©2005 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
To determine whether CDK2-selective inhibitors can be used for cancer therapy, we
evaluated the effects of GW8510, a commercially available synthetic CDK inhibitor that
is relatively selective for CDK2,7on six NSCLC cell lines (A549, H1299, H460, H226,
H358 and H322) and normal human fibroblast (NHFB). We found that GW8510
induced apoptosis and cell cycle arrest in a time- and dose-dependent manner in the panel
of NSCLC cell lines but not in the normal cell line. We also found that downregulation
of X-linked inhibitor of apoptosis (XIAP) contributes to induction of apoptosis by
Downregulation of XIAP and Induction of Apoptosis by the Synthetic
Cyclin-Dependent Kinase Inhibitor GW8510 in Non-Small Cell Lung
[Cancer Biology & Therapy 5:2, 165-170, February 2006]; ©2006 Landes Bioscience
John J. Davis2
1Department of Metabolism and Endocrinology; The First Affiliated Hospital;
Zhejiang University; People's Republic of China
2Department of Thoracic and Cardiovascular Surgery; The University of Texas MD
Anderson Cancer Center; Houston, Texas USA
*Correspondence to: Bingliang Fang; Department of Thoracic and Cardiovascular
Surgery; Unit 445; The University of Texas MD Anderson Cancer Center; 1515
Holcombe Boulevard; Houston, Texas 77030 USA; Tel.: 713.563.9147; Fax:
713.794.4901; Email: email@example.com
Received 10/04/05; Accepted 11/10/05
Previously published online as a Cancer Biology & Therapy E-publication:
cyclin-dependent kinases, small molecule,
apoptosis, non-small lung cancer, X-linked
inhibitor of apoptosis
We thank Don Norwood for editorial review of
the manuscript. National Cancer Institute grants
RO1 CA 092487-01A1 and RO1 CA 098582-
01A1 (both to B. Fang), National Cancer Institute
Lung Specialized Program of Research Excellence,
and National Institutes of Health Core Grant CA-
Small-molecule inhibitors of cyclin-dependent kinases (CDKs) are known to induce cell
cycle arrest and apoptosis in certain cancer cells. In order to evaluate the antitumor activity
of one such inhibitor, GW8510, against human lung cancers, we analyzed the effects of
GW8510 on six nonsmall cell lung cancer (NSCLC) cell lines (A549, H1299, H460,
H226, H358 and H322) and normal human fibroblast (NHFB). We treated the cells with
GW8510 at concentrations of 0-10 µM, and found that it suppressed cell growth in vitro
in all the lung cancer cells but not in NHFB. Subsequent study showed that GW8510
induced apoptosis and cell cycle arrest in the A549, H1299 and H460 cells in a time-
and dose-dependent manner. Western blot analysis showed that GW8510 downregulated
the expression of X-linked inhibitor of apoptosis (XIAP) but had no detectable effect on the
expression of Bax, Bak, or Bcl2. GW8510 also downregulated XIAP mRNA level,
suggesting that downregulation of XIAP expression occurs at the transcriptional level.
Moreover, ectopic XIAP expression diminished growth inhibition and apoptosis induction
by GW8510. Importantly, GW8510 was not capable of inducing apoptosis of NHFB
cells. These results suggest that GW8510 might provide a treatment strategy for human
NSCLC and XIAP is an important target for GW8510-induced apoptosis of NSCLC cells
that occurs through inhibition of XIAP mRNA transcription.
Lung cancer is the most common cancer worldwide and the leading cause of cancer-
related death.1Histologically, lung cancer is classified as small cell lung cancer (SCLC) or
non-small cell lung cancer (NSCLC), the latter of which includes squamous cell carcinoma,
adenocarcinoma, and large cell carcinoma. SCLC usually responds well to both radiother-
apy and chemotherapy. However, treatment of NSCLC is often challenging, because it is
generally less responsive or even not responsive to such therapies.2Consequently, the prog-
nosis for patients with NSCLC is often poor, with a 5-year survival rate of 10% and long
survival durations limited mainly to patients with operable early-stage disease.1,2
Therefore, development of new strategies for the treatment of NSCLC is highly desirable.
Deregulation of the cell cycle, a process controlled by various cyclins, cyclin-dependent
kinases (CDKs), CDK inhibitors, and certain tumor suppressor gene products, is known
to be one of the critical events that drive cancer cells into uncontrolled proliferation.
Molecular changes, including overexpression of cyclins and CDKs and loss of CDK
inhibitors, are frequently detected in tumor cells. For example, CDK2 orchestrates the
orderly progression of the eukaryotic cell cycle and plays a key role in the progression from
late G1to late G2phase.3Furthermore, deregulation of cyclin E, an activator of CDK2,
has been found to be associated with a broad spectrum of human malignancies.4,5Cyclin
E has also been reported to be a strong independent prognostic indicator in patients with
early-stage NSCLC.6High levels of cyclin E expression correlate significantly with short
mean survival times. Thus, CDK2 may serve as a potential target for therapeutic inter-
vention in patients with NSCLC.
Cancer Biology & Therapy
GW8510. These findings may impact future devel-
opment of CDK inhibitors for the treatment of
MATERIALS AND METHODS
Agents. GW8510 (Fig. 1A) and dimethyl sulfoxide
(DMSO) were obtained from Sigma (St. Louis, MO).
GW8510 was dissolved in DMSO at a concentration of
10 mM as a stock solution and stored at 4˚C. Unless
otherwise indicated, DMSO alone was used as a control.
RPMI 1640, high-glucose Dulbecco’s modified Eagle’s
medium, and Ham’s F12 medium were purchased from
Mediatech, Inc. (Herndon, VA). Fetal bovine serum
(FBS), and TRIzol LS reagent were purchased from
Invitrogen Corporation (Carlsbad, CA). The sulforho-
damine B (SRB) protein-binding dye and propidium
iodide (PI) reagent were purchased from Sigma.
Polyclonal rabbit antisera specific for Bax, Bak, Bcl2, and
caspase-3 were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). A murine monoclonal XIAP antibody
was purchased from Transduction Laboratories (San Diego,
CA), and a monoclonal antibody specific for β-actin was
purchased from Sigma. The ECL antimouse and antirabbit
antibodies, hybond-ECL membrane and ECL solution
were purchased from Amersham Biosciences (Arlington
Heights, IL). The primers were synthesized by Sigma.
The Nucleofector Kit V was obtained from Amaxa
(Cologne, Germany). The Advantage RT-for-PCR Kit
was purchased from BD Biosciences Clontech (Mountain
View, CA ).
Cell lines and cell cultures. A549, H1299, H460,
H226, H358 and H322 were maintained in our labora-
tory. A549 was cultured in Ham’s F12 medium containing
10% (v/v) heat-inactivated FBS and 1% (v/v) antibiotics.
The other NSCLC cell lines were maintained in RPMI
1640 medium containing 10% (v/v) heat-inactivated
FBS and 1% (v/v) antibiotics. NHFB (fourth passage)
was maintained in Dulbecco’s modified Eagle’s medium containing 10%
(v/v) heat-inactivated FBS and 1% (v/v) antibiotics. Cells were cultured at
37˚C in a humidified incubator containing 5% CO2.
SRB assay. Cell viability was determined by using the SRB colorimetric
assay as described previously.8Briefly, after fixation of adherent cells with
trichloroacetic acid in a 96-well microplate, protein was stained with SRB,
and the optical density was determined at 570 nm to reflect the number of
stained cells, which showed the cell viability. The relative cell viability was
determined by referring to the cell viability of the DMSO control which was
set at 100%. Each experiment was performed in quadruplicate and repeated
at least three times.
Flow cytometry analysis. Both floating and attached cells (use trypsin)
were collected and washed twice with cold phosphate-buffered saline (PBS).
The cells were then fixed with cold 70% ethanol and kept overnight at 4˚C.
Thirty minutes before the assay, PI staining (1 mL of PI, 10 µL of RNase,
9 mL of PBS; 50 µg/mL PI) was performed. Flow cytometric evaluation of
the cell cycle status and apoptosis were performed according to a previously
described method.9,10The percentage of cells at G1, S, G2, and M phase was
calculated by using the Cell Quest software program (Becton-Dickinson,
San Jose, CA).
Western blot assay. A total of 2-4 × 105cells were lysed in lysis buffer
(20 mM HEPES, pH 7.9, 150 mM NaCl, 0.5 mM dithiothreitol, 10 mM
KCl, 0.2 mM ethylenediaminetetraacetic acid, 10% glycerol, complete
proteinase inhibitors). Lysates were sonicated with the use of a sonicating
machine for 2 x 30 seconds at setting 6 on ice and then spun down at
15,000 rpm for 15 minutes at 4°C; the supernatant was then carefully
collected. The protein concentration was assessed by the BCA protein assay
method. Total extracts (50 µg/lane) were normalized and subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (12% gels) immunoblot
assay. Blotting was performed on a hybond-ECL membrane, and the signal
was detected by using an ECL solution. Western blot analysis of XIAP, Bax,
Bak, Bcl2, caspase-3 and β-actin was performed with specific antisera or
monoclonal antibodies as described previously.9,10Horizontal scanning
densitometry of Western blots was performed by using acquisition into the
Adobe Photoshop software program (Adobe Systems, San Jose, CA).
Expression of β-actin was used as a control.
Reverse transcriptase-polymerase chain reaction.Total RNA was isolated
by using TRIzol according to the manufacturer’s instructions, mixed with a
random hexamer primer, and incubated at 70˚C for five minutes.
Single-strand cDNA was synthesized from 0.5 µg of total RNA by using
moloney murine leukemia virus reverse transcriptase (RT) and deoxynucle-
oside triphosphate at 42˚C for 1 hour and 90˚C for five minutes; synthesis
was stopped at 4˚C. The mRNAs for XIAP were amplified by polymerase
chain reaction (PCR) with specific primers. The sequence of the sense and
antisense primer for XIAP was 5'-TGAATCTGATGCTGTGAGTT and
5'-CCTCAAGTGAATGAGTTAAA, respectively. The level XIAP mRNA
was then determined by a semi-quantitative PCR with serial-diluted cDNA
samples. The conditions for the XIAP PCR reactions were as follows: 1 x at
94˚C for three minutes; 35 x at 94˚C for 45 seconds, 50˚C for 45 seconds,
and 72˚C for 1 minute; and 1 x at 72˚C for ten minutes. Detection of glyc-
eraldehyde-3-phosphate dehydrogenase (GAPDH) was conducted as the
internal control, and the sense and antisense primers for GAPDH were
combined with the Advantage RT-for-PCR kit. The conditions for GAPDH
PCR reactions were as follows: 1 x at 94˚C for three minutes; 35 x at 94˚C
Downregulation of XIAP by a CDK2 Inhibitor
Figure 1. The structure of GW8510 and its cell-killing effects on NSCLC cell lines. (A) Chemical
structure of GW8510. (B) Cell-killing effects of GW8510 on the six NSCLC cell lines and NHFB
after treatment for 72 hours. Cell viability was determined by using the SRB assay. Cells treated
with the same amount of DMSO (v/v, 0.1%) whose viability was set at 1 were used as controls.
The viability of the NSCLC cells but not NHFB was significantly suppressed by GW8510 after
the concentration of GW8510 reached 5 µM (p < 0.05). The data represent four quadrupli-
cate assays with similar results. The values represent the mean ± standard deviation. (C)
Time-response to GW8510. H1299, and A549 cells were treated with 5 µM GW8510. Cell
viability was then determined over time as indicated. Significant suppression was found on the
first day after treatment (compared with DMSO treatment; p < 0.05). The data represent three
quadruplicate assays with similar results. The values represent the mean ± standard deviation.
Cancer Biology & Therapy
2006; Vol. 5 Issue 2
Cancer Biology & Therapy
whose cell viability after treatment was set at 1
as controls. In comparison with PBS, DMSO
(0.1% of the final concentration) did not have
any detectable effects on cell viability (data not
shown). In contrast, we found a dose-dependent
decline in cell viability in all six NSCLC cell
lines after treatment with GW8510 (Fig. 1B).
Interestingly, GW8510 had minimal effects on
the growth of NHFB (1–10 µM GW8510
could not suppress the growth of the normal
cells). The mean drug concentration that inhib-
ited cell growth by 50% in the NSCLC cell
lines was 4.22 µM; the concentrations ranged
from 3.67 µM in H460 to 5.28 µM in A549.
To test whether the effects of GW8510 on the
NSCLC cell lines were time dependent, we
treated A549 and H1299 with 5 µM GW8510
and then determined the cell viability of these
cell lines over 24-96 hours. The results showed
that GW8510 induced a time-dependent
decrease in cell viability in both cell lines (Fig. 1C).
Induction of apoptosis by GW8510.
Cancer cell viability may be reduced by either
suppressing cell growth or cell necrosis. To
determine whether the GW8510-reduced cell
viability was associated with apoptosis and cell
cycle arrest, we performed cell cycle analysis.
We treated H460, H1299 and A549 cells with
GW8510 at a concentration of 5 µM and 10 µM
for 24 hours and 72 hours respectively. We then
harvested cells and analyzed them by using a
flow cytometric assay. The results showed that
treatment with GW8510 induced apoptosis in
all three cell lines, as evidenced by a marked
increase in the percentage of cells at sub-G1
phases of the cell cycle at both 24 and 72 hours
(Fig. 2A). The apoptosis induction was most
profound in H460 cells, which is consistent
with the data from the cell viability assay (Fig. 1B).
In addition to induction of apoptosis, treatment
with GW8510 resulted in an apparent increase
in the proportion of cells at G2phase (Fig. 2B). Taken together, these results
indicated that GW8510 induces apoptosis and cell cycle arrest at G2.
GW8510 downregulated expression of XIAP. To characterize molecular
mechanisms underlying the induction of apoptosis by GW8510, we meas-
ured protein levels of several molecules that are important in apoptotic
pathways, including Bax, Bak, Bcl2 and XIAP. For this purpose, we treated
A549 and H460 cells with 1–10 µM GW8510 for 48 hours. We then har-
vested cells for Western blot analysis. Treatment with GW8510 did not
induce obvious changes in the expression of Bax, Bak, or Bcl2 at any of the
doses tested. In contrast, it dramatically downregulated the expression of
XIAP. This downregulation was dose-dependent and more dramatic in
H460 cells than in A549 cells, the earlier of which are more sensitive to
GW8510 than A549 (Fig. 3A). In contrast, we observed no obvious change
in XIAP expression in NHFB treated by GW8510 (Fig. 3B). A time-course
study showed that downregulation of the expression of XIAP by GW8510
was time dependent in both A549 and H460 cells (Fig. 3C). Moreover, the
downregulation of XIAP expression did not recover even when we removed
GW8510 from the medium for 48 hours (Fig. 3D).
To further analyze the mechanisms of GW8510-mediated downregulation
of XIAP expression, we measured XIAP mRNA level by using an RT-PCR
assay. We treated cells with GW8510 at a concentration of 5 µM and 10 µM
for eight hours and 16 hours, respectively. We then detected XIAP mRNA
level by using RT-PCR. The results showed that the XIAP mRNA level was
dramatically reduced after eight hours of exposure to 5 µM GW8510 and
for 45 seconds, 65˚C for 45 seconds, and 72˚C for one minute; and 1 x at
72˚C for ten minutes. PCR products were analyzed by using agarose gel
electrophoresis and visualized with the use of ethidium bromide.
Electroporation assay. H1299 and A549 cells (2 x 106) were suspended
in 100 µL of Nucleofector Kit solution V mixed with 4 µg of XIAP plasmid
(a gift from Dr. John C. Reed, the Burnham Institute, La Jolla, CA)11and
nucleotransfected with the use of a Nucleofector program (Amaxa) according
to the manufacturer’s instructions. Green fluorescent protein (GFP) plasmid
was transfected as a control. Twenty-four hours later, some of the transfectants
were lysed for Western blot analysis, and some were treated with GW8510.
Statistical analysis. Differences in the experimental groups were analyzed
by using analysis of variance with the Statistica software program (StatSoft,
Tulsa, OK). A P ≤ 0.05 was considered statistically significant. The drug
concentration that inhibited cell growth by 50% was calculated by using the
CurveExpert 1.3 software program (Hyams Development, Starkville, MS).
Cytotoxic effects of GW8510 on NSCLC cell lines. To evaluate the
effect of CDK2 inhibitors on lung cancer cells, we analyzed the cytotoxic
effect of the CDK2 inhibitor GW8510 on the growth of the six NSCLC
cell lines. For this purpose, we treated cells with GW8510 at concentrations
ranging from 1–10 µM. We then determined cell viability by using the SRB
assay at 72 hours after the treatment. We used cells treated with DMSO
Downregulation of XIAP by a CDK2 Inhibitor
Figure 2. Apoptosis induction in NSCLC cell lines and NHFB cells. (A) Apoptosis of NHFB, A549,
H1299, and H460 cells after 24 hours and 72 hours of exposure to DMSO, 5 µM GW8510, or 10 µM
GW8510. The apoptotic percentage was demonstrated by the percentage of cells at sub-G1phase
which was determined by fluorescence-activated cell sorter analysis. No significant difference in the
sub-G1percentage of NHFB cells was found (p > 0.05), whereas NSCLC cells showed a significant
increase in the sub-G1percentage, even at 5 µM (p < 0.05). The data represent three experiments with
similar results. (B) Cell cycle in A549 and H460 cells after 24 hours and 72 hours of exposure to DMSO,
5 µM GW8510, or 10 µM GW8510. The percentage of cells at sub-G1phases and G2phase is shown:
the percentage of cells at G2phase increased, whereas that of cells at G1phase decreased. The data
were obtained from one of three experiments with similar results.
Figure 3. Effect of GW8510 on apoptosis. (A) Western blot
analysis of XIAP, Bax, Bak, Bcl2, and caspase-3 in A549 and
H460 cells 48 hours after treatment with control DMSO (lane 1),
1.0 µM GW8510 (lane 2), 2.5 µM GW8510 (lane 3), 5.0 µM
GW8510 (lane 4), 7.5 µM GW8510 (lane 5), or 10.0 µM
GW8510 (lane 6). The expression of β-actin served as the loading
control. The data were obtained from one of three experiments
with similar results. (B) Western blot analysis of XIAP in NHFB 48
hours after treatment as described above. (C) Time-response to
GW8510. Cells were treated with DMSO (-) or 5 µM GW8510 (+)
for the indicated times (24, 48, and 72 hours [h]). (D) Continually
effect on cells after removal of GW8510. A549 and H460 cells
were treated with DMSO (-) or 5 µM GW8510 (+) for 72 hours.
DMSO and GW8510 were then removed from the medium. Cells
were harvested at different time points after removal (12, 24 and
48 hours [h]). (E) RT-PCR analysis of XIAP mRNA in H460 after
treatment with DMSO (lane 1), 5 µM GW8510 (lane 2), or 10 µM
GW8510 for 8 and 16 hours (h). PCR products were analyzed
by using 1.2% agarose gels electrophoresis.
was dose dependent. We found the same dose-dependent reduc-
tion after 16 hours of exposure, as well (Fig. 3E). These data
showed that the reduced expression of XIAP caused by GW8510
might occur at the transcriptional level.
Diminished GW8510-induced apoptosis as a result of XIAP
overexpression.To determine the role of XIAP downregulation in
GW8510-induced cell death, we transfected A549 and H1299
cells with an XIAP-expressing plasmid11by electroporation with
the use of an Amaxa machine. Twenty-four hours after electropo-
ration, we measured the XIAP expression in the transfected cells
by using Western blot analysis. The results revealed that trans-
fected cells had an approximately twofold increase in XIAP
expression when compared with parental cells or cells transfected
with a control plasmid expressing GFP (Fig. 4A). When we treat-
ed these cells with GW8510 at a concentration of 5 µM and 10
µM for 24 hours and 72 hours respectively, the percentage of
apoptotic cells was dramatically reduced among cells transfected
with XIAP when compared with parental cells or cells transfected
with GFP (Fig. 4B). Similarly, a cell viability assay showed that
transfection of the XIAP-expressing plasmid resulted in reduced
susceptibility to GW8510 when compared with parental cells or
cells transfected with GFP (Fig. 4C). These results indicated that
downregulation of XIAP correlates with induction of apoptosis by
GW8510 in cancer cells.
The findings presented here suggest that GW8510
inhibits the growth of the lung cancer cell lines, correlating
with the downregulation of the mRNA and protein level of
XIAP. GW8510 is a commercially available synthetic CDK
inhibitor, which is a selective inhibitor of CDK2. The
CDKs are increasingly recognized as important targets for
therapeutic intervention for various proliferative disease
states, including cancer.12Several small-molecule CDK inhibitors
have been evaluated clinically for treatment of cancer.13For exam-
ple, flavopiridol, a pan-CDK inhibitor, was the first CDK inhibitor
to be tested clinically for cancer therapy. By inhibiting a number of
protein kinases, and with the greatest inhibitory activity directed
toward CDK, flavopiridol inhibited cell proliferation in 60 National
Cancer Institute human tumor cell lines with no obvious
tumor-type selectivity.14However, clinical studies have shown that
the activity of flavopiridol administered alone is not effective and
that combination with other therapeutic agents is required to pro-
duce a detectable clinical response,14indicating the need for devel-
opment of new CDK inhibitors. Nevertheless, studies on flavopiri-
dol-mediated antitumor activity have revealed several molecular
events elicited by CDK inhibitors, including cell cycle arrest and
p53-independent apoptosis,15,16upregulation of E2F1 and repres-
sion of Mcl-1,17,18and downregulation or inhibition of XIAP,
Bcl-XL, p21 or Mdm2.18,19
Downregulation of XIAP by a CDK2 Inhibitor
Cancer Biology & Therapy
2006; Vol. 5 Issue 2
Cancer Biology & Therapy
In the present study, we investigated the biological effects of the
CDK2 inhibitor GW8510 on cancer and normal cells. This
compound was once reported to be able to prevent chemotherapy-
induced alopecia in rats.20However, the authors of that study
retracted their findings because they were not able to reproduce the
prevention of alopecia by this compound in a neonatal rat model of
chemotherapy-induced alopecia,21even though the compound has
the correct chemical structure and CDK2 inhibition as reported
previously.7Our results showed that GW8510 suppressed cell
growth and induced apoptosis in NSCLC cells but not in normal
cells. We also found that GW8510-mediated cell- growth inhibition
and apoptosis induction may at least be partially explained by down-
regulation of XIAP by this compound. We also found that Mcl-1
was downregulated by GW8510, however, no dramatic changes
were observed on levels of p53, p21 or Bcl-XL(data not shown).
Inhibitor of apoptosis proteins (IAPs) play an evolutionarily
conserved role in regulating programmed cell death in diverse
species, including humans.22Several members of this family of
proteins have been identified, including XIAP, human IAP-1, human
IAP-2, neuronal apoptosis inhibitory protein, and surviving.23,24
These proteins share a common caspase recruitment domain and an
NH2-terminal baculovirus inhibitor of apoptosis repeat motif. With
the exception of neuronal apoptosis inhibitory protein and survivin,
these proteins also include a COOH-terminal RING zinc finger
domain important for protein-protein and protein-nucleic acid
interactions.25Human XIAP, a key member of the IAP family, has
been shown to be a direct inhibitor of caspase-3 and caspase-726and
to interfere with the Bax/cytochrome-c pathway by
inhibiting caspase-9.22,27Overexpression of XIAP
has been shown to protect cells from apoptosis.24
Also, there is evidence that XIAP plays an important
role in the oncogenesis and progression of
NSCLC.28,29Therefore, downregulation of XIAP by
GW8510 or other CDK inhibitors may be a useful
approach for treatment of NSCLC. Nevertheless, the
application of GW8510 and its analogs will also
depend on their in vivo pharmacokinetic properties,
including absorption, distribution, metabolism, and excretion.
Thus, if sufficient GW8510 could be obtained, it would be interest-
ing to test in vivo activity of this compound.
Downregulation of XIAP in human breast cancer cell lines and
chronic lymphocytic leukemia cells by other small-molecule CDK
inhibitors has been reported.18Interestingly, expression of XIAP is
regulated at the translational level by a rare cap-independent mech-
anism mediated by an internal ribosome entry sequence in its
5'-untranslated region, which facilitates the antiapoptotic function
of XIAP in response to cellular stresses such as irradiation and
chemotherapy.30In the present study, we found that XIAP mRNA
level was downregulated by GW8510 and that XIAP protein
expression was continually suppressed after removal of GW8510,
suggesting that the suppression of XIAP by GW8510 was very effec-
tive. Whether the downregulation of XIAP mRNA was mediated by
inhibition of CDK2 is not yet clear. Nevertheless, ectopic XIAP
expression diminished GW8510-mediated apoptosis, indicating that
downregulation of XIAP expression correlates with induction of
apoptosis by this compound. This information may have an impact
on the design of combination therapy regimens for multimodality
treatment of cancer. Indeed, small-molecule CDK inhibitors such as
flavopiridol have been shown to work synergistically with the pro-
teasome inhibitor PS341 and the histone deacetylase inhibitor
suberoylanilide hydroxamic acid.31,32Whether similar combination
effects can be induced with GW8510 remains to be determined.
Downregulation of XIAP by a CDK2 Inhibitor
Figure 4. Diminished GW8510-induced cell death as a
result of overexpression of XIAP. (A) XIAP expression in
A549 and H1299 at 24 hours after transient transfection of
solution alone (lane 1), GFP plasmid (lane 2), and XIAP
plasmid (lane 3) by electroporation with the use of an
Amaxa machine. β-actin was used as the loading control.
The data were obtained from one of three experiments with
similar results. (B) Apoptosis of the transfectants. Twenty-four
hours after transfection, transfectants were treated with
DMSO, 5 µM GW8510 or 10 µM GW8510. The percentage
of cells at sub-G1phases reflected the apoptotic percentage
and was determined by fluorescence- activated cell sorter
analysis. The data represent three experiments with similar
results. The standard errors of the mean are shown. (C)
Cell-killing effects of GW8510 on XIAP-transfected H1299
and A549. Twenty-four hours after transfection, transfectants
were treated with 0–10 µM GW8510. Seventy-two hours
after treatment, cell viability was determined by using the
SRB assay as described in Figure 1B. The cell-killing effect
of GW8510 was significantly diminished in XIAP-transfected
cells when compared with that in parental cells or GFP-trans-
fected cells at 5 µM (p < 0.05). The data represent four
quadruplicate assays with similar results. The standard
errors of the mean are shown.
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1. Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ.
Cancer statistics, 2005. Ca: A Cancer J Clin 2005; 55:10-30.
2. Cortes-Funes H. New treatment approaches for lung cancer and impact on survival.
Seminar Oncol 2002; 29:26-9.
3. van den Heuvel S, Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle con-
trol. Science 1993; 262:2050-4.
4. Hubalek MM, Widschwendter A, Erdel M, Gschwendtner A, Fiegl HM, Muller HM,
Goebel G, Mueller-Holzner E, Marth C, Spruck CH, Reed SI, Widschwendter M. Cyclin
E dysregulation and chromosomal instability in endometrial cancer. Oncogene 2004;
5. Moberg KH, Bell DW, Wahrer DC, Haber DA, Hariharan IK. Archipelago regulates
Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature 2001;
6. Muller-Tidow C, Metzger R, Kugler K, Diederichs S, Idos G, Thomas M,
Dockhorn-Dworniczak B, Schneider PM, Koeffler HP, Berdel WE, Serve H. Cyclin E is
the only cyclin-dependent kinase 2-associated cyclin that predicts metastasis and survival
in early stage nonsmall cell lung cancer. Cancer Res 2001; 61:647-53.
7. Bramson HN, Corona J, Davis ST, Dickerson SH, Edelstein M, Frye SV, Gampe Jr RT,
Harris PA, Hassell A, Holmes WD, Hunter RN, Lackey KE, Lovejoy B, Luzzio MJ,
Montana V, Rocque WJ, Rusnak D, Shewchuk L, Veal JM, Walker DH, Kuyper LF.
Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): Design, synthesis, enzy-
matic activities, and X-ray crystallographic analysis. J Med Chem 2001; 44:4339-58.
8. Pauwels B, Korst AE, de Pooter CM, Pattyn GG, Lambrechts HA, Baay MF, Lardon F,
Vermorken JB. Comparison of the sulforhodamine B assay and the clonogenic assay for in
vitro chemoradiation studies. Cancer Chemother Pharmacol 2003; 51:221-6.
9. Zhu H, Guo W, Zhang L, Davis JJ, Teraishi F, Cao X, Smythe WR, Fang B. Enhancing
TRAIL-induced apoptosis by Bcl-XL siRNA. Cancer Biol Ther 2005; 4:393-7.
10. Wu S, Zhu H, Gu J, Zhang L, Teraishi F, Davis JJ, Jacob DA, Fang B. Induction of apop-
tosis and downregulation of Bcl-XL in cancer cells by a novel small molecule
2[[3-(2,3-dichlorophenoxy)propyl]amino] ethanol (2,3-DCPE). Cancer Res 2003;
11. Deveraux QL, Takahashi R, Salvesen GS, Reed JC. X-linked IAP is a direct inhibitor of
cell-death proteases. Nature 1997; 388:300-4.
12. Sielecki TM, Boylan JF, Benfield PA, Trainor GL. Cyclin-dependent kinase inhibitors:
Useful targets in cell cycle regulation. J Med Chem 2000; 43:1-18.
13. Senderowicz AM, Sausville EA. Preclinical and clinical development of cyclin-dependent
kinase modulators. J Natl Cancer Inst 2000; 92:376-87.
14. Dai Y, Grant S. Small molecule inhibitors targeting cyclin-dependent kinases as anticancer
agents. Current Oncol Reports 2004; 6:123-30.
15. Litz J, Carlson P, Warshamana-Greene GS, Grant S, Krystal GW. Flavopiridol potently
induces small cell lung cancer apoptosis during S phase in a manner that involves early
mitochondrial dysfunction. Clin Cancer Res 2003; 9:4586-94.
16. Shapiro GI, Koestner DA, Matranga CB, Rollins BJ. Flavopiridol induces cell cycle arrest
and p53-independent apoptosis in nonsmall cell lung cancer cell lines. Clin Cancer Res
17. Ma Y, Cress WD, Haura EB. Flavopiridol-induced apoptosis is mediated through upregu-
lation of E2F1 and repression of Mcl-1. Mol Cancer Ther 2003; 2:73-81.
18. Wittmann S, Bali P, Donapaty S, Nimmanapalli R, Guo F, Yamaguchi H, Huang M, Jove
R, Wang HG, Bhalla K. Flavopiridol downregulates antiapoptotic proteins and sensitizes
human breast cancer cells to epothilone B-induced apoptosis. Cancer Res 2003; 63:93-9.
19. Demidenko ZN, Blagosklonny MV. Flavopiridol induces p53 via initial inhibition of
Mdm2, p21 and, independently of p53, sensitizes apoptosis-reluctant cells to tumor necro-
sis factor. Cancer Res 2004; 64:3653-60.
20. Davis ST, Benson BG, Bramson HN, Chapman DE, Dickerson SH, Dold KM, Eberwein
DJ, Edelstein M, Frye SV, Gampe JR, Griffin RJ, Harris PA, Hassell AM, Holmes WD,
Hunter RN, Knick VB, Lackey K, Lovejoy B, Luzzio MJ, Murray D, Parker P, Rocque WJ,
Shewchuk L, Veal JM, Walker DH, Kuyper LF. Prevention of chemotherapy-induced
alopecia in rats by CDK inhibitors. Science 2001; 291:134-7.
21. Davis ST, Benson BG, Bramson HN, Chapman DE, Dickerson SH, Dold KM, Eberwein
DJ, Edelstein M, Frye SV, Gampe Jr RT, Grifffen RJ, Harris PA, Hassell AM, Holmes
WD, Hunter RN, Knick VB, Lackey K, Lovejoy B, Luzzio MJ, Murray D, Parker P,
Rocque WJ, Shewchuk-Chapman L, Veal JM, Walker DH, Kuyper LF. Retraction.
Retraction of Science 2001; 291:134-7, (PMID: 11141566. Science 2002; 298:2327).
22. Deveraux QL, Reed JC. IAP family proteins—suppressors of apoptosis. Genes Develop
23. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in can-
cer and lymphoma. Nature Med 1997; 3:917-21.
24. Liston P, Roy N, Tamai K, Lefebvre C, Baird S, Cherton-Horvat G, Farahani R, McLean
M, Ikeda JE, MacKenzie A, Korneluk RG. Suppression of apoptosis in mammalian cells by
NAIP and a related family of IAP genes. Nature 1996; 379:349-53.
25. Asselin E, Mills GB, Tsang BK. XIAP regulates Akt activity and caspase-3-dependent cleav-
age during cisplatin-induced apoptosis in human ovarian epithelial cancer cells. Cancer Res
26. Takahashi R, Deveraux Q, Tamm I, Welsh K, Assa-Munt N, Salvesen GS, Reed JC. A sin-
gle BIR domain of XIAP sufficient for inhibiting caspases. J Biol Chem 1998;
27. Deveraux QL, Roy N, Stennicke HR, Van Arsdale T, Zhou,Q, Srinivasula SM, Alnemri ES,
Salvesen GS, Reed JC. IAPs block apoptotic events induced by caspase-8 and cytochrome
c by direct inhibition of distinct caspases. EMBO J 1998; 17:2215-23.
28. Pardo OE, Lesay A, Arcaro A, Lopes R, Ng BL, Warne PH, McNeish IA, Tetley TD,
Lemoine NR, Mehmet H, Seckl MJ, Downward J. Fibroblast growth factor 2-mediated
translational control of IAPs blocks mitochondrial release of Smac/DIABLO and apoptosis
in small cell lung cancer cells. Mol Cell Biol 2003; 23:7600-10.
29. Ferreira CG, Van DV, Span SW, Jonker JM, Postmus PE, Kruyt FA, Giaccone G.
Assessment of IAP (inhibitor of apoptosis) proteins as predictors of response to chemother-
apy in advanced nonsmall-cell lung cancer patients. Ann Oncol 2001; 12:799-805.
30. Holcik M, Lefebvre C, Yeh C, Chow T, Korneluk RG. A new internal-ribosome-entry-site
motif potentiates XIAP-mediated cytoprotection. Nature Cell Biol 1999; 1:190-2.
31. Almenara J, Rosato R, Grant S. Synergistic induction of mitochondrial damage and apop-
tosis in human leukemia cells by flavopiridol and the histone deacetylase inhibitor suberoy-
lanilide hydroxamic acid (SAHA). Leukemia 2002; 16:1331-43.
32. Dai Y, Rahmani M, Pei XY, Dent P, Grant S. Bortezomib and flavopiridol interact syner-
gistically to induce apoptosis in chronic myeloid leukemia cells resistant to imatinib mesy-
late through both Bcr/Abl-dependent and -independent mechanisms. Blood 2004;
Cancer Biology & Therapy
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