ICANCKR RESEARCH 53. 652-657. February I. 1993]
Suramin Inhibits Growth and Yet Promotes Insulin-like Growth Factor II
Expression in HepG2 Cells1
Andrea Kraft, Lola \1. Reid, and Isabel Zvibel2
Alben Einslein College of Medicine, DciHinments of Molecular Pharmacology ¡A.K,, L M. R.. I. Z.¡unti Microbiology and Immunology ¡L.M. R.¡.Bronx. New York 104dl
growth factors to their receptors, including platelet-derived growth
factor, epidermal growth factor, TGF-ß,1interleukin 2, and IGF I
(3-7). Suramin is able to block the internal autocrine transformation
and "normalize" the phenotype of .m-transformed cells, as well as that
ABSTRACT
Suramin, a drug shown to inhibit the growth of some tumor cell types
in vivo and in vitro, was found to strongly inhibit the proliferation of the
human hepatoma cell line, HepG2, grown in either serum-supplemented
medium or a serum-free, hormonally defined medium tailored for
hepatoma cells. In parallel, suramin induced the expression of the 6.0kilobasc transcript of insulin-like growth factor II (IGF II) but had no
of basic fibroblast growth factor-transformed cells, by an as yet un
known mechanism (8, 9). Suramin inhibits the activity of a wide
variety of enzymes, including DNA polymerase, reverse transcriptase
(10, II), and enzymes responsible for the degradation of glycosaminoglycans (12). This last effect is responsible for the bleeding disor
ders in suramin-treated patients, in whom there are high circulating
levels of dermatan and heparan sulfates (2). Suramin inhibits the
growth of many tumor cell lines in vitro ( 1, 13) and is a differentiation
signal for many cells (14, 15).
Insulin-like Growth Factors and Differentiation. IGF I and IGF
II are of great importance for proliferation of a variety of cell types
(16). IGF II is most likely involved in fetal development (16) and in
tumorigenesis (17). Elevated levels have been found in various tu
mors, some of them of fetal origin (17-20). Human hepatocarcinomas
express the fetal transcripts of IGF II. and many tumors that express
IGF II also express early markers such as a-fetoprotein (20-22).
Metastatic potential in a panel of human hepatoma cell lines was
found to be inversely correlated with expression and matrix regulatability of IGF II and TGF-ß(23). These findings complement many
others that support the hypothesis that hepatomas are transformants of
early progenitor cells in the liver (24).
Despite the apparent importance of IGFs as regulators of growth
and differentiation, little is known about the capacity of distinct
chemotherapeutic drugs to interact with IGF II pathways. The heparinlike pharmacology of suramin held our attention, as did its docu
mented ability to interfere with growth factors or their receptors. We
questioned whether suramin. like heparins, could directly regulate the
synthesis or abundance of mRNAs encoding growth factors, such as
IGF II and TGF-ß,and whether its known influence on the growth and
differentiation of cells is mediated via these factors.
significant effect on transforming growth factor ß,mRNA levels in these
cells. The induction in the abundance of IGF II mRNA was posttranscriptionally regulated. The growth-inhibitory effect of suramin was not me
diated through IGF II, since addition of IGF II directly to the medium
mildly stimulated the growth of HepG2 cells in a dose-responsive manner.
Treatment of cells with suramin for 24 h resulted also in increased
albumin mKNA levels in both HepG2 cells and normal rat hepatocytes.
Suramin's effect on albumin occurred only in cells in medium supple
mented with serum and in freshly plated cells, i.e., cells that had not been
in culture long enough to form their own extracellular matrix substratum.
We hypothesize that, as for heparin, suramin can bind serum factor(s),
adversely affecting the stability of albumin mRNA.
Addition of either IGF I or IGF II directly to cells resulted in an
increase in albumin mRNA in HepG2 cells after 4 days in culture, impli
cating a role for these factors in differentiation. Vet they showed no effect
unless the cells were grown for 2 days in serum-supplemented medium and
then switched to a hormonally defined medium. Thus, both mitogenic and
differentiation effects of IGFs were observed, and the qualitative re
sponses of the cells to K.I s were dictated by other variables.
Neither suramin nor Idi II had an effect on total sulfation levels in cells
or medium conditioned by HepG2 cells and rat hepatocytes, suggesting
that they have few, if any, effects on glycosaminoglycan synthesis or the
extent of sulfation. Therefore, at present, suramin's potent biological ef
fects on the growth and differentiation of HepG2 cells and rat hepatocytes
are clearly complex and mediated through as yet unclear mechanisms.
INTRODUCTION
Suramin. a polysulfonuted naphthylurea broadly used in the treat
ment of trypanosomiasis and oncoceriasis since 1920, is currently
being investigated as an antitumor agent for the treatment of advanced
cancer, especially prostate cancer ( 1). The drug exerts a wide variety
of biological effects ( 1). Although its mechanism of action is not fully
understood, current investigations by Stein et al. (2) have found that
its chemical structure and pharmacology have many parallels with
heparins. It is a symmetric molecule composed of two naphthalene
rings and two aromatic rings linked by carbonyl amides with peptide
bonds and replete with O-linked sulfates. There are multiple points in
suramin's structure permitting extensive rotation and therefore allow
MATERIALS
AND METHODS
Animals
Rat hepatocytes from Sprague Dawley rats (200-250 g) (purchased from
Taconic Farms (Germantown. NY) were prepared by the standard liver perfu
sion procedures of Berry and Friend (25), using the buffer and perfusion
mixture of Leffert et al. (26).
Cells
ing various conformations potentially conducive to binding to specific
proteins. Suramin has been shown to block the binding of numerous
Early passage HepG2 cells, derived from a well-differentiated,
static. human hepatocellular carcinoma (27), were obtained
Knowles (Wistar Institute. Philadelphia. PA).
Received 7/13/92; accepted 11/12/92.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 Funding for these studies derived from American Cancer Society Grants BC-349 and
BC-675. from NIH Grants P30-CA13330 and AM 17702. and from a grant from the
Council for Tobacco Research (1897). Salary support for A. K. derived from a DFG
fellowship from the Deutsche Forschungsgemeinschaft, and partial salary support for I. Z.
came from the Richard Molin Foundation.
2 To whom requests for reprints should be addressed, at Albert Einstein College of
Medicine, Department of Molecular Pharmacology. 1300 Morris Park Avenue. Chanin
Building. Room 601. Bronx. NY 10461.
nonmeta-
from Barbara
Culture Conditions
Substrata.
Cells were plated directly onto 100- or 150-mm tissue culture
plastic dishes (Falcon).
'The abbreviations used are: TGF-ß.transforming growth factor ß;IGF I and II.
insulin-like growth factor I and II: GAG. glycosoaminoglycan; SSM. serum-supplemented
media: HDM. hormonally defined media: cDNA. complementary DNA.
652
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SURAMIN
REGULATION
Media. The hepatomas and hepatocytes were cultured in RPMI 1640 (Gib-
Measurement of GAG Sulfation
co. Grand Island, NY) and supplemented with penicillin (I(X) ug/ml) and
streptomycin (100 ug/ml). This medium was further supplemented with l()7r
fetal bovine serum (Gibco) to produce SSM or with a defined mixture of trace
elements, hormones, and growth factors to produce a serum-free HDM. The
HepG2 cells and hepatocytes were plated onto 35-cm: dishes at a density of
10" and 6 X IO5 cells/dish, respectively. HepG2 cells were plated in SSM for
2 days and the hepatocytes for 6 h. HepG2 cells were then changed to serumfree RPMI 1640 containing antibiotics and either 10 ug/ml insulin. 50 ng/ml
IGF II. or 10 ug/ml insulin ±0.5 imi suramin. The hepatocytes were changed
to HDM designed for hepatocytes (29) ±0.1 mM suramin or HDM supple
mented with 50 ng/ml IGF II ±0.1 mM suramin. One hundred uCi/plate of
H,35SO4 (New England Nuclear. Boston. MA) was added for 18 h to the cells.
HDM used for the hepatomas was designed for the growth of hepatoma cells
on tissue culture plastic and has been described in detail elsewhere (28). The
HDM for hepatocytes has been previously described (29).
Experimental
Conditions
After IX h. both media and the total cellular proteins, obtained by extraction of
the cell layer with phosphate-buffered saline and 0.5% Nonidet P-40. were
collected. GAGs were precipitated overnight at 4°Cwith 0.3 Msodium acetate
HepG2 cells were plated at a density of 6 X lO'VlSO-crrr dish in SSM and
changed after 6 h to the appropriate HDM with or without suramin (Mobay
Corporation, New York. NY) at a concentration of 0.5 HIM.Total RNA was
extracted after the cells were cultured for 24 or 96 h.
For the experiments in which IGF I, IGF II. and TGF-ßeffects were tested.
HepG2 cells were plated on 150-cm2 plates in SSM and changed to the
and 3 volumes of ethanol. The next day, the GAGs were precipitated by
centrifugation at 3(XK)rpm for 30 min in a Sorvall preparation centrifuge. The
incorporated counts were read using a Beekman beta scintillation counter.
appropriate condition after 6 h or 2 days. The growth factors were added to
RPMI 1640 supplemented with antibiotics in the following concentrations: IGF
I at 10 ng/ml (Amgen, Thousand Oaks, CA); IGF II at 50 ng/ml (Bachern,
Philadelphia, PA); or TGF-ßat 1 ng/ml (R & D Systems, Minneapolis. MN)
in the absence or presence of 50 ng/ml bovine lung-derived heparin (lot
53F-0532; Sigma, St. Louis, MO).
Hepatocytes were plated at a density of 6 x IO6 cells/ 100-cm2 dish. After
RESULTS
Suramin's
6 h in SSM, the cells were changed to HDM. Suramin was added at a nontoxic
concentration of 0.1 mM. Total RNA was extracted after 24 h.
Growth Curves
Cells were plated in triplicate for each condition at 10s cells/well in 24-well
plates (Costar) in SSM. After 12 h. the plates were rinsed with phosphatebuffered saline and changed to HDM with or without 0.5 HIMsuramin, IGF II
(1, 10. 50 ng/ml) or TGF-ß(1 ng/ml). The medium was replaced on the second
day. Cell counts were done using a Coulter counter 2M (Coulter Counter
Electronics, Ltd.).
Molecular Hybridization
Assays
Nuclear transcript run-on assays and Northern blots (see below) were used
to determine the synthesis and abundance of tnRNA encoding IGF II, TGF-ß,
or albumin. Internal controls included hybridizations for genes that proved
constitutive under the experimental conditions. In the nuclear transcript runons, dihydrofolate reducÃ-asewas used: for the Northern blots, 18S ribosomal
RNA was used. The absorbance was measured on a Quantimat densitometer
(model 920: Manufacturer's Cambridge Instrument) for each autocrine growth
OF LIVER CELLS
Influence
on the Synthesis
and Abundance
of
mRNAs Encoding Growth Factors. Our prior studies (23) indicated
that heparins cooperate with specific peptide hormones to regulate the
synthesis and abundance of mRNAs for all of the known fetal tran
scripts of IGF II, as well as TGF-ß,,in the minimally deviant tumor
cell line HepG2. Since suramin has a heparin-like pharmacology, it
was logical to suppose that it might also affect the expression of IGFs
and TGF-ß,in HepG2. Indeed, as shown in Fig. 1, 0.5 m\i suramin
induced a 6- and 8-fold increase in the mRNA abundance of the
6.0-kilobase transcript species of IGF II in HepG2 cells grown in SSM
and HDM. respectively. However, unlike heparins. suramin had no
effect on the lower-molecular-weight
transcripts (4.5 and 3.7
kilobases) of IGF II. After 4 days of exposure to suramin in SSM.
HepG2 cells showed only a 3-fold increase in the 6.0-kilobase tran
script of IGF II. Also distinct from heparins. suramin showed no effect
on TGF-ß,mRNA expression after either 24 h or 4 days of treatment.
Transcriptionally. suramin caused a very minor increase in IGF II
synthesis ( 1.5-fold) and had no effect on TGF-ßmRNA synthesis after
24 h of culture in HDM (Fig. 2). Thus, the increase in abundance of
24h
factor gene or for albumin. The absorbance for these genes was divided by that
for dihydrofolate reducÃ-ase (nuclear transcript runons) or by that for 18S
(Northern blots), which were used as internal controls.
96h
IGF n
Northern Blots
Total RNA was extracted using the guanidinium isothiocyanate method, as
described by Chomc/ynski and Sacchi (30). RNA samples were resolved by
electrophoresis through l"7r agarose formaldehyde gels in 3-(A/-morpholino)propanesulfonic acid buffer (31). RNA was transferred to Gene Screen (New
England Nuclear. Boston, MA), and the RNA-containing filters were prehy-
„¿
TGFß
bridi/ed and then hybridi/.ed with the appropriate probes. The cDNA clones
complementary to specific mRNAs (listed below) were radioactively labeled
by primer extension with [12P]dCTP as described by Feinberg and Vogelstein
(32). The cDNAs used in hybridi/.ation were the human prepro-IGF II cDNA
(33), the human TGF-ß, cDNA obtained from the American Type Culture
Collection (34), human albumin cDNA (a kind gift of Richard Lawn. Genen-
18 S
tech), and rat albumin (a kind gift of Mark Zern. Brown University, Provi
dence. RI), a rat cDNA for the IGF II receptor (American Type Culture
Collection), a hamster probe for dihydrofolate reducÃ-ase(obtained from Peter
Melera, Sloan Kettering Cancer Institute, New York, NY), and a mouse probe
for 18S (obtained from James Darnell. Rockefeller University, New York. NY).
Suramin: -
SSM HDMSSM HDM
Nuclear Transcript Run-on Assays
The method is a modification of the Clayton and Darnell procedure and is
described in detail elsewhere (23).
Fig. I. Northern blots of total RNA ( 10 ug/sloi) extracted from HepG2 cells cultured
for 24 and % h in the presence or absence of 0.5 mM suramin in SSM or HDM. The same
blot was sequentially probed with "P-labeled probes for IGF [I. TGF-ß.and 18S.
653
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SURAMIN
HOM
REGULATION
Suramin
pGEM
DHFR
IGF1
OF LIVER CELLS
I and IGF II. It is possible that the serum requirement permitted the
cells to deposit a matrix with which IGF II and IGF I could act
synergistically or bind to and be stabilized. Even though suramin
increases IGF II levels in HepG2 cells cultured in either SSM or HDM
after 24 h, it increased albumin expression only in cells grown in
SSM.
IGFIrec
TGF/9
Fig. 2. Transcriptional run-ons of HepG2 cells cultured for 24 h ¡nHDM and HDM
supplemented with 0.5 mu suramin.
the 6.0-kilobase transcript of IGF II was primarily by posttranscriptional mechanisms.
The Effect of Suramin, IGF II, and TGF-ßon the Proliferation
of HepG2 Cells. Suramin is known to inhibit the proliferation of a
variety of cell types ( 1). We wanted to test whether it inhibits the
growth of HepG2 cells and whether this effect could be mimicked by
IGF II on its own. As shown in Fig. 3A, suramin had a strong
antiproliferative effect on HepG2 cells grown in either SSM or in
HDM. IGF II, on the other hand, showed a dose-dependent stimula
tory effect on HepG2 cells; 1 ng/ml IGF II had a modest growthstimulatory effect, whereas 50 ng/ml IGF II increased the growth of
HepG2 cells to levels comparable to those of cells in SSM. TGF-ß( 1
ng/ml) slightly inhibited HepG2 proliferation; this effect showed a lag
of 24 h. We conclude that IGF II, on its own, does not have a
growth-inhibitory effect, and therefore, suramin's effect on growth is
not merely via induction of IGF II expression. Since suramin did not
significantly induce TGF-ß,transcriptionally or posttranscriptionally,
and since TGF-ß,showed no significant direct effect on its own on
growth, it has also been eliminated as the mechanism by which
suramin inhibits HepG2 cell growth. There remains the possibility that
suramin, as has been postulated for heparin (23), alters the response of
cells to specific growth factors. This is probable since IGF II on its
own was mitogenic, whereas suramin-treated cells, with a resultant
elevation of IGF II mRNA abundance, are growth inhibited.
The Effect of Suramin and Various Growth Factors on Tissuespecific Gene Expression in HepG2 Cells and Rat Hepatocytes.
Albumin was selected as a representative differentiation marker in
HepG2 cells and rat hepatocytes. Suramin increased albumin mRNA
abundance 6-fold in HepG2 cells plated for 24 h in SSM followed by
24 h in SSM with 0.5 min suramin but not in cells maintained under
serum-free conditions for the second 24 h or in cells cultured for 4
days in either SSM or HDM (Fig. 4).
We tested whether IGF II, as well as IGF I and TGF-ß,could affect
albumin expression in HepG2 cells and whether this effect could be
enhanced in the presence of bovine lung heparin. IGF I and IGF II
caused a 6- and 4-fold increase, respectively, in albumin mRNA
abundance in HepG2 cells. This increase was not further augmented
by the presence of bovine lung heparin (Fig. 5A). Interestingly, this
increase in albumin mRNA was observed only if the cells were cul
tured in SSM for 2 days prior to the addition of the growth factors. If
the cells were plated and changed to serum-free medium containing
these growth factors on the same day, no effect of IGF I and IGF II on
albumin mRNA levels was apparent even up to 4 days of treatment
(Fig. 5ß).Therefore, a period of time in SSM and then a switch to
serum-free conditions proved necessary for the cells to respond to IGF
Fig. 3. A. growth curve of HepG2 cells grown in SSM or HDM in the absence or
presence of 0.5 imi suramin; B, growth curve of HepG2 cells grown in HDM and HDM
supplemented with various doses of IGF II and TGF-ß.
24h
96h
ALB
18 S
Suramin:
—¿-f
SSM
-
+
11
HDM SSM
HDM
Fig. 4. Northern blots of total RNA ( 10 pg/slot) extracted from HepG2 cells cultured
for 24 and 96 h in SSM or HDM in the absence or presence of 0.5 HIMsuramin.
654
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SURAMIN
REGULATION
OF LIVER CELLS
Insulin
Insulin IGF II IGF I
IGF I
TGFß
alb
- 1
alb
IGF II
18s
18s
+
Heparin:
Heparin:
I No heparm
I With heparm
No heparm
ith h«parm
IGF II
Fig. 5. A. Northern blot of tola! RNA ( 10 pg/slot) extracted from HepG2 cells grown for 4 days in RPMI 1640 in the presence of one of the following factors, added separately:
1(X)|jg/ml insulin; 10 ng/ml IGF I; 50 ng/ml IGF II: or l ng/ml TGF-ßin the absence or presence of 50 (ig/ml bovine lung heparin. The cells were changed lo these conditions after
they were cultured for 2 days in SSM. ß,as in A, except that the cells were changed to conditions 6 h after plating in SSM. Belim- each figure is a histogram representing albumin
expression normalized to 18S expression for each condition.
In rat hepatocytes as well (Fig. 6), suramin increases albumin
mRNA expression after 24 h. However, mature rat hepatocytes ex
press IGF I but little or no IGF II. We tested IGF II effects on albumin
expression in the rat hepatocytes and found no effect.
The Effect of Suramin and IGF II on GAG Sulfation in HepG2
Cells and Rat Hepatocytes. Various studies showed that suramin
inhibits enzymes that degrade GAGs and therefore lead to the accu
mulation of GAGs (12). IGF I and IGF II have been shown to cause
the differentiation of chondrocytes by increasing the synthesis and the
sulfation of GAGs in these cells (35). Highly sulfated GAGs were
shown to stop proliferation and cause differentiation in many systems
(36-41). We tested whether the effects of suramin and of IGF II on
differentiation were mediated through increased sulfation levels that
could reflect either increased amounts of GAGs and/or increased
sulfation of the GAGs of both secreted and cell-associated forms in
both rat hepatocytes and HepG2 cells. Table 1 shows representative
data of several experiments providing preliminary evidence that
suramin or IGF II have no effect on the total sulfation levels in the
medium or in the cells of either HepG2 cells or hepatocytes. Similarly,
we found no difference in extent of sulfate incorporation in cells or
medium in the cells treated with IGF II for 1 to 4 days (data not
shown). We conclude that the effects of suramin and IGF II, if any, on
levels of sulfation in cell-associated or secreted components are not
detectable by these assays.
DISCUSSION
We investigated the effect of the drug suramin on proliferation, on
the expression of two autocrine growth factors, and on a marker of
differentiation in the human hepatoma cell line HepG2 and in normal
rat hepatocytes. Suramin's effects on all three biological responses
ALB
18 S
Suramin:
Fig. 6. Northern blot of total RNA ( 10 ug/slot) extracted from rat hepatocytes, cultured
for 24 h in HDM in the absence or presence of O.I m\i suramin.
were dissociable from each other and in each case proved to be
multifactorial. It strongly inhibited the proliferation of HepG2 cells
under all culture conditions, induced IGF II expression under a subset
of the culture conditions, and induced albumin expression in HepG2
and hepatocytes under yet a different subset of culture conditions. The
data suggest multiple, complex mechanisms regulated or influenced
by suramin and indicate many parallels with heparins.
Suramin was similar to heparins (23) in its impact on autocrine
growth factors in HepG2 cells. Like heparins, it increased IGF II
mRNA expression when the cells were grown in either SSM or HDM.
However, unlike heparins, suramin did not affect TGF-ß, mRNA
expression either transcriptionally or posttranscriptionally. Heparin's
effects were observed within 24 h and were sustained for more than 96
h (23), whereas suramin's influence on IGF II expression peaked by
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Sl'RAMIN
RKCil'I.AÕÕON OÃ- I.IVKR O.I.I.S
negative effects of serum on the stability of liver-specific mRNAs.5 In
addition, the serum factors adversely affecting liver-specific mRNAs
have been partially purified and found to be inactivated by a sulfatecontaining detergent, sodium dodecyl sulfate.6 With this as back
Table 1 Effect of IGF lìanil Hiiwnin on the sulfation of GAGs in HepG2 cella ami nit
hcntttocyles
HepG2 cells und rat hepatocytes were plated onto 35-mrrr dishes. HepG2 cells were
grown for 2 days in SSM and rat hepatocytes for 6 h. after which the cells were changed
to HDM (hepatocytes) or RPMI supplemented with insulin, in the presence or absence of
IGF II or suramin (HepG2 cells). Hv5 SOj ( 100 uCi/plate) was added to the culture media.
ground, it is reasonable to hypothesize that suramin. like heparins and
the carrageenans. might bind the factor(s) and, thereby, neutralize
their adverse effects on the stability and abundance of liver-specific
mRNAs. This hypothesis would also explain why suramin had no
effect on albumin mRNA abundance in cultures in HDM (having no
serum) or in 4-day cultures (cultures that would have a matrix sub
stratum to bind the serum factors). It is known from earlier studies
(41) that different media cause the cells to produce distinct extracel
lular matrices and that growth factors affect the cell response differ
ently when presented with a matrix of given chemistry. Therefore, one
can speculate that suramin's action is determined by its interaction
After 18 h of incubation, media and cell extracts were prepared, the total GAGs were
precipitated with 0.3 M sodium acetate and 3 volumes of ethanol, and the counts incor
porated were read using a beta scintillation counter.
Culture conditions
Cell extract
Media
Hepatocytes
(epm x l06/6 x IO5 cells)
HDM
HDM + 50 ng/ml IGF II
HDM + O.I mM suramin
1.12 ±0.08
1.42 ±0.09
1.38 ±0.19
3.2 ±0.42
3.4 ±I.I
3.7 ±1.0
HepG2
(cpm x lO'VI.S x IO6 cells)
Insulin. 10 ug/ml
IGF II, 50 ng/ml
Insulin + 0.5 m\i suramin
1.65 ±0.16
1.49 ±0.27
1.86 ±0.27
3.4 ±0.18
3.3 ±0.26
3.6 ±0.22
24 h and then waned. In contrast to heparin, suramin caused an
increase in the 6.0-kilobase IGF II transcript by posttranscriptional
mechanisms only and had no effect on the abundance of the smaller
transcripts.
Also, suramin. like heparins (23), was strongly inhibitory of the
proliferation of HepG2 cells. Its inhibition of cell proliferation was not
mediated via IGF II. since IGF II was mitogenic for HepG2 cells in a
dose-responsive manner. Rather, the possible mechanism(s) related to
its effects on growth are likely to involve binding of growth factors or
growth factor receptors. Suramin has been shown to prevent the
binding of many growth factors such as platelet-derived growth factor,
TGF-ß,interleukin 2, and epidermal growth factor to their receptors
(3-7) and was recently shown to block the mitogenic effect of IGF I
on a human osteosarcoma cell line (5). IGF II is known to bind to and
stimulate proliferation of cells either through its own receptor, the IGF
II/mannose-6-phosphate receptor, or through the IGF I receptor (42).
Suramin may increase IGF II expression but inhibit this growth factor
binding to the IGF I or IGF II receptors and therefore inhibit cell
proliferation, or. similar to heparin, it could change the receptor num
ber of the IGF. We have found that HepG2 cells showed decreased
IGF II binding after 4 days of culture in the presence of bovine
lung-derived heparin due to a reduction in the number of cell surface
receptors; the addition of heparin directly during the binding assay had
no effect on IGF II binding.4
Our finding that suramin induces albumin expression both in nor
mal rat hepatocytes and in HepG2 corroborates the findings of others
that suramin can have an impact on the expression of tissue-specific
genes (14, 15). Our data are interpreted most readily as an indirect
effect in which suramin is complexing serum factors that adversely
affect the expression of tissue-specific mRNAs. Serum is known to
inhibit mRNA synthesis and to lower the mRNA stability of tissuespecific mRNAs in hepatocytes and hepatomas (43). The active fac
tors in serum can be neutralized by plating cells on a complex matrix
substratum (28, 44) or by allowing cells to generate a matrix in
culture, requiring 3-4 days in culture in SSM (44). It has been pro
posed that matrix binds the serum factors and therefore diminishes or
eliminates their effects on tissue-specific mRNAs. This hypothesis is
supported by findings that purified matrix components can bind the
serum factors and neutralize their biological effects. Heparins and
carrageenans, sulfated polymers of galactose derived from seaweed
and having heparin-like pharmacology, have been shown to block the
with yet unknown components in the extracellular matrix, which in
return are able to induce tissue-specific gene expression under special
conditions and developing states. The matrix chemistry of a cell plays
a crucial role in drug effects. We found that indeed both IGF I and II
can increase albumin mRNA expression in HepG2 cells, if the cells
were grown in SSM for 2 days, which allowed them to deposit a
matrix with which IGF I and II could act synergistically or bind to and
be stabilized. Our results suggest that suramin's effect on albumin
expression in HepG2 cells is mediated through the neutralization of
negative serum factors. The IGFs have been shown to be both growth
and maturation factors for embryonal tissues (16). If one considers
hepatomas to be transformed liver cell progenitors, one expects IGF II
to be both a growth factor and a differentiation factor for these cells
and to have no effect on mature hepatocytes.
The differentiation and proliferation of various types of cells, in
cluding liver cells, have been shown to be regulated by highly sulfated
GAG species (36-41). Moreover, Ishihara et al. (45) have shown that
growth-arrested hepatocytes produce a highly sulfated, heparin-like
heparan sulfate species, which is secreted, endocytosed, and translo
cated to the nucleus and which is thought to be relevant to the
growth-arrested state of the cells. Studies from our laboratory have
demonstrated that heparins cooperate with various hormones to affect
tissue-specific gene expression in hepatocytes (39^1) and autocrine
growth factor expression in hepatoma cells (23). Which genes are
induced by heparins appears to be dependent on whether the cells are
immature precursors or adult cells. That is. the stage of maturity of the
cells, reflective of their position within the liver lineage, dictates the
repertoire of genes available for regulation (24). Suramin, acting
effectively as a heparinoid molecule, appears to also induce different
genes in hepatomas, transformants of precursor cells, and in hepato
cytes, the mature cells. In hepatomas, suramin regulates early genes
such as IGF II and albumin, whereas in hepatocytes it regulates
adult-specific genes such as connexin 32.
Both suramin and IGF I and IGF II can modulate the amount and
sulfation of cellular GAGs in some systems. Suramin was shown to
cause an accumulation of GAGs, particularly in the liver of the in
jected animals, due to its inhibition of the GAG-degradative enzymes
iduronate sulfatase, ß-glucuronidase, and hyaluronidase. The main
GAGs that accumulate in these animals are heparan sulfates and
dermatan sulfates ( 12). It was shown many years ago that IGF I and
IGF II increase the production of GAGs in the developing cartilage
(35). We tested whether either IGF II or suramin caused an increase in
the incorporation of sulfate intracellularly or in secreted components
and found no effects, data that are strongly suggestive but inconclu
sive about effects on GAG synthesis or sulfation.
5 M. Fujita and L. Reid, unpublished data.
fi D. E. Johnston and D. M. Jefferson, unpublished data.
4 I. Zvibel and L. Reid, unpublished data.
656
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SURAMIN
REGULATION
This is the first study in which the effect of the antitumor drug
suramin on the expression of autocrine growth factors has been in
vestigated. It will be interesting to see whether suramin or other
polysulfated heparin-like molecules can affect the production of au
tocrine growth factors in other tumors or in normal precursor cells.
OF LIVER CELLS
21.
22.
ACKNOWLEDGMENTS
23.
We wish to thank Elaine Malay. Patricia Hoist, and Dinish Williams for
technical support; Patricia Hoist for her many efforts as laboratory manager;
and Rosina Passela for excellent secretarial assistance.
24.
25.
REFERENCES
26.
1. La Rocca. R. V.. Stein. C. A., and Myers. C. E. Suramin: prototype of a new
generation of antitumor compounds. A review. Cancer Cells (Cold Spring Harbor). 2:
106-115, 1990.
2. Stein. C. A., La Rocca, R. V.. Thomas, R., McAtee. N., and Myers, C. E. Suramin: an
anti-cancer drug with a unique mechanism of action. J. Clin. Oncol.. 7: 499. 1989.
3. Hosang. M. Suramin binds to platelet-derived growth factor and inhibits its biological
activity. J. Cell. Biochem., 29: 265-273. 1985.
4. Berns, E. M. J. J., Schurmans, A. L. G.. Bolt, J.. Lamb. D. J.. Foekens, J. A., and
Mulder, E. Antiproliferative effects of suramin on androgen responsive tumour cells.
Eut J. Cancer Clin. Oncol.. 26: 4760-474, 1990.
5. Pollak, M.. and Richard, M. Suramin blockade of insulin-like growth factor I stim
ulated proliferation of human osteosarcoma cells. J. Nati. Cancer Inst., 82: 13491352. 1990.
6. Coffey. R. J.. Leof. E. B., Shipley, G. D.. and Moses. H. L. Suramin inhibition of
growth factor receptor binding and mitogenicity in AKR-2B cells. J. Cell. Physiol..
132: 143-148, 1987.
7. Mills. G. B.. Zhang. N., May, C.. Hill. M.. and Chung. A. Suramin prevents binding
of interleukin 2 to its cell surface receptor: a possible mechanism for immunosuppression. Cancer Res.. 50: 3036-3042. 1990
8. Betsholt/. C., Johnsson, A., Heldin, C-H.. and Westermark, B. Efficient reversion of
simian sarcoma virus transformation and inhibitions of growth factor-induced mitogenesis by suramin. Proc. Nati. Acad. Sci. USA, 83: 6440-6444. 1986.
9. Yayon, A., and Klagsbrun. M. Auiocrine transformation by chimeric signal peptidebasic fibroblast growth factor: reversal by suramin. Proc. Nati. Acad. Sci. USA, 87:
5346-5350. 1990.
10. Wills. E. D.. and Wormall. A. Studies on suramin: the action of the drug on some
enzymes. Biochem. J.. 47: 158-170. 1950.
11. DeClercq. E. Suramin: a potent inhibitor of the reverse transcriptase of RNA tumor
viruses. Cancer Lett., 8: 9-22. 1979.
12. Constantopoulos. G.. Rees, S.. Cragg. B. G.. Ban-anger, J. A., and Brady, R. O.
Experimental animal model for mucopolysaccharidosis: suramin-induced glycosaminoglycan and sphingolipid accumulation in the rat. Proc. Nati. Acad. Sci. USA, 77:
3700-3704. 1980.
13. Myers, C. E., Stein, C.. La Rocca, R.. Cooper, M., Cassidy, J.. and McAtee, N.
Suramin: an antagonist of heparin-binding tumor growth factors with tumor activity
against a broad spectrum of human tumors. Proc. Annu. Meet. Am. Soc. Clin. Oncol.,
8: A256. 1989.
14. Fantini, J.. Rognoni, J-B.. Roccabianca, M., Pommier. G., and Maryaldi, J. Suramin
inhibits cell growth and glycolytic activity and triggers differentiation of human colic
adenocarcinomas cell clone HT29-D4. J. Biol. Chem., 264: 10282-10286, 1989.
15. Hensey, C. E., Boscoboinik, D., and Azzi, A. Suramin. an anti-cancer drug, inhibits
protein kinase C and induces differentiation in neuroblastoma cell clone NB2a. FEBS
Lett., 258: 156-158. 1989.
16. Zapf. J., Schmid. C., and Froesch. E. R. Biological and immunological properties of
insulin-like growth factors I and II. Clin. Endocrino!. Metab., 13: 3-30, 1984.
17. Reeve, A. E., Eccles, M. R., Wilkins. R. J.. Bell. G. I., and Millow, L. J. Expression
of insulin-like growth factor II transcripts in Wilm's tumour. Nature (Lond.), 317:
27.
28.
29.
30.
Expression of insulin-like growth factor I (IGF-I) and IGF-II mRNA during hepatic
development, proliferation and carcinogenesis in the rat. Carcinogenesis (Lond.), 9:
209-213. 1988.
Lamas, E., LeBail, B., Housset, C.. Boucher. O.. and Brecho!, C. Localization of
insulin-like growth factor II and hepatitis B virus mRNAs and proteins in human
hepatocellular carcinomas. Lab. Invest.. 64: 98-104, 1991.
Fu. X. X., Su. C. Y. Lee, Y. Hintz. R., Biempica. L.. Synder. R., and Rogler, C. E.
Insulin-like growth factor II expression and oval cell proliferation associated with
hepatocarcinogenesis in woodchuck hepatitis virus carriers. J. Virol., 62: 3422-3430,
1988.
Zvibel. I.. Halay. E., and Reid. L. M. Heparin and hormonal regulation of mRNA
synthesis and abundance of autocrine growth factors: relevance to clonal growth of
tumors. Mol. Cell. Biol.. //: 108-116. 1981.
Sigal. S.. Brill. S.. Fiorino. A., and Reid, L. M. The liver as a stem cell and lineage
system. Am. J. Physiol.. 26.?: GI39-G148. 1992.
Berry. M.. and Friend. D. High-yield preparation of isolated rat liver parenchymal
cells. J. Cell Biol.. 43: 506-520, 1969.
Leffert, H.. Koch. K. S., Moran, T.. and Williams, M. Liver cells. Methods Enzymol.,
58: 536-544. 1979.
Knowles. B. B., Howe. C. C.. and Aden. D. P. Human hepatocellular carcinoma cell
lines secrete the major plasma proteins and hepatitis B surface antigen. Science
(Washington DC). 209: 497^499, 1980.
Gatmattan. Z., Jefferson, D. M.. Ruiz-Opazo, N., Biempica. L.. Arias. !.. Dudas. G.,
Leinwand, L. A., and Reid, L. M. Regulation of growth and differentiation of a rat
hepatoma cell line by the synergistic interactions of hormones and collagenase sub
strata. J. Cell Biol., 97: 1179-1190. 1983.
Enat. R., Jefferson, D. M.. Ruiz-Opazo. N., Gatmaitan. Z.. Leinwand. L., and Reid.
L. M. Hepatocyte proliferation in vitro: its dependence on the use of serum-free,
hormonally defined medium and substrata of extracellular matrix. Proc. Nail. Acad.
Sci. USA, 8/: 1411-1415, 1984.
Chomczynski. P.. and Sacchi. N. Single-step method of RNA isolation by acid
-.•u.mulinimi
thiocyanate-phenol-chloroform
extraction. Anal. Biochem.. 162: 156-
159, 1987.
31. Maniatis. T, Fritsch, E. F.. and Sambrook. J. Molecular Cloning: A Laboratory
Manual, pp. 191-193. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
1982.
32. Feinberg, A. P., and Vogelstein. B. A technique for radiolabeling DNA restriction
endonuclease fragments to high specific activity. Anal. Biochem., 137: 266-267,
1984.
33. Bell, G. I.. Merryweather, J. P.. Sánchez-Pescador. R.. Stempien, M. M.. Priestley, L..
Scott. J.. and Rail. L. B. Sequence of a cDNA clone encoding human preproinsulinlike growth factor II (Letter). Nature (Lond.). 310: 775, 1984.
34. Kasid. A.. Bell, G., and Director. E. Effects of TGF-ßon human lymphokine activated
killer cells precursors. J. Immunol.. 141: 690-698. 1988.
35. Salmon, W. D., Jr., and Daughaday, W. H. A hormonally controlled serum factor
which stimulates sulfate incorporation by cartilage in vitro. J. Lab. Clin. Med., 49:
825-836. 1957.
36. Castello!. J. J., Addonizio, M. L.. Rosenberg, R. D.. and Karnovsky. M. J. Cultured
endotheliat cells produce a heparin-like inhibitor of smooth muscle cell growth. J.
Cell Biol., 90: 372-379, 1981.
37. Reilly. C. F Rat vascular smooth muscle cells immortalized with SV40 large T
antigen possess defined smooth muscle cell characteristics including growth inhibi
tion by heparin. J. Cell. Physiol., 142: 342-351, 1990.
38. Elenius, K.. Maata. A.. Salmivirta. M., and Jalkanen, M. Growth factors induce 3T3
cells Io express bFGF-binding syndecan. J. Biol. Chem.. 267: 6435-6441, 1992.
39. Doerr. R., Zvibel. !.. Chiuten. D.. D'Olimpio. J.. and Reid. L. M. Clonal growth of
40.
41.
258-260, 1985.
18. Tricoli. J. V.. Rail. L. B.. Karakousis, C. P.. Herrera. L., Petrelli, N. J.. Bell. G. I., and
Shows, T. B. Enhanced levels of insulin-like growth factor messenger RNA in human
colon carcinomas and liposarcomas. Cancer Res., 46: 6169-6180, 1986.
19. Hoppener. J. W. M.. Mosselman, S.. Roholl. P. J. M., Lamhrechts. C., Slebos. R. J. C..
dePagter-Holhuizen. P.. Lips. C. J. M.. Jansz. H. S.. and Sussenbach. J. S. Expression
of insulin-like growth factor I and II genes in human smooth muscle tumour. EMBO
J., 7: 1379-1385, 1988.
20. Norsledt, G. A., Levinovitz. A.. Moller, C.. Eriksson. L. C., and Andersson. G.
42.
43.
44.
45.
tumors on tissue-specific biomatrices and correlation with organ site specificity of
métastases.Cancer Res.. 49: 384-392. 1989.
Spray. D.. Fujita. M.. Saez. J.. Choi. H.. Watanabe. T.. Hertzberg. E.. Rosenberg. L.,
and Reid. L. M. Proteoglycans and glycosaminoglycans induce gapjunction synthesis
and function in primary liver cultures. J. Cell Biol.. 105: 541-551. 1987.
Reid. L. M., Abreu, S. L., and Montgomery. K. Extracellular matrix and hormonal
regulation of synthesis and abundance of mRNAs in cultured liver cells. In: I. M.
Arias, W. B. Jakoby, H. Popper. D. Schachter, and D. A. Shafritz (eds.). The Liver:
Biology and Pathobiology, Ed. 2. pp. 717-737. New York: Raven Press. 1988.
Roth. R. A. Structure of the receptor for insulin-like growth factor II: the puzzle
amplified. Science (Washington DC). 239: 1269-1271. 1988.
Jefferson. D. M., Clayton, D. F, Darnell, J. E., Jr., and Reid, L. M. Posttranscriptional
modulation of gene expression in cultured rat hepatocytes. Mol. Cell. Biol.. 4:
1929-1934. 1984.
Reid. L. M. Defining hormone and matrix requirements for differentiated epithelia.
Methods Mol. Biol.. 5: 237-276, 1990.
Ishihara. M.. Fedarko. N., and Conrad, H. Transport of heparan sulfate into the nuclei
of hepatocytes. J. Biol. Chem.. 261: 13575-13580, 1986.
657
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Suramin Inhibits Growth and Yet Promotes Insulin-like Growth
Factor II Expression in HepG2 Cells
Andrea Kraft, Lola M. Reid and Isabel Zvibel
Cancer Res 1993;53:652-657.
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