The mechanism of the cytotoxicity of Ricinus communis phytoagglutinin toward rat ascites tumor cells

BIOCHEMICAL

Vol. 48, No. 4, 1972

THE

MECHANISM

OF THE

PHYTOAGGLUTININ

Kikuo

Onozaki,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CYTOTOXICITY

TOWARD

Motowo

Tomita,

RAT

Yoshio

Faculty of Pharmaceutical Sciences, and Department of Chemotheraphy, Toshima-ku, Received

July

OF RICINUS ASCITES

Sakurai,

COMMUNIS

TUMOR

CELLS

and the late

Tyunosin

Ukita

Bunkyo-ku, Nishisugamo,

Tokyo

University of Tokyo, Cancer Institute, Tokyo, Japan

3,1972

Summary: Ricinus communis phytoagglutinin having a high cytotoxicity toward rat ascites tumor cells inhibited the protein and DNA synthesis, but not RNA synthesis, of the cultured cells in vitro. Phytoagglutinin covalently attached to large polymers of Sepharose also showed the inhibition of DNA synthesis and the inhibition was counteracted by addition of galactose. The results indicate that interaction of Ricinus communis agglutinin with surface membrane may cause the cellular metabolic alterations. It is known

that

on the cell

surface

Previously

we prepared

for

terminal

highly

Ricinus toward

purified

communis

cultured

of the cytotoxicity

toward

the rat ascites

Ricinus Thus,

the crude

column

two protein

Copyright All rights

extract

of Sepharose

galactose

peak

communis

tumor

fraction

was

peaks which

RCS.

The

by filtration had a high

@ 1972 by Academic Press, of reproduction in any form

Inc. reserved.

tumor

lines l-4

cell

which

are

specific

affinity

column

the phytoagglutinins

precatorius --in vitro

possess 5.

This

strong report

communis

deals

phyto-

cells.

were

purified

communis

4B and the protein

named

that

moieties

and Methods

agglutinins of Ricinus

toward

of the Ricinus

Materials

to sugar

by use of a specific

and Abrus cells

bind

phytoagglutinins,

and observed

rat ascites

the mechanism

which

cytotoxicity

residues,

of Sepharose,

from

agglutinin

exhibit

g-galactose-like

cytotoxicity with

phytoagglutinins

membrane

chromatography obtained

some

peak

beans obtained

RCS fraction through

was

a column

agglutinating

783

as described

previously5.

was

on a

by eluting

further of BioGel

activity

charged

toward

with

subdivided P150. human

0.1 into

The 0

first

M

.

Vol. 48, No. 4, 1972

erythrocytes were

and the second

named The

method

and RCS-II,

agglutinin

was

reacted

Sepharose

complex

was

was

lactose

washed

and with

it was

further

(2.88

(adult lo6

female parent

four

cells.

culture

medium

was

cells

were

the solution

was

added

ice-cold

the reaction

0.3

at 37”. with

mixture

cells

cultured

When

YS cells

during

were

the incubation

decreased not affected

even

was

which

conjugated

containing

0.1

(14 Ci/mmole)

and

M use,

PS.

harvested

days

after

intraperitoneal in assay

England

essential

medium

YS cells

(2 x lo5

of the radio

The

(MEM)

rats of

cells)

which

was

in 0. 9 ml of with

the same

compound

was

(pH 7.4)

and 0.1 ml

reaction

was

insoluble

by the method

donor

in serum-free

diluted

saline

Corp.

inoculation

isotopic

medium.

Nuclear

from

tubes

of C13CCOOH-

terminated

fraction

of Takeishi

of

et al.

of 7

.

and Discussion RNA

and protein

and the results

with

RCS,

almost

at 3 hr.

by CNBr

before

were

Each

to the

Immediately

ml of phytoagglutinin

studied

incubated

at 3 hr.

at 4’.

cells

determined

was

(PS)

(YS)

Radioactivity

significantly

saline

stored

according

RCS-Sepharose

New

of RCS on the DNA,

--in vitro

The

from

to the culture

was

effect

added.

cultured

Results The

of protein

phosphate-buffered

PS.

activity

activated

amount

uridine-3H

minimal

added

to 10 pCi/ml

by adding

3-4

was

purchased

To the harvested

and incubated

diluted

strain) The

agglutinating

to Sepharose

(40 ml)

The

with

sarcoma

or serum-free

of valine.

medium

times

were

ascites

devoid

mg).

(6. 7 Ci/mmole),

Donryu

199 medium

Sepharose

and then

Ci/mmole)

Yoshida

attached

2 1 of physiological

10 1 of PS, washed

had a weak

% of the protein

with

Thymidine-‘H valine-3H

covalently

RCS (400 99.4

which

respectively.

et al. 6

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

fraction

RCS-I

of Kristiansen

and was with

BIOCHEMICAL

inhibitory

784

given of DNA

hand, effect

the RNA

of YS

in Table

I.

synthesized

at 2 hr as the control,

On the other

A strong

are

the amount

the same

synthesis

but

synthesis

of RCS was

observed

was

Vol. 48, No. 4, 1972

Table

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

YS cells were cultured in 199 medium with or without RCS (2. 5 pg/ml). Th.ymidine-3H , uridine-3H or valine-3H was added and the radioactivity incorporated into the acid insoluble fraction of the cells was measured. Each value represents an average of results of three experiments differing from the mean by less than 10 %. TABLE Effects

of Ricinus

communis

on the macromolecular

DNA synthesis RNA synthesis Protein synthesis

for

the protein

the early effect

on protein

effects

may

communis

ascites

cells

Radioactivity incorporated at incubation time of

(cpm)

1 hr

2 hr

3 hr

- RCS

230

693

1,759

3,277

+ RCS

251

813

1,532

1,964

- RCS

1, 524

3,239

7,792

15,111

+ RCS

2,202

3,801

6,916

15,365

- RCS

87

130

265

289

+ RCS

54

94

143

161

a significant

of the incubation. synthesis

inhibition

It thus seemed

first,

and then

the mechanism

was

noted

already

that RCS exerted

inhibited

DNA

of the cytotoxic

from inhibitory

synthesis.

action

These

of Ricinus

agglutinins.

to galactose

we prepared

RCS can agglutinate moieties

that

structure

Sepharose

of Yoshida

0.5 hr

constitute

Since

possible

phytoagglutinin

synthesis

synthesis:

stage

I

on the cell

an interaction

is the trigger

has the cytotoxicity

cannot

surface

of the agglutinin of the cytotoxicity.

the phytoagglutinin which

YS cells5,

enter

covalently the YS cells

or not.

785

it will

have

membrane with

and therefore surface

to large

and studied

sites it is

membrane

To investigate bound

the binding

this

possibility

polymers

whether

of

the derivative

Vol. 48, No. 4, 1972

Table

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

II

YS cells were incubated in 199 medium with the phytoagglutinin-Sepharose complex in the presence and absence of sugar (3 mg/ml) for 3 hr at 37”. The mixture was then pulse-labeled for 1 hr with thymidine-3H. The radioactivity incorporated in the control (- RCS) experiment was 1.25 x 104. TABLE Effect

of phytoagglutinin-Sepharose

on DNA

synthesis

complex

of Yoshida

Radioactivity Sugar added

II

ascites

incorporated

RCS-Sepharose 0.015 0.15

(pi/ml)

(% of control)

Con A-Sepharose 0.15

1:5

None

94

72

25

95

galactose

98

99

98

-

glucose

93

67

33

-

Microscopic on the surface

examination

of the RCS-Sepharose

presence

of galactose

presence

of glucose

that

decreased

dependent action

adsorption

by the presence

on the amount of the complex

The

was

the polymer

inhibition. activity

This was

during possibility

present

-

(data

can be adsorbed

not shown).

In the

whereas

in the

in the adsorption,

indicating

to the D_-galactose-like

residue

manner. of DNA

synthesized

of the RCS-Sepharose

counteracted

of glucose.

possibility

bind

of the complex

was

82

the YS cells

observed

the amount

not by the presence

from

was

in a specific

(pi/ml) 1.5

did not take place,

RCS can still

II shows,

that

complex

no inhibition

surface

As Table was

this

the polymer-bound

on the cell

showed

cells.

was

in the solution

complex,

Furthermore,

by the presence

considered

incubation

added.

that

obtained

786

by the fact by filtering

and it was the inhibitory

of galactose,

the agglutinins

and the resulting excluded

in the YS cells

free that

may

but

be detached

RCS caused little

the

inhibitory

the reaction

mixture

BIOCHEMICAL

Vol. 48, No. 4, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 INCUBATION

Fig. 1 Effect RCS-II.

of galactose

2 TIME(

on the protein

3 hr)

synthesis

of YS cells

treated

with

YS cells were cultured in MEM medium which was devoid of valine with or without RCS-II (2. 5 pg/ml). To the medium, galactose was added to 0.1 M concentration 30 min or 1 hr after the start of the incubation. M: control (- RCS-1I);OI-C) : + RCS-II; w-a : + RCS-II, + galactose at 30 min; O---O : + RCS-II, + galactose at 1 hr

through

millipore

Sepharose collected

in varying from

synthesis: mixture Further,

filter.

each

[ DNA before

amounts mixture

the filtrate

agglutinin that

inhibited

RCS-15, inhibitory

likewise

complex

the DNA

observed

has about

and we examined effect

from

its ability

of RCS-II

with

the DNA

of RCS-Sepharose

in the

90.5

was

residues. only

RCS-Sepharose

ten times

also

containing

higher

This

only

by the same

was

extent,

indicating

not due to an artifact bound

cytotoxicity

of galactose.

to be specific

polymer-bound

to a small

synthesis

18’7

prepared is known

the polymer

if the protein by addition

@/0/l. 5 (pi/ml).

effect.

; the phytoagglutinin

of preparing

and RCS-

to inhibit

the medium

complex

synthesis

YS cells

3 hr at 37” and the filtrate

/[ amount

had no inhibitory

in the process RCS-II

for for

% of control]

and g-mannose-like

the effect

produced

incubated examined

A-Sepharose

way as RCS-Sepharose

containing

; 100 %6/O. 15 (pi/ml),

complex

g-glucose-

was

obtained

Concanavalin

the mixture

was

synthesis,

filtration]

RCS-Sepharose

for

Thus,

agglutinin. toward

could

YS cells

be rescued

As illustrated

than

from in Fig.

the I,

BIOCHEMICAL

Vol. 48, No. 4, 1972

when

galactose

the start When

was added

was

of the protein

communis

chains

Although

it has

without

known

whether

et al.

have

and

recently

been

shown8

was hindered. was

that inhibits

ricin

that

relate

isolated.

‘It should

Nicolson

from

Ricinus

no stimulation

also

communis

We thank

of the manuscript.

for

discussions.

synthesis.

10

that

during

Dr.

University Dr.

Hikoya

Toshiaki

method

Hayatsu

of Tokyo,

action.

Lin

toward

murine

for Osawa

et al. 9 ascites it is not

the toxin

the preparation

purification

by the same

toxin

protein

contains

reported

membrane.

However,

preparation

to

communis

cell-free

cytotoxicity

Ricinus

by attaching

the Ricinus

to its --in vivo

be noted

that

on the cell

inhibit

and DNA

and Blaustein

Sciences,

ricin,

has a strong

the protein

YS cells

residues

can directly

not necessarily

preparation valuable

or 1 hr after

to suggest

toward

g-galactose-like

activity,

Acknowledgements: Pharmaceutical

there

it is tempting

or not our phytoagglutinin

manuscript

agglutinins

30 min

of RCS-II

YS cells,

its cytotoxicity

containing

demonstrated cells

results

exerts

it does

tumor

this

cells

effect

to the control

of these

hemagglutinating

synthesis,

the inhibitory

added

agglutinin

the sugar

treated

synthesis.

On the basis

have

to the RCS-II

of the incubation,

galactose

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Lin of

of phyto-

as ours 5 .

of the Faculty his helping is also

of us in the

acknowledged

References 1. Anderew, F. D. and Gabourel, J. D. : Fed. Proc., 25 : 561 (1966) 2. Datta, S. P., Cerine, M., Ghorse, T. and Cerini, J. : Brit. J. Cancer, 23 : 616 (1969) 3. Dent, P. B. : J. Nat. Cancer Inst., 46 : 763 (1971) 4. Shoham, J., Inbar, M. and Sachs, L. : Nature, 227 : 1244 (1970) 5. Tomita, M., Kurokawa, T., Onozaki, K., Ichiki, N., Osawa, T. and Ukita, T. : Experientia, 28 : 84 (1972) 6. Kristiansen, T., Sundberg, L. and Porath, J. : Biochim. Biophys. Acta, 184 : 93 (1969) 7. Takeishi, K. , Ukita, T. and Nishimura, S. : J. Biol. Chem., 243 : 5761 (1968) 8. Olsnes, S. and Pihl, A. : FEBS letters, 20 : 327 (1972) 9. Lin, J. Y., Lin, K., Chen, C. C. and Tung, T. C. : Cancer Res., 31 : 921 (1971) 10. Nicolson, G. L. and Blaustein, J. : Biochim. Biophys. Acta, 266 : 543 (1972)

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