TITLE:
Gemcitabine Inhibits Murine Head and Neck Squamous Cell Carcinoma Growth via Proteasome-Dependent Degradation of Chk1 Leading to Cell Cycle Arrest and Apoptosis
AUTHORS:
Anren Song, Jean Wu, Emily Whitaker, Nadarajah Vigneswaran
KEYWORDS:
Chk1; Gemcitabine; Head and Neck Cancer; Apoptosis; siRNA
JOURNAL NAME:
Journal of Cancer Therapy,
Vol.3 No.5,
October
18,
2012
ABSTRACT: Head and neck squamous cell carcinoma (HNSCC) is a common tumor worldwide that often presents at an advanced stage with poor prognosis. Advanced HNSCC frequently exhibits resistance to chemotherapy limiting its efficacy. Gemcitabine is a pyrimidine-based analog that is currently used for the treatment of metastatic pancreatic cancer. In this study, we examined the anti-tumor effects of gemcitabine in a highly aggressive murine model of HNSCC (LY2). In vitro cell viability and in vivo tumor growth inhibitory assays were carried out to determine the sensitivity of LY2 cells to gemcitabine. Immunohistochemical, Western blotting, RT-PCR and RNAi-mediated silencing assays were used to characterize effects of gemcitabine cell proliferation, DNA synthesis, apoptosis, pro-survival and DNA damage response signaling pathways. LY2 cells treated with gemcitabine undergo apoptosis mediated by the activation of both caspase-3 and -9. Gemcitabine on treatment induces rapid phosphorylation of checkpoint kinase 1 (Chk1) in LY2 cells and subsequent degradation in a time and dose dependent manner. Proteasome inhibitor MG132 blocks Chk1 degradation and decreases LY2 cells susceptibility to gemcitabine. Inhibition of Chk1 function, either using inhibitor PD 407824 or small interfering RNA (siRNA), increases the sensitivity of LY2 cells to gemcitabine. Gemcitabine treatment resulted in significant reduction in tumor growth relative to saline-treated control in a syngeneic orthotopic murine model of HNSCC. Gemcitabine-induced DNA replication stress in LY2 cells activates Chk1 by phosphorylation and promotes Chk1 degradation via the ubiquitin-proteasome pathway. Depletion of Chk1 terminates S-phase check point in LY2 cells resulting in apoptotic cell death. Our data provides an important rationale for integrating gemcitabine to optimize chemotherapeutic efficacy in HNSCC.