Thioridazine enhances sensitivity to carboplatin in human head and neck cancer cells through downregulation of c-FLIP and Mcl-1 expression

Carboplatin is a less toxic analog of cisplatin, but carboplatin also has side effects, including bone marrow suppression. Therefore, to improve the capacity of the anticancer activity of carboplatin, we investigated whether combined treatment with carboplatin and thioridazine, which has antipsychotic and anticancer activities, has a synergistic effect on apoptosis. Combined treatment with carboplatin and thioridazine markedly induced caspase-mediated apoptosis in head and neck squamous cell carcinoma (AMC-HN4) cells. Combined treatment with carboplatin and thioridazine induced downregulation of Mcl-1 and c-FLIP expression. Ectopic expression of Mcl-1 and c-FLIP inhibited carboplatin plus thioridazine-induced apoptosis. We found that augmentation of proteasome activity had a critical role in downregulation of Mcl-1 and c-FLIP expression at the post-translational level in carboplatin plus thioridazine-treated cells. Furthermore, carboplatin plus thioridazine induced upregulation of the expression of proteasome subunit alpha 5 (PSMA5) through mitochondrial reactive oxygen species (ROS)-dependent nuclear factor E2-related factor 2 (Nrf2) activation. In addition, combined treatment with carboplatin and thioridazine markedly induced apoptosis in human breast carcinoma (MDA-MB231) and glioma (U87MG) cells, but not in human normal mesangial cells and normal human umbilical vein cells (EA.hy926). Collectively, our study demonstrates that combined treatment with carboplatin and thioridazine induces apoptosis through proteasomal degradation of Mcl-1 and c-FLIP by upregulation of Nrf2-dependent PSMA5 expression.

transcriptional activity. However, oxidative stress modifies the cysteine residue of Keap-1, and then, Nrf2 is released. 29 The translocation of Nrf2 from the cytosol to the nucleus induces gene expression by binding to the antioxidant response element (ARE). 29 In our study, we investigated whether combined treatment with carboplatin and thioridazine induces apoptosis and examined the molecular mechanisms of apoptosis in head and neck squamous cell carcinoma (AMC-HN4) cells.

Results
Combined treatment with carboplatin and thioridazine induces apoptosis. To determine whether there is an apoptotic effect between carboplatin and thioridazine, AMC-HN4 cells were treated with carboplatin in the absence or presence of thioridazine. Carboplatin alone and thioridazine alone had no effect on apoptosis, but combined treatment with carboplatin and thioridazine induced apoptosis and PARP cleavage, which is a substrate of caspase-3 ( Figure 1a). Next, we investigated whether combined treatment with carboplatin and thioridazine had synergistic effects. Isobologram analysis suggested that carboplatin plus thioridazine had synergistic effects ( Figure 1b). Furthermore, carboplatin plus thioridazine increased chromatin damage in the nucleus (Figure 1c) and cytoplasmic histone-associated DNA fragments (Figure 1d). In addition, we examined whether caspase activation is involved in apoptosis in carboplatin plus thioridazine-treated cells. Combined treatment markedly increases caspase-3 activation (Figure 1e), and a pancaspase inhibitor (z-VAD) blocked carboplatin and thioridazine-induced apoptosis as well as PARP cleavage Upregulation of PSMA5 expression is associated with carboplatin plus thioridazine-induced apoptosis. Downregulation of c-FLIP and Mcl-1 protein expression is mainly regulated by the activation of the Ubiquitin-proteasome pathway (UPP). 30,31 As shown in Figure 3a, proteasome inhibitors (MG132 and lactacystine) inhibited carboplatin plus thioridazine-mediated downregulation of c-FLIP and Mcl-1 protein expression, and proteasome activity was markedly increased by the combined treatment with carboplatin and thioridazine ( Figure 3b). Therefore, we investigated whether carboplatin plus thioridazine modulates the expression of the key proteasome subunit, 20 S proteasome subunit alpha type 5 (PSMA5). PSMA5 expression is markedly upregulated by the carboplatin plus thioridazine treatment ( Figure 3c). Next, we examined whether upregulation of PSMA5 expression has a critical role in carboplatin plus thioridazine-mediated apoptosis. As shown in Figure 3d, downregulation of PSMA5 by siRNA inhibited apoptosis and cleavage of PARP in carboplatin plus thioridazine-treated cells. In addition, the combined treatment-mediated downregulation of c-FLIP and Mcl-1 was recovered by knockdown of PSMA5 (Figure 3d). The combined treatment with carboplatin and thioridazine upregulated the expression of PSMA5 at the protein and mRNA level in a time-dependent manner ( Figure 3e). The PSMA5 promoter activity was also increased with the carboplatin plus thioridazine treatment ( Figure 3f). Therefore, our data indicate that upregulation of PSMA5 has a critical role in carboplatin plus thioridazine-mediated apoptosis by downregulating expression of c-FLIP and Mcl-1.
The Nrf2/ARE signaling pathway is largely indispensable for carboplatin plus thioridazine-mediated upregulation of PSMA5 expression. Previous studies reported that the Nrf2/ARE signaling pathway is associated with the regulation of PSMA5 expression. 28 Therefore, we investigated whether the Nrf2/ARE pathway is associated with PSMA5 expression in carboplatin plus thioridazine-treated cells. As shown in Figure 4a, carboplatin plus thioridazine induced upregulation of Nrf2 expression. Furthermore, nuclear translocation of Nrf2 is induced within 1 h and increased up to 6 h after carboplatin plus thioridazine treatment (Figure 4b). Combined treatment with carboplatin plus thioridazine induced the transcriptional activity of ARE (Figure 4c). To identify the importance of Nrf2/ARE signaling on carboplatin plus thioridazine-induced apoptosis, cells were treated with Nrf2 siRNA. Downregulation of Nrf2 expression by siRNA inhibited apoptotic cell death and upregulated PSMA5 expression ( Figure 4d). Therefore, these data suggest that the combined treatment triggers upregulation of PSMA5 expression in an Nrf2/ARE signaling pathway-dependent manner.
Generation of ROS is critically required for carboplatin plus thioridazine-induced apoptosis. As the Nrf2/ARE signaling pathway is activated by reactive oxygen species (ROS) production, 32 we investigated whether combined treatment with carboplatin and thioridazine induces ROS production. Combined treatment with carboplatin and thioridazine induced ROS production within 1 h (Figure 5a). In addition, we determined intracellular ROS by measuring the expression level of peroxiredoxin (Prx)-SO3. As shown in Figure 5b, peroxiredoxin (Prx)-SO3 was detected within 1 h in carboplatin plus thioridazine-treated cells. Next, we investigated whether ROS are involved in the activation of Nrf2/ARE signaling. We found that ROS scavengers (N-acetylcysteine (NAC), glutathione-ethyl-ester, and trolox) inhibited the nuclear translocation of Nrf2 as well as ARE transcriptional activity in carboplatin plus thioridazine-treated cells ( Figure  5c and d). The carboplatin plus thioridazine-induced PSMA5 promoter activity was also reduced by ROS scavengers (Figure 5e). Furthermore, ROS scavengers inhibited apoptotic cell death, downregulated c-FLIP and Mcl-1 expression, and upregulated PSMA5 expression in carboplatin plus thioridazine-treated cells (Figure 5f). Therefore, our results suggest that ROS have a critical role in carboplatin plus thioridazine-induced apoptosis.
Mitochondrial ROS have an important role in carboplatin plus thioridazine-induced apoptosis. As shown in Figure 5f, ROS have an important role in apoptosis induced by carboplatin plus thioridazine. Therefore, we investigated the source of ROS production induced by carboplatin plus thioridazine. ROS are mainly produced by NADPH oxidase and the mitochondrial electron transport chain. 33 Cells were treated with NADPH oxidase inhibitors (diphenyleneiodonium (DPI) and apocynin) and a mitochondrial complex I inhibitor (rotenone). 34,35 Rotenone reduced carboplatin plus thioridazine-induced apoptotic cell death and cleavage of PARP, but DPI and apocynin did not ( Figure 6a). In addition, rotenone reversed the upregulation of Nrf2 and PSMA5 expression and downregulated c-FLIP and Mcl-1 expression ( Figure 6a). As shown in Figure 6b, combined treatment with carboplatin and thioridazine

Discussion
In this study, we demonstrated that carboplatin plus thioridazine induces apoptosis in cancer cells, but not in normal cells. Combined treatment with carboplatin and thioridazine downregulated c-FLIP and Mcl-1 expression at the post-translational level in a proteasome-dependent manner. Along with the augmentation of proteasome activity, mitochondrial ROS activated the Nrf2/ARE signaling pathway and induced upregulation of PSMA5 expression in carboplatin plus thioridazine-treated cells.
Multiple mechanisms are involved in the anticancer activity of thioridazine. First, thioridazine inhibits PI3K/Akt signaling, which is important for cancer cell survival. Thioridazine inhibits cell viability and induces cell death through inhibition of the PI3K/Akt signaling pathway in ovarian cancer and in cervical and endometrial cancer cells. 11,12 In addition, thioridazine inhibits angiogenesis and tumor growth in ovarian cancer xenografts by inhibiting PI3K/Akt signaling. 17 In our study, thioridazine also inhibited Akt phosphorylation (Supplementary Figure S1a). However, PI3K/Akt inhibitors (LY294002 and wortmanin) plus carboplatin did not induce apoptosis in head and neck cancer cells (Supplementary Figure S1b). Furthermore, both PI3K/Akt inhibitors had no  Figure S1b). Therefore, the anticancer effects of thioridazine are independent of the inhibition of the PI3K/Akt signaling pathways in head and neck cancer AMC-HN4 cells. Next, upregulation of ROS by thioridazine treatment also has a critical a role on the anticancer effect. Min et al. 25 reported that thioridazine inhibited Mcl-1 and c-FLIP expression in a ROSdependent manner, resulting in induction of apoptosis in TRAIL-treated human renal carcinoma Caki cells. However, thioridazine alone did not increase intracellular ROS levels ( Figure 6c) and had no effect on the downregulation of c-FLIP and Mcl-1 expression (Figure 1g). Interestingly, a high concentration of carboplatin (450 μM) induced ROS production in cardiomyocytes, 36 AMC-HN3 cells, 37 and human laryngeal carcinoma cells. 38 However, a low concentration of carboplatin (200 nM) did not increase ROS production in our experiment. In addition, Rodrigues et al. 39 reported that thioridazine interacts with the membrane of mitochondria, acquiring antioxidant activity. By contrast, 10 μM thioridazine induced ROS production in Caki cells. However, 10 μM thioridazine had no effect on ROS production in AMC-HN4 cells. These effects of thioridazine on ROS production may result from the different cellular ROS statuses and cell contexts. Although thioridazine alone did not induce ROS production, combined treatment with carboplatin and thioridazine induced apoptosis by downregulating the expression of c-FLIP and Mcl-1 in a ROS-dependent manner. Finally, thioridazine inhibits the focal adhesion kinase (FAK) and Src signaling pathway. In ovarian cancer cells, thioridazine inhibits VEGF-induced angiogenesis by inhibiting FAK and Src phosphorylation. 18 However, identification of the anticancer molecular mechanism of thioridazine requires further study.
When anticancer drugs induce apoptosis in cancer cells, the expression of apoptosis-related proteins is often regulated at the post-translational level. 25,40 Protein degradation is mainly regulated by the ubiquitin-proteasomal or lysosomal pathways. In this studies, combined treatment with carboplatin and thioridazine induced proteasome activity (Figure 3b), followed by downregulation of c-FLIP and Mcl-1 expression (Figure 3a). When proteins are degraded at the post-translational level, there are critical two steps: (1) augmentation of proteasome activity. In our study, combined treatment with carboplatin and thioridazine markedly increased proteasome activity. For the augmentation of proteasome activity, carboplatin plus thioridazine upregulated PSMA5 expression at the transcriptional level, which is a protein that has a 20 S proteasome catalytic core (Figure 3e). In previous studies, upregulation of the proteasome subunit was shown to be regulated by the Nrf2/ ARE signaling pathways. 28,41 Combined treatment with carboplatin and thioridazine induced translocation of Nrf2 into  Figure S2). Therefore, upregulation of PSMA5 has a critical role on augmentation of proteasome activity. (2) upregulation of specific E3 ligase expression. As E3 ligase attaches ubiquitin to target proteins, protein degradation by the ubiquitin-proteasome system has high specificity. For example, four E3 ligases of Mcl-1 are known, including Mcl-1 ubiquitin ligase E3, 30 beta transducin-containing protein (β-TrCP), 42 F-box and WD repeat domain-containing 7, 43 and Tripartite motif containing 17 (Trim17). 44 Casitas B-lineage lymphoma (Cbl), 45 and Itch 46 work as an E3 ligase of c-FLIP. We also investigated the E3 ligase expression of c-FLIP and Mcl-1. However, combined treatment with carboplatin and thioridazine did not change E3 ligase expression of β-TrCP, Cbl, and Itch (Supplementary Figure S3). Further investigation is required to determine whether other E3 ligases or unknown E3 ligases might be involved in the degradation of c-FLIP and Mcl-1 expression.
Collectively, these results suggest that carboplatin plus thioridazine induces apoptosis in human head and neck cancer cells. The augmentation of proteasome activity by mitochondrial ROS-mediated PSMA5 expression induced downregulation of c-FLIP and Mcl-1 expression in carboplatin plus thioridazine-treated cells. Therefore, the combined modality approach with a low concentration of carboplatin and thioridazine may reduce the occurrence of unexpected side effects and improve the drug's anticancer effects.  leupeptin, 10 μg/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 western blotting kit according to the manufacturer's instructions.
Determination of the synergy and cell viability assay. The XTT assay was employed to measure cell viability using a WelCount Cell Viability Assay Kit (WelGENE, Daegu, Korea). In brief, the reagent was added to each well and was then measured with a multi-well plate reader (at 450 nm/690 nm).
4′,6′-Diamidino-2-phenylindole (DAPI) staining for nuclei condensation and fragmentation. To examine the 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 DAPI 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. A 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, carboplatin-treated cells, or cells that were treated with a combination of carboplatin and thioridazine. In brief, each culture plate was centrifuged for 10 min at 200 × g, the supernatant was removed, and the cell pellet was lysed for 30 min. Then, the plate was centrifuged again at 200 × g for 10 min and the supernatant, which 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 were measured by spectrophotometry at 405 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 the DEVDase activity, cell lysates were prepared after their respective treatments with carboplatin in the presence or absence of thioridazine. Assays were performed in 96-well microtiter plates by incubating 20 μg of the cell lysates in 100 μl 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 μM. The lysates were incubated at 37°C for 2 h. Thereafter, the absorbance at 405 nm was measured with a spectrophotometer.  Preparation of cytosol and nuclear extracts. Following the required treatments, AMC-HN4 cells were trypsinized and suspended in buffer A (10 mM HEPES at pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). After incubation on ice for 30 min, the cells were centrifuged at 2500 rpm for 3 min to obtain a nuclear pellet. The supernatant fractions were collected as the cytosol extract. Buffer C (20 mM HEPES at pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF) was added, followed by a rotation for 30 min at 4°C. The resulting lysates were centrifuged at 12 000 rpm at 4°C for 5 min. The supernatant fractions were collected as the nuclear extract.
Small interfering RNAs. The GFP (control), Nrf2, and PSMA5 small interfering RNA (siRNA) duplexes used in this study were purchased from Santa