Bortezomib-induced apoptosis in HNSCC cells is both p38/JNK pathways-dependent activation

Objectives: Bortezomib (PS-341) represents a new class of anticancer drugs which has been shown to potently inhibit the growth and/or progression of human cancers, including head and neck squamous cell carcinoma (HNSCC). In this study, we present evidence for the involvement of MAP kinase signalling pathways, JNK and p38 in the modulation of bortezomib-induced apoptosis in HNSCC cells for the first time. Methods: MTT assay, Flow Cytometry Analysis (FACS), Western blot, Kinase-assay, Electrophoretic Mobility Shift Assay (EMSA), Immune-fluorescence-staining (IF), Confocal Laser Scan Microscopy (CLSM) and H&E staining. Results: To determine whether the MAP kinase signalling pathways JNK, p38 and ERK are involved in the regulation of bortezomib-induced cell death of HNSCC, the HNSCC cell lines CLS-354 and SCC nasal septum were exposed to bortezomib for 24h. Using MTT assay, we could confirm the ability of bortezomib to kill HNSCC cells. Whereas, the analysis of bortezomib-induced cell death in HNSCC cells by flow cytometry using annexin/PI staining demonstrated that bortezomib-induced cell death of HNSCC cells is mediated by an apoptotic mechanism as evidenced by PARP cleavage. Data obtained from in vitro kinase assay revealed that bortezomib-induced apoptosis is associated with the activation of the three MAP kinase signalling pathways JNK, p38 and ERK. In addition, data obtained from EMSA demonstrated the enhancement of DNA-binding activity of AP-1, ATF-2 and ELK-1, the physiological substrats of JNK, p38 and ERK, respectively. However, the inhibition of the MAP kinase pathways JNK and p38, but not ERK, showed the involvement of both JNK and p38 pathways in the modulation of apoptosis induced by bortezomib in HNSCC cells. Conclusions: Our data obtained from this study demonstrate the activation of both JNK and p38 pathways that are essential for the modulation of bortezomib-induced apoptosis in HNSCC cells and may be clinically relevant for the treatment of HNSCC.