Effects And Possible Mechanism Of Arsenious Acid On Inducing Differentiation Of The Hepatoma Cell Lines BEL-7402

Aims: To investigate the roles of arsenious acid (As2O3) on inducing differentiation of hepatoma cell line BEL-7402 and to provide experimental data for clinic utilization.Methods: Hepatoma cell lines BEL-7402 were treated with different concentrations of As2O3 in vitro and RPMI 1640 was used as medium. In order to observe morphologic changes of BEL-7402 cells, Giemsa stain was used. The activities of tyrosine aminotransferase (TAT), y-glutamyl transpeptidase (y-GT) and alkaline phosphatase (ALP) were determined with colorimetry. The content of a -fetoprotein (AFP) in the supernatant of cultured BEL-7402 cells was detected by radioimmunoassay. The growth curve was made to reflect the condition of cell growth and soft agarose cloning technique was used to assess the malignant degree of hepatoma cells after treating with As2O3. Furthermore, The expression of hepatocyte neclear factor 4 (HNF 4) was analyzed by reverse transcription polymerase chain reaction (RT-PCR).Results: When the concentration of As2O3 was over 0.25 umol/L, the shape of BEL-7402 cells had many changes on day 5, becoming small and round and cell nuclear turning small and round also. Nuclear notch almost dispeared. Meanwhile, the volume ratio of nucleus to plasma was decreased, the nucleolus became few or unconspicuous. The excreation of AFP became fewer and fewer with time and increased dose of As2O3, showing dose- and time-dependent effects after culturing for 2 days (P<0.01) and became lowest on day 6 at the concentration of 0.5 and 1.0mol/L. The activity of y-GT also had dose- dependent and time-dependent effect and became lowest on day 7 at the concentration of 0.5 and 1.0 umol/L. The activity of TAT increased. On day 4, the cells treated with 0.25, 0.5 and 1.0 umol/L of As2O3 had higher activity compared with control group (P<0.05) and it continued afterwhile. The activity of ALP increased obviously on day 3 or day 4 then decreased after this. The activity of ALP in control, 0.06 umol/L, 0.12 umol/L group peaked on day 7, while 0.5 umol/L, 1 mol/L group on day 4. On the whole, 0.25, 0.50 and 1.0 mol/Lof As2O3 inhibited the growth of hepatoma cells obviously with a dose-dependent. However, the inhibitory effect of As2O3 was weak at the concentration of 0.06 and 0.12 mol/L and no difference was showed compared with the group without As2O3 treatment. Compared with control group, the percentage of colony formation of hepatoma cells decreased obviously treated with 0.5 and 1.0 umol/L As2O33 (P<0.5). The mRNA expression of HNF 4a increased with the treatment of As2O3 at the concentration of 0.5 and 1.0 umol/L As2O3 (P<0.01).Conclusion: Asenious acid (0.25, 0.5 umol/L) could induce the differentiation of hepatoma cell line BEL-7402 in which HNF4a may play a pivotal role.