The Growth Inhibitory Effect And Mechanism Of Mesenchymal Stem Cells On Liver Cancer Cells

ABSTRACT As one of adult stem cells, mesenchymal stem cells (MSCs) had multi-differentiation potential, wide sources, easy purification, low immunogenicity, strong proliferation, migration to tumor and other characteristics and were the important seed cells for stem cell therapy. At present, MSCs has been used in the treatment of tissue defects and organ degenerative diseases, which had made important progress. On the other hand, MSCs are tried to use for cancer prevention and treatment. This study investigated the inhibition effect and mechanism of MSCs on HepG2 cells in vitro and in vivo.In vitro, to explore the inhibition effect of MSCs conditional media treated HepG2 or co-cultured with HepG2 cells by MTT experiments. The conditional media groups included MSCs or HEK 293 conditional media groups, specific groups as follows: HepG2 media control group, MSCs media control group,20% conditional media group, 40% conditional media group,60% conditional media group,80% conditional media group,100% conditional media group; co-cultured groups included MSCs or HEK 293 cell lines (as control) and HepG2 were co-cultured, divided into four proportion groups (10:1,5:1,1:1,1:5), to test the inhibitory effect of MSCs on HepG2 cells respectively at 24 h,48 h and 72 h. The apoptosis of HepG2 induced by MSCs was analyzed by flow cytometry. After HepG2 was treated with MSCs or MSCs conditional media, the expressions of Wnt signaling pathway related factors (bcl-2, c-Myc, (3-catenin and survivin) were investigated by RT-PCR. After HepG2 was treated with MSCs conditional media, the expression of bcl-2 was investigated by Western blot. The content of Dkk-1 secreted from MSCs was analysed by ELISA kit. In vivo, MSCs or HEK 293 and HepG2 cells at different proportion were co-injected into nude mice under the arms, the growth of tumor was observed; producted the frozen sections and HE staining of tumor tissue, examinated the pathological of tumor tissue and the inhibition effect of MSCs on HepG2 cells was observed.From the MTT experiments,40%,60%,80% and 100% MSCs conditional media groups all showed significant inhibition effect (P<0.05), only 20% MSCs conditional media had no inhibition effect (P>0.05). After HepG2 were co-cultured with MSCs, 10:1,5:1 and 1:1 experimental groups showed significant inhibition effect at 24 h,48 h and 72 h; 1:5 and 1:10 experimental groups had no significant inhibition effect. In the flow cytometry experiments, MSCs induced significant apoptosis of HepG2 cells in MSCs:HepG2=5:1and 1:1 co-culture groups at 48 h and 72 h. After HepG2 cells were treated by MSCs conditional media, the expression in mRNA level of bcl-2, c-Myc,β-catenin, and survivin all decreased at 24 h,48 h and 72 h. After HepG2 were co-cultured with MSCs at 24 h,48 h and 72 h compared with control, the expression in mRNA level of bcl-2, c-Myc,β-catenin, and survivin was downregulated in MSCs:HepG2=5:1 group and MSCs:HepG2=1:1 group at 24 h,48 h and 72 h; in MSCs:HepG2=1:5 group, the expression in mRNA level of bcl-2, c-Myc,β-catenin and survivin did not decrease. In Western blot experiment, the expression of bcl-2 was downregulated compared with control. ELISA result showed that Dkk-1 content from the supernatants of MSCs was much higher than the supernatants of HEK 293. In vivo, after MSCs or HEK 293 and HepG2 were co-injected in nude mouse, compared with control, the tumor growth was significantly inhibited in MSCs:HepG2 2:1 and 1:2 groups, and was not inhibited in HEK293:HepG2 1:1 and 1:2 groups. The result of tumor tissue slices showed that there were more connective tissue and less tumor cells in MSCs:HepG2=2:1 group, and that there were exuberant tumor cells with thick color in HEK 293:HepG2=2:1 group and HEK 293:HepG2=1:1 group.MSCs could inhibit proliferation and promote apoptosis of hepatoma cell line HepG2 with quantity and time dependence. MSCs could secrete Dkk-1 and inhibit expressions of Wnt signaling pathway related factors (bcl-2, c-Myc,β-catenin and survivin) of tumor cells, and then inhibit the proliferation and promote apoptosis of HepG2. Therefore, this study provides a new approach and experimental basis for the treatment in cancer by MSCs, and expands the stem cell applications.