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Bakuchiol sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins
Biochemical and Biophysical Research Communications 473 (2016) 586e592
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Bakuchiol sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins Mi Hee Park a, b, *, Jong Han Kim c, Young-Ho Chung b, Seung Ho Lee c, ** a b c
The Hormel Institute, University of Minnesota, United States Division of Life Science, Korea Basic Science Institute, Daejeon, Republic of Korea Botanical Drug Laboratory, Korea Ginseng Corporation, Yuseong-gu, Daejeon, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2016 Accepted 27 March 2016 Available online 28 March 2016
We investigated whether bakuchiol, an analog of resveratrol enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. Bakuchiol enhanced expression of cell death receptor (DR) in TRAIL-sensitive and -resistant colon cancer cells in a dosedependent manner. A combination of bakuchiol with TRAIL significantly inhibited cell growth of TRAIL sensitive HCT116 and TRAIL resistant HT-29 cells. The expression of TRAIL receptors; DR4 and DR5 was significantly increased by treatment of bakuchiol, however, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed. Moreover, the expression of apoptosis related proteins such as cleaved caspase-3, -8, -9 and PARP was increased by combination treatment of bakuchiol and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory effects of bakuchiol in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the bakuchiol induced cell growth inhibitory effects. The collective results suggest that bakuchiol facilitates TRAILinduced apoptosis in colon cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals. © 2016 Elsevier Inc. All rights reserved.
Keywords: Bakuchiol TRAIL Death receptor JNK ROS
1. Introduction Tumor necrosis factor (TNF)-a-related apoptosis-inducing ligand (TRAIL), a member of the TNF ligand superfamily, is considered a potential agent for cancer therapeutics due to its ability to induce apoptosis selectively in a variety of cancer cells without toxicity in normal human cells [1,2]. Although many cancer cells express functional TRAIL receptors; death receptor 4 (DR4) and DR5, resistance to TRAIL is common because decreased level or mutation of DR4 and DR5 or the loss of distal signaling cascades [3,4]. For these reasons TRAIL alone may not be sufficient to treat many malignant tumors. Sensitization of cancer cells to TRAIL can be restored by * Corresponding author. The Hormel Institute, University of Minnesota, United States; Division of Life Science, Korea Basic Science Institute, Daejeon, Republic of Korea. ** Corresponding author. Botanical Drug Laboratory, Korea Ginseng Corporation, Yuseong-gu, Daejeon, Republic of Korea. E-mail addresses: [email protected], [email protected] (M.H. Park), [email protected] (S.H. Lee). http://dx.doi.org/10.1016/j.bbrc.2016.03.127 0006-291X/© 2016 Elsevier Inc. All rights reserved.
treatment with subtoxic concentration of chemotherapeutic drugs through upregulation of TRAIL receptors, DR4 and DR5 [5,6]. Several recent studies have suggested that the DR4 and DR5 TRAIL receptors are up-regulated by different mechanisms such as MAPKs including extracellular signal-regulated kinases (ERK)1/2, p38 MAPK and cJun NH2-terminal kinase (JNK), reactive oxygen species (ROS) and C/ EBP homologous transcription factor (CHOP) [7e9]. Bakuchiol, a prenylated phenolic monoterpene isolated from the seeds of Psoralea corylifolia L. (Leguminosae) and commonly used in traditional Chinese and Indian folkloric medicine as a kidneytonifying agent for alleviating asthma, diarrhea and osteoporosis osteoporosis [10e12]. Bakuchiol also induces caspase-3-dependent apoptosis through the activation of JNK (c-jun N-terminal kinase), followed by Bax translocation into the mitochondria in rat liver myofibroblasts [13]. Recent study has demonstrated that bakuchiol has anti-tumor effect in lung cancer cells through S phase arrest, caspase 9/3 activation, p53 and Bax up-regulation and Bcl-2 downregulation by reactive oxygen species-related apoptosis [12]. Especially, bakuchiol is an analog of resveratrol (3,5,4-trihydroxy-
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trans-stilbene) that is a phytoalexin found in grapes, berries, and peanuts, and is one of the most promising agents for cancer prevention [14e16]. Moreover, resveratrol enhances antitumor activity of TRAIL in prostate cancer xenografts through activation of FOXO transcription factor [17], resveratrol sensitized melanomas to TRAIL through modulation of antiapoptotic gene expression [18], resveratrol-mediated sensitization to TRAIL-induced apoptosis depends on death receptor and mitochondrial signaling [19] and resveratrol is a potent sensitizer of tumor cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through p53-independent induction of p21 and p21-mediated cell cycle arrest associated with survivin [20]. We hypothesized that the bakuchiol that is an analog of resveratrol can enhance the sensitivity of cancer cells to TRAIL. In this study, we demonstrated that the bakuchiol enhances antitumor activity of TRAIL in colon cancer cells.
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Peprotech (Rocky Hill, NJ). 2.1.1. Extraction of bakuchiol from P. corylifolia Dried seeds of P. corylifolia L were extracted with boiling distilled water for 3 h and the extract concentrated, and partitioned with n-butanol. The butanol fraction was dissolved with methanol, and partitioned with CH2CL2 to give a CH2CL2 fraction. The bioactive CH2CL2 fraction was separated by sephadex LH-20 column chromatography to obtain 3 fractions (AeC). The fraction B was chromatographed on a Sephadex LH-20 column with CH2Cl2: MeOH (40:1) to give 3 fractions (B1eB3). Fraction B1 was separated by silica gel column chromatography (n-hexane: EtOAc (4:1)) to yield (S)-bakuchiol. All extracts were quantitatively analyzed by HPLC system and a UV detector. The detector wavelength was set at 245 nm. 2.2. Cell culture and regents
2. Materials and methods 2.1. Materials Soluble Recombinant human Apo2L/TRAIL was purchased from
HCT116 (human colorectal carcinoma) and HT-29 (human colorectal adenocarcinoma) cancer cells were obtained from the American Type Culture Collection (Manassas, VA). HCT116 and HT29 cancer cells were grown at 37 C in 5% CO2 humidified air in
Fig. 1. Bakuchiol enhanced TRAIL-induced cytotoxicity and apoptosis in colon cancer cells. A, Cell viability was determined by direct counting viable cells after pretreated with bakuchiol (5 mg/ml) for 24 h and exposed to TRAIL in HCT116 and HT-29 cells as described in materials and methods. B, Apoptosis was analyzed by Annexin V/FITC assay. Data means ± SD expressed as percentage of control value, which is set to 100%. At least three independent experiments were carried out in triplicate. *, p < 0.05, significantly different from control cells.
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RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. RPMI 1640, penicillin, streptomycin and FBS were purchased from Gibco Life Technologies (Grand Island, NY, USA).
2.3. Cell viability To determine viable cell numbers, HCT116 and HT-29 cancer cells were seeded onto 24-well plates (5 104 cells/well). The cells were trypsinized, pelleted by centrifugation for 5 min at 1500 rpm, resuspended in 10 ml of phosphate-buffered saline (PBS), and 0.1 ml of 0.2% trypan blue was added to the cell suspension in each solution (0.9 ml each). Cells that showed signs of trypan blue uptake were considered to be dead, whereas those that excluded trypan blue were considered to be viable. Each assay was carried out in triplicate.
2.4. Evaluation of apoptotic cell death The apoptotic ratios of cells were determined with the Annexin V-FITC apoptosis detection kit (Roche). Briefly, after 24 h bakuchiol treatment, the cells were collected and washed twice with cold PBS buffer, resuspended in 100 mL of binding buffer, incubated with 10 mL of Annexin V conjugated to FITC and 10 mL PI for 15 min at room temperature, and analyzed by flow cytometry. Cells treated with DMSO were used as the negative control. 2.5. Measurement of ROS Generation of ROS was assessed by 2, 7- dichlorofluorescein diacetate (DCF-DA, Sigma Aldrich, St Louis, MO, USA), an oxidationsensitive fluorescent probe. Briefly, cells were plated in black 96 well plates (1 104 cells/well), and subconfluent cells were subsequently treated with 10 mM DCFH-DA at 37 C for 30 min, and
Fig. 2. Effect of bakuchiol on the expression of DR4 and DR5. A, HCT116 and HT-29 cells were treated with bakuchiol (5 mg/ml) for 24 h, then equal amounts of total proteins (50 mg/ lane) were subjected to 12% SDS-PAGE. Expression of DR4, DR5 and b-actin was detected by Western blotting using specific antibodies. B, HCT116 and HT-29 cells were transfected with DR4 siRNA, DR5 siRNA or control siRNA (100 nM). After 24 h, cells were pretreated with bakuchiol for 24 h, and then incubated with TRAIL for 24 h. Cell viability was measured by direct counting. Data means ± SD expressed as percentage of control value, which is set to 100%. At least three independent experiments were carried out in triplicate. *, p < 0.05, significantly different from control cells. #, p < 0.05, significantly different from bakuchiol and TRAIL combination treated cells. b-actin protein was used as internal control. Each band is representative for three experiments.
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the remained DCF-DA was washed with HBSS (HEPES buffered salt solution). And then the cells were treated with bakuchiol and TRAIL for 1 h. The fluorescence intensity of DCF was measured in a microplate-reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
software (GraphPad Software, La Jolla, CA). The differences in all data were assessed by one-way analysis of variance (ANOVA). A value of P < 0.05 was considered to be statistically significant.
2.6. Western blotting
3.1. Bakuchiol potentiates TRAIL-induced apoptosis in both TRAILsensitive and -resistant colon cancer cells
The membranes were immunoblotted with the following primary antibodies: mouse monoclonal antibodies directed against cleaved caspase-8 (1:1000 dilutions; Cell Signaling Technology, Beverly, MA) and rabbit polyclonal antibodies directed against ERK, phospho-ERK and JNK (1:500 dilutions; Santa Cruz Biotechnology), and against PARP, XIAP, survivin, bcl2, cleaved caspase-3, -9 and phospho-JNK (1:1000 dilutions; Cell Signaling Technology, Beverly, MA). The blot was then incubated with the corresponding antirabbit/goat immunoglobulin G-horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology Inc.). Immunoreactive proteins were detected with the Enhanced Chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech, Inc., Buckinghamshire, United Kingdom). 2.7. Transfection Colon cancer cells (5 104 cells/well) were plated in 24-well plates and transiently transfected with 0.4 mg of the empty vector or the constitutively activated 100 nM of negative siRNA, DR4 or DR5 siRNA per well, using a mixture of plasmid and the lipofectamine reagent in OPTI-MEM, according to the manufacturer's specification (Invitrogen, Carlsbad, CA, USA). 2.8. Statistical analysis The data were analyzed using the GraphPad Prism 4 ver. 4.03
3. Results
To investigate whether bakuchiol enhances the sensitivity of cancer cells to TRAIL, we first examined the effect of bakuchiol on TRAIL-induced apoptosis. We showed that HCT116 cell was moderately sensitive to TRAIL alone, however, HT-29 cancer cell was resistant to TRAIL even after treatment of TRAIL up to 50 or 100 ng/ml (data not shown). Both of these cell lines were sensitive toward bakuchiol (Fig. 1A). To investigate whether the combination treatment of bakuchiol and TRAIL has an effect on resistant cancer cell growth, we treated these cancer cells with half doses of IC50 of bakuchiol (The IC50 value of bakuchiol on HCT116 and HT-29 was 14.2 mg/ml and 10 mg/ml, respectively) with TRAIL. Pretreatment (24 h) with bakuchiol significantly enhanced TRAIL-induced cytotoxicity in TRAIL-sensitive HCT116 cells and TRAIL-resistant HT29 cells (Fig. 1A). We also showed that the bakuchiol significantly enhanced TRAIL-induced apoptosis in both cell lines. Annexin V/PI data indicated that the apoptosis index was much increased in the combination treated group of bakuchiol and TRAIL (65.62% in HCT116 cell; 77.15% in HT29 cell) compared with only bakuchiol (19.96% in HCT116 cell; 11.83% in HT29 cell) or TRAIL (26.82% in HCT116 cell; 9.3% in HT29 cell) treated group (Fig. 1B). 3.2. Effect of bakuchiol on the expression of death receptors in colon cancer cells Bakuchiol induced both DR4 and DR5 in a dose dependent
Fig. 3. Effect of bakuchiol on the expression of survival proteins and apoptosis related proteins. HCT116 and HT-29 cells were treated with bakuchiol (5 mg/ml) for 24 h, then equal amounts of total proteins (50 mg/lane) were subjected to 12% SDS-PAGE. A, HCT116 and HT-29 cells were treated with bakuchiol (5 mg/ml) for 24 h, Equal amounts of total proteins (50 mg/lane) were subjected to 12% SDS-PAGE. Expression of survivin, XIAP, FLIP, Bcl2 and b-actin was detected by Western blotting using specific antibodies. b-actin protein was used as internal control. Each band is representative for three experiments. B, HCT116 and HT-29 cells were pretreated with bakuchiol for 24 h, after that the cells were treated with TRAIL for 24 h, and whole cell extracts were analyzed by western blotting using antibodies against caspase-3, caspase-8, caspase-9, PARP and b-actin. b-actin protein was used as internal control. Each band is representative for three experiments.
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manner in TRAIL-sensitive HCT116 and TRAIL-resistant HT-29 cells (Fig. 2A). We also found that transfection of cells with DR4 or DR5 siRNA but not with the control siRNA reduced the combination of bakuchiol and TRAIL-induced cell growth inhibition (Fig. 2B). These results indicate that both DR4 and DR5 play an important role in sensitizing the effect of bakuchiol on TRAIL-induced cell death. 3.3. Effect of bakuchiol on the expression of survival proteins and apoptosis related proteins in colon cancer cells Several studies have shown that various anti-apoptotic proteins such as survivin, cFLIP, Bcl2 and XIAP induce resistance to TRAIL [21e23]. As shown in Fig. 3A, we showed that bakuchiol decreases expression of survivin, cFLIP, Bcl2 and XIAP, suggesting that the bakuchiol can overcome the TRAIL resistance through over-
expression of DR4 and DR5, as well as down regulation of survival proteins. Next, we compared the effect of bakuchiol and/or TRAIL on the activation of caspase-8, caspase-3, caspase-9 cleavage and PARP in the TRAIL sensitive colon cancer (HCT116) cell and in the TRAIL-resistant colon cancer (HT-29) cell. Although bakuchiol and TRAIL alone had little effect on the activation of caspases cleavage, the combination treatment significantly increased expression of the apoptotic cell death regulatory proteins (Fig. 3B). 3.4. Bakuchiol activates JNK and ROS and induction of TRAIL receptors is JNK and ROS dependent pathway TRAIL death receptors are activated through MAPK pathway. So, we investigated whether bakuchiol induces the MAPK pathway in the colon cancer cell lines, thereafter induces the death receptors.
Fig. 4. Effect of JNK and ROS on upregulation of DR4 and DR5 and apoptosis induced by bakuchiol and TRAIL. A, HCT116 and HT-29 cells were treated with bakuchiol (5 mg/ml) for 24 h, then equal amounts of total proteins (50 mg/lane) were subjected to 12% SDS-PAGE. Expression of MAPK pathway proteins was detected by Western blotting using specific antibodies. B, HCT116 and HT-29 cells were pretreated with SP600125 (5e10 mM) for 1 h, and then cells were treated with bakuchiol for 24 h and equal amounts of total proteins (50 mg/lane) were subjected to 12% SDS-PAGE. Expression of DR4 and DR5 was detected by Western blotting using specific antibodies. C, HCT116 and HT-29 cells were pretreated with SP600125 (5e10 mM) for 1 h, and then cells were treated with bakuchiol for 24 h and then treated with TRAIL for another 24 h. Cell viability was measured by direct counting. Data means ± SD expressed as percentage of control value, which is set to 100%. At least three independent experiments were carried out in triplicate. D, After treatment of bakuchiol and TRAIL for 30 min, the HCT116 and HT-29 cells were incubated with 10 mM DCF-DA at 37 C for 4 h, and then washed twice with PBS. The fluorescence intensity of DCF was measured in a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. E, HCT116 and HT-29 cells were pretreated with NAC (10 mM) for 1 h, and then cells were treated with bakuchiol for 24 h and then treated with TRAIL. Cell viability was measured by direct counting. Data means ± SD expressed as percentage of control value, which is set to 100%. At least three independent experiments were carried out in triplicate. *, p < 0.05, significantly different from control cells.
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We showed that the bakuchiol activated JNK phosphorylation in both colon cancer cell lines (Fig. 4A). Further, we showed that the treatment of JNK inhibitor, SP600125 reversed the expression of death receptors (Fig. 4B). From these results, we suggested that the expression of DRs by bakuchiol was JNK pathway dependent. Furthermore, we examined whether the suppression of JNK activation by its inhibitor could abrogate the cytotoxicity induced by the combination treatment of bakuchiol and TRAIL. We showed that cell growth inhibition by the combination treatment of bakuchiol and TRAIL was completely reversed by JNK inhibitor (10 mM) (Fig. 4C). Several studies demonstrated that the DRs pathway is dependent on the ROS pathway [24,25]. And previous study has been showed that the resveratrol induced ROS release in several cancer cells [26,27], but the effect of bakuchiol has not been studied yet. So we investigated whether the up-regulation of DRs induced by bakuchiol could be regulated by ROS. First, we showed that the ROS generation was increased by treatment of bakuchiol in a dose dependent manner in both cell lines (Fig. 4D). Moreover, we also showed that pretreatment of NAC (10 mM) reversed the effect of the bakuchiol induced cytotoxic effect of TRAIL (Fig. 4E). 4. Discussion We demonstrated here that bakuchiol can overcome TRAIL resistance in colon cancer cells. First, we found that the combination of bakuchiol and TRAIL increased apoptotic cell death and induced expression of apoptosis related proteins in both TRAIL sensitive HCT116 cells and TRAIL resistant HT-29 cells. TRAIL mediates apoptotic cell death through enhanced expression of two death receptors, DR4 and DR5 which are expressed on the surface of cancer cells, and the binding of TRAIL to DR4 and DR5 leads to the activation of caspase 8 and 10, which in turn cleaves and activates executioner caspases that mediate apoptosis [28,29]. Thus, we hypothesized that the cancer inhibitory effect of bakuchiol could be related with increased DR4 and DR5 expression, resulting enhanced apoptotic effect to TRAIL. In this study, we showed that the expression of DR4 and DR5 was increased by treatment of bakuchiol in both TRAIL sensitive and TRAIL resistant cancer cells. Among the several factors, intracellular levels of survival proteins are related with TRAIL resistance [21e23]. Thus, in this study, we showed that the expression of survival proteins such as Bcl2, cFLIP, survivin and XIAP was down regulated by bakuchiol correlated with the increased expression of DR4 and DR5. From these results, we suggest that bakuchiol can enhance TRAIL-induced apoptosis and can overcome TRAIL-resistance through down-regulation of cell survival proteins and up-regulation of death receptors. Next we investigated the molecular mechanisms of the bakuchiol induced expression of DRs. Several studies indicated that the expression of death receptor is up-regulated by p53, CHOP and MAPK pathway [30,31]. Bakuchiol already known to induce MAPK pathway in the cancer cell lines [13], so we investigated whether MAPK pathways are related with DR induction by bakuchiol. Here, we showed that bakuchiol induce the JNK phosphorylation in both cell lines, but not induced ERK and p38 phosphorylation. We also showed that the JNK inhibitor, SP600125 was abolished the bakuchiol induced DR4 and DR5 expression and suppressed the growth inhibitory effect induced by bakuchiol and TRAIL. Similar with our result, simvastatin (SVA) was shown to up-regulate expression of death receptor-5 (DR5), CCAAT/enhancer binding protein homologous protein (CHOP) and phosphorylated c-Jun N-terminal kinase (pJNK) in human breast cancer cell lines. siRNA knockdown of DR5, CHOP or JNK significantly blocked SVA-induced apoptosis, demonstrating the importance of JNK/CHOP/DR5 signaling pathway in SVA-induced apoptosis [32]. Other group also showed that gossypol enhances TRAIL-induced apoptosis through the
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down-regulation of cell survival proteins and the up-regulation of TRAIL death receptors through the ROS-ERK-CHOP-DR5 pathway [11]. Recent study also demonstrated that g-tocotrienol suppressed tumor growth in a syngeneic implantation mouse mammary cancer model by inhibiting cell proliferation and inducing apoptosis by activation of caspase-3, -8, and -9 through induction of DR5 and JNK and p38 MAPK in human breast cancer cell lines [33]. Similar with other reports, our finding demonstrated that bakuchiolinduced DR expression could be mediated with JNK pathway for enhancing the apoptotic sensitivity to TRAIL. ROS generation could be involved in DRs [34,35]. In this study, we also showed whether bakuchiol-induced death receptor upregulation is mediated with ROS in both cells. The treatment of bakuchiol induced ROS generation, but the antioxidant NAC abolished the upregulation of DR4 and DR5 induced by the treatment of bakuchiol. The potentiated effect of bakuchiol on TRAIL-induced apoptosis was also neutralized by the NAC. In agreement with these observations, previous studies using zerumbone and celastrol have shown the role of ROS and MAPK in the up-regulation of DRs and the potentiation of [36,37]. Our results indicate that like several other anti-cancer agents, bakuchiol also induces DRs through ROS generation for sensitization of cancer cells against TRAIL. Taken together, this study indicates that bakuchiol sensitizes TRAIL resistant colon cancer cells to the TRAIL, and suggests that the combination treatment of bakuchiol and TRAIL could be applicable as an anti-colorectal cancer agent. Conflict of interest The authors have declared that no conflict of interest exists. Acknowledgment This work was supported by grants to Y.H.C. from the Korea Basic Science Institute (D34402, T34417), and by the Creative Fusion Research Program through the Creative Allied Project funded by the National Research Council of Science & Technology (CAP-12-1-KIST). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.03.127. References [1] J. Lemke, S. von Karstedt, J. Zinngrebe, H. Walczak, Getting TRAIL back on track for cancer therapy, Cell Death Differ. 21 (2014) 1350e1364. [2] S.R. Wiley, K. Schooley, P.J. Smolak, W.S. Din, C.P. Huang, J.K. Nicholl, G.R. Sutherland, T.D. Smith, C. Rauch, C.A. Smith, Identification and characterization of a new member of the TNF family that induces apoptosis, Immunity 3 (1995) 673e682. [3] F.C. Kischkel, D.A. Lawrence, A. Chuntharapai, P. Schow, K.J. Kim, A. Ashkenazi, Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5, Immunity 12 (2000) 611e620. [4] P.T. Daniel, T. Wieder, I. Sturm, K. Schulze Osthoff, The kiss of death: promises and failures of death receptors and ligands in cancer therapy, Leukemia 15 (2001) 1022e1032. [5] A. Mühlethaler-Mottet, K.B. Bourloud, K. Auderset, J.M. Joseph, N. Gross, Drugmediated sensitization to TRAIL-induced apoptosis in caspase-8complemented neuroblastoma cells proceeds via activation of intrinsic and extrinsic pathways and caspase-dependent cleavage of XIAP, Bcl-xL and RIP, Oncogene 23 (2004) 5415e5425. [6] S. Lacour, O. Micheau, A. Hammann, V. Drouineaud, J. Tschopp, E. Solary, M.T. Dimanche-Boitrel, Chemotherapy enhances TNF-related apoptosisinducing ligand DISC assembly in HT29 human colon cancer cells, Oncogene 22 (2003) 1807e1816. [7] S. Prasad, J.H. Kim, S.C. Gupta, B.B. Aggarwal, Targeting death receptors for TRAIL by agents designed by Mother Nature, Trends Pharmacol. Sci. 35 (2014) 520e536.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Bakuchiol sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins Mi Hee Park a, b, *, Jong Han Kim c, Young-Ho Chung b, Seung Ho Lee c, ** a b c
The Hormel Institute, University of Minnesota, United States Division of Life Science, Korea Basic Science Institute, Daejeon, Republic of Korea Botanical Drug Laboratory, Korea Ginseng Corporation, Yuseong-gu, Daejeon, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2016 Accepted 27 March 2016 Available online 28 March 2016
We investigated whether bakuchiol, an analog of resveratrol enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. Bakuchiol enhanced expression of cell death receptor (DR) in TRAIL-sensitive and -resistant colon cancer cells in a dosedependent manner. A combination of bakuchiol with TRAIL significantly inhibited cell growth of TRAIL sensitive HCT116 and TRAIL resistant HT-29 cells. The expression of TRAIL receptors; DR4 and DR5 was significantly increased by treatment of bakuchiol, however, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed. Moreover, the expression of apoptosis related proteins such as cleaved caspase-3, -8, -9 and PARP was increased by combination treatment of bakuchiol and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory effects of bakuchiol in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the bakuchiol induced cell growth inhibitory effects. The collective results suggest that bakuchiol facilitates TRAILinduced apoptosis in colon cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals. © 2016 Elsevier Inc. All rights reserved.
Keywords: Bakuchiol TRAIL Death receptor JNK ROS
1. Introduction Tumor necrosis factor (TNF)-a-related apoptosis-inducing ligand (TRAIL), a member of the TNF ligand superfamily, is considered a potential agent for cancer therapeutics due to its ability to induce apoptosis selectively in a variety of cancer cells without toxicity in normal human cells [1,2]. Although many cancer cells express functional TRAIL receptors; death receptor 4 (DR4) and DR5, resistance to TRAIL is common because decreased level or mutation of DR4 and DR5 or the loss of distal signaling cascades [3,4]. For these reasons TRAIL alone may not be sufficient to treat many malignant tumors. Sensitization of cancer cells to TRAIL can be restored by * Corresponding author. The Hormel Institute, University of Minnesota, United States; Division of Life Science, Korea Basic Science Institute, Daejeon, Republic of Korea. ** Corresponding author. Botanical Drug Laboratory, Korea Ginseng Corporation, Yuseong-gu, Daejeon, Republic of Korea. E-mail addresses: [email protected], [email protected] (M.H. Park), [email protected] (S.H. Lee). http://dx.doi.org/10.1016/j.bbrc.2016.03.127 0006-291X/© 2016 Elsevier Inc. All rights reserved.
treatment with subtoxic concentration of chemotherapeutic drugs through upregulation of TRAIL receptors, DR4 and DR5 [5,6]. Several recent studies have suggested that the DR4 and DR5 TRAIL receptors are up-regulated by different mechanisms such as MAPKs including extracellular signal-regulated kinases (ERK)1/2, p38 MAPK and cJun NH2-terminal kinase (JNK), reactive oxygen species (ROS) and C/ EBP homologous transcription factor (CHOP) [7e9]. Bakuchiol, a prenylated phenolic monoterpene isolated from the seeds of Psoralea corylifolia L. (Leguminosae) and commonly used in traditional Chinese and Indian folkloric medicine as a kidneytonifying agent for alleviating asthma, diarrhea and osteoporosis osteoporosis [10e12]. Bakuchiol also induces caspase-3-dependent apoptosis through the activation of JNK (c-jun N-terminal kinase), followed by Bax translocation into the mitochondria in rat liver myofibroblasts [13]. Recent study has demonstrated that bakuchiol has anti-tumor effect in lung cancer cells through S phase arrest, caspase 9/3 activation, p53 and Bax up-regulation and Bcl-2 downregulation by reactive oxygen species-related apoptosis [12]. Especially, bakuchiol is an analog of resveratrol (3,5,4-trihydroxy-
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trans-stilbene) that is a phytoalexin found in grapes, berries, and peanuts, and is one of the most promising agents for cancer prevention [14e16]. Moreover, resveratrol enhances antitumor activity of TRAIL in prostate cancer xenografts through activation of FOXO transcription factor [17], resveratrol sensitized melanomas to TRAIL through modulation of antiapoptotic gene expression [18], resveratrol-mediated sensitization to TRAIL-induced apoptosis depends on death receptor and mitochondrial signaling [19] and resveratrol is a potent sensitizer of tumor cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through p53-independent induction of p21 and p21-mediated cell cycle arrest associated with survivin [20]. We hypothesized that the bakuchiol that is an analog of resveratrol can enhance the sensitivity of cancer cells to TRAIL. In this study, we demonstrated that the bakuchiol enhances antitumor activity of TRAIL in colon cancer cells.
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Peprotech (Rocky Hill, NJ). 2.1.1. Extraction of bakuchiol from P. corylifolia Dried seeds of P. corylifolia L were extracted with boiling distilled water for 3 h and the extract concentrated, and partitioned with n-butanol. The butanol fraction was dissolved with methanol, and partitioned with CH2CL2 to give a CH2CL2 fraction. The bioactive CH2CL2 fraction was separated by sephadex LH-20 column chromatography to obtain 3 fractions (AeC). The fraction B was chromatographed on a Sephadex LH-20 column with CH2Cl2: MeOH (40:1) to give 3 fractions (B1eB3). Fraction B1 was separated by silica gel column chromatography (n-hexane: EtOAc (4:1)) to yield (S)-bakuchiol. All extracts were quantitatively analyzed by HPLC system and a UV detector. The detector wavelength was set at 245 nm. 2.2. Cell culture and regents
2. Materials and methods 2.1. Materials Soluble Recombinant human Apo2L/TRAIL was purchased from
HCT116 (human colorectal carcinoma) and HT-29 (human colorectal adenocarcinoma) cancer cells were obtained from the American Type Culture Collection (Manassas, VA). HCT116 and HT29 cancer cells were grown at 37 C in 5% CO2 humidified air in
Fig. 1. Bakuchiol enhanced TRAIL-induced cytotoxicity and apoptosis in colon cancer cells. A, Cell viability was determined by direct counting viable cells after pretreated with bakuchiol (5 mg/ml) for 24 h and exposed to TRAIL in HCT116 and HT-29 cells as described in materials and methods. B, Apoptosis was analyzed by Annexin V/FITC assay. Data means ± SD expressed as percentage of control value, which is set to 100%. At least three independent experiments were carried out in triplicate. *, p < 0.05, significantly different from control cells.
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RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. RPMI 1640, penicillin, streptomycin and FBS were purchased from Gibco Life Technologies (Grand Island, NY, USA).
2.3. Cell viability To determine viable cell numbers, HCT116 and HT-29 cancer cells were seeded onto 24-well plates (5 104 cells/well). The cells were trypsinized, pelleted by centrifugation for 5 min at 1500 rpm, resuspended in 10 ml of phosphate-buffered saline (PBS), and 0.1 ml of 0.2% trypan blue was added to the cell suspension in each solution (0.9 ml each). Cells that showed signs of trypan blue uptake were considered to be dead, whereas those that excluded trypan blue were considered to be viable. Each assay was carried out in triplicate.
2.4. Evaluation of apoptotic cell death The apoptotic ratios of cells were determined with the Annexin V-FITC apoptosis detection kit (Roche). Briefly, after 24 h bakuchiol treatment, the cells were collected and washed twice with cold PBS buffer, resuspended in 100 mL of binding buffer, incubated with 10 mL of Annexin V conjugated to FITC and 10 mL PI for 15 min at room temperature, and analyzed by flow cytometry. Cells treated with DMSO were used as the negative control. 2.5. Measurement of ROS Generation of ROS was assessed by 2, 7- dichlorofluorescein diacetate (DCF-DA, Sigma Aldrich, St Louis, MO, USA), an oxidationsensitive fluorescent probe. Briefly, cells were plated in black 96 well plates (1 104 cells/well), and subconfluent cells were subsequently treated with 10 mM DCFH-DA at 37 C for 30 min, and
Fig. 2. Effect of bakuchiol on the expression of DR4 and DR5. A, HCT116 and HT-29 cells were treated with bakuchiol (5 mg/ml) for 24 h, then equal amounts of total proteins (50 mg/ lane) were subjected to 12% SDS-PAGE. Expression of DR4, DR5 and b-actin was detected by Western blotting using specific antibodies. B, HCT116 and HT-29 cells were transfected with DR4 siRNA, DR5 siRNA or control siRNA (100 nM). After 24 h, cells were pretreated with bakuchiol for 24 h, and then incubated with TRAIL for 24 h. Cell viability was measured by direct counting. Data means ± SD expressed as percentage of control value, which is set to 100%. At least three independent experiments were carried out in triplicate. *, p < 0.05, significantly different from control cells. #, p < 0.05, significantly different from bakuchiol and TRAIL combination treated cells. b-actin protein was used as internal control. Each band is representative for three experiments.
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the remained DCF-DA was washed with HBSS (HEPES buffered salt solution). And then the cells were treated with bakuchiol and TRAIL for 1 h. The fluorescence intensity of DCF was measured in a microplate-reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
software (GraphPad Software, La Jolla, CA). The differences in all data were assessed by one-way analysis of variance (ANOVA). A value of P < 0.05 was considered to be statistically significant.
2.6. Western blotting
3.1. Bakuchiol potentiates TRAIL-induced apoptosis in both TRAILsensitive and -resistant colon cancer cells
The membranes were immunoblotted with the following primary antibodies: mouse monoclonal antibodies directed against cleaved caspase-8 (1:1000 dilutions; Cell Signaling Technology, Beverly, MA) and rabbit polyclonal antibodies directed against ERK, phospho-ERK and JNK (1:500 dilutions; Santa Cruz Biotechnology), and against PARP, XIAP, survivin, bcl2, cleaved caspase-3, -9 and phospho-JNK (1:1000 dilutions; Cell Signaling Technology, Beverly, MA). The blot was then incubated with the corresponding antirabbit/goat immunoglobulin G-horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology Inc.). Immunoreactive proteins were detected with the Enhanced Chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech, Inc., Buckinghamshire, United Kingdom). 2.7. Transfection Colon cancer cells (5 104 cells/well) were plated in 24-well plates and transiently transfected with 0.4 mg of the empty vector or the constitutively activated 100 nM of negative siRNA, DR4 or DR5 siRNA per well, using a mixture of plasmid and the lipofectamine reagent in OPTI-MEM, according to the manufacturer's specification (Invitrogen, Carlsbad, CA, USA). 2.8. Statistical analysis The data were analyzed using the GraphPad Prism 4 ver. 4.03
3. Results
To investigate whether bakuchiol enhances the sensitivity of cancer cells to TRAIL, we first examined the effect of bakuchiol on TRAIL-induced apoptosis. We showed that HCT116 cell was moderately sensitive to TRAIL alone, however, HT-29 cancer cell was resistant to TRAIL even after treatment of TRAIL up to 50 or 100 ng/ml (data not shown). Both of these cell lines were sensitive toward bakuchiol (Fig. 1A). To investigate whether the combination treatment of bakuchiol and TRAIL has an effect on resistant cancer cell growth, we treated these cancer cells with half doses of IC50 of bakuchiol (The IC50 value of bakuchiol on HCT116 and HT-29 was 14.2 mg/ml and 10 mg/ml, respectively) with TRAIL. Pretreatment (24 h) with bakuchiol significantly enhanced TRAIL-induced cytotoxicity in TRAIL-sensitive HCT116 cells and TRAIL-resistant HT29 cells (Fig. 1A). We also showed that the bakuchiol significantly enhanced TRAIL-induced apoptosis in both cell lines. Annexin V/PI data indicated that the apoptosis index was much increased in the combination treated group of bakuchiol and TRAIL (65.62% in HCT116 cell; 77.15% in HT29 cell) compared with only bakuchiol (19.96% in HCT116 cell; 11.83% in HT29 cell) or TRAIL (26.82% in HCT116 cell; 9.3% in HT29 cell) treated group (Fig. 1B). 3.2. Effect of bakuchiol on the expression of death receptors in colon cancer cells Bakuchiol induced both DR4 and DR5 in a dose dependent
Fig. 3. Effect of bakuchiol on the expression of survival proteins and apoptosis related proteins. HCT116 and HT-29 cells were treated with bakuchiol (5 mg/ml) for 24 h, then equal amounts of total proteins (50 mg/lane) were subjected to 12% SDS-PAGE. A, HCT116 and HT-29 cells were treated with bakuchiol (5 mg/ml) for 24 h, Equal amounts of total proteins (50 mg/lane) were subjected to 12% SDS-PAGE. Expression of survivin, XIAP, FLIP, Bcl2 and b-actin was detected by Western blotting using specific antibodies. b-actin protein was used as internal control. Each band is representative for three experiments. B, HCT116 and HT-29 cells were pretreated with bakuchiol for 24 h, after that the cells were treated with TRAIL for 24 h, and whole cell extracts were analyzed by western blotting using antibodies against caspase-3, caspase-8, caspase-9, PARP and b-actin. b-actin protein was used as internal control. Each band is representative for three experiments.
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manner in TRAIL-sensitive HCT116 and TRAIL-resistant HT-29 cells (Fig. 2A). We also found that transfection of cells with DR4 or DR5 siRNA but not with the control siRNA reduced the combination of bakuchiol and TRAIL-induced cell growth inhibition (Fig. 2B). These results indicate that both DR4 and DR5 play an important role in sensitizing the effect of bakuchiol on TRAIL-induced cell death. 3.3. Effect of bakuchiol on the expression of survival proteins and apoptosis related proteins in colon cancer cells Several studies have shown that various anti-apoptotic proteins such as survivin, cFLIP, Bcl2 and XIAP induce resistance to TRAIL [21e23]. As shown in Fig. 3A, we showed that bakuchiol decreases expression of survivin, cFLIP, Bcl2 and XIAP, suggesting that the bakuchiol can overcome the TRAIL resistance through over-
expression of DR4 and DR5, as well as down regulation of survival proteins. Next, we compared the effect of bakuchiol and/or TRAIL on the activation of caspase-8, caspase-3, caspase-9 cleavage and PARP in the TRAIL sensitive colon cancer (HCT116) cell and in the TRAIL-resistant colon cancer (HT-29) cell. Although bakuchiol and TRAIL alone had little effect on the activation of caspases cleavage, the combination treatment significantly increased expression of the apoptotic cell death regulatory proteins (Fig. 3B). 3.4. Bakuchiol activates JNK and ROS and induction of TRAIL receptors is JNK and ROS dependent pathway TRAIL death receptors are activated through MAPK pathway. So, we investigated whether bakuchiol induces the MAPK pathway in the colon cancer cell lines, thereafter induces the death receptors.
Fig. 4. Effect of JNK and ROS on upregulation of DR4 and DR5 and apoptosis induced by bakuchiol and TRAIL. A, HCT116 and HT-29 cells were treated with bakuchiol (5 mg/ml) for 24 h, then equal amounts of total proteins (50 mg/lane) were subjected to 12% SDS-PAGE. Expression of MAPK pathway proteins was detected by Western blotting using specific antibodies. B, HCT116 and HT-29 cells were pretreated with SP600125 (5e10 mM) for 1 h, and then cells were treated with bakuchiol for 24 h and equal amounts of total proteins (50 mg/lane) were subjected to 12% SDS-PAGE. Expression of DR4 and DR5 was detected by Western blotting using specific antibodies. C, HCT116 and HT-29 cells were pretreated with SP600125 (5e10 mM) for 1 h, and then cells were treated with bakuchiol for 24 h and then treated with TRAIL for another 24 h. Cell viability was measured by direct counting. Data means ± SD expressed as percentage of control value, which is set to 100%. At least three independent experiments were carried out in triplicate. D, After treatment of bakuchiol and TRAIL for 30 min, the HCT116 and HT-29 cells were incubated with 10 mM DCF-DA at 37 C for 4 h, and then washed twice with PBS. The fluorescence intensity of DCF was measured in a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. E, HCT116 and HT-29 cells were pretreated with NAC (10 mM) for 1 h, and then cells were treated with bakuchiol for 24 h and then treated with TRAIL. Cell viability was measured by direct counting. Data means ± SD expressed as percentage of control value, which is set to 100%. At least three independent experiments were carried out in triplicate. *, p < 0.05, significantly different from control cells.
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We showed that the bakuchiol activated JNK phosphorylation in both colon cancer cell lines (Fig. 4A). Further, we showed that the treatment of JNK inhibitor, SP600125 reversed the expression of death receptors (Fig. 4B). From these results, we suggested that the expression of DRs by bakuchiol was JNK pathway dependent. Furthermore, we examined whether the suppression of JNK activation by its inhibitor could abrogate the cytotoxicity induced by the combination treatment of bakuchiol and TRAIL. We showed that cell growth inhibition by the combination treatment of bakuchiol and TRAIL was completely reversed by JNK inhibitor (10 mM) (Fig. 4C). Several studies demonstrated that the DRs pathway is dependent on the ROS pathway [24,25]. And previous study has been showed that the resveratrol induced ROS release in several cancer cells [26,27], but the effect of bakuchiol has not been studied yet. So we investigated whether the up-regulation of DRs induced by bakuchiol could be regulated by ROS. First, we showed that the ROS generation was increased by treatment of bakuchiol in a dose dependent manner in both cell lines (Fig. 4D). Moreover, we also showed that pretreatment of NAC (10 mM) reversed the effect of the bakuchiol induced cytotoxic effect of TRAIL (Fig. 4E). 4. Discussion We demonstrated here that bakuchiol can overcome TRAIL resistance in colon cancer cells. First, we found that the combination of bakuchiol and TRAIL increased apoptotic cell death and induced expression of apoptosis related proteins in both TRAIL sensitive HCT116 cells and TRAIL resistant HT-29 cells. TRAIL mediates apoptotic cell death through enhanced expression of two death receptors, DR4 and DR5 which are expressed on the surface of cancer cells, and the binding of TRAIL to DR4 and DR5 leads to the activation of caspase 8 and 10, which in turn cleaves and activates executioner caspases that mediate apoptosis [28,29]. Thus, we hypothesized that the cancer inhibitory effect of bakuchiol could be related with increased DR4 and DR5 expression, resulting enhanced apoptotic effect to TRAIL. In this study, we showed that the expression of DR4 and DR5 was increased by treatment of bakuchiol in both TRAIL sensitive and TRAIL resistant cancer cells. Among the several factors, intracellular levels of survival proteins are related with TRAIL resistance [21e23]. Thus, in this study, we showed that the expression of survival proteins such as Bcl2, cFLIP, survivin and XIAP was down regulated by bakuchiol correlated with the increased expression of DR4 and DR5. From these results, we suggest that bakuchiol can enhance TRAIL-induced apoptosis and can overcome TRAIL-resistance through down-regulation of cell survival proteins and up-regulation of death receptors. Next we investigated the molecular mechanisms of the bakuchiol induced expression of DRs. Several studies indicated that the expression of death receptor is up-regulated by p53, CHOP and MAPK pathway [30,31]. Bakuchiol already known to induce MAPK pathway in the cancer cell lines [13], so we investigated whether MAPK pathways are related with DR induction by bakuchiol. Here, we showed that bakuchiol induce the JNK phosphorylation in both cell lines, but not induced ERK and p38 phosphorylation. We also showed that the JNK inhibitor, SP600125 was abolished the bakuchiol induced DR4 and DR5 expression and suppressed the growth inhibitory effect induced by bakuchiol and TRAIL. Similar with our result, simvastatin (SVA) was shown to up-regulate expression of death receptor-5 (DR5), CCAAT/enhancer binding protein homologous protein (CHOP) and phosphorylated c-Jun N-terminal kinase (pJNK) in human breast cancer cell lines. siRNA knockdown of DR5, CHOP or JNK significantly blocked SVA-induced apoptosis, demonstrating the importance of JNK/CHOP/DR5 signaling pathway in SVA-induced apoptosis [32]. Other group also showed that gossypol enhances TRAIL-induced apoptosis through the
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down-regulation of cell survival proteins and the up-regulation of TRAIL death receptors through the ROS-ERK-CHOP-DR5 pathway [11]. Recent study also demonstrated that g-tocotrienol suppressed tumor growth in a syngeneic implantation mouse mammary cancer model by inhibiting cell proliferation and inducing apoptosis by activation of caspase-3, -8, and -9 through induction of DR5 and JNK and p38 MAPK in human breast cancer cell lines [33]. Similar with other reports, our finding demonstrated that bakuchiolinduced DR expression could be mediated with JNK pathway for enhancing the apoptotic sensitivity to TRAIL. ROS generation could be involved in DRs [34,35]. In this study, we also showed whether bakuchiol-induced death receptor upregulation is mediated with ROS in both cells. The treatment of bakuchiol induced ROS generation, but the antioxidant NAC abolished the upregulation of DR4 and DR5 induced by the treatment of bakuchiol. The potentiated effect of bakuchiol on TRAIL-induced apoptosis was also neutralized by the NAC. In agreement with these observations, previous studies using zerumbone and celastrol have shown the role of ROS and MAPK in the up-regulation of DRs and the potentiation of [36,37]. Our results indicate that like several other anti-cancer agents, bakuchiol also induces DRs through ROS generation for sensitization of cancer cells against TRAIL. Taken together, this study indicates that bakuchiol sensitizes TRAIL resistant colon cancer cells to the TRAIL, and suggests that the combination treatment of bakuchiol and TRAIL could be applicable as an anti-colorectal cancer agent. Conflict of interest The authors have declared that no conflict of interest exists. Acknowledgment This work was supported by grants to Y.H.C. from the Korea Basic Science Institute (D34402, T34417), and by the Creative Fusion Research Program through the Creative Allied Project funded by the National Research Council of Science & Technology (CAP-12-1-KIST). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.03.127. References [1] J. Lemke, S. von Karstedt, J. Zinngrebe, H. Walczak, Getting TRAIL back on track for cancer therapy, Cell Death Differ. 21 (2014) 1350e1364. [2] S.R. Wiley, K. Schooley, P.J. Smolak, W.S. Din, C.P. Huang, J.K. Nicholl, G.R. Sutherland, T.D. Smith, C. Rauch, C.A. Smith, Identification and characterization of a new member of the TNF family that induces apoptosis, Immunity 3 (1995) 673e682. [3] F.C. Kischkel, D.A. Lawrence, A. Chuntharapai, P. Schow, K.J. Kim, A. Ashkenazi, Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5, Immunity 12 (2000) 611e620. [4] P.T. Daniel, T. Wieder, I. Sturm, K. Schulze Osthoff, The kiss of death: promises and failures of death receptors and ligands in cancer therapy, Leukemia 15 (2001) 1022e1032. [5] A. Mühlethaler-Mottet, K.B. Bourloud, K. Auderset, J.M. Joseph, N. Gross, Drugmediated sensitization to TRAIL-induced apoptosis in caspase-8complemented neuroblastoma cells proceeds via activation of intrinsic and extrinsic pathways and caspase-dependent cleavage of XIAP, Bcl-xL and RIP, Oncogene 23 (2004) 5415e5425. [6] S. Lacour, O. Micheau, A. Hammann, V. Drouineaud, J. Tschopp, E. Solary, M.T. Dimanche-Boitrel, Chemotherapy enhances TNF-related apoptosisinducing ligand DISC assembly in HT29 human colon cancer cells, Oncogene 22 (2003) 1807e1816. [7] S. Prasad, J.H. Kim, S.C. Gupta, B.B. Aggarwal, Targeting death receptors for TRAIL by agents designed by Mother Nature, Trends Pharmacol. Sci. 35 (2014) 520e536.
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