Apoptotic inhibition of head and neck squamous cell carcinoma cells by tumor necrosis factor alpha.

OBJECTIVES

To evaluate the effect and mechanism of action of tumor necrosis factor alpha (TNF-alpha) on head and neck squamous cell carcinoma (HNSCC) cell lines. Specifically, to find out whether TNF-alpha induces apoptotic (programmed) cell death.

DESIGN

Cytotoxicity and kinetics were evaluated through the use of a methylene blue colorimetric assay. The mechanism of cell death was evaluated through gel electrophoresis of extracted DNA, double-stranded DNA fragmentation, and Hoechst 33342-propidium iodide double staining. TARGET CELLS: Four HNSCC cell lines were investigated: UMSCC-1, UMSCC-8, UMSCC-19, and CAL-27.

RESULTS

Tumor necrosis factor alpha at a concentration of 10000 U/mL induced cytotoxic effects in all four cell lines with varying sensitivities. Significant cytotoxic effects required 3 to 4 days of exposure to TNF-alpha. Further experiments demonstrated that TNF-alpha induced double-stranded DNA fragmentation of targets, which preceded the detection of actual cytotoxic effects. Treated HNSCC cell lines had their DNA fragmented into a ladder pattern of endonucleolytic cleavage of multiples of 180 to 200 base pairs, consistent with apoptosis. Propidium iodide-Hoechst 33342 double staining confirmed that TNF-alpha-induced nuclear alterations precede permeabilization of the plasma membrane, supporting cell death in these tumor cells by apoptosis.

CONCLUSION

Tumor necrosis factor alpha exposure results in variable cytotoxic reactions in HNSCC cell lines, with DNA fragmentation preceding significant cell death. The mechanism of cell death is apoptosis, with typical morphological features. Further studies are needed to better elucidate the role of TNF-alpha in the treatment of in vivo HNSCC.