The human runt-related
transcription factor 3 gene (RUNX3) is considered to be a candidate tumor suppressor gene in gastric
carcinoma. However, the role of RUNX3 in the regulation of cell proliferation remains unclear. In the present study, we constructed an adenoviral vector encoding human RUNX3
cDNA under the control of a Tet-responsive promoter (Ad-Tet-FLAG-RUNX3), which regulates the expression of RUNX3 in the presence or absence of
doxycycline. A recombinant adenoviral expression vector encoding LacZ (Ad-Tet-LacZ) was used as a negative control. The effect of the transduction of RUNX3 on cell growth was examined using the Tet-On system in a human gastric
carcinoma cell line, MKN-1. Exogenous RUNX3 expression was induced successfully by Ad-Tet-FLAG-RUNX3, but not Ad-Tet-LacZ, in the presence of
doxycycline in the MKN-1 cells. At 72 h after
infection, the proliferative activity in RUNX3-expressing cells was 55% or less of that of the control cells. Flow cytometry revealed that the sub-G(1) peak was increased in cells expressing RUNX3 (34.11%), indicating that the inhibition of cell growth was due to apoptosis, which was confirmed based on
Hoechst 33258 staining, the release of
cytochrome c from mitochondria into the cytosol, and detection of cleaved
caspase-3 by western blotting in MKN-1 cells. Comprehensive analysis using a
cDNA microarray showed that RUNX3 upregulated 17 apoptosis-related genes (including FADD,
TRAF6,
caspase-2, ING1, ING4,
Calpain 10, and DNase1) and downregulated 135 apoptosis-related genes (including FLIP, PEA15, TXN2, HSPD1, IKK, and TIAL1) in MKN-1 cells. Pathway analyses to generate functional networks of the genes suggested that promotion of the formation of the death-inducing signaling complex and activation of the mitochondria-mediated pathway were associated with RUNX3-induced apoptosis. In conclusion, our findings suggest that exogenous RUNX3 expression suppressed cell proliferation by inducing apoptosis via the
death-receptor mitochondria-mediated pathway in MKN-1 cells.