Butein Sensitizes Human Hepatoma Cells to TRAIL-Induced Apoptosis via Extracellular Signal-Regulated Kinase/Sp1–Dependent DR5 Upregulation and NF-κB Inactivation

Tumor necrosis factor (TNF) family members have limited use in anticancer therapy because they cause severe cytotoxicity to normal cells (1). However, for a couple of decades, TNF-related apoptosis-inducing ligand (TRAIL) has been thought to be a new candidate for anticancer therapy because TRAIL selectively suppresses tumor growth in vivo and in vitro but has little or no effect on normal cells (2, 3). TRAIL can bind to two death receptors (DR), DR4 and DR5, which contain a cytoplasmic functional death domain. After TRAIL binds to DRs, TRAIL triggers cell death through at least two fundamental apoptotic pathways, referred to as the extrinsic pathway and the intrinsic pathway, depending on the cell type (4). TRAIL-induced apoptosis initiated by the extrinsic pathway involves DR engagement, death-inducing signaling complex formation, and proteolytic activation of caspase-8 (5). Proteolytic caspase-8 further activates Bid, which, in turn, translocates to the mitochondria and activates the intrinsic pathway (6). Activated caspase-8 is released into the cytoplasm and induces a protease cascade that activates effector caspases such as caspase-3 and caspase-7 (7). DR4 and DR5 not only give the apoptosis signal through the Fas-associated death domain and caspase-8 but also activate NF-κB, which regulates the expression of survival factors such as members of the inhibitor of apoptosis (IAP) family (IAP1, IAP2, and XIAP) and Bcl-xL.

Many studies have attempted to find new anticancer agents, which could augment apoptosis induced by TRAIL, because some cancer cells are resistant to the apoptotic effects of TRAIL (8–10). The mechanism underlying the augmentation of TRAIL-induced apoptosis is largely related to their ability to upregulate the expression level of TRAIL receptors (i.e., DR4 and DR5). Therefore, agents that upregulate the expression of DR4 and/or DR5 may have potential for clinical management of cancer. Because DR4 and DR5 can be regulated by either a p53-dependent or a p53-independent mechanism (11–13), it is possible that augmentation of TRAIL-induced apoptosis by anticancer agents may be either p53 dependent or p53 independent.

Natural products have played a highly significant role over the years in the discovery of new drugs. This is particularly evident in the treatment of cancers and infectious diseases in which more than 60% and 75% of drugs, respectively, are of natural origin (14). Therefore, a natural product with strong synergistic activity with TRAIL but minimal toxicity could be a new tool for cancer therapy. Butein is one such agent that has been identified from numerous plants, including the stem bark of Semecarpus anacardium, the heartwood of Dalbergia odorifera, and the traditional Chinese and Tibetan medicinal herbs Caragana jubata and Rhus verniciflua Stokes. Previous studies suggested that butein sensitizes apoptosis in human promyelocytic leukemia cells and B16 melanoma cells (15, 16). In the in vitro experiment, butein also suppressed the proliferation of many human cancers, including breast carcinoma, colon carcinoma, osteosarcoma, and hepatic stellate cells (17–20). But the anticancer mechanisms of butein in TRAIL-mediated apoptosis are little understood.

In this study, we showed DR5 expression and NF-κB–mediated mechanism underlying butein-sensitized apoptosis induced by TRAIL in human hepatoma cancer cells.

Antibodies against Sp1, nucleolin, p50, p65, Bid, caspase-3, caspase-8, caspase-9, poly(ADP-ribose) polymerase (PARP), DR4, DR5, IAP-1, IAP-2, XIAP, Bcl-2, Bcl-xL, Bax, and Bad were purchased from Santa Cruz Biotechnology. Antibodies against c-Jun NH₂-terminal kinase (JNK), phospho-JNK, extracellular signal-regulated kinase (ERK), phospho-ERK, p38, and phospho-p38 were purchased from Cell Signaling. The antibody against β-actin was purchased from Sigma. Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulin, recombinant human TRAIL/Apo2 ligand (the nontagged 19-kDa protein, amino acids 114–281), and TNF-α were purchased from KOMA Biotechnology. Antibodies for Fas and PP2A were purchased from Upstate Biotechnology. The blocking antibody against DR5 was purchased from R&D Systems. 6-Carboxy-2′,7′-dichlorofluorescein diacetate (H₂DCFDA) and 3,3′-dihexyloxacarbocyanine iodide (DiOC₆) were purchased from Molecular Probes. Glutathione, N-acetyl-l-cysteine (NAC), pyrrolidine dithiocarbamate (PDTC), MG132, and PS341 were purchased from Sigma. Butein was also purchased from Sigma and dissolved in DMSO (vehicle). PD98059, SP600125, SB239063, z-VAD-fmk, and z-IETD-fmk were purchased from Calbiochem.

Human hepatoma cell lines Hep3B and HepG2 were cultured in DMEM (Life Technologies). PC3, HCT116, and U937 cells were cultured in RPMI 1640 (Life Technologies) supplemented with 10% fetal bovine serum and antibiotics (Sigma). MTT assays were used to determine cell viability.

Cells were fixed with 1 unit/mL of RNase A (DNase free) and 10 μg/mL of propidium iodide (Sigma) overnight at room temperature in the dark. A FACSCalibur flow cytometer (Becton Dickinson) was used to analyze the level of apoptotic cells containing sub-G₁ DNA content. For Annexin V and DiOC₆ staining, live cells were incubated with Annexin V (R&D Systems) and DiOC₆.

Cells were lysed in a buffer containing 10 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, and 0.5% Triton X-100 for 30 minutes on ice. Lysates were vortexed and cleared by centrifugation at 10,000 × g for 20 minutes. Fragmented DNA in the supernatant was extracted with an equal volume of phenol/chloroform/isoamyl alcohol mixture (25:24:1) and analyzed.

Total cell extracts were prepared using PRO-PREP protein extraction solution (iNtRON Biotechnology). The preparation of cytoplasmic and nuclear extracts was conducted using NE-PER nuclear and cytosolic extraction reagents (Pierce). Total cell extracts were separated on polyacrylamide gels, and then standard procedures were used to transfer them to the nitrocellulose membranes. The membranes were developed using an enhanced chemiluminescence reagent (Amersham).

Cells were plated at a density of 5 × 10⁴, allowed to attach for 24 hours, and exposed to 5 mmol/L NAC alone, 5 mmol/L glutathione alone, 10 μmol/L butein alone, or NAC or glutathione plus butein for 1 hour. The cells were stained with 10 μmol/L H₂DCFDA for 10 minutes at 37°C, and flow cytometry was used to determine the fluorescence intensity.

A caspase activation kit was used according to the manufacturer's protocol (R&D Systems) to measure the activity of caspase-like protease.

Total RNA was extracted using the Trizol reagent (Invitrogen), and reverse transcription-PCR (RT-PCR) was conducted. The sense primer 5′-GTCTGCTCTGATCACCCAAC-3′ and the antisense primer 5′-CTGCAACTGTGACTCCTATG-3′ were used to amplify human DR5 mRNA. For glyceraldehyde-3-phosphate dehydrogenase, the sense primer 5′-CGTCTTCACCATGGAGA-3′ and the antisense primer 5′-CGGCCATCACGCCCACAGTTT-3′ were used.

Indirect staining with primary rabbit anti-human DR4 or DR5 followed by FITC-conjugated IgG was used to analyze cells for the surface expression of DR4 and DR5. Flow cytometry was used to analyze the expressions of these DRs.

The pDR5/SacI plasmid [containing DR5 promoter sequence (−2,500/+30)] and pDR5/−605 [containing DR5 promoter sequence (−605/+3)] were gifts from Dr. T. Sakai (Kyoto Prefectural University of Medicine, Kyoto, Japan). To analyze the promoter regions responsible for butein, the reporter constructs containing single (mSp1-1 and mSp1-2), double (mSp1-3 and mSp1-4), or triple (mSp1-5 and mSp1-6) point mutations at putative Sp1-binding sites of the DR5 promoter were used. The detailed procedure to generate these mutants has been previously described (21).

The 25-nucleotide small interfering RNA (siRNA) duplexes used in this study were purchased from Santa Cruz Biotechnology. The cells were transfected with siRNA oligonucleotides using FuGENE 6 Transfection Reagent according to the manufacturer's recommendations.

DNA-protein binding assays were carried out with a nuclear extract. Synthetic complementary Sp1 (5′-ATTCGATCGGGGCGGGGCGAGC-3′) and NF-κB (5′-AGTTGAGGGGACTTTCCCAGGC-3′) binding oligonucleotides (Santa Cruz Biotechnology) were 3′-biotinylated using the biotin 3′-end DNA labeling kit (Pierce) according to the manufacturer's instructions.

The chromatin immunoprecipitation (ChIP) assay was done using the EZ-Chip assay kit according to the manufacturer's protocol (Upstate Biotechnology). The primers used for the amplification of the Sp1-binding site of DR5 promoter region were 5′-GCCAGGGCGAAGGTTA-3′ (sense) and 5′-GGGCATCGTCGGTGTAT-3′ (antisense; 276-bp DNA product; ref. 22).

All data were derived from at least three independent experiments. The images were visualized with Chemi-Smart 2000 (Vilber Lourmat). Images were captured using Chemi-Capt (Vilber Lourmat) and transported into Photoshop. Scion Imaging software (23) was used to quantify. Statistical analyses were conducted using SigmaPlot software (version 6.0). Values were presented as mean ± SD. Significant differences between the groups were determined using the unpaired Student's t test. Statistical significance was regarded at P < 0.05.

Antiproliferative activity of TRAIL was first analyzed in four human cancer cell lines: Hep3B, HepG2, HCT116, and U937. Treatment with 100 ng/mL TRAIL induced limited inhibition of cell proliferation (<20%) at 24 hours, suggesting that these cells are resistant to the apoptotic effects of TRAIL (Fig. 1A). Next, we examined the antiproliferative effects of butein alone or in combination with TRAIL in these cells. Butein alone did not significantly induce any morphologic signs of cell death up to 5 μmol/L, although the cellular activity to reduce MTT formazan was slightly decreased at this concentration. However, cell proliferation was significantly reduced by treatment with a combination of butein and TRAIL when the concentration of TRAIL (100 ng/mL) was fixed. These results show that treatment with a combination of butein and TRAIL effectively inhibits cell proliferation in TRAIL-resistant hepatoma cells in a cell type–nonspecific manner. Next, we investigated whether the combination treatment is dependent on apoptosis. As shown in Fig. 1B, the treatment of Hep3B cells with a combination of 5 μmol/L butein and 100 ng/mL TRAIL for 24 hours significantly increased the accumulation of sub-G₁ phase cells (top) and Annexin V staining (middle), whereas treatment with butein or TRAIL alone slightly increased. Pretreatment with a pan-caspase inhibitor z-VAD-fmk significantly blocked the accumulation of sub-G₁ phase cell populations and Annexin V induced by treatment with butein and TRAIL. When phase-contrast microscopy was used to examine the change of cell morphology, the cells treated with a combination of 5 μmol/L butein and 100 ng/mL TRAIL for 24 hours displayed decreased cell numbers with some apoptotic shrinkage (bottom) compared with the untreated control cells (Fig. 1B). Furthermore, DNA fragmentation analysis also showed a typical ladder pattern of internucleosomal DNA fragmentation in hepatoma cells treated with a combination of butein and TRAIL but not in cells treated with butein or TRAIL alone (Fig. 1C). These apoptosis phenomena were also completely blocked by the pretreatment with z-VAD-fmk. Taken together, these results suggest that butein stimulates TRAIL-induced apoptosis through caspase activation but is not cell specific.

Hepatoma cells had been shown to be resistant to TRAIL because of insufficient activation of the extrinsic pathway and, more importantly, because of the blocking of death signaling at the mitochondrial level (4). Therefore, Western blot analyses of proapoptotic and antiapoptotic factors were done; it was presumed that the inhibitory factors in mitochondria were suppressed by treatment with a combination of butein and TRAIL. As shown in Fig. 2A, time-dependent cleavage of caspase-8 and Bid was observed by treatment with the combination of butein and TRAIL. In contrast, little or no change was observed in cells treated with a single agent. Similarly, caspase-9, caspase-3, and PARP were also activated after the combined treatment. Next, apoptotic events in the mitochondria were evaluated by measuring the mitochondrial membrane potential using DiOC₆. Marked reduction in the mitochondrial membrane potential had occurred in cells treated with butein plus TRAIL (Fig. 2B). In addition, this process was accompanied by the release of cytochrome c from the mitochondria into the cytosol (Fig. 2C). However, pretreatment with caspase-8 inhibitor z-IETD-fmk normalized mitochondrial membrane potential and completely blocked the release of cytochrome c into cytosol (Fig. 2C). Furthermore, these caspases activity was significantly increased by the combined treatment (Fig. 2D). The combined treatment also degraded the expression of Bcl-2, XIAP, IAP-1, and IAP-2. In contrast, the level of Bad expression was upregulated by the combined treatment (Fig. 2E). These results indicated that treatment with a combination of butein and TRAIL reduces the expression of multiple proteins associated with cell survival through the extrinsic and intrinsic apoptotic signal pathway.

As the TNF superfamily members share similar protein structures and DR-mediated apoptotic signaling pathways (24), we next examined whether butein sensitizes TNF-α–mediated apoptosis, possibly targeting the common component(s) of these DR-mediated apoptotic pathways. Consistent with the TRAIL results, TNF-α alone had no significant influence on cell proliferation in Hep3B cells (Fig. 3A). Interestingly, treatment with a combination of these agents plus butein had no effect on cell death in Hep3B cells, which were very sensitive to the butein/TRAIL-combined treatment. These results indicate that butein selectively facilitates the inhibition of cell growth induced by TRAIL but not the other tested death ligand. Because TRAIL is known to trigger apoptotic signals via two types of DRs, DR4 and DR5, we next examined whether the modulation of DR4 and/or DR5 protein levels by butein might be involved in its sensitizing effect on TRAIL-induced apoptosis. We found that treatment of Hep3B cells with butein induces a time-dependent (left) and dose-dependent (right) increase in the protein levels of DR5 but did not affect the levels of DR4 (Fig. 3B). Fluorescence-activated cell sorting (FACS) analysis also showed that the butein-induced surface expression of DR5 but not DR4 was also significantly increased in Hep3B cells (Fig. 3C). Consistent with this, treatment with butein significantly increased DR5 protein levels in other human cancer cell lines, HepG2, HCT116, PC3, and U937 (Fig. 3D and E). To confirm the functional role of DR5 in the sensitization of TRAIL-induced apoptosis by butein, we also examined the effect of DR5-specific blocking chimera antibody on butein/TRAIL-induced apoptosis. The addition of DR5-specific blocking antibody dose dependently reversed the inhibition of butein/TRAIL-induced cell proliferation in Hep3B cells (Fig. 3F). Similarly, treatment with anti-DR5 antibody in Hep3B cells decreased the DNA fragmentation as a marker of apoptosis (Fig. 3G). Furthermore, suppression of DR5 expression by transfection of Hep3B cells with DR5 siRNA also effectively inhibited butein-stimulated TRAIL-induced growth inhibition (Fig. 3H), supporting the idea that butein-induced upregulation of DR5 is critical for the enhancement of TRAIL sensitivity in Hep3B cells.

To examine whether butein-induced DR5 upregulation is controlled at the transcriptional level, we did RT-PCR analysis of DR5. After the cells were treated with 10 μmol/L butein, we observed a gradual increase in DR5 mRNA levels of Hep3B (Fig. 4A). We further explored the effects of butein on the promoter activities of reporter constructs containing 2.5- and 0.6-kb fragments of the DR5 gene promoter region (pDR5/SacI and pDR5/−605, respectively; ref. 25) in Hep3B cells. We found that butein significantly increases the promoter activities of both pDR5/SacI (data not shown) and pDR5/−605 (Fig. 4B), suggesting that butein-responsive elements are localized within the smaller fragment (605 bp). Previously, Yoshida et al. (26) showed that the region of the DR5 promoter spanning nucleotides −605 to +3 contains typical transcription factor binding sites, including four Sp1 sites and a TATA-like box site (Fig. 4B). To examine which Sp1 site(s) in the DR5 promoter are critical for butein-induced DR5 upregulation, we analyzed luciferase assays using reporter constructs with several Sp1 mutants of the promoter. As shown in Fig. 4B, transfection with double mutant mSp1-3 (mutated at the −305 and −300 Sp1 sites) construct significantly decreased butein-induced DR5 promoter activity compared with the wild-type (pDR5/−605) construct. Furthermore, the DR5 promoter activity derived from the triple mutant constructs mSp1-5 and mSp1-6 was significantly decreased by treatment with butein. However, single mutant constructs mSp1-1 and mSp1-2 and double mutant mSp1-4 had only a slight effect on DR5 promoter activity. These results suggest that the two putative Sp1-binding sites present at −305/−300 may play an important role in butein-induced enhancement of DR5 promoter activity. To determine whether butein has an effect on the DNA-binding activity of Sp1, we did electrophoretic mobility shift assay (EMSA) by incubating nuclear extracts from cells exposed to butein with a biotin-labeled Sp1 response element containing oligonucleotides. As shown in Fig. 4C, treatment with 10 μmol/L butein time dependently increased the specific DNA-binding activity of Sp1 in HepG2 and Hep3B cells. Furthermore, Sp1 DNA-binding complexes were undetected when a mutant Sp1 oligonucleotide was used in the binding reaction, and supershift assays using specific Sp1 antibody confirmed that the slowest migrating complex represents Sp1 (data not shown). Western blotting assay detected that treatment with 10 μmol/L butein increased Sp1 translocation to the nucleus (Fig. 4D). To further investigate the exact mechanism of butein on the regulation of DR5 expression, a ChIP assay was done to examine the binding of Sp1 on the DR5 promoter regions. As shown in Fig. 4E, butein increased Sp1 binding to the promoter regions of DR5. To additionally investigate whether butein-induced Sp1 DNA-binding activity was due to Sp1 phosphorylation, the nuclear extract was incubated with the serine-threonine phosphatase PP2A before the binding reaction. As observed in Fig. 4F, the increased Sp1 DNA-binding activity due to butein was blunted in the samples treated with PP2A. These data suggest that butein activates DR5 transcription by increasing Sp1 phosphorylation and consequent Sp1 DNA-binding activity.

The phosphorylation of Sp1 has been widely studied and the results showed that some kinases phosphorylate Sp1, which affects transactivation, but some other kinases phosphorylate Sp1, which affects its DNA binding affinity (27). Serine or threonine residues could be phosphorylated by different kinases, including DNA-dependent protein kinase, mitogen-activated protein kinase (MAPK), casein kinase II, and cyclin-dependent kinase 2. To investigate whether butein-mediated Sp1 DNA binding is MAPK dependent, Western blotting analysis was used to test the change in phosphorylation of JNK, ERK, and p38. As shown in Fig. 5A, 10 μmol/L butein time dependently increased the phosphorylation of JNK, ERK, and p38. ERK, JNK1/2, and p38 inhibition abrogated butein-mediated Sp1-binding activity (Fig. 5B). Inhibition of ERK, JNK1/2, and p38 inhibited Sp1 translocation to the nucleus (Fig. 5C). However, only inhibition of ERK by PD98059 significantly blocked butein-induced DR5 upregulation and inhibited cleavage of caspase-3 (Fig. 5D) and induction of sub-G₁ population induced by treatment with butein plus TRAIL (Fig. 5E). FACS analysis also showed that PD98059 significantly suppresses the combined treatment-induced surface expression of DR5 in Hep3B cells (Fig. 5F). Moreover, pretreatment of 20 μmol/L PD98059 for 1 hour reduced DR5 promoter (pDR5/−605) activity induced by butein (Fig. 5G). Additionally, we investigated whether reactive oxygen species (ROS) are involved in butein-induced apoptosis in hepatoma cells treated with 5 μmol/L butein. H₂DCFDA-based FACS detection revealed that intracellular ROS levels slightly increased following treatment with butein (Fig. 5H). The butein-induced increases in ROS levels were completely blocked by pretreatment with antioxidants, NAC, and glutathione. We next tested whether scavenging of ROS attenuates the growth inhibition induced by the treatment of the combination of butein and TRAIL. Pretreatment with NAC and glutathione did not block the antiproliferative effects induced by the combination of butein and TRAIL (Fig. 5H). These results suggest that ERK may be involved in butein-induced DR5 expression via Sp1 activation.

Because TRAIL can trigger both death and survival signaling (e.g., NF-κB), thus regulating the expression of several factors involved in cell survival, we examined the effect of butein on TRAIL-mediated NF-κB by EMSA assay. As shown in Fig. 6A, 50 ng/mL TRAIL induced a strong increase in binding of NF-κB within 0.5 hour and was sustained for 3 hours. Pretreatment with butein inhibited TRAIL-induced NF-κB activation in a dose-dependent manner (Fig. 6B). Especially, at a dose of 10 μmol/L, butein significantly inhibited NF-κB activation induced by TRAIL as much as NF-κB inhibitors [PDTC, MG-132, and bortezomib (PS341)] in Hep3B cells (Fig. 6B). It is well known that NF-κB activation by TRAIL is mediated by phosphorylation and degradation of IκBα. As shown in Fig. 6C, butein blocked TRAIL-dependent IκBα degradation. Butein also repressed nuclear translocation of p65 and p50 induced by TRAIL. To further confirm the role of NF-κB in butein/TRAIL-induced apoptosis, cells were pretreated for 30 minutes with PDTC, MG132, and PS341. The cells were then treated with butein and TRAIL for an additional 24 hours, and flow cytometry was used to assess the sub-G₁ cell populations. As shown in Fig. 6D, cells incubated with TRAIL or PDTC, MG132, and PS341 alone resulted in a slight increase in the sub-G₁ percentage. However, treatment with a combination of the NF-κB inhibitors and TRAIL or TRAIL/butein markedly increased the accumulation of sub-G₁ phase cells. In addition, as observed by MTT assay (data not shown), butein-mediated repressed cell viability is enhanced by blocking of the NF-κB signal pathway. These results suggest that butein treatment is likely to inhibit the NF-κB pathway and that this pathway is likely involved in apoptosis.

Although TRAIL is thought to be a highly promising candidate for cancer treatment, use of TRAIL has major limits in clinical applications because of resistance against TRAIL in a variety of cancer cells (2, 3). Nevertheless, many scientists have reported that conventional cytotoxic drugs such as antioxidants and novel molecular-targeted agents or irradiation markedly sensitize TRAIL-induced apoptosis in TRAIL-resistant cancer cells (8–10). Thus, efforts to identify agents that activate DRs or block antiapoptotic effectors may improve anticancer therapeutic design. Here, we provide evidence that butein is capable of triggering TRAIL-induced apoptosis at least in TRAIL-resistant hepatoma cells.

DRs have a highly conserved extracellular region containing cysteine-rich repeats and a conserved intracellular region of 80 amino acids termed the death domain (28). The domain is essential for the rising death signal, resulting in the activation of caspase-8 (29). Therefore, it has been thought that regulation of DRs causes effective death of cancer cells through direct binding with TRAIL. Some scientists reported that a few chemicals or proteins sensitize TRAIL-induced apoptosis through overexpression of DR4 or DR5 (11–13). In this study, we also found that butein mainly increases the expression of DR5 at the transcriptional level but maintains the expression of DR4. Additionally, for the functional study of DR5, DR5-specific blocking chimeric antibody and transient knockdown of DR5 expression by siRNA duplexes significantly inhibited the growth-inhibitory effects induced by treatment with a combination of butein and TRAIL in Hep3B cells. Nevertheless, we cannot rule out the possibility that the upregulation of DR5 is not the only way for sensitizing to TRAIL because knockdown of DR5 caused only a modest decrease in TRAIL-induced apoptosis despite the almost complete loss of DR5 expression (8). Recently, it is well known that the tumor suppressor gene p53 is a main regulator for the expression of genes engaged in apoptosis (30). Many scientists have reported that overexpression of p53 transactivates DR4 and DR5 gene-induced DNA damage–inducing agents, suggesting that DR4 and DR5 definitely are important regulators in p53-dependent apoptosis (11, 13). Nevertheless, there has been conflicting evidence about the involvement of p53 in regulating the expression of DR4 and DR5 until now (17). In this study, butein induced the expression of DR5, regardless of p53 status. These results may imply that upregulation of DR5 is functionally essential for butein-sensitized apoptosis at least in TRAIL-resistant hepatoma cells through a p53-independent mechanism.

Sp1 is well known to bind to G-rich elements such as GC-box (GGGGCGGGG) and GT-box (GGTGTGGGG; ref. 31). Recently, it has been well known that binding of Sp1 in the promoter regions tightly regulates DR5 transcription in a variety of cancer cells, and the required specific Sp1 site was mainly found at −195/−159 to the transcription start site (21, 22). In this study, we found that treatment with butein significantly induced expression of Sp1 in HepG2 and Hep3B cells that was correlated to the induction of DR5. Reporter gene analysis also showed two regions (−305 and −300) of the DR5 promoter that seemed to be important in butein regulation of DR5 promoter activity. Furthermore, the ChIP assay showed that Sp1 can directly bind to the DR5 promoter regions and regulated transcriptional expression. These results suggest that induction of Sp1 significantly sensitizes butein/TRAIL-dependent apoptosis through DR5 expression. Nevertheless, the regulation of DR5 expression is much less understood in the combined treatment. Currently, DR5 expression has been regulated by Sp1 transcription factor activity (21, 22). It was reported that ROS generation is a major target for triggering and amplifying TRAIL-dependent apoptosis through DR5 (32, 33). JNK directly is activated by ROS and stimulates DR5 expression (25, 34). Therefore, we hypothesized that the combined treatment sensitizes the ROS-JNK-Sp1 signal cascades to activate DR5 expression; however, pretreatment of NAC or glutathione has no significant effect on cell growth inhibition and DR5 promoter activity. Nevertheless, our previous study reported that high doses of butein (<40 μmol/L) increase G₂-M phase arrest and cell growth inhibition through the ROS-JNK pathway (35). However, low doses of butein (<10 μmol/L) induced G₁ phase arrest without cell growth inhibition in a ROS-independent manner. Only the ERK inhibitor PD98059 significantly blocked butein-mediated Sp1 activation and cell death. The results indicate that Sp1 is a butein-regulated transcription factor, and ERK is an intermediary kinase in this relationship. Additionally, JNK could be correlated with DR5 expression via Sp1 activation because it is well known that pretreatment with SP600125 moderately decreased DR5 expression and cell death (8, 25, 34). We did further study to elucidate if JNK and p38 have influence on the DR5 promoter activity. SP600125 itself significantly increased promoter activity of DR5, but SP600125 evidently downregulated its activity in the presence of butein (data not shown). These data indicated that the JNK signal pathway may be also related to DR5 regulation in butein-induced apoptosis. Nevertheless, we cannot rule out the possibility of EKR inhibition by 20 μmol/L SP600125 because SP600125 quite selectively blocks JNK at nanomolar concentrations. Additionally, although SB203580 suppressed the expression of Sp1 and proapoptotic proteins, we cannot find any differences of DR5 promoter activity in the presence of SB203580. Therefore, more prudent experiments will be needed for the effects of p38 on TRAIL-induced apoptosis.

The NF-κB transcription factor family consists of several structurally related proteins such as c-Rel, Rel-A (p60), Rel-B, p50/p105, and p52/p100, which form homodimers or heterodimers with each other and regulate the expression of a number of genes (36). TRAIL induced additional binding activity of NF-κB composed of a Rel-A/p50 heterodimer in these cells. Ravi et al. (37) have recently reported that Rel-A^−/− mouse fibroblasts are highly sensitive to TRAIL-induced apoptosis and that anti-CD40–mediated activation of NF-κB, including Rel-A, effectively blocked TRAIL-induced apoptosis. Therefore, NF-κB composed of Rel-A/p50 seems to play a key role in the resistance of Hep3B and HepG2 cells to TRAIL-induced apoptosis. Indeed, pretreatment with butein inhibited TRAIL-mediated activation of Rel-A/p50 NF-κB that was sensitized to TRAIL-induced apoptosis by butein. Similar observations have been reported that butein suppressed activation of Rel-A/p50 NF-κB induced by TNF-α (38). Taken together, these results indicate that butein could possibly sensitize to TRAIL-induced apoptosis by inhibiting the activation of NF-κB, including Rel-A. In contrast, recently important findings showed that the activation status of NF-κB is not sufficient to determine the fate of a cell with respect to TRAIL-induced apoptosis in hepatocellular carcinoma (10). Braeuer et al. (39) also reported that constitutively activated NF-κB, but not induced NF-κB, leads to TRAIL resistance by upregulation of XIAP in human cancer cells. Therefore, the effects of NF-κB will be investigated in TRAIL resistance. In addition, suppression of NF-κB activity by butein may also be involved in the stimulation of caspase-8 activity because the NF-κB–induced products, IAP-1, IAP-2, and XIAP, are known to cooperatively block caspase-8 activity (40).

In summary, we showed that butein sensitizes apoptosis in TRAIL-resistant cells via upregulation of DR5 expression. We also showed that DR5 expression is tightly regulated by Sp1 activity in the promoter regions of the gene. However, p53 is not necessary for butein-induced DR5 expression and triggers apoptosis in TRAIL-stimulated cells. We also found that the JNK inhibitor SP600125 partially suppresses Sp1 and DR5 expression and cell death; however, the ERK inhibitor PD98059 significantly abrogated the effect. Consequently, butein p53 independently upregulates DR5 expression through the ERK and Sp1 mechanism and sensitizes apoptosis in TRAIL-resistant cells.

No potential conflicts of interest were disclosed.

Grant Support: Basic Science Research Program through the National Research Foundation funded by the Ministry of Education, Science and Technology (No. 2009-0068626; G-Y. Kim).

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.