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Mol Cancer Ther. 2006;5:2970-2976
© 2006 American Association for Cancer Research

Research Articles: Therapeutics, Targets, and Development

Blockade of transforming growth factor-ß-activated kinase 1 activity enhances TRAIL-induced apoptosis through activation of a caspase cascade

Min-Kyung Choo1,2, Noritaka Kawasaki1, Pattama Singhirunnusorn1, Keiichi Koizumi1, Shintaro Sato3, Shizuo Akira3,4, Ikuo Saiki1,2 and Hiroaki Sakurai1,2

1 Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama; 2 21st Century COE Program, University of Toyama, Toyama, Japan; and 3 Akira Innate Immunity Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency; 4 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan

Requests for reprints: Hiroaki Sakurai, Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. Phone: 81-76-434-7636; Fax: 81-76-434-5058. E-mail: hsakurai{at}inm.u-toyama.ac.jp

Abstract

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF-{alpha} ligand family that selectively induces apoptosis in a variety of tumor cells. To clarify the molecular mechanism of TRAIL-induced apoptosis, we focused on transforming growth factor-ß-activated kinase 1 (TAK1) mitogen-activated protein kinase (MAPK) kinase kinase, a key regulator of the TNF-{alpha}-induced activation of p65/RelA and c-Jun NH2-terminal kinase/p38 MAPKs. In human cervical carcinoma HeLa cells, TRAIL induced the delayed phosphorylation of endogenous TAK1 and its activator protein TAB1 and TAB2, which contrasted to the rapid response to TNF-{alpha}. Specific knockdown of TAK1 using small interfering RNA (siRNA) abrogated the TRAIL-induced activation of p65 and c-Jun NH2-terminal kinase/p38 MAPKs. TRAIL-induced apoptotic signals, including caspase-8, caspase-3, caspase-7, and poly(ADP-ribose) polymerase, were enhanced by TAK1 siRNA. Flow cytometry showed that the binding of Annexin V to cell surface was also synergistically increased by TRAIL in combination with TAK1 siRNA. In addition, pretreatment of cells with 5Z-7-oxozeaenol, a selective TAK1 kinase inhibitor, enhanced the TRAIL-induced cleavage of caspases and binding of Annexin V. The TAK1-mediated antiapoptotic effects were also observed in human lung adenocarcinoma A549 cells. In contrast, TAK1-deficient mouse embryonic fibroblasts are resistant to TRAIL-induced apoptosis, and treatment of control mouse embryonic fibroblasts with 5Z-7-oxozeaenol did not drastically promote the TRAIL-induced activation of a caspase cascade. These results suggest that TAK1 plays a critical role for TRAIL-induced apoptosis, and the blockade of TAK1 kinase will improve the chances of overcoming cancer. [Mol Cancer Ther 2006;5(12):2970–6]

Introduction

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand or Apo 2 ligand (TRAIL/Apo2L) is a typical member of the TNF-{alpha} ligand family that induces apoptosis in various types of tumor cells but not in most normal cells (1–3). TRAIL is known to bind to five different receptors (4–6): DR4/TRAIL-R1 and DR5/TRAIL-R2, two death receptors that can trigger the death signal by TRAIL (7, 8), and DcR1 (TRAIL-R3), DcR2 (TRAIL-R4), and osteoprotegerin, decoy receptors that control the binding of TRAIL to DR4/DR5 (9–11). TRAIL triggers apoptosis by binding to DR4 and DR5, which leads to the formation of the death-inducing signaling complex. This complex consists of trimerized receptors, the death domain-containing adaptor protein Fas-associated death domain, and caspase-8 (12–14). Activation of caspase-8 leads directly to the activation of caspase-7 and caspase-3 and, subsequently, apoptotic cell death (15). In contrast, evidence is accumulating that TRAIL has the ability to activate stress response pathways, including nuclear factor-{kappa}B (NF-{kappa}B) p65, c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), through adaptor molecules, such as TRAF2, RIP1, and NF-{kappa}B essential modulator (16–19).

Transforming growth factor-ß-activated kinase 1 (TAK1) was originally identified as a MAPK kinase kinase (MAP3K), which can be activated by transforming growth factor-ß, bone morphologic protein (20). Rather, TAK1 was recently found to be a crucial regulator of the JNK, p38, and p65 pathways in response to TNF-{alpha}, interleukin-1, and ligands of Toll-like receptor, such as lipopolysaccharide (21–26). TAK1 has also been shown to participate in intracellular events involving the T-cell receptor, B-cell receptor, and LMP1 oncogene product of EBV (27–29). We have recently reported that intermolecular autophosphorylation at Thr187 is involved in the TNF-{alpha}-induced activation of TAK1 in HeLa cells (30), and the activation of TAK1 is essential for TNF-{alpha}-promoted pulmonary metastasis in murine colon carcinoma cells (31). Recently, it has been reported that suppression of the p65, JNK, and p38 signaling pathways in a kinase-inactive mutant of TAK1 increased the sensitivity of cells to apoptosis, indicating that TAK1 plays a crucial role in cell survival signaling (32). However, the role of TAK1 in the TRAIL-induced p65/JNK/p38 pathways and caspase-mediated apoptotic cell death remains to be investigated.

TRAIL is a novel apoptosis-inducing antitumor agent, and a clinical evaluation of which is under way (33–35). Therefore, mechanistic insights into the proapoptotic and antiapoptotic signals from DR4/DR5 would provide information with which to improve the effectiveness and safety of TRAIL. In the present study, we investigated the role of TAK1 in TRAIL-induced apoptosis and found that TAK1 is an upstream kinase regulating the TRAIL-induced activation of p65/JNK/p38 pathways and suppression of TAK1 activity enhanced TRAIL-induced apoptosis through the activation of the caspase-mediated death signals.

Materials and Methods

Antibodies and Reagents
The anti-phosphorylated TAK1 (Thr187) antibody was reported previously (30). Antibodies against phosphorylated p65 (Ser536), phosphorylated JNK (Thr183/Thr185), phosphorylated p38 (Thr180/Tyr182), caspase-8, caspase-7, caspase-3, and anti-poly(ADP-ribose) polymerase (PARP) were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against TAK1 (M-579), TAB1 (C-20), TAB2 (K-20), p65 (C-20), p38 (C-20), JNK (FL), and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant human TRAIL/Apo2L was purchased from PeproTech (London, United Kingdom). 5Z-7-Oxozeaenol, a specific inhibitor of TAK1, was kindly provided by Dr. Koichiro Ono (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan).

Cell Culture
HeLa cells, TAK1-deficient mouse embryonic fibroblasts (MEFs; ref. 36), and normal MEFs were maintained in DMEM (high glucose) supplemented with 10% FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin. A549 cells were maintained in RPMI 1640 supplemented with 10% FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cultures were kept at 37°C in a humidified atmosphere of 5% CO2/95% air. Cells were treated with TRAIL in medium containing 10% FCS.

Small Interfering RNAs and Transfection
Duplex small interfering RNAs (siRNA) with a two-nucleotide overhang at the 3'-end of the sequence were designed at iGENE Therapeutics (Tsukuba, Japan) and synthesized at Hokkaido System Science Co. Ltd. (Sapporo, Japan) The target sequences were as follows: UGGCUUAUCUUACACUGGA (TAK1) and CGUACGCGGAAUACUUCGA [firefly luciferase (GL2)]. HeLa cells were transfected with siRNAs at a final concentration of 20 nmol/L using LipofectAMINE reagents (Invitrogen Life Technologies, Carlsbad, CA). At 72 h after transfection, cells were stimulated.

Immunoblotting
After stimulation or transfection, whole-cell lysates were prepared with lysis buffer [25 mmol/L HEPES (pH 7.7), 0.3 mol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.1% Triton X-100, 20 mmol/L ß-glycerophosphate, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L DTT, 10 µg/mL aprotinin, 10 µg/mL leupeptin]. Cell lysates or immunoprecipitates described below were resolved by SDS-PAGE and transferred to an Immobilon-P nylon membrane (Millipore, Bedford, MA). The membrane was treated with BlockAce (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) at 4°C for 2 h and probed with antibodies. The primary antibodies were detected using horseradish peroxidase–conjugated anti-rabbit, anti-goat, or anti-mouse IgG (DAKO, Glostrup, Denmark) and visualized with the enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ).

Immunoprecipitation
Cell lysates were diluted with an equal volume of dilution buffer [20 mmol/L HEPES (pH 7.7), 2.5 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.05% Triton X-100, 20 mmol/L ß-glycerophosphate, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L DTT, 10 µg/mL aprotinin, 10 µg/mL leupeptin]. After centrifugation, lysates were incubated with anti-TAB2 antibody (Santa Cruz Biotechnology) on ice for 1.5 h and then rotated with protein G-Sepharose (Amersham Biosciences) at 4°C for 2 h. The Sepharose beads were washed thrice with wash buffer (a 1:1 mixture of whole-cell lysate buffer and dilution buffer).

Annexin V Apoptosis
Annexin V apoptosis (MBL, Nagoya, Japan) was detected according to the manufacturer's instructions. Cells were treated with 5Z-7-oxozeaenol and/or TRAIL and centrifuged at 14,000 rpm for 10 min. Cell lysates were resuspended with 500 µL of 1x binding buffer. Enhanced green fluorescent protein–conjugated Annexin V was added to the cells for 5 min at room temperature. After incubation, the cells binding to Annexin V were analyzed using the FACScan analysis system (Becton Dickinson, Franklin Lakes, NJ).

Results

TRAIL Activates TAK1-Mediated NF-{kappa}B p65 and JNK/p38 MAPK Signaling Pathways
TRAIL is a member of the TNF-{alpha} ligand family, which regulates apoptosis by binding to its death receptor (1–5). TRAIL is also known to trigger survival signaling pathways, including NF-{kappa}B p65 and JNK/p38 MAPKs (16–19). To address TRAIL-induced signaling pathways in cancer cells, we confirmed the TRAIL-induced phosphorylation of p65, JNK, and p38 in human cervical cancer HeLa cells. Figure 1 shows that TRAIL induced the activation of these signaling pathways. However, the activation was not observed until 30 min after the stimulation, a delay compared with the responses to TNF-{alpha} (data not shown).


Figure 1
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  		Figure 1. Activation of TAK1-mediated NF-{kappa}B, JNK, and p38 MAPK signaling pathways by TRAIL. HeLa cells were treated with TRAIL (200 ng/mL) or TNF-{alpha} (10 ng/mL) for the period indicated. Whole-cell lysates were analyzed by Western blotting with antibodies against phosphorylated p65 (pp65), p65, phosphorylated JNK (pJNK), JNK, phosphorylated p38 (pp38), and p38. Whole-cell lysates were immunoprecipitated (IP) with anti-TAB2 antibody and then immunoblotted with antibodies against phosphorylated TAK1 (pTAK1), TAK1, TAB1, and TAB2.

 
We have recently identified Thr187 as a critical autophosphorylation site in the TNF-{alpha}-induced activation of TAK1 (30). To investigate the role of TAK1 in the TRAIL-induced signaling pathways, HeLa cells were treated with TRAIL and cell lysates were immunoprecipitated with anti-TAB2 antibody. As reported previously, the immunoprecipitates contained TAK1 and TAB1, confirming stable complex formation of TAK1-TAB1-TAB2. Immunoblotting with a phosphorylated-specific anti-TAK1 antibody showed that TRAIL induced a transient activation of TAK1. The phosphorylation at Thr187 was detected at 30 min after stimulation, paralleling the delayed activation of p65/JNK/p38 described above. TAB1 and TAB2, two TAK1-binding proteins, were also phosphorylated from 30 min. Similar results were obtained when lysates were immunoprecipitated with anti-TAK1 antibody (data not shown). In contrast, TNF-{alpha} induced activation of the TAK1-TAB1-TAB2 complex within 10 min (Fig. 1). These kinetic analyses suggest that TAK1 is a kinase regulating the TRAIL-induced activation of the p65, JNK, and p38 signaling pathways.

RNA Interference with TAK1 siRNA Sensitizes Cells to TRAIL-Induced Apoptosis
To investigate the role of TAK1 in TRAIL-induced apoptosis, we used RNA interference to down-regulate the expression of endogenous TAK1. Endogenous expression as well as the phosphorylated band of TAK1 disappeared without affecting the expression of p65, JNK, and p38 in cells transfected with the siRNA for TAK1 but not the luciferase siRNA. The knockdown of TAK1 blocked the TRAIL-induced activation of p65, JNK, and p38 (Fig. 2A ). These results suggest that TAK1 is a major MAP3K that regulates the TRAIL-induced activation of the p65 and JNK/p38 signaling pathways. This is consistent with our previous findings that the TNF-{alpha}-induced activation of the p65 and JNK/p38 signaling pathways was abrogated by the TAK1 siRNA (30).


Figure 2
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  		Figure 2. Enhancement of TRAIL-induced apoptosis by TAK1 siRNA. HeLa cells were transfected with siRNA (20 nmol/L) against TAK1 and luciferase as a control. At 72 h after transfection, the following assays were done. A, cells were stimulated with TRAIL (200 ng/mL) for the period indicated, and immunoblotting (IB) assays were done as described above. B, cells were treated with TRAIL (200 ng/mL) for 3 h, and whole-cell lysates were immunoblotted with antibodies against caspase-8, caspase-3, caspase-7, PARP, and TAK1. C, cells were treated with TRAIL (50 ng/mL) for 9 h, and apoptotic cells were detected by monitoring enhanced green fluorescent protein (EGFP)–conjugated Annexin V binding to the cell surface using the FACScan analysis system.

 
We next evaluated the effects of TAK1 siRNA on TRAIL-induced apoptosis (Fig. 2B and C). Figure 2B shows that down-regulation of TAK1 expression by siRNA enhanced the activation of TRAIL-induced sequential apoptotic cascades involving caspase-8, caspase-3, caspase-7, and PARP. Apoptotic cells were also detected by monitoring the binding of enhanced green fluorescent protein–fused Annexin V to the cell surface using the fluorescence-activated cell sorting system. TAK1 siRNA only slightly induced binding to Annexin V; however, it synergistically enhanced the apoptosis caused by TRAIL (Fig. 2C). These results show that TAK1 regulated a cell survival signal and suppression of TAK1 enhanced TRAIL-induced apoptosis.

Effects of a TAK1 Inhibitor on TRAIL-Induced Apoptosis
To confirm the role of TAK1 in TRAIL-induced apoptosis, we tried to investigate the effects of 5Z-7-oxozeaenol, a selective inhibitor of TAK1, on the apoptosis. We tried to determine the inhibitory activity of this compound in the cell. TNF-{alpha}-induced phosphorylation of TAK1 at Thr187 reflects intracellular TAK1 activity because it is mediated by autophosphorylation activity. Figure 3A shows that 5Z-7-oxozeaenol inhibited the TNF-{alpha}-induced phosphorylation of TAK1 in a dose-dependent manner and completely suppressed it at 300 nmol/L. Blockade of TAK1 kinase by 5Z-7-oxozeaenol sensitized cells to TNF-{alpha}-induced apoptosis as reported in cells transfected with kinase-inactive mutant of TAK1 (Fig. 3B).


Figure 3
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  		Figure 3. Suppression of TAK1 activity by 5Z-7-oxozeaenol. A, HeLa cells were pretreated with 5Z-7-oxozeaenol (5Z-7) at the indicated concentration for 30 min and then stimulated with TNF-{alpha} (10 ng/mL) for 5 min. Whole-cell lysates were immunoprecipitated with anti-TAB2 antibody and then immunoblotted with antibodies against phosphorylated TAK1, TAK1, TAB1, and TAB2. B, HeLa cells were treated with 5Z-7-oxozeaenol (300 nmol/L) for 2 h and then simulated with TNF-{alpha} (10 ng/mL) for 8 h. Whole-cell lysates were immunoblotted as described above.

 
Next, we examined the effects of 5Z-7-oxozeaenol on TRAIL-induced apoptosis (Fig. 4 ). Pretreatment of cells with 300 nmol/L 5Z-7-oxozeaenol blocked the TRAIL-induced phosphorylation of TAK1 as well as downstream signaling pathways (Fig. 4A). Pretreatment of cells with 5Z-7-oxozeaenol increased the TRAIL-mediated activation of the proapoptotic caspase cascade (Fig. 4B). The TRAIL-induced binding of Annexin V was also enhanced by inhibiting TAK1 kinase activity (Fig. 4C). We also detected enhanced degradation of genomic DNA by the inhibitor (data not shown). These results indicate that the inhibition of TAK1 improves the chances of cancer cells undergoing apoptosis in response to TRAIL.


Figure 4
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  		Figure 4. Enhancement of TRAIL-induced apoptosis by 5Z-7-oxozeaenol. A, HeLa cells were pretreated with 5Z-7-oxozeaenol at 300 nmol/L for 30 min and stimulated with TRAIL (200 ng/mL) for the period indicated. Whole-cell lysates and immunoprecipitates were immunoblotted as described above. B, HeLa cells were pretreated with 5Z-7-oxozeaenol (300 nmol/L) for 2 h and then treated with TRAIL (200 ng/mL) for 3 h. Whole-cell lysates were immunoblotted with antibodies against caspase-8, caspase-3, caspase-7, and PARP. C, HeLa cells were pretreated with 5Z-7-oxozeaenol (300 nmol/L) for 30 min and then treated with TRAIL (50 ng/mL) for 9 h. The binding of Annexin V to cell surface was determined as described above.

 
Effects of a TAK1 Inhibitor on TRAIL-Induced Apoptosis in A549 Cells and TAK1-Deficient Fibroblasts
We next evaluated the effects of TAK1 on TRAIL-mediated apoptosis in human lung adenocarcinoma A549 cells. Figure 5A shows that, as in HeLa cells, TRAIL induced the delayed phosphorylation of TAK1. TAK1-mediated p65, JNK, and p38 MAPK signaling pathways were also activated by TRAIL (data not shown). An enhancement of cleavage of caspase-8/caspase-3/caspase-7 and PARP by 5Z-7-oxozeaenol was observed in TRAIL-stimulated A549 cells (Fig. 5B). Pretreatment of A549 cells with 5Z-7-oxozeaenol also slightly increased TRAIL-induced binding of Annexin V (data not shown).


Figure 5
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  		Figure 5. Enhancement of apoptosis by a combination of 5Z-7-oxozeaenol and TRAIL in A549 cells. A, A549 cells were treated with TRAIL (200 ng/mL) for the period indicated. Cell lysates were immunoprecipitated with anti-TAB2 antibody and then immunoblotted with anti-phosphorylated TAK1 and anti-TAK1 antibodies. B, cells were treated with 5Z-7-oxozeaenol (300 nmol/L) for 2 h and then stimulated with TRAIL (200 ng/mL) for 8 h. Whole-cell lysates were immunoblotted as describe above.

 
TRAIL has been shown to induce apoptosis in cancer cells but not in most normal cells. The selective induction of apoptosis through the suppression of TAK1 was investigated using MEFs. As reported previously (37), we confirmed that TAK1-deficient MEFs were sensitive to the TNF-{alpha}-induced activation of caspase-3 and PARP and susceptible to TNF-{alpha}-induced cell death (Fig. 6A ). In contrast, although mRNA expression of DR5 TRAIL receptor was comparable in normal MEFs and TAK1-deficient MEFs (data not shown), TRAIL failed to induce apoptosis in TAK1-deficient MEFs. In addition, pretreatment of 5Z-7-oxozeaenol did not drastically sensitize control MEFs to the TRAIL-induced apoptosis compared with the response of TNF-{alpha}-induced apoptotic cell death (Fig. 6B).


Figure 6
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  		Figure 6. Resistance to TRAIL-mediated apoptosis in MEFs. A, TAK1-deficient MEFs and normal MEFs were stimulated with TRAIL (200 ng/mL) for the period indicated or TNF-{alpha} (10 ng/mL) for 2 h. Cell lysates were immunoblotted with anti-caspase-3 and anti-PARP antibodies. B, wild-type MEFs were pretreated with 5Z-7-oxozeaenol (300 nmol/L) for 2 h and then stimulated with TRAIL (200 ng/mL) for 4 or 8 h or TNF-{alpha} (10 ng/mL) for 2 h. Whole-cell lysates were immunoblotted as described above.

 
Discussion

TAK1, one of the best-characterized members of the MAP3K family, is activated by various cellular stressors, including TNF-{alpha}, interleukin-1, and lipopolysaccharide (21–31). TAK1 participates in various cellular functions and is an upstream stimulatory molecule of the JNK, p38, and I{kappa}B kinase signaling pathways (21–26). The crucial role of TAK1 in cell survival is recognized nowadays (32, 37). NIH3T3 cells stably transfected with a kinase-inactive mutant of TAK1 were susceptible to TNF-{alpha}-induced apoptosis (32). As confirmed in Fig. 6, TAK1-deficient embryonic fibroblasts are also highly sensitive to TNF-{alpha}-induced apoptosis. In addition, blockade of TAK1 kinase activity by 5Z-7-oxozeaenol sensitized both HeLa and MEF cells to TNF-{alpha}-induced apoptosis (Figs. 3B and 6B). In the present study, we focused on the role of TAK1 in TRAIL signaling pathways and showed a novel function of TAK1 in TRAIL-induced apoptosis, the modulation of the caspase-mediated apoptotic program.

The signaling from DR4/DR5 has been characterized, and the delayed response is a unique feature of TRAIL-induced stress signaling pathways. We have previously identified Thr187 as an autophosphorylation site triggering the activation of TAK1 in response to TNF-{alpha} (30). In the present study, we found that TRAIL also induces delayed phosphorylation at Thr187 at 30 min, which paralleled the activation of p65 and MAPKs. However, as was seen in the TNF-{alpha}-induced phosphorylation of TAK1, the phosphorylation of TAK1 in response to TRAIL was also transient. We have recently reported that the rapid dephosphorylation is associated with a p38/TAB1-mediated feedback inhibition of TAK1 (30). Therefore, the transient phosphorylation of TAK1 by TRAIL is assumed to be the result of the p38/TAB1-mediated feedback control. The phosphorylation was blocked by a TAK1 inhibitor, indicating that it is mediated by autophosphorylation. More importantly, down-regulation of TAK1 expression by RNA interference or 5Z-7-oxozeaenol revealed that TAK1 is a major MAP3K regulating the TRAIL-induced p65, JNK, and p38 signaling pathways. We previously reported that receptor activator of NF-{kappa}B ligand, another member of the TNF-{alpha} ligand family, activated TAK1 via TRAF6 (38). These results indicate the possibility that TAK1 is a common MAP3K regulating the stress signaling pathways from the TNF-{alpha} receptor superfamily.

TRAIL has recently emerged as a novel anticancer agent based on its ability to potently induce apoptosis in tumor cells while exhibiting no toxicity in most normal cells (33–35). The combination of TRAIL and a chemotherapeutic agent, such as 5-fluorouracil, cis-diamminedichloroplatinum, doxorubicin, or CPT-11, was shown to augment apoptosis in some human cancer cells (6, 39). In addition, several natural compounds, including dietary flavonoids, sensitize malignant tumor cells to TRAIL (40). The molecular basis for the sensitization of tumor cells by these agents may involve up-regulation of DR4/DR5 (41), down-regulation of the antiapoptotic molecules Bcl-2, Bcl-xL, and c-FLIP, or up-regulation of proapoptotic molecules, including Bax, caspases, and Fas-associated death domain (42). In the present study, we showed that a blockade of TAK1 kinase activity enhanced TRAIL-induced apoptosis through activation of the caspase cascade. However, up-regulation of the death receptors was not detected in HeLa cells (data not shown). On the other hand, suppression of TAK1 led to impaired activation of NF-{kappa}B and MAPKs. NF-{kappa}B is widely accepted as an antiapoptotic factor in the death receptor signaling involving TNF-{alpha} and TRAIL (43). In contrast, the roles of JNK and p38 in TRAIL-induced apoptosis are still controversial (44). Investigation of main pathways to contribute to the sensitization of tumor cells to TRAIL and identification of target genes supporting the antiapoptotic function will help our understanding of the functional significance of TAK1 in TRAIL-mediated apoptosis.

In terms of therapeutic strategy, a selective effect of TRAIL on tumor cells is very important. The experiments using normal mouse fibroblasts showed that this might be achieved through the use of a TAK1 inhibitor. However, additional experiments using human cancer cells from multiple organs as well as normal human epithelial cells are necessary to show the tumor selectivity of the targeted cancer therapy of TRAIL in combination with TAK1 inhibitors. Collectively, these findings suggest that blocking TAK1 activity selectively sensitizes DR4/DR5-expressing tumor cells to TRAIL-induced apoptosis by suppressing the antiapoptotic pathways but the up-regulation of DR4/DR5 expression does not. It is therefore helpful to evaluate the apoptosis-enhancing properties of TAK1 inhibitors in a wide variety of tumor cells that are resistant to TRAIL-induced apoptosis but express DR4/DR5.

In summary, we showed that TAK1 is a critical MAP3K regulating the TRAIL-induced activation of NF-{kappa}B p65 and MAPKs and inhibition of TAK1 activity sensitizes tumor cells to TRAIL-induced apoptosis. These findings show the potential of TAK1 inhibitors as chemotherapeutic agents especially in combination with TRAIL.

Footnotes

Grant support: Grant-in-Aids for Scientific Research (C) no. 17590055 and 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/28/06; revised 9/13/06; accepted 10/18/06.

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