Effects Of Differentiation Induced By SAHA On The Human Hepatoma Cells

Objective An inhibitor of histone deacetylase (HDAC) enzymatic activity, SAHA (suberoylanilide hydroxamic acid), was used to investigate its effects on the differentiation of the human hepatoma SMMC-7721 cells.Methods The hepatoma SMMC-7721 cells were treated with various concentrations of SAHA in different duration in vitro. Cell morphology and ultrastructure were examined by the light microscope and transmission electron microscopy (TEM). Cell viability was analyzed by MTT assay. The expression of Alpha-fetoprotein (AFP) and proliferating cell nuclear antigen (PCNA) was determined with immunocytochemistry. Flow cytometry (FCM) was used to analyze the cell cycle. The expression of p21WAFl mRNA was detected by semi-quantitative RT-PCR.Results SAHA inhibited the proliferation of SMMC-7721 cells in dose-dependent and time-dependent manners. Light microscope and transmission electron microscopy showed that SMMC-7721 cells induced by SAHA had undergone the restorational alteration in morphology and ultrastructure, which were different from those of nontreated cells but were similar to those of normal cells, and the changes were as follows: the cells turned to be flat and spread; the nucleo-cytoplasmic ratio lessened and nuclear shape became rather regular; the number of nucleolus reduced and its volume lessened; euchromatin increased while heterochromatin decreased in nucleus; in the cytoplasm, mitochondria grew in number with relatively consistent structure and well-developed mitochondria cristae; Golgi complex turned to be well-developed and typical; rough endoplasmic reticulum increased. Immunocytochemistry assay showed that the expression of AFP and PCNA were declined significantly. FCM analysis showed SAHA could arrest SMMC-7721 cells in G0/G1 phase, with an accumulation of the cells in G0/G1 phase while a decrease of cells in S phase. Semi-quantitative RT-PCR detection revealed that the expression of p21WAFl mRNA was upregulated remarkably in the cells treated with SAHA.Conclusion SAHA could inhibit the proliferation of the hepatoma SMMC-7721 cells and induce differentiation of the cells, which were associated with inhibition of enzymatic activity of HDAC and with upregulation of the expression of p21WAFl mRNA as well as arrest of the hepatoma cells in GO/Gl phase.