Objective: To investigate the effects of 5-Aza-deoxycytidine (5-Aza-CdR) on the amount of exosomes and immuno-associated proteins produced in hepatoma HepG2 and Hep3B cells.
Methods: Exosomes derived from HepG2 and Hep3B cells with or without treatment by 5-Aza-CdR were isolated and purified by combination of ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under the electron microscope. The concentration of proteins in exosomes was detected by BCA. The expression of HSP70, HLA-I and NY-ESO-1 proteins in exosomes was assayed by Western blot and immuno-electron microscopy. The mRNA expression of p53 gene was observed by reverse transcription-polymerase chain reaction (RT-PCR).
Results: The mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-Aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were significantly increased after treatment by 5-Aza-CdR (P < 0.05). The immuno-electron microscopy and Western blotting showed that after treatment with 5-Aza-CdR, the contents of HSP70, HLA-I and NY-ESO-1 proteins were increased in exosomes in both HepG2 and Hep3B hepatoma cells.
Conclusion: 5-Aza-CdR, an inhibitor of DNA methyltransferase, can increase the amount of exosomes and exosome-containning immuno-associated proteins secreted by hepatoma cells. It may be contributed by up-regulation of p53 gene and demethylation mechanism of 5-Aza-CdR.