Antipsychotic agent thioridazine sensitizes renal carcinoma Caki cells to TRAIL-induced apoptosis through reactive oxygen species-mediated inhibition of Akt signaling and downregulation of Mcl-1 and c-FLIP(L)

Thioridazine has been known as an antipsychotic agent, but it also has anticancer activity. However, the effect of thioridazine on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitization has not yet been studied. Here, we investigated the ability of thioridazine to sensitize TRAIL-mediated apoptosis. Combined treatment with thioridazine and TRAIL markedly induced apoptosis in various human carcinoma cells, including renal carcinoma (Caki, ACHN, and A498), breast carcinoma (MDA-MB231), and glioma (U251MG) cells, but not in normal mouse kidney cells (TMCK-1) and human normal mesangial cells. We found that thioridazine downregulated c-FLIP(L) and Mcl-1 expression at the post-translational level via an increase in proteasome activity. The overexpression of c-FLIP(L) and Mcl-1 overcame thioridazine plus TRAIL-induced apoptosis. We further observed that thioridazine inhibited the Akt signaling pathway. In contrast, although other phosphatidylinositol-3-kinase/Akt inhibitors (LY294002 and wortmannin) sensitized TRAIL-mediated apoptosis, c-FLIP(L) and Mcl-1 expressions were not altered. Furthermore, thioridazine increased the production of reactive oxygen species (ROS) in Caki cells, and ROS scavengers (N-acetylcysteine, glutathione ethyl ester, and trolox) inhibited thioridazine plus TRAIL-induced apoptosis, as well as Akt inhibition and the downregulation of c-FLIP(L) and Mcl-1. Collectively, our study demonstrates that thioridazine enhances TRAIL-mediated apoptosis via the ROS-mediated inhibition of Akt signaling and the downregulation of c-FLIP(L) and Mcl-1 at the post-translational level.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates the extrinsic apoptotic pathway. TRAIL binds to death receptors (DRs; DR4 and DR5) and then recruits caspase-8 into a death-inducing signaling complex (DISC). The activation of caspase-8 directly activates caspase-3 or induces mitochondria-mediated caspase-3 activation, which results in apoptosis. Previous studies have shown that cancer cells highly express DRs, whereas normal cells highly express the decoy receptors. 13 Moreover, the level of cellular FLICE-inhibiting protein (c-FLIP(L)), which inhibits caspase-8 recruitment to DISC, is higher in normal cells than in tumor cells. 14 Accordingly, TRAIL had been known as a promising anticancer drug. 15 However, many cancer cells represent resistance to TRAIL-mediated apoptosis via multiple mechanisms. The downregulation of DRs and/or upregulation of antiapoptotic proteins (c-FLIP(L), the Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1) and the inhibitor of apoptosis proteins (IAPs)) have been demonstrated to be associated with TRAIL resistance. [16][17][18][19][20] Previous studies have shown that TRAILresistant cancer cells can be sensitized by combination treatment. Thus, the development of TRAIL sensitizers is needed to increase sensitivity against TRAIL.
In the present study, we assessed the sensitizing effects of thioridazine on TRAIL-induced apoptosis in human renal carcinoma Caki cells. Combination treatment with TRAIL and thioridazine could facilitate the development of an effective strategy for cancer treatment.

Results
Thioridazine sensitizes to TRAIL-mediated apoptosis in human renal carcinoma, breast carcinoma, and glioma cells, but not normal cells. Thioridazine has anticancer effects on multiple cancer cells. [5][6][7][8][9][10] Therefore, we investigated the ability of thioridazine to sensitize human renal carcinoma Caki cells to TRAIL. To determine whether thioridazine plus TRAIL induce apoptosis, an FACS analysis to measure the DNA content and western blotting to detect the cleavage of PARP, a substrate of caspase-3, were performed. Thioridazine or TRAIL did not affect apoptosis, but a combined treatment with thioridazine plus TRAIL markedly increased the sub-G1 population and PARP cleavage (Figure 1a). In addition, thioridazine plus TRAIL induced chromatin damage in the nuclei ( Figure 1b) and cytoplasmic histone-associated DNA fragments (Figure 1c). Combined treatment with thioridazine and TRAIL increased caspase-3 activation (Figure 1d), and z-VAD-fmk (z-VAD), a pan-caspase inhibitor, markedly inhibited thioridazine plus TRAIL-induced apoptosis (Figure 1e, upper panel). The cleavage of PARP and caspase-3 was also blocked by z-VAD treatment (Figure 1e, lower panel). To identify the mechanisms underlying sensitization to TRAIL by thioridazine, we examined the expression levels of apoptosis-related proteins, including the Bcl-2 family, IAP family, constituents of DISC (FADD and cellular FLICE-inhibitory protein (c-FLIP(L)), and DRs in thioridazine-treated Caki cells. Thioridazine markedly downregulated c-FLIP(L) and Mcl-1 expression, but the expression of other proteins remained unchanged (Figure 1f). These results indicate that thioridazine sensitizes Caki cells to TRAIL-mediated apoptosis.
Next, we used other human renal carcinoma (ACHN and A498), human breast carcinoma (MDA-MB231), and human glioma (U251MG) cells to investigate the anticancer effect of thioridazine on other carcinoma cells. As shown in Figure 2a, combined treatment with thioridazine plus TRAIL induced apoptosis and PARP cleavage in ACHN, A498, MDA-MB231, and U251MG cells. In contrast, thioridazine plus TRAIL induced neither cellular shrinkage nor apoptosis in normal cells (mouse kidney cells (TMCK-1) and human mesangial cells), whereas cellular shrinkage and blebbing was detected in Caki cells (Figure 2b). Furthermore, thioridazine induced the downregulation of c-FLIP(L) and Mcl-1 expression in carcinoma cells, but not in normal cells (Figure 2c). These data indicate that thioridazine can sensitize cancer cells other than Caki cells to TRAIL. Previous studies reported that c-FLIP(L) and Mcl-1 are degraded by the proteasome-ubiquitin system. 21,22 Therefore, we investigated whether the downregulation of c-FLIP(L) and Mcl-1 is associated with proteasome activity. First, we examined the ability of proteasome inhibitors (MG132 and lactacystin) to reverse the thioridazine-mediated downregulation of c-FLIP(L) and Mcl-1 expression. As shown in Figure 4a, proteasome inhibitors blocked the downregulation of c-FLIP(L) and Mcl-1 expression. Next, we measured the chymotrypsin-like activity of proteasome with Suc-Leu-Leu-Val-Tyr-AMC as the proteasome substrate to investigate the ability of thioridazine to increase proteasome activity.
As expected, thioridazine increased the chymotrypsin-like activity of the proteasome within 12 h (Figure 4b). We used the proteasome sensor vector, ZsProSensor-1, to confirm the induction of proteasome activity by thioridazine. The vector encodes a destabilized green fluorescence protein (ZsGreen), which is rapidly degraded by proteasomes. For example, when proteasomes are inhibited, the fluorescent protein accumulates, which allows green fluorescence to be detected by fluorescence microscopy. Importantly, thioridazine decreased green fluorescence, whereas MG132 increased green fluorescence (Figure 4c). We further examined the ability of thioridazine to modulate the protein expression of two critical proteasome subunits: 20S proteasome subunit alpha type 5 (PSMA5) and 19S proteasome non-ATPase regulatory subunit 4 (PSMD4/S5a). 23 However, the increase in the proteasome activity was not associated with PSMA5 and PSMD4/S5a expression in thioridazinetreated Caki cells. Next, we investigated the importance of the downregulation of c-FLIP(L) and Mcl-1 due to proteasome activation to thioridazine plus TRAIL-mediated apoptosis. When proteasome inhibitors (MG132 and lactacystin) reversed the downregulation of c-FLIP(L) and Mcl-1, the sub-G1 population and PARP cleavage were markedly inhibited in thioridazine plus TRAIL-treated cells (Figure 4e). These data suggest that the downregulation of c-FLIP(L) and Mcl-1 expression due to the induction of proteasome activity is critical in thioridazine plus TRAIL-mediated apoptosis.
Inhibition of Akt signaling is involved in thioridazinemediated TRAIL sensitization. Thioridazine has been known to exert anticancer effects via the inhibition of the PI3K/Akt signaling pathways. 5,6 To examine the involvement of the PI3K/Akt pathway in thioridazine-mediated TRAIL sensitization, we tested the effect of PI3K/Akt inhibitors (LY294002 and wortmannin) on c-FLIP(L) and Mcl-1 expression. Although LY294002 and wortmannin, similar to . The cytoplasmic histoneassociated DNA fragments were determined by a DNA fragmentation detection kit (c). Caspase activities were determined with colorimetric assays using caspase-3 (DEVDase) assay kits (d). (e) Caki cells were treated with 10 mM thioridazine plus 50 ng/ml TRAIL for 24 h in the presence or absence of 50 mM z-VAD-fmk (z-VAD). The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, procaspase-3, cleaved caspase-3, and actin were determined by western blotting. The level of actin was used as a loading control. (f) Caki cells were treated with the indicated concentrations of thioridazine for 24 h. The protein expression levels of DR5, FADD, c-FLIP(L), XIAP, Bcl-2, Bcl-xL, Mcl-1, Bax, Bim, Puma, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a, c, d, and e represent the mean±S.D. from three independent samples. *Po0.001 compared with the thioridazine treatment alone. # Po0.001 compared with the co-treatment of thioridazine and TRAIL. The data represent three independent experiments thioridazine, markedly inhibited Akt phosphorylation within 3 h, LY294002 and wortmannin did not reduce the c-FLIP(L) and Mcl-1 expression levels ( Figure 5a). The present study demonstrates that thioridazine, unlike LY294002 and wortmannin, exhibits an additional activity in downregulating c-FLIP(L) and Mcl-1 expression beyond the inhibition of Akt signaling. Therefore, we hypothesized that thioridazine could be a stronger TRAIL sensitizer than LY294002 and wortmannin. Our data indicated that the increased sensitivity to TRAIL-mediated apoptosis was most pronounced in thioridazine-treated cells ( Figure 5b). These data collectively suggest that thioridazine, via the downregulation of c-FLIP(L) and Mcl-1 expression, could serve as a more effective agent to induce TRAIL-mediated apoptosis.
Thioridazine increased ROS production. The effect of thioridazine on reactive oxygen species (ROS) production is controversial. Thioridazine increased the levels of ROS in myelin and mitochondria in rats, 24 but also had antioxidant activity via an interaction with the inner membrane of mitochondria in rat liver mitochondria. 25 We examined the ability of thioridazine to increase ROS production in Caki cells. As shown in Figures 6a and b, thioridazine markedly induced intracellular ROS production, and pretreatment with ROS scavengers (N-acetyl-L-cysteine (NAC), glutathione ethyl ester (GEE), and trolox) inhibited the increase in the sub-G1 population and PARP cleavage in thioridazine plus TRAIL-treated cells. Furthermore, all ROS scavengers reversed the downregulation of c-FLIP(L) and Mcl-1 and prevented the inhibition of Akt phosphorylation in thioridazine plus TRAIL-mediated cells. As shown in Figures 3 and 4, both c-FLIP(L) and Mcl-1 expression were downregulated at the post-translational levels via a proteasome degradation pathway. Therefore, Caki cells were pretreated with ROS scavengers followed by the addition of thioridazine to further examine the association between ROS and the induction of

Discussion
In this study, we demonstrated the mechanism that underlies the sensitization to TRAIL-mediated apoptosis due to thioridazine. Thioridazine induced the downregulation of c-FLIP(L) and Mcl-1 expressions at the post-translational levels via the upregulation of proteasome activity and decreased Akt phosphorylation. In addition, thioridazine increased intracellular ROS production, which is associated with the induction of proteasome activity and inhibition of Akt signaling. Therefore, our results suggest that thioridazine increased TRAIL-mediated apoptosis via the ROS-mediated inhibition of Akt signaling and downregulation of Mcl-1 and c-FLIP(L) (Figure 6d). These findings support that thioridazine could be an attractive drug for TRAIL sensitization.
Thioridazine is well known to exert anticancer effects via the inhibition of the PI3K/Akt singling pathway. 5,6 Previous studies reported that PI3K/Akt activation protected human leukemia HL60 cells from TRAIL-mediated apoptosis, 26 and the inhibition of PI3K/Akt augmented TRAIL sensitization in human leukemia, 27 neuroblastoma, 28 and colon cancer cells. 29 In our study, thioridazine also inhibited Akt phosphorylation (Figure 5a). Previous studies reported that the PI3K/Akt signaling pathway is associated with c-FLIP(L) and Mcl-1 expression in murine B lymphocytes and ovarian cancer cells, respectively. 30,31 In addition, PI3K/Akt modulates c-FLIP(L) and Mcl-1 protein stability. Moumen et al. 32 reported that PI3K/Akt maintains c-FLIP(L) protein stability via Met signaling in hepatocytes, and granulocyte-macrophage colony-stimulating factor enhances Mcl-1 stability via the activation of PI3K/Akt in neutrophils. 33 However, other PI3K/ Akt inhibitors (LY294002 and wortmannin) did not affect or downregulate c-FLIP(L) and Mcl-1 expression (Figure 5a) or the induction of proteasome activity (data not shown). The thioridazine-mediated downregulation of c-FLIP(L) and Mcl-1 expression is probably independent of PI3K/Akt signaling in human renal carcinoma Caki cells ( Figure 5). Thus, thioridazine likely acts as a stronger sensitizer than LY294002 and wortmannin (Figure 5b).
ROS are important signaling molecules that modulate cellular responses. Thioridazine markedly increased ROS production (Figure 6a), and ROS was associated with thioridazine plus TRAIL-mediated apoptosis (Figure 6b). Furthermore, we detected that ROS modulated the downregulation of c-FLIP(L) and Mcl-1 expression (Figure 6b). According to previous reports, ROS have dual positive and negative functions on proteasome activity. In neutrophils and hepatocytes, ROS inhibited proteasome activity. 34,35 In contrast, ROS increased proteasome activity in skeletal muscle myotubes and lens epithelial cells. 36,37 In our study, the induction of proteasome activity by thioridazine depended on ROS production, and ROS scavengers reversed thioridazine-mediated c-FLIP(L) and Mcl-1 downregulation (Figure 6b). However, the mechanism by which thioridazine modulates proteasome activity is unclear. We examined the effect of thioridazine on the protein expression of two critical proteasome subunits: PSMA5 and PSMD4/S5a. 23 However, thioridazine did not alter either protein (Figure 4d). Therefore, the mechanism by which thioridazine modulates proteasome activity requires further evaluation.
Taken together, our results suggest that thioridazine sensitizes cancer cells, but not normal cells, to TRAILmediated apoptosis via the ROS-mediated inhibition of Akt signaling and downregulation of c-FLIP(L) and Mcl-1 Western blot analysis. For the western blotting experiments, the cells were washed with cold phosphate-buffered saline (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 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml pepstatin, and 2 mM EDTA). The lysates were centrifuged at 10 000 Â g for 10 min at 4 1C, and the supernatant fractions were collected. The proteins were separated by SDS-PAGE electrophoresis and transferred to Immobilon-P membranes (Millipore Corporation, Billerica, MA, USA). The specific proteins were detected using an enhanced chemiluminescence western blotting kit according to the manufacturer's instructions. 4 0 ,6 0 -Diamidino-2-phenylindole staining 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 fixation, the cells were washed with PBS and a 300 nM 4 0 ,6 0 -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.
The DNA fragmentation assay. The cell death detection ELISA plus kit (Boerhringer Mannheim; Indianapolis, IN, USA) was used to determine the level of apoptosis by detecting fragmented DNA within the nuclei of thioridazine-treated cells, TRAIL-treated cells, or cells that had been treated with a combination of thioridazine and TRAIL. Briefly, each culture plate was centrifuged for 10 min at 200 Â g, the supernatant was removed, and the cell pellet was lysed for 30 min. The plate was then centrifuged again at 200 Â g for 10 min, and the supernatant that contained the cytoplasmic histone-associated DNA fragments was collected and incubated with an immobilized anti-histone antibody. The reaction products were incubated with a peroxidase substrate for 5 min and measured by spectrophotometry at 405 nm and 490 nm (reference wavelength) with a microplate reader. The signals in the wells containing the substrate alone were subtracted as the background.
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 thioridazine. Assays were performed in 96-well microtiter plates by incubating 20 mg of cell lysates in 100 ml of reaction buffer (1% NP-40, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol) 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; ttp://rsb.info.nih.gov/ij).
Statistical analysis. The data were analyzed using a one-way ANOVA and post-hoc comparisons (Student-Newman-Keuls) using the Statistical Package for Social Sciences 8.0 software (SPSS Inc., Chicago, IL, USA).