Cyclosporin a inhibits biological effects of tumor promoting phorbol esters

Vol. 126, No. 1, 1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 327-332

January 16, 1965

CYCLOSPORIN A INHIBITS

BIOLOGICAL

M. Gschwendt,

EFFECTS OF TUMOR PROMOTING PHORBOL ESTERS

W. Kittstein,

F. Horn,

and F. Marks

Deutsches Krebsforschungszentrum, Institut fiir Im Neuenheimer Feld 280, D-6900 Heidelberg, Received

November

Biochemie, F.R.G.

26, 1984

The antilymphocytic and antiphlogistic agent cyclosporin A (CsA) inhibits in vivo various effects induced by the tumor promoting phorbol ester 12-O-tetXZf&anoylphorbol-13-acetate (TPA). These include the edema of the mouse ear, the alkaline phosphatase (AP) activity and the ornithine decarboxylase (ODC) activity in mouse epidermis as well as the generation of a specific arachidonic acid (AA) metabolite in mouse epidermis. AA metabolism in an epidermal cell-free system of mouse epidermis was not suppressed by CsA. According to thin layer chromatography the TPA-induced and as yet unidentified AA metabolite exhibits a polarity between that of 5-HETE and 12-/15-HETE. Studies with inhibitors indicate it to be a lipoxygenase product. 0 1985 Academic Press, Inc.

CsA is biology

for

the

the

production

of this

inhibitory

the

phospholipid property

whether

this

activity

whether

both

activities

pound.

Very

activation

metabolism

further

of CsA is

recently

of prostaglandins

to these known.

its

lymphocyte

antiphlogistic

of CsA is

related

are entirely it

the

to inhi-

(3-5).

activity

(1).

CsA recep-

properites

that changes

(7). It

in

A

is not known

antilymphocytic

be due to an inhibition

The mode

the early membrane

suppression

as to

was reported

plasma

independent

was shown that

by CsA might

with

to its

most

ability

however,

and it

by interfering

of the

The immuno-

Probably

lymphokines

(6)

(1).

1 and 2, as well

Recently,

on human lymphocytes

lymphocyte

(2).

of CsA is its

interleukin

e.g.

is not yet

activity

by Thomson

capacity

of T cells

action

demonstrated

CsA inhibits

recently

of lymphokines,

the responsiveness

were

antilymphocytic

immunosuppressive

inhibit

tors

with

of CsA has been reviewed

important bit

an immune suppressant

activity of the

of tissue

or

com-

levels

of phospholipase

A2 (8). Tumor sumably

promotion from

is a single

a complex cell

process that

in which

has been

327

a visible

initiated

tumor

develops

by a carcinogen.

pre-

The

0006-291X/85 $1.50 Copyright 0 I985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 126, No. 1, 1985

most bol

thoroughly esters

effects

studied

esters

of tumor

elucidation

generation other

in this (9).

of phorbol

mechanism.

of an AA metabolite

effects

of tumor

a still

esters

increasing

Inhibitors should

Here we report

phorbol

MATERIAL

promotion

by phornumber

has been described,

as yet.

and on the

promoting

is tumor

and --in vitro

is unknown

effects

of this

respect

Although

--in vivo

promotion

and of various

the

system

in mouse epidermis

of phorbol

mechanism tion

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

promo-

tools

for

on the TPA-induced

inhibition esters

the

of tumor

be valuable

of

by CsA of this on mouse epidermis

and --in vivo.

AND METHODS

Materials:

doz, Basel, Switzerland. German Cancer Research Animals:

Female

NMRI mice

Edema induction Alkaline Ornithine

7 to 8 weeks)

and measurement:

as described

previously

(10).

as described

previously

(11).

(AP)

decarboxylase

assay:

(ODC) assay:

of protein: and analysis

were

Dr.

(age

phosphatase

Determination Generation

TPA was kindly supplied by Prof. Center, Heidelberg, F.R.G.

as described

was carried

out

of arachidonic

according

acid

used in all

by O'Brien to Lowry

E. Hecker, experiments.

et al.

(12).

et al.

(13).

(AA) metabolites:

Epidermal specimens were prepared from frozen back skin of mice and homogenized as described previously (11). The homogenate was centrifuged at 8,000 x g for 1 min. One ml of the supernatant (epidermal cell-fre system; 800 pg protein/ml) was incubated with 10 pl of a 1:l mixture of [I&]AA and unlabeled AA at 370C for 45 min. After cooling in ice, 3 ml of chloroform/ methanol (19:l) were added. After extensive mixing, the phases were separated by centrifugation at 10,000 x g for 5 min. 2.5 ml of the chloroform/methanol phase were evaporated to dryness and redissolved in 30 pl chloroform/methanol (2:l). A 25 pl aliquot was analyzed by thin layer chromatograph on silica 60 (Merck/Darmstadt) using ether/hexane/acetic acid (60:40:1 7 as developing solvent. The chromatographic analysis was recorded by a radiochromatogram scanner (Berthold). RESULTS AND DISCUSSION The inhibition The edema of the

of various mouse ear,

TPA effects which

in vivo

was found

328

by CsA is

to be maximal

shown in 6 h after

Table the

1.

Vol.

126,

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

No. 1, 1985

Table

1:Inhibition

of

various

TPA-effect

Edema

TPA effects

Dose

(mouse

ear)a

of

by CsA in

CsA

Inhibition

25

(mouse

ODC activity

acid (mouse

A single

dose

of

::

skin)b

(mouse

Arachidonic metabolite

(%)

31 75

La 50 w 100 IN 250 u9 AP activity

vivo

1.5 2.5

mg mg

60 100

2.0

mg

92

2.5

mg

100

skin)c

skin)d

CsA,

as

indicated,

was

applied

together

with

a The edema was measured 6 h after TPA treatment. Each mean of 4 determinations. With 1 nmole TPA alone the ear plug increased from 10.45 to 21.54 mg (difference: 0% inhibition). For the procedure see ref. 10.

TPA.

value is the weight of the 11.09 mg =

b AP activity was measured 22 h after TPA treatment. Each value is the mean of 6 determinations. With 20 nmole TPA alone the absorbance at 405 nm increased from 0.77 to 8.80/mg protein (difference: 8.03/mg protein = 0% inhibition). For the procedure see ref. 11. '

ODC activity was measured mean of 2 determinations. increased from 80 to 4537 For the procedure see ref.

d

see

Figure

application

our

1

of 1 nmole TPA (lo),

CsA. Besides activity

the

recent

tested

back

of

20 nmole

mice.

of skin

all

tumor

CsA. The other

Because

of

the

been

promotion

effects

larger

single

treatment

effect

was

mouse

completely

epidermis

of the

skin abolished

was

with

area

increased

tumor if

2.5

329

90% by 100 pg of

demonstrated

inhibitors

of TPA and up to 2.5 mg of CsA were

in

AP activity

with

that

to about

of CsA, an antiphlogistic

has already

we proposed

inhibitors

presently

properties

inflammation

findings,

edema are also

was suppressed

immunosuppressive

in chronic

skin

4 h after TPA treatment. The value is the With 20 nmole TPA alone the radioactivity cpm (difference: 4457 cpm = 0% inhibition). 12.

used. more

promoting mg CsA

of the TPA-induced This

(10).

of TPA were for

were

Based on

(1).

the

hypothesis

determined

in the

topical

application,

Recently,

we found

than

phorbol applied

lo-fold

22

esters together

is

that

h after

a

This

(11). with

Vol. 126,No.

1, 1985

TPA. As discussed induced that

capillary

several

by TPA.

resistance.

If

wellknown

was also

suppressed

the TPA-induced vivo

(Fig.

layer

TPA effect,

this

holds

To our

the

1 and Table

chromatography

indicated

from

that

leaking

true,

knowledge,

increase

CsA should however,

in epidermal

by CsA. Finally, of an AA metabolite

l).The

as yet

unidentified

between

5- and 12-/15-HETE

the TPA-

endothelial be able

cells to

a stabilizing

has not been described

very efficiently generation

data

arose

of CsA on the microvasculature

Another

thin

(ll),

in mouse epidermis

had been damaged

effect

in

previously

AP activity

increase

tely

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

as yet.

ODC activity CsA inhibited

(12), comple-

in mouse epidermis metabolite (Figure

migrated 1). To our

I

TPA

TPAlCsA

Figure mis in

1: Radiochromatogram vitro.

of

AA metabolites

generated

in

mouse

epider-

Two hundred pl acetone alone (control), 20 nmole TPA in acetone or 20 nmole TPA/2.5 mg CsA in acetone were applied to the shaved back skin of 2 mice each. 24 hours later the mice were sacrificed. Epidermal specimens were prepared from the back skin, homogenized and used for the generation of AA metabolites in vitro as described in Methods. Extraction of metabolites, separation by thin layer chromatography and analysis of the radiochromatogram were performed as described in Methods. [3H]-5-HETE, [3H]-12-HETE and [3H]-15-HETE served as standards. The profiles were reproduced in 5 independent experiments.

330

on

Vol. 126,No.

1, 1985

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

the

by TPA of such a metabolite

knowledge previously. lite not

induction

Lysed

in vitro, shown).

invading that

the epidermis

system.

by the

but

This

secondly,

in tumor

cell-free

system

promotion

(14),

Recently,

by directly

is

however,

an indirect

possible

that

by CsA in vivo.

immunosuppressive effects.

One possibility

leading CsA. CsA is

agent

to these It

remains based

also

various

Taken

CsA is is

that

effects

an open question on the

inhibition

to play

a very potent there

cell-free

respect

whether

the

of this 331

even at in vivo

Furthermore,

it

is

of PKC by TPA was results

inhibitor

this

of the

does not exclude,

indicate

process

unknown

the

TPA process

can be inhibited

immunosuppressive as yet

that

of several

is a common TPA-induced

and that

epidermal

the TPA effects

stimulation the

a central

a 50% inhibition

PKC. This

together,

pathway

of PKC by TPA

in this

CsA inhibited

the

metabolite

by CsA in the

caused

not

inhibitor.

in an epidermal

phloretin

(data

cyclooxygenase

of PKC by CsA in vivo.

following

howacid

the TPA-induced

on the stimulation

TPA-stimulated

inhibition

completely,

is thought

CsA was ineffective

the

a process

which

inhibitors

that

the

indomethacin

the

it

cell-

including

as a lipoxygenase

was not suppressed

not likely

inhibiting

than

act

C (PKC),

2x10e5M

PKC-activity. it

simply

by various

system

epidermal

nordihydroguaiaretic

that

rather

we reported

For instance,

Thus

suppressed

not

kinase

TPA-stimulated 5x10m4M.

CsA did

suppression

(15).

lipoxygenase

exclude

and that

in the

inhibitor

place

we cannot

such "TPA-activated"

quercetin,

first

leucocytes

with

was suppressed

cyclooxygenase

protein

system.

and on its

the

that

TPA-stimulated

metabolite

(data

metabolite

of AA in this

in the

metabo-

this

metabolism

not by the

along

to produce

inhibitors

indicates

was generated

Nevertheless,

AA metabolism

lipoxygenase

this

by "ordinary"

to inhibit

TPA-induced

not produce

generated

due to a contamination

The in vitro of the

was not

did

AA to HETEs efficiently

TPA treatment.

CsA was unable

and sesamol,

role

metabolized

leucocytes

in our assay

generation

and,

after

TPA caused

leucocytes.

shown).

they

Thus the metabolite

was found

ever,

from mouse blood

even though

in vivo

free

leucocytes

has not been reported

activity process.

by of

Vol. 126, No. 1, 1985

BIOCHEMICALAND

BIOPHYSICALRESEARCH

COMMUNICATIONS

ACKNOWLEDGEMENT We are

very

grateful

to Sandoz,

Basel,

for

the

gift

of cyclosporin

A.

REFERENCES 1

34 5 6

9 10 11 12 13 14 15

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