Download Human hepatocellular carcinoma cell lines exhibit multidrug

Human hepatocellular carcinoma cell lines exhibit multidrug resistance
unrelated to MDR\ gene expression
D.-W. SHEN1'3, YUAN-G. LU3, KHEW-V. CfflN 1 ,1. PASTAN2 and M. M. GOTTESMAN1'*
1
3
Laboratory of Cell Biology, 2Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA,
Shanghai Institute of Cell Biology, China
•Author for correspondence at: Laboratory of Cell Biology, Building 37, Room 2E18, National Cancer Institute, Bethesda,
MD20892, USA
Summary
Multidrug resistance of human cancer cells may
result from expression of P-glycoprotein, the product
of the MDRl gene, acting as an energy-dependent
drug efflux pump. However, direct evidence that
expression of the MDRl gene contributes to the
multidrug resistance of human liver carcinomas has
not been established. In this study, we tested five cell
lines derived from human hepatocellular carcinomas
for sensitivity to a variety of drugs used widely as
anticancer agents: these included vinblastine, doxorubicin, actinomycin D, mitomycin C, 5-fluorouracil,
6-mercaptopurine, melphalan, methotrexate, cis-platinum and etoposide (VP-16). All five hepatoma cell
lines were resistant at different levels to these
chemicals compared to human KB cells. Although it
has been demonstrated that resistance to vinblastine,
colchicine, doxorubicin and actinomycin D in human
multidrug-resistant cells is associated with overexpression of P-glycoprotein, very little expression of
P-glycoprotein was found in these human hepatoma
cells. Neither verapamil nor quinidine, inhibitors of
the drug efflux pump, were able to overcome
multidrug resistance in hepatoma cells. These results
indicate that the multidrug resistance phenotype in
human hepatocellular carcinoma cells cannot be
attributed to expression of the MDRl gene, but that
novel mechanisms may account for the resistance of
these cancer cells.
Introduction
existing studies indicate that the disease is resistant to
most chemotherapeutic regimens tested. The intrinsic
mechanisms by which liver carcinomas resist chemotherapy or acquire drug resistance after treatment remain
to be determined.
In the present study, five cell lines derived from human
hepatocellular carcinomas (Chen et al. 1980; Shen and
Chen, 1985) were used to explore the association between
multidrug resistance and expression of the MDRl gene in
human liver carcinoma cells.
The phenotype of multidrug resistance (MDR) has been
studied extensively in tissue culture cells as well as in
human cancers (Pastan and Gottesman, 1987; Endicott
and Ling, 1989; Shen et al. 1986a; Goldstein et al. 1989),
where it is commonly associated with expression of the
MDRl gene, which encodes the 1700003fr membrane
glycoprotein, acting as an ATP-dependent efflux pump to
prevent accumulation of drugs in resistant cells (Gottesman and Pastan, 1988). It has been reported recently that
MDRl RNA is expressed at substantial levels in human
colon, kidney, small intestine and liver (Fojo et al. 19876;
Gottesman et al. 1989), and at increased levels in rat liver
induced by carcinogens, hepatectomy and malignant
transformation (Thorgeirsson et al. 1987; Fairchild et al.
1987; Burt et al. 1988). In the case of renal adenocarcinomas, correlative evidence and data based on the use of
inhibitors of the multidrug transporter suggest that the
MDRl gene contributes directly to the intrinsic multidrug
resistance of this cancer (Fojo et al. 1987a; Kakehi et al.
1988; Kanamaru et al. 1989).
Despite the large number of patients throughout the
world who die of primary liver cancer, the number of
patients with this tumor who have entered carefully
planned clinical chemotherapy regimens from which valid
conclusions can be drawn remains limited. However, most
Journal of Cell Science 98, 317-322 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
Key words: P-glycoprotein, hepatoma cells, doxorubicin, cisplatinum.
Materials and methods
Cell lines and cell culture
Five human hepatoma cell lines, BEL-7402, BEL-7404, BEL7405, QGY-7703 and SMMC-7721, were derived from different
specimens of primary liver cell carcinomas not subjected to
chemotherapy prior to surgery. Their biological characteristics
have been previously described in detail (Shen and Chen, 1985;
Chen et al. 1980). These hepatoma cell lines were all grown as
monolayer cultures at 37 °C in 5 % CO2, using Dulbecco's modified
Eagle's medium with 4.5 gl" 1 of glucose (Gibco), supplemented
with 15 % fetal bovine serum (Whittaker, M. A. Bioproducta), Lglutamine, penicillin and streptomycin.
KB-3-1, a human KB epidermoid carcinoma cell line, and its
colchicine-selected derivative, KB-8-5, which was maintained in
colchicine, lOngml" 1 , were used for comparison (Akiyama et al.
317
1985; Shen et al. 1986a). The culture conditions for the KB cell
lines were the same as for the human hepatoma cell lines
described above.
Drug sensitivity assay
The dose—response curves of the hepatoma cell lines and the KB3-1 cell line that served as a drug-sensitive control, were
determined by seeding 6 x 104 cells in 1 ml medium in each well of
24-well dishes. At the time of seeding, the chemicals at desired
concentrations were introduced into the cell cultures. After
incubation for 3 days, the cells were counted with a Coulter
Counter. The ICM value was determined as the concentration of
drug inhibiting cell growth to 50 % of that in control (drug-free)
medium. The relative resistance factor was determined by
dividing the mean IC^ value of the drug for the hepatoma cell
lines by that for the KB 3-1 cell line that served as their control.
The values are means of triplicate determinations.
Most of the chemicals tested in this study were purchased from
Sigma, except mitomycin C (Calbiochem) and cis-platinum
(platinal; Bristol Myers Laboratories).
Nucleic acid hybridization and protein gel
High molecular weight genomic DNAs and total RNAs were
prepared as described (Shen et al. 19866). RNA slot blots and
Southern blots were hybridized with a 1383 base-pair insert of the
MDR1 cDNA probe pHDR 5A (Ueda et al. 1987), double labeled by
nick translation (Lofstrand Labs). Restriction endonucleases
£coRI and HindBl (Bethesda Research Laboratories) were used
for digestion of genomic DNAs. Southern and slot-blot hybridizations were performed as described previously (Shen et al. 19866).
Crude membrane fractions labeled with [ S]methionine were
extracted from cells by differential centrifugation (Germann et al.
1989). P170 membrane protein was immunoprecipitated with
polyclonal antibody 4007 (Tanaka et al. 1990). Samples were run
on SDS-6 % polyacrylamide gels.
Results and discussion
Multidrug resistance phenotype
Human liver carcinoma is one of the most common cancers
in males and females in the world (Yu, 1985; Munoz and
Busch, 1987). However, no chemotherapeutic agents have
been found to provide a clinically effective treatment of
this disease. In this study we screened a variety of
anticancer drugs or cytotoxic agents, including natural
products affected by the multidrug resistance phenotype,
using five human hepatocellular carcinoma-derived cell
lines as an in vitro model.
Colchicine, vinblastine, actinomycin D, doxorubicin and
etoposide (VP-16), drugs known to be transported by the
P-glycoprotein efflux pump (Pastan and Gottesman 1987;
Endicott and Ling 1989; Gottesman and Pastan, 1988),
were tested for their toxicity against five human hepatoma
cell lines as compared to KB-3-1 cells. Table 1 shows that
trie BEL-7404 cell line was 9.6 times more resistant to
colchicine than KB-3-1 cells, while the other four cell lines
exhibited only slightly higher resistance to this drug. All
five liver cell lines showed a similar level of resistance to
vinblastine, actinomycin D and doxorubicin, which was 2to 5-fold greater than for KB-3-1 cells. All the hepatoma
cell lines were even more resistant to VP-16, except for the
SMMC-7721 cells.
We also examined the toxicity of some anti-cancer
agents that are poor substrates for P-glycoprotein, such as
cis-platinum, mitomycin C, melphalan, methothrexate, 5fluorouracil (5FU) and 6-mercaptopurine (6MP). The
results are shown in Table 2. All five hepatoma cell lines
were quite resistant to mitomycin C, with 11- to 15-fold
more resistance than KB-3-1 cells. Most of the cell lines
also showed substantial resistance to all of the other
agents tested. Thus, the five hepatoma cell lines displayed
a broad multidrug resistance phenotype as evidenced by
resistance to all of the chemicals tested here including
drugs known to be affected by the multidrug transporter,
or unrelated compounds. These results confirm the clinical
impression that hepatocarcinomas are resistant to the
commonly used anticancer agents (Falkson and Coetzer,
1987; Kamiyama and Tobe, 1987). The broad spectrum of
resistance suggest that the resistance cannot be explained
by expression of the MDR1 gene, which encodes part of a
Table 1. Relative drug resistance of human hepatocarcinoma cells to MDR drugs
Relative resistance
Chemicals
KB3-1
BEL7402
QGY7703
BEL7404
BEL7405
SMMC7721
Colchicine
Vinblastine
Actinomycin D
Doxorubicin
VP-16
1.0
1.0
1.0
1.0
1.0
1.8
2.7
1.2
1.7
2.2
3.6
4.5
6.4
3.0
8.7
9.6
3.1
2.4
5.0
6.4
1.5
2.2
1.9
4.0
4.5
1.3
2.7
4.8
2.4
1.4
Relative resistance is expressed as the ICjo of the hepatoma cell lines divided by the
unity (see Materials and methods)
of KB-3-1 cells, which were arbitrarily assigned a value of
Table 2. Relative drug resistance of human hepatocarcinoma cells to non-MDR drugs
Relative resistance
KBChemicals
3-1
cis-platinum
Mitomycin C*
Melphalan
Methothrexate
5FU
6MP
1.0
1.0
1.0
1.0
1.0
1.0
BEL7402
QGY7703
BEL7404
2.7
10.3
11.1
4.7
1.0
5.8
13.3
15.6
11.1
12.1
2.7
13
2.6
2.5
2.3
1.3
60
2.8
3.3
1.8
1.1
3.3
1.2
3.2
1.8
14.5
BEL7405
SMMC7721
3.4
13.0
2.8
ND
Relative resistance is expressed as the ICJO of the hepatoma cell lines divided by the ICw of KB-3-1 cells, which is arbitrarily assigned a value of
unity (see Materials and methods).
• Mytomycin C may be poorly transported by P-glycoprotein.
318
D.-W. Shen et al.
120
0.1
1
10
Colchicine (ngml" 1 )
100
1
100
10
Mitomycin C (ngml" 1 )
10
100
1000
cis-platinum (ngml" 1 )
Fig. 1. Effect of verapamil on in vitro sensitivity of BEL-7404 cells to colchicine, cis-platinum and mitomycin C. The concentration
of verapamil used was 10/igml" 1 . (B) -Verapamil; ( • ) +verapamil.
transport system with specificity for hydrophobic natural
products only. The data presented here might also indicate
that inherent drug resistance is associated with these liver
carcinoma cells, since the five cell lines were all obtained
from patients without chemotherapy before surgery.
Whether acquired multidrug resistance developed during
passage in vitro might be determined by comparing our
results with primary cultures from untreated patients.
The effect of reversing agents
We reported that quinidine, at a clinically achievable
concentration, enhanced sensitivity to vinblastine in cells
from several renal cell lines and primary renal cell
cultures that are naturally multidrug resistant (Fojo et al.
1987a; Kakehi et al. 1988; Kanamaru et al. 1989). Several
calcium-channel blockers (i.e. verapamil), and many other
agents (i.e. reserpine, phenothiazines, cyclosporin A) are
also known to reverse the multidrug resistance phenotype,
due to expression of the MDRI gene ire vitro (Tsuruo,
1988). To determine whether the MDR phenotype in
hepatoma cells could be overcome by reversing agents,
verapamil, quinidine, reserpine and thioridazine were
tested. The results are shown in Figs 1 and 2.
Verapamil was effective at reducing resistance of renal
cell lines as well as resistance of KB colchicine-resistant
cells at a concentration of lO^gml" 1 (Fojo et al. 1987a).
However, verapamil failed to overcome resistance in the
hepatoma cell line BEL-7404 to the P-glycoprotein
substrate colchicine, or to cis-platinum or mitomycin C
when the same concentration was used (Fig. 1). Fig. 2A
and B shows that the resistance of QGY-7703 cells to
colchicine or mitomycin C was not overcome by quinidine
at 7.5/igml~1, a concentration known to reverse drug
resistance in many cell lines. Neither reserpine nor
thioridazine at concentrations indicated in Fig. 2C and D
enhanced the sensitivity of the hepatoma cell line BEL7404 to colchicine. These results indicated that the
mechanism(s) involved in the multidrug resistance phenotype in hepatoma cells was probably not associated with
overexpression of P-glycoprotein.
Expression and amplification of the MDRi gene
The most common form of resistance to multiple chemotherapeutic agents results from expression of a 170 000 MT
membrane protein (P-glycoprotein, P170), encoded by the
MDRI gene. We determined the level of P-glycoprotein in
each of the hepatocarcinoma cell lines by immunoprecipitation. As shown in Fig. 3, although a very light band of
unknown origin is seen with a molecular weight of
170000Afr, this band is much lighter than the P170 band
found in KB-8-5 cells which are 3- to 6-fold drug-resistant
compared to KB-3-1. These data, taken together with the
evidence presented above that inhibitors of P-glycoprotein
do not reverse the MDR phenotype in hepatoma cells,
indicate that the drug resistance of these cells cannot be
attributed to expression of P-glycoprotein.
To determine with greater specificity whether the
hepatomas express the MDRI gene, MDRI mRNA levels
were measured by slot blot hybridization with a 32 Plabeled MDRI cDNA probe. Only one of the five hepatoma
cell lines, QGY-7703, showed slightly higher MDRI RNA
levels than that of the other hepatoma cell lines or the KB3-1 cells, but still less than the low-level multidrugresistant cell line KB-8-5 (Fig. 4). No evidence of MDRI
gene rearrangement or amplification was detected in any
of the hepatoma cell lines (data not shown).
The broad range of multidrug resistance of these
hepatoma cells, and their failure to express significant
levels of MDRI RNA, or P-glycoprotein, or for their
resistance to be overcome by verapamil, indicate that
novel mechanisms of multidrug resistance are responsible
for their phenotype. The general resistance of the
hepatomas may be derived in some way from mechanisms
related to the important role that the liver plays in
MDR phenotype in human hepatoma lines
319
1
1
10
10
Mitomycin C ( n g m P 1 )
Colchicine ( n g m P 1 )
120"
F i g . 2. (A,B) Effect of q u i n i d i n e
on in vitro s e n s i t i v i t y of QGY-
1
10
100
1000
0
0.1
Colchicine ( n g m P 1 )
detoxification of xenobiotics and chemical toxins in vivo.
Recently, an increasing body of evidence has shown
atypical or multiple patterns of drug resistance in human
leukemia cell lines that fail to overexpress P-glycoprotein
(Norris et al. 1989; Finalay et al. 1990), but none of these
320
D.-W. Shen et al.
7703 cells to colchicine and
mitomycin C. The concentration
of quinidine used was
7.5/igml" 1 . (C,D) Effect of
reserpine and thioridazine on in
vitro sensitivity of BEL-7404
cells to colchicine. The
concentrations of the reversing
agents used were: (0) 0, ( • )
1/JM, (•) 10/iM, ( ~ O ~ )
100 /flu reserpine (C); and (B) 0,
1
10
Colchicine ( n g m P 1 )
( • ) 1 / M , (WIO/IM, (
O
)
100/<M thioridazine (D).
resistance patterns corresponds to those observed in the
hepatoma cells. Increased levels of glutathione-S transferase and decreased topoisomerases I and II are thought to
be associated with drug resistance in human breast cancer
cells (Batist et al. 1986; Cazenave et al. 1989) and in some
LU
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•
IC-772
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7405
7404
m
•
-7703
m
1
7402
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to
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j
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other cell lines (Per e* a/. 1987; Beck, 1989; Tan et al. 1989).
Continued analysis of these hepatoma cell lines should
yield valuable information about these and other novel
mechanisms of drug resistance.
CO
I
O
The authors thank Dr Lori Goldstein for providing probes,
Joyce Sharrar and Dwayne Eutsey for secretarial assistance, and
Steven Neal for photographic help.
X
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(Received 14 September 1990 - Accepted 28 November 1990)