Antiproliferative Activity in Vitro and in Vivo of the Spicamycin ...

Vol. 3, 455-463,

March

Clinical

1997

Antiproliferative Analogue

Activity

KRN5500

with

in Vitro

and

in Vivo

Altered

Glycoprotein

of the

Cancer

Research

455

Spicamycin

Expression

in Vitro

Angelika

M.

Melinda

Burger,

Gurmeet

Hollingshead,

Kunio

Randy

Nagashima,

Kimberly

Louis

L. K.

Edward

(indicating

Kaur, T. Fischer,

A. Sausville’

have

Laboratory Division

of Biological

Chemistry

of Basic Sciences, 20892-7458, and

and

[G. K., K. L. K. D., E. A. S.],

National Cancer SAIC Frederick

concentrations

of drug,

toxicity.

effects

Maryland Response Modifiers Program [R. T. F.], Frederick Cancer Research and Development Center, Frederick, Maryland 2 1702-1201

novelty

of this

germ

3-N-acetyl revealed

gluco-

of mi.

loss

inflated. Our findcells exposed to SPA

appeared the possibffity that

processing after exposure to low prior to the occurrence of overt cyto-

glycoprotein

These

tenninal

microscopy

are

consistent

effect of SPA on the enzymatic tant for proper glycoprotem

Institute, NIH, Bethesda, [K. N.] and Biological

and

acid

apparatus

raise

in the

cell

Electron

the Golgi

altered

per

sialic

residues).

crovilli, and ings, therefore,

Biological Testing Branch [A. M. B., M. H.], Laboratory of Pharmaceutical Chemistry EL. M.], Developmental Therapeutics Program, Division of Cancer Treatment, Diagnosis and Centers,

bound

(detecting

samine

and

was noted, but a decrease was noted for wheat

mannose)

of lectin

agglutinin

Maispeis,

Duncan,

tenninal

amount

agent’s

with

a prominent

machinery processing mechanism

likely

early

or organelles

impor-

the

and emphasize of action.

INTRODUCTION Spicamycin was nucleoside-like

ABSTRACT The spicamycin analogue KRN5500 (NSC 650426; SPA) is derived from Streptomyces alanosinicus. The unique structure contains a purine, an aminoheptose sugar, glycine, and a tetradecadiene fatty acid. SPA potently inhibits the growth of certain human tumor cell lines in vitro (IC50 for growth < 100 nM) and displays marked activity in vivo in Cob 205 colon carcinoma xenografts. Selective inhibition of labeled precursor incorporation was not evident at 1 or 4 h of exposure to the drug, but at 8 h, [3H]leucine incorporation was inhibited by approximately 40% at or below the IC50 for cell growth. Because of the structural similarity of SPA to inhibitors of glycoprotein processing, we examined the effect of SPA on indicators of glycoprotein synthesis and processing

in

colon

205 mm)

HL6OTB

carcinoma

to SPA

promyelocytic

cells.

at the IC50

Brief

for growth

of [3Hjmannose. When examined prolonged (40-48 h) incubation mannose-containing carbohydrates, tinin of

and concanavalin mannose-containing

HL6OTB

cells.

glycoprotein flow

crease

cytometry

Significant expression using

in the number

leukemia increased

glycoproteins

was

changes in intact

in the cells

fluorescence-labeled of cells binding G.

1, differs

the purine

(3).

U.S.C.

Section

through

The

cules

original

with

fatty

purine;

1 , SAN-gly)

soon

The

An inagglutinin

solely

to

and cellular

and

a decrease

in protein

related

activity

cells

inhibit gly

2

this

to yield

protein

The

of protein

of

semi-

documented murine

in

synthesis.

a rabbit

used

is a matter

for

are: SPA,

these

in

vitro

spicamycin

in not

system,

SANin

effects

analogue

8720; Fax: (301) 402-0831.

ConA,

fluorescence

intensity.

at -2

KRN5500;

I To whom requests for reprints should be addressed, at Developmental Therapeutics Program, DCTDC, NCI, Executive Plaza North, Rm. 843, 6130 Executive Blvd., Rockville, MD 20852-7458. Phone: (301) 496-

mean

inhibi-

occurred synthesis,

amino nucleoside; SAN-gly, spicamycin to glycine; NCI, National Cancer Institute; fluorescein-conjugated; GNA, Galanthus nivalis agglutinin; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; every day; WGA, wheat germ agglutinin; CFU, colony-forming

A; MFI,

to

could

puromycin

protein

of have

exposure

KRNS500

to

(5)

of KRNS500

reticulocyte

potency

will States.

et a!.

following

Although

However, (IC,0

of SPA

synthesis

SPA

in the United

SAN, spicamycin nucleoside linked

concanavalin

mole-

different

including

to the hydrolysis

comparable

concentrations

abbreviations

moieties

purified

have

Kamishohara

SAN-gly.

synthesis

demonstrated

with

trials

pharmacology

investigation.

SPA tumor

linked

acid

properties,

I clinical

described and

All

1 , SAN),

models

pharrnaceutic

for Phase

detailed interest

group.

to many

studies vivo

amino

a mixture

linked

KRN5500 in

of

tumor xenografts in athyrnic mice (4, 5). demonstrated antitumor activity and fa-

toxicological be available

current

by

in several

linkage

fatty

was

core

activity

vorable

Fig.

antibiotic

SAN-gly

in

in that

amino-nucleoside

to different

spicarnycin

a common

acid

spicamycin

plus (Fig.

the purine

common

leukemia and human Based on empirically

of surface

1734

glycine

nucleosides

antitumor

Received 6/12/96; revised 12/29/96; accepted 12/30/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked 18

the

(L-mannohepto-pyranose

higher

with

occurs

contain

through

shown

conventional

to sugar

spicarnycins

tion

advertisement in accordance indicate this fact.

from

Cob

demonstrated

lectins.

Fig.

(-30

in

of

of the semisynthetic

glycero-3-L-mannoheptapyranosyl]arnino-9H-purine),

Subsequent

observed

nivalis

structure

including

in the pattern

pattern

were

(1, 2). The

as a mixture from Streptomyces

(6-[4-deoxy-4-(2E,4E-tetradecadienoylglycyl])amino-L-

components.

incorporation

change

SPA2

described isolated

spicamycins

by Western blotting after with lectins that target Galanthus nivalis agglu-

A, a qualitative

879-MT3

alanosinicus

originally components

synthetic

and

of exposure

periods

unique

amino FITC,

MU, qd, unit;

456

SPA Inhibits

Cell

Growth

and

Glycoprotein

Processing

I

I

SAN-gly

I

MA);

all other

Chemical from

OH

(N

HTHH

NH

portions

of the molecule

temperature.

rpm

3 mm,

for

natural line

products screen

those

KRN5SOO

can

16

p.M).

related

to inhibition

dolichol

to form

nose

residue

is contributed

UDP

carrier.

The

processed

to the

by

remains

itor of the initial

(6-8).

pothesis

that

centrations found that concentrations

relevant

protein

of oligosaccharide

synthesis

the

structure

of SPA

of tunicamycin,

could

affect

therefore,

in vitro;

as the

Tunicamycin,

is being an inhib(transfer

of

to dolia sugar to (a]-

addressed

the

hy-

processing

at con-

tumor cell growth inhibition. alter glycoprotein expression those with antiproliferative

an effect

an early

via a to the

is reminiscent

we

glycoprotein

relevant to human SPA does indeed that approximate

be considered

donor transferred

reticulum.

distinct) SPA

The man-

from UDP-N-acetylglucosamine a fatty acid tail linked through

Because

clearly

donor.

is then

complex

stages

mannose and other to the lipid carrier,

oligosaccharide unit

in the endoplasrnic

though

proceeds, fashion

oligosaccharide

N-acetylglucosarnine chyl-P), also possesses

fects

synthesis

an oligosaccharide

peptide-polyribosome

uracil

inhibition

in sensitive cell types, the action of spicarnycin

of protein

processing in a stepwise

phosphate,

target

growth

question.

As glycoprotein are added

sugars

cell

vitro

be simply

an open

with

(IC50 for growth Thus, how or whether

in

0.03-0.

associated

on glycoprotein

consequence

to its antiproliferative

of SPA

We at ef-

synthesis action,

must

potentially

mechanism.

protein-based described

AND

Drugs I) was

from

solutions Jackson were

Brewery

Therapeutics the Drug

were

Synthesis

prepared

Laboratory, obtained

Muskegan,

from

(Indianapolis,

Vector

mmol)

was

purchased

[3H]thymidine ic activity, Ci/mmol)

Cell culture nologies,

(specific 46

Ci/mmol),

were

from

media Inc.

Ml). were

from activity,

ICN

Inc. from

were NCI.

Burdick

plemented

[3H]leucine

Amersham

Corp.

activity, (Irvine,

with

SPA

on tumor

described lines.

was

days

obtained

Costar

were

after

number

size

of viable

Lomb,

calculated

from

of CPUs

greater

than

were

conducted

regulations ated

against

human

of athymic

fragments

mice

using

tored

by in situ

were

a tumor

(rng)

caliper were

using

(L x W2)/2].

calculated

Service.

tumor

xenografts mice.

in the

sum

studies

care SPA

and

was

growing

Briefly,

in the

region

growth

of the

was

to determine the length

and

mass.

width

meas(mg)

for a prolate

ellipsoid

[Weight

efficacy

assessed

by:

Median

tumor

weight

tumor

weight

.

The

of treated Xl00

.

Median

rnoni-

tumor

Compound

was

use

evalu-

20-30-mg

the formula

%T/C=

was

of volumes

efficacy

axillary

Tumor

from

analyzer

inhibition

humane

Health

measurement

the

following

(13).

the

trocar.

at I or 4 SPA,

image

All in vivo

implanted implant

cells/plate);

recorded

Growth

(nu/nu)

cell

of

on the total

with

as

HL6OTB

( l0

FAS-Il

in Vivo.

of the U. S. Public

for activity

were

in diameter

p.M

performed,

and

addition

NY).

in compliance

compartment

tumor

60

Activity

of

conver-

experiments

the

of SPA

cells

effects

MU

was plates

an Omnicon

the effects

Antitumor

after

Rochester,

The

205

in 35-mm

colonies

by using

and

HL6OTB

using

nM) in separate days

Tusup-

1640

(1 1).

the Cob

in agar

fibroblast

Research

FCS.

assayed

cell

human

promyelo-

in RPM!

assay

(1 2), with

an in

human

Cancer

10%

colony-forming

Six

reduction

(Bausch

TB human

previously

cell

uses 205

monolayers.

also

examines tumor

for viable

Cob

MRC-5

and

were

(0. 1-1000

stain The

and cultured were

NC! effect

(clone and

glutamine

plating. and

MU

MD)

plated

added

CA),

with

a %T/C

indicative

25 Ci/

were

CA);

mg.

(where

initiated NSC

when The

compound

80.

Heights,

IL).

days

Life

Tech-

(Cambridge,

of 20 mice 5 only.

the was

as five apart

growth

650426

either

(specif-

T is treated

of tumor

Tween

from

Co.

agar

The

in a human

the Frederick

all others

elsewhere

Cells

SPA

from

as described

soft

153

(specific

and

at 10,000

(10).

HL6OTB

cell growth

sion to formazan, The

line,

2 mti

as a suspension;

activity,

[3H]uridine

were

1h

to remove

B protein

and the WI-38

(Frederick,

ac-

Mannheim

(Arlington

of

for

dialyzed

Vitro.

in detail

obtained

grew

=

rng

of control

lectins

Boehringer

Radiochemicals

cell line),

rnor Repository

Stock

and

(Burlingame,

(specific

and

MD)

drugs

Branch,

85 Ci/mmol),

and supplements

650426; Fig. Japan) to the

Fluorescein-labeled

[2-3H]o-mannose

(Gaithersburg,

Other

(American

Laboratories,

lectins IN).

NCI.

and Chemistry

in DMSO

and digoxigenin-labeled Co.

Co., Ltd.

Program,

NSC (Tokyo,

incubated

of antiproliferative

sulforhodamine

were

urernents

SPA (KRN5SOO;

by Kirin

provided

Developmental quired

METHODS

and Reagents.

p.1

1 .25

centrifuged

was

compounds

previously

carcinoma lines

s.c.

chlo-

Thirty-five

and

was

was

in

synthetic

Quantitation

leukemia

Weights

MATERIALS

(9).

vitro

cell

solution supernatant

Activity and

mass, cytic than

The the

sodium

M

FITC-Celite.

colon p.M)

9.5)

and the mixture

Antiproliferative

(see text).

(pH

Sigma

5 rng of lectin

in 0. 1 5 of 10 mg/mI.

buffer

and

from

otherwise.

GNA-FITC,

dissolved

concentration

added,

at room excess

Fig. I Structure of spicamycin analogue KRN5500 (NSC 650426), 6-[4-deoxy-4-(tetradeca-2(E),4(E)-dienoylglycyl)amino-L-g/vcero-3L-manno-heptapyransoyl]amino-9H-purine, SPA. The overlines indicate

the SAN and SAN-gly

were

purchased

indicated

lectin,

NaHCO3/Na2CO3

M

FITC-Celite

(N[1

0

1

to a final

were

unless

were

nivalis

solution

of

chemicals MO),

the FITC

Galanthus

ride H

and

(St. Louis,

To synthesize

-I

SAN

drugs

Co.

(q4d was

daily

doses

and C is control)

suppression.

median

weights

in saline

suspension (qd

x 3) to groups vehicle

tumor

suspended

x

5)

were

was

Tween

40,

treatments 150

containing

± 50 0.05%

administered

or as three

of six mice.

(saline/0.05%

of less than

Compound

doses

The

i.p. spaced

control

80) treated

4

group qd x

Clinical

Macromolecular Uptake. following

Synthesis

uridine,

or leucine,

cells

after

of

scintillation in cells

respectively,

exposure

collection

cells

using

on

cell

were

HL6OTB, flasks

and 50 or were

untreated

tunicamycin, was added three were

ml of Aquasure

LSC

MA)

analyzer

flasks

using

Co.,

lyl-phosphate. Blue

cells RPMI,

Flow

205,

ing 0.1% lected

The

exceeding

were detached counted, and

SPA

containing Control

1500

Tri-Carb

cells in 10

Systems,

liquid

scintillation

formed,

buffer

containing

ride,

1%

v/v

50 msi

med

using

1 mM

fluoride,

10 p.g/ml

ylmethylsulfonyl

EDTA,

using

Twenty-five

p.g

leupeptin,

the of

BCA cell

using

and

a nitrocellulose

Inc.,

Keene,

Ref.

to carbohydrates

specificity

when

cellulose

used

SR

(where

antibodies

We containing

in the

acid

pathway,

residues

Lectins

used

that were:

mannose-mannose tam a high content mannose

residues

anntennery

transferred

normally

as lectins

added

be used used

great

binding

onto

nitro-

in a conven-

to detect

GNA,

late

effects

of

which

to detect mannoseat an early stage of directed

against

(21);

WGA, and

chains

(20).

thus,

sialic

inhibitory

leukemia

HL6OTB 205 was

flM.

In the

ICim lines,

high

acid

mannose,

Lectins

were

conjugated

a

samples

cells 30

effects

2) cells.

-500

±

Of 68 cell lines

tested

line

screen,

colon

and

carcinoma

OVCAR-5;

striking

48-h

clinically to NC!. may

(SE),

the

IC50

assessment

when

tested

lines tested 10 p.M. The

of sensitivity? was unique, not

possess

a novel

lines

and

following

The

nM, respectively.

the

of SPA

to delineate

MU

assay, was

15 ± 5.6

cumulative

IC50 and ICim Colony

95

±

the

two human fibroblast to the growth-inhibup to 20

volume

were

for Cob

counts

the IC50 300

nM, and

at concentrations

for HL6OTB

growth

studied the SPAthe promyelocytic

colony-forming assay, SPA potently ability of both HL6OTB and Cob

ICim

mech-

the striking

and the ICim was

to SPA

used agent or to The pattern in the

To confirm cell

and

sensitivity

in the routine

In a 6-day

in vitro.

line,

of SPA

When

IC50

six (HL6OTB,

carcinoma

in certain

10 ni

perexam-

microscope.

cell

SPA

(23).

prepawas

were

intermediate degrees of SPA in the screen

that

in I .25% Cell

studies

subpanel;

flM)

of SPA

In a soft agar the colony-forming

fixed

buffer.

of -

CFUs

>60

10 and

-100

205 were

decreased

p.M.

inhibited 205 (Fig.

-50

and

in a similar

or termi-

which hybrid,

and

and

was 52 ± 8.2 nM. Interestingly, WI-38 and MRC-5, were insensitive

nM, respectively.

sialic

PBS

(22),

to any current agent available

HL6OTB

of terminal

and ConA,

100

inhibition

for Cob

binds

binds

col-

San Jose,

was that with

Seventeen of the 68 tumor to concentrations as high as

potency

p.m.

(20);

were

cells).

cacodylate

demonstrated

suggests

of growth

processing.

which

residues

staining

Inhibition.

cell lines had of the activity

screen,

with

tumor




was associated any hypothesis

Cancer

clarify

and other enzymes

our results

do raise

of expressed glycoproteins could marker of the effect of SPA in the

Phase

I trials

with

swainsonine

462

SPA Inhibits

Cell Growth

and Glycoprotein

Processing

mv

I

;

I

Lc

my”

.

-

.

‘

*‘

.

.

i: .1. .

-

.,

,.,i,

..

.,

...

.,..

:

n ?‘;t.\\.

-

‘

.‘ ,‘

ti..

.__;...

.:

,

‘

:

-:.,

-:-;.

-

.:

.

.::

-

,,

. ..

-i--

.-. -4

1

;:

.,l’

L’

‘

.

#{149}#{149}‘

t

:d47

Fig. 8 SPA-induced g, Golgi apparatus;

ic,

-

y___

-5

‘

.

.__,;

ultrastructural modifications. A. Cob 205 cells were exposed to vehicle control; B, 50 vesicular compartment. The insert in each panel shows details of Golgi apparatus; bar,

nM;

0.5

C’,

500

p.M.

nM

SPA.

my,

microvilli;

Clinical

The

basis

apparent, anticancer resistant cell

for the differential

because

cytotoxicity

growth

there 100