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.
Highlights
Preservation of the genomic DNA from extrinsic or intrinsic damage is crucial for maintaining normal cellular function and to prevent cellular transformation
It was evident that increasing concentrations of gemcitabine in the culture medium inhibited the growth of LY2 cells in a dose-dependent manner (Figure 1(a))
Since growth inhibitory effect of gemcitabine against tumor cells can be mediated by apoptosis or by triggering cell cycle arrest [13,21,25], we examined the effect of gemcitabine on induction of apoptosis in LY2 cells
Summary
Preservation of the genomic DNA from extrinsic or intrinsic damage is crucial for maintaining normal cellular function and to prevent cellular transformation. DNA checkpoint pathways are activated in mitotically active cells in response to DNA damage caused by extrinsic (i.e. radiation or chemotherapeutic agents) or intrinsic (i.e. DNA replication stress) factors. Phosphoinositide 3-kinase related kinases (PI3K) namely; ataxia telangiectasia and rad-3-related kinase (ATR) and ataxia-telangiectasia mutated (ATM) coordinate DNA damage responses via activation of Chk and Chk, respectively [4,5,6]. ATR/ Chk are activated by UV and other intrinsic factors that induce DNA replication stress whereas the activation ATM/Chk signaling is trigged by DNA double strand breaks [7]. The ATM or ATR cascades become activated within minutes of a DNA damage signal resulting in phosphorylation and activation of the transcription factor p53, either directly or indirectly, via the activation of Chk2 [8]. Cells with damaged DNA that have escaped G1 checkpoint are halted at G2
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