IGF-1 promotes the growth and metastasis of hepatocellular carcinoma via the inhibition of proteasome-mediated cathepsin B degradation

Figure 4 IGF-1 impeded cathepsin B degradation by inhibiting ubiquitin-proteasome system activity. A: IGF-1 did not affect the mRNA level of cathepsin B (CTSB). Hep1-6 and H22 cells were treated with IGF-1 for 12 h. The mRNA expression levels of CTSB and actin were determined by RT-PCR; B: IGF-1 impeded CTSB degradation. Hep1-6 and H22 cells were incubated with CHX (10 g/mL) for the indicated time points after IGF-1 stimulation. CTSB and actin protein levels were detected by immunoblotting; C: CTSB degradation is dependent on ubiquitin-proteasome system (UPS) activity. Hep1-6 cells were treated with CHX plus an autophagy inhibitor (bafilomycin, 200 nmol/L) or UPS inhibitor (MG132, 10 μmol/L) at the indicated time points. Cell lysates were isolated for immunoblotting; D: IGF-1 inhibited UPS activity. Hep1-6 cells were transfected with Ub^G76V-GFP plasmid. At 24 h after the cells were transfected, they were treated with IGF-1 for another 12 h, and Ub^G76V-GFP expression was detected immunoblotting with anti-GFP antibody or by flow cytometry; E: PA28γ overexpression reversed the IGF-1-induced inhibition of UPS activity. Hepa 1-6 cells were infected with lentivirus containing PA28γ. After 24 h, the cells were treated with IGF-1 for 12 h. Ub^G76V-GFP expression was detected by immunoblotting with anti-GFP antibody or by flow cytometry; F: PA28γ overexpression recovered CTSB degradation. Hepa 1-6 cells were infected with lentivirus containing PA28γ. After 24 h, the cells were treated with IGF-1 for 12 h, and CTSB expression was detected by immunoblotting with anti-CTSB antibody. Data are presented as the mean ± SE of 3 independent assays. ^aP < 0.05.