Luteolin Induces Apoptosis Via Death Receptor 5 Upregulation in Human Malignant Tumor Cells

Luteolin induces apoptosis via death receptor 5 upregulation in human malignant tumor cells

Mano Horinaka1,2, Tatsushi Yoshida1, Takumi Shiraishi1,3, Susumu Nakata1, Miki Wakada1, Ryoko Nakanishi1, Hoyoku Nishino4, Hiroshi Matsui2 and Toshiyuki Sakai*,1

1Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan; 2Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan; 3Department of Urology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan; 4Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan

Luteolin, a naturally occurring flavonoid, induces apop- et al., 2003). We previously reported that luteolin arrests tosis in various cancer cells. Little is known however the cell cycle in the G1 phase in human gastric cancer concerning the underlying molecular mechanisms respon- cells (Matsukawa et al., 1993). In addition, luteolin sible for this activity. In this report, we reveal a novel induces apoptosis in human myeloid leukemia cells (Ko mechanism by which luteolin-induced apoptosis occurs, et al., 2002), where it was observed by the detection of and show for the first time that the apoptosis by luteolin is morphologic alterations and DNA fragmentation, mediated through death receptor 5 (DR5) upregulation. although it was not elucidated how luteolin caused this Luteolin markedly induced the expression of DR5, along effect. Thus, luteolin is an important cancer chemopre- with Bcl-2-interacting domain cleavage and the activation ventive agent that induces apoptosis in tumor cells, but of caspase-8, -10, -9 and -3. In addition, suppression of little is known about the molecular and biochemical DR5 expression with siRNA efficiently reduced luteolin- mechanisms responsible for this activity. induced caspase activation and apoptosis. Human recom- Death receptor 5 (DR5), also called tumor necrosis binant DR5/Fc also inhibited luteolin-induced apoptosis. factor-related apoptosis-inducing ligand (TRAIL)-R2, is On the other hand, luteolin induced neither DR5 protein a member of the tumor necrosis factor-receptor (TNF- expression nor apoptosis in normal human peripheral R) family (MacFarlane et al., 1997; Walczak et al., 1997) blood mononuclear cells. These results suggest that DR5 and a receptor for TNF-related apoptosis-inducing induced by luteolin plays a role in luteolin-induced ligand (TRAIL) (Wiley et al., 1995; Pitti et al., 1996). apoptosis, and raises the possibility that treatment with TRAIL selectively induces apoptosis in cancer cells in luteolin might be promising as a new therapy against vitro and in vivo with little or no toxicity toward normal cancer. cells (Ashkenazi et al., 1999; Walczak et al., 1999; Oncogene (2005) 24, 7180–7189. doi:10.1038/sj.onc.1208874; Lawrence et al., 2001). DR5 and DR4 mediate TRAIL- published online 11 July 2005 induced apoptosis through the formation of a death- inducing signaling complex (DISC) containing the death Keywords: luteolin; DR5; apoptosis; caspase receptor, adapter proteins such as Fas-associated death domain (FADD), and initiator caspases such as pro-caspase-8 (Hersey and Zhang, 2001) or pro-caspase- Introduction 10 (Kischkel et al., 2001; Wang et al., 2001). Conse- quently, pro-caspase-8 or pro-caspase-10 is activated by Luteolin is a naturally occurring flavone found in edible autoproteolytic processing, which then cleaves and plants such as broccoli, celery, perilla leaf and seeds, and activates downstream effector caspases, such as carrots at high concentrations (Miean and Mohamed, caspase-3. Additionally, the Bcl-2-interacting domain 2001). The cancer preventing effect of luteolin has been (Bid) is also cleaved by caspase-8 (Yamada et al., 1999). examined in terms of the incidence of fibrosarcoma Truncated-Bid causes the loss of mitochondrial mem- induced by a mutagen in mice, and was found to bring brane potential and caspase-9 cleavage, resulting in about a significant reduction in the incidence of tumors apoptosis. Thus, truncated-Bid is a mediator that and death of mice fed luteolin-mixed diets (Elangovan transduces the proapoptotic signal from the extrinsic et al., 1994). A potent preventive effect via topical pathway to the intrinsic pathway. application of luteolin was also observed for the DR5 is a downstream gene of the p53 tumor- antitumor promotion of skin papillomas in mice (Ueda suppressor gene (Wu et al., 1997; Takimoto and El- Deiry, 2000), and inactivation of DR5 significantly *Correspondence: T Sakai; E-mail: [email protected] increases tumor growth in vitro and in vivo (Wang Received 8 December 2004; revised 16 May 2005; accepted 24 May 2005; and El-Deiry, 2004). In addition, DR5 is a specific published online 11 July 2005 target gene of conventional anticancer drugs such as Inductions of apoptosis and DR5 expression by luteolin M Horinaka et al 7181 doxorubicin and etoposide (Sheikh et al., 1998; Gibson first investigated the effects of luteolin on the expression et al., 2000; Wen et al., 2000). Accordingly, DR5 is a of death receptor-related genes using an RNase protec- specific target for effective cancer therapy. tion assay. As shown in Figure 3a, luteolin significantly In this study, we elucidated for the first time one of upregulated DR5 mRNA in HeLa cells. Caspase-8, the mechanisms by which luteolin induces apoptosis in Fas ligand, Fas, DR4, TRAIL and RIP expression was cancer cell lines, and discovered that it is a flavonoid slightly increased, but DcR1 and DcR2 expression was found in edible plants that causes apoptosis through not detected. We next carried out a Northern blot DR5-mediated apoptosis signaling. Moreover, induced analysis to confirm the upregulation of DR5 mRNA by apoptosis is selectively induced in cancer cells but not in luteolin (Figure 3b). Luteolin treatment increased DR5 normal human peripheral blood mononuclear cells mRNA in a dose- and time-dependent manner. Up- (PBMC). Our results show the novel effects of luteolin regulation of DR5 mRNA was remarkable after 12 h and raise the possibility that it can be used as a with 10 mM luteolin treatment. promising strategy for the treatment of cancer. We then examined the effect of luteolin on DR5 promoter activity using a DR5 promoter-luciferase reporter plasmid as previously described (Yoshida Results et al., 2001) with a transient luciferase assay. As shown in Figure 3c, luteolin significantly increased DR5 Luteolin induces apoptosis in HeLa cells promoter activity, indicating that it increases DR5 We examined the effect of luteolin on the increase of cell expression through the DR5 promoter in HeLa cells. number in human cervical cancer HeLa cells by The DR5-promoter plasmid we used does not contain counting the cells. As shown in Figure 1a, the increase an p53-binding site, because a functional p53-binding of cell number was suppressed by luteolin in a dose- site is present in intron 1. Therefore, this indicates that dependent manner. Figure 1b shows the morphological luteolin upregulates DR5 at the transcriptional level in features of HeLa cells treated with luteolin. Luteolin- a p53-independent manner. treated cells exhibited characteristic features of apoptosis including chromatin condensation and nuclear Luteolin treatments induce DR5 and DR4 protein fragmentation. expression, Bid cleavage and the activation We showed quantitative data on luteolin-induced of caspase-8, -10, -9 and -3 apoptosis (Figure 1c and d). To investigate cell cycle behavior, logarithmically proliferating HeLa cells were As shown in Figure 4a, we also found that luteolin incubated in the absence or presence of various increased DR5 protein expression in a dose- and time- concentrations of luteolin for 12, 24 or 48 h and dependent manner by Western blot analysis, for which examined by flow cytometric analysis. Treatment with the level of protein was increased by 10 mM or more. luteolin increased the percentage of cells in the sub-G1 Time course studies showed that DR5 protein started to phase (Figure 1d). increase 12 h after treatment with 10 mM luteolin (Figure 4a). These band patterns for DR5 protein are consistent with the results of a previous report (Griffith Luteolin-induced apoptosis is interrupted by the inhibition et al., 1999), and we confirmed that they could be of several caspases detected by immunoblot analysis reprobed with another To determine the effectors active in luteolin-induced specific goat anti-DR5 antibody (Calbiochem, La Jolla, apoptotic pathways, we examined the role of caspases CA, USA) reactive toward a different epitope (data not activated during apoptosis using several caspase inhibi- shown). Moreover, to confirm the activation of apop- tors. As shown in Figure 2a, the pan-caspase inhibitor totic signals when HeLa cells were treated with the zVAD-fmk and caspase-3, -8 and -10 inhibitors inter- indicated concentrations of luteolin, we performed rupted apoptosis induced by luteolin. Specifically, Western blot analyses of caspase-8, -10, -9 and -3 and caspase-10 inhibitor blocked apoptosis to the same Bid. Luteolin induced the activation of the caspases degree as that inhibited by zVAD-fmk. In addition, (Figure 4b) and Bid cleavage (Figure 4c) at the caspase-9 inhibitor slightly but not significantly blocked concentration of 10 mM or more, which was correlated apoptosis induced by luteolin. These data indicates that with the induction of DR5 protein. Next, we performed luteolin-induced apoptosis is dependent on caspases Western blot analysis of TRAIL and DR4. TRAIL related to the extrinsic pathway. expression was not markedly induced by luteolin In addition, we carried out cell counting assay using (Figure 4d), but luteolin slightly increased DR4 protein zVAD-fmk. As shown in Figure 2b, zVAD-fmk expression (Figure 4d), which was correlated with the recovered the decrease of cell number by luteolin. results of the RNase protection assay (Figure 3a).

Luteolin induces DR5 mRNA expression and increases DR5 siRNA blocks apoptosis induced by luteolin DR5 promoter activity in HeLa cells We next examined whether or not upregulation of DR5 Since caspase-8 and -10 inhibitors effectively inhibited expression by luteolin is related to its effect on apoptosis apoptosis induced by luteolin, we hypothesized that in HeLa cells. The induction of DR5 protein was death receptor pathways are involved in apoptosis. We efﬁciently reduced by transiently transfected DR5

Oncogene Inductions of apoptosis and DR5 expression by luteolin M Horinaka et al 7182

Figure 1 Luteolin induces apoptosis in HeLa cells. (a) Effect of luteolin on the increase of the cell number. At 1 day after inoculation of HeLa cells, luteolin at 5 (’), 10 (m), 20( Â )or40() mM was added, and the cell number was compared with control culture with equivalent DMSO (~) by counting the cells. The values shown are means (n ¼ 3); bars, 7s.d. (b) DAPI staining of HeLa cells. HeLa cells were untreated or treated with 20 mM luteolin for 24 h, and nuclear morphology was then visualized using DAPI stain under a ﬂuorescence microscope. (c) Cells were analyzed for DNA content by PI staining using ﬂow cytometry as described in Materials and methods. HeLa cells were incubated with or without 20 mM luteolin for 24 h. (d) Sub-G1 populations of the cells were treated with luteolin at the indicated concentrations shown. The data represents the means of triplicate experiments. UT: untreatment, CT or control: treatment with 0.02% DMSO

Oncogene Inductions of apoptosis and DR5 expression by luteolin M Horinaka et al 7183 siRNA (Figure 5a). In addition, the suppression of DR5 expression (Figure 5a), but did not suppress the luteolin- expression by siRNA prevented the activation of induced apoptosis in HeLa cells (Figure 5b). Thus, the caspase-9 and -3 (Figure 5a), and apoptosis induced enhancement of apoptosis caused by luteolin is specifi- by luteolin (Figure 5b). These data indicates that cally due to DR5 but not DR4. upregulation of DR5 accounts at least in part for apoptosis caused by the luteolin treatment of HeLa cells. At the same time, we performed an experiment using DR5/Fc chimera protein and anti-TRAIL antibody block siRNA of DR4 gene to downregulate the expression of apoptosis induced by luteolin DR4. The siRNA of DR4 effectively reduced DR4 To confirm that the induction of apoptosis by luteolin is mediated through DR5, we used a human recombinant DR5/Fc chimera protein, which has dominant negative function against DR5. DR5/Fc chimera protein most effectively inhibited the luteolin-induced apoptosis at 1 mg/ml or more (data not shown), and we used DR5/Fc chimera protein at 1 mg/ml in this study. As shown in Figure 6a, DR5/Fc chimera protein partially blocked apoptosis caused by luteolin treatment. On the other hand, the chimera protein of another death receptor, TNF-R/Fc, could not block it. Moreover, in order to indicate more clearly that TRAIL is involved in luteolin-induced apoptosis, we conducted an experiment using neutralizing anti-TRAIL antibody (Figure 6b). Anti-TRAIL antibody weakly but significantly inhibited luteolin-induced apoptosis; however, anti-EWS antibody as a control could not block luteolin-induced apoptosis. Thus, these results demon- strated that the enhancement of apoptosis caused by luteolin is at least partially mediated via the interaction between TRAIL and DR5.

Luteolin also induces DR5 expression and apoptosis in other human malignant tumor cells We next investigated whether luteolin induces DR5 protein in other human malignant tumor cells, using DU145 prostate cancer cell line. Luteolin induced DR5 expression and apoptosis in a dose-dependent manner (Figure 7a and b), and that suppression of DR5 expression with siRNA efﬁciently reduced luteolin- induced apoptosis in DU145 as well as in HeLa cells (Figure 7c and d). These observations suggest that luteolin can induce apoptosis via DR5 upregulation not only in HeLa cells but also in other human malignant tumor cells.

Luteolin does not induce DR5 protein expression and apoptosis in normal human PBMC Figure 2 Caspase inhibitors block apoptosis enhanced by luteolin. (a) HeLa cells were treated with 10 mM luteolin for 24 h with or Since we found that luteolin upregulates DR5 expres- without various inhibitors. À, DMSO only; C-3, treated with sion and induces apoptosis in HeLa cells and DU145 zDEVD-fmk (caspase-3 inhibitor); C-8, treated with zIETD-fmk cells, we next examined the effect of luteolin treatment (caspase-8 inhibitor); C-9, treated with zLEHD-fmk (caspase-9 on normal human cells. The side effects of chemother- inhibitor); C-10, treated with zAEVD-fmk (caspase-10 inhibitor); apeutic agents on hematopoietic cells are a major VAD, treated with zVAD-fmk (pan-caspase inhibitor). All caspase inhibitors were applied at 20 mM. Sub-G1 populations (apoptotic problem during clinical treatment. Thus, we used cells) were detected by ﬂow cytometry as described in Materials and normal human PBMC as a model. The level of DR5 methods. *Po0.05 when compared to treatment with only 10 mM expression was signiﬁcantly lower in normal human luteolin. (b) HeLa cells were treated with 10 mM luteolin for 24 h PBMC compared with HeLa cells, and luteolin did not with or without 20 mM zVAD-fmk (pan-caspase inhibitor). Cell number was counted as described in Materials and methods. The induce DR5 protein expression (Figure 8a). Further- cell number with solvents only was estimated as 100%. À, solvents more, it did not induce apoptosis in normal human only. *Po0.05. The values shown are means (n ¼ 3); bars, 7s.d. PBMC (Figure 8b).

Oncogene Inductions of apoptosis and DR5 expression by luteolin M Horinaka et al 7184 Discussion mechanism responsible for this has not been elucidated. In this study, we found that apoptosis induced by Luteolin exhibits antitumor effects by inducing apop- luteolin is mediated by the upregulation of DR5. tosis (Ueda et al., 2003). However, the molecular We showed for the first time that luteolin increases DR5 expression in HeLa cells (Figure 3a and b). Moreover, it enhances DR5 promoter activity (Figure 3c). The tumor-suppressor gene p53 transacti- vates the DR5 gene through an intronic p53-binding site (Takimoto and El-Deiry, 2000). We used the 50-flanking region of the DR5 gene without a p53-binding site for the luciferase reporter assay. In addition, HeLa cells have a functionally inactivated-p53 protein, which forms a complex with the E6 oncoprotein encoded by human papilloma viruses (May et al., 1991). Moreover, we observed increased expression of DR5 by luteolin in DU145 cells harboring p53 mutation (Figure 7a). These observations suggest that luteolin induces DR5 expression in human malignant tumor cells in a p53- independent manner. Several observations suggest that luteolin enhances the apoptosis through the DR5 upregulation. (i) Caspase-8 and -10 were activated by luteolin treatment (Figure 4b). In addition, caspase-8 and -10 inhibitors efficiently blocked luteolin-induced apoptosis (Figure 2). Caspase- 8 and -10 are important direct downstream mediators of DR5. (ii) Bid was cleaved by luteolin treatment (Figure 4c). Bid interconnects the extrinsic apoptotic pathway initiated by DR5 to the intrinsic apoptotic pathway (Yamada et al., 1999), where caspase-9 is activated as a secondary effect by Bid. (iii) Luteolin upregulated DR5, and the upregulation was correlated with the induction of apoptosis (Figures 1, 3 and 4a). In addition, the activation of mediator caspases was correlated with the upregulation of DR5 (Figures 3 and 4). Previous reports showed that the overexpression of DR5 leads to the apoptotic death of cancer cells

Figure 3 Luteolin treatment induces DR5 mRNA expression and increases DR5 promoter activity in HeLa cells. (a) RNase protection assay showing the induction of DR5 mRNA and its cognate receptors by luteolin treatment in HeLa cells. Lane 1, 1/50 probes not treated with RNase; lane 2, RNase-protected probes following hybridization with yeast tRNA; lanes 3 and 4, Total RNA from HeLa cells treated with only 0.02% DMSO or 20 mM luteolin for 12 h, hybridized with probes, and then digested with RNase as described in Materials and methods. The housekeeping genes glycelaldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L32 are shown as loading controls. (b) Northern blot analysis shows the induction of DR5 mRNA expression by luteolin in HeLa cells. HeLa cells were cultured in the presence of various concentrations of luteolin for 12 h (upper) or in the presence of 10 mM luteolin for the indicated times (lower), and DR5 mRNA induction by luteolin was then examined. In all, 10mg of total RNA was probed with human DR5 cDNA. Ethidium bromide staining of 28S and 18S rRNA is shown as loading controls. The relative fold induction is shown as the RI intensity/ 28S intensity in comparison with CT. (c) The activation of DR5 promoter activity in HeLa cells by luteolin. HeLa cells were transiently transfected with a DR5 promoter-luciferase reporter plasmid (pDR5PF) or vacant vector pGVB2 for luciferase assay. At 24 h after transfection, the cells were treated with 10 mM luteolin for 12 h and then harvested and assayed as described in Materials and methods. Relative luciferase activity is shown as raw light units (RLU) in cell lysate per microgram of protein. CT or À; DMSO only. The values shown are means (n ¼ 3); bars, 7s.d. *Po0.02

Oncogene Inductions of apoptosis and DR5 expression by luteolin M Horinaka et al 7185

Figure 4 The effects of luteolin on DR5, caspases, Bid, TRAIL and DR4 proteins in HeLa cells. (a) Luteolin induced increase in the level of DR5 protein in HeLa cells. Cells were treated with various concentrations of luteolin for 12 h (left) or in the presence of 10 mM luteolin for the indicated times (right), and then subjected to Western blot analysis. (b) Luteolin induced activation of caspases in HeLa cells. Cells were treated with various concentrations of luteolin for 12 h, and then used for Western blot analysis of caspases. Decreases in the levels of pro-caspases indicate their activation. (c) Luteolin-induced activation of Bid protein in HeLa cells. Decreased levels of Bid protein reﬂect Bid cleavage and activation. (d) Luteolin slightly increased DR4 protein expression in HeLa cells. There was no effect of luteolin on the expression of TRAIL. To examine comparable loading, the same blot was probed with anti-b-actin antibody. CT or À; DMSO only

(Sheridan et al., 1997; Wu et al., 1997). (iv) Most induce ligand-independent apoptosis (Sheikh and importantly, only the suppression of DR5 expression by Huang, 2004). We therefore raise a possibility that siRNA blocked luteolin-induced caspase activation and luteolin might induce apoptosis through other mechan- -apoptosis, indicating that DR5 upregulation induced by isms such as a ligand-independent manner rather than luteolin is responsible for the induction of apoptosis in the interaction between TRAIL and DR5. HeLa cells and DU145 cells (Figures 5 and 7). On the For a chemotherapeutic agent, it is important to other hand, the suppression of DR4 expression by efﬁciently kill cancer cells but not normal cells. In siRNA could not block the luteolin-induced-apoptosis. particular, the damage of hematopoietic cells is a serious (v) Finally, DR5/Fc chimera protein partially blocked problem. We found that luteolin has no cytotoxicity apoptosis induced by treatment with luteolin (Figure 6a). against human normal PBMC (Figure 8). Luteolin On the other hand, anti-TRAIL antibody very weakly treatment did not induce apoptosis in PBMC because blocked the apoptosis (Figure 6b). Several studies have DR5 expression was not induced by it, where the level of reported that death receptors such as Fas and TNF-R DR5 expression was originally very low (Figure 8a). can induce apoptosis in a ligand-independent manner Some studies also reported that DR5 is more strongly (Beltinger et al., 1999; Fumarola et al., 2001; Qiao et al., expressed in cancer cells than in normal cells (Ichikawa 2001). Furthermore, DR5 is also believed to be able to et al., 2001; Koornstra et al., 2003).

Oncogene Inductions of apoptosis and DR5 expression by luteolin M Horinaka et al 7186

Figure 5 DR5 siRNA blocks apoptosis enhanced by luteolin, but DR4 siRNA does not affect the apoptosis. (a) Western blot analysis. The efﬁciency of DR5 siRNA and DR4 siRNA was tested in HeLa cells. HeLa cells were transfected with DR5 siRNA, DR4 siRNA or LacZ control siRNA as described in Materials and methods. At 24 h after transfection, the cells were treated with or without 20 mM luteolin for 24 h. Western blot analysis was carried out with anti-DR5, DR4, caspase-3 and -9 antibodies. To examine comparable loading, the same blot was probed with an anti-b-actin antibody. The mock shows that transfection was performed without siRNA. À; solvents only. (b) siRNA targeted towards DR5 resulted in the inhibition of apoptosis by luteolin. Sub-G1 populations (apoptotic cells) were detected by ﬂow cytometry as described in Materials and methods. The values shown are means (n ¼ 3); bars, 7s.d. *Po0.05

Previous studies in vivo have shown the biological manner. p53 mutations are present in more than half of effects of luteolin, such as preventing carcinogenesis all malignancies (Greenblatt et al., 1994), and mutations in mice (Elangovan et al., 1994; Ueda et al., 2003). in the DR5 gene are rare (Pai et al., 1998; Jeng and Hsu, When rats were orally given luteolin, the total 2002; Dechant et al., 2004). Taken together, this raises luteolin concentration was 15.5 mM in the blood plasma the possibility that luteolin might be effective against 30 min after dosing (Shimoi et al., 1998). This humoral various types of malignancies associated with p53 level of luteolin was similar to the concentration used in mutations. our study. For humans, luteolin is obtained on a daily In summary, luteolin activates the DR5 gene through basis through edible plants. Furthermore, luteolin can p53-independent regulation and induces apoptosis via be orally or topically administrated without harm DR5 upregulation. In addition, it does not induce DR5 (Merfort et al., 1994; Shimoi et al., 1998). Problems of protein expression and apoptosis in normal human side effects during luteolin treatment have not pre- PBMC. These results highlight a new mechanism viously been reported. Luteolin has antimutagenic responsible for luteolin-induced apoptosis, and raise activity (Huang et al., 1983), and conventional antic- the possibility that luteolin might be promising as a new ancer agents that activate the DR5 gene exhibit cancer therapy. genotoxicity (Sheikh et al., 1998; Gibson et al., 2000; Wen et al., 2000). Our ﬁndings imply that luteolin may be utilized as an agent that is safer than these Materials and methods conventional anticancer agents. Moreover, we found that luteolin selectively induces apoptosis in HeLa and Reagents DU145 cells compared with PBMC, and that this is Luteolin (Wako, Osaka, Japan) was dissolved in dimethyl dependent on DR5 expression in a p53-independent sulfoxide (DMSO) to a ﬁnal concentration of 0. 02% in media

Oncogene Inductions of apoptosis and DR5 expression by luteolin M Horinaka et al 7187

Figure 7 Luteolin also induces DR5 expression and apoptosis in other human malignant tumor cells, DU145. (a) Western blot analysis. DU145 cells were treated with luteolin at the indicated concentrations for 24 h. To examine comparable loading, the same blot was probed with anti-b-actin antibody. (b) Sub-G1 populations of DU145 cells were treated with luteolin at the indicated concentrations. The data represents the means of triplicate experiments. (c) Western blot analysis. The efﬁciency of DR5 siRNA was tested in DU145 cells. DU145 cells were transfected with DR5 siRNA or LacZ control siRNA as described in Materials and methods. At 24 h after transfection, the cells were treated with or without 20 mM luteolin for 24 h. Western blot analysis was carried out with anti-DR5 antibody. To examine comparable loading, the same blot was probed with an anti-b-actin antibody. The mock shows that transfection was performed without siRNA. (d) siRNA targeted towards DR5 resulted in the inhibition of apoptosis by luteolin. Sub-G1 populations (apoptotic cells) were detected by ﬂow cytometry as described in Materials and methods. The data represents the means of triplicate experiments. The values shown are means (n ¼ 3); bars, 7s.d. *Po0.05. CT or À;DMSO only

Figure 6 DR5/Fc chimera and anti-TRAIL antibody block apoptosis enhanced by luteolin. (a) HeLa cells were treated with Cell culture and cell counting assay 20 mM luteolin for 24 h with or without 1 mg/ml DR5/Fc chimera protein or TNF-R/Fc chimera protein. (b) HeLa cells were treated Human cervical cancer HeLa cells provided from Dr Y Shiio with 20 mM luteolin for 24 h with or without 100 ng/ml anti-TRAIL of the University of Tokyo were maintained in Dulbecco’s- antibody. Anti-EWS antibody was used as a control antibody. Sub- Modified Eagle Medium (DMEM) supplemented with 10% G1 populations (apoptotic cells) were detected by flow cytometry fetal bovine serum, 4 mM glutamine, 100 U/ml penicillin and as described in Materials and methods. À; solvents only. The values 100 mg/ml streptomycin, and incubated at 371C in a humidified shown are means (n ¼ 3); bars, 7s.d. *Po0.05 atmosphere of 5% CO2. Human prostate cancer DU145 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin without causing cytotoxicity. Goat polyclonal neutralizing and 100 mg/ml streptomycin, and incubated at 371Cina anti-human TRAIL antibody, human recombinant DR5 humidified atmosphere of 5% CO2. Normal human PBMC (TRAIL-R2)/Fc, TNF-R/Fc chimera protein, and caspase were isolated as previously described (Nakata et al., 2004). inhibitors such as zVAD-fmk, zDEVD-fmk, zIETD-fmk, PBMC were acquired from a healthy volunteer after obtaining zLEHD-fmk and zAEVD-fmk were purchased from R&D informed consent. Systems (Minneapolis, MN, USA). Goat polyclonal anti-EWS For the cell counting assay, cells were plated at a density of antibody was purchased from Santa Cruz Biotechnology 4 Â 104 cells in 12-well dishes. At 24 h after cell plating, various (Santa Cruz, CA, USA). Caspase inhibitors were subsequently concentrations of luteolin were added to the culture medium. diluted in DMSO before use. Cells were counted 24 and 48 h after luteolin treatment.

Oncogene Inductions of apoptosis and DR5 expression by luteolin M Horinaka et al 7188 as a probe for Northern blot analysis. Northern blot analysis and RNase protection assay were carried out as previously described (Nakata et al., 2004).

Luciferase assay HeLa cells (1 Â 105 cells) were seeded in six-well plates, and 1.0 mg of pDR5PF ( ¼ pDR5/SacI; Yoshida et al., 2001) or vacant vector plasmid was transfected using a CellPhect transfection kit (Amersham Pharmacia Biotech Inc., Piscat- away, NJ, USA). After 24 h of incubation, the cells were treated with or without luteolin, and 48 h after the transfection they were collected for luciferase assay. The luciferase assay was performed as previously described (Yoshida et al., 2001). All luciferase assays were carried out in triplicate, and each assay was performed at least three times. The data were analyzed using the Student’s t-test. Differences were consid- ered to be statistically signiﬁcant from the controls for Po0.05.

Western blot analysis Western blot analysis was carried out as previously described (Nakata et al., 2004). Rabbit polyclonal anti-DR5 antibody (Millennium Biotechnology, Ramona, CA, USA), rabbit polyclonal anti-DR4 antibody (ProSci, Poway, CA, USA), rabbit polyclonal anti-TRAIL antibody (Cayman Chemical, Ann Arbor, MI, USA), mouse monoclonal anti-Bid, anti- caspase-8, -9, -10 antibodies (MBL, Nagoya, Japan), mouse monoclonal anti-pro-caspase-3 antibody (Immunotech, Mar- seille, France) and mouse monoclonal anti-b-actin (Sigma, St Figure 8 Upregulation of DR5 expression and induction of Louis, MO, USA) were used as primary antibodies. Signals apoptosis induced by luteolin was not observed in normal human were detected using an ECL or ECL Advance Western blot PBMC. (a) Luteolin did not enhance DR5 protein expression in analysis system (Amersham Pharmacia Biotech Inc., Piscat- normal human PBMC. HeLa cells and normal human PBMC were away, NJ, USA). treated with various concentrations of luteolin for 12 h, and Western blot analysis was then carried out with an anti-DR5 antibody. To examine comparable loading, the same blot was siRNAs probed with an anti-b-actin antibody. (b) Luteolin did not induce apoptosis in normal human PBMC. Normal human PBMC were The DR5 and LacZ siRNA sequences used were previously treated with 20 mM luteolin for 24 h. Sub-G1 populations (apoptotic described (Wang and El-Deiry, 2004), and DR4 siRNA cells) were detected using a flow cytometry as described in sequences used were previously described (Ren et al., 2004) Materials and methods. À; DMSO only. The values shown are (synthesized by Proligo, Kyoto, Japan), where LacZ siRNA means (n ¼ 3); bars, 7s.d. *Po0.05 was used as a siRNA control. In brief, 1 day prior to transfection, cells were seeded without antibiotics to a density of 30–40%. DR5, DR4 and LacZ siRNA (1 nM in HeLa, 10 nM in DU145) were transfected into cells using a modified oligofectamine protocol (Invitrogen, Carlsbad, CA, USA), for Analysis of apoptosis which the volume of oligofectamine was reduced to one-third Cells grown in six-well plates were left untreated, incubated the recommended volume to limit toxic effects. After 24 h, the with DMSO or with luteolin for 24 h. They were then washed cells were treated with 20 mM luteolin for 24 h and then in PBS, fixed in methanol, incubated with 40-diamidino-2- harvested. phenylindole (DAPI) solution for 30 min in the dark, and then analyzed using a fluorescent microscope (Olympus, Tokyo, Japan) at 420 nm. Unsynchronized cells were exposed to the agent for 12, 24 or Abbreviations 48 h and then harvested from the culture dishes. After washing DR5, death receptor 5; TNF, tumor necrosis factor; TNF-R, with PBS, the cells were treated with 0.1% Triton X-100 and tumor necrosis factor-receptor; TRAIL, tumor necrosis factor- RNase A, and their nuclei were then stained with propidium related apoptosis-inducing ligand; Bid, Bcl-2-interacting do- iodide (PI) before their DNA content was measured as main; PBMC, peripheral blood mononuclear cells; GAPDH, previously described (Nakata et al., 2004). For each experi- glycelaldehyde-3-phosphate dehydrogenase. ment, 10 000 cells were collected. Acknowledgements We thank Dr Takafumi Kimura (Department of Hygiene, Northern blot analysis and RNase protection assay Kyoto Prefectural University of Medicine) for very helpful Total RNA from HeLa cells was isolated using Sepasol-RNA I experimental advice. This work was supported in part by the (Nacalai Tesque Inc., Kyoto, Japan) according to the Ministry of Education, Culture, Sports, Science and Techno- manufacturer’s instructions. Full-length DR5 cDNA was used logy of Japan.

Oncogene Inductions of apoptosis and DR5 expression by luteolin M Horinaka et al 7189 References

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