Downregulation of XIAP and Induction of Apoptosis by the Synthetic ...

[Cancer Biology & Therapy 5:2, 165-170, February 2006]; ©2006 Landes Bioscience

Downregulation of XIAP and Induction of Apoptosis by the Synthetic Cyclin-Dependent Kinase Inhibitor GW8510 in Non-Small Cell Lung Cancer Cells Research Paper

ABSTRACT

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 timeand 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.

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 protected]

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Received 10/04/05; Accepted 11/10/05

Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/abstract.php?id=2316

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INTRODUCTION

Lung cancer is the most common cancer worldwide and the leading cause of cancerrelated death.1 Histologically, 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 radiotherapy and chemotherapy. However, treatment of NSCLC is often challenging, because it is generally less responsive or even not responsive to such therapies.2 Consequently, the prognosis 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 G1 to late G2 phase.3 Furthermore, deregulation of cyclin E, an activator of CDK2, has been found to be associated with a broad spectrum of human malignancies.4,5 Cyclin E has also been reported to be a strong independent prognostic indicator in patients with early-stage NSCLC.6 High levels of cyclin E expression correlate significantly with short mean survival times. Thus, CDK2 may serve as a potential target for therapeutic intervention in patients with NSCLC. 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 CDK2,7 on 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

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KEY WORDS

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of Metabolism and Endocrinology; The First Affiliated Hospital; Zhejiang University; People's Republic of China

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1Department

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Fengqin Dong1,2 Wei Guo2 Lidong Zhang2 Shuhong Wu2 Fuminori Teraishi2 John J. Davis2 Bingliang Fang2,*

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cyclin-dependent kinases, small molecule, apoptosis, non-small lung cancer, X-linked inhibitor of apoptosis

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ACKNOWLEDGEMENTS

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We thank Don Norwood for editorial review of the manuscript. National Cancer Institute grants RO1 CA 092487-01A1 and RO1 CA 09858201A1 (both to B. Fang), National Cancer Institute Lung Specialized Program of Research Excellence, and National Institutes of Health Core Grant CA16672.

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Cancer Biology & Therapy

165

Downregulation of XIAP by a CDK2 Inhibitor

GW8510. These findings may impact future development of CDK inhibitors for the treatment of NSCLC.

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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 sulforhodamine 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. Figure 1. The structure of GW8510 and its cell-killing effects on NSCLC cell lines. (A) Chemical The Nucleofector Kit V was obtained from Amaxa structure of GW8510. (B) Cell-killing effects of GW8510 on the six NSCLC cell lines and NHFB (Cologne, Germany). The Advantage RT-for-PCR Kit after treatment for 72 hours. Cell viability was determined by using the SRB assay. Cells treated was purchased from BD Biosciences Clontech (Mountain with the same amount of DMSO (v/v, 0.1%) whose viability was set at 1 were used as controls. View, CA ). The viability of the NSCLC cells but not NHFB was significantly suppressed by GW8510 after Cell lines and cell cultures. A549, H1299, H460, the concentration of GW8510 reached 5 µM (p < 0.05). The data represent four quadrupliH226, H358 and H322 were maintained in our labora- cate assays with similar results. The values represent the mean ± standard deviation. (C) tory. A549 was cultured in Ham’s F12 medium containing Time-response to GW8510. H1299, and A549 cells were treated with 5 µM GW8510. Cell 10% (v/v) heat-inactivated FBS and 1% (v/v) antibiotics. viability was then determined over time as indicated. Significant suppression was found on the The other NSCLC cell lines were maintained in RPMI first day after treatment (compared with DMSO treatment; p < 0.05). The data represent three 1640 medium containing 10% (v/v) heat-inactivated quadruplicate assays with similar results. The values represent the mean ± standard deviation. FBS and 1% (v/v) antibiotics. NHFB (fourth passage) was maintained in Dulbecco’s modified Eagle’s medium containing 10% method. Total extracts (50 µg/lane) were normalized and subjected to sodium (v/v) heat-inactivated FBS and 1% (v/v) antibiotics. Cells were cultured at dodecyl sulfate-polyacrylamide gel electrophoresis (12% gels) immunoblot 37˚C in a humidified incubator containing 5% CO2. assay. Blotting was performed on a hybond-ECL membrane, and the signal SRB assay. Cell viability was determined by using the SRB colorimetric was detected by using an ECL solution. Western blot analysis of XIAP, Bax, assay as described previously.8 Briefly, after fixation of adherent cells with Bak, Bcl2, caspase-3 and β-actin was performed with specific antisera or trichloroacetic acid in a 96-well microplate, protein was stained with SRB, monoclonal antibodies as described previously.9,10 Horizontal scanning and the optical density was determined at 570 nm to reflect the number of densitometry of Western blots was performed by using acquisition into the stained cells, which showed the cell viability. The relative cell viability was Adobe Photoshop software program (Adobe Systems, San Jose, CA). determined by referring to the cell viability of the DMSO control which was Expression of β-actin was used as a control. set at 100%. Each experiment was performed in quadruplicate and repeated Reverse transcriptase-polymerase chain reaction. Total RNA was isolated at least three times. by using TRIzol according to the manufacturer’s instructions, mixed with a Flow cytometry analysis. Both floating and attached cells (use trypsin) random hexamer primer, and incubated at 70˚C for five minutes. were collected and washed twice with cold phosphate-buffered saline (PBS). Single-strand cDNA was synthesized from 0.5 µg of total RNA by using The cells were then fixed with cold 70% ethanol and kept overnight at 4˚C. moloney murine leukemia virus reverse transcriptase (RT) and deoxynucleThirty minutes before the assay, PI staining (1 mL of PI, 10 µL of RNase, oside triphosphate at 42˚C for 1 hour and 90˚C for five minutes; synthesis 9 mL of PBS; 50 µg/mL PI) was performed. Flow cytometric evaluation of was stopped at 4˚C. The mRNAs for XIAP were amplified by polymerase the cell cycle status and apoptosis were performed according to a previously chain reaction (PCR) with specific primers. The sequence of the sense and described method.9,10 The percentage of cells at G1, S, G2, and M phase was antisense primer for XIAP was 5'-TGAATCTGATGCTGTGAGTT and calculated by using the Cell Quest software program (Becton-Dickinson, 5'-CCTCAAGTGAATGAGTTAAA, respectively. The level XIAP mRNA San Jose, CA). was then determined by a semi-quantitative PCR with serial-diluted cDNA Western blot assay. A total of 2-4 × 105 cells were lysed in lysis buffer samples. The conditions for the XIAP PCR reactions were as follows: 1 x at (20 mM HEPES, pH 7.9, 150 mM NaCl, 0.5 mM dithiothreitol, 10 mM 94˚C for three minutes; 35 x at 94˚C for 45 seconds, 50˚C for 45 seconds, KCl, 0.2 mM ethylenediaminetetraacetic acid, 10% glycerol, complete and 72˚C for 1 minute; and 1 x at 72˚C for ten minutes. Detection of glycproteinase inhibitors). Lysates were sonicated with the use of a sonicating eraldehyde-3-phosphate dehydrogenase (GAPDH) was conducted as the machine for 2 x 30 seconds at setting 6 on ice and then spun down at internal control, and the sense and antisense primers for GAPDH were 15,000 rpm for 15 minutes at 4°C; the supernatant was then carefully combined with the Advantage RT-for-PCR kit. The conditions for GAPDH collected. The protein concentration was assessed by the BCA protein assay PCR reactions were as follows: 1 x at 94˚C for three minutes; 35 x at 94˚C

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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 inhibited 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 Figure 2. Apoptosis induction in NSCLC cell lines and NHFB cells. (A) Apoptosis of NHFB, A549, harvested cells and analyzed them by using a H1299, and H460 cells after 24 hours and 72 hours of exposure to DMSO, 5 µM GW8510, or 10 µM flow cytometric assay. The results showed that GW8510. The apoptotic percentage was demonstrated by the percentage of cells at sub-G1 phase treatment with GW8510 induced apoptosis in which was determined by fluorescence-activated cell sorter analysis. No significant difference in the all three cell lines, as evidenced by a marked sub-G1 percentage of NHFB cells was found (p > 0.05), whereas NSCLC cells showed a significant increase in the percentage of cells at sub-G1 increase in the sub-G1 percentage, even at 5 µM (p < 0.05). The data represent three experiments with phases of the cell cycle at both 24 and 72 hours similar results. (B) Cell cycle in A549 and H460 cells after 24 hours and 72 hours of exposure to DMSO, (Fig. 2A). The apoptosis induction was most 5 µM GW8510, or 10 µM GW8510. The percentage of cells at sub-G1 phases and G2 phase is shown: the percentage of cells at G2 phase increased, whereas that of cells at G1 phase decreased. The data profound in H460 cells, which is consistent with the data from the cell viability assay (Fig. 1B). were obtained from one of three experiments with similar results. In addition to induction of apoptosis, treatment with GW8510 resulted in an apparent increase for 45 seconds, 65˚C for 45 seconds, and 72˚C for one minute; and 1 x at in the proportion of cells at G2 phase (Fig. 2B). Taken together, these results 72˚C for ten minutes. PCR products were analyzed by using agarose gel indicated that GW8510 induces apoptosis and cell cycle arrest at G2. electrophoresis and visualized with the use of ethidium bromide. GW8510 downregulated expression of XIAP. To characterize molecular Electroporation assay. H1299 and A549 cells (2 x 106) were suspended mechanisms underlying the induction of apoptosis by GW8510, we measin 100 µL of Nucleofector Kit solution V mixed with 4 µg of XIAP plasmid ured protein levels of several molecules that are important in apoptotic (a gift from Dr. John C. Reed, the Burnham Institute, La Jolla, CA)11 and pathways, including Bax, Bak, Bcl2 and XIAP. For this purpose, we treated nucleotransfected with the use of a Nucleofector program (Amaxa) according A549 and H460 cells with 1–10 µM GW8510 for 48 hours. We then harto the manufacturer’s instructions. Green fluorescent protein (GFP) plasmid vested cells for Western blot analysis. Treatment with GW8510 did not was transfected as a control. Twenty-four hours later, some of the transfectants induce obvious changes in the expression of Bax, Bak, or Bcl2 at any of the were lysed for Western blot analysis, and some were treated with GW8510. doses tested. In contrast, it dramatically downregulated the expression of Statistical analysis. Differences in the experimental groups were analyzed XIAP. This downregulation was dose-dependent and more dramatic in by using analysis of variance with the Statistica software program (StatSoft, H460 cells than in A549 cells, the earlier of which are more sensitive to Tulsa, OK). A P ≤ 0.05 was considered statistically significant. The drug GW8510 than A549 (Fig. 3A). In contrast, we observed no obvious change concentration that inhibited cell growth by 50% was calculated by using the in XIAP expression in NHFB treated by GW8510 (Fig. 3B). A time-course CurveExpert 1.3 software program (Hyams Development, Starkville, MS). 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 RESULTS GW8510 from the medium for 48 hours (Fig. 3D). Cytotoxic effects of GW8510 on NSCLC cell lines. To evaluate the To further analyze the mechanisms of GW8510-mediated downregulation effect of CDK2 inhibitors on lung cancer cells, we analyzed the cytotoxic of XIAP expression, we measured XIAP mRNA level by using an RT-PCR effect of the CDK2 inhibitor GW8510 on the growth of the six NSCLC assay. We treated cells with GW8510 at a concentration of 5 µM and 10 µM cell lines. For this purpose, we treated cells with GW8510 at concentrations for eight hours and 16 hours, respectively. We then detected XIAP mRNA ranging from 1–10 µM. We then determined cell viability by using the SRB level by using RT-PCR. The results showed that the XIAP mRNA level was assay at 72 hours after the treatment. We used cells treated with DMSO dramatically reduced after eight hours of exposure to 5 µM GW8510 and

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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 reduction 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 plasmid11 by electroporation with the use of an Amaxa machine. Twenty-four hours after electroporation, we measured the XIAP expression in the transfected cells by using Western blot analysis. The results revealed that transfected 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 treated 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.

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DISCUSSION The findings presented here suggest that GW8510 inhibits the growth of the lung cancer cell lines, correlating E 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.12 Several small-molecule CDK inhibitors have been evaluated clinically for treatment of cancer.13 For example, 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.14 However, clinical studies have shown that the activity of flavopiridol administered alone is not effective and 168

that combination with other therapeutic agents is required to produce a detectable clinical response,14 indicating the need for development of new CDK inhibitors. Nevertheless, studies on flavopiridol-mediated antitumor activity have revealed several molecular events elicited by CDK inhibitors, including cell cycle arrest and p53-independent apoptosis,15,16 upregulation of E2F1 and repression of Mcl-1,17,18 and downregulation or inhibition of XIAP, Bcl-XL, p21 or Mdm2.18,19




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-G1 phases 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-transfected 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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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 chemotherapyinduced alopecia in rats.20 However, 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,21 even though the compound has the correct chemical structure and CDK2 inhibition as reported previously.7 Our 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 downregulation 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.22 Several 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.25 Human XIAP, a key member of the IAP family, has been shown to be a direct inhibitor of caspase-3 and caspase-726 and www.landesbioscience.com

to interfere with the Bax/cytochrome-c pathway by inhibiting caspase-9.22,27 Overexpression 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.28,29 Therefore, 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 interesting 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.18 Interestingly, expression of XIAP is regulated at the translational level by a rare cap-independent mechanism 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.30 In 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 effective. 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 proteasome inhibitor PS341 and the histone deacetylase inhibitor suberoylanilide hydroxamic acid.31,32 Whether similar combination effects can be induced with GW8510 remains to be determined.


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