5-FU Induces Apoptosis of Fas (+), HepG2 Cells Via Activation of Fas-mediated Caspase and Mitochondria Dysfunction.

PURPOSE In order to investigate the role of Fas on the chemosensitivity of cancer cells in regards to chemotherapeutic agents, the Fas/FasL signaling pathway of apoptosis was explored in human hepatoma cells. MATERIALS AND METHODS Fas expression of hepatoma cells including Chang, Huh7, HepG2, and Hep3B cells, was determined by RT-PCR and flow cytometry analysis. Cell viability was measured by MTT assay and apoptosis was assessed by DNA fragmentation assay. The catalytic activity of the caspase-family proteases including caspase-3, 6, 8, and 9 proteases, was tested using fluorogenic biosubstrates. The expression of apoptotic mediators including cytochrome c, PARP, and Bcl2 family proteins were measured from cytosolic and mitochondrial compartments. Mitochondrial membrane potential was measured by fluorescence staining with JC-1, rhodamine 123. RESULTS Fas mRNA was constitutively expressed in Chang and HepG2 as defined as Fas (+) cells, but not in Huh7 and Hep3B cells, defined as Fas (-) cells. Fas (+) cells were markedly sensitive to 5-FU whereas Fas (-) cells were resistant and able to survive. 5-FU increased Fas expression of Fas (+) HepG2 cells and simultaneously resulted in apoptotic death, characterized by the ladder-pattern fragmentation of genomic DNA. Moreover, it increased the catalytic activity of caspase-8 protease, which eventually cleaved the Bid into truncated Bid which translocated into mitochondria only in Fas (+) cells. It also increased the caspase-9 protease activity with Bax expression, cytosolic release of cytochrome c, and mytochondrial dysfunction only in Fas (+) HepG2 cells. Furthermore, 5-FU increased the enzymatic activity of caspase-3 protease with PARP digestion in HepG2 cells. CONCLUSION 5-FU exerted cytotoxicity against hepatoma cells via activation of Fas-mediated apoptotic signaling including caspase cascades and mytochondrial dysfunction. Our data suggests that Fas may be an important modulator of the chemosensitivity of cancer cells vis- -vis anticancer chemotherapeutic agents.