Prostaglandin D2, a potential antineoplastic agent

Vol. 105, No. 3, 1982 April

BIOCHEMICAL

AND BIOPHYSICAL

14, 1982

RESEARCH COMMUNICATIONS Pages 956-964

PROSTAGLANDIN D2, A POTENTIAL ANTINEOPLASTIC Masanori

AGENT*

Fukushima, Taket'oshi Kato, Ryuzo Ueda, Kazuo Ota, Shuh Narumiyat, and Osamu Hayaishif Department of Internal Medicine, and Laboratory of Chemotherapy, Aichi Cancer Center, Chikusa-ku, Nagoya 464, Japan and tDepartment of Medical Chemistry, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606, Japan

Received

March

2,

1982

Cytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D2 (PGD2) was found most active. PGD2 exerted a dose dependent inhibition of L1210 cell growth over 3.6 PM. At 14.3 PM growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC58 value of PGD2 on L1210 cell growth was calculated to be 6.9 UM (2.4 pg/ml), and at this concentration the DNA synthesis in 2a hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD2 was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA2 showed a growth inhibitory accomparable, and PGE2 a less but significant while PGB2, PGF2iX and PG12 had no such effects on cell tivity, These results suggest a proliferation at 14.3 PM concentration. potential antineoplastic activity of PGD2. INTRODUCTION Prostaglandin of platelet its

biological

prostaglandins

D2 (PGD2) has been described (l-3)

aggregation function

is

and a neuromodulator

not yet

of many lines

of tumor

for

PGD2, an analogue

review).

fully

both

understood.

in vitro --

and -in vivo

However, Since

1972,

the growth (see 5, 6

of PGE2, has been demonstrated

* This work was supported in part Research and for Scientific Research tion, Science and Culture, Japan. Abbreviations; PG, prostaglandin. for 50% growth inhibition. 0006-291X/82/070956-09$01.00/0 Copyrighi 0 1982 bv Academrc Press. Inc. .AII ri~qhrsof reproducrion VI 0n.vjorrn reserved.

(4).

are known to inhibit

of E and A series cells

as an inhibitor

956

Grants-in-aid from the Ministry by

1C50, concentration

for of

to

Cancer Educaneeded

Vol. 105, No. 3, 1982

BIOCHEMICAL

inhibit

of B-16 melanoma cells,

metastasis

been ascribed believed

inhibition

tumor

reported

that

was inhibited

by the

These reports

tumor-growth

metastasis the

(7,8).

this

effect

aggregation Recently

of cultured

of

has which

is

Kawamura and

mastocytoma

cells

PGD2, a major

PG in these

cells

PGD2 might

have a general

that

activity.

Here we present

activity

such as L1210 mouse leukemia

kemia cells

but

growth

suggested

cytotoxic

RESEARCH COMMUNICATIONS

of platelet

addition

inhibitory

PGD2 has a potent cells

its

to favor

Koshihara

(9).

to

AND BIOPHYSICAL

on several cells

evidence

lines

and several

that

of tumor human leu-

in vitro.

MATERIALS AND METHODS Chemicals-Synthetic prostaglandins, PGA2, PGB2, PGD2, PGE2, PGFza, and PG12eNa, were the generous gifts from Ono PharmaThe purities of PGD2 and other ceutical Co. Ltd., Osaka, Japan. PGs were over 96.9% and 99.9%, respectively, as judged by hiqh [6-3H]Thymidine (21 Ci/mmolj pressure liquid chromatography. [6-3H]uridine (15 Ci/mmol) and L-[4,5-3Hlleucine (150 Ci/mmolj were purchased from the Radiochemical Centre, Amersham, England. Fetal calf serum was from GIBCO. All other chemicals were of analytical grade. Stability of PGD2 in aqueous media was determined by incubation in phosphate buffer, pH 7.0, at 37". As judged by high pressure liquid chromatographic profile, the remaining PGD2 was incubation of 3, 6, 24, and 71.9%, 57.7%, 15.3%, and 7.2% after 48 hr, respectively. Cell Culturedell lines used were L1210 mouse leukemia cellsmaintained in suspension in our laboratory since 1976, and human leukemia cell lines such as NALL-1 (nonT nonB cell origin) (lo), RPM1 8402 (T cell origin) (ll), RPM1 8226 (myeloma cell origin) (12), and Sk-Ly-16 (B cell origin) which were kindly supplied by Dr. L. 3. Old, Memorial Sloan-Kettering Cancer Center, N.Y. Cells were cultured in RPM1 1640 medium supplemented with 10% fetal calf serum. PGs were dissolved in 99.5% ethanol and diluted in culture media immediately before use with a final concentration of 0.1% ethanol. Media containing PGs were sterilized by Millipore filtration. Control media contained 0.1% ethanol. L1210 cells were plated at a density of 1 X 105 cells/ml in the medium containing PG, and grown in suspension for-4 days. Human leukemia cells were plated at a density of 5 X 103 cells/well and grown in culture plates (24 wells, Falcon) with 2 nl of media under the humidified atmosphere of 5% CO2 in air. Media containing PG were changed every 3 days. Cell viability was measured by trypan blue exclusion at times indicated. Measurement of DNA, RNA, and Protein Syntheses---Gne ml aliquots of L1210 culture cells were taken into c'1 culture flask (Falcc )n 2003) at indicated times and incubated for 2 hr with 1 LlCi of I:3~~1thymidine, L3Hluridine, or L3H]leucine at 37' ..-in a 1 -c _^^_ L--n -c CD c.n 1- -1 -~ -.. 957

Vol. 105, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

24

0

48

Culture

72 Time

RESEARCH COMMUNICATIONS

96

(hrs)

Fiq. 1. Growth curve of L1210 cells in the presence and absence of PGD2. Cells were cultured as described under Materials and Methods. o-------o: with 5 ug/ml with 2.5 uq/ml of PGDz, A----A: of PGD2, .B: control.

by the addition of ice-cold 0.85% NaCl and cells were washed twice with saline. The radioactivity incorporated into acid-inBiosoluble materials were counted as described previously (13). syntheses of these macromolecules were expressed in terms of 1 X lo5 viable cells. RESULTS Fig.

Effect

of PGD2 _ on Proliferation of -- L1210 Cells. in the presence shows the growth curve of L1210 cells of

L1210 cells

PGD2.

grew exponentially

with

a doubling

time

of approximately

then

the

rate

decreased.

growth

of L1210 cells 7.1 @I (2.5 14.3

p'M_(5

lated

~JM -

uM, - cells

Over

10.6

with

irregular

plasm,

PGD2 for

was observed to be 6.9

12 hr until

level

villi

over (2.4

4 days, 3.6

under

showed degenerative formation

and the number of viable

3 days,

control

the

958

at

completely

almost

inhibition

conditions

at

various

ICso value

conof

was calcuwe examined.

changes microscopically

and many vacuoles cells

rate

cells

were grown with

2).

and

the growth

a dose-dependent

@I (Fig.

pg/ml)

of the

the growth

When L1210 cells

ug/ml).

of

of the

or absence

a 24 hr lag phase

PGD2 decreased

and inhibited

pg/ml),

centrations growth

to a half

after

1

decreased

in their during

cyto-

culture

at

BIOCHEMICAL

Vol. 105, No. 3, 1982

2

1

AND BIOPHYSICAL

5 Prostoglandin

RESEARCH COMMUNICATIONS

10 20 D2 (PM)

cells Fig. 2. Dose response curve of PGD2. ~1210 leukemia were cultured in the presence of various concentration of PGD2 as After 4 days culture, number of described Materials -and Methods. In this experiment, cells which exclude trypan blue was co nted. 2 100% represent cell number of 281 X 10 cells/ml.

24.8

uM; the number decreased -

to

after

7 X lo4 cells/ml

4 days of

culture. Specificity cytotoxic

of PGs for -----___

activities

of other

and PG12 were examined PGD2 (Table

Ll210

The ICso value

which less

was almost potent

equal

than

(5 uq/ml).

The

Inhibition.

PGs such as PGA2, PGB2, PGE2, PGFzU,

reported

and compared with with

PGA2 and PGE2 showed a significant cells.

Growth

on L1210 cells,

As already

I).

Cell

cytotoxic

other

cell

lines

activity about

to that

PGE2 was about

PGD2 in this

system

with

(5,6),

PM - (2.5

IC50 around

and PGI2 had no effects

PGB2, PGF2,,

jlq/ml)

two times

14.3

PM_

the

pres-

under

ent conditions.

When PG12 or PGFZn were added simultaneously

with

same amounts,

PGD2 in the

inhibition did

not

of L1210 cells, interfere

Spectrum Lines.

The

with

the

-of Cytotoxicity

effect

no change was found

suggesting action of --

that

either

of PGD2 at the

of

on L1210

of PGA2 was found of PGD2.

7.1

that

in the

growth

PG12 or PGF2n cellular

level.

PGD2 ______ among Human Leukemia Cell of PGD2 was also examined on human leukemia 959

Vol. 105, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL Table

Effect ___-

of prostaglandins

I

on -- L1210

leukemia

ilg/ml

X

lo4

none

tested,

of cells

PGF2

5

277

285

PGB2

5

211

218

PGE2

5

138

155

10

26

30

117

132

5

11

17

5

19

26

+PGI2

5/5

20

22

+PGF2

5/5

13

14

survived

indicated

cells

a strong

of

seen at

of culture in are described

(5 ug/ml).

number decreased

observations

remaining

4 days Details

DM - concentration

the culture

was not

By microscopic all

14.3

the cell

and recovery

288 303

Cell numbers were counted after presence of various prostaglandins. Materials and Methods. ______

lines

276 281

PGD2

at

cells/ml

5

2.5

of

PGD2

cell

only

culture

6 to 40% of PGD2,

(Table

II).

was observed

in

These results

in L1210 cells.

action

the under

all

6 days in the presence

a marked degeneration

cytocidal

In

remarkably;

the 8th day of

as observed

growth

2

PG12

PGA2

cells

cell -___

Cell number Exp. 1 Exp.

Addition

culture

RESEARCH COMMUNICATIONS

on

human

leukemia

Syntheses

of --

cells.

Effect Cells.

of PGD2 on DNA, RNA, and __ Protein In order to determine the inhibition

on cell

growth,

L3H]leucine

studied. suppressed

the

effects

incorporation

As shown in Table to

on into

III,

mechanism of PGD2

[3H]thymidine,

[3H]uridine

acid-insoluble

materials

both

54% and 70% of the control 960

and were

DNA and RNA syntheses level,

L1210

respectively,

were

BIOCHEMICAL

Vol. 105, No. 3, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table II of PGD2 on the growth of human leukemia culture ~___ -

Effect

Cell line

Total

Origin

cell

number (X lo4 cells) day 8

day 6 control

cells

control

NALL-1

MonT, B

103

PGD2 3

186

PGD2 1

RPM1 8402

T cell

127

12

245

11

RPM1 8226

Myeloma

78

20

114

25

Sk-Ly-16

B cell

385

7

499

5

Cells, 5 x 105, were plated in 2 ml of culture medium and grown in culture well for indicated times. The medium containing 14.3 PM PGD2 (5 ug/ml) was changed every 3 days. Details are describea under Materials and Methods.

after

24 hr culture

with

56%, respectively,

with

and RNA syntheses ture,

the

supression

proceeded,

but

at

24 hr.

stage

versible

14.3

I.IM_(5 ug/ml).

of the

with

7.1

such recovery

ed to PGD2 both early

UM - PGD2 (2.5

The protein

centration. extent

rates

7.1

As evidenced

after

synthesis

was also suggested

and that

by DNA

48 and 72 hr of cul-

was not observed

DNA and RNA biosyntheses

14.3

and to 57% and

UM - PGD2 was reversed

These results

of exposure

above

cells

ug/ml),

as the

with

14.3

inhibited

that

JJM con-

to a lesser

in the

cells

were interfered

such interference

culture

expos-

in the

became irre-

FM - PGD2 concentration.

DISCUSSION From the a potent cells of same

results

cytotoxic

including

activity cells

PGD2 on L1210 cell concentration

ent experimental

described

of

above,

we conclude

on a wide range human leukemia

growth

as the conditions

act ion of PGD2 was reported

was 2.4

ICSo value (1 to on

2.5

mastocytoma

961

of cultured

origins.

\lg/ml

PGD has 2 leukemia

The ICSO value

which

of bleomycin ug/ml).

that

was about under

Similar cultured

the

the prescytotoxic

ce 11s recent-

Vol. 105, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table III Effect ---2

of PGD on DNA RNA and -' ~ protein - --'

Culture

syntheses of -_ L1213 leukemia cells

PGD2 (ug/ml)

time

0

2.5

5.0

0

2.5

Thymidine

Uridine cells

cpm/105 0

time

2,368

--

--

1,042

--

1,469

1,026

24

hr

3,183

1,726

1,840

48

hr

1,730

2,949

1,182

72

hr

701

5.0

937

384

0

2.5

Leucine

* --

268

--

--

824

282

275

190

122

199

150

111

132

801

734

285

344

373

198

87

* The values represent means of duplicate experiments. DNA, RNA and Protein syntheses were measured as described rials and Methods. -__

ly

Fitzpatrick

19).

action

and Stringfellow

of PGD2 on B-16 melanoma cells

ascribed

to the

inhibition

view

of our present

tory

activity

inhibition tion.

and the

of PGD2 on rodent by PGD2 in vivo

--In vivo

experiments

of PGD2 (0.5

might

remarkably

inoculated

subcutaneously

mice:

tumor

was 1.79

g in PGD2-treated

p < 0.005)

(Kato,

T.,

also

(14),

i.p.

M.,

growth

injection

the growth regions mice,

and Ota,

K.,

of Ehrlicl in ICR

and 0.32 -+

2 weeks of treatment

Fukushima,

in

in our laboratory,

in the axillar

after

However,

the direct

the daily

decreased

they

to such preven-

+ 1.05 q in control

mice

which

weak antiaggrega-

contribute

showed that

tumor

size

relatively

are now in progress

results

mq/kg/day)

(7,8),

aggregation.

platelets

under Mate

an anti-metastatic

in vivo -~

of platelet

findings

and the preliminary

0.26

reported

5.0

(n = 10,

unpublished

ob-

servation). Among other significant with

prostaglandins

growth

the previous

inhibition studies

tested, (Table

reporting

stag land ins of E and A series

the

PGE2 and PGA2 showed

These results

I).

of other 962

only

growth cell

inhibition

types

(5,6).

agreed by proThese

BIOCHEMICAL

Vol. 105, No. 3, 1982

AND BIOPHYSICAL

prostaqlandins

were also

macromolecules

such as DNA, RNA and protein

cultured

(15,16).

cells

reported

to inhibit

Consistent

with

inhibited

the DNA and RNA biosyntheses

synthesis

to a lesser

PGD2 has less

safely

NADH-linked

that

Furthermore,

metabolizing

enzyme,

PGD2 or its

derivatives

which

in several

of lines

reports,

considerably

of

PGD2 also

and protein

III). on

than hiqh

systemic

other

par-

be administered

PGD2 is not metabolized

dehydrogenase,

is abundant

circulation

prostaqlandins

dosescan

because

15-hydroxyprostaglandin

biosyntheses

these

actions

and arrhythmia

PGA2, indicating in vivo. -__

(Table

catastrophic

such as hypotension ticularly

extent

RESEARCH COMMUNICATIONS

in various useful

may be a promising

a major tissues

by PG

(171,

antineoplastic

agent. ACKNOWLEDGEMENT We thank tic their

prostaqland excellent

Ono Pharmaceutical ins,

and Miss

technical

Co. Ltd.

Kyomi Ito

for

supplying

and Ke iko

Nishida

us authenfor

assistance.

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

12. Matsuoka, Y., Moore, G. E., Yagi, Y., and Pressman, D. (1967) Proc. Sci. Exp. Biol. Med.,%, 1246-1250. 13. Fukushima, F Ota,K.,u]imoto, D., and Horiuchi, K. (1980) Biochem. Biophys. Res. Commun. 92, 1409-1414. ~___ 14. Whittle, B. J. R., Moncada, S., and Vane, J. R. (1978) Prostaqlandins 16, 373-388. 15. Eisenbarth, G. S., Wellman, D. K., and Lebovitz, H. E. (1974) Biochem. Biophys. Res. ____ Commun. 60, 1302-1308. 16. Honn, K. V., Dunn II, J. R., Morgan, L. R., Bienkowski, M., Res. and Marnett, L. J. (1979) Biochem. Biophys. -_____Commun. 87, 795-801. 12, 647-679. 17. Hansen, H. S. (1976) Prostaglandins

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