Abstract
BackgroundRecent studies indicate an increase in tumor progression and recurrence in head and neck squamous cell carcinomas (HNSCC) of cancer patients taking recombinant human erythropoietin (rhEpo) for anemia. This study was undertaken to investigate the potential role of rhEpo in invasion, proliferation, and cisplatin-induced cell death in HNSCC cell lines.MethodsThe following experiments were performed with two HNSCC cell lines, UMSCC-10B and UMSCC-22B. Presence of EpoR in both cell lines was determined by western blot and quantitative PCR. Colorimetric MTS assays and clonogenic assays were used to study the effect of rhEpo at pharmacologically relevant doses on cell proliferation. Matrigel invasion assays were performed in order to determine effects of exogenous rhEpo on invasive abilities. Clonogenic assays were also used to study potential cytoprotective effects of rhEpo against cisplatin. Immunoblotting was done to analyze the effect of rhEpo on Akt phosphorylation. Finally, MTS and TUNEL assays were performed to test our hypothesis that Akt activation by PI3K was involved in rhEpo-mediated cisplatin resistance.ResultsHNSCC cell lines were shown to express Epo receptor (EpoR). RhEpo increased invasion 1.8-fold in UMSCC-10B and 2.6-fold in UMSCC-22B compared to control. RhEpo at 10 U/ml increased cell proliferation by 41% and 53% in UMSCC-10B and UMSCC-22B, respectively, and colony formation by 1.5-fold and 1.8-fold. UMSCC-10B treated with cisplatin and exposed to rhEpo at 1 and 10 U/ml resulted in a 1.7-fold and 3.0-fold increase in colony number compared to control, respectively. UMSCC-22B treated with cisplatin and rhEpo at 1 or 10 U/ml resulted in ~2.5-fold increase in colony number. A TUNEL assay demonstrated a 30.5% and 76.5% increase in survival in UMSCC-10B and UMSCC-22B cells, respectively, in cisplatin and rhEpo-treated cells compared to cisplatin alone. MTS assay showed similar cytoprotective effects. Western blot revealed increased phosphorylation of Akt upon exposure of HNSCC cell lines to rhEpo. MTS assay and TUNEL analyses implicate Akt as a likely contributor to regulation of rhEpo-mediated cytoprotection.ConclusionsThe results demonstrate that, in HNSCC cells expressing functional EpoR, rhEpo promotes invasion, cell proliferation, and induces resistance to cisplatin, which may contribute to tumor progression.
Highlights
Recent studies indicate an increase in tumor progression and recurrence in head and neck squamous cell carcinomas (HNSCC) of cancer patients taking recombinant human erythropoietin for anemia
In both HNSCC cell lines, Epo receptor (EpoR) protein was expressed at relatively high levels, which correlated with mRNA data (Figure 1)
The effect of recombinant human erythropoietin (rhEpo) on cell invasion was significant (P < 0.05) at a concentration of 1 U/ml, substantially less than serum stimulation (Figure 3b). These findings indicate that exposure of the established HNSCC cell lines to rhEpo for 40 h can increase cell invasion capabilities, consistent with findings reported by other investigators that used the UMSCC-22B cell line
Summary
Recent studies indicate an increase in tumor progression and recurrence in head and neck squamous cell carcinomas (HNSCC) of cancer patients taking recombinant human erythropoietin (rhEpo) for anemia. Recombinant human epoetin alfa (rhEpo) is a glycoprotein (30.4 kDa) produced by recombinant DNA technology, and has the same biologic effects as the endogeneous erythropoietin produced by the kidneys. RhEpo has been used since 1993 for the treatment of anemia, including those associated with chemo- and radiation therapy in cancer patients. Recent studies have shown expression and function of Epo and EpoR in a variety of human cancers, including solid tumors and tumor cell lines [1,2,3]. Given the precise role of rhEpo in human cancers, tumor progression and recurrence, is not well understood, clinical and basic research studies are still necessary to define signaling pathways activated by rhEpo/EpoR within nonhematopoietic cancer cells
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