SAS cells were pretreated with or without bortezomib (20 nM) 1 h before cisplatin treatment (8
SAS cells were pretreated with or without bortezomib (20 nM) 1 h before cisplatin treatment (8.0 g/mL). CYLD SAR-7334 HCl expression is involved in cisplatin resistance in OSCC and elucidated its molecular mechanism. In this study, to assess the effect of CYLD down-regulation on cisplatin resistance in human OSCC cell lines (SAS), we knocked-down the CYLD expression by using CYLD-specific siRNA. In cisplatin treatment, cell survival rates in CYLD knockdown SAS cells were significantly increased, indicating that CYLD down-regulation caused cisplatin resistance to SAS cells. Our results suggested that cisplatin resistance caused by CYLD down-regulation was associated with the mechanism through which both the reduction of intracellular cisplatin accumulation and the suppression of cisplatin-induced apoptosis via the NF-B hyperactivation. Moreover, the combination of cisplatin and bortezomib treatment exhibited significant anti-tumor effects on cisplatin resistance caused by CYLD down-regulation in SAS cells. These findings suggest the possibility that loss of CYLD expression may cause cisplatin resistance in OSCC patients through NF-B hyperactivation and may be associated with poor prognosis in OSCC patients. ( 0.05 and ** 0.01 vs siCon group. ?? 0.01 vs cisplatin treated siCYLD group. 2.2. CYLD Down-Regulation Induced Cisplatin Resistance through NF-B Hyperactivation It was reported that CYLD, a deubiquitinating enzyme, negatively regulates the NF-B signaling pathway in the process of malignant progression [7,8,9,10,11,17,18]. NF-B activation is known to be involved in the development of resistance to anticancer drugs such as cisplatin and 5-FU in OSCC [26,27]. Since previous clinical studies also showed the NF-B hyperactivation in OSCC tissues [23,24,25], we next sought to determine whether NF-B hyperactivation is usually involved in cisplatin resistance induced by CYLD down-regulation in SAS cells. Consistent with the previous reports about CYLD function as a negative regulator of NF-B signaling pathway, CYLD knockdown by CYLD-specific siRNA significantly enhanced the NF-B activity in SAS cells (Physique 2a). Moreover, BAY 11-7085, an irreversible NF-B inhibitor, significantly suppressed cisplatin resistance induced by CYLD knockdown in a dose-dependent manner (Physique 2b). These results suggested that CYLD down-regulation induced cisplatin resistance through NF-B hyperactivation in OSCC cells. Open in a separate window Physique 2 CYLD down-regulation induced cisplatin resistance through NF-B hyperactivation. (a) NF-B activity was assessed by luciferase reporter assay 48 h after transfection with CYLD-specific siRNA. (b) Cell survival rates of SAS cells against cisplatin treatment (8.0 g/mL) with or without NF-B inhibitor (BAY 11-7085) were assessed. Values are means SD of triplicate samples. ** 0.01 vs siCon SAR-7334 HCl group. ?? 0.01 vs cisplatin treated siCYLD group. N.S.: not significant. 2.3. Effect of CYLD Down-Regulation on Intracellular Cisplatin Accumulation and Cisplatin-Induced Apoptosis in OSCC Cells One of the main factors for cisplatin resistance has been the reduction of intracellular cisplatin accumulation [28,29,30]. We thus tried to determine the level of cisplatin accumulation in CYLD-knockdown SAS cells by using ICP-MS. As shown in Physique 3a, the intracellular cisplatin accumulation was significantly reduced by CYLD knockdown at any cisplatin concentration. In addition, the reduced intracellular cisplatin accumulation in CYLD-knockdown SAS cells was recovered, at least in part, by NF-B inhibitor (BAY 11-7085) treatment (Physique 3b). Those results suggested that this reduction of intracellular cisplatin accumulation caused by NF-B hyperactivation was partially involved in SAR-7334 HCl cisplatin resistance caused by CYLD down-regulation in SAS cells. Open in a separate window Physique 3 Effect of CYLD down-regulation on intracellular cisplatin accumulation and cisplatin-induced apoptosis in OSCC cells. (a,b) Measurement of intracellular cisplatin accumulation in SAS cells using ICP-MS. Cells were treated with various cisplatin concentrations (0C10 g/mL) for 6 h (a) and pretreated with or without NF-B inhibitor (BAY 11-7085, 0C10 M) 1 h before cisplatin treatment (b). (cCe) The cisplatin-induced apoptosis was assessed by Annexin-V/7-AAD staining using flow cytometry. Cells were pretreated with or without 10C15 M NF-B inhibitor (BAY 11-7085) 1 h before cisplatin treatment (8.0 g/mL) (e). Values are means SD of triplicate samples. * 0.05 and ** 0.01 vs. siCon group. ?? 0.01 vs cisplatin treated siCYLD group. N.S.: not significant. It is well-documented that DNA damage and subsequent induction of apoptosis may be the primary cytotoxic mechanism of cisplatin [31,32]. In contrast, it has been also reported that one of the mechanisms of cisplatin resistance is usually suppression of cisplatin-induced apoptosis in various types of malignant tumors [28,29]. Since previous studies showed that loss of CYLD function leads to SAR-7334 HCl the suppression of apoptosis by NF-B hyperactivation in malignant Dig2 tumors [7,8,9,10,11], we next sought to determine whether the suppression of apoptosis was involved in cisplatin resistance caused by CYLD down-regulation in SAS cells. The percentage of apoptotic SAS cells after cisplatin treatment was evaluated by Annexin-V/7-AAD staining using flow cytometry analysis. As shown in Figure 3c,d, the percentage of apoptotic cells treated with 8.0 g/mL.