Synergism of Fas-mediated apoptosis and tumor necrosis factor on adriamycin cytotoxicity to transitional cell carcinoma.

OBJECTIVE

To explore the accurate Fas antigenic expression in transitional cell carcinoma (TCC) cells and and compare its synergistic modulation on adriamycin cytotoxicity with alpha-tumor necrosis factor (TNF-alpha).

METHODS

The expression of Fas antigen in normal urothelium and TCC cell lines was measured by flow cytometry. The Fas/Fas ligand reaction-induced cytotoxic changes in tumor cells were evaluated by microculture MTT technique. The synergism efficacy of Fas-mediated apoptosis and TNF-alpha on adriamycin cytotoxicity of TCC cells was analyzed. The Fas/Fas ligand-induced apoptosis in tumor cells was studied by annexin-V apoptotic cell expression and DNA ladder fragmentation analysis.

RESULTS

75% of TCC cell lines showed Fas antigen expression. The Fas expression was not influenced by in vitro growth density of tumor cells. Apoptosis of TCC cells was observed during 48 h of treatment after activation of the Fas/Fas ligand pathway. TNF-alpha had a higher cytotoxicity activity on TCC cells than Fas ligand (17 vs. 0.7%) while combination of both reagents had 30% increased synergistic cytotoxicity. The increasing rate of apoptotic body after 3 days of treatment was 74% in adriamycin, 32% in Fas ligand, 60% in TNF-alpha, 69% in Fas and adriamycin combination and Fas and TNF-alpha combination, 103% in combination of TNF-alpha and adriamycin, and 136% in combination of these three reagents individually. In the DNA ladder analysis, Fas ligand had a 1.5-fold increase of DNA fragmentation when compared with adriamycin while TNF-alpha had a 4.4-fold increase and triple combination had a 5.5-fold increase after 3 days of treatment.

CONCLUSIONS

Although the Fas antigen expression is common in TCC cells and apoptosis can be activated by the Fas/Fas ligand pathway activation, the synergistic cytotoxicity of adriamycin by the Fas/Fas ligand pathway is lower than that of TNF-alpha.