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Cancer Research 68, 8918, November 1, 2008. doi: 10.1158/0008-5472.CAN-08-1120
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Baicalein Overcomes Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Resistance via Two Different Cell-Specific Pathways in Cancer Cells but not in Normal Cells

Hiroya Taniguchi1,2, Tatsushi Yoshida1, Mano Horinaka1, Takashi Yasuda1,3, Ahmed E. Goda1,4, Masako Konishi1, Miki Wakada1, Keisho Kataoka2, Toshikazu Yoshikawa2 and Toshiyuki Sakai1

1 Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science; Departments of 2 Gastroenterology and 3 Urology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, Japan; and 4 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tanta University, Tanta, Egypt

Requests for reprints: Toshiyuki Sakai, Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. Phone: 81-75-251-5339; Fax: 81-75-241-0792; E-mail: tsakai{at}koto.kpu-m.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. A current problem is that some cancers still remain resistant to TRAIL. We show for the first time that a naturally occurring flavonoid, baicalein, overcomes TRAIL resistance in cancer cells. The combination of baicalein and TRAIL effectively induced apoptosis in TRAIL-resistant colon cancer SW480 cells. Baicalein up-regulated the expression of death receptor 5 (DR5) among TRAIL receptors at the mRNA and protein levels. Suppression of this up-regulation with small interfering RNA (siRNA) efficiently reduced the apoptosis induced by TRAIL and baicalein, suggesting that the sensitization was mediated through DR5 induction. Moreover, baicalein also overcame TRAIL resistance with DR5 up-regulation in prostate cancer PC3 cells. Of note, the combination of TRAIL and baicalein hardly induced apoptosis in normal human cells, such as blood cells and hepatocytes. Baicalein increased DR5 promoter activity, and this enhanced activity was diminished by mutation of a CCAAT/enhancer-binding protein homologous protein (CHOP)–binding site in SW480 cells. In SW480 cells, CHOP siRNA blocked both functions of baicalein. CHOP expression was induced by baicalein in SW480 cells; however, in PC3 cells, baicalein scarcely induced CHOP and mutation of the CHOP-binding site did not abrogate the DR5 promoter activation by baicalein. Interestingly, baicalein induced reactive oxygen species (ROS) and a ROS scavenger prevented DR5 expression and TRAIL sensitization in PC3 but not SW480 cells. These results indicate that, using two different pathways, baicalein exposes cancer surveillance of TRAIL and overcomes TRAIL resistance in cancer cells. [Cancer Res 2008;68(21):8918–27]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Baicalein is a flavonoid derived from the root of Scutellaria baicalensis, widely used in Chinese herbal medicine. Baicalein is also known as a selective 12-lipoxygenase (12-LOX) inhibitor (1), which induces antiproliferation and apoptosis in various cancer cells (2–4). However, this flavonoid also has other activities, such as antibacterial, antiviral, antioxidative, and prooxidative effects (5, 6). Thus, the molecular mechanism of the antitumor effect is still unclear.

Reactive oxygen species (ROS) are critical signaling molecules and play important roles in a variety of normal biochemical functions and abnormal pathologic processes.

In cancer cells, some chemotherapeutic agents and radiation therapy generate ROS and lead to necrotic or apoptotic cell death (7, 8). Thus, a therapeutic strategy is proposed to use ROS-generating compounds in cancer therapy. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2L) selectively induces apoptosis in various cancer cells in vitro and in vivo, with little or no toxicity in normal cells (9–12). Endogeneous TRAIL is expressed in immune cells and contributes to immunosurveillance for cancer (13). Furthermore, TRAIL deficiency in mice accelerates malignant tumors (14). Therefore, TRAIL is one of the most promising agents for cancer therapeutics. Death receptor 5 (DR5; also called TRAIL-R2 or KILLER) is a receptor for TRAIL. TRAIL interacts with DR5 or DR4 (also called TRAIL-R1), leading to aggregation of the receptors, recruitment of the adaptor molecule FADD, and activation of initiator caspase-8 or caspase-10, which can directly evoke cleavage of downstream effector caspases (15, 16). On the other hand, Bid, a proapoptotic Bcl-2 family member, is also cleaved by caspase-8 or caspase-10 and then activates the mitochondrial apoptotic signaling pathway. Decoy receptor 1 (DcR1) and DcR2 are also TRAIL receptors and have dominant negative effects by competing with DRs for TRAIL interaction (16). Interestingly, some studies have reported that DR5 is expressed more abundantly in cancer cells than in normal cells (17, 18). Accordingly, the TRAIL-DR5 pathway is considered to be an attractive candidate for cancer chemotherapy, and clinical trials with recombinant human TRAIL and agonistic DR5 antibodies are ongoing (19). However, some tumors remain tolerant to TRAIL-induced apoptosis (20). Thus, it is important to overcome this resistance to expand the ability of TRAIL in cancer therapy.

Here, we report, for the first time, that baicalein overcomes TRAIL resistance in cancer, but not normal cells, through DR5 up-regulation mediated by two different pathways, ROS induction and up-regulation of a transcription factor, CCAAT/enhancer-binding protein homologous protein (CHOP/GADD153).


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Reagents. Baicalein and 12-hydroxyeicosatetraenoic acid (12-HETE) were purchased from Cayman Chemical and dissolved in DMSO. Soluble recombinant human TRAIL/Apo2L (untagged TRAIL, amino acids 114–281) was purchased from PeproTech. Human recombinant DR5/Fc chimera and caspase inhibitors, such as zVAD-fmk, were purchased from R&D Systems. Catalase was purchased from Sigma-Aldrich.

Cell culture. Human colon cancer SW480 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 4 mmol/L glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Human prostate cancer PC3 cells were maintained in RPMI 1640 with 10% FBS, 2 mmol/L glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Normal peripheral blood mononuclear cells (PBMC) were isolated using a Ficoll-Paque PLUS (Amersham Biosciences) density gradient according to the manufacturer's instructions and maintained in X-VIVO 20 (Cambrex) serum-free medium with 0.2% bovine serum albumin (BSA; Sigma). Normal human hepatocytes (Cell System-Hc cells) were obtained from Cell Systems and maintained in SFM CS-C medium according to the instructions of the supplier. All cells were incubated at 37°C, with humidity and 5% CO2.

Plasmid preparation. As previously described (21), the digested SacI-NocI fragment from the DR5 promoter region of the human genomic DNA was subcloned into the SacI-NcoI site of the pGVB2 luciferase assay vector (Toyo Ink) to produce pDR5PF. Deletion mutants of pDR5PF, termed pDR5/BamHI, pDR5/–605, pDR5/–347, and pDR5/–198 were generated using the Mungbean-Exonuclease III system from the Kilo-sequence deletion kit (Takara). The construct pDR5/mtCHOP, which has a mutation in the CHOP-binding site at –272/–269, was generated by site-directed mutagenesis using the Quick Change XL Site-Directed Mutagenesis kit (Stratagene) from pDR5/–347. The reporter plasmid CHOP 3K, which contains 3 kbp of the CHOP promoter, was previously described (22).

Transfection and luciferase assay. A series of DR5 reporter plasmids and vacant vector plasmid (1.0 µg) were transfected into cells (1.0 x 105) using the DEAE-dextran method (GE Healthcare). After 24 h, the cells were treated with or without baicalein (40 µmol/L) for 24 h and then harvested. Levels of luciferase activity were detected in triplicate, and the experiments were repeated several times.

RNA isolation and quantification analysis by real-time reverse transcription–PCR. DR5 mRNA expression was determined by reverse transcription–PCR (RT-PCR). Total cellular RNA was extracted from cells using the Sepasol RNAI (Nakalai Tesque), and cDNA was synthesized from 2 µg of total RNA using M-MLV reverse transcriptase (Promega) and amplified by PCR using Taqman Probes purchased from Applied Biosystems.

Detection of apoptosis. DNA fragmentation was quantified by the percentage of hypodiploid DNA (sub-G1). In brief, cells were pretreated with the indicated concentration of baicalein and/or TRAIL for 24 h. After being washed with PBS, the collected cells were suspended in 0.1% Triton X-100/PBS solution. Cells were then treated with RNase A (Sigma), and the nuclei were stained with propidium iodide (Sigma). The DNA content was measured using FACSCalibur (Becton Dickinson). For each experiment, 10,000 events were analyzed. Cell Quest software (Becton Dickinson) was used to analyze the data.

For morphologic features, SW480 cells were washed in PBS, fixed in methanol, incubated with 4',6-diamidino-2-phenylindole solution for 30 min in the dark, and then analyzed using a fluorescent microscope (Olympus) at 420 nm.

Western blot analysis. Whole cell lysate containing 50 µg of protein was separated on a 10% to 12.5% SDS-polyacrylamide gel for electrophoresis and blotted onto polyvinylidene difluoride membranes (Millipore). Rabbit polyclonal DR5 and DR4 (Prosci), DcR1 and DcR2 (Imgenex), caspase-3 (Cell Signaling Technology), CHOP (GADD153), Bcl-XL, cellular inhibitor of apoptosis protein (cIAP-1) and Bax (Santa Cruz Biotechnology), survivin, cIAP-2 (R&D Systems) antibodies and mouse monoclonal X-linked inhibitor of apoptosis protein (XIAP; R&D Systems), caspase-8, caspase-9, and caspase-10 antibodies (MBL), Bcl-2 (Santa Cruz), and β-actin antibody (Sigma) were used as the primary antibodies. The signal was detected with an enhanced chemiluminescence Western blot analysis system (GE Healthcare).

Small interfering RNAs. The DR5, DR4, CHOP, and LacZ small interfering RNA (siRNA) was described previously (23, 24) and synthesized by Proligo. One day before transfection, the cells were seeded into the medium without antibiotics at a density of 30% to 40%. The siRNA was transfected with Oligofectamine (Invitrogen). Twenty-four hours after transfection, the cells were treated with baicalein and/or TRAIL and then harvested.

Measurement of intercellular ROS. Cells were pretreated with 40 µm baicalein or DMSO for 12 h and treated with 10 µmol/L 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) dye (Molecular Probes). After 30 min of incubation, the increase in fluorescence was measured by flow cytometry. The mean fluorescence intensity at 530 nm was calculated.

Statistical analysis. Tests for statistical significance were performed using a two-tailed, paired Student's t test. Samples were considered significantly different when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Baicalein overcomes TRAIL resistance in SW480 colon cancer cells. At first, we investigated the effect of baicalein on TRAIL-induced apoptosis by measuring the sub-G1 population. Baicalein or exogenous recombinant human TRAIL alone slightly induced apoptosis in SW480 colon cancer cells (Fig. 1A ). However, the combined treatment with TRAIL and baicalein markedly induced apoptosis in a dose-dependent manner.


Figure 1
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  		Figure 1. Baicalein overcomes TRAIL resistance in colon cancer SW480 cells. A, SW480 cells were treated with 20 ng/mL of TRAIL and/or various concentrations of baicalein for 24 h. The population of apoptotic cells with sub-G1 DNA contents was analyzed by flow cytometry, as described in Materials and Methods. DMSO was used as a control. Columns, mean (n = 3); bars, SD. B, SW480 cells were treated with 20 ng/mL of TRAIL, 40 µmol/L baicalein, and/or various inhibitors for 24 h. C-3, treated with zDEVD-fmk caspase-3 inhibitor; C-8, with zIETD-fmk caspase-8 inhibitor; C-9, with zLEHD-fmk caspase-9 inhibitor; C-10, with zAEVD-fmk caspase-10 inhibitor; zVAD, zVAD-fmk pan-caspase inhibitor. All caspase inhibitors were applied at 20 µmol/L. Sub-G1 populations were detected using flow cytometry. C, SW480 cells were treated with 20 ng/mL of TRAIL and/or 40 µmol/L baicalein with or without 1 µg/mL of DR5/Fc chimeric protein for 24 h. Apoptosis (sub-G1) was analyzed by flow cytometry. Columns, mean (n = 3); bars, SD.

 
DR5/Fc chimeric protein and caspase inhibitors block apoptosis enhanced by the combination of TRAIL and baicalein. As shown in Fig. 1B, we showed that the pan-caspase inhibitor zVAD-fmk and caspase-3, caspase-8, caspase-9, and caspase-10 inhibitors attenuated the increase in the sub-G1 population induced by the combination of TRAIL and baicalein, suggesting that the sub-G1 population reflects caspase-dependent apoptosis. To confirm that the apoptosis caused by the combination of baicalein and TRAIL is mediated through death receptors, we used DR5/Fc chimeric protein, which has a dominant-negative function against the TRAIL receptors. The DR5/Fc protein efficiently blocked apoptosis caused by TRAIL and baicalein in SW480 cells (Fig. 1C). Figure 2A shows the morphologic features of SW480 cells treated with TRAIL and baicalein. Combined treatment with TRAIL and baicalein forced most of the cells to float, and the phenomena were efficiently blocked by cotreatment with zVAD-fmk whereas each agent alone did not cause marked change. The cells treated with both TRAIL and baicalein exhibited the characteristic features of apoptosis, including chromatin condensation and nuclear fragmentation (Fig. 2B), whereas zVAD-fmk efficiently blocked the morphologic change caused by TRAIL and baicalein. We also showed that the combination of baicalein and TRAIL cleaved caspase-3, caspase-8, caspase-9, caspase-10, and poly(ADP-ribose) polymerase (PARP), whereas zVAD-fmk blocked this cleavage (Fig. 2C). These results indicate that baicalein strongly enhanced TRAIL-induced apoptosis in a synergistic fashion through a caspase pathway.


Figure 2
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  		Figure 2. Baicalein and TRAIL cooperate to activate apoptotic signaling. A and B, SW480 cells were treated with 20 ng/mL of TRAIL and/or 40 µmol/L baicalein with or without 20 µmol/L zVAD-fmk (zVAD). The cells were observed under a microscope (A), and nuclear morphology was visualized using 4'-diamidino-2-phenylindole staining under a fluorescence microscope (B). C, each cell lysate from the dishes treated as described above was analyzed by Western blotting with anti–caspase-3, caspase-8, caspase-9, and caspase-10 and anti-PARP antibodies. β-Actin was used as a loading control. DM, treated with DMSO.

 
Baicalein induces DR5 mRNA and protein expression in SW480 cells. To elucidate the underlying mechanism by which baicalein enhanced TRAIL-induced apoptosis, we examined DR5 up-regulation by baicalein in SW480 cells. First, we carried out Western blotting to investigate the induction of DR5 protein. Baicalein considerably increased DR5 protein in a dose-dependent manner (Fig. 3A ). Moreover, we investigated the cell surface expression of DR5, which was functionally active, using immunostaining and flow cytometry. As shown in Supplementary Fig. S1A, baicalein promoted the cell surface expression of DR5. We also examined the increase of DR5 mRNA by real-time PCR. Baicalein substantially up-regulated DR5 mRNA expression in a dose-dependent manner (Fig. 3B).


Figure 3
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  		Figure 3. Baicalein up-regulates DR5 expression and reduction of DR5 protein by siRNA prevents the apoptosis induced by TRAIL and baicalein. A, whole cell lysates were prepared from SW480 cells treated with various concentrations of baicalein for 24 h. The lysates were analyzed by Western blotting of anti-DR5 antibody. β-Actin is shown as the loading control. B, the induction of DR5 mRNA by baicalein was analyzed by real-time RT-PCR, as described in Materials and Methods. Columns, mean (n = 3); bars, SD. Each sample was standardized against the signal of a loading control, glyceraldehyde-3-phosphate dehydrogenase. C, whole cell lysates were prepared from SW480 cells treated with various concentrations of baicalein for 24 h. The lysates were analyzed by Western blotting of indicated proteins. β-Actin was used as a loading control. (–), not treated; DM, treated with DMSO. D, SW480 cells were treated with 20 nmol/L LacZ (Lac), DR5, DR4, or DR4 + DR5 siRNA. OF, treatment with the transfection reagent Oligofectamine only. Left, 24 h after transfection, cells were treated with 40 µmol/L baicalein or DMSO. Cell lysates were analyzed by Western blotting. Right, 24 h after transfection, cells were treated with 40 µmol/L baicalein and/or 20 ng/mL of TRAIL. Apoptosis (sub-G1) was analyzed by flow cytometry. Columns, means (n = 3); bars, SD. Data was analyzed using Student's t test. *, P < 0.05.

 
Baicalein does not markedly affect the protein expression of other TRAIL receptors, IAPs, and Bcl-family proteins in SW480 cells. To investigate whether other TRAIL receptors are relevant to the sensitization of the TRAIL-induced apoptosis by baicalein, we carried out Western blotting. As shown in Fig. 3C, baicalein did not change the expression of DR4, DcR1, and DcR2. We also examined the expression of IAP family proteins, which can suppress caspase activity and protect cells from apoptosis. Protein levels of XIAP, cIAP-1, and survivin in Western blotting were not significantly changed by baicalein treatment in SW480 cells. cIAP-2 was weakly decreased by baicalein treatment. Next, we carried out Western blotting of several Bcl family proteins associated with the mitochondrial apoptotic pathway. The expression of Bcl-2, Bcl-xL, and Bax proteins was not influenced by baicalein. Moreover, the protein levels of cFLIP were not altered by baicalein (data not shown). These findings suggest that baicalein did not markedly affect the protein expression of TRAIL receptors, except for DR5, IAP family proteins, Bcl-2, Bcl-xL, and Bax in SW480 cells.

Down-regulation of DR5 prevented the apoptosis induced by the combination of baicalein and TRAIL. We showed that baicalein up-regulated DR5 expression and sensitized cancer cells to TRAIL-induced apoptosis. To confirm that the up-regulation by baicalein is responsible for sensitization to TRAIL, we used a siRNA targeting DR5 and blocked the induction of DR5 expression by baicalein. As shown in Fig. 3D, the DR5 siRNA efficiently down-regulated DR5 and prevented the sensitizing effect of baicalein on TRAIL-induced apoptosis. On the other hand, down-regulation of DR4 did not block the apoptosis induced by the combination of baicalein and TRAIL. These results indicate that baicalein sensitizes SW480 cells to TRAIL-induced apoptosis due to the up-regulation of DR5.

Baicalein also sensitizes other human malignant tumor cells to TRAIL-induced apoptosis through DR5 up-regulation. We next investigated whether baicalein enhanced TRAIL-induced apoptosis in other human malignant tumor cells using human prostate cancer PC3 cells. Combined treatment with baicalein and TRAIL induced apoptosis more strongly in PC3 cells than did baicalein or TRAIL alone (Fig. 4A ). As shown in Fig. 4B, caspase inhibitors and DR5/Fc protein efficiently blocked apoptosis caused by TRAIL and baicalein in PC3 cells. Moreover, baicalein dose-dependently increased protein levels of DR5 (Fig. 4C) and enhanced cell surface expression of DR5 in PC3 cells (Supplementary Fig. S1B), suggesting that baicalein sensitizes PC3 cells to TRAIL-induced apoptosis through a DR5-caspase pathway.


Figure 4
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  		Figure 4. Baicalein also enhances TRAIL-induced apoptosis in PC3 cells, but not in PBMCs or hepatocytes. A, human prostate cancer PC3 cells were treated with 5 ng/mL of TRAIL and/or various concentrations of baicalein for 24 h. The populations of apoptotic cells with sub-G1 DNA contents were analyzed by flow cytometry. DMSO was used as a control. Columns, mean (n = 3); bars, SD. B, PC3 cells were treated with 5 ng/mL of TRAIL, 40 µmol/L baicalein, and/or 20 µmol/L of various inhibitors (top) or 1 µg/mL of DR5/Fc chimeric protein (bottom) for 24 h. C-3, treated with zDEVD-fmk caspase-3 inhibitor; C-8, treated with zIETD-fmk caspase-8 inhibitor; C-9, treated with zLEHD-fmk caspase-9 inhibitor; C-10, treated with zAEVD-fmk caspase-10 inhibitor; zVAD, zVAD-fmk pan-caspase inhibitor. Sub-G1 populations were detected using flow cytometry. C, whole cell lysates were prepared from PC3 cells treated with various concentrations of baicalein for 24 h. The lysates were analyzed by Western blotting of anti-DR5 antibody. β-Actin is shown as the loading control. –, not treated; DM, treated with DMSO. D, PBMCs, human hepatocytes, and SW480 were treated with 20 ng/mL of TRAIL and/or 40 µmol/L baicalein for 24 h. Apoptosis (sub-G1) was analyzed by flow cytometry. Columns, mean (n = 3); bars, SD. The hepatocytes were observed under a microscope.

 
Baicalein does not enhance TRAIL-induced apoptosis in normal PBMCs or in hepatocytes. Next, we investigated whether the combination of baicalein and TRAIL is toxic to normal human cells. We used normal human PBMCs and hepatocytes as models. Interestingly, the combination of baicalein and TRAIL scarcely induced apoptosis in PBMCs and hepatocytes, although it markedly induced apoptosis in SW480 cells (Fig. 4D). In a microscopic examination, apoptotic morphologic changes were rarely detected in hepatocytes treated with TRAIL and baicalein. Moreover, we investigated the effects of TRAIL and baicalein on PBMCs or hepatocytes from two more individuals. As shown in Supplementary Fig. S2, the cotreatment with baicalein and TRAIL hardly enhanced apoptosis in PBMCs and hepatocytes.

CHOP is increased by baicalein and responsible for DR5 up-regulation in SW480 cells. Furthermore, we investigated whether baicalein could activate the DR5 promoter using a series of DR5 reporter plasmids. We found that the promoter activity of pDR5PF, a luciferase reporter plasmid containing a 2.5-kbp fragment of the DR5 promoter region, was increased by baicalein in SW480 cells (Fig. 5A ). This result suggests that baicalein regulates DR5 expression through transcription.


Figure 5
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  		Figure 5. CHOP is associated with the baicalein-mediated induction of DR5. A, baicalein induces DR5 promoter activity, and mutation of the CHOP-binding site attenuates the DR5 promoter activity enhanced by baicalein in SW480 cells. SW480 cells were transiently transfected with the reporter plasmids. Twenty-four hours after transfection, the cells were treated with 40 µmol/L baicalein or DMSO for 24 h, and cell lysates were harvested for luciferase assays, as described in Materials and Methods. Columns, means of triplicate experiments; bars, SD; *, P<0.05. B, baicalein up-regulates CHOP protein expression and CHOP promoter activity. The whole cell lysates from SW480 cells treated with various concentrations of baicalein for 24 h were analyzed by Western blotting of anti-CHOP antibody. β-Actin is shown as the loading control. –, not treated; DM, treated with DMSO. The luciferase assay was performed as described in A and Materials and Methods, using the reporter plasmid containing the CHOP promoter and the luciferase gene (CHOP3K) or control plasmid (pGVB2). Columns, means of triplicate experiments; bars, SD; *, P<0.05. C, the luciferase assay of DR5 promoter in PC3 cells was performed as described in A. Columns, means of triplicate experiments; bars, SD; *, P < 0.05. D, Western blotting of anti-CHOP antibody and the luciferase assay of CHOP promoter in PC3 cells were performed as described in B. β-Actin is shown as the loading control. –, not treated; DM, treated with DMSO. Columns, means of triplicate experiments; bars, SD; *, P < 0.05.

 
In SW480 cells, luciferase activity from pDR5/–347, as well as pDR5PF, was increased by baicalein, whereas that from pDR5/–198 was not. This observation suggests that the major baicalein-responsive elements are located within a 149-bp region between –347 and –198 relative to the first base of the translation initiation codon. This region contains a potential CHOP-binding site. To determine whether CHOP is associated with transactivation of the DR5 promoter by baicalein, we introduced a site-directed mutation into the CHOP-binding site to generate pDR5/mtCHOP from pDR5/–347. Luciferase assays with these plasmids showed that the mutation abrogated the activation of the DR5 promoter by baicalein (Fig. 5A). Moreover, baicalein facilitated CHOP binding to DR5 promoter evaluated by chromatin immunoprecipitation (ChIP) assay in SW480 cells (Supplementary Fig. S3). These results indicate that CHOP is responsible for DR5 up-regulation by baicalein in SW480 cells. Western blotting showed that CHOP protein was markedly increased by baicalein treatment in a dose-dependent manner (Fig. 5B). Moreover, baicalein also enhanced CHOP promoter activity, indicating that CHOP is also induced by baicalein at a transcriptional level through the promoter in SW480 cells.

ROS are responsible for DR5 up-regulation in PC3 cells, but not in SW480 cells. The luciferase assay in PC3 cells showed different findings from that in SW480 cells. Luciferase activity from pDR5/PF and pDR5/-347 was increased by baicalein, but the mutation at the CHOP-binding site did not completely abrogate the activation by baicalein in PC3 cells, although baicalein moderately increased the CHOP protein level and CHOP promoter activity in PC3 cells compared with SW480 cells (Fig. 5C and D). Additionally, ChIP assay revealed that baicalein hardly promotes CHOP binding to DR5 promoter in PC3 cells (Supplementary Fig. S3). These findings indicate that DR5 up-regulation by baicalein in PC3 cells is almost independent of CHOP, and other mechanisms may participate in the up-regulation.

Baicalein is known to have ROS-generating ability (25–27). Thus, we investigated whether ROS are related to DR5 up-regulation by baicalein. As shown in Fig. 6A , 12 h after treatment with 40 µmol/L baicalein, the intracellular ROS level rose moderately in PC3 cells, but not in SW480 cells. Then, a hydrogen peroxide scavenger, catalase, efficiently blocked the apoptosis caused by TRAIL and baicalein in PC3 cells, but not in SW480 cells (Fig. 6B). Meanwhile, a metabolite of 12-LOX activity, 12-HETE, could not attenuate the apoptosis in either cell line. Western blotting revealed that catalase attenuated the expression of DR5 protein induced by baicalein in PC3 cells, but not in SW480 cells. As shown in Fig. 6C, siRNA targeting CHOP attenuated DR5 induction by baicalein and efficiently blocked the sensitizing effect on TRAIL-induced apoptosis in SW480 cells, although siRNA did not completely block them. However, a similar effect was not observed in PC3 cells (data not shown). These findings imply that DR5 induction by baicalein may be independent of the intracellular ROS level in SW480 cells, whereas DR5 up-regulation by baicalein in PC3 cells is due to an increase of the intracellular ROS level (Fig. 6D). We additionally performed the similar experiments using four other cell lines. Baicalein up-regulated DR5 expression mainly through CHOP and enhanced TRAIL-induced apoptosis not only in SW480 cells but also colon cancer HCT116-(p53–/–) and hepatoma HLE cells (Supplementary Fig. S4). Moreover, baicalein up-regulated DR5 expression and enhanced TRAIL efficacy mainly via ROS induction not only in PC3 cells but also T-cell leukemia Jurkat cells and prostate cancer DU145 cells (Supplementary Fig. S5).


Figure 6
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  		Figure 6. ROS are responsible for DR5 up-regulation in PC3 cells, but not SW480 cells. A, SW480 or PC3 cells were treated with DMSO or 40 µmol/L baicalein with or without 500 units/mL of catalase. Twelve hours after the treatment, ROS levels were analyzed by a flow cytometer using CM-H2DCFDA, as described in Materials and Methods. B, top, SW480 or PC3 cells were treated with 40 µmol/L baicalein and/or TRAIL with or without catalase or 12-HETE for 24 h. The concentration of TRAIL in SW480 or PC3 cells was 20 and 5 ng/mL, respectively. The population of apoptotic cells with sub-G1 DNA contents was analyzed by flow cytometry. Columns, mean (n = 3); bars, SD. Bottom, the whole cell lysate from SW480 or PC3 cells, treated with 40 µmol/L baicalein with or without 500 units/mL of catalase for 24 h, was analyzed by Western blotting for anti-DR5 antibody. Asterisk, background band. β-Actin is shown as the loading control. DM, treated with DMSO; Bai, treated with 40 µmol/L baicalein; Bai + CAT, treated with 40 µmol/L baicalein and 500 units/mL catalase. C, SW480 cells were treated with 100 nmol/L LacZ or CHOP siRNA. OF, treatment with the transfection reagent Oligofectamine only. Top, 24 h after the transfection, cells were treated with 40 µmol/L baicalein or DMSO. Cell lysates were analyzed by Western blotting. Bottom, 24 h after the transfection, cells were treated with 40 µmol/L baicalein and/or 20 ng/mL of TRAIL. Apoptosis (sub-G1) was analyzed by flow cytometry. Columns, means (n = 3); bars, SD. Data were analyzed using Student's t test. *, P < 0.05. D, novel mechanisms of enhanced apoptosis by a combination of baicalein and TRAIL in SW480 or PC3 cells. Baicalein overcomes TRAIL resistance by up-regulating DR5 expression through two different cellular stress pathways. In SW480-type cells, baicalein up-regulates DR5 expression via CHOP induction and then enhances TRAIL-induced apoptosis. In PC3-type cells, baicalein up-regulates DR5 expression through the generation of ROS and enhances TRAIL-induced apoptosis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
TRAIL is one of the most attractive candidates for cancer treatment (15–18) and phase I/phase II studies are ongoing with recombinant human TRAIL and agonistic TRAIL receptor antibodies (19). Considerable numbers of cancer cells, however, are resistant to apoptosis induced by TRAIL (20). Thus, researchers are currently seeking TRAIL sensitizers to overcome resistance to TRAIL in cancer cells. In this report, we showed for the first time that a flavonoid, baicalein, can up-regulate DR5 expression and overcome TRAIL resistance in cancer cells. Baicalein has also been reported to suppress the proliferation of various cancer cells by causing the down-regulation of Bcl-2 expression (3, 28). However, in this study, Bcl-2 protein was not changed by baicalein. The siRNA targeting DR5, but not DR4, clearly reduced the enhancement of TRAIL-induced apoptosis by baicalein in SW480 cells, suggesting that the sensitization of TRAIL-induced apoptosis by baicalein is mainly due to the up-regulation of DR5 expression, which is more important than DR4 for the TRAIL-induced apoptosis in our case. However, slight decrease of cIAP-2 may partially contribute to the enhancement by baicalein, because DR5 blockage cannot completely inhibit apoptosis.

The most attractive feature of TRAIL is its tumor-selective killing, with little or no toxicity in normal cells. Previous reports, however, have shown that histidine-tagged TRAIL is toxic to human hepatocytes (10, 11), and also other classical chemotherapeutic agents are known to induce liver injury and leukocytopenia. We therefore used human normal hepatocytes and PBMCs to examine the toxic effect of an untagged form of TRAIL in this study. If baicalein sensitizes normal cells to TRAIL-induced apoptosis, the combination would not be valuable. To date, many agents for use with TRAIL have been reported; however, few reports have analyzed the toxicity of such combinations in normal cells. Indeed, histone deacetylase inhibitor, proteasome inhibitor, and cisplatin increased cytotoxicity in hepatocytes, as well as cancer cells in combination with TRAIL (11, 29, 30). We showed that baicalein did not enhance TRAIL-induced apoptosis in human PBMCs and hepatocytes. This property of baicalein is very useful for TRAIL treatment against cancer.

All of the cancer cell lines we used in the present study harbor mutations in the tumor suppressor p53 gene. Mutations of the p53 gene exist in more than a half of all malignant tumors and confer resistance to conventional antitumor agents (31). In addition, DR5 is a target of p53 (32). Our results indicate that baicalein induces DR5 expression in a p53-independent manner, and the treatment of TRAIL with baicalein is effective even in malignant tumors with an inactivated p53.

Baicalein is a bioactive flavonoid derived from the root of S. baicalensis GEORGI, a traditional herbal medicine in China and Japan. Previous reports have shown that baicalein has a growth inhibitory effect on cancer cells in vitro and in vivo (1–4). This anticancer effect of baicalein is considered to be mainly due to inhibition of 12-LOX activity and 12-HETE production, which play important roles in tumor progression and metastasis (33–35). Recent studies indicated that 12-HETE reversed baicalein-induced apoptosis and growth inhibition (2, 3). In this study, however, 12-HETE could not block apoptosis by TRAIL and baicalein, suggesting that DR5 up-regulation and the sensitization of TRAIL-induced apoptosis by baicalein are not related to the inhibition of 12-LOX in cancer cells. As mentioned above, baicalein itself has been considered to be an attractive anticancer agent. Our results suggest that, when combined with TRAIL, baicalein becomes a more powerful tool in cancer treatment.

In this report, we also showed that baicalein up-regulates CHOP expression followed by the induction of DR5 expression in SW480 cells. CHOP is involved in growth arrest and apoptosis after DNA damage and a variety of stresses, such as nutrient deprivation, endoplasmic reticulum stress, and anticancer agents (36–38). On the other hand, in PC3 cells, up-regulation of DR5 expression by baicalein primarily depends on an increase of the intracellular ROS level. Catalase could effectively attenuate the increase, indicating that the intracellular ROS produced by baicalein include H2O2. However, catalase could not completely block the up-regulation of DR5 and enhancement of TRAIL-induced apoptosis by baicalein, suggesting that other ROS or molecules may be related to the phenomena in PC3 cells. Although several previous reports indicated that ROS induce CHOP expression (39, 40), the CHOP level in SW480 was increased by 40 µmol/L baicalein without an increase in the intracellular ROS level, indicating that the up-regulation of CHOP expression in SW480 cells occurs independent of the ROS induced by baicalein.

From our knowledge, this is the first report that an agent sensitizes cancer cells to TRAIL through different cell-specific pathways. As mentioned in Fig. 6D, baicalein induces DR5 expression and overcomes TRAIL resistance dependent on CHOP induction in cancer cells, such as SW480. Even if the level of CHOP expression is low, baicalein generates ROS and consequently facilitates DR5 up-regulation and TRAIL-induced apoptosis in cancer cells, such as PC3. Although TRAIL-resistant cancer cells escape TRAIL surveillance, baicalein can trigger two cellular stress pathways in different types of cancer cells and expose cancer cells to TRAIL by the double check systems. Oncogenic stress involving the activation of Ras and overexpression of Myc can up-regulate DR5 expression (41, 42). Taken together, DR5 acts as a stress sensor in cancer cells and consequently exposes cancer cells to TRAIL surveillance. In addition, baicalein facilitates stress in cancer cells and stimulates the DR5 sensor. Recently, DR5 deficiency in mice has been reported to promote tumorigenesis (43, 44). These results support our consideration.

In conclusion, we showed that baicalein overcomes TRAIL resistance through the up-regulation of DR5 expression via two cell-specific pathways, ROS induction and CHOP up-regulation, with low toxicity in normal cells. Baicalein may be useful to increase TRAIL efficacy in the treatment of malignant tumors.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Japanese Ministry of Education, Culture, Sports, Science and Technology.

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.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 4/ 9/08. Revised 8/13/08. Accepted 8/18/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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