[CANCER RESEARCH 47, 4601-4607, September 1, 1987]
Resistance of Human Melanoma Cells against the Cytotoxic and
Complement-enhancing Activities of Doxorubicin1
Mounanandham Panneerselvam, Reinhard Bredehorst, and Carl-Wilhelm Vogel2
Departments of Biochemistry and Medicine, and the Vincent T. Lombardi Cancer Research Center, Georgetown University, Washington, DC 20007
ABSTRACT
The anticancer agent doxorubicin has two different effects on human
SK-MEL-170 melanoma cells: the well known direct cytotoxicity and a
marked enhancement of their susceptibility to killing by the R24 mono
clonal anti-Go] ganglioside antibody and human complement. This com
plement-enhancing effect of doxorubicin is also present after covalent
immobilization onto glycerol-coated glass beads preventing cellular up
take of the drug (M. Panneerselvam, R. Bredehorst, and C-YV.Vogel,
Proc. Nati. Acad. Sci. USA, «5:9144-9148,1986). In order to investigate
the effect of doxorubicin resistance on the complement-enhancing activity
of the drug, we have established a doxorubicin-resistant SK-MEL-170
subline. The development of drug resistance in these melanoma cells was
associated with multiple phenotypical changes including an increased
expression of at least four high molecular weight plasma membrane
proteins or glycoproteins with molecular weights of approximately
220,000,180,000,150,000, and 130,000, respectively. The drug-resistant
cells accumulated doxorubicin at approximately 2-fold lower amounts in
accordance with a 2-fold higher efflux of doxorubicin from these cells.
The basal complement susceptibility of the doxonibicin-resistant cells
was reduced by more than 60% presumably as a result of the observed
reduced expression of GDJ sites and decreased binding of Ob. Most
importantly, the doxorubicin-resistant cells were also resistant against
the complement-enhancing effect of the free and immobilized drug. The
two doxorubicin resistance phenomena, the resistance to the cytotoxic
and to the complement-enhancing activities of the drug, seem to be
fundamentally different: (a) to achieve comparable cytotoxic and comple
ment-enhancing effects on drug-sensitive and -resistant SK-MEL-170
cells, the drug-resistant cells required 178-fold higher concentrations of
free doxorubicin for the cytotoxic effect but only 5-fold higher amounts
of the free drug and 12-fold higher amounts of the immobilized drug for
the complement-enhancing effect; (A) resistance against the cytotoxic
activity of doxorubicin is associated with reduced intracellular drug
accumulation, while the data obtained with immobilized doxorubicin
indicate that resistance to the complement-enhancing activity of the drug
is independent of cellular drug uptake. These data suggest that different
mechanisms are responsible for the two resistance phenomena in doxo
rubicin-resistant melanoma cells.
Associated with pleiotropic drug resistance is the increased
expression of high molecular weight plasma membrane protein
species (13-21), which have been postulated to be involved in
the altered drug accumulation.
Recently, we reported a novel cell surface effect of doxorub
icin: the enhancement of killing of human melanoma cells by
monoclonal antibody plus complement (22). Doxorubicin in
hibits in an oxygen radical-mediated process the degradation of
cell surface-bound C3b (22-24) which we had previously iden
tified as a protective mechanism of complement-resistant mel
anoma cells (25). Most important was the observation that
doxorubicin exerted the same effect after immobilization onto
glycerol-coated glass beads by which the cellular uptake of the
drug was prevented (22-24). Since these data demonstrate that
the complement-enhancing activity of doxorubicin is independ
ent of membrane transport mechanisms, it was interesting to
study the effect of immobilized doxorubicin on drug-resistant
cells.
In the present study we generated a doxorubicin-resistant
human SK-MEL-170 melanoma cell line and characterized
phenotypical changes as compared to the parent doxorubicinsensitive cell line. The complement susceptibility of these cell
lines prior to and after treatment with free and immobilized
doxorubicin was determined using the R24 monoclonal anti
body to the GD3 ganglioside antigen of human melanoma cells
for sensitization (26, 27) and human serum as a source of
complement. This murine IgG3 antibody has been shown to be
an efficient activator of human complement (28). Our data
demonstrate that doxorubicin-resistant SK-MEL-170 cells are
also resistant to the oxygen radical-mediated cell surface effect
of free and immobilized doxorubicin of enhancing the comple
ment susceptibility. Resistance to the cytotoxic effect of doxo
rubicin and resistance to the complement-enhancing effect of
the drug differ fundamentally, and several lines of evidence
suggest that both types of resistance may be caused by different
mechanisms.
INTRODUCTION
The anthracycline glycoside doxorubicin (former generic
name: Adriamycin) is a widely used antineoplastic agent in the
treatment of human malignancies (for review, see Ref. l). One
major problem with doxorubicin treatment is the frequent
development of drug resistance of malignant cells. Resistance
to doxorubicin has also been reported in association with crossresistance to other chemotherapeutic agents, a phenomenon
termed pleiotropic drug resistance (2-6). Studies with different
cell lines suggest that this type of resistance is due to both a
membrane restriction of drug uptake (5, 7, 8) and an enhanced
efflux mechanism which extrudes drug from the cell (5, 8-12).
Received 11/24/86; revised 5/4/87; accepted 6/8/87.
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.
1Part of this work was presented at the 71st Annual Meeting of the Federation
of American Societies for Experimental Biology, Washington, DC, March/April
1987 (47). This work was supported by NIH grant CA 35525 to C-W. V.
3 Recipient of Research Career Development Award CA 01039 from the
National Cancer Institute, Department of Health and Human Services.
MATERIALS
AND METHODS
Selection of a Doxorubicin-resistant Subline of SK-MEL-170 Mela
noma Cells. Human SK-MEL-170 melanoma cells were kept in contin
uous culture using minimal essential medium supplemented with 10%
(v/v) heat-inactivated fetal calf serum, 1% (v/v) nonessential amino
acids, 1% (v/v) sodium pyruvate, 2% (v/v) L-glutamine, and 10 IHM4(2-hydroxyethyl)-l-piperazineethanesulfonic
acid (GIBO). Grand Is
land, NY) (25). The first step of selection was accomplished by growing
the cells in the presence of 0.1 pg/ml doxorubicin (Sigma, St. Louis,
MO). Subsequently, the cells were grown in the presence of 0.25 ng/
ml doxorubicin. Further selection steps were performed by doubling
the concentration of doxorubicin up to 2 ¿tg/ml.
Doxorubicin Accumulation and Efflux. To assay cellular accumulation
of doxorubicin, parent and doxorubicin-resistant SK-MEL-170 cells
were harvested with trypsin (M. A. Byproducts, Walkersville, MD)
from confluent flasks, pelleted, and resuspended in PBS3 at 23°C.For
each time point 0.5 ml of the cell suspension (1 x 10' cells) was mixed
'The abbreviations used are: PBS, phosphate-buffered
saline; NK, natural
killer.
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with 0.5 ml PBS containing S ¿ig
of doxorubicin and incubated at 23°C. solutions of radiolabeled proteins had specific activities of 4.5 x 10s
cpm//jg (C3), 4 x IO6 cpm//ig (monoclonal anti-C3 antibodies), and
At time intervals as indicated the cells were pelleted, washed once with
cold PBS, and sonicated in 1 ml of 50% ethanol containing 0.3 N HC1. 3.7 X 10* cpni/fig (monoclonal R24 antibody).
Binding of I2sl-labeled R24 Antibody, I2sl-Iabeled C3, and I25I-labeled
After centrifugation the clear supernatant was assayed by fluorometry
at an excitation wavelength of 470 nm and an emission wavelength of anti-O Antibodies to Parent and Doxorubicin-resistant SK-MEL-170
Cells. The binding of I25l-labeled R24 antibody to the melanoma cells
580 nm. Blanks consisted of control samples to which no doxorubicin
was determined by incubating 1 x 10* cells for 20 min at 37°Cwith
had been added. All experiments were performed in triplicate. The
500 ¡tiR24 ascites [1/10 diluted with 3.5 mM 5,5-diethylbarbituric acid
doxorubicin concentration was determined from a standard curve.
(Sigma)-143 HIMNaCl-0.1% (w/v) gelatin (Difco Laboratories, Detroit,
To determine the efflux of intracellular doxorubicin, the cells were
MI)-0.15 mM CaCl2, 0.5 mM MgCl2, pH 7.4] (corresponding to ap
incubated in the presence of doxorubicin for 16 min under conditions
proximately 25 /ig R24 antibody) supplemented with 0.24 /ig I25Iare described above, pelleted, washed once with cold PBS, resuspended
in PBS (1 x 10' cells/ml), and then incubated at 23°C.After different
labeled R24 antibody (9 x 10s cpm). Separation of free and bound
periods of incubation the cells were pelleted, washed once in cold PBS,
antibody was performed by centrifugation through 12% (w/v) sucrose
and then assayed for intracellular doxorubicin as described above.
(Sigma) as described recently (25). Calculations of antibody molecules
Cell Survival by Colony Formation. To determine the dose response
per cell are based on the specific activity of the radiolabeled protein
curves to doxorubicin of parent SK-MEL-170 melanoma cells and of and a molecular weight of 155,000 for murine IgG. Binding studies
the doxorubicin-resistant subline the cells (102-104) were plated in 25- with human I2sl-labeled C3 and the radiolabeled monoclonal antibodies
ml tissue culture flasks in 5 ml of complete culture medium. After 20
h, various amounts of doxorubicin in complete culture medium were
added. After incubation for 8-10 days at 37'C the colonies were
counted. The I>-„
value is the concentration of drug which reduced the
plating efficiency to 50% of the control without drug. Relative resistance
was determined by dividing the I)M.value of the doxorubicin-resistant
SK-MEL-170 cells by the D»value of the parent SK-MEL-170 cells.
The average plating efficiencies of the parent doxorubicin-sensitive SKMEL-170 cells and of the doxorubicin-resistant subline were virtually
identical with 29 and 31%, respectively.
Complement Cytotoxicity Assay. Cytotoxicity mediated by the com
plement-activating R24 murine IgG3 monoclonal antibody (generously
provided by Dr. L. J. Old) plus human serum as complement source
was determined as described recently (25). Briefly, parent and doxorub
icin-resistant SK-MEL-170 cells were harvested with trypsin from
confluent flasks. Cells (5 x 10s) were sensitized with R24 monoclonal
antibody (200 n\ of 1:5 diluted R24 ascites from BALB/c mice). After
15 min at 37°Cthe cells were washed once and incubated with 500 n\
of normal human serum (stored at -70°C until use; 1:2 dilution) for
60 min at 37'C. Cytotoxicity was determined by dye exclusion with
0.4% (w/v) trypan blue in a hemocytometer. Unsensitized parent and
doxorubicin-resistant SK-MEL-170 cells were used as controls.
Immobilization of Doxorubicin. Immobilization of doxorubicin via
the amino group in the carbohydrate moiety onto carbonyldiimidazoleactivated glycerol-coated controlled pore glass beads (74-125 iim di
ameter) (Pierce, Rockford, IL) was carried out as described previously
(22). Briefly, 10 mg of doxorubicin in 10 ml of 0.1 M sodium borate,
pH 8.0, were incubated with 1.5 mg of dry carbonyldiimidazole-activated glycerol-coated controlled pore glass beads for 72 h at 4°C.After
washing with 0. l M sodium borate, pH 8.0, the beads were incubated
with 0.1 M hydroxylamine, pH 8.0, for l h at 4°Cto block unreacted
to human C3 were performed as described previously (25). The two
murine monoclonal antibodies to human C3 were a generous gift from
Dr. H. J. Müller-Eberhard(29). Antibody I (designated P3C3 204.10)
recognizes an epitope in the C3c domain and antibody II (designated
Ab 130) recognizes a neoantigen on iC3b and C3dg (C3d).
Other Methods. Plasma membranes were isolated as described (30)
and subjected to sodium dodecyl sulfate polyacrylamide (7%, w/v) gel
electrophoresis (extracts obtained from 10s cells were applied per lane)
with subsequent Coomassie blue staining or autoradiography. Human
C3 was purified from fresh frozen plasma as described (31).
RESULTS
Selection of a Doxorubicin-resistant SK-MEL-170 Cell Line.
Doxorubicin-resistant
human SK-MEL-170 melanoma cells
were isolated in a series of single steps. The doxorubicin con
centration of the selection medium was gradually increased
from 100 ng/ml to 2 Mg/ml. Fig. 1 shows the dose-survival
curves of the parent doxorubicin-sensitive SK-MEL-170 cells
and a doxorubicin-resistant SK-MEL-170 subline grown for 6
months in the presence of 2 ng/m\ doxorubicin. The doxorub
icin concentration by which the plating efficiency of the doxo
rubicin-resistant cells is reduced by 50% (D50, 3.16 ng/m\
doxorubicin) is 178-fold higher than that for the parent SKMEL-170 cells (Dso, 0.0178 Mg/ml).
Doxorubicin Accumulation and Efflux from Drug-resistant and
-sensitive SK-MEL-170 Cells. As shown in Fig. 2, accumulation
100
imidazolyl carbamate groups. Subsequently, the beads were washed
excessively with 0. l M sodium borate, pH 8.0, acetonitrile, and methanol for 4-5 days at 4"( ' until free doxorubicin could no longer be
detected. As determined by acid-catalyzed release of the aglycone
moiety (22), approximately 30 /¿gdoxorubicin were immobilized per
100 ill of packed wet beads. Control beads were only treated with
hydroxylamine.
Treatment of Cells with Free and Immobilized Doxorubicin. Parent or
doxorubicin-resistant SK-MEL-170 cells (5 x 10s) were incubated at
37'C for 15 h in 1 ml of complete culture medium containing varying
amounts of either free or immobilized doxorubicin. Controls were run
in the absence of free doxorubicin or with control beads. Prior to the
Cytotoxicity assay, free doxorubicin was removed by washing, and
immobilized doxorubicin by differential sedimentation of the beads and
the cells (after vortexing). All experiments were performed in duplicate.
Radioactive Labeling. Cells were metabolically labeled for 2-4 days
during exponential growth phase with [35S]methionine (800 Ci/mmol;
50 jiiCi/ml medium) (Amersham, Arlington Heights, IL) or with [I4C]glucosamine (200 mCi/mmol; 2 /¿Ci/mlmedium) (Amersham) in com
plete medium. Human C3, the monoclonal antibodies to human C3,
and the monoclonal R24 antibody were radiolabeled with NaI2SI(Amer
sham) by using immobilized chloramine-T (lodo-Beads; Pierce). Stock
10
0
0.01
0.1
1
10
Doxorubicin (fig/ml)
Fig. 1. Colony-forming ability of doxorubicin-sensitive and -resistant SKMEL-170 cells as a function of doxorubicin concentration. The plating efficiency
of parent (•)and doxorubicin-resistant (O) SK-MEL-170 cells was determined
in duplicate experiments. Bars, SD.
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RESISTANCE AGAINST COMPLEMENT-ENHANCEMENT
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presence of at least four high molecular weight proteins with
molecular weights of approximately 220,000, 180,000,
150,000, and 130,000, respectively (Fig. 3, left, lane B). Plasma
membranes of the parent doxorubicin-sensitive SK-MEL-170
cells contain these proteins in much lower or undetectable
quantities (Fig. 3, left, lane A). Differences between the drugresistant and -sensitive cells were also observed when the plasma
membrane proteins of both cell lines were analyzed after met
abolic labeling with [35S]methionine.As shown in Fig. 3 (mid
dle, lane B), in doxorubicin-resistant cells significant amounts
of the 35Slabel were incorporated into proteins with a molecular
weight ranging between 150,000 and 220,000. The autoradiography of 35S-labeled membrane extracts obtained from the
parent doxorubicin-sensitive cells revealed no radioactivity or
only faintly labeled bands in the same molecular weight range
(Fig. 3, middle, lane A). Incubation of the doxorubicin-resistant
20
30
40
SO
cells with [>4C]glucosamineresulted in significant labeling of a
Incubation at 23°C (min)
protein at the M, 220,000 position (Fig. 3, right, lane B). A
Fig. 2. Doxorubicin accumulation and efflux from drug-sensitive and -resistant
very faint [l4C]glucosamine label is also seen in some other
SK-MEL-170cells. Doxorubicin accumulation and efflux from drug-sensitive (•)
and -resistant (O) SK-MEL-170 cells was performed in phosphate-buffered saline
proteins with molecular weights ranging between 130,000 and
at 23"C. [, removal of extracellular doxorubicin. All experiments were performed
200,000. Plasma membranes of the parent doxorubicin-sensi
in triplicate. Bars, SD.
tive cells exhibited in the high molecular weight range only a
very faint 14Clabel at the M, 220,000 position (Fig. 3, right,
of doxorubicin in the drug-resistant SK-MEL-170 cells was
lane A ).
reduced by a factor of approximately two as compared to the
Effect of Doxorubicin Resistance on Basal Complement Sus
parent drug-sensitive melanoma cells. The experiments were ceptibility and C3 Binding. Of the parent SK-MEL-170 mela
performed at 23°Cin PBS to slow the accumulation rate (32)
noma cells, 33 ±2% (SD) are lysed by the R24 monoclonal
so that early time points could be examined. This difference in antibody and human complement. The complement suscepti
intracellular doxorubicin accumulation is in accordance with bility is decreased to 10 ±3% after the development of doxo
the observation that the rate of efflux of doxorubicin from the rubicin resistance. Associated with the higher complement re
drug-resistant cells was twice as high as from the drug-sensitive sistance of this SK-MEL-170 subline is a reduced expression
cells. Within 32 min at 23°Cthe intracellular doxorubicin
of GD3sites as measured by the uptake of labeled R24 antibody.
concentration decreased by 25% in the drug-sensitive cells and The number of bound R24 antibody molecules per cell is
by 50% in the drug-resistant cells. Identical results were ob decreased from 390,000 ±53,000 in the parent SK-MEL-170
tained when net accumulation and efflux of doxorubicin were cell line to 260,000 ±24,000 in the doxorubicin-resistant
determined in the presence of 15 mM glucose. These data subline. After complement activation the binding of C3 to the
suggest that accelerated efflux of doxorubicin across the plasma R24-sensitized doxorubicin-resistant SK-MEL-170 cells was
membrane is a major factor contributing to the resistance also reduced (by approximately 55%) as compared to the parent
against the cytotoxic activity of the drug in the doxorubicin- cell line. Kinetic analyses of binding of C3 to sensitized parent
and doxorubicin-resistant SK-MEL-170 cells showed that this
resistant cell line.
Changes in Plasma Membrane Protein Expression in Doxo- decrease in C3 binding is not caused by a different pattern of
rubicin-resistant SK-MEL-170 Cells. Analyses by sodium do- C3 binding and degradation. Both cell lines exhibited similar
decyl sulfate gel electrophoresis of isolated plasma membrane patterns with maximal binding of C3 occuring after 15 min of
proteins of the doxorubicin-resistant SK-MEL-170 cells (ex incubation (Fig. 4).
For the characterization of the forms of C3 bound to parent
posed for 6 months to 2 Mg/ml doxorubicin) revealed the
200 K —
92 K
220
180
150
130
K
K
K
K
220 K
200 K •*
200 K
116 K
.
92 K
-.
220 K
116 K
92 K
66 K -~
66 K —
45 K
45 K -~
45 K —
Fig. 3. Membrane proteins of doxorubicin-sensitive (lanes A) and -resistant (lancx B) SK-MEL-170 cells. Left, plasma membranes obtained from 1(1"cells subjected
to sodium dodecyl sulfate polyacrylamide gel electrophoresis with subsequent Coomassie blue staining; middle, plasma membranes obtained from 10s cells after
metabolic labeling with [35S]methionine subjected to sodium dodecyl sulfate gel electrophoresis with subsequent autoradiography; right, plasma membranes obtained
from 10s cells after metabolic labeling with ['4C]glucosamine subjected to sodium dodecyl sulfate gel electrophoresis with subsequent autoradiography. K, thousands.
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RESISTANCE
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BY DOXORUBICIN
FREE DOXORUBICIN
60
40
15
30
Incubation
I
ui
45
80
IMMOBILIZED DOXORUBICIN
at 37 C (min)
Fig. 4. Kinetics or binding of human '"I-labeled C3 to doxorubicin-sensitive
(•)and -resistant (O) SK-MEL-170 cells. Each time point was determined in
triplicate. Bars, SD.
60
40
DOXORUBICIN-RESISTANT
CELLS
20
25 -
20-
50
100
150
DOXORUBICIN (><g/ml)
Fig. 6. Effect of anthracycline resistance on doxorubicin-induced enhancement
of complement susceptibility. Doxorubicin-sensitive (•)and -resistant (O) SKMEL-170 cells were treated with increasing concentrations of free (top) or
immobilized (bottom) doxorubicin. Complement-mediated cytotoxicity was deter
mined as described under "Materials and Methods." Bars, SD.
60
INCUBATION
10
20
60
AT 37°C (mm)
Fig. 5. Characterization of the forms of C3 on doxorubicin-sensitive (left) and
-resistant («'«An
SK-MEL-170 cells by monoclonal antibodies to C3. Antibody I
(
) recognizes an epitope on C3c, thereby detecting C3b and iC3b. Antibody
111
) is specific for a neoantigen that is formed upon inactivation of C3b
by Factor I, thereby detecting iC3b, C3dg, and C3d. Bars, SD.
doxorubicin-resistant cells developed also resistance against the
complement-enhancing activity of free and immobilized doxo
rubicin. The complement susceptibility of parent doxorubicinsensitive cells could be enhanced from approximately 30->70%
by treatment with 40 tig/ml of free doxorubicin or 15 Mg/ml of
immobilized doxorubicin. Glass beads without immobilized
doxorubicin (control beads) had no complement-enhancing ef
fect. Significantly higher concentrations of free and immobi
lized doxorubicin were required to enhance the complement
susceptibility of the doxorubicin-resistant SK-MEL-170 cells
(Fig. 6). For example, to achieve a 30% enhancement (drugsensitive cells, from 33-63% cytotoxicity; drug-resistant cells,
from 10-40% cytotoxicity) the drug-resistant cells required
approximately S-fold higher concentrations of free doxorubicin
and 12-fold higher concentrations of immobilized doxorubicin.
and doxorubicin-resistant SK-MEL-170 cells, two monoclonal
antibodies against different antigenic determinants of human
C3 were applied. Antibody I binds to an epitope in the C3c
domain of C3, thereby detecting C3b and iC3b on the cell
surface. Antibody II recognizes a neoantigen that appears upon
inactivation of C3b by Factor I. This monoclonal antibody
detects iC3b, C3dg, and C3d on the cell surface. As shown in
Fig. 5, antibody II recognizing the degradation products of C3b DISCUSSION
is the predominant antibody at all times on the parent and the
doxorubicin-resistant cells indicating the rapid inactivation of
The present study demonstrates that the development of
C3b on the surface of both cell lines. On doxorubicin-resistant
doxorubicin resistance in human SK-MEL-170 melanoma cells
cells, the inactivation of C3b may occur slightly faster since the is associated with multiple phenotypical changes. The drugrelative amount of intact C3b appears to be somewhat lower resistant cells showed a significantly accelerated efflux of dox
than on the parent drug-sensitive cells. In addition, at all times
orubicin across the plasma membrane and a corresponding
the total number of anti-C3 antibodies (antibodies I plus II) is decrease in the intracellular accumulation of the drug. Several
less on the doxorubicin-resistant cells than on the parent dox
high molecular weight plasma membrane proteins were ex
pressed at significantly higher levels in the drug-resistant than
orubicin-sensitive cells which is in accordance with the de
creased total amount of surface-bound '-M-labeled C3 on the in the drug-sensitive cells. Associated with a reduced expression
doxorubicin-resistant cells (compare above).
of CM,i ganglioside sites on the cell surface and a decreased
Enhancement of Complement Susceptibility by Free and Im
binding of the complement component C3b, the susceptibility
of the resistant cells to killing by the R24 monoclonal anti-G,,,
mobilized Doxorubicin. Fig. 6 shows the effect of different
concentrations of free and immobilized doxorubicin on the R24
antibody and human complement was reduced by more than
antibody and human complement-mediated lysis of parent and
60%. Most importantly, the doxorubicin-resistant melanoma
doxorubicin-resistant SK-MEL-170 cells. It is evident that the cells were also resistant against the complement-enhancing
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activity of free and immobilized doxorubicin. At present, it is
unknown whether the development of resistance to the cytotoxic activity of doxorubicin is necessarily linked to the occur
rence of a decreased basal complement susceptibility and to the
development of resistance to the complement-enhancing activ
ity of free and immobilized doxorubicin. Since the selection of
the doxorubicin-resistant subline took place over six months in
the continuous presence of the mutagenic drug, the possibility
exists that the observed multiple phenotypical changes are due
to independent genetic events.
Most likely, different cellular mechanisms are responsible for
the increased resistance to lysis by the R24 antibody plus
complement, the resistance to the cytotoxic activity of doxo
rubicin, and the resistance to the complement-enhancing activ
ity of the drug. The decreased basal complement susceptibility
appears to be a consequence of the reduced expression of the
GD.Iganglioside which serves as antigen for the complementactivating R24 monoclonal antibody used for sensitization of
the cells. Thereby, less C3b is deposited on the cell surface
which limits the efficiency of the complement attack. The
reduced expression of the GD3 ganglioside in the doxorubicinresistant cell line is in accordance with the observation that
daunorubicin-resistant Chinese hamster cells are blocked at the
level of hematosides (GM3) (17), the biosynthetic precursors of
GD3 (33). However, it should be mentioned that other factors
may also contribute to the decreased basal complement suscep
tibility, e.g., interference of the overexpressed high molecular
weight plasma membrane proteins with the binding of comple
ment components. Furthermore, it has been reported that drugresistant human K562 leukemia cells also developed resistance
to NK cell-mediated lysis (34). It remains to be investigated
whether the decreased basal susceptibility of drug-resistant
tumor cells to NIC-mediated lysis and complement-mediated
lysis is coincidental or whether it reflects similar phenotypical
membrane changes that result in a decreased susceptibility to
both immune effector mechanisms since both NK cells and
complement use pore-forming proteins with structural and
functional similarities as killer molecules (35).
Resistance to the cytotoxic activity of doxorubicin of different
anthracycline-resistant cells has been attributed to the presence
of an energy-dependent active extrusion pump (5, 8-11). We
have also observed a significantly accelerated efflux of doxorub
icin from the drug-resistant SK-MEL-170 melanoma cells re
sulting in a decreased intracellular drug accumulation. Overexpressed high molecular weight plasma membrane proteins
may be involved in the mechanism of altered membrane trans
port of the drug. In multidrug-resistant Chinese hamster ovary
cells a M, 170,000 P-glycoprotein has been correlated with the
reduced accumulation of colchicine (36). Using radiolabeled
photoactive analogues of vinblastine, specific photoaffinity la
beling of a Mr 150,000-180,000 membrane protein in multidrug-resistant Chinese hamster lung cells (37) and of a M,
150,000-170,000 membrane protein in multidrug-resistant KB
cells (38) has been demonstrated. The photoaffinity labeling
could be blocked by the calcium channel blocker verapamil (38,
39) which has been shown to reverse multidrug resistance (38,
40-42). Recently, modulation of vincristine and actinomycin D
transport by a monoclonal antibody to a Mr 170,000-180,000
glycoprotein in the cell membrane of human myelogenous
leukemia cells and human ovarian cancer cells both resistant to
doxorubicin has been described (43). In doxorubicin-resistant
SK-MEL-170 melanoma cells we observed the increased
expression of at least four high molecular weight proteins with
molecular weights of approximately
220,000, 180,000,
BY DOXORUBICIN
150,000, and 130,000, respectively. Membrane extracts of the
parent doxorubicin-sensitive cell line contain these proteins in
much lower or undetectable quantities. The M, 220,000 protein
could be significantly labeled with [l4C]glucosamine, while the
glycoprotein character of the other high molecular weight pro
teins is questionable. It should be mentioned that metabolic
labeling was performed for at least two doubling times (approx
imately, 48 h) to avoid differences in the incorporation of the
label due to intracellular pool differences. However, under these
conditions it cannot be excluded that due to metabolic interconversions of the carbohydrate label to amino acids some of
the bands represent proteins rather than glycoproteins. Based
on these data, none of the high molecular weight proteins in
the doxorubicin-resistant SK-MEL-170 melanoma cell mem
branes appears to be identical with the M, 170,000 P-glycopro
tein found in drug-resistant Chinese hamster ovary cells (13,
19). However, in other drug-resistant cell lines proteins similar
to those found in the drug-resistant SK-MEL-170 melanoma
cells have been identified. For example, plasma membranes of
doxorubicin-resistant Chinese hamster lung cells contain a M,
180,000 and a M, 220,000 protein both of which are expressed
in drug-sensitive cells only in very low levels (20). However, in
contrast to our results, the M, 220,000 protein could not be
radioactively labeled after incubation of resistant cells with
[14C]glucosamine, whereas the M, 180,000 protein was labeled
under these conditions (20). A M, 150,000 glycoprotein is
present in cell membrane preparations from daunorubicin-re
sistant Chinese hamster lung cells (17), and in Vinca alkaloidresistant human leukemic lymphoblasts an overexpression of
three cell surface membrane glycoproteins with molecular
weights of 180,000, 155,000, and 130,000, respectively, has
been reported (42). The two resistance-associated glycoproteins
with molecular weights of 155,000 and 130,000, respectively,
have been related to cellular differentiation events, since their
expression was found to be reduced after a differentiation
stimulus of the lymphoblasts with 12-0-tetradecanoylphorbol13-acetate (42). It remains to be investigated whether the high
molecular weight membrane proteins expressed in doxorubicinresistant SK-MEL-170 melanoma cells are involved, directly
or indirectly, in the altered drug transport mechanism or in
other drug resistance-related effects such as decreased C3b
binding.
Several lines of evidence suggest that the resistance to the
cytotoxic activity of doxorubicin and the resistance to the
complement-enhancing activity of the drug may be caused by
different mechanisms. To begin with, equitoxic and equicomplement-enhancing doxorubicin concentrations differ funda
mentally. A decrease of the plating efficiency by a factor of two
requires a doxorubicin concentration of 0.0178 ¿/g/mlfor the
drug-sensitive and 3.16 /ug/nil for the drug-resistant cells. On
the other hand, to increase the complement susceptibility for
example by 30% (drug-sensitive cells, from 33-63% cytotoxicity; drug-resistant cells, from 10-40% cytotoxicity) the drugsensitive cells have to be incubated with 30 Mg/m' of
doxorubicin and the drug-resistant cells with 153
Thereby, the equitoxic doxorubicin concentrations differ by a
factor of 178 but the equicomplement-enhancing
concentra
tions by only a factor of five. The most likely explanation for
this striking difference between equitoxic and equicomplementenhancing doxorubicin concentrations is that the complementenhancing effect does not require cellular uptake of doxorubicin
while the drug has to accumulate within the cell to exert its
cytotoxic activity. Support for this hypothesis derives from
recent studies in our laboratory providing evidence for mem-
4605
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RESISTANCE
AGAINST COMPLEMENT-ENHANCEMENT
brane components as main targets for the complement-enhanc
ing activity of doxorubicin (24), and from experiments with
doxorubicin immobilized onto glycerol-coated glass beads (22).
Such doxorubicin beads efficiently enhance the complement
susceptibility of SK-MEL-170 melanoma cells. Involvement of
cellular uptake mechanisms such as endocytosis can be excluded
since the diameter of the glass beads is approximately twice as
large as the diameter of the melanoma cells. Furthermore,
immobilized doxorubicin enhances the complement suscepti
bility even at concentrations where the free drug is no longer
active, indicating that this effect is not due to release of drug
from the beads. In accordance with the data obtained with free
doxorubicin. equicomplement-enhancing concentrations of the
immobilized drug differ also by not more than a factor of 12.
Recently, we could demonstrate that the complement-en
hancing activity of free and immobilized doxorubicin is medi
ated by oxygen radicals including hydrogen peroxide, superoxide anión radical, and hydroxyl radical (23, 24). Therefore,
doxorubicin-resistant SK-MEL-170 cells may have developed
a mechanism rendering them resistant to oxygen radical-medi
ated membrane injury. For example, in the doxorubicin-resist
ant cells the synthesis of scavengers for reactive oxygen species
such as antioxidant defense enzymes could be increased. Pos
sible support for this hypothesis comes from a recent report
that five times as much intracellular anthracycline glycoside
was required to kill a doxorubicin-resistant sarcoma cell line
versus the parent cell line (44) indicating that resistance to
doxorubicin cannot be only due to impaired drug accumulation.
In human MCF-7 breast cancer cells resistance to the cytotoxic
effect of doxorubicin has been reported to involve at least in
part a significant increase of intracellular glutathione-S-transferase activity (45). Alternatively, the resistance to the comple
ment-enhancing activity of doxorubicin-resistant cells could be
caused by an altered susceptibility of the membrane target(s)
for oxygen radical attack, e.g., due to the presence of the
overexpressed high molecular weight proteins, or by a drasti
cally reduced generation of oxygen radicals in the presence of
the drug-resistant cells, or by a decreased binding of doxorubicin
to membrane components of doxorubicin-resistant SK-MEL170 cells. In a recent study, specific binding of doxorubicin to
isolated macromolecular lipids from doxorubicin-sensitive and
-resistant mouse leukemia cells was compared (46). In the
resistant cells doxorubicin binding of the lipids was altered such
that up to 10 times more doxorubicin was required for satura
tion. Further work in our laboratory is directed toward better
understanding the molecular basis for the resistance against the
complement-enhancing activity of free and immobilized doxo
rubicin.
8.
9.
10.
11.
12.
13.
14.
15.
17.
18.
20.
21.
22.
24
26.
ACKNOWLEDGMENTS
We thank Dr. Lloyd J. Old for his continuous interest and support
of this work and for the generous supply of the R24 monoclonal
antibody. We also thank Dr. H. J. Müller-Eberhard for generously
providing the monoclonal anti-C3 antibodies.
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Resistance of Human Melanoma Cells against the Cytotoxic and
Complement-enhancing Activities of Doxorubicin
Mounanandham Panneerselvam, Reinhard Bredehorst and Carl-Wilhelm Vogel
Cancer Res 1987;47:4601-4607.
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