Inhibition Effect Of 5-azacytidine On HSG Cells In Vitro And In Vivo

5-azacytidine is originally developed as an anticancer drug, and has considerable activity in the acute nonlymphoblast leukemias. 5-azaC is a pyrimidine ring analog in which ring carbon 5 is replaced with nitrogen when incorporated into DNA. 5-azaC cannot be methylated and causes complete demethylation of genomic DNA. Methylation may provide a mechanism for a heritable alteration of a gene structure. It represses gene activity, perhaps by altering some aspects of chromatin structure. The hypomethylation of gene sequences is generally correlated with an open chromatin structure and their transition into an active state. 5-azaC analogs inhibit the condensation of definite chromosome regions when incorporated into replicating DNA.In an attempt to develop a new therapy method for salivary gland tumor, the present study was undertaken to evaluate, in vitro and in vivo, the effect of 5-azaC on human salivary intercalated ductal cell line, HSG. HSG cells were treated with different concentration of 5-azaC, morphological changes were observed, growth curve and growth inhibition rate of 5-azaC on HSG cells was calculated, tumor inhibition rate was undertaken by culturing the HSG cells and 5-azaC treated HSG cells on agar, and inoculating the HSG cells and 5-azaC treated HSG cells into nude mice. To determinate the mechanisms of 5-azaC induced apoptosis to HSG cells, immunohistochemical methodwas used with anti-p53, anti-bcl-2, anti-FAP-1 antibodies, antisense FAP-1 was transfected into HSG cells, and apoptosis effect of 5-azaC on HSG cells was determinated by flowcytometry, electron microscopy.The results were as follows: the growth of HSG cells was inhibited after treated with 5umol/L and 10umol/L 5-azaC, MI, AgNORs decreased, results of colony forming ability of HSG cells showed that the colony forming ability decreased after treated with 5-azaC, flowcytometry results indicated that cell cycle was inhibited at Gl phase, morphology changes occurred after treatment with 5-azaC, granule could be seen in cytoplasm, RT-PCR results showed that a light strap could be seen at 201bp, immunohistochemical results showed that in contrast with control group, PCNA, p53, bcl-2, CyclinDl, and FAP-1 expression decreased after treated with 5-azaC. Flowcytometry results indicated that apoptosis peak could be seen before Gl phase, and apoptosis detected by microscope, electron microscope and DNA ladder occurred after treated with 5umol/L 5-azaC. HSG cells were more sensitive to 5-azaC after tranfected with antisense FAP-1, a higher rate of growth inhibition was seen, and more HSG cells undertook apoptosis after transfected with FAP-1 when treated with 5-azaC.All results above indicated that 5-azaC can inhibit the growth of HSG cells, induce HSG cells differentiate into acinar, induce apoptosis of HSG cells, and FAP-1 may play a role in 5-azaC induced apoptosis. Besides, bcl-2 and p53 may also play a role in 5-azaC induced apoptosis. Thus, 5-azaC may be a promising therapeutic method for salivary gland tumor with further study in the future.