OBJECTIVETo investigate the effect of combined CsA and FK506 with 5-FU on hepatocellular carcinoma rats.

METHODSA syngeneic rat model of hepatocellular carcinoma was used. Control group (A) underwent 4 ml 5% GS. Treatment group was divided into 3 groups namely, group B: only 5-FU and 5% GS; group C: 5-FU, CsA and 5% GS; group D: 5-FU, FK506 and 5%GS. Cell cycle, apoptosis, necrosis and mitochondrial transmembrane potential were measured by flow cytometry, laser scanning confocal microscopy, and electron transmission microscopy. Statistical analysis was performed by SPSS 10.0 for Windows software. Statistical comparisons were made with ANOVA followed by Dunnett's T3 or LSD test.

RESULTSCompared to the control group, the percentage of apoptotic cells including trifle necrotic cells was significantly higher, and among the treatment group, group D was the highest, and group C was higher than group B. In the treatment group, cell cycle of hepatoma cells was mainly arrested at S phase, but in group D, G0/G1 phase cells were significantly decreased and S phase cells significantly increased. Compared to the control group, mitochondrial transmembrane potential was significantly decreased in the treatment group, among with, group B was the lowest, group C was higher than group D. Morphological changes demonstrated by electron microscopy included dispersed nuclear chromatin, loss of nucleoli, membrane bleeding, cell shrinkage, typical apoptotic bodies and marked swelling of mitochondria in the treatment group. In the control group, however, they were characterized by normal cell ultrastructure.

CONCLUSIONSThe present study reveals that 5-FU combined with CsA or FK506 demonstrated a synergistic effect on hepatocellular carcinoma rats. For FK506, the powerful mutual effect is related to the increase of tumor cell's quantity in S phase. Both CsA and FK506 can provide protection on mitochondrial transmembrane potential reduction against hepatoma cells damage from 5-FU.