSA
inoculation
1.0•~101, HSA
the
the
were
which
reduced
the
of
cells
group
effect cells.
results
1.2•~107
inoculum in
no
summarizes
experiment,
Fig
inoculatio with
on
Ehrlich
No before
the . 4
antitumor in oculation tumo r cells.
the
survival
carci
noma. I
effect or
was in
that
When time
n of the tumor cells HSA b efore inoculation
the became
.
How of
the
Antitumor
Table 2 demonstrates tion with HSA. which
received
more
markedly
the results
The survival
alone was longer,
when HSA
Activity
from
BCG
251
of the effect of cyclophosphamide
time of the mice which had received
compared
with those
in addition
prolonged
of Extract
to
than
of the control
group.
cyclophosphamide,
in the
in combina
cyclophosphamide
the
In the
survival
cyclophosphamide-alone
groups
time
was
groups.
TABLE 2.
Effect of combined regimen of cyclophosphamide and HSA on Ehrlich carcinoma
CY, cyclophosphamide.
Groups 2 and 3 received 2 mg and 4 mg, respectively, of cyclopho sphamide by intraperitoneal injection 2 days after intraperitoneal inoculation of 2 X 108 tumor cells. Groups 4 and 5 received tion to cyclophosphamide 2 and 3.
0.3 mg of HSA intraperitoneally in addi given in the same manner as in Groups
DISCUSSION It
is
well
effects.
Hiu
adjuvant
on
(1973,
known
1975),
(1974)
demonstrated
antitumor
al.
found
activity
repeated
several
effect ther
of effective was the
after made
when very
combined
cyclophosphamide
slight
inoculated
1 •~ 105
cells
when
is due
likely
immunotherapy
of
to
HSA a
cells plus
decrease
al.
the
low
of
duration
for
of the
present
not
study
by
large HSA
but
the
known
Mitomycin
cells
have a
inoculated,
as
water material
satisfactory
when
is
was exerts
There
effective
tumor
effect
yield of
such
the
mycobacterial no
It
al.
reported
adjuvant
were
drugs number
as
quantity
inoculated.
et
concerning
from
the
very
Juy
prepared
In
a
al.
macrophages
reports
less
et
only
experiment.
1974).
antitumor in
and this
carcinoma were
Mathe
experiment,
is
et
Ehrlich
was few
a large
in
of
peritoneal
water-soluble
dose
water-soluble
showed
Presumably
the
(Bast
the
this
the
established
1.2•~107 of
in
prepare
reasons be
of
antitumor
a
injection.
adjuvants
once that
to
Hiu
single
a
injections.
that
are
effect
is
similar
stating cells
only
difficult
not
only
to
of
reports
a
which
been
water-soluble
repeated it
could
reports tumor
it
For
HSA
the given
Thus
has
with
of was
injections.
treatment
number
vivo
only
adjuvants
effect
have
the of
adjuvant
there
HSA
activity
soluble
antitumor
water-soluble
Sarcoma-180.
antitumor
was
in
When
on
been
the
However
to
by
related
effect
substance
administered
vitro
closely
According
water-soluble
in
is
antitunior
cells.
when
an by
et
activity
remarkable
tumor
activity
preparations.
for
leukemic
stimulated Adam
adjuvant a
mycobacterial
antitumor
by
the
reported
L1210
slight
when
that
(1972)
the
whe C and effect
of
252
H. Sato et al.
cytostatic which
agents
had
been
or to the
recovery
depressed
by antitumor
of the
function
of reticuloendothelial
systems
drugs.
The reason why the injection of HSA prior to the inoculation of tumor cells was ineffective remains to be clarified. Experiments are now being carried out along this line. References 1)
Adam, a
A.,
Ciorbaru,
Adam, F.,
smegmatis. A.,
Parant, of
Bast,
R.C.,
Bekierkunst,
5)
A.
factor
57,
963-964.
Hiu,
I.J.
Juy,
(1972)
un
&
from
Immun.,
Bona,
C.
&
H.J.
of
New
Chedid,
L.
properties
cell
wall
of
of
My
851-854. L.,
Lamensans,
A.,
Preparation
delipidated
and
cells
of
Parant, biological
Mycobacterium
855-861.
(1974)
BCG
and
cancer
(First
of
two
1413-1420.
in
cancer
nonliving
mycobacteria
medium.
fraction
238,
J.
from
BCG
and
nat.
Cancer
with
adjuvant
Inst.,
and
241-242.
Effet
d'origine
with
aqueous
lipid-free
Biol.,
(1974)
hydrosoluble
69,
(1973)
7,
and
from
Chedid,
fractions
and
Nature
Isolation
(Wash.), E., F.M.
Rapp,
290,
Water-soluble
adjuvant
Sci.
Immunotherapy
activity.
D.,
par
T.
Med.,
(1976)
(1972) fraction
Berger,
Infect.
Borsos, J.
E.
Lederer,
(trehalose-6,6•Œ-dimycolate)
antitumor 6)
Acad.
&
opaca.
B.,
Engl.
Lederer,
J.F.,
adjuvant
Nocardia
Zbar,
nat.
J.F.
water-soluble
New
cord
&
immunoadjuvant
Petit,
Rosselet,
and
parts). 4)
R.,
M.,
smegmatis
J.F.
Proc.
Ciorbaru,
properties
3)
Petit,
water-soluble
cobacterium 2)
R.,
macromolecular,
antitumoral
de
mycobacterienne.
macrophages
C.R.
peritoneaux
Acad.
Sci.
(Paris),
278,
2859-2862. 7)
Mathe,
G.,
Kamel,
experimental
M.,
immunotherapy. 8)
Mathe, I.J.
Bourut, for
Meyer, from
11)
J.
Sato,
H.,
low
K.
&
cells
of
the
conjugates
by
Cellular
Immunol.,
500-508.
M.A.,
Lett.,
(1973)
H.,
V. by
, H., biological
bovis
response
a
25,
Lett.,
Nagai
and
strain
of with
7,
(1973) in
Kamel,
EORTC-ICIG
Cancer,
11,
Biologically regression
An cancer
of
M.,
Hiu,
experimental 801-807. active
components
strain-2
guinea
adjuvant-active simple
pig
& guinea the
mycobacterial
extraction
technique
of
the
301-304.
Hydrosoluble
FEBS
pretreatment
FDeutsch,
guinea-pigs
and
by
M .
immune
bacilli.
in
Arai,
J.
A hydrosoluble,
FEBS
Mycobacterium
hapten
munity
(1972)
P.
M.,
of
B . (1974)
. preparation
Chemical
C.
applicable
103-111.
Constanti-Sourojon, of
Wainberg,
52, P.
Bourut,
Bruley-Rosset,
Europ.
Zbar,
Suppression
weight.
A.,
&
II.
Jolles,
molecular
(1976)
B.E.,
modulation
adjuvant. I.
Peurois).
Yokosawa,
Shlomo,
generation
Inst.,
& Jolles,
(strain D.
of
Konno,
second
-peptidoglycan' cells
delipidated
14)
D.
I.,
The
Azuma,
Cancer
Migliore-Samour,
&
13)
nat.
&
immunity"
1987-1997. Florentin,
wall.
0. of
0.,
immunity E.E.,
Halle-Pannenko, adjuvants
33,
(1975)
cell
Migliore-Samour, "polysaccharide
fractions 12)
Ribi,
mycobacterial
bacterial
Res.,
systemic
T.J.,
hepatoma. 10)
C.
M.,
"systemic
Halle-Pannenko,
screening 9)
for
Cancer
G., &
Dezfulian,
screening
35,
adjuvant-active
mycobacterial
317-321.
Kurita,
K.,
Kumano,
properties BCG Weiss,
of
. Tohoku D.W.
pigs MER
to
N.,
hot J.
exp.
(1973) poorly
fraction
of
Motomiya,
water
M.
extract
Med.,
from
120,
75-81.
Potentiation
and
imnunogenic
protein
attenuated
tubercle
370-379. & Weiss
methanol
, D.W. extraction
(1976) residue
Stimulation of BCG
of . Brit.
anti-tumor
im
J.
34,
Cancer,
J. exp.
Med.,
1978,
Antitumor
Activity
Delipidated
BCG
HIROSI
125, 247-252
of
Hot-Water
Extract
SATO, ATSUNOBU YOKOSAWA, HIDEO
NAGAI,
NOBUKO
KIYOSHI
KONNO
KUMANO,
MASAKICHI
from
ARAI,
HIROMI
MOTOMIYA
and
The Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai 980
SATO, H., YOKOSAWA,A., ARAI, H., NAGAI, H., KUMANO, N., MOTOMIYA, M. and KONNO, K. Antitumor Activity of Hot Water Extract from. Delipidated BCG. Tohoku J. exp. Med., 1978, 125 (3), 247-252The antitumor activity of hot water extract of delipidated BCG was investigated in mice inoculated with Sarcoma-180 cells and Ehrlich carcinoma cells, respectively. The hot-water extract was found to be effective when administered after and ineffective when administered before the inoculation of tumor cells. When this extract was given with anticancer drugs, such as Mitomycin C and cyclophosphamide, a combined effect was obtained in the treatment of Sarcoma-180 and of Ehrlich carcinoma. water-soluble adjuvant; BCG
The mycobacterial preparations such as whole bacilli of BCG, cell wall skeleton (Meyer et al. 1974), methanol extraction residue (Shlomo et al. 1973), and cord factor (trehalose dimycolate) (Bekierkunst 1976), which are all adjuvant-active, have been employed in the treatment of neoplasms in recent years. Also the adjuvant activity of these preparations is known to be essential for their antitumor activity. Since 1972, several water-soluble substances from delipidated mycobacteria (Adam et al. 1973; Migliore-Samour and Jolles 1972, 1973) and cell walls (Adam et al. 1972) have been reported to possess adjuvant activity. The hot-water extract obtained from defatted Mycobacterium bovis strain BCG was found to have adjuvant activity and was designated as HSA (Hot-water Soluble Adjuvant) in our previous paper (Sato et al. 1976). In the inoculum
present
study
of Sarcoma-180
the
antitumor
and
Ehrlich
effect
of the
carcinoma
HSA
in mice
on the
intraperitoneal
was investigated.
MATERIALS AND METHODS Throughout
the
experiments,
4-week-old
ddI
mice
of both
sexes
weighing
22-24
g
were employed. Sarcoma-180 and Ehrlich carcinoma in ascitic form have been maintained in ddI mice by weekly inoculations. In each experiment the suspension of the tumor cells was diluted with saline immediately inoculated intraperitoneally. The test. Received
for publication,
before viability
December
use and the desired number of the cells were of cells was examined by the dye exclusion
7, 1977. 247
248
H. Sato et al.
The hot-water extract was prepared as previously reported taken up in 0.25 ml of saline and injected intraperitoneally.
(Sato et al. 1976) and was
Antitumor effect on Sarcoma-180 Experiment received of
3•~108
HSA as
I.
Mice
were
intraperitoneal tumor
for a
cells.
The
7 consecutive
control
(Fig.
divided
injections
The
after
3
equal
HSA
for
group
was
inoculation
number
of
the
of
mice.
was
The
days
with
tumor
survivors
20
consecutive
treated
of
the
groups 7
first
before
intraperitoneal
cells.
The
recorded
in
group
inoculation injections
third the
group
above
of
was
three
used groups
1). Experiment ‡U.
Eighty
mice
four
groups
Group
1
The
fourth
before
(control, group
and
(Group
after
3 in
inoculation
with
3•~108
saline
tumor
at
1,
received The
7
cells.
3,
5
group groups.
days
after
used
as
above
cells
Sixty
of
7
size
consecutive
of
for
control
5
4 mg
inoculation the
days
from
compared
a
the
day
for
5
of
20
of
Mitomycin
tumor
after
in
of
the
Experiment
I.
consecutive
days
the
mice
were
cells.
The
inoculation same
inoculated
C (1
of
the
as
in
manner
mg/kg)
second
in group
tumor the
cells. first
and
control.
the
manner
to
days
among
after
The in
groups
HSA
every
same
dose
third
time
were
group
terms
of day of
days
20
mice.
before HSA
received
of
the
2
and
experimental
were
other two of
saline
received
saline
The former
the after
of
mice
for
7
saline
was
Group
A
inoculation
of
consecutive
alone
compared
and
was
among
the
two
groups
tumor and
the
of
cells.
20
mice
Group
each. 1
inoculation
of
tumor
inoculation
of
the
inoculation to
groups and
the
groups
In
this
received cells.
tumor
HSA Group
3
cells.
The
alone.
an given
groups
4
1.0•~10,
after
before
injections
in
into to
Group
hundred
the
divided reduced
days
inoculation
four
equal
of the
cells.
and
cells.
as in
3
mg
received
was
received
tumor
into
0.25
survival
cyclophosphamide
2
1
HSA
divided
B
mice
One
of same
in
1).
prior
group
of
mice. Three as
HSA
injection
of
as
of
tumor
conseuctive
Experiment ‡V. and
of
consisting an
from and
served
inoculum
days
HSA
fourth
HSA
were
the
Eighty
the
received
of 20
treated
cells. each
received
C
groups
were
injection
inoculation
of
mean
(Table
Experiment ‡U.
for
2)
an
mice
after
Group
The
groups
experiment
group
mice
(1.2•~107).
control.
three
first
4 equal
Fig.
tumor of
injections
inoculation a
in
effect on Ehrlich carcinoma
intraperitoneal
tumor
3•~108
group
into
2
received
Mitomycin
fourth
I.
7
2)
injections
The
Experiment received
Fig.
7 days
received
Antitumor
the
and
divided
Group
groups The
consecutive
third
second
Four
were
and
of
Experiment ‡V.
in
the
second
days
group.
into
of
two
of groups
were
2•~106 of
injected
were
treated
cells.
The
control
with with
at
cells 2
.
days
2
after
cyclophosphamide
HSA
numbers
(Table
tumor
mice
for of
7 consecutive survivors
were
2).
RESULTS
Antitumor effect on Sarcoma-180 Fig. 1 shows the results of the experiment in which HSA was injected for 7 consecutive days each before or after inoculation of Sarcoma-180 cells. The survival time of the mice received HSA before inoculation of the tumor cells was prolonged only slightly. On the other hand an increase of survival time was evident in the mice treated with HSA after inoculation of the sarcoma, hells The results summarized mice
that
of another
in Fig. 2. had
received
experiment
In this HSA
in which
experiment before
survival
inoculation
HSA
alone
was administered
time was not prolonged of
the
group which received HSA for 5 consecutive days before the tumor cells only a very slight effect was observed .
tumor and
are in the
cells . In after inoculation
the of
Antitumor
Fig.
1.
Antitumor
Group
1,
activity
injected
Fig.
inoculation
0.25
106
tumor
2.
Antitumor
Group
mg
of
Fig.
3.
for
activity
1,
received
0.3
7
from
Sarcoma-180
3•~106
0.25
tumor
BCG
249
. mg
of
cells;
consecutive
of
HSA
mg
of
inoculation
cells;
Group and
Effect
of
1,
7
given
intraperitoneal
3,
after
HSA
for
Group
days
7 consecutive
2
, injected intraperitoneal
after
days
before
intraperitoneally inoculation of
3•~
mg
tumor
cells;
Group
I
and
Group Group
2.
tumor
0.3
of
of
consecutive
3,
given
of
HSA
both
C and
3•~
days Mitomycin
cells;
after
for
106
tumor
on at
and
HSA
mg
cells,
2,
3,
5
and
given
0.25
inoculation in
before of
of 5
HSA 3•~106
consecutive respectively.
Sarcoma-180. 1,
intraperitoneal C
0.3
inoculation
HSA
Group
days
received
intraperitoneally of
C intraperitoneally tumor
consecutive
2,
intraperitoneal
Mitomycin
3•~106
7
after
7
Group
inoculation
Mitomycin of
mg
for
cells;
days
received
inoculation for
intraperitoneally
consecutive
regimen
0.02
Sarcoma-180.
3•~106
intraperitoneal
combined
intraperitoneally
on HSA
of
for
before
Group
on
with
of
HSA
intraperitoneally
days
HSA
of Extract
cells.
intraperitoneal
tumor
of
intraperitoneally
intraperitoneal with
Activity
the
same
7
days mg of
manner
after of
HSA
3•~106 as
in
250
H. Sato
The combined experiment
III
C alone. Mitomycin
effect of HSA
the survival
et al.
plus Mitomycin
time became
longer
C is demonstrated in the mice treated
In addition it was found that in the group treated C the survival time became markedly prolonged.
in Fig. 3.
In
with Mitomycin
with
both
HSA
and
TABLE 1.
Antitumor activity of HSA on Ehrlich carcinoma
Group
A:
Injected for
intraperitoneally
7
inoculation Group
B:
4.
Antitumor
Group
1,
activity injected
7
intraperitoneal t oneally with of
of
HSA
on
0.3
mg
tumor
of
HSA
days
for
7
Group
before
and
after
mg
of
HSA
cells.
of
tumor
injected
HSA
for
cells;
consecutive
3,
mg
intraperitoneal
carcinoma 0.3
1.0•~105
0.25
after tumor
Ehrlieh with
of
cells;
1.2•~107
of HSA
cells.
with days
mg
intraperitonal
tumor
consecutive
intraperitoneally inoculation
1.0•~105
consecutive
of
1.2•~107
0.25
before
intraperitoneally
inoculation
Fig.
days
of
Injected for
with
consecutive
7
days
days
. injected intraperitoneal
after
before
2
intraperitoneally
intraperitoneal
consecutive
Group
inoculation
with
0
of
1
.3 mg .0•~105
intraperi inoculation of
HSA
for
tumor
5
cells
respectively.
Antitumor effect on Ehrlich carcinoma Table n
this
1
obtained which size longer ever tumor
either was
of
the
in
the
treated
with was
HSA
mice
which
was
observed
received in
had
after to
group
effect
of
inoculated received
as after treated
of in
HSA .
HSA
inoculation
1.0•~101, HSA
the
the
were
which
reduced
the
of
cells
group
effect cells.
results
1.2•~107
inoculum in
no
summarizes
experiment,
Fig
inoculatio with
on
Ehrlich
No before
the . 4
antitumor in oculation tumo r cells.
the
survival
carci
noma. I
effect or
was in
that
When time
n of the tumor cells HSA b efore inoculation
the became
.
How of
the
Antitumor
Table 2 demonstrates tion with HSA. which
received
more
markedly
the results
The survival
alone was longer,
when HSA
Activity
from
BCG
251
of the effect of cyclophosphamide
time of the mice which had received
compared
with those
in addition
prolonged
of Extract
to
than
of the control
group.
cyclophosphamide,
in the
in combina
cyclophosphamide
the
In the
survival
cyclophosphamide-alone
groups
time
was
groups.
TABLE 2.
Effect of combined regimen of cyclophosphamide and HSA on Ehrlich carcinoma
CY, cyclophosphamide.
Groups 2 and 3 received 2 mg and 4 mg, respectively, of cyclopho sphamide by intraperitoneal injection 2 days after intraperitoneal inoculation of 2 X 108 tumor cells. Groups 4 and 5 received tion to cyclophosphamide 2 and 3.
0.3 mg of HSA intraperitoneally in addi given in the same manner as in Groups
DISCUSSION It
is
well
effects.
Hiu
adjuvant
on
(1973,
known
1975),
(1974)
demonstrated
antitumor
al.
found
activity
repeated
several
effect ther
of effective was the
after made
when very
combined
cyclophosphamide
slight
inoculated
1 •~ 105
cells
when
is due
likely
immunotherapy
of
to
HSA a
cells plus
decrease
al.
the
low
of
duration
for
of the
present
not
study
by
large HSA
but
the
known
Mitomycin
cells
have a
inoculated,
as
water material
satisfactory
when
is
was exerts
There
effective
tumor
effect
yield of
such
the
mycobacterial no
It
al.
reported
adjuvant
were
drugs number
as
quantity
inoculated.
et
concerning
from
the
very
Juy
prepared
In
a
al.
macrophages
reports
less
et
only
experiment.
1974).
antitumor in
and this
carcinoma were
Mathe
experiment,
is
et
Ehrlich
was few
a large
in
of
peritoneal
water-soluble
dose
water-soluble
showed
Presumably
the
(Bast
the
this
the
established
1.2•~107 of
in
prepare
reasons be
of
antitumor
a
injection.
adjuvants
once that
to
Hiu
single
a
injections.
that
are
effect
is
similar
stating cells
only
difficult
not
only
to
of
reports
a
which
been
water-soluble
repeated it
could
reports tumor
it
For
HSA
the given
Thus
has
with
of was
injections.
treatment
number
vivo
only
adjuvants
effect
have
the of
adjuvant
there
HSA
activity
soluble
antitumor
water-soluble
Sarcoma-180.
antitumor
was
in
When
on
been
the
However
to
by
related
effect
substance
administered
vitro
closely
According
water-soluble
in
is
antitunior
cells.
when
an by
et
activity
remarkable
tumor
activity
preparations.
for
leukemic
stimulated Adam
adjuvant a
mycobacterial
antitumor
by
the
reported
L1210
slight
when
that
(1972)
the
whe C and effect
of
252
H. Sato et al.
cytostatic which
agents
had
been
or to the
recovery
depressed
by antitumor
of the
function
of reticuloendothelial
systems
drugs.
The reason why the injection of HSA prior to the inoculation of tumor cells was ineffective remains to be clarified. Experiments are now being carried out along this line. References 1)
Adam, a
A.,
Ciorbaru,
Adam, F.,
smegmatis. A.,
Parant, of
Bast,
R.C.,
Bekierkunst,
5)
A.
factor
57,
963-964.
Hiu,
I.J.
Juy,
(1972)
un
&
from
Immun.,
Bona,
C.
&
H.J.
of
New
Chedid,
L.
properties
cell
wall
of
of
My
851-854. L.,
Lamensans,
A.,
Preparation
delipidated
and
cells
of
Parant, biological
Mycobacterium
855-861.
(1974)
BCG
and
cancer
(First
of
two
1413-1420.
in
cancer
nonliving
mycobacteria
medium.
fraction
238,
J.
from
BCG
and
nat.
Cancer
with
adjuvant
Inst.,
and
241-242.
Effet
d'origine
with
aqueous
lipid-free
Biol.,
(1974)
hydrosoluble
69,
(1973)
7,
and
from
Chedid,
fractions
and
Nature
Isolation
(Wash.), E., F.M.
Rapp,
290,
Water-soluble
adjuvant
Sci.
Immunotherapy
activity.
D.,
par
T.
Med.,
(1976)
(1972) fraction
Berger,
Infect.
Borsos, J.
E.
Lederer,
(trehalose-6,6•Œ-dimycolate)
antitumor 6)
Acad.
&
opaca.
B.,
Engl.
Lederer,
J.F.,
adjuvant
Nocardia
Zbar,
nat.
J.F.
water-soluble
New
cord
&
immunoadjuvant
Petit,
Rosselet,
and
parts). 4)
R.,
M.,
smegmatis
J.F.
Proc.
Ciorbaru,
properties
3)
Petit,
water-soluble
cobacterium 2)
R.,
macromolecular,
antitumoral
de
mycobacterienne.
macrophages
C.R.
peritoneaux
Acad.
Sci.
(Paris),
278,
2859-2862. 7)
Mathe,
G.,
Kamel,
experimental
M.,
immunotherapy. 8)
Mathe, I.J.
Bourut, for
Meyer, from
11)
J.
Sato,
H.,
low
K.
&
cells
of
the
conjugates
by
Cellular
Immunol.,
500-508.
M.A.,
Lett.,
(1973)
H.,
V. by
, H., biological
bovis
response
a
25,
Lett.,
Nagai
and
strain
of with
7,
(1973) in
Kamel,
EORTC-ICIG
Cancer,
11,
Biologically regression
An cancer
of
M.,
Hiu,
experimental 801-807. active
components
strain-2
guinea
adjuvant-active simple
pig
& guinea the
mycobacterial
extraction
technique
of
the
301-304.
Hydrosoluble
FEBS
pretreatment
FDeutsch,
guinea-pigs
and
by
M .
immune
bacilli.
in
Arai,
J.
A hydrosoluble,
FEBS
Mycobacterium
hapten
munity
(1972)
P.
M.,
of
B . (1974)
. preparation
Chemical
C.
applicable
103-111.
Constanti-Sourojon, of
Wainberg,
52, P.
Bourut,
Bruley-Rosset,
Europ.
Zbar,
Suppression
weight.
A.,
&
II.
Jolles,
molecular
(1976)
B.E.,
modulation
adjuvant. I.
Peurois).
Yokosawa,
Shlomo,
generation
Inst.,
& Jolles,
(strain D.
of
Konno,
second
-peptidoglycan' cells
delipidated
14)
D.
I.,
The
Azuma,
Cancer
Migliore-Samour,
&
13)
nat.
&
immunity"
1987-1997. Florentin,
wall.
0. of
0.,
immunity E.E.,
Halle-Pannenko, adjuvants
33,
(1975)
cell
Migliore-Samour, "polysaccharide
fractions 12)
Ribi,
mycobacterial
bacterial
Res.,
systemic
T.J.,
hepatoma. 10)
C.
M.,
"systemic
Halle-Pannenko,
screening 9)
for
Cancer
G., &
Dezfulian,
screening
35,
adjuvant-active
mycobacterial
317-321.
Kurita,
K.,
Kumano,
properties BCG Weiss,
of
. Tohoku D.W.
pigs MER
to
N.,
hot J.
exp.
(1973) poorly
fraction
of
Motomiya,
water
M.
extract
Med.,
from
120,
75-81.
Potentiation
and
imnunogenic
protein
attenuated
tubercle
370-379. & Weiss
methanol
, D.W. extraction
(1976) residue
Stimulation of BCG
of . Brit.
anti-tumor
im
J.
34,
Cancer,
