Effect of 5-aza-2'-deoxycytidine on immune-associated proteins in exosomes from hepatoma - PubMed (original) (raw)
Effect of 5-aza-2'-deoxycytidine on immune-associated proteins in exosomes from hepatoma
Wen-Hua Xiao et al. World J Gastroenterol. 2010.
Abstract
Aim: 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.
Conclusion: 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.
Figures
Figure 1
Immunoelectron microscopy showing colloidal gold HSP70 labeled HepG2 exosomes in control group (A) and in experiment group (B), colloidal gold HLA-I labeled HepG2 exosomes in control group (C) and experiment group (D), colloidal gold NY-ESO-1 labeled HepG2 exosomes in control group (E) and experiment group (F), colloidal gold HSP70 labeled Hep3B exosomes in control group (G) and experiment group (H), colloidal gold HLA-I labeled Hep3B exosomes in control group (I) and experiment group (J), colloidal gold NY-ESO-1 labeled Hep3B exosomes in control group (K) and experiment group (L) (×140 000).
Figure 2
Immune molecules Western blotting results (A), semi-quantities results of Western blotting (B). 1: HepG2 control; 2: HepG2 experiment; 3: Hep3B control; 4: Hep3B experiment.
Figure 3
RT-PCR results of p53 gene expression (A), semi-quantities results of RT-PCR (B). M: Marker; C: Control group; E: Experiment group.
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