[CANCER RESEARCH 51. 6629-6635, December 15, 1991|
Disruption of Cellular Energy Balance by Suramin in Intact Human Prostatic
Carcinoma Cells, a Likely Antiproliferative Mechanism
Randall Rago,1 Jacque Mîtchen,Ann-LiîCheng,2 Terry Oberley, and George Wilding3
Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, Wisconsin 53792 ¡R.R., J. M,, A-L. O, G. W.¡;Departments of Medicine
and Pathology, Wm. S. Middleton Memorial Veteran's Hospital, Madison, Wisconsin 53705 ¡R.R., J. M., T. O., G. W.J
ABSTRACT
The antiparasitic drug, suramin, has antiproliferative effects in human
carcinoma cells. It has been suggested that this occurs through blockade
of growth factor-receptor interactions. Three types of evidence that
suramin rapidly inhibits cellular respiration or disrupts cellular energy
balance in intact cells of the human prostate carcinoma cell line, 1)1 145,
are presented. Beginning at approximately 10' M, suramin rapidly causes
dose-dependent
inhibition
of tetrazolium
conversion
by mitochondria!
dehydrogenases in intact cells, demonstrating an inhibition of respiration.
This effect is reversed by exchange with suramin-free media but not by
pretreatment with serum, epidermal growth factor, insulin-like growth
factor I, acidic and basic fibroblast growth factors, or calcium. Rhodamine
123 (10 Mg/ml) uptake by mitochondria in intact DU145 cells is inhibited
in the presence of IO"3 M suramin. Treatment with 10"4-10'-' M suramin
causes the loss of rhodamine 123 from cells with mitochondria prestained
with rhodamine 123, indicating that suramin is acting as an ¡onophoreor
respiratory poison. Also shown by electron microscopy are progressive
toxic changes in mitochondria of DU145 cells within l h after treatment
with 10"* M suramin.
These data indicate that in intact 1)1 145 cells Id'"' M suramin rapidly
disrupts cellular energy balance or respiration as seen by three studies
of mitochondria! state. Disruption of energy balance or respiration rep
resents a likely antiproliferative mechanism, as is thought to be a primary
mechanism for the action of suramin in parasitic diseases. This proposed
mechanism of action for suramin can explain the most prominent observed
clinical toxicities of nephrotoxicity, adrenal toxicity, coagulopathy, and
demyelinating neuropathy.
INTRODUCTION
Suramin is a polysulfonated naphthylurea traditionally used
to treat human trypanosomiasis (African sleeping sickness)
caused by Trypanosoma bmcei and human onchocerciasis
caused by Onchocerca volvulus (1). Suramin has recently been
shown to have antitumor effects in human tumors (2, 3).
Antiproliferative effects are seen with numerous human cancer
cell lines, including human prostate carcinoma cells (4). A
favorable response was seen in patients with prostate cancer (5)
which led to our interest in the drug and further examination
of its activity in prostate carcinoma cell culture.
The antiproliferative mechanism(s) of suramin is not known.
Suramin, at low concentrations, has been shown to interfere
with numerous assays, including those of of isolated enzymes
(1,6) and growth factor-receptor binding (7-9). It is likely that
this compact polyanion binds nonspecifically on the basis of
charge at regulatory sites or active sites of enzymes and macromolecules, thereby interfering with function. For example,
Received 6/3/91; accepted 10/4/91.
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.
' Fellowship recipient under grant T32 CA096I4.
; Current address: Department of Internal Medicine, National Taiwan Univer
sity Hospital, Taipei, Taiwan.
' Recipient of an American Cancer Society Career Development Award and
supported in part by National Cancer Institute grant CA50590 and the Veterans
Administration. To whom requests for reprints should be addressed, at Depart
ment of Human Oncology, University of Wisconsin Clinical Cancer Center, K4/
666 CSC, 600 Highland Avenue, Madison, WI 53792.
suramin competes for the ATP-binding site of the trypanosomal
protein kinase I (10). Nuclear magnetic resonance analysis of
the yeast phosphoglycerate kinase indicates that suramin binds
to both the ATP- and triose-substrate-binding sites (11). These
experiments indicate that suramin may, in particular, inhibit
enzymes that are energy dependent (ATP) or that utilize
charged substrates.
The ability to inhibit many isolated assays nonspecifically
makes it difficult to determine a specific antiproliferative mech
anism^) for suramin. For this reason, in this paper, the pro
posed mechanism of action of suramin in parasites will be
reviewed briefly, and similar effects in intact cells of the human
prostatic cell line DU 145 (12) will be discussed.
In 1904, Ehrlich demonstrated antitrypanosomal activity of
trypan red (1). Suramin was developed as a colorless derivative
of trypan red by Bayer in 1916 and bears a close structural
relationship to trypan blue and trypan red (13). Trypan blue
has been used in low concentrations in mammalian cell viability
studies, but at higher concentrations it is toxic and effects
respiration (14-17). Trypan blue can inhibit cellular hydrolytic
activity within l h after injection into mice, thus demonstrating
a rapid in vivo enzymatic inhibition by a polyanionic substance
structurally similar to suramin (18).
A major antitrypanosomal mechanism of suramin is inhibi
tion of glycolysis and respiration. It has been known since 1928
and 1933 that suramin inhibits glycolysis and respiration, re
spectively, in trypanosomes. This is believed to occur primarily
by the inhibition of two key glycolytic enzymes in T. brucei,
glycerol-3-phosphate oxidase and glycerol-3-phosphate dehydrogenase (19). Interestingly, both T. brucei (bloodstream
forms) and O. volvulus are unusual in that they depend exclu
sively on glycolysis to obtain energy (20, 21). In a bovine
trypanosomiasis treated with suramin, Trypanosoma evansi, an
altered malic dehydrogenase confers high level resistance to
suramin (22). Suramin resistance via mutation in malic dehy
drogenase substantiates interference with respiration or energy
balance as a prime site of action for suramin in T. evansi.
Studies of isolated mammalian cell preparations and cells
also have shown that suramin effects respiration and energy
balance. Suramin inhibits oxygen consumption and ATP syn
thesis in intact rat liver mitochondria! preparations by inhibi
tion of succinate dehydrogenase, the ATPase complex, and/or
adenine nucleotide translocase (23). The H+-ATPase in organelle preparations of bovine adrenal medulla and rat liver
lysosomes and mitochondria is inhibited by suramin (24). Sur
amin has also been shown to inhibit glycolysis in intact human
colon adenocarcinoma cells (25).
Knowing that suramin can affect respiration or energy bal
ance in parasites, and in mammalian organelle preparations
and cells, we further examined its effect on respiration or energy
balance in intact cells by three different assays: (a) conversion
of tetrazolium by mitochondria! dehydrogenases, (b) uptake
and retention of rhodamine 123 by functional mitochondria,
and (c) ultrastructural examination of mitochondria by electron
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DISRUPTION
OF CELLULAR ENERGY BALANCE BY SURAMIN
microscopy. These results were presented earlier in abstract
form (26).
MATERIALS
AND METHODS
Cell Culture. The human prostate carcinoma cell line, DU145, was
used in experiments. Cells were incubated at 4°Cwith 5% CO2 in
DME4 media with F5 (both from Gibco BRL, Grand Island, NY). Cells
were serially passed into 10-cm tissue culture dishes after trypsinization
with two rinses of 0.5% trypsin (Gibco BRL) and then diluted in
DMEF5. Cells were counted using a standard hemacytometer. For
assays, cells were seeded into 6-well or 96-welI plates in 1 ml or 100 M'
DMEF5, respectively, 24 h prior to the addition of test substances.
Assays in the Presence of Suramin. A stock solution of 10~2M surain in
from FBA, Federal Republic of Germany, was made fresh for each
experiment by dissolving in DME. Serial dilutions of this stock in
DME or DMEF5 were added to wells containing DME or DMEF5
and cells to yield the desired final concentrations. Cell proliferation
was measured at points of interest by MTT, DNA, and anchoragedependent colony formation assays.
MTT Assay. At time points of interest, 96-well microculture plates
were assayed by the MTT assay (27, 28). MTT (30 M' of 5 mg/ml;
Sigma Chemical Co., St. Louis, MO) was added in phosphate-buffered
saline to microculture wells containing cells and media. After 4 h
incubation at 37.5°Cwith 5% CO2, the plates were emptied, and 100
ß\of dimethyl sulfoxide (Sigma) was added to each well. After the
plates were mixed, they were measured for absorbance at 515 nm on a
96-well plate reader (Biotek Microplate, Winooski, VT). The wells used
as blanks contained no cells.
For the measurement of inhibition of tetrazolium conversion by
suramin, the MTT assay was performed as above, immediately after
the addition of suramin to microculture plates. Also, various serum,
EGF, acidic and basic FGF, IGF, and calcium concentrations were
tested for the ability to reverse the inhibition of tetrazolium conversion
by various concentrations of suramin. Each was added l h before the
addition of suramin, and then tetrazolium conversion was measured by
the MTT assay. Serum from Gibco BRL was added to microculture
wells for final concentrations of 5-20%. EGF from Collaborative
Research Inc. (Bedford, MA) was reconstituted in DMEF5 and added
to microculture wells for final added concentrations of 0-200 ng/ml.
Likewise, acidic FGF (0.5, 5, and 50 ng/ml), basic FGF (1, 10, and 50
ng/ml), and IGF (0.5, 5, and 100 ng/ml) (all from Collaborative
Research Inc.) were added as final concentrations to microculture wells
in DME (serum free). Calcium chloride (Fisher Scientific, Fair Lawn,
NJ) was dissolved in DME and added to microculture wells for final
concentrations of 1-11 mivi.
DNA Assay. At time points of interest, 96-well microculture plates
were assayed by a fluorometric DNA assay (29) using the fluorochrome
bisbenzimidazole (Hoechst 33258; Hoefer Scientific, San Francisco,
CA). Plates containing cell cultures to be assayed were emptied of
medium and were stored frozen until the time of assay. Then, 100 M'
distilled water was added to each well and incubated for 1 h. The plates
were again frozen and thawed. Next, 100 M' of 10 Mg/ml bisbenzimi
dazole in Tris-buffered, 2 M NaCl was added to each well. After 1 h,
fluorescence was measured on a 96-well fluorescent plate reader (Flow
Fluoroskan I, McLean, VA). The wells used as blanks contained no
cells.
Anchorage-dependent Colony-forming Assay. DU 145 cells (500/well)
were seeded in 1 ml DMEF5 on day -1 into 6-well plates (Corning,
Corning, NY). On day 0, suramin was added in 1 ml DMEF5 to achieve
the final concentration of 10~3M. Untreated control wells received no
suramin. Suramin was removed by exchange with 2 ml suramin-free
media at 1 h and 1 and 3 days. Colonies were counted after 8 days in
the cultures in which the suramin was not removed and in the untreated
control cultures. In the cultures in which the suramin was removed, the
colonies were counted 8 days after the removal of the suramin. At time
points of interest, wells were stained with crystal violet, and colonies
estimated to contain >40 cells were counted with an inverted lens
microscope.
Rhodamine 123. Rhodamine 123 (Eastman Kodak, Rochester, NY)
was made fresh and used (30) at a final concentration of 10 jig/ml in
DMEF5. Cells were grown to 75% confluency on glass coverslips in
DMEF5. The coverslips were incubated (incubation media) for 10 min
in DMEF5 with rhodamine 123 (10 /ug/ml) with or without 10~3 M
suramin. Coverslips were rinsed several times with DMEF5. Next,
coverslips were inverted over slides with depression wells containing
DMEF5 (observation media) with no additives or 1, 0.5, 0.25, and 0.1
x 10~3 M suramin. These slides were observed for initial fluorescence,
as well as subsequent changes in fluorescence while incubated at 37°C
with 5% CÛ2.Observations were made with a fluorescent microscope
at /4485nm.Suramin and rhodamine 123 did not form a precipitate over
the concentration ranges used in these experiments.
Electron Microscopy after Suramin Treatment. DU 145 cells were
grown in DMEF5 to approximately 50% confluency in 10-cm tissue
culture plates. Media was exchanged with media that contained IO"4 M
suramin in 10 ml DMEF5. At time points of interest, cells were
trypsinized from plates by 2 rinses with 0.5% trypsin and prepared for
electron microscopy as described previously (31). Briefly, horizontal
ultrathin sections were cut, double stained with lead citrate and uranyl
acetate, and then viewed in a Phillips 301 electron microscope. Repre
sentative cells were photographed by an electron microscopist who was
unaware of the treatments. Electron micrographs were then reviewed
by a second electron microscopist who noted and recorded significant
findings.
RESULTS
Antiproliferative Effect of Suramin. The antiproliferative ef
fect of suramin was demonstrated using the MTT assay (Fig.
1A), DNA assay (Fig. IB), and anchorage-dependent colony
formation assay (Fig. 2). These three assays show that there
was no proliferation of DU 145 cells after 8 days at a concen
tration of IO'3 M suramin. The antiproliferative effect was dose
dependent, and at 10~5 M suramin there was no inhibition of
growth by the MTT and DNA assays. There was good corre
lation between the MTT and DNA assays for cell proliferation.
Effect of Suramin on Tetrazolium Conversion. The MTT assay
measures a tetrazolium salt conversion to a colored formazan
product by mitochondrial enzyme activity and is commonly
used to estimate cell number during a period of days (27).
However, Fig. 3 demonstrates that suramin rapidly reduced the
conversion of tetrazolium dye during 4 h in a dose-dependent
fashion. After 4 h, the reduced production of formazan crystals
in the mitochondria of DU 145 cells can be seen by light
microscopy. Fig. 4A shows formazan crystals deposited in mi
tochondria after 4 h in the MTT assay; Fig. 4B shows reduced
formazan formation during 4 h with the addition of 10~3 M
suramin. Suramin begins to inhibit tetrazolium conversion at
approximately 10~4 M (143 Mg/ml) in this system. This inhibi
tion was reversed by exchange with medium not containing
suramin. At 10~3 M suramin, tetrazolium conversion in 10"
DU 145 cells was 98.6 ±7.7% of control after exchange with
suramin-free media, as compared to 44.0 ±8.3% of control if
suramin was not removed.
Table 1 demonstrates that 10~3 M suramin inhibits tetrazo
lium conversion to the same degree compared to controls after
the addition of serum to 16%, EGF to 20 ng/ml, or calcium to
11 mM. Additionally, EGF concentrations to 200 ng/ml or
' The abbreviations used are: DME. Dulbecco's modified Eagle's; F5, 5% fetal
serum
concentrations to 20% were also tested and could not
bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoIium
bro
reverse the effect of suramin on tetrazolium conversion. Fur
mide; EGF, epidermal growth factor; FGF. fibroblast growth factor; IGF. insulinthermore, under serum-free conditions, inhibition of tetrazolike growth factor I: ECGF. endothelial cell growth factor.
6630
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DISRUPTION
1.000
OF CELLULAR ENERGY BALANCE BY SURAMIN
SURAMIN CONCENTRATION
O
OCONTROL
•¿
- -»10-3 M
0.100--
Effect of Suramin on Rhodamine 123 Uptake and Retention.
We further examined the ability of suramin to inhibit mito
chondria! function by demonstrating altered rhodamine 123
uptake and retention by the mitochondria of DU 145 cells.
Suramin interfered with rhodamine 123 (10 ng/m\) uptake and
retention (Table 2). There was little or no fluorescence devel
oped by rhodamine 123 in the presence of 10"' M suramin,
indicating interference with the ability of mitochondria to ac
cumulate rhodamine 123. If suramin (1, 0.5, 0.25, and 0.1 X
10~' M) was added after cells had accumulated rhodamine 123,
E
o
0.010-0.005
-1
100
4
5
DAYS
SURAMIN CONCENTRATION
O
O CONTROL
•¿-•10-3 M
A
A 10-4 M
A- -A 10-5 M
Fig. 1. Proliferation with IO"3, IO"4, and IO'5 M suramin. DU145 cells (IO3/
well) were seeded in 100 ¡ADM E media with F5 in 96-well microculture plates
on day —¿1.
On day 0, suramin was added to wells in 100 n\ DMEF5 for final
concentrations of O, IO"3, 10~4, and 10"' M. Cell proliferation was measured by
(A) MTT assay or (A) DNA fluorometric assay on days O(MTT assay done prior
to addition of suramin), 3, 6, and 8. Point, average of 8 determinations; bar, SD.
150T
the fluor was released into the surrounding media, demonstrat
ing disruption of the ability of the mitochondria to retain
rhodamine 123.
Effect of Suramin on Mitochondria! Ultrastructure. Finally,
10~4 M suramin caused toxic mitochondrial changes within l h
as seen by electron microscopy in Fig. 5. These changes became
progressively more pronounced with time. There was advanced
cell damage by 3 days, as evidenced by the presence of multivesicular bodies that were not observed in control cells.
Reversal of Effects of Suramin. The inhibition by suramin of
tetrazolium conversion was reversible by exchange with medium
that did not contain the drug as seen by the MTT assay.
Inhibition of tetrazolium conversion could not be reversed by
the addition of serum, multiple growth factors, or calcium. The
reversibility of the antiproliferative effect of suramin after re
moval of suramin was also demonstrated by anchorage-depend
ent colony formation at the concentration IO"3 M in Fig. 2. If
suramin was removed after l h or 1 or 3 days by exchange with
fresh medium, there was a return of colony formation. However,
total colony count decreased with increased time of suramin
exposure, indicating some irreversible toxicity at the concentra
tion 10~' M. The reversibility of the antiproliferative effect of
suramin by medium exchange and some irreversible toxicity at
high suramin concentrations were also seen with the DNA and
MTT assays (results not shown).
DISCUSSION
In addition to measuring cell proliferation, the MTT assay
was used in this study to measure the immediate inhibition of
tetrazolium conversion after the addition of suramin. The con
version of tetrazolium to the formazan product occurs by re
duction with NADH or NADPH. At physiological pH and
temperature, this is a specific enzymatic reaction that directly
NO
REMOVE REMOVE REMOVE SURAMIN
SURAMIN SURAMIN SURAMIN SURAMIN NOT
1 HOUR 1 DAY 3 DAYS REMOVED
0.300 T
Fig. 2. Colony formation assay with suramin or suramin removed. DU 145
cells (5 x lOVwell) were seeded in 1 ml DMEF5 in 6-well culture plates on day
—¿
1.On day 0, suramin was added to wells in 1 ml DMEF5 for a final concentration
of IO"3 M; control wells received no suramin. Suramin was removed and replaced
with 2 ml fresh media at l h and 1 or 3 days. Colonies were counted after 8 days
when suramin was not removed and in cultures of untreated controls. If suramin
was removed by exchange, colonies were counted 8 days after removal of suramin.
Column, 3 determinations: bar, SD.
lium conversion by concentrations
of 1, 0.5, and 0.25 x 10~3M
0.050
suramin was not effected by IGF (1, 10, and 100 ng/ml), acidic
10
15
25
X 10-* M SURAMIN
or basic FGF (0.5, 5, and 50 ng/ml), or calcium (2, 5, and 10
HIM)(results not shown). Thus, none of these could competi
Fig. 3. Reduction in tetrazolium conversion by suramin. DU 145 cells (IO4/
tively reverse the inhibition by suramin of tetrazolium conver
well) were seeded in 100 jjl DMEF5 in 96-well microculture plates on day —¿1.
One day later suramin was added to wells in 100 ii\ DMEF5 for final concentra
sion. The order in which suramin and these other test substances
tions of 0, 1.25, 2.5, 3.75, 5, 12.5, and 25 x IO'4 M. After a 1-h incubation,
were added did not make a difference in the inhibition of tetrazolium conversion was measured by a 4-h MTT assay. Point, 8 determina
tetrazolium conversion.
tions; bar, SD.
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DISRUPTION
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•¿
Fig. 4. Light micrographs of suramintreated cells and MTT assay. Light micro
graphs of DU 145 cells after 4 h incubation for
MTT assay without (A) and with 10~3 M (B)
suramin.
Table 1 Reduction of tetrazolium conversion during 4 h by I0~'.» summit! in the
presence of serum, epidermal growth factor, or calcium
DU 145 cells (5 x 10s) were pretreated for l h with 10~3 M suramin (as
described for Fig. 3). After 1 h, fetal calf serum, EGF, or Ca2* were added in 100
¿j|DMEF5 to achieve final concentrations. At this time, an MTT assay was
performed, and tetrazolium conversion was measured after 4 h. Tetrazolium
conversion by A is expressed as a percentage of a control that did not contain
suramin. Each entry represents the average of eight determinations ±SD.
TreatmentFetal
control59.7
A vs.
(%)5710121416EGF
calf serum
6.751.3
±
±7.647.9
7.648.7
±
9.247.9
±
10.149.6
±
14.358.8
±
(ng/ml)0110IS20Ca2*
5.955.5
±
±4.252.1
±5.050.4
5.045.4
±
5.951.3
±
(mM)"12311%
3.651.6
±
2.552.7
±
3.350.5
±
±2.5
1A total of 2 x IO4cells were plated.
Table 2 Rhodamine 123 fluorescence in the presence suramin
DU 145 cells were plated on glass coverslips and allowed to proliferate to about
75% confluency in DMEF5. Coverslips were incubated for 10 min in DMEF5
(incubation media) containing rhodamine 123 (10 ng/ml), suramin (10~3 M), or
both. Coverslips were then washed with several rinses of DMEF5. Next, coverslips
were inverted over slides with depression wells containing DMEF5 (observation
media) with no additives or suramin (1,0.5, 0.25, or 0.1 x IO~3M).
Additives to incubation
mediaRhodamine
to
observation
mediaNone
(+)
Rhodamine + Suramin
None
0(0)
+ (becomes 0)
RhodamineAdditives
SuraminFluorescence"+
°+, positive fluorescence: 0, negative fluorescence. Fluorescence was observed
by fluorescent microscopy at the excitation wavelength 485 nm. The initial
presence or absence of fluorescence is indicated, and subsequent loss or develop
ment of fluorescence at 4 h is indicated in parentheses.
reflects activity of cellular dehydrogenases and is not affected
by glutathione or other strong reducing substances in the cell
(32, 33). The MTT assay measures dehydrogenase activity in
the respiratory chain in at least two sites (34). The NADH
dehydrogenases are involved primarily in the energy-producing
reactions of the respiratory chain: glycolysis, TCA cycle, and
oxidative phosphorylation. The NADPH dehydrogenases are
primarily involved in biosynthetic reductive reactions such as
the hepatic P-450 system and the production of adrenal steroid
hormones (35).
These dehydrogenases are located primarily in the mitochon
dria and, in some cells, to a small degree in the endoplasmic
reticulum. Mosmann (27) demonstrated that up to 200,000
RBCs, metabolically active cells that contain no mitochondria
or endoplasmic reticulum, convert no or little tetrazolium. This
correlates with the deposition of the insoluble formazan crystals
in the mitochondria (32) and can be seen in DU 145 cells in
Fig. 4A. Dehydrogenase activity directly reflects cellular respi
ration and has been used in the past to study the effect of drugs
on respiration (36, 37).
To our knowledge this is the first study to use the MTT assay
to evaluate the effect of a drug on respiration or energy balance.
The rapid and reversible inhibition of tetrazolium conversion
by suramin in intact DU 145 cells demonstrates inhibition of
dehydrogenase activity, a direct reflection of diminished cellular
respiration and mitochondrial activity. This inhibition of tet
razolium conversion by suramin begins at approximately IO"4
M (143 Ã-Ã-g/ml),
which is below clinically desired plasma levels
(>200 Mg/ml) at which antitumor effects are seen (3).
The inhibition of tetrazolium conversion by suramin is a
rapid event, occurring during <4 h. Various substances known
to stimulate DU 145 cell growth or substances known to be
antagonized by suramin were tested for their ability to reverse
the rapid inhibition of tetrazolium conversion by suramin. In
our laboratory, serum (1-10%) stimulates DU145 cell growth.
EGF and EGF receptors are present in DU 145 cells and EGF
stimulates proliferation in DU 145 cells (38-40). IGF and heparin-binding growth factor-stimulated proliferation are antag
onized by suramin in other cell systems (41, 42), and calcium
has been shown to reverse suramin inhibition of growth (43).
None of these substances reversed the rapid inhibition of tet
razolium conversion by suramin in DU 145 cells when they were
tested over a broad range of concentrations. In addition, we
have examined >12 primary prostate epithelial cultures and
have not been able to reverse the effects of suramin with the
addition of EGF, ECGF, basic FGF, calcium, bovine pituitary
extract, or transforming growth factor ß
(44). Thus, inhibition
of tetrazolium conversion was not competitively reversed by the
addition of serum, multiple growth factors, or calcium. The
rapid inhibition by suramin of tetrazolium conversion in intact
DU 145 cells indicates disruption of cellular energy balance or
respiration at suramin concentrations
that are routinely
achieved in human plasma.
The ability of mitochondria to accumulate and retain the
fluorescent compound, rhodamine 123, is dependent on main
tenance of the energy-dependent transmembrane potential of
the mitochondria (30). Demonstrated in this report is rapid
inhibition by suramin of the uptake and retention of rhodamine
123 by mitochondria of DU 145 cells. This indicates the loss of
the energy-dependent transmembrane potential of the mito-
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DISRUPTION
OF CELLULAR ENERGY BALANCE BY SURAMIN
M
M
—¿
M
M
*
e
d
Fig. 5. Electron micrographs of suramin Incaled DU145 cells, a, untreated control cells at 0 h. Mitochondria (M) have normal cristae (x 18,000). ft, 10 ' M
•¿-immun
at l h. Loss of cristae is already present (*) (x 18.000). c, 10~' M suramin at 2 h. Mitochondria show extreme loss of cristae (arrow) (x 18.000). d, \0~' M
suramin at 24 h. Swollen mitochondria are present (arrowhead) (x 23.700). e, 10~4 M suramin at 3 days. Multivesicular bodies are now evident in the cytoplasm
(arrow) (x 18,000)./
control at 3 days. Neither multivesicular bodies nor swollen mitochondria (M) are present (x 13,000).
and showed its effect on phospholipid metabolism. The studies
we performed with tetrazolium conversion also show noncom
petition with respect to suramin and growth factors. We believe
that suramin may inhibit growth factor-stimulated proliferation
suramin, a concentration that can be achieved in human plasma.
but at a site beyond the binding of the growth factor to its
The rapid disruption by suramin of rhodamine 123 uptake and receptor.
The effects seen with suramin at low concentrations on
retention by mitochondria in intact DU 145 cells indicates that
suramin acts as a respiratory poison or ionophore in intact cells respiration and energy balance of intact cells occurs so rapidly
at suramin concentrations that are achievable in human plasma.
that we think it may involve interference with surface membrane
Finally, our findings document the rapid toxic mitochondrial
structures. Our data from the rhodamine 123 studies show that
changes by electron microscopy, further demonstrating an effect suramin may act as an ionophore. Further studies are under
by IO"4 M suramin on cellular energy balance. These effects
way in our laboratory to answer this question. In addition,
occur rapidly, within 1 h, again, at suramin concentrations that
Dunn and Blakely (47) showed that suramin functionally is a
reversible antagonist of cell surface P^-purinoreceptors. Inhibi
are achievable in human plasma.
Kopp and Pfeiffer (46) demonstrated that suramin had a tion of intracellular calcium release and growth factor-induced
noncompetitive interaction with EGF receptors in HT-29 cells increases in cytoplasmic free calcium (48) and phosphoinositide
6633
chondria. Interference with rhodamine 123 staining of this type
has been described as characteristic of inhibitors of electron
transport in the respiratory chain or ionophores (45). Again,
these effects can be demonstrated at 143 mg/ml (10~4 M)
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DISRUPTION
OF CELLULAR ENERGY BALANCE BY SURAMIN
synthesis (46) illustrates how suramin could inhibit growth
factor-stimulated second messenger signaling. Extramitochondrial calcium concentrations have been shown to regulate activ
ity of calcium-dependent mitochondria! dehydrogenases (49).
Interference by suramin at any of these sites could explain how
suramin might inhibit growth factor stimulation of respiration
and, hence, proliferation.
The inhibition of glycolysis and respiration by suramin in
trypanosomiasis and onchocerciasis provides a model to suggest
a mechanism of action of suramin on intact organisms. It is
probable that the antiproliferative mechanism of suramin on
intact DU 145 cells involves the inhibition of respiration or
disruption of cellular energy balance as seen by three different
studies in this report. The inhibition by suramin of glycolysis
in a human colon adenocarcinoma cell line also suggests that
suramin inhibits respiration or energy balance in intact mam
malian cells (25).
The disruption of cellular energy balance or respiration may
explain the most prominently observed clinical toxicities of
nephropathy, adrenal toxicity, coagulopathy, and demyelinating neuropathy. The function of mitochondrial dehydrogenases
are crucial to the function of the kidney and adrenal gland. The
adrenal mitochondrial P-450 enzymes are responsible for ste
roid biosynthesis and have been shown to be inhibited by
suramin (50). Coagulation factors must undergo microsomal
enzymatic carboxylation in order to have functional activity
(51). Demyelinating neuropathy may reflect the effect of sura
min on the oligodendroglia cells which are especially sensitive
to cytotoxic anoxic damage (52).
In summary, it is apparent that suramin may act as an
antiproliferative agent, at least in part, via the disruption of
respiration or cellular energy balance. Energy balance and cell
ular respiration will need to be considered with regard to drug
interactions, toxicities, and therapy.
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Disruption of Cellular Energy Balance by Suramin in Intact
Human Prostatic Carcinoma Cells, a Likely Antiproliferative
Mechanism
Randall Rago, Jacque Mitchen, Ann-Lii Cheng, et al.
Cancer Res 1991;51:6629-6635.
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