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Oridonin enhances TRAIL-induced apoptosis through GALNT14-mediated DR5 glycosylation
Biochimie 165 (2019) 108e114
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Research paper
Oridonin enhances TRAIL-induced apoptosis through GALNT14-mediated DR5 glycosylation Mi-Yeon Jeon a, Seung Un Seo a, Seon Min Woo a, Kyoung-jin Min a, Hee Sun Byun b, Gang Min Hur b, Sun Chul Kang c, Taeg Kyu Kwon a, * a b c
Department of Immunology, School of Medicine, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu, 42601, South Korea Department of Pharmacology, College of Medicine, Chungnam National University, 266 Munhwa-ro, Daejeon, 35015, South Korea Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbuk, 38453, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 March 2019 Accepted 17 July 2019 Available online 20 July 2019
Oridonin is a diterpenoid isolated from the Rabdosia rubescens and has multiple biological effects, such as anti-inflammation and anti-tumor activities. In present study, we revealed that the sensitizing effect of oridonin on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in several cancer cells, but not in normal cells. Oridonin enhanced death-signaling inducing complexes (DISC) formation and DR5 glycosylation without affecting expression of downstream intracellular apoptosis-related proteins. Oridonin upregulated peptidyl O-glycosyltransferase GALNT14 in a dose- and time-dependent manner. Knockdown of GALNT14 by siRNA and Endo H treatment reduced oridonininduced DR5 glycosylation. Furthermore, treatment with inhibitor of glycosylation (benzyl-a-GalNAc) blocked oridonin plus TRAIL-induced apoptosis. Collectively, our results suggest that oridonin-induced DR5 glycosylation contributes to TRAIL-induced apoptotic cell death in cancer cells. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: Oridonin DR5 Glycosylation TRAIL Apoptosis
1. Introduction Tumor necrosis-factor related apoptosis-inducing ligand (TRAIL) is one of the cytokines of the TNF superfamily. Its binding to death receptor (DR) 4 and/or DR5 can induce the formation of deathsignaling inducing complexes (DISC), which is critical for activation of caspase and induction of apoptosis [1e3]. The expressional and functional modification of DR5 regulates sensitivity of TRAILmediated apoptosis [1e3]. O-glycosylation is one of the major post-translational modifications and regulates various biological processes [4]. Glycosylation modulates biochemical and functional properties of cell surface proteins, such as conformation, multimerization, protein stability and signaling events [4]. Sensitivity to TRAIL correlates with expression levels of O-glycosyltransferase [5].
Abbreviations: TRAIL, Tumor necrosis factor-related apoptosis-inducing ligand; DISC, Death-signaling inducing complexes; DR, Death receptor; GALNT14, Polypeptide N-acetylgalactosaminyltransferase 14; MAPK, mitogen-activated protein kinase; NF-kB, Nuclear factor-kappa B; PARP, Poly (ADP-ribose) polymerase; STAT, signal transducer and activator of transcription; MMP, Mitochondrial membrane potential. * Corresponding author. Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu, 42601, South Korea. Tel.: þ82 53 258 7358/þ82 258 7355. E-mail address: [email protected] (T.K. Kwon).
O-glycosylation of DR5 increases TRAIL-induced apoptosis through augment of death receptor clustering and DISC formation [6]. Oridonin, an active diterpenoid compound isolated from medicinal herb Rabdosia rubescens, possess potent antitumor effect on various cancers, such as colon cancer cells, lymphoma cells and breast cancer cells [7e10]. Recently, it has been reported that oridonin synergizes with various anticancer agents by modulating the expression levels of apoptotic proteins and/or promoting apoptosis signaling pathway [11e13]. Furthermore, oridonin induces autophagy through inhibition of glucose metabolism in colorectal cancer cells [14]. In addition, oridonin also had anti-inflammatory properties in endothelial cells through the suppression of MAPK and NF-kB activation [15]. Many studies have been investigated mechanisms of the antitumor effect of oridonin. However, the potential molecular mechanisms of oridonin are still unclear. In present study, for the first time, we provide mechanistic evidence that oridonin exerted a sensitizing effect on TRAIL-induced apoptosis through up-regulation of DISC formation by DR5 glycosylation. 2. Materials and methods 2.1. Cells and materials American type Culture Collection (Manassas, VA, USA) supplied
https://doi.org/10.1016/j.biochi.2019.07.015 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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the human cancer cell lines including renal (Caki and A498) and lung (A549) carcinoma, and normal TCMK-1. Normal human renal mesangial cells (NHMCs) were purchased from Lonza (Basel, Switzerland). The cells were cultured with Dulbecco's Modified Eagle's Medium including 10% fetal bovine serum, 1% penicillinstreptomycin and 100 mg/ml gentamycin (Thermo Fisher Scientific, Waltham, MA, USA). Oridonin was purchased from Enzo life Sciences (Farmingdale, NY, USA). R&D system (Minneapolis, MN, USA) supplied z-VAD-fmk, TRAIL and anti-survivin antibody, and Cell Signaling Technology (Beverly, MA, USA) supplied antibodies, including anti-DR5, anti-phospho-p38, anti-p38, anti-phosphoAKT, anti-AKT, anti-phospho-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phospho-STAT1 (S727), anti-STAT1, anti-caspase8 and anti-PARP. Anti-FADD, anti-Bax and anti-XIAP antibodies were obtained from BD Biosciences (San Jose, CA, USA). Anti-Mcl-1, anticIAP2, anti-Bcl2, anti-Bcl-xL, anti-p53, anti-phosph-STAT3 (S727, Y705), anti-STAT3 and anti-GALNT14 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-c-FLIP and anti-DR4 antibodies were obtained from ALEXIS Corporation
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(San Diego, CA, USA) and Abcam (Cambridge, UK), respectively. Endoglycosidase H (Endo H) was purchased from New England Biolabs (Ipswich, MA, USA). Anti-actin antibody and Benzyl-aGalNAc were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Flow cytometry analysis Using 100 ml of phosphate-buffered saline (PBS), cells were suspended, and then were added 200 ml of 100% ethanol. After incubation at 4 C for 1 h, cells were centrifuged for 2e3 min (3000g). Pellet (cells) were washed with PBS and added 150 ml of RNase (12.5 mg/ml) including 1.12% sodium citrated buffer (pH 8.4). After 30 min at 37 C, 150 ml of propidium iodide solution (50 mg/ml) added to the cells. The cells were analyzed using fluorescentactivated cell sorting on a FACScan flow cytometry. 2.3. Western blot analysis Cells were lysed in ERK lysis buffer (50 mM Tris-HCl, 1 mM
Fig. 1. Oridonin induces TRAIL-mediated apoptosis in cancer cells, not normal cells. (AeD) Caki cells were treated with oridonin (3, 5 mM) in the absence or presence of TRAIL (30e50 ng/ml) for 24 h. Flow cytometry was used to detect the sub-G1 population (graph and histogram), and the protein expression was determined by Western blotting (A). Cell morphological change was detected using interference light microscopy (B). Damage of the nuclei were detected by DAPI staining (C), and caspase activities were determined with colorimetric assays using caspase-3 (DEVDase) assay kits (D). (E) Caki cells were treated with 5 mM oridonin plus 50 ng/ml TRAIL in the presence or absence of 20 mM z-VAD-fmk (zVAD) for 24 h. Flow cytometry was used to detect the sub-G1 population (graph and histogram). The protein expression levels of PARP and actin were determined by Western blotting. (F) ACHN and A549 cells were treated with the indicated concentrations with oridonin in the absence or presence of 50 ng/ml TRAIL for 24 h. Flow cytometry was used to detect the sub-G1 population (graph and histogram). (G) Caki, TCMK-1 and mesangial cells (MC) were with the 5 mM oridonin in the absence or presence of 50 ng/ml TRAIL for 24 h. Flow cytometry was used to detect the sub-G1 population. The values in A, D, E, F, and G represent the mean ± SD from three independent experiments. *p < 0.01 compared to the control. #p < 0.01 compared to oridonin plus TRAIL.
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EGTA, 1% TritonX-100, 1 mM Phenylmethylsulfonyl fluoried, pH 7.5), and then centrifuged for 15 min (10,000g, 4 C). The supernatant transferred into new tube, and then determined protein concentration using BCA Protein Assay Kit (Thermo Fisher Scientific, Walsham, USA). The proteins were separated on 8e12% SDS-PAGE and transferred to an Immobilon-P membrane (GE Healthcare Life Science, Pittsburgh, PO, USA). After blocking, membranes were incubated with specific primary antibodies for overnight. After washed, membranes were attached with secondary antibody for 1 ~ 2 h. Specific proteins were detected using an enhanced chemiluminescence (ECL) Western blot kit (EMD Millipore, Darmstadt, Germany). 2.4. 40 ,60 -diamidino-2-phenylindole staining (DAPI) for nuclei condensation and fragmentation The cells were fixed with 1% paraformaldehyde for 15 min at room temperature and then washed with PBS. The nuclei were stained with a 300 nM DAPI solution (Roche, Mannheim, Germany) for 30 min, and the cells were examined by fluorescence microscopy. 2.5. Aps-glu-val-asp-ase (DEVDase) activity assay Cell lysates were incubated with 100 ml of reaction buffer (1% NP-40, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol) including a 5 mM caspase substrate [Asp-Glu-Val-Asp-chromophore-p-nitroanilid (DEVD-pNA)]. The absorbance at 405 nm was determined with a spectrophotometer (BMG Labtceh, Ortenberg, Germany).
2.6. Detection of activated bax Cells were harvested by trypsinization, and then fixed with 4% paraformaldehyde for 30 min. After washed two times with PBS (1% Fetal calf serum), and incubated at 4 C with the Bax(6A7) antibody (1:100) in PBS (1% FCS and 0.1% saponine). After 1 h, cells were washed two times with PBS (1% FCS), and incubated for 1 h at 4 C with secondary antibody (1:500) in PBS (1% FAS, 0.1% saponine). After washing, the cells were resuspended in PBS (1% FCS). The Bax activation was determined using flow cytometry. 2.7. Measurement of mitochondrial membrane potential by rhodamine 123 Rhodamine 123 (Molecular Probes Inc., Eugene, OR) uptake by mitochondria is directly proportional to its membrane potential. After treatment, cells were incubated with rhodamine 123 (5 mM) for 5 min in the dark at 37 C. The cells were harvested, suspended in PBS, and then analyzed using a flow cytometer. 2.8. DISC immunoprecipitation After treatment, cells were washed with PBS, and then lysed in RIPA lysis buffer including 10 mM NEM, 1 mM PSMF, protease inhibitor cocktail. After centrifuged for 15 min (13,000 rpm, 4 C), the supernatant transferred a new e-tube and analyzed protein amount using BCA kit. Lysates were added caspase-8 antibody and were rotated for overnight. The protein G bead were washed using RIPA lysis buffer 3 times and added protein sample including primary antibody. The samples were rotated for 2 h at 4 C, and washed with
Fig. 2. Oridonin has no effect on expression of apoptosis-related proteins. (AeB) Cells were treated with the indicated concentrations of oridonin for 24 h. The protein expression was determined by Western blotting. (C) Caki cells were treated 5 mM oridonin for the indicated time periods, and then cells were stained for active Bax using conformation-specific antibodies (Bax 6A7). The fluorescence intensity was detected by flow cytometry (graph and histogram). (pc: positive control; PP242 plus curcumin). (D) Caki cells were treated with 5 mM oridonin for the indicated time periods. The mitochondrial membrane potential was measured using a flow cytometry (graph and histogram). The values in C and D represent the mean ± SD of three independent experiments. *p < 0.01 compared to control. ns ¼ not significance.
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RIPA lysis buffer. After washing, 5 x cooking buffer added samples were heated 95 C for 10 min and then the caspase-8 interacting proteins were detected in 10% SDS-PAGE for immunoblot analysis. 2.9. Quantitative PCR (qPCR) Total RNA was isolated using the Trizol reagent (Life Technologies; Gaithersburg, MD), and we obtatined the cDNA was prepared using M-MLV reverse transcriptase (Gibco-BRL; Gaithersburg, MD). The cDNA and forward/reverse primers were added to 2 TAKARA SYBR Fast master mix, and reactions were performed on LightCycler 480 real-time amplification instrument (Roche, Basel, Switzerland). The following primers were used for the amplification of human GALNT14 and actin: GALNT14 (forward) 50 -CAT TGC TGT CGG TCA TCT -30 and (reverse) 50 -TGT CAG TCA CCT TCT TC -30 . actin (forward) 50 - CTA CAA TGA GCT GCG TGT G-30 and (reverse) 50 -TGG GGT GTT GAA GGT CTC -30 . Threshold cycle number (Ct) of each gene was calculated, and actin was used as reference genes. Delta-delta Ct values of genes were presented as relative fold induction. 2.10. Small interfering RNA (siRNA) The green fluorescent protein (GFP) (control) siRNA and GALNT14 siRNA were purchased from Bioneer (Daejeon, Korea). Cells were transfected with siRNA using Oligonucleotides Reagent (Invitrogen). 2.11. Endo H digestion
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induces caspase-dependent apoptotic cell death. As shown in Fig. 1F, we observed that combined treatment induced apoptosis in other renal carcinoma cells (ACHN) and lung carcinoma cells (A549). However, combined treatment did not affect on apoptosis in normal mouse kidney cells (TCMK-1) (Fig. 1G). 3.2. Effect of oridonin on expression levels of apoptosis-related proteins To elucidate the underlying mechanisms of TRAIL sensitization of RCC cells by oridonin, we examined the effect of oridonin on expression levels of apoptosis-related proteins. Oridonin did not change the expression levels of apoptosis-related proteins (antiapoptotic proteins: Bcl-xL, Bcl-2, Mcl-2, cIAP2, XIAP, Survivin, cFLIP, and pro-apoptotic proteins: Bax, DR4, DR5, p53) and phosphorylation status and expression levels of STAT1/3 and MAPKs (Fig. 2A and B). Since Bax activation and mitochondrial membrane potential (MMP) play a critical role in mitochondria-mediated intrinsic apoptosis [17,18], we examined the effect of oridonin on Bax activation and loss-of-MMP. Oridonin did not induce Bax activation and loss-of-MMP (Fig. 2C and D). Furthermore, to identify the molecular mechanisms leading to apoptotic cell death in combined treatment with oridonin and TRAIL, we tested expression levels of apoptosis-related proteins by combined treatment. As shown in Fig. 3, oridonin plus TRAIL also did not alter tested any proteins. These data suggest that the apoptotic effect of oridonin plus TRAIL on RCC cells is not associated with mitochondriamediated intrinsic apoptotic pathway.
Samples were incubated with 1500 U/ml Endo H (New England Biolabs, Ipswich, MA) according to the manufacturer's instruction. After 5 h at 37 C, the digested samples were subjected to Western blot analysis. 2.12. Statistical analysis We repeated experiments at least three times in our studies, and all data were represented as the means. Statistical analysis was performed by a one-way ANOVA and post hoc comparisons (Student-NewmaneKeuls) using the SPSS (Statistical Package for the Social Sciences, version 22.0) (SPSS Inc.; Chicago, IL). The p-values <0.05 were considered significant. 3. Results 3.1. Oridonin enhances TRAIL-induced apoptosis in human renal carcinoma caki cells and A549 cells Renal cell carcinoma (RCC) is the most common cancer found within the kidney, and it is resistant to chemotherapeutic agents, compared with other cancers [16]. Therefore, to find the way to increase sensitivity of RCC cells to anti-cancer drugs could enhance cell death. To determine the sensitizing effects of oridonin on TRAIL-induced apoptosis, clear cell RCC Caki cells were treated with oridonin alone, TRAIL alone and combined treatment. Combined treatment with oridonin and TRAIL increased the sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP) (Fig. 1A), and showed typical apoptotic morphologies (Fig. 1B) and chromatin condensation (Fig. 1C). However, single treatment with oridonin or TRAIL did not induce apoptosis. Next, we investigated whether combined treatment induces caspase-dependent apoptosis. Combined treatment significantly induced caspase-3 activation (Fig. 1D). PARP cleavage and induction of sub-G1 population were inhibited by pan-caspase inhibitor, z-VAD-fmk (Fig. 1E). Our data indicated that combined treatment with oridonin and TRAIL
Fig. 3. Combined treatment with oridonin plus TRAIL did not change the expression of apoptosis-related proteins. Caki cells were treated with 5 mM oridonin in the absence of presence of 50 ng/ml TRAIL for 24 h. The protein expression was determined by Western blotting.
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3.3. Enhancement of TRAIL-induced apoptosis by oridonin is associated with DISC formation and DR5 glycosylation DISC formation plays a critical role in death receptor-mediated extrinsic and/or intrinsic apoptosis pathway [1]. We examined whether oridonin affects the DISC formation. An immunoprecipitation assay using an anti-caspase 8 antibody showed FADD, cleaved forms of caspase-8 and DR5 in the isolated DISC after oridonin plus TRAIL treatment (Fig. 4A). The peptidyl O-glycosyltransferase GALNT14-mediated DR5 O-glycosylation promotes sensitivity of cancer cells to TRAIL-induced apoptosis [6]. Therefore, we examined whether upregulation of DR5 glycosylation contributes to oridonin plus TRAIL-induced apoptosis. Oridonin induced upregulation of GALNT-14 protein expression in a dose- and timedependent manner in Caki and A549 cells (Fig. 4B). Also, oridonin upregulated GALNT14 mRNA expression in a time-dependent manner (Fig. 4C). Combined treatment induced glycosylated-high molecular weight DR5 form in Caki and A549 cells (Fig. 4D). Treatment of these cells with GALNT14 siRNA or digestion of oridonin plus TRAIL-treated Caki cell extracts with EndoH reduced expression levels of glycosylated DR5 (Fig. 4E and F). Furthermore,
to investigate the significance of glycosylation of DR5 in sensitization of RCC cells to TRAIL by oridonin., we examined whether benzyl-a-GalNAc (a general inhibitor of O-glycosylation) inhibits oridonin plus TRAIL-mediated apoptosis. Benzyl-a-GALNAc inhibited apoptosis and PARP cleavage in oridonin plus TRAIL-treated Caki and A549 cells (Fig. 4G and H). Therefore, our data indicated that DISC formation and DR5 glycosylation plays a critical role in sensitization of RCC cells to TRAIL by oridonin. 4. Discussions In present study, we showed mechanistic evidence that oridonin enhanced TRAIL-induced apoptosis in cancer cells. Oridonin enhanced DISC formation without affecting downstream intracellular apoptosis-related proteins. Oridonin induced DR5 glycosylation in a GALNT14-dependent manner, and a pharmacological inhibitor of glycosylation (benzyl-a-GalNAc) inhibited orionin plus TRAIL-induced apoptosis. Oridonin inhibited the expression of several anti-apoptotic proteins, such as Bcl-2, Mcl-1, and XIAP in hepatocellular cancer cells [19]. Wu et al. reported that a novel oridonin analog
Fig. 4. Oridonin enhances DISC formation via upregulation of DR5 glycosylation. (A) Caki cells were treated with 5 mM oridonin in the absence of presence of 50 ng/ml TRAIL for 24 h. After immunoprecipitation with anti-caspase 8 antibody, the protein expression was determined by Western blotting. (B) Caki and A549 cells were treated with the indicated concentrations of oridonin for 24 h (left panel) or the indicated times (right panel). The protein expression was determined by Western blotting. (C) Caki cells were treated with 5 mM oridonin for the indicated time periods. The mRNA expression was determined by qPCR. (D) Caki and A549 cells were treated with the indicated concentrations of oridonin in the absence of presence of 50 ng/ml TRAIL for 24 h. The protein expression was determined by Western blotting. (E) Caki cells were transfected with control siRNA (siCont) and GALNT14 (siGALNT14), and the treated with 5 mM oridonin plus 50 ng/ml TRAIL for 24 h. The protein expression was determined by Western blotting. (F) Caki cells were treated with 5 mM oridonin plus 50 ng/ml TRAIL for 24 h. After treatment, lysates were subjected to Endo H, and then protein expression was determined by Western blotting. (G and H) Caki (G) and A549 (H) cells were pretreated with the indicated concentrations of Benzyl-a-GalNAc for 30 min, and then treated with the indicated concentraions of oridonin plus 50 ng/ml TRAIL for 24 h. Flow cytometry was used to detect the sub-G1 population, and the protein expression was determined by Western blotting. The values in C, G, and H represent the mean ± SD of three independent experiments. *p < 0.05 compared to control. #p < 0.05 compared to the oridonin plus TRAIL.
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suppresses triple-negative breast cancer cells and tumor growth though the induction of DR5 [20]. In our study, oridonin did not affect expression levels of apoptosis-related proteins and death receptors (DR4 and DR5). Oridonin markedly enhanced the DISC formation in combined treated with oridonin and TRAIL (Fig. 4A). Previous study reported that O-glycosylation of DR5 is important for sensitivity to the TRAIL [6]. Wagner et al. found that expression of GALNT14 mRNA is markedly higher in TRAIL-sensitive pancreatic, NSCLC and melanoma cell lines, compared with resistant cell lines. Pharmacologic inhibition of O-glycosylation enzymes or siRNA knockdown of GALNT14 in sensitive cells reduces sensitivity to TRAIL, and GALNT14 overexpression in resistant cells increases glycosylation of DR5 and TRAIL-induced apoptosis. Glycosylation of DR5 by GALNT14 promotes ligand-induced clustering of DR5, which in turn increases DISC recruitment and caspase-8 activation [6]. In contrast, glycosylation of DR5 has no effect on TRAIL binding affinity and cell surface expression of DR5 [6]. In case of DR4, Dufour et al. reported that N-linked glycosylation plays a critical role for DR4 [21]. Oridonin-induced TRAIL sensitization of cancer cells was caused through a GALNT14-mediated DR5 glycosylation and upregulation of DISC formation. Oridonin induced GALNT14 mRNA and protein expression (Fig. 4B and C). Knockdown by GALNT14 siRNA or treatment with Endo H inhibited oridonin plus TRAIL-induced DR5 glycosylation (Fig. 4E and F). We demonstrated that inhibitor of O-glycosylation markedly inhibited oridonin plus TRAIL-induced apoptosis and PARP cleavage (Fig. 4G and H). Overall, our findings provide insights into the molecular mechanisms of TRAIL sensitization through which oridonin enhances the ability of O-glycosylation of DR5 through up-regulating GALNT14 expression. Nevertheless, how oridonin specifically regulates transcriptional activation of GALNT14 is a critical question for further development of TRAIL-based chemotherapeutic strategies. We found that the glycosylated DR5 band by oridonin alone was relatively low levels compared to the combination of oridonin plus TRAIL (Fig. 4D). There is one of possibility for this result. When TRAIL binds to DR5 at the membrane, it activates the downstream signal and then undergoes endocytosis. After endocytosis, DR5 is recycled or degraded by lysosome [22]. Alternatively, up-regulation of the ubiquitination by E3 ligase, such as c-Cbl, induces degradation by proteasome [23]. However, since the glycosylation prevents endocytosis of death receptor, DR5 remains in the membrane for a long time without degradation [21]. On the other hand, in the absence of ligand, glycosylated DR5 is mainly present in cytosol [24], and cytosolic protein is preferentially degraded by proteasome [25]. Therefore, it is likely that the band detection of glycosylated DR5 by oridonin in the absence of TRAIL was difficult. To confirm this possibility, we detected glycosylated DR5 in the presence of proteasome inhibitor, MG132. We identified the increased glycosylated DR5 by oridonin, which was similar to the level found in combined treatment with oridonin and TRAIL (data not shown). These results indicate that oridonin may improve treatment results of combined treatment with TRAIL in the future clinical application. Conflicts of interest The authors declare no conflicts of interest. Author contributions M.Y.J. K.-j.M and T.K.K. designed the project and wrote the paper. M.Y.J., S.U.S., S.M.W., and K.-j.M performed the experimental work in the study. H.S.B., G.M.H., S.C.K., and T.K.K. analyzed data and critically revised manuscript. All the authors read and approved the final manuscript.
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[22] J.D. Twomey, S.R. Kim, L. Zhao, W.P. Bozza, B. Zhang, Spatial dynamics of TRAIL death receptors in cancer cells, Drug Resist. Updates 19 (2015) 13e21. [23] J.J. Song, M.J. Szczepanski, S.Y. Kim, J.H. Kim, J.Y. An, Y.T. Kwon, M.A. Alcala Jr., D.L. Bartlett, Y.J. Lee, c-Cbl-mediated degradation of TRAIL receptors is responsible for the development of the early phase of TRAIL resistance, Cell. Signal. 22 (2010) 553e563. [24] Y. Liang, W. Xu, S. Liu, J. Chi, J. Zhang, A. Sui, L. Wang, Z. Liang, D. Li, Y. Chen,
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Research paper
Oridonin enhances TRAIL-induced apoptosis through GALNT14-mediated DR5 glycosylation Mi-Yeon Jeon a, Seung Un Seo a, Seon Min Woo a, Kyoung-jin Min a, Hee Sun Byun b, Gang Min Hur b, Sun Chul Kang c, Taeg Kyu Kwon a, * a b c
Department of Immunology, School of Medicine, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu, 42601, South Korea Department of Pharmacology, College of Medicine, Chungnam National University, 266 Munhwa-ro, Daejeon, 35015, South Korea Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbuk, 38453, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 March 2019 Accepted 17 July 2019 Available online 20 July 2019
Oridonin is a diterpenoid isolated from the Rabdosia rubescens and has multiple biological effects, such as anti-inflammation and anti-tumor activities. In present study, we revealed that the sensitizing effect of oridonin on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in several cancer cells, but not in normal cells. Oridonin enhanced death-signaling inducing complexes (DISC) formation and DR5 glycosylation without affecting expression of downstream intracellular apoptosis-related proteins. Oridonin upregulated peptidyl O-glycosyltransferase GALNT14 in a dose- and time-dependent manner. Knockdown of GALNT14 by siRNA and Endo H treatment reduced oridonininduced DR5 glycosylation. Furthermore, treatment with inhibitor of glycosylation (benzyl-a-GalNAc) blocked oridonin plus TRAIL-induced apoptosis. Collectively, our results suggest that oridonin-induced DR5 glycosylation contributes to TRAIL-induced apoptotic cell death in cancer cells. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: Oridonin DR5 Glycosylation TRAIL Apoptosis
1. Introduction Tumor necrosis-factor related apoptosis-inducing ligand (TRAIL) is one of the cytokines of the TNF superfamily. Its binding to death receptor (DR) 4 and/or DR5 can induce the formation of deathsignaling inducing complexes (DISC), which is critical for activation of caspase and induction of apoptosis [1e3]. The expressional and functional modification of DR5 regulates sensitivity of TRAILmediated apoptosis [1e3]. O-glycosylation is one of the major post-translational modifications and regulates various biological processes [4]. Glycosylation modulates biochemical and functional properties of cell surface proteins, such as conformation, multimerization, protein stability and signaling events [4]. Sensitivity to TRAIL correlates with expression levels of O-glycosyltransferase [5].
Abbreviations: TRAIL, Tumor necrosis factor-related apoptosis-inducing ligand; DISC, Death-signaling inducing complexes; DR, Death receptor; GALNT14, Polypeptide N-acetylgalactosaminyltransferase 14; MAPK, mitogen-activated protein kinase; NF-kB, Nuclear factor-kappa B; PARP, Poly (ADP-ribose) polymerase; STAT, signal transducer and activator of transcription; MMP, Mitochondrial membrane potential. * Corresponding author. Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu, 42601, South Korea. Tel.: þ82 53 258 7358/þ82 258 7355. E-mail address: [email protected] (T.K. Kwon).
O-glycosylation of DR5 increases TRAIL-induced apoptosis through augment of death receptor clustering and DISC formation [6]. Oridonin, an active diterpenoid compound isolated from medicinal herb Rabdosia rubescens, possess potent antitumor effect on various cancers, such as colon cancer cells, lymphoma cells and breast cancer cells [7e10]. Recently, it has been reported that oridonin synergizes with various anticancer agents by modulating the expression levels of apoptotic proteins and/or promoting apoptosis signaling pathway [11e13]. Furthermore, oridonin induces autophagy through inhibition of glucose metabolism in colorectal cancer cells [14]. In addition, oridonin also had anti-inflammatory properties in endothelial cells through the suppression of MAPK and NF-kB activation [15]. Many studies have been investigated mechanisms of the antitumor effect of oridonin. However, the potential molecular mechanisms of oridonin are still unclear. In present study, for the first time, we provide mechanistic evidence that oridonin exerted a sensitizing effect on TRAIL-induced apoptosis through up-regulation of DISC formation by DR5 glycosylation. 2. Materials and methods 2.1. Cells and materials American type Culture Collection (Manassas, VA, USA) supplied
https://doi.org/10.1016/j.biochi.2019.07.015 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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the human cancer cell lines including renal (Caki and A498) and lung (A549) carcinoma, and normal TCMK-1. Normal human renal mesangial cells (NHMCs) were purchased from Lonza (Basel, Switzerland). The cells were cultured with Dulbecco's Modified Eagle's Medium including 10% fetal bovine serum, 1% penicillinstreptomycin and 100 mg/ml gentamycin (Thermo Fisher Scientific, Waltham, MA, USA). Oridonin was purchased from Enzo life Sciences (Farmingdale, NY, USA). R&D system (Minneapolis, MN, USA) supplied z-VAD-fmk, TRAIL and anti-survivin antibody, and Cell Signaling Technology (Beverly, MA, USA) supplied antibodies, including anti-DR5, anti-phospho-p38, anti-p38, anti-phosphoAKT, anti-AKT, anti-phospho-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phospho-STAT1 (S727), anti-STAT1, anti-caspase8 and anti-PARP. Anti-FADD, anti-Bax and anti-XIAP antibodies were obtained from BD Biosciences (San Jose, CA, USA). Anti-Mcl-1, anticIAP2, anti-Bcl2, anti-Bcl-xL, anti-p53, anti-phosph-STAT3 (S727, Y705), anti-STAT3 and anti-GALNT14 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-c-FLIP and anti-DR4 antibodies were obtained from ALEXIS Corporation
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(San Diego, CA, USA) and Abcam (Cambridge, UK), respectively. Endoglycosidase H (Endo H) was purchased from New England Biolabs (Ipswich, MA, USA). Anti-actin antibody and Benzyl-aGalNAc were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Flow cytometry analysis Using 100 ml of phosphate-buffered saline (PBS), cells were suspended, and then were added 200 ml of 100% ethanol. After incubation at 4 C for 1 h, cells were centrifuged for 2e3 min (3000g). Pellet (cells) were washed with PBS and added 150 ml of RNase (12.5 mg/ml) including 1.12% sodium citrated buffer (pH 8.4). After 30 min at 37 C, 150 ml of propidium iodide solution (50 mg/ml) added to the cells. The cells were analyzed using fluorescentactivated cell sorting on a FACScan flow cytometry. 2.3. Western blot analysis Cells were lysed in ERK lysis buffer (50 mM Tris-HCl, 1 mM
Fig. 1. Oridonin induces TRAIL-mediated apoptosis in cancer cells, not normal cells. (AeD) Caki cells were treated with oridonin (3, 5 mM) in the absence or presence of TRAIL (30e50 ng/ml) for 24 h. Flow cytometry was used to detect the sub-G1 population (graph and histogram), and the protein expression was determined by Western blotting (A). Cell morphological change was detected using interference light microscopy (B). Damage of the nuclei were detected by DAPI staining (C), and caspase activities were determined with colorimetric assays using caspase-3 (DEVDase) assay kits (D). (E) Caki cells were treated with 5 mM oridonin plus 50 ng/ml TRAIL in the presence or absence of 20 mM z-VAD-fmk (zVAD) for 24 h. Flow cytometry was used to detect the sub-G1 population (graph and histogram). The protein expression levels of PARP and actin were determined by Western blotting. (F) ACHN and A549 cells were treated with the indicated concentrations with oridonin in the absence or presence of 50 ng/ml TRAIL for 24 h. Flow cytometry was used to detect the sub-G1 population (graph and histogram). (G) Caki, TCMK-1 and mesangial cells (MC) were with the 5 mM oridonin in the absence or presence of 50 ng/ml TRAIL for 24 h. Flow cytometry was used to detect the sub-G1 population. The values in A, D, E, F, and G represent the mean ± SD from three independent experiments. *p < 0.01 compared to the control. #p < 0.01 compared to oridonin plus TRAIL.
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EGTA, 1% TritonX-100, 1 mM Phenylmethylsulfonyl fluoried, pH 7.5), and then centrifuged for 15 min (10,000g, 4 C). The supernatant transferred into new tube, and then determined protein concentration using BCA Protein Assay Kit (Thermo Fisher Scientific, Walsham, USA). The proteins were separated on 8e12% SDS-PAGE and transferred to an Immobilon-P membrane (GE Healthcare Life Science, Pittsburgh, PO, USA). After blocking, membranes were incubated with specific primary antibodies for overnight. After washed, membranes were attached with secondary antibody for 1 ~ 2 h. Specific proteins were detected using an enhanced chemiluminescence (ECL) Western blot kit (EMD Millipore, Darmstadt, Germany). 2.4. 40 ,60 -diamidino-2-phenylindole staining (DAPI) for nuclei condensation and fragmentation The cells were fixed with 1% paraformaldehyde for 15 min at room temperature and then washed with PBS. The nuclei were stained with a 300 nM DAPI solution (Roche, Mannheim, Germany) for 30 min, and the cells were examined by fluorescence microscopy. 2.5. Aps-glu-val-asp-ase (DEVDase) activity assay Cell lysates were incubated with 100 ml of reaction buffer (1% NP-40, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol) including a 5 mM caspase substrate [Asp-Glu-Val-Asp-chromophore-p-nitroanilid (DEVD-pNA)]. The absorbance at 405 nm was determined with a spectrophotometer (BMG Labtceh, Ortenberg, Germany).
2.6. Detection of activated bax Cells were harvested by trypsinization, and then fixed with 4% paraformaldehyde for 30 min. After washed two times with PBS (1% Fetal calf serum), and incubated at 4 C with the Bax(6A7) antibody (1:100) in PBS (1% FCS and 0.1% saponine). After 1 h, cells were washed two times with PBS (1% FCS), and incubated for 1 h at 4 C with secondary antibody (1:500) in PBS (1% FAS, 0.1% saponine). After washing, the cells were resuspended in PBS (1% FCS). The Bax activation was determined using flow cytometry. 2.7. Measurement of mitochondrial membrane potential by rhodamine 123 Rhodamine 123 (Molecular Probes Inc., Eugene, OR) uptake by mitochondria is directly proportional to its membrane potential. After treatment, cells were incubated with rhodamine 123 (5 mM) for 5 min in the dark at 37 C. The cells were harvested, suspended in PBS, and then analyzed using a flow cytometer. 2.8. DISC immunoprecipitation After treatment, cells were washed with PBS, and then lysed in RIPA lysis buffer including 10 mM NEM, 1 mM PSMF, protease inhibitor cocktail. After centrifuged for 15 min (13,000 rpm, 4 C), the supernatant transferred a new e-tube and analyzed protein amount using BCA kit. Lysates were added caspase-8 antibody and were rotated for overnight. The protein G bead were washed using RIPA lysis buffer 3 times and added protein sample including primary antibody. The samples were rotated for 2 h at 4 C, and washed with
Fig. 2. Oridonin has no effect on expression of apoptosis-related proteins. (AeB) Cells were treated with the indicated concentrations of oridonin for 24 h. The protein expression was determined by Western blotting. (C) Caki cells were treated 5 mM oridonin for the indicated time periods, and then cells were stained for active Bax using conformation-specific antibodies (Bax 6A7). The fluorescence intensity was detected by flow cytometry (graph and histogram). (pc: positive control; PP242 plus curcumin). (D) Caki cells were treated with 5 mM oridonin for the indicated time periods. The mitochondrial membrane potential was measured using a flow cytometry (graph and histogram). The values in C and D represent the mean ± SD of three independent experiments. *p < 0.01 compared to control. ns ¼ not significance.
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RIPA lysis buffer. After washing, 5 x cooking buffer added samples were heated 95 C for 10 min and then the caspase-8 interacting proteins were detected in 10% SDS-PAGE for immunoblot analysis. 2.9. Quantitative PCR (qPCR) Total RNA was isolated using the Trizol reagent (Life Technologies; Gaithersburg, MD), and we obtatined the cDNA was prepared using M-MLV reverse transcriptase (Gibco-BRL; Gaithersburg, MD). The cDNA and forward/reverse primers were added to 2 TAKARA SYBR Fast master mix, and reactions were performed on LightCycler 480 real-time amplification instrument (Roche, Basel, Switzerland). The following primers were used for the amplification of human GALNT14 and actin: GALNT14 (forward) 50 -CAT TGC TGT CGG TCA TCT -30 and (reverse) 50 -TGT CAG TCA CCT TCT TC -30 . actin (forward) 50 - CTA CAA TGA GCT GCG TGT G-30 and (reverse) 50 -TGG GGT GTT GAA GGT CTC -30 . Threshold cycle number (Ct) of each gene was calculated, and actin was used as reference genes. Delta-delta Ct values of genes were presented as relative fold induction. 2.10. Small interfering RNA (siRNA) The green fluorescent protein (GFP) (control) siRNA and GALNT14 siRNA were purchased from Bioneer (Daejeon, Korea). Cells were transfected with siRNA using Oligonucleotides Reagent (Invitrogen). 2.11. Endo H digestion
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induces caspase-dependent apoptotic cell death. As shown in Fig. 1F, we observed that combined treatment induced apoptosis in other renal carcinoma cells (ACHN) and lung carcinoma cells (A549). However, combined treatment did not affect on apoptosis in normal mouse kidney cells (TCMK-1) (Fig. 1G). 3.2. Effect of oridonin on expression levels of apoptosis-related proteins To elucidate the underlying mechanisms of TRAIL sensitization of RCC cells by oridonin, we examined the effect of oridonin on expression levels of apoptosis-related proteins. Oridonin did not change the expression levels of apoptosis-related proteins (antiapoptotic proteins: Bcl-xL, Bcl-2, Mcl-2, cIAP2, XIAP, Survivin, cFLIP, and pro-apoptotic proteins: Bax, DR4, DR5, p53) and phosphorylation status and expression levels of STAT1/3 and MAPKs (Fig. 2A and B). Since Bax activation and mitochondrial membrane potential (MMP) play a critical role in mitochondria-mediated intrinsic apoptosis [17,18], we examined the effect of oridonin on Bax activation and loss-of-MMP. Oridonin did not induce Bax activation and loss-of-MMP (Fig. 2C and D). Furthermore, to identify the molecular mechanisms leading to apoptotic cell death in combined treatment with oridonin and TRAIL, we tested expression levels of apoptosis-related proteins by combined treatment. As shown in Fig. 3, oridonin plus TRAIL also did not alter tested any proteins. These data suggest that the apoptotic effect of oridonin plus TRAIL on RCC cells is not associated with mitochondriamediated intrinsic apoptotic pathway.
Samples were incubated with 1500 U/ml Endo H (New England Biolabs, Ipswich, MA) according to the manufacturer's instruction. After 5 h at 37 C, the digested samples were subjected to Western blot analysis. 2.12. Statistical analysis We repeated experiments at least three times in our studies, and all data were represented as the means. Statistical analysis was performed by a one-way ANOVA and post hoc comparisons (Student-NewmaneKeuls) using the SPSS (Statistical Package for the Social Sciences, version 22.0) (SPSS Inc.; Chicago, IL). The p-values <0.05 were considered significant. 3. Results 3.1. Oridonin enhances TRAIL-induced apoptosis in human renal carcinoma caki cells and A549 cells Renal cell carcinoma (RCC) is the most common cancer found within the kidney, and it is resistant to chemotherapeutic agents, compared with other cancers [16]. Therefore, to find the way to increase sensitivity of RCC cells to anti-cancer drugs could enhance cell death. To determine the sensitizing effects of oridonin on TRAIL-induced apoptosis, clear cell RCC Caki cells were treated with oridonin alone, TRAIL alone and combined treatment. Combined treatment with oridonin and TRAIL increased the sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP) (Fig. 1A), and showed typical apoptotic morphologies (Fig. 1B) and chromatin condensation (Fig. 1C). However, single treatment with oridonin or TRAIL did not induce apoptosis. Next, we investigated whether combined treatment induces caspase-dependent apoptosis. Combined treatment significantly induced caspase-3 activation (Fig. 1D). PARP cleavage and induction of sub-G1 population were inhibited by pan-caspase inhibitor, z-VAD-fmk (Fig. 1E). Our data indicated that combined treatment with oridonin and TRAIL
Fig. 3. Combined treatment with oridonin plus TRAIL did not change the expression of apoptosis-related proteins. Caki cells were treated with 5 mM oridonin in the absence of presence of 50 ng/ml TRAIL for 24 h. The protein expression was determined by Western blotting.
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3.3. Enhancement of TRAIL-induced apoptosis by oridonin is associated with DISC formation and DR5 glycosylation DISC formation plays a critical role in death receptor-mediated extrinsic and/or intrinsic apoptosis pathway [1]. We examined whether oridonin affects the DISC formation. An immunoprecipitation assay using an anti-caspase 8 antibody showed FADD, cleaved forms of caspase-8 and DR5 in the isolated DISC after oridonin plus TRAIL treatment (Fig. 4A). The peptidyl O-glycosyltransferase GALNT14-mediated DR5 O-glycosylation promotes sensitivity of cancer cells to TRAIL-induced apoptosis [6]. Therefore, we examined whether upregulation of DR5 glycosylation contributes to oridonin plus TRAIL-induced apoptosis. Oridonin induced upregulation of GALNT-14 protein expression in a dose- and timedependent manner in Caki and A549 cells (Fig. 4B). Also, oridonin upregulated GALNT14 mRNA expression in a time-dependent manner (Fig. 4C). Combined treatment induced glycosylated-high molecular weight DR5 form in Caki and A549 cells (Fig. 4D). Treatment of these cells with GALNT14 siRNA or digestion of oridonin plus TRAIL-treated Caki cell extracts with EndoH reduced expression levels of glycosylated DR5 (Fig. 4E and F). Furthermore,
to investigate the significance of glycosylation of DR5 in sensitization of RCC cells to TRAIL by oridonin., we examined whether benzyl-a-GalNAc (a general inhibitor of O-glycosylation) inhibits oridonin plus TRAIL-mediated apoptosis. Benzyl-a-GALNAc inhibited apoptosis and PARP cleavage in oridonin plus TRAIL-treated Caki and A549 cells (Fig. 4G and H). Therefore, our data indicated that DISC formation and DR5 glycosylation plays a critical role in sensitization of RCC cells to TRAIL by oridonin. 4. Discussions In present study, we showed mechanistic evidence that oridonin enhanced TRAIL-induced apoptosis in cancer cells. Oridonin enhanced DISC formation without affecting downstream intracellular apoptosis-related proteins. Oridonin induced DR5 glycosylation in a GALNT14-dependent manner, and a pharmacological inhibitor of glycosylation (benzyl-a-GalNAc) inhibited orionin plus TRAIL-induced apoptosis. Oridonin inhibited the expression of several anti-apoptotic proteins, such as Bcl-2, Mcl-1, and XIAP in hepatocellular cancer cells [19]. Wu et al. reported that a novel oridonin analog
Fig. 4. Oridonin enhances DISC formation via upregulation of DR5 glycosylation. (A) Caki cells were treated with 5 mM oridonin in the absence of presence of 50 ng/ml TRAIL for 24 h. After immunoprecipitation with anti-caspase 8 antibody, the protein expression was determined by Western blotting. (B) Caki and A549 cells were treated with the indicated concentrations of oridonin for 24 h (left panel) or the indicated times (right panel). The protein expression was determined by Western blotting. (C) Caki cells were treated with 5 mM oridonin for the indicated time periods. The mRNA expression was determined by qPCR. (D) Caki and A549 cells were treated with the indicated concentrations of oridonin in the absence of presence of 50 ng/ml TRAIL for 24 h. The protein expression was determined by Western blotting. (E) Caki cells were transfected with control siRNA (siCont) and GALNT14 (siGALNT14), and the treated with 5 mM oridonin plus 50 ng/ml TRAIL for 24 h. The protein expression was determined by Western blotting. (F) Caki cells were treated with 5 mM oridonin plus 50 ng/ml TRAIL for 24 h. After treatment, lysates were subjected to Endo H, and then protein expression was determined by Western blotting. (G and H) Caki (G) and A549 (H) cells were pretreated with the indicated concentrations of Benzyl-a-GalNAc for 30 min, and then treated with the indicated concentraions of oridonin plus 50 ng/ml TRAIL for 24 h. Flow cytometry was used to detect the sub-G1 population, and the protein expression was determined by Western blotting. The values in C, G, and H represent the mean ± SD of three independent experiments. *p < 0.05 compared to control. #p < 0.05 compared to the oridonin plus TRAIL.
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suppresses triple-negative breast cancer cells and tumor growth though the induction of DR5 [20]. In our study, oridonin did not affect expression levels of apoptosis-related proteins and death receptors (DR4 and DR5). Oridonin markedly enhanced the DISC formation in combined treated with oridonin and TRAIL (Fig. 4A). Previous study reported that O-glycosylation of DR5 is important for sensitivity to the TRAIL [6]. Wagner et al. found that expression of GALNT14 mRNA is markedly higher in TRAIL-sensitive pancreatic, NSCLC and melanoma cell lines, compared with resistant cell lines. Pharmacologic inhibition of O-glycosylation enzymes or siRNA knockdown of GALNT14 in sensitive cells reduces sensitivity to TRAIL, and GALNT14 overexpression in resistant cells increases glycosylation of DR5 and TRAIL-induced apoptosis. Glycosylation of DR5 by GALNT14 promotes ligand-induced clustering of DR5, which in turn increases DISC recruitment and caspase-8 activation [6]. In contrast, glycosylation of DR5 has no effect on TRAIL binding affinity and cell surface expression of DR5 [6]. In case of DR4, Dufour et al. reported that N-linked glycosylation plays a critical role for DR4 [21]. Oridonin-induced TRAIL sensitization of cancer cells was caused through a GALNT14-mediated DR5 glycosylation and upregulation of DISC formation. Oridonin induced GALNT14 mRNA and protein expression (Fig. 4B and C). Knockdown by GALNT14 siRNA or treatment with Endo H inhibited oridonin plus TRAIL-induced DR5 glycosylation (Fig. 4E and F). We demonstrated that inhibitor of O-glycosylation markedly inhibited oridonin plus TRAIL-induced apoptosis and PARP cleavage (Fig. 4G and H). Overall, our findings provide insights into the molecular mechanisms of TRAIL sensitization through which oridonin enhances the ability of O-glycosylation of DR5 through up-regulating GALNT14 expression. Nevertheless, how oridonin specifically regulates transcriptional activation of GALNT14 is a critical question for further development of TRAIL-based chemotherapeutic strategies. We found that the glycosylated DR5 band by oridonin alone was relatively low levels compared to the combination of oridonin plus TRAIL (Fig. 4D). There is one of possibility for this result. When TRAIL binds to DR5 at the membrane, it activates the downstream signal and then undergoes endocytosis. After endocytosis, DR5 is recycled or degraded by lysosome [22]. Alternatively, up-regulation of the ubiquitination by E3 ligase, such as c-Cbl, induces degradation by proteasome [23]. However, since the glycosylation prevents endocytosis of death receptor, DR5 remains in the membrane for a long time without degradation [21]. On the other hand, in the absence of ligand, glycosylated DR5 is mainly present in cytosol [24], and cytosolic protein is preferentially degraded by proteasome [25]. Therefore, it is likely that the band detection of glycosylated DR5 by oridonin in the absence of TRAIL was difficult. To confirm this possibility, we detected glycosylated DR5 in the presence of proteasome inhibitor, MG132. We identified the increased glycosylated DR5 by oridonin, which was similar to the level found in combined treatment with oridonin and TRAIL (data not shown). These results indicate that oridonin may improve treatment results of combined treatment with TRAIL in the future clinical application. Conflicts of interest The authors declare no conflicts of interest. Author contributions M.Y.J. K.-j.M and T.K.K. designed the project and wrote the paper. M.Y.J., S.U.S., S.M.W., and K.-j.M performed the experimental work in the study. H.S.B., G.M.H., S.C.K., and T.K.K. analyzed data and critically revised manuscript. All the authors read and approved the final manuscript.
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