5-Fluorouracil Mediates Apoptosis and G1/S Arrest in Laryngeal Squamous Cell Carcinoma via a p53-Independent Pathway

Abstract

5-Fluorouracil (5-FU) is a commonly used chemotherapeutic agent in the treatment of laryngeal squamous cell carcinoma. 5-FU can induce cell cycle arrest or apoptosis in various cancers via either a p53-dependent or a p53-independent pathway; however, its pathway of action in laryngeal carcinoma is unknown. In this study, we aim to investigate the role that p53 plays in the cytotoxic effect of 5-FU on laryngeal squamous carcinoma cells. We employed two human laryngeal squamous carcinoma cell lines with different p53 statuses-one (UMSCC12) had truncated non-functional p53 and the other (UMSCC11A) had mutant but functional p53. Cell death was detected using cytotoxicity assay and Annexin V staining. Cell cycles were analyzed by flow cytometry. Western blot was used to analyze the protein expression. 5-FU induces apoptosis in both UMSCC12 and UMSCC11A cells in a dose- and time-dependent manner, suggesting that the pathway was p53-independent. 5-FU induced the accumulation of retinoblastoma protein and a cyclin dependent kinase inhibitor, p21WAF1/CIP1, in both UMSCC12 and UMSCC11A cells. However, 5-FU did not induce p53 expression in either UMSCC12 or UMSCC11A cells. In addition, G1/S cell cycle phase arrest was associated with antiproliferative activity of 5-FU in both cell lines. In order to gain an insight into the role p53 plays in response to 5-FU treatment in laryngeal carcinoma, we further transfected either a wildtype p53 plasmid or an empty pcDNA3.1 vector into UMSCC12 cells. We found that 5-FU increased pRb and p21WAF1/CIP1 expression in both p53-transfected and vector-transfected cells without the significant accumulation of p53. Our results suggest that 5-FU mediates apoptosis and G1/S cell cycle phase arrest in laryngeal carcinoma via a p53-independent but p21WAF1/CIP1-dependent or p21WAF1/CIP1-Rb-dependent pathway. While p53 does not seem to be involved in 5-FU induced apoptosis and cell cycle arrest in laryngeal carcinoma, further studies are needed to examine the roles of retinoblastoma protein and p21WAF1/CIP1 in laryngeal carcinoma receiving chemotherapy.