Clinical. Cancer. Research. 455. Antiproliferative. Activity in Vitro and in Vivo of the ...... Cancer. Res., 51: 1247-1256,. 1991. 13. Alley, M. C., and Lieber, M. M. ...
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