Galangin sensitizes TRAIL-induced apoptosis through down-regulation of anti-apoptotic proteins in renal carcinoma Caki cells

Galangin, bioflavonoids, has been shown anti-cancer properties in various cancer cells. In this study, we investigated whether galangin could enhance TRAIL-mediated apoptosis in TRAIL resistant renal carcinoma Caki cells. Galangin alone and TRAIL alone had no effect on apoptosis, while combined treatment with galangin and TRAIL significantly induced apoptosis in renal carcinoma (Caki, ACHN and A498) but not normal cells (normal mouse kidney cells and human normal mesangial cells). Galangin induced down-regulation of Bcl-2 protein at the transcriptional level via inhibition of NF-κB activation but not p53 pathway. Furthermore, galangin induced down-regulation of cFLIP, Mcl-1 and survivin expression at the post-translational levels, and the over-expression of Bcl-2, cFLIP, Mcl-1 and survivin markedly reduced galangin-induced TRAIL sensitization. In addition, galangin increased proteasome activity, but galangin had no effect on expression of proteasome subunits (PSMA5 and PSMD4). In conclusion, our investigation suggests that galangin is a potent candidate for sensitizer of TRAIL resistant cancer cell therapy.


Results
Galangin enhances TRAIL-mediated apoptosis in human renal carcinoma. We examined whether galangin could sensitize TRAIL resistant Caki cells. In FACS analysis, combined treatment with TRAIL and galangin markedly increased sub-G1 population and PARP cleavage in a dose dependent manner, but no increase in treatment with TRAIL alone or galangin alone ( Fig. 1A and Supplementary Information Fig. S1). Next, we examined whether combined treatment with galangin and TRAIL have synergistic effects. Galangin plus TRAIL markedly reduced cell viability in various concentrations of galangin and TRAIL. The isobologram analysis suggested that combined treatment with galangin and TRAIL have synergistic effects (Fig. 1B). Combined treatment with galangin and TRAIL caused chromatin damaged in the nuclei (Fig. 1C), and cytoplasmic histone-associated DNA fragmentation (Fig. 1D). Furthermore, combination treatment with galangin and TRAIL induced caspase-2 and 3 activation ( Fig. 1E and Supplementary Information Fig. S2), and pan-caspase inhibitor (z-VAD) blocked galangin plus TRAIL-induced apoptosis and cleavage of PARP and caspase-3 (Fig. 1F). Next, we investigated whether the alteration in expression levels of apoptotic regulatory proteins might be associated  Supplementary Fig. S4). (b) Isoboles were obtained by plotting the combined concentrations of each drug required to produce 50% cell death. The straight line connecting the IC 50 values obtained for two agents when applied alone corresponds to an additivity of their independent effects. Values below this line indicate synergy, whereas values above this line indicate antagonism. (c-e) Caki cells were treated with 50 ng/ml of TRAIL with or without 30 μ M galangin for 24 h. The condensation and fragmentation of the nuclei were detected by 4' , 6'-diamidino-2-phenylindole staining (c). The cytoplasmic histone-associated DNA fragments were determined by a DNA fragmentation detection kit (d). Caspase activity was determined with colorimetric assays using caspase-3 (DEVDase) assay kit (e). (f) Caki cells were treated with 30 μ M of galangin and 50 ng/ml of TRAIL for 24 h in the presence or absence of 50 μ M z-VAD-fmk (zVAD). The sub G1 population was measured by flow cytometry. PARP and cleaved caspase-3 levels were determined by western blot analysis. Actin was used as a loading control. (cropped, fulllength blots are in Supplementary Fig. S4). (g) Caki cells were treated with indicated concentrations of galangin for 24 h. The protein level of apoptosis related factors were examined by western blot analysis, such as DR4, DR5, cFLIP, Bcl-2, Bcl-xL, Mcl-1, CIAP2, XIAP and survivin. Actin was used as a loading control. (cropped, full-length blots are in Supplementary Fig. S4). *p < 0.01 compared to the control. **p < 0.01 compared to the co-treatment of galangin and TRAIL.
Galangin down-regulates Bcl-2 expression at the transcriptional level through inhibition of NF-κB. We next explored the underlying mechanisms of galangin-mediated down-regulation of Bcl-2 expression. Expression levels of Bcl-2 protein and mRNA were down-regulated by galangin in a time-dependent manner ( Fig. 2A). To examine transcriptional regulation of Bcl-2, Caki cells were transfected with Bcl-2 promoter (Bcl-2/-3254) plasmid. Galangin down-regulated Bcl-2 promoter activity in a dose-dependent manner (Fig. 2B). Previous studies reported that tumor suppressor p53 is a negative regulator of Bcl-2 16 . Thus, we examined whether galangin modulates Bcl-2 transcriptional regulation through p53. We observed that pifithrin-α (p53 inhibitor) and p53 siRNA had no effect on down-regulation of Bcl-2 expression and apoptosis in galangin-treated Caki cells (Fig. 2C,D, and Supplementary Information Fig. S3). Furthermore, galangin did not alter the expression level of p53 protein (Fig. 2D). These data suggested that galangin-induced Bcl-2 down-regulation is not associated with the expression amount of p53. Next, we examined whether galangin inhibits NF-κ B-mediated transcriptional activity, since activation of NF-κ B induces Bcl-2 mRNA expression 17 . Galangin inhibited NF-κ B promoter activity (Fig. 2E). In addition, galangin-mediated inhibitory effect of Bcl-2 promoter was reversed in p65 overexpressed Caki cells (Fig. 2F). To investigate the importance of down-regulation of Bcl-2 expression on galangin plus TRAIL-induced apoptosis, Bcl-2 protein was over-expressed in Caki cells. Overexpression of Bcl-2 markedly inhibited galangin-induced increase of sub-G1 cell population and PARP cleavage (Fig. 2G). These results indicate that NF-κ B-dependent and p53-independent down-regulation of Bcl-2 may be involved in galangin plus TRAIL-mediated apoptosis.
Galangin decreased cFLIP at the post-transcriptional level in Caki cells. Next, we examined the mechanism of cFLIP down-regulation in galagin-treated cells. Since we observed that cFLIP protein but not mRNA level was down-regulated in galangin-treated Caki cells (Fig. 3A), we examined the effect of galangin on the protein stability of cFLIP. Combined treatment with cycloheximide (CHX) and galangin more rapidly degraded cFLIP protein (Fig. 3B). Previous studies reported that cFLIP was mainly degraded by ubiquitn-proteasome pathway 18 . Therefore, we assessed whether ubiquitin-proteasome system is involved in degradation of cFLIP by galangin treatment. Galangin-induced cFLIP down-regulation was inhibited by proteasome inhibitor (lactacystin) (Fig. 3C). cFLIP is degraded via the Cbl and Itch E3 ligase-mediated ubiquitin-proteasome pathways 18,19 . Therefore, we examined protein expression levels of Cbl and Itch in galangin treated cells. As shown in Fig. 3D, galangin did not change expression levels of both E3 ligases. Overexpression of cFLIP markedly inhibited galangin-induced increase of sub-G1 cell population and PARP cleavage (Fig. 3E). These data collectively support that down-regulation of c-FLIP by proteasome but not by Cbl and Itch E3 ligase involves galangin plus TRAIL-induced apoptosis. . Therefore, we investigated whether galangin induced proteasome activity. Galangin increased proteasome activity within 3 h, which sustained up to 24 h (Fig. 5A). Therefore, we examined expression levels of two critical proteasome subunits, 26S proteasome non-ATPase regulatory subunit 4 (PSMD4/S5a) and 20 S proteasome subunit alpha type 5 (PSMA5). However, galangin did not alter the expression levels of both proteins (Fig. 5B). Next, we investigated whether reactive oxygen species (ROS) is involved in galangin-mediated TRAIL sensitization. As shown in Fig. 5C,D, galangin did not induce ROS production, and ROS scavengers (NAC and GEE) had no effect on galangin plus TRAIL-induced apoptosis. Therefore, galangin-mediated TRAIL sensitization is independent of ROS signaling.
Galangin induces TRAIL-mediated apoptosis in other renal cell carcinoma, but not normal cells. The effect of galagin on TRAIL sensitization was examined in other renal carcinoma (ACHN and A498 cells) and normal cells. As shown in Fig. 6A,B, combined treatment with galangin and TRAIL markedly increased sub-G1 population and PARP cleavage in both cell lines. However, combined treatment with galangin plus TRAIL no produced morphological changes and induction of the sub-G1 population in human mesangial cells (MC) and mouse renal tubular epithelial (TMCK-1) cells (Fig. 6C,D). These data suggest that galangin enhances TRAIL-mediated apoptosis in renal cancer cells, but not in normal cells.

Discussion
To overcome TRAIL resistance in various cancer cells, identification of sensitizer for TRAIL is required for the establishment of more effective TRAIL-based cancer therapies. In this study, we show that galangin sensitizes  Supplementary Fig. S5). (g) Vector cells harboring empty vector (Caki/vec) and Bcl-2 overexpressing cells (Caki/Bcl-2) were treated with 50 ng/ml TRAIL and 30 μ M galangin for 24 h. The sub G1 population was measured by flow cytometry. The protein levels of PARP and Bcl-2 were determined by western blot analysis. Actin was used as a loading control. (cropped, full-length blots are in Supplementary Fig. S5). *p < 0.01 compared to the control. **p < 0.01 compared to the galagin in Caki/vector. Caki cells to TRAIL-mediated apoptosis through down-regulation of Bcl-2, cFLIP, Mcl-1 and survivin expression. Down-regulation of Bcl-2 expression is probably associated with inhibition of NF-κ B transcriptional activity. In addition, galangin increased proteasome activity, and induced down-regulation of cFLIP, Mcl-1 and survivin expression at the post-translational levels. Furthermore, galangin enhanced TRAIL-induced apoptosis in other renal carcinoma, but not normal cells. Therefore, our data suggest that galangin could be an attractive candidate for the TRAIL sensitizer.
Previously several reports have shown that galangin induces apoptosis in various cancer cells by different pathways [20][21][22] . Galangin inhibits proliferation of hepatocellular carcinoma cells by inducing endoplasmic reticulum stress, activating AMPK and mitochondrial dysfunction via up-regulation of Bax and down-regulation of Bcl-2 [20][21][22] . High concentration of galangin (130 μ M) induced autophagy through upregulation of p53 in HepG2 cells 15 . Transcription factor p53 has been shown to negatively regulate Bcl-2 transcription levels 16 . However, in our study, low concentration of galangin (30 μ M) did not induce up-regulation of p53 (Fig. 2D). In addition, siRNA-mediated p53 knockdown and pretreatment with pifithrin-α (a p53 inhibitor) did not reverse galangin-mediated  Supplementary Fig. S6). (e) Vector cells harboring empty vector (Caki/vec) and cFLIP overexpressing cells (Caki/cFLIP) were treated with 50 ng/ml TRAIL and 30 μ M galangin for 24 h. The sub G1 population was measured by flow cytometry. The protein levels of PARP and cFLIP were determined by western blot analysis. Actin was used as a loading control. (cropped, full-length blots are in Supplementary Fig. S6).
Galangin induced down-regulation of cFLIP, Mcl-1 and survivin expression at the post-translational levels (Figs 3 and 4). These proteins are mainly degraded by ubiquintin-proteasome pathway. We found that proteasome inhibitor (lactacystin) markedly reversed galangin-mediated down-regulation of cFLIP, Mcl-1 and survivin expression (Figs 3 and 4) and proteasome activity was increased by treatment with galangin (Fig. 5). E3 ligase, such as Cbl and Itch, induces ubiquitination of c-FLIP and induces its proteasomal degradation. Chang et al. reported that TNFα -mediated JNK activation accelerates turnover of the cFLIP through activation of the E3 ubiquitin ligase Itch 19 . Mcl-1, anti-apoptotic Bcl-2 family members, is regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Mcl-1 is known to be a short-lived protein 24 . Mcl-1 is modulated by the ubiquitin-proteasome system such as four different E3 ubiquitin-ligases (Mule, SCFβ -TrCP, SCFFbw7 and Trim17) and one deubiquitinase (USP9X) 25 . Recently, Ren et al. have suggested that E3 ubiquitin ligases β -TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation 26 . Survivin protein is tightly post-translationally modified by ubiquitylation and phosphorylation 27 . Survivin is also regulated by XIAP-XAF1 complex 28 . Tecleab et al. have reported that depletion of K-Ras promotes proteasome degradation of survivin 29 . Proteasome subunits of PSMA5 and PSMD4/S5a play a critical role in proteasomal activity. However, galangin did not alter expression levels of proteasome subunits and E3 ligases in Caki cells (Figs 3D and 5B). Although we  Supplementary Fig. S7).
did not prove the regulatory mechanism of galangin in down-regulation of cFLIP, Mcl-1 and survivin, we suggest that galangin could potentially modulate the protein stability. Taken together, our results suggest that galangin sensitizes TRAIL-induced apoptosis through the down-regulation of anti-apoptotic proteins (Bcl-2, cFLIP, Mcl-1 and survivin) expression. Therefore, galangin might be a potential sensitizer for the treatment of TRAIL-resistant renal cancer.  Western blot analysis. For the Western blotting experiments, the cells were washed with cold PBS and lysed on ice in modified RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM Na 3 VO 4 , and 1 mM NaF) containing protease inhibitors (100 μ M phenylmethylsulfonyl fluoride, 10 μ g/ml leupeptin, 10 μ g/ml pepstatin, and 2 mM EDTA). The lysates were centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatant fractions were collected. The proteins were separated by SDS-PAGE electrophoresis and transferred to Immobilon-P membranes. The specific proteins were detected using an enhanced chemiluminescence (ECL) Western blotting kit according to the manufacturer's instructions.

Materials and Methods
Determination of synergy and cell viability assay. The possible synergistic effect of galangin and TRAIL was evaluated using the isobologram method. In brief, the cells were treated with different concentrations of galangin and TRAIL alone or in combination. After 24 h, XTT assay was employed to measure the cell viability using WelCount Cell Viability Assay Kit (WelGENE, Daegu, Korea). In brief, reagent was added to each well and was then measured with a multi-well plate reader (at 450 nm/690 nm). Relative survival was assessed and the concentration effect curves were used to determine the IC 50 (the half-maximal inhibitory concentration) values for each drug alone and in combination with a fixed concentration of the second agent 30 . 4′,6′-Diamidino-2-phenylindole staining (DAPI) for nuclei condensation and fragmentation. To examine cellular nuclei, the cells were fixed with 1% paraformaldehyde on glass slides for 30 min at room temperature. After the fixation, the cells were washed with PBS and a 300 nM 4′ ,6′ -diamidino -2-phenylindole solution (Roche, Mannheim, Germany) was added to the fixed cells for 5 min. After the nuclei were stained, the cells were examined by fluorescence microscopy. Asp-Glu-Val-Asp-ase (DEVDase) Activity Assay. To evaluate DEVDase activity, cell lysates were prepared after their respective treatments with TRAIL in the presence or absence of galangin. Assays were performed in 96-well microtiter plates by incubating 20 μ g of cell lysates in 100 μ l of reaction buffer (1% NP-40, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol) containing a caspase substrate [Asp-Glu-Val-Asp-chromophore-p-nitroanilide (DVAD-pNA)] at 5 μ M. Lysates were incubated at 37 °C for 2 h. Thereafter, the absorbance at 405 nm was measured with a spectrophotometer.

Construction of Bcl-2, cFLIP and Mcl-1 stable cells. The Caki cells were stably transfected with
pMAX-Bcl-2 (provided by Dr. Rakesh Srivastava, NIH/NIA), pcDNA 3.1-cFLIP, pcDNA 3.1-Mcl-1 or control plasmid pcDNA 3.1 vector using LipofectAMINE2000 as recommended by the manufacturer (Invitrogen Carlsbad, CA). After 48 h of incubation, transfected cells were selected in cell culture medium containing 700 μ g/ ml G418 (Invitrogen). After 2 or 3 weeks, to eliminate the possibility of clonal differences between the generated stable cell lines, the pooled clones were tested for Bcl-2, cFLIP(L) and Mcl-1 expression by immunoblotting, and the cells were used in this study.
DNA transfection and luciferase assay. Transient transfection was performed in 6-well plates. One day before the transfection, Caki cells were plated at approximately 60 to 80% confluence. The NF-κ B promoter plasmid or Bcl-2/-3254 promoter plasmid was transfected into the cells using Lipofectamine TM 2000 (Invitrogen; Carlsbad, CA). To assess the promoter-driven expression of the luciferase gene, the cells were collected and disrupted by sonication in lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM EDTA, 1% Triton X-100, and 10% glycerol), and aliquots of the supernatant were used to analyze the luciferase activity according to the manufacturer's instructions (Promega; Madison, WI).

Measurement of reactive oxygen species (ROS). Intracellular accumulation of ROS was determined
using the fluorescent probes 2′ , 7′ -dichlorodihydrofluorescein diacetate (H 2 DCFDA). H 2 DCFDA is commonly used to measure ROS generation. Caki cells were treated with galangin, and then cells were stained with the fluorescent dye H 2 DCFDA for an additional 10 min. Then, cells were trypsinized and resuspended in PBS, and fluorescence was measured at specific time intervals with a flow cytometer (Becton-Dickinson; Franklin Lakes, NJ, USA).
Densitometry. The band intensities were scanned and quantified using the gel analysis plugin for the open source software ImageJ 1.46 (Imaging Processing and Analysis in Java; http://rsb.info. nih.gov/ij). Statistical analysis. The data were analyzed using a one-way ANOVA followed by post-hoc comparisons (Student-Newman-Keuls) using the Statistical Package for Social Sciences version 22.0 (SPSS Inc., Chicago, IL, USA).