Effect of 5-aza-2'-deoxycytidine on immune-associated proteins in exosomes from hepatoma.
OBJECTIVE
To study the effect of 5-aza-2'-deoxycytidine (5-aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-I (HLA-I) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B.


METHODS
Exosomes derived from HepG(2) and Hep3B cells treated with or without 5-aza-CdR were isolated and purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under electron microscope. Concentration of proteins in exosomes was measured by bicinchoninic acid protein assay. Expression of HSP70, HLA-I and NY-ESO-1 proteins in exosomes was detected by Western blotting and immunoelectron microscopy. mRNA expression of p53 gene was detected by reverse transcription polymerase chain reaction.


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 increased significantly after treatment with 5-aza-CdR (P < 0.05). After treatment with 5-aza-CdR, immunoelectron microscopy and Western blotting showed that the HSP70, HLA-I and NY-ESO-1 proteins were increased in exosomes produced by both hepatoma cell lines.


CONCLUSIONS
5-Aza-CdR, an inhibitor of DNA methyltransferase, can increase exosomes produced by hepatoma cells and immune-associated protein component of exosomes, which may be mediated by p53 gene up-regulation and 5-aza-CdR demethylation.
Xiao
Wen-Hua
WH
Department of Oncology, First Affiliated Hospital, Chinese PLA General Hospital, Beijing 100048, China. w_hxiao@hotmail.com
Sanren
Gao-Wa
GW
Zhu
Jian-Hua
JH
Li
Qiu-Wen
QW
Kang
Huan-Rong
HR
Wang
Ru-Liang
RL
Song
Lin-Ping
LP
Ye
Ming
M
D000951:Antigens, Neoplasm / Q000378:metabolism; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D002499:Centrifugation, Density Gradient; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D055354:Exosomes / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology; D006680:HLA Antigens / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D008565:Membrane Proteins / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D015854:Up-Regulation
D000951:Antigens, Neoplasm; C104948:CTAG1B protein, human; D004791:Enzyme Inhibitors; D006680:HLA Antigens; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine