Alpha-fetoprotein-specific transfer factors downregulate alpha-fetoprotein expression and specifically induce apoptosis in Bel7402 alpha-fetoprotein-positive hepatocarcinoma cells.
OBJECTIVE
To investigate the mechanisms of AFP-specific transfer factors (AFP-TF) in induced Bel7402 cells apoptosis. Further, we investigate the interaction between AFP-TF and AFP in the apoptosis.


METHODS
Bel7402 and HepG2 AFP-positive hepatocarcinoma cell lines, SK-Hep-1 AFP-negative hepatocarcinoma cell line and Changliver normal liver cell line are used. Cell viability is evaluated by MTT assay and apoptosis is measured by Hoechst33342 staining and TUNEL assay. FACS is used to analyze the cell cycle. AFP expression is examined by RT-PCR, Western blotting and immunocytochemistry. The interaction between AFP-TF and AFP in the apoptosis is investigated by addition of AFP in cultures or AFP transfection in Bel7402 cells prior to AFP-TF treatment. Mitochondrial membrane potential (DeltaPsi(m)) and intracellular Ca2+ concentration are respectively measured by Rhodamine123 and Fluo-3 AM Ester. Western blotting detects the involvement of several apoptosis-related proteins. Finally, caspase-3 and Caspase-9 activity are respectively examined.


RESULTS
AFP-TF can induce apoptosis in Bel7402 and HepG2 AFP-positive hepatocarcinoma cells, but not SK-Hep-1 and Changliver cells. AFP-mRNA level changes little in apoptotic Bel7402 cells; while AFP expression is downregulated and uniformly dispersed throughout the whole cell. Addition of exogenous AFP or overexpression of intracellular AFP can reduce such apoptotic effect. Besides, apoptotic Bel7402 cells show a disruption of DeltaPsi(m), an immediate elevation of Ca2+ concentration, a prominently decreased ratio of bcl-2 to bax, a release of cytochrome c from mitochondria to cytosol, and ultimately an activation of caspase-9 and caspase-3.


CONCLUSIONS
AFP-TF induced Bel7402 cells apoptosis is mitochondrial-dependent and is mediated by the interaction of AFP-TF with intracellular AFP.
Zhang
Hui
H
School of Life Science, Shandong University, Jinan, China.
Bai
Zengliang
Z
Chen
Jing
J
Wang
Ze
Z
Li
Juan
J