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Center for Molecular Biology of the University of Heidelberg (ZMBH), Heidelberg, Germany (T.E.); Department of Pediatrics, University of Jena, Jena, Germany (A.S.); Hospital Pharmacy, University of Aachen, Aachen, Germany A.O.); Institute of Human Genetics (E.G.) and Laboratory of Molecular Tumor Biology, Department for Dermatology (P.R., H.O.W.), University of Erlangen-Nuremberg, Erlangen, Germany; Institute of Toxicology, University of Mainz, Mainz, Germany (J.G.H.); Department of Neurosurgery, University of Göttingen, Göttingen, Germany (M.-E.H.); German Cancer Research Center, Heidelberg, Germany (M.V.); Department of Pharmacology, Fox Chase Center, Philadelphia, Pennsylvania (K.D.T.); University of Maryland Greenebaum Cancer Center, Department of Medicine, University of Maryland School of Medicine, and the Baltimore Veterans Affairs Medical Center, Baltimore, Maryland (D.D.R.); and Department for Oncology Research, Merck KGaA, Darmstadt, Germany (J.O.F.)
Received January 30, 2003; accepted April 28, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-glutamylcysteine synthetase increased resistance to ART. The
conditional expression of the CDC25A gene using a tetracycline
repressor expression vector increased sensitivity toward ART.
Multidrug-resistant cells differentially expressing the MDR1,
MRP1, or BCRP genes were not cross-resistant to ART. ART
acts via p53-dependent and- independent pathways in isogenic p53+/+
p21WAF1/CIP1+/+, p53-/- p21WAF1/CIP1+/+, and p53+/+
p21WAF1/CIP1-/- colon carcinoma cells.
). They are
recommended by the World Health Organization as salvage treatment options for
otherwise unresponsive Plasmodium falciparum and Plasmodium
vivax strains. Large clinical studies with malaria patients have shown
that ART is well tolerated, with few and insignificant side effects
(Hien et al., 1992). In
addition to the well known antimalarial activity of ART, we have previously
identified a profound cytotoxic action of ART against cancer cell lines of
different tumor types (Efferth et al.,
2001).
Although progress in understanding of the antimalarial mechanism of
artemisinin has been made (Walker et al.,
2000), the molecular action of ART toward tumor cells is still
unexplored. The aim of the present investigation was to analyze the modes of
ART's anticancer action. In collaboration with the U.S. National Cancer
Institute (NCI; Bethesda, MD), ART has been tested in 55 tumor cell lines
(Efferth et al., 2001). Now,
we have analyzed these data, together with the mRNA expression measured by
microarray hybridization (Scherf et al.,
2000) and with cell cycle and proliferation parameters
(O'Connor et al., 1997) by
Kendall's 
test and hierarchical cluster analysis. For target validation,
cell lines transduced with genes identified by cluster analysis were taken as
examples to prove a causative role of these genes for the response to ART.
Furthermore, we analyzed the activity of ART against multidrug-resistant (MDR)
tumor cells that differentially expressed the MDR-conferring MDR1,
multidrug resistance-related protein 1 (MRP1), or BCRP
genes.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cells and Cell Lines.
Cell Lines of the Developmental Therapeutics Program of the NCI The
panel for the present investigations consisted of 55 human tumor cell lines
representing leukemia, melanoma, non-small cell lung cancer, colon cancer,
renal cancer, ovarian carcinoma, tumors of the central nervous system,
prostate carcinoma, and breast cancer. The cell lines and their cultivation
have been described previously (Alley et
al., 1988). Cells were assayed by means of a sulforhodamine B
assay (Rubinstein et al.,
1990).
Transduced Cell Lines. MSV-HL13 cells transduced with pcDNA3
expression vectors harboring cDNAs for heavy and light subunits of
-glutamylcysteine synthetase and MSV-PC4 cells carrying a pcDNA3 mock
control expression vector were handled as described
(O'Brien et al., 2000). MSV
cells are a clonal variant of NIH3T3 cells. The establishment of human
glioblastoma multiforme U-87MG cell lines transduced with a mock control
expression vector (U-87MG.Lux) or an expression vector harboring an epidermal
growth factor receptor (EGFR) gene with a genomic deletion of exons 2
through 7 (U-87MG.
EGFR) has been reported previously
(Huang et al., 1997). The cell
lines were kindly provided by Dr. W. K. Cavenee (Ludwig Institute for Cancer
Research, San Diego, CA) and cultured as described
(Nagane et al., 1996). Rat
embryo R12 cells containing the tetracycline (Tet) repression expression
vector pUHD 15-1 and a hygromycin resistance vector were previously described.
CDC25A expression was inducible after tetracycline removal for 48 h,
resulting in a 5-fold increase in CDC25A phosphatase activity
(Blomberg and Hoffmann,
1999).
Multidrug-Resistant Tumor Cell Lines. Leukemic CCRFCEM and HL-60
cells were maintained in RPMI 1640 medium (Invitrogen, Eggenstein, Germany)
supplemented with 10% fetal calf serum in a humidified 5% CO2
atmosphere at 37°C. Cells were passaged twice weekly. All experiments were
performed with cells in the logarithmic growth phase. The multidrug resistance
gene 1 (MDR1)-expressing CEM/ADR5000 was maintained in 5000 ng/ml
doxorubicin. The MRP1-expressing HL-60/AR subline was continuously
treated with 100 nM daunorubicin. The establishment of the resistant subline
has been described (Kimmig et al.,
1990; Brügger et al.,
1999). Sensitive and resistant cells were kindly provided by Dr.
J. Beck (Department of Pediatrics, University of Greifswald, Greifswald,
Germany). Breast cancer cells transduced with control vector
(MDA-MB-231-pcDNA3) or with cDNA for the breast cancer resistance protein
BCRP (MDA-MB-231-BCRP clone 23) were maintained under standard
conditions as described above for CCRF-CEM and HL-60 cells. The generation of
the cell lines followed a published protocol
(Doyle et al., 1998). The cell
lines were continuously maintained in 800 ng/ml geneticin (Invitrogen,
Karlsruhe, Germany).
Knockout Cell Lines. Human wild-type HCT116 colon cancer cells
(p53+/+ p21WAF1/CIP1+/+) as well as knockout clones (p53-/-
p21WAF1/CIP1+/+, and p53+/+ p21WAF1/CIP1-/-) derived by
homologous recombination (Waldman et al.,
1995; Bunz et al.,
1998; Weber et al.,
2002) were a generous gift from Dr. B. Vogelstein and H. Hermeking
(Howard Hughes Medical Institute, Baltimore, MD). The cell lines were
propagated in McCoy's 5A medium (Invitrogen) supplemented with 10% fetal calf
serum, and 1% penicillin/streptomycin (Invitrogen). The cells were passaged
twice weekly.
Growth Inhibition Assay
The in vitro response to cytostatic drugs was evaluated by means of a
growth inhibition assay. Aliquots of 5 x 105 cells/ml were
seeded in culture medium, and drugs were immediately added at different
concentrations. Cells were counted up to 10 days after seeding. The resulting
growth curves represent the net outcome of cell proliferation and cell death.
Cell numbers were counted in each eight independent determinations.
Reverse Transcribed-Polymerase Chain Reaction (RTPCR) Assay
Total RNA was extracted by using the RNeasy Mini Kit (QIAGEN GmbH, Hilden,
Germany) and the RNase-free DNase set (QIAGEN), and dissolved in
diethylpyrocarbonate-treated H2O. Two micrograms of RNA were
reverse-transcribed using 20 pmol of oligo(dT) primer and the Omniscript
reverse transcriptase kit (QIAGEN) in a final volume of 40 µl. The reverse
transcription mixture (50 µl) was diluted with the same volume of
H2O, and 2 µl (100 ng) of cDNA was added to 48 µl of PCR mix.
The PCR mix contained 5 µl of 10x reaction buffer, 5 µl of
deoxynucleoside-5'-triphosphate mix (2 mM for each
deoxynucleoside-5'-triphosphate), 5 µl of each primer (5 pmol/µl),
0.25 µl(5U/µl) Taq Polymerase (Roche Diagnostics, Mannheim,
Germany), and 27.75 µl of H2O. A 5-min denaturation step at
94°C was followed by 29 cycles (MDR1), 26 cycles (MRP1),
and 24 cycles (BCRP) of amplification, respectively. Each cycle was
composed of denaturation (94°C, 30 s), primer annealing (55°C, 30 s),
and primer extension (72°C, 30 s). The PCR was completed by a final
extension step (72°C, 10 min). The cycle number for each gene was
determined by estimation of the linear region of gene amplification by
subjecting a cDNA mix to an increasing number of PCR cycles. The negative
controls were constructed by using the cDNA synthesis mixture as described
above, without addition of reverse transcriptase or containing water instead
of cDNA. PCR products were electrophoretically separated onto 2.0% agarose
gels and stained with ethidium bromide.
The primers were: MDR1 (ABCB1), 1524 5'-ATT GGT GTG GTG AGT CAG GAA-3' 1544 and 1908 5'-TGA CGT CAG CAT TAC GAA CT-3' 1889; MRP1 (ABCC1), 2296 5'-CGT GTA CTC CAA CGC TGA C-3' 2314 and 2621 5'-CTG GAC CGC TGA CGC CCG TG-3' 2602; BCRP (ABCG2), 1025 5'-AGA CTT ATG TTC CAC GGG CC-3' 1044 and 2138 5'-CCA AGG CCA CGT GAT TCT TC-3' 2119.
Immunoblotting and Kinase Assays
The procedures have been described
(Funk et al., 1997). Briefly,
whole-cell lysates were prepared by brief sonication in lysis buffer
containing 50 mM HEPES, pH 7.5, 450 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10%
glycerol, 1 mM NaF, 0.1 mM sodium orthovanadate, 10 mM
-glycerophosphate, and 0.1% Tween 20. For immunoblotting, 20 µg of
whole-cell extract were loaded. Antibodies were sourced as follows: anti-p53
and anti-p21 (Oncogene Science, Cambridge MA), anti-RB (BD Biosciences
PharMingen, Heidelberg, Germany), anti-CDK2 (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), anti-cyclin B1 (GNS-1; BD Biosciences PharMingen), and
anti-CDC25A (Santa Cruz Biotechnology). In vitro kinase assays were performed
as described previously in detail
(Hermeking et al., 1995). For
immunoprecipitation, antibodies against CDK2 or cyclin B1 were used.
Flow Cytometry
Cell samples were fixed in 70% ethanol in phosphate-buffered saline after
labeling with bromodeoxyuridine (BrdU; Sigma Chemie, Deisenhofen, Germany),
stained with 50 µg/ml propidium iodide (Sigma Chemie), and subjected to
flow cytometry using a Becton Dickinson FacScan II (BD Biosciences).
Statistical Analyses
Objects were classified by calculation of distances according to the
closeness of between-individual distances by means of hierarchical cluster
analysis. All objects were assembled into a cluster tree (dendrogram). The
merging of objects with similar features leads to the formation of a cluster,
where the length of the branch indicates the degree of relation. The distance
of a subordinate cluster to a superior cluster represents a criterion for the
closeness of clusters as well as for the affiliation of single objects to
clusters. Thus, objects with tightly related features appear together, whereas
the separation in the cluster tree increases with progressive dissimilarity.
Recently, cluster models have been applied for gene expression profiling and
for approaching molecular pharmacology of cancer
(Efferth et al., 1997;
Scherf et al., 2000;
Volm et al., 2002). Cluster
analyses applying the complete-linkage method were done with the WinSTAT
program (Kalmia Company, Inc., Cambridge, MA). Missing values were
automatically omitted by the program, and the closeness of two joined objects
was calculated by the number of data points they contained. To calculate
distances between all variables included in the analysis, the program
automatically standardizes the variables by transforming the data with a mean
= 0 and a variance = 1.
Kendall's test was used to calculate significance values (P
values) and rank correlation coefficients (R values) as a relative
measure for the linear dependence of two variables. This test was implemented
into the WinSTAT program (Kalmia). Kendall's test determines the
correlation of rank positions of values. Ordinal and metric scaling of data
are suited for the test and are transformed into rank positions. There is no
condition regarding normal distribution of the data set for the performance of
Kendall's test.
The Mann-Whitney U test is an implement of the WinSTAT program
(Kalmia). The test was used to analyze two rows of values for significant
differences. It is a parameter-free test without need for normal distribution.
The 
2 test (WinSTAT, Kalmia) was applied to bivariate
frequency distributions of pairs of nominal scaled variables.
COMPARE analyses were performed with software implemented into the web site
of the NCI
(http://dtp.nci.nih.gov).
COMPARE analyses yielded rank-ordered lists of compounds. Every compound of
the Standard Agent Database of the NCI was ranked for similarity between its
modulation of in vitro cell growth patterns and the modulation of in vitro
cell growth patterns of a selected seed or probe compound
(Paull et al., 1989). To
obtain COMPARE rankings, a scale index of similarity between the seed compound
cell growth pattern and the pattern for each of the COMPARE data base
compounds is created. This methodology is used to identify the presumable mode
of action of investigational drugs by comparing their IC50 profiles
using the NCI cell lines with those of drugs with well characterized
mechanisms of action.
In addition to the calculation of P and R values, the
problem of multiple hypothesis testing was addressed. The probability of type
I errors increases as the number of tests increases
(Hochberg and Benjamini, 1990;
Keselman et al., 2002).
Therefore, a step-up resampling multicomparison procedure was applied to
control the false discovery rate (FDR) among the significant correlations at
significance levels of 0.05. This program has been developed by Reiner et al.
(2003) and is available on
http://www.math.tau.ac.il.
The FDR is the expected proportion 
of erroneous rejections among all
rejections of the null-hypothesis (Reiner
et al., 2003).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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).
The IC50 values were in a range from 246 nM to 
100 µM
(log10, -6.609 to -4). These data were subjected to COMPARE
analysis. The COMPARE computations of IC50 values for ART against
171 agents included in the Standard Agent Database of the NCI did not reveal
any information regarding the mode of action of ART (data not shown). Thus,
ART does not seem to belong to any traditional class of antitumor drugs (e.g.,
topoisomerase I/II inhibitors, tubulin poisons, DNA/RNA inhibitors, etc.).
Therefore, we searched for other cellular and molecular determinants of ART's
action against tumor cells.
Proliferative Activity and Cell Cycle Distribution. A large number
of cellular and molecular parameters have been analyzed in the cell lines of
the NCI's screening panel
(http://dtp.nci.nih.gov)
(O'Connor et al., 1997;
Scherf et al., 2000). We first
correlated IC50 values of ART to various proliferation parameters.
As shown in Fig. 1a, there was
a significant positive correlation between cell doubling times of untreated
cells and IC50 values for ART (P = 0.00132; R =
0.27879). Thus, rapidly growing cell lines were more susceptible to ART than
slowly growing ones. Next, the cell cycle distribution of the 55 cell lines in
relation to ART's activity was analyzed. We observed a positive correlation
between IC50 values of ART and percentage of cells in the
G0/G1 phase of the cell cycle (P = 0.02244;
R = 0.20232) and an inverse correlation between ART's IC50
values and S-phase proportions of the cell lines (P = 0.03567;
R = -0.18190). Cell lines with a low percentage of
G0/G1 cells and a high percentage of S-phase cells had a
high proliferative activity and were most sensitive to ART. G2/M
phases did not significantly correlate with the inhibitory action of ART
(Fig. 1d)
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For comparison, the relationship between proliferation and
growth-inhibiting activity was also analyzed for standard anticancer agents.
Table 1 shows the P
values obtained by Kendall's test. The IC50 values for
doxorubicin, vinblastine, 5-fluorouracil, and methotrexate correlated
significantly with the doubling times of the cell lines of the NCI panel,
whereas those for carboplatin, dacarbazine, and ifosfamide did not. If the
cell cycle distribution was compared with the IC50 values, the
S-phase fractions correlated significantly with the IC50 values for
5-fluorouracil and methotrexate. The cell fractions in G2/M phase
were significantly associated with the IC50 values for doxorubicin,
etoposide, vinblastine, 5-fluorouracil, and methotrexate. The
G0/G1 phases did not correlate with the IC50
values for the drugs analyzed. Furthermore, carboplatin, dacarbazine, and
ifosfamide did not correlate to any cell cycle phase.
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mRNA Expression Profiling. IC50 values of ART were then
correlated with the constitutive mRNA expression levels measured by microarray
hybridization. We selected expression data of 465 genes deposited in the NCI's
data base
(http://dtp.nci.nih.gov).
These genes belong to six categories of different biological functions (drug
resistance genes, DNA damage response and repair genes, oncogenes and tumor
suppressor genes, apoptosis-regulating genes, proliferation-associated genes,
and cytokines and cytokine-associated genes). These categories of genes were
chosen because they are frequently involved in the response of tumor cells to
established antineoplastic drugs. For this reason, the data base of the
Developmental Therapeutics Program of the NCI was searched for several text
strings (resistance, damage, repair, oncogene, tumor suppressor,
proliferation, cell cycle, growth, mitosis, apoptosis, death, cytokine,
interferon, interleukin). Furthermore, genes were included in the analysis
that are well known from the literature to affect chemosensitivity and
chemoresistance. After correlation analysis by means of Kendall's test,
the significance level was adjusted to 0.05 for FDR calculation. The resulting
-value of 0.00573 indicated that only 54 of 465 genes (12%) correlated
significantly to ART response, with an error probability of 
5%
(Table 2).
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The baseline mRNA expression data were subjected to hierarchical cluster
analyses (Fig. 2). The
dendrogram of the 55 cell lines was divided into three main clusters. To
examine whether these clusters were associated with the response to ART, these
clusters were correlated to the IC50 data for ART that had not been
included before the cluster analysis. The median IC50 value of all
55 cell lines tested (log10 = -5.335 M) was used as the cut-off
threshold to separate sensitive from resistant cell lines. Interestingly,
cluster 1 (n = 6) and cluster 2 (n = 25) contained
significantly more ART-sensitive tumor cell lines, whereas cluster 3
(n = 24) was enriched with ART-resistant ones
(Table 3). As calculated by
means of the 2 test, the distribution of sensitive and
resistant tumor cell lines in these clusters is significantly different
(P = 0.00017; Table
3). Cluster 1 contained only leukemia cell lines, whereas clusters
2 to 3 consisted only of cell lines of solid tumors.
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Effect of ART on Transduced Cell Lines. To start analyzing the
functional role of some of these genes in the cellular response to ART, target
validation experiments were performed in selected cases by treating transduced
cell lines with ART. We selected three genes from the panel of 60 that belong
to three different functional groups, e.g., detoxification of radical
molecules and reactive oxygen species (glutamate-cysteine ligase, regulatory
(30.8 kDa), -glutamylcysteine synthetase, GLCLR), oncogenic
transformation (epidermal growth factor receptor, EGFR), or cell
cycle regulation (CDC25A). The three genes were chosen by the
following criteria: 1) an error probability of 5% in FDR calculations, 2)
a significance value of P < 0.005, and 3) the availability of
suitable cell models.
In the NCI cell line panel, a significant correlation between mRNA
expression of GLCLR and IC50 values for ART was found
(Fig. 3a, P = 0.00051;
r = 0.30452). To corroborate the relationship of ART to
GLCLR, we treated MSV-HL13, transduced with the
-glutamylcysteine synthetase heavy and light subunits, and MSV-PC4,
transduced with mock control vector. These transduced cell lines have been
previously described (O'Brien et al.,
2000). The IC50 values calculated from the
dose-response curves in Fig. 3d showed a 3.5-fold increase in drug resistance in MSV-HL13 cells compared with
MSV-PC4 cells.
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The EGFR mRNA expression of the NCI cell line panel correlated
significantly with the IC50 values for ART
(Fig. 3b; P = 0.00390;
R = 0.24678). Therefore, we opted to analyze the relationship between
cellular response to ART and expression of EGFR. U-87MG cells
transduced with a truncated EGFR (U-87MG.EGFR) or with control
expression vector (U-87MG.Lux) have been reported elsewhere
(Huang et al., 1997). Both
cell lines were exposed to ART. The IC50 values calculated from the
dose-response curves in Fig. 3e
showed that U-87MG.EGFR cells were 13.6-fold more resistant to ART than
were the U-87MG.Lux cells, indicating a causative role of the EGFR
for resistance of tumor cells to ART.
An inverse correlation between CDC25A mRNA expression and the
IC50 values for ART in the 55 NCI cell lines was observed
(Fig. 3c; P = 0.00055;
R = -0.30264). Furthermore, we used R12 cells transduced with the Tet
repressor expression vector pUHD 15-1 containing CDC25A. The
repression of CDC25A expression in this Tet-off model has been
described (Blomberg and Hoffmann,
1999). Aliquots of R12 cells were cultured in the presence or
absence of tetracycline. Removal of tetracycline for 48 h sensitized R12 cells
6.9-fold toward ART in comparison to tetracycline-pretreated cells
(Fig. 3f). With this set of
experiments, 3 of 60 genes identified by correlation analyses
(Table 2) were validated as
causally related to the cellular response toward ART.
For comparison, a parallel experimentation was set up for doxorubicin as a
standard anticancer drug. A significant correlation was observed between the
IC50 values for doxorubicin and mRNA expression of EGFR in
the NCI cell line panel (Fig.
4b, P = 0.01546; R = 0.18948) but not for
GLCLR or CDC25A (Fig. 4,
a and c). Furthermore, over-expression of EGFR in
U-87MG.EGFR cells resulted in a 3.2-fold resistance to doxorubicin as
compared with U-87MG.Lux control cells
(Fig. 4d). The
GLCLR-transfectant MSV-HL13 cells were 1.4-fold resistant to
doxorubicin (Fig. 4e), whereas
CDC25A did not have any influence on doxorubicin sensitivity in R12
Tet-off cells (Fig. 4f).
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Comparison of ART to Established Anticancer Drugs. Next, we compared the number of genes correlating with the IC50 values for ART to those correlating to 15 established anticancer agents. Figure 5 shows the number of genes in the NCI cell line panel in which mRNA expression correlated to the IC50 values of the various anticancer drugs. Interestingly, ART was among the drugs that correlated with most genes of all gene categories except for drug resistance genes. This indicates that these gene categories may play a less important role in determining ART's inhibitory action on tumor cells than genes of the other gene groups analyzed.
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Multidrug Resistance. The drug comparison analysis in Fig. 5 stimulated us to investigate the role of drug resistance in determining ART's cellular effects in more detail. For this reason, the effects of ART on different multidrug-resistant tumor cells was investigated. As shown by reverse-transcribed polymerase chain reaction (RT-PCR), these cell lines expressed individual mRNA species for MDR-conferring ATP-binding cassette (ABC) transporter genes (Fig. 6c). Doxorubicin-resistant CEM/ADR5000 cells over-expressed MDR1 (ABCB1), doxorubicin-resistant HL-60/AR cells over-expressed the MDR-related protein 1 (MRP1; ABCC1), and MDA-MB-231-BCRP clone 23 cells over-expressed the breast cancer resistance protein (BCRP, ABCG2). The corresponding drug-sensitive cell lines did not over-express any of these ABC transporter genes. Trace amounts of MRP1 mRNA were detected in both sensitive and resistant CEM cells. The drug resistance phenotype of these cell lines was verified with doxorubicin using growth inhibition assays (Fig. 6a). As calculated from IC50 values of sensitive and resistant cells, CEM/ADR5000 cells revealed 833-fold, HL-60/AR cells 149-fold, and MDA-MB-231-BCRP clone 23 cells 10-fold resistance toward doxorubicin. Interestingly, ART did not encounter cross-resistance in any of these resistant cell lines (Fig. 6b), indicating that ART is not subject to MDR mechanisms constituted by these ABC transporter genes.
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p53-Mediated Chemoresistance. Since the tumor suppressor p53 is another important factor of chemoresistance, we also analyzed the possibility that p53 affects the response of tumor cells to ART. Interestingly, p53 was not among the genes identified in Table 2. Comparing the p53 mutational status of the NCI cell line panel (http://dtp.nci.nih.gov) to the IC50 values for ART did not show a significant correlation (Fig. 7; Mann-Whitney U test). Furthermore, we tested the p53-dependent downstream gene p21WAF1/CIP1. Again, we did not find a correlation of the IC50 values for ART and the constitutive mRNA expression of p21WAF1/CIP1 (data not shown).
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Considering the paramount importance of the p53 pathway for drug
resistance, we analyzed p53 and p21WAF1/CIP1 in more detail. For
this reason, we used human wild-type HCT-116 colon cancer cells (p53+/+
p21WAF1/CIP1+/+) and isogenic knockout clones (p53-/-
p21WAF1/CIP1+/+ and p53+/+ p21WAF1/CIP1-/-) derived by
homologous recombination (Waldman et al.,
1995; Bunz et al.,
1998; Weber et al.,
2002). First, we measured the incorporation of BrdU in the three
cell lines without and with 10-5 M ART for 24 or 48 h by
flow cytometry (Fig. 8a). ART
inhibited the BrdU incorporation in all three cell lines in a time-dependent
manner and to a similar extent. This indicates that the two knockout cell
lines were similarly sensitive to ART-induced inhibition of proliferation as
wild-type HCT-116 cells. Using immunoblotting and kinase assays, we analyzed
the protein expression and kinase activity of cell cycle regulating genes in
wild-type cells and knockout mutants (Fig.
8, b and c). Treatment with ART induced the expression of p53
protein after 12 and 24 h in wild-type cells but not in p53+/+
p21WAF1/CIP1-/- knockout cells
(Fig. 8b). The
p21WAF1/CIP1 protein was strongly induced in wild-type cells and
very weakly induced in p53-/- p21WAF1/CIP1 +/+ cells upon ART
treatment. Hypophosphorylation of the tumor suppressor protein RB coincided
with a down-regulation of CDK2 kinase activity in response to ART
treatment that is indicative of G1/S arrest. Protein expression and
kinase activity of the G2/M regulator cyclin B1 declined after
treatment of all three cell lines with ART for 12 and 24 h. Because
CDC25A, which governs the entry of G1 cells into the S
phase was causally related to ART sensitivity
(Fig. 3f), we also analyzed
this protein in the knockout cell lines. Expression of CDC25A protein
was down-regulated 24 and 48 h after treatment with ART
(Fig. 8c).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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100 µM (log10, -6.609
to -4). In clinical antimalaria studies, plasma peak concentrations of
2640 ± 1800 µg/ml (= 6.88 ± 4.69 mM) have been measured upon
intravenous application of 2 mg/kg ART in patients
(Batty et al., 1996). Since the
IC50 values for ART of the 55 cell lines are about 3 orders of
magnitude lower than these plasma levels, it is reasonable to speculate that
sufficient concentrations may be achievable to inhibit tumor cell growth in
human beings.
A survey of 465 genes showed that the constitutive expression of 54 genes
correlated significantly with the response to ART at a FDR probability of
5%. This was a surprisingly high number of genes. It should, however, be
considered that the correlation coefficients for these 54 genes were rather
small. From this analysis it is still not clear whether the correlations
reflect causative relationships or simply epiphenomena. Nevertheless, this
type of analysis represents a valuable source to produce testable
hypotheses.
The expression of several drug resistance genes correlated with the
IC50 values of ART, especially of genes that are involved in the
detoxification of electrophilic compounds (glutathione-associated enzymes).
This is in accordance with the reaction of ART's endoperoxide bridge to form
reactive oxygen species (ROS) and/or ART carbon-centered free radicals, both
of which affect cellular proteins and lipids of the parasites
(Asawamahasakda et al., 1994;
Berman and Adams, 1997). A role
for the endoperoxide bridge can also be assumed for the inhibitory activity in
tumor cells. The glutathione S-transferase enzyme family catalyzes
the conjugation of radical molecules and ROS to reduced glutathione. Cellular
glutathione content is regulated by GLCLR, which is crucial for the cellular
detoxification capacity of a number of xenobiotics. Because GLCLR was one of
the 54 candidate genes, we analyzed its importance in determining ART
cytotoxicity in more detail. Indeed, transduced cells were more resistant to
ART than were control cells. Of interest, there was only a mild influence of
GLCLR on doxorubicin cytotoxicity. This could imply that in the transfectant,
the quinone formed from doxorubicin is not as good an electrophilic substrate
as the endoperoxide bridge of artesunate.
In comparison with established anticancer drugs, ART correlated with fewer
drug resistance genes. This indicates that drug resistance genes may be a less
important issue if ART is established as a viable anticancer drug. This
observation fits well with a recent investigation
(Efferth et al., 2002a). ART
was more potent than the artemisinin derivatives arteether and artemether, and
the number of drug resistance genes in which mRNA expression correlated with
the IC50 values of ART was lower than that for arteether or
artemether. In the present investigation, the multidrug resistance-conferring
genes MDR1, MRP1, and BCRP did not influence resistance to
ART. Recently, we also found that MDR1-over-expressing
doxorubicin-resistant CEM/ADR5000 cells or MRP1-over-expressing
epirubicin-resistant CEM/E1 cells were not cross-resistant to ART
(Efferth et al., 2002b).
CEM/ADR5000 cells were used as controls in the present study. The lack of
cross-resistance of MRP1-over-expressing cells was confirmed here
using doxorubicin-resistant HL-60 cells. Now, we have extended this concept
using BCRP-over-expressing cells. These findings give reason to hope
that ART may be suited to treat refractory, multidrug-resistant tumors in a
clinical setting. This is also consistent with ART's activity against
multidrug-resistant Plasmodium strains
(Price, 2000) and against
ganciclovir-resistant human cytomegaloviruses
(Efferth et al., 2002c). The
correlations of the IC50 values for ART with the expression of DNA
topoisomerase I should be interpreted with some caution. ART did not correlate
with DNA topoisomerase I-inhibiting drugs (camptothecin, topothecan)
(Efferth et al., 2002a). Since
DNA topoisomerases are involved not only in multidrug resistance but also in
proliferation, these correlations may reflect ART's relationship with
proliferative index.
Some DNA damage and repair genes were correlated with ART activity, e.g.,
excision repair genes (ERCC5), damage recognition proteins of the
high mobility group (HMG), and others. DNA repair represents a well
known mechanism of drug resistance
(Martin, 2001). DNA
recognition proteins prevent repair of drug-induced DNA cross-links
(Ohndorf et al., 1999).
Previous investigations on ART's antimalarial activity focused only on protein
alkylation and lipid peroxidation
(Asawamahasakda et al., 1994;
Berman and Adams, 1997). ART
has not yet been shown to induce DNA damage. Therefore, the importance of the
correlations of IC50 values for ART to DNA damage and repair gene
expression remains unproven.
As shown previously, ART induces apoptosis in KG-1a leukemia cells
(Efferth et al., 1996). In the
present study, a number of apoptosis-regulating genes correlated with cellular
response toward ART. This fits well with the concept that key molecules of the
apoptosis cascade are important for chemotherapy-induced cell death
(Johnstone et al., 2002).
A role for oncogenes and tumor suppressor genes for the cellular response
to established anticancer drugs has been described
(el Deiry, 1997). In the
present investigation, several oncogenes and tumor suppressor genes were
associated with sensitivity of tumor cells to ART. Interestingly, the p53
mutational status of the 55 NCI cell lines did not correlate with the
IC50 values for ART. The loss of p53 function may, therefore, not
result in ART resistance as previously shown for other anticancer drugs
(O'Connor et al., 1997).
Isogenic p53-/- cells derived from parental HCT116 cells and knockout cells of
the p53-downstream gene p21WAF1/CIP1 were not resistant to ART.
After exposure to ART, p53 protein expression was induced in p53+/+ cells,
indicating that ART does stimulate a p53-dependent stress-response pathway.
Immunoblot and kinase assays of p53-dependent proteins that govern the
progression through G1 and G2 cell cycle phases did not
provide an explanation for a different regulation of these proteins in
knock-in and knockout cells. Thus, p53-independent pathways must also be
operative as a response pattern to ART. Interestingly, the expression of the
CDC25A gene product that is a p53-independent regulator of
G1/S progression was down-regulated in knock-in and knockout cells
after ART exposure. This is a clue that ART may act via
CDC25A-dependent pathways in cancer cells.
EGFR expression correlates with resistance to established drugs
(Wosikowski et al., 1997), and
we also found a correlation with ART resistance. In addition, we used tumor
cells transduced with an EGFR gene truncated in its extracellular
domain through a deletion of exons 2 to 7 (U-87MG.EGFR). This truncated
EGFR renders cells resistant to cisplatin, paclitaxel, and
vincristine (Nagane et al.,
1998). Transduction of the same tumor cell line with wild-type or
tyrosine kinase-deficient EGFR genes did not result in drug
resistance. In the present investigation, U-87MG.EGFR cells were
13.6-fold more resistant to ART and 3.2-fold more resistant to doxorubicin
than were U-87MG.Lux cells transduced with a mock control expression vector.
The fact that EGFR confers resistance to ART and several other standard
anticancer drugs confirms a general role for EGFR in drug resistance. This
gene is, therefore, not a unique target for ART. Although EGFR and
CDC25A, both of which have been tested in our study, are considered
as growth-promoting genes, they showed opposing effects. EGFR induced
resistance, whereas CDC25A caused sensitivity to ART. This
contradiction may be explainable by their effect on apoptosis. Besides its
influence on proliferation, EGFR induces expression of the
antiapoptotic BCL-2 and BCL-XL genes,
down-regulates proapoptotic BAX expression, and inhibits apoptosis
induced by diverse stimuli including cytostatic drugs
(Nagane et al., 1998;
Wang et al., 1999).
CDC25A is a target gene of the protooncogene MYC, and
CDC25A accelerates apoptosis
(Galaktionov et al., 1996).
Interestingly, MYC was also among the genes the expression of which
correlated to sensitivity toward ART in our analysis.
Many of these 54 genes regulate proliferation, e.g., cell cycle genes
(e.g., CDKs, CDCs), mitotic spindle motors (i.e., kinesins,
dynein), and growth factors (e.g., CTGF, VEGFC). We have validated
the role of CDC25A in a tetracycline repressor cell model.
CDC25A is a rate-limiting controller for the transition from
G1 to S phase of the cell cycle. CDC25A dephosphorylates
and activates the cyclin-CDK complexes that are active during
G1 (Blomberg and Hoffmann,
1999). Since the IC50 values of ART correlated with the
G0/G1- and S-phase fractions in the 55 NCI cell lines,
CDC25A may be an important regulator of cellular response to ART.
This is compatible with the fact that ART is more cytotoxic in cell lines with
a higher proliferative index. The fact that expression of CDC25A
sensitized cells to ART but not to doxorubicin provides another hint that
CDC25A might be a specific target for ART. This coincides with the
down-regulation of CDC25A after ART exposure in p53 and p21 knock-in
and knockout HCT-116 cell lines. Repression of CDC25A in the R12
Tet-off cell model reduces the entry from G1 into S phase
(Blomberg and Hoffmann, 1999).
Although the precise mechanism is yet unknown, it could be that cells in the
transition from G1 to S phase are more vulnerable than cells in
other phases of the cycle. Furthermore, it could be speculated that mechanisms
in addition to S-phase entry may also be important. In a recent study, Wang et
al. (2002) demonstrated that
CDC25A interacts with EGFR. The authors linked
CDC25A to EGFR downstream mitogenic signaling routes.
Becuase both CDC25A and EGFR mRNA expression in the NCI cell
line panel correlated significantly with the IC50 values for ART,
this novel CDC25A-EGFR pathway may be important for cellular response
to ART.
Cytokines and cytokine-associated genes were also tested in our approach.
In addition to signaling proliferation and differentiation, cytokines
contribute to the persistence of tumors following chemotherapeutic challenge
(Löwenberg et al., 1993).
Cytokines may influence drug resistance by stimulation of proliferation,
apoptosis-regulating (i.e., BCL-XL), drug
resistance, and detoxification (e.g., GST-
) gene expression
(Mizutani et al., 1995). All
of these cytokine functions may also contribute to growth-inhibitory actions
of ART on cancer cells.
|
Acknowledgements |
|---|
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Footnotes |
|---|
ABBREVIATIONS: ART, artesunate; NCI, National Cancer Institute; MDR,
multidrug-resistant; MRP, multidrug resistance-related protein; BCRP, breast
cancer resistance protein; MSV, Moloney sarcoma virus; Tet, tetracycline;
BrdU, bromodeoxyuridine; FDR, false discovery rate; GLCLR, glutamate-cysteine
ligase, regulatory (30.8 kDa), -glutamylcysteine synthetase; EGFR,
epidermal growth factor receptor; RT-PCR, reverse transcribed-polymerase chain
reaction; ABC, ATP-binding cassette.
1 Present address: Institute for Clinical and Molecular Virology, University
of Erlangen-Nuremberg, Erlangen, Germany. 
2 Present address: The Beaston Institute for Cancer Research, Glasgow,
UK.
Address correspondence to: Thomas Efferth, Center for Molecular Biology of the University of Heidelberg (ZMBH), Heidelberg, Germany. E-mail: thomas.efferth{at}web.de
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