Abstract 4329: In vitro and in vivo antitumor effects of gemcitabine in a murine head and neck squamous cell carcinoma (HNSCC) model

Abstract

Abstract Background: Gemcitabine (2’2’-difluorodeoxycytidine/Gem) is a nucleoside analog and exerts its cytotoxicity by incorporating into the DNA during replication which leads to termination of chain elongation and tumor cell apoptosis. Chemotherapy with Gem is the standard of care for patients with pancreatic cancer and it has shown clinical efficacy and favorable toxicity profile in patients with advanced HNSCC. Goal: We evaluated the molecular aspects of Gem's anti-tumor effect in a highly metastatic orthotropic model of HNSCC, generated using a murine SCC cells (LY2) in BALB/c mice. Methods: Exponentially growing LY2 cells were treated with Gem (100nM-1µM) and its effect on cell survival, proliferation, apoptosis and signaling pathways involved cell growth were examined using MTT assay, Western blotting and immunohistochemical (IHC) assays for cleaved caspase 3 and proliferating cell nuclear antigen (PCNA). LY2 cells were intraorally implanted in BALB/c mice, after one week, tumor bearing mice were randomly divided into experimental (n=8) and control (n=8) groups and treated with intraperitoneal injections of Gem (100mg/Kg, twice a week for 3-weeks) and saline, respectively. Primary tumor growth rates were monitored weekly and tumor metastases were analyzed by histological examination. Results: LY2 cells were susceptible to killing by Gem (IC50 = 500 nM). The extracellular signal-regulated kinase (ERK) activation in LY2 cells was blocked immediately after the treatment with Gem. The activation of apoptosis as measured by the expression levels cleaved caspase 3, reached the highest level at 24 hours after the treatment. Gem treatment down-regulated the expression checkpoint kinase (CHK1) that protects cells from DNA damage induced cell death in LY2 cells. The mean apoptosis rate (% of cleaved caspase 3 positive cells detected by IHC) of LY2 cells treated with Gem was significantly higher (21%) compared to untreated control cells (< 5%). Interestingly, LY2 cells treated with Gem revealed significantly higher PCNA positive cells (76%) than untreated control cells (28%). Gem treatment of tumor bearing mice resulted in significant reduction tumor growth rate and incidence of regional lymph node metastases compared to the control group. Conclusion: The increase in PCNA positive cells within the LY2 cells treated with Gem compared to untreated control is counterintuitive to the anti-proliferative effect of Gem. Increased PCNA positive fraction of cells in Gem treatment group is most likely caused by S-phase arrest which has been reported to occur in cancer cells treated with Gem. Hence, PCNA cannot be used to determine the cell proliferation rates of tumor cells treated with Gem. Our data suggest that Gem has multiple innovative modes of anti-tumor activity against HNSCC which include DNA replication defect, loss of ERK activation and CHK1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4329. doi:10.1158/1538-7445.AM2011-4329