Pretreatment of Acetylsalicylic Acid Promotes Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis by Down-regulating BCL-2 Gene Expression*

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues. However, not all cancers are sensitive to TRAIL-mediated apoptosis. Thus, TRAIL-resistant cancer cells must be sensitized first to become responsive to TRAIL. In this study, we observed that pretreatment by acetylsalicylic acid (ASA) augmented TRAIL-induced apoptotic death in human prostate adenocarcinoma LNCaP and human colorectal carcinoma CX-1 cells. Western blot analysis showed that pretreatment of ASA followed by TRAIL treatment activated caspases (8, 9, and 3) and cleaved poly(ADP-ribose) polymerase, the hallmark feature of apoptosis. Most interestingly, at least 12 h of pretreatment with ASA was prerequisite for promoting TRAIL-induced apoptosis and was related to down-regulation of BCL-2. Biochemical analysis revealed that ASA inhibited NF-κB activity, which is known to regulate BCL-2 gene expression, by dephosphorylating IκB-α and inhibiting IKKβ activity but not by affecting the HER-2/neu phosphatidylinositol 3-kinase-Akt signal pathway. Overexpression of BCL-2 suppressed the promotive effect of ASA on TRAIL-induced apoptosis and changes in mitochondrial membrane potential. Taken together, our studies suggested that ASA-promoted TRAIL cytotoxicity is mediated through down-regulating BCL-2 and by decreasing mitochondrial membrane potential.

Tumor cells develop resistance to apoptotic stimuli induced by various therapeutic agents, such as drugs, irradiation, and immunotherapy, because most of their primary cytotoxic effects are through apoptosis (1,2). After the initial response to these therapies, tumor cells develop resistance and/or are selected for resistance to apoptosis. Therefore, new therapeutic strategies are needed to reverse resistance to apoptosis.
Recent studies have also revealed that TRAIL, 2 which is constitutively expressed on murine natural killer cells in the liver, plays an important role in surveillance of tumor metastasis (3). The apoptotic signal of TRAIL is transduced by binding to the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5), which are members of the tumor necrosis factor-␣ receptor superfamily. Both DR4 and DR5 contain a cytoplasmic death domain that is required for TRAIL receptor-induced apoptosis. TRAIL also binds to TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2), which act as decoy receptors by inhibiting TRAIL signaling (4 -7). Unlike DR4 and DR5, DcR1 does not have a cytoplasmic domain, and DcR2 retains a cytoplasmic fragment containing a truncated form of the consensus death domain motif (8). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic molecule that has been shown to exert, selectively, anti-tumor cytotoxic effects both in vitro and in vivo with minimal toxicity to normal tissues (9,10). TRAIL has been considered a new therapeutic agent, and preclinical studies demonstrate its antitumor activity alone or in combination with drugs (10 -13). However, many tumor cells have been shown to be resistant to TRAIL (14,15). Several researchers have reported that TRAIL resistance can be overcome by various sensitizing agents like chemotherapeutic drugs (16,17), cytokines (18), and matrix metalloprotease inhibitors (19) that are able to render TRAIL-resistant tumor cells sensitive to TRAIL apoptosis.
In recent studies, nonsteroidal anti-inflammatory drugs (NSAIDs), such as acetylsalicylic acid (aspirin; ASA), have been used as chemopreventive agents of cancers to induce apoptosis or to reduce the incidence of tumor formations in a variety of organs, i.e. colon (20), lung (21), stomach (22), and colorectum (23). ASA is known to act by directly suppressing the cyclooxygenase enzyme (COX-1 and COX-2), the ratelimiting enzyme catalyzing the biosynthesis of prostaglandins, thereby blocking the production of proinflammatory prostaglandins. ASA was also shown to be effective in the inhibition of ultraviolet radiation and carcinogen-induced tumor formations in animal models (24,25).
Cell Culture and Survival Assay-Human prostate adenocarcinoma LNCaP and DU-145 cell lines, human colorectal carcinoma CX-1 cell line, and normal prostate YPEN cell line were obtained from the American Tissue Type Culture Collection (Manassas, VA). LNCaP, CX-1, and YPEN cells or DU-145 cells were cultured in RPMI 1640 medium (Invitrogen) or Dulbecco's modified Eagle's medium (Invitrogen), respectively, with 10% fetal bovine serum (HyClone, Logan, UT), 1 mM L-glutamine, and 26 mM sodium bicarbonate for monolayer cell culture. The dishes containing cells were kept in a 37°C humidified incubator with a mixture of 95% air and 5% CO 2 . One day prior to the experiment, cells were plated in 60-mm dishes. For trypan blue exclusion assay (26), trypsinized cells were pelleted and resuspended in 0.2 ml of medium, 0.5 ml of 0.4% trypan blue solution, and 0.3 ml of phosphate-buffered saline solution (PBS). The samples were mixed thoroughly, incubated at room temperature for 15 min, and examined under a light microscope. At least 300 cells were counted for each survival determination.
Production of Recombinant TRAIL-A human TRAIL cDNA fragment (amino acids 114 -281) obtained by RT-PCR was cloned into pET-23d (Novagen, Madison, WI) plasmid, and His-tagged TRAIL protein was purified using the QIAexpress protein purification system (Qiagen, Valencia, CA).
TUNEL Assay-For detection of apoptosis by the TUNEL method, cells were plated in slide chambers. After treatment, cells were fixed with 70% ethanol in PBS. Cells were washed once, permeabilized by incubating with 100 l of 0.1% Triton X-100, 0.1% sodium citrate, and then washed twice in PBS. The TUNEL reaction was carried out at 37°C for 1 h with 0.3 nmol of fluorescein isothiocyante-12-dUTP, 3 nmol of dATP, 2 l of CoCl 2 , 25 units of terminal deoxynucleotidyltransferase, and TdT buffer (30 mM Tris, pH 7.2, 140 mM sodium cacodylate) in a total reaction volume of 50 l. The reaction was stopped with 2 l of 0.5 M EDTA. Cells were observed under a fluorescence microscope.
RNA Interference by siRNA of COX-2-To down-regulate the COX-2, COX-2 siRNA (Santa Cruz Biotechnology) was used. COX-2 siRNA was transfected into LNCaP cells and incubated for 36 h. The interference of COX-2 protein expression was confirmed by immunoblot using anti-COX-2 antibody (Cayman Chemical).
Transfection-In order to generate Bcl-2 overexpressing LNCaP cells and CX-1 cells, cells were transfected with pcDNA3-Bcl-2 or pcDNA3-neo using Lipofectamine Plus (Invitrogen). Transfected cells were selected for 3 weeks in growth medium containing 0.5 mg of G-418 (geneticin; Invitrogen) per ml. The clone expressing the highest level of Bcl-2 was used for this study. The expression level was determined by immunoblot analysis. Protein Extracts and PAGE-Cells were lysed with 1ϫ Laemmli lysis buffer (2.4 M glycerol, 0.14 M Tris, pH 6.8, 0.21 M SDS, 0.3 mM bromphenol blue) and boiled for 10 min. Protein content was measured with BCA Protein Assay Reagent (Pierce). The samples were diluted with 1ϫ lysis buffer containing 1.28 M ␤-mercaptoethanol, and equal amounts of protein were loaded on 8 -12% SDS-polyacrylamide gels. SDS-PAGE analysis was performed according to Laemmli (27) using a Hoefer gel apparatus.
Immunoblot Analysis-Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% nonfat dry milk in PBS/Tween 20 (0.1%, v/v) at 4°C overnight. The membrane was incubated with primary antibody (diluted according to the manufacturer's instructions) for 2 h. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG was used as the secondary antibody. Immunoreactive protein was visualized by the chemiluminescence protocol (ECL, Amersham Biosciences).
Measurement of Mitochondrial Membrane Depolarization-The mitochondria-specific dye TMRM (Molecular Probe, Eugene, OR) was used to measure the mitochondrial potential. CX-1 cells were grown in 6-well plates and were pretreated with 1 mM aspirin in the presence or absence of TRAIL (200 ng/ml). After treatment, the cells were collected, washed in PBS, and resuspended in 500 l of FASC buffer. Cells were incubated for 20 min with 200 nM TMRM (Molecular Probe) at 4°C in the dark, washed in cold PBS twice, and then resuspended in 500 l of PBS buffer. The cells were visualized by flow cytometry. Positive samples were stained with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (mitochondrial membrane-depolarized inducer), and the surface markers were analyzed by an EPICS XL-MCL flow cytometer with a single argon laser at 488 nm (Beckman Coulter, Inc., Hialeah, FL).
Isolation of Nuclear Proteins-Nuclear extracts were prepared by the modified procedure of Dignam et al. (28). Following treatment with ASA for 20 h, LNCaP cells were washed three times with PBS and incubated on ice for 15 min in hypotonic buffer A (10 mmol/liter HEPES, pH 7.9, 10 mmol/liter KCl, 0.1 mmol/liter EDTA, 0.1 mmol/ liter EGTA, 1 mmol/liter DTT, 0.5 mmol/liter phenylmethylsulfonyl fluoride, and 0.6% Nonidet P-40). Cells were vortexed gently for lysis, and the nuclei were separated from the cytosol by centrifugation at 12,000 ϫ g for 1 min. Nuclei were resuspended in buffer C (20 mmol/ liter HEPES, pH 7.9, 25% glycerol, 0.4 mol/liter NaCl, 1 mmol/liter EDTA, 1 mmol/liter EGTA, 1 mmol/liter DTT, and 0.5 mmol/liter phenylmethylsulfonyl fluoride) and shaken for 30 min at 4°C. Nuclear extracts were obtained by centrifugation at 12,000 ϫ g, and protein concentration was measured by Bradford assay (Bio-Rad). NF-B in nuclear extracts was detected by Western blotting as described above. Electrophoretic Mobility Shift Assay-LNCaP cells were treated with various concentrations of ASA (0.01-1 mM) or 200 ng/ml TRAIL for 20 h, and nuclear extract was prepared as described above. The nuclear extract (10 g of protein) was incubated with binding buffer (20 mM HEPES, pH 7.6, 1 mM EDTA, 1 mM ammonium sulfate, 1 mM DTT, 30 mM KCl, 0.2% Tween 20) and 1 g of poly(dI-dC) for 10 min on ice. Biotin-labeled probe NF-B-specific oligonucleotide (5Ј-AGTT-GAGGGGACTTTCCCAGGC-3Ј) was used. The reaction was incubated at room temperature for 30 min. The negative control consisted of free probe only. A competition control was set up by adding non-biotinlabeled cold probe to the reaction. The samples separated on a 6% native polyacrylamide gel in 0.5% TBE for 50 min at 120 V. The samples were then transferred in 0.5% TBE onto a nylon membrane at 300 mA for 40 min. After transfer, the sample was fixed on the membrane by UV crosslinking. The membrane was first blocked with 1% blocking reagent (Roche Applied Science) at room temperature for 30 min. The biotinlabeled probe was then detected with streptavidin-horseradish peroxidase diluted 1: 20,000 (Pierce). After washing three times and equilibrating in buffer, the membrane was overlaid with lumino/enhancer and substrate for 5 min. The image was acquired using a Kodak X-Omat 2000A (Eastman Kodak, Rochester, NY).
Flow Cytometry-Cells were treated with ASA for the indicated time points with or without TRAIL. After washing, cells were blocked for 30 min with 1% bovine serum albumin in PBS. Cells were then incubated with 1 g of primary antibodies to DR4 or DR5 (Alexis) in 1% bovine serum albumin for 30 min followed by washing with PBS. Finally, cells were incubated with Alexa 488-conjugated goat anti-mouse IgG (Molecular Probe) for 30 min. After washing, the cells were analyzed on a FACScan flow cytometer. Matched isotype using control IgG antibodies was included.   RT-PCR Analysis of Bcl-2 mRNA Levels-Total cellular RNA was extracted using the Trizol method (Invitrogen) according to the manufacturer's instructions. For each RT-PCR, 1 g of total RNA was used with Novagen One-step RT-PCR kit (EMD Bioscience). The following sense and antisense primers were used at 0.5 M for each: Bcl-2 primer, sense, 5Ј-CGACGACTTCTCCCGCCGCTACCGC-3, and antisense,   DECEMBER 9, 2005 • VOLUME 280 • NUMBER 49

RESULTS
ASA Promotes TRAIL-induced Cytotoxicity-To investigate the effect of ASA on TRAIL-induced cytotoxicity, human prostatic adenocarcinoma LNCaP cells were pretreated with ASA and treated with TRAIL in the presence of ASA. Fig. 1, A and B, shows that little or no cytotoxicity was observed with 1 mM ASA alone or 200 ng/ml TRAIL alone. However, pretreatment of ASA promoted TRAIL-induced cytotoxicity that was dependent upon concentrations of ASA (Fig. 1A) and TRAIL (Fig. 1B). Similar results were observed with TUNEL staining (Fig. 1C). Data from TUNEL assays show that apoptotic cell death occurred when LNCaP cells were pretreated with ASA followed by TRAIL.
Effect of ASA on TRAIL-induced Apoptosis-Additional studies were designed to examine whether pretreatment with ASA followed by treatment with TRAIL causes PARP cleavage, the hallmark feature of apoptosis, in prostate cancer LNCaP and DU-145 cells and normal prostate YPEN cells. Fig. 2 shows that PARP (116 kDa) was cleaved yielding a characteristic 85-kDa fragment in the presence of TRAIL (50 -200 ng/ml) and ASA (1-10 mM) in prostate cancer cells, but not in normal prostate cells. The cleavage of PARP was not observed by treatment with ASA alone. These results were similar to the observations of cytotoxicity (Fig. 1, A and B). Western blot analysis shows that procaspase-8 (55 kDa) was cleaved to the intermediates (41 and 43 kDa) by pretreatment with ASA and treatment with TRAIL in LNCaP and DU-145 cells. The combined treatment of TRAIL and ASA also resulted in an increase in caspase-9 activation as well as caspase-3 activation in LNCaP and DU-145 cells (Fig. 2, A and B). The precursor form of caspase-9 and -3 was cleaved to the active form of 37 and 17 kDa, respectively. ASA alone did not activate caspases. We extended our studies to investigate a time course and dose response on PARP cleavage. Fig. 3A shows that at least 12 h of pretreatment with ASA was required for PARP cleavage in the presence of TRAIL. Fig. 3, B and C, shows that a minimal amount of 10 ng/ml TRAIL or 0.01 mM ASA was required for PARP cleavage in the presence of 1 mM ASA or 200 ng/ml TRAIL, respectively, in LNCaP cells. We further investigated whether treatment with ASA is a prerequisite. Fig. 4 shows that combined treatment with TRAIL and ASA without pretreatment with ASA caused little or no cytotoxicity and PARP cleavage. Taken together, these results suggest that pretreatment

FIGURE 9. Effect of acetylsalicylic acid on IKK activity (A), IB-␣ phosphorylation (B), or NF-B (D and E) translocation in LNCaP cells, and purity of the nuclear extracts (C).
A, cells were lysed, and IKK proteins were purified by immunoprecipitation (IP). The purified IKK proteins were incubated with or without 1 mM ASA for 30 min at 4°C, and in vitro kinase assay was performed at 30°C for 30 min with GST-IB-␣ as substrate. B, cells were treated with various concentrations of ASA (0.01-1 mM) or 200 ng/ml TRAIL for 20 h and lysed. Equal amounts of protein (20 g) were separated by SDS-PAGE and immunoblotted (IB) with anti-phospho-IB-␣, anti-IB-␣, or antiactin antibody. Lane C, control. C-E, cells were treated with various concentrations of ASA (0.01-1 mM) or 200 ng/ml TRAIL for 20 h, and nuclear proteins were extracted (C). D, equal amounts of nuclear protein (20 g) were separated by SDS-PAGE and immunoblotted with anti-NF-B antibody. E, the nuclear extracts were incubated with biotin-labeled oligonucleotide at room temperature for 30 min. Gel mobility shift assays were performed as described under the "Experimental Procedures." with ASA for 12 h is essential for inducing apoptotic death in the presence of TRAIL.
Role of COX in TRAIL-induced Apoptosis-It is well known that ASA inhibits only COX-1 at low concentrations (IC 50 ϭ 44 M) but both COX-1 and COX-2 at higher concentrations (IC 50 ϭ 1100 M) (29). To examine whether the promotive effect of ASA on TRAIL-induced apoptosis is mediated through inhibiting COX, LNCaP cells were pretreated with various NSAIDs and then treated with TRAIL. Unlike ASA, Fig. 5, A-C, shows that no significant cleavage of PARP was observed by treatment with various concentrations of sulindac sulfide (IC 50 5D shows that the expression of COX-2 was effectively inhibited by siCOX-2. However, knock-down of COX-2 expression did not promote TRAIL-induced apoptosis. Nonetheless, pretreatment with ASA promoted TRAIL-induced apoptosis regardless of the presence or the absence of COX-2. These results suggest that COX is not involved in ASA-promoted TRAIL cytotoxicity. Effect of ASA on the Level of TRAIL Receptor Family and Anti-apoptotic Proteins-It is well known that TRAIL can interact with death receptors (DR4 and DR5), which trigger apoptotic signals (4). Such signals may be blocked by expression of the antagonistic decoy receptors (DcR1 and DcR2). Previous studies demonstrate that increased DR5 levels are induced by chemotherapeutic agents (30). Thus, we examined whether pretreatment with ASA affects the level of TRAIL receptors and anti-apoptotic proteins, and consequently promotes apoptosis by treatment with TRAIL. LNCaP cells were pretreated with ASA (1-10 mM) and treated with 200 ng/ml TRAIL in the presence of ASA. Data from Western blot analysis reveal that ASA treatment did not significantly alter the total cellular levels of the TRAIL receptors (DR4, DR5, and DcR2) and anti-apoptotic proteins (FLIP L , FLIP S , IAP-1, IAP-2, and Bcl-X L) (Fig. 6, A and C). Data from flow cytometric analysis show that TRAIL induced surface expression of DR5 but not DR4 (Fig. 6B). However, ASA treatment did not enhance the DR5 expression. Most interestingly, ASA treatment resulted in a decrease in the level of Bcl-2 (Fig.  6C). The reduction of Bcl-2 during treatment with 1 mM ASA was dependent upon exposure time (Fig. 6B). To confirm the effect of ASA on BCL-2 gene expression, LNCaP or DU-145 prostatic cancer cells were treated with various concentrations of ASA, and expression of BCL-2 was examined. Fig. 7A shows that ASA reduced the level of Bcl-2 in both cell lines. Data from RT-PCR and Northern blot assay in Fig. 7, B and C, show that the level of BCL-2 mRNA was significantly decreased during treatment with ASA. The reduction of BCL-2 mRNA was dependent upon ASA concentration. These results suggest that the reduction of Bcl-2 levels during treatment with ASA was because of suppression of BCL-2 gene transcription.   DECEMBER 9, 2005 • VOLUME 280 • NUMBER 49

JOURNAL OF BIOLOGICAL CHEMISTRY 41053
Effect of ASA on the HER-2/neu-PI3K-Akt-NF-B Signal Transduction Pathway-It is well known that BCL-2 expression is regulated by NF-B, a dimeric transcription factor (31). We postulated that ASA inhibits NF-B activity, which subsequently decreases transcription of BCL-2. To examine this possibility, the effect of ASA on upstream signal transduction of NF-B was investigated. Fig. 8 shows that ASA treatment did not change the level of HER-2/neu, PI3K, and Akt or alter the phosphorylation of these proteins. In contrast, ASA treatment inhibited IKK␤ activity, dephosphorylated IB-␣, increased the level of IB-␣, and prevented NF-B nuclear translocation (Fig. 9). These results suggest that ASA down-regulates BCL-2 gene expression by inhibiting the IKK␤-IB-␣-NF-B signal transduction pathway.
Role of Bcl-2 in ASA-enhanced TRAIL Cytotoxicity-To determine whether ASA-mediated down-regulation of BCL-2 plays an important role in the augmentation of TRAIL-induced apoptotic death, LNCaP cells or human colorectal carcinoma CX-1 cells were stably transfected with either an empty control vector (pcDNA 3-neo) or vector contain-ing BCL-2 (pcDNA3-Bcl-2). Figs. 10 and 11 show that pretreatment with ASA followed by treatment with TRAIL caused PARP cleavage, activation of caspases, as well as cytotoxicity in control vector transfected cells. However, overexpression of BCL-2 protected LNCaP and CX-1 cells from ASA-enhanced TRAIL cytotoxicity. These results suggest that ASA-promoted TRAIL cytotoxicity is mediated by down-regulating BCL-2.
Overexpression of BCL-2 Prevents Alteration of Mitochondrial Membrane Potential by Treatment with ASA and TRAIL-Bcl-2 is an antiapoptotic protein that inhibits the release of cytochrome c from mitochondria into the cytoplasm, thereby down-regulation of BCL-2 may promote intrinsic mitochondria-mediated apoptosis (32,33). To investigate whether ASA disrupts mitochondrial membrane potential and overexpression of BCL-2 protects cells from this disruption, CX-1/Bcl-2 or CX-1/neo cells were pretreated with ASA and treated with TRAIL. We used the mitochondria-specific dye TMRM to measure the mitochondrial membrane potential. Fig. 12 shows that overexpression of BCL-2 inhibited the loss of mitochondrial membrane potential during treatment with ASA alone or ASA in combination with TRAIL.
A Model for the Effect of ASA on the TRAIL-induced Apoptotic Pathway- Fig. 13 shows a schematic diagram of a model that is based on the literature and our data. ASA blocks the Akt-NF-B survival signal pathway by inhibiting IKK␤. The inhibition of this pathway results in suppression of the expression of BCL-2, an anti-apoptotic molecule.

DISCUSSION
Aspirin (acetylsalicylic acid) is a nonsteroidal anti-inflammatory drug (NSAID) widely used for its anti-pyretic and analgesic properties. ASA is also known to induce gastrointestinal side effects, mainly in the form of gastric and duodenal ulcerations or erosions. However, epidemiological findings have revealed that ASA reduces the risk of colorectal cancer and adenoma (34,35). In this study, we demonstrate that pretreatment with ASA promotes TRAIL-induced apoptotic death. The enhancement of apoptosis by treatment with ASA is probably because of downregulation of BCL-2, activation of caspases, induction of conformational change, translocation of Bax, and cytochrome c release (Figs. 2, 6, and 7) (36 -40). Our observations were similar to previous reports (41). Previous studies have shown that aspirin inhibits the transcription factor  NF-B (42,43), which is critical for the expression of several anti-apoptotic genes including C-IAP1, C-IAP2, BCL-X L , FLIP, and BCL-2 (31, 44 -46). The inhibition of NF-B activity is mediated through preventing the phosphorylation and degradation of the inhibitory subunit IB (Fig. 9) (47). Although the expression of these Bcl-2 family and IAP family proteins is known to be regulated by NF-B, our data show that ASA inhibits preferentially BCL-2 gene expression (Fig. 6). Thus, a fundamental question that remains unanswered is how ASA inhibits selectively the expression of the BCL-2 gene among BCL-2 family and IAP family genes. It is well known that the NF-B family of proteins, including NF-B1, NF-B2, RelA, RelB, and c-Rel, can form homo-and heterodimers in vitro, except for RelB. In mammals, the most widely distributed NF-B is a heterodimer composed of p50 and p65 (also called RelA) subunits (48). NF-B activity is regulated by the IB family of proteins that interacts with and sequesters the transcription factor in the cytoplasm. IB proteins become phosphorylated by the multisubunit IKK complex, which subsequently targets IB for ubiquitination and degradation by the 26 S proteasome (49). At this time only speculations can be made concerning the role of NF-B in the down-regulation of BCL-2 gene expression during treatment with ASA. One possibility is that differential activation of NF-B may be responsible for selective inhibition of BCL-2 gene expression. As mentioned above, the inhibition of five members of the NF-B family may differ during treatment with ASA, and this differential inhibition of the various members of NF-B family causes a selective inhibition of BCL-2 gene expression (50). We believe that many critical questions still remain to be answered in order to understand the mechanisms of the regulation of BCL-2 gene expression by ASA. However, this model will also provide a framework for future studies.
Previous studies have shown that ASA inhibits tumor necrosis factor-␣-and interleukin-1-induced NF-B activation and sensitizes HeLa cells to apoptosis (51). In this study, we observed that ASA augments TRAIL cytotoxicity in TRAIL-resistant LNCaP cells that contain high levels of HER-2/neu. It is well known that HER-2/neu has an intrinsic tyrosine kinase activity that activates PI3K in the absence of ligand (52). PI3K consists of a regulatory subunit (p85) that binds to an activated growth factor/cytokine receptor and undergoes phosphorylation, which results in the activation of its catalytic subunit (p110) (53). PI3K phosphorylates phosphoinositides at the 3Ј-position of the inositol ring, and its major lipid product is phosphatidylinositol 3,4,5-triphosphate (54). Phosphatidylinositol 3,4,5-triphosphate facilitates the recruitment of Akt to the plasma membrane through binding with the pleckstrin homology domain of Akt (54). Akt is activated by phosphoinositide-dependent kinase-1 (PDK1) through phosphorylation at threonine 308 and serine 473 (55). A number of pro-apoptotic proteins have been identified as direct Akt substrates, including BAD, caspase-9, and Forkhead transcription factors (56 -61). The pro-apoptotic function of these molecules is suppressed upon phosphorylation by Akt. Recent studies also show that Akt induces the degradation of IB by promoting IKK␣ activity and subsequently stimulating the nuclear translocation of NF-B (62). In this study, we have revealed that ASA does not affect the HER-2/neu-PI3K-Akt signal transduction pathway (Fig. 8). However, ASA can interrupt the Akt-NF-B signal transduction pathway by inhibiting IKK␤ activity (Fig. 9) (47). A previous study shows that 1 mM aspirin treatment inhibits 75% of endogenous IKK kinase activity, even though more than 90% of IKK␤ activity is inhibited without altering IKK␣ activity in the presence of aspirin (47). These results suggest that a small percentage of total IKK␣ forms IKK␣ homodimers that still contain kinase activity in the presence of aspirin. In this study, we believe that blockade of HER-2/neu-mediated survival signals by inhib-iting IKK␤ activity can sensitize TRAIL-resistant tumor cells. We also believe that this study will provide information to improve the efficacy of TRAIL-based clinical therapy.