Recombinant soluble gp130 protein reduces DEN-induced primary hepatocellular carcinoma in mice

Abstract

IL-6 (interleukin 6) plays an important role in the development and growth of hepatocellular carcinoma (HCC) via both classic signaling and trans-signaling pathways. Soluble gp130 (sgp130) is known to be a natural inhibitor of the trans-signaling pathway. In the present study, our goal was to investigate whether recombinant sgp130 could suppress the initiation and progression of HCC in mouse models. Our results demonstrate that sgp130 induced an apoptosis of HepG2 cells and inhibited the clonogenicity of HepG2 in vitro. Moreover, the IL-6 trans-signaling pathway is significantly suppressed by sgp130 as reflected by the decrease in the level of STAT3 phosphorylation and other inflammatory factors both in vitro and in vivo. In the DEN-induced HCC mouse model, intravenous injection of sgp130 attenuated hepatic fibrosis at 16 weeks and reduced the initiation and progression of primary HCC at 36 weeks. Furthermore, our results also demonstrate that intravenous administration of sgp130 significantly suppressed the growth and metastasis of xenograft human HCC in NOD/SCID mice.

Figure 1. Sgp130 influences the growth and apoptosis of HepG2 cells.

(a) IL-6 increases the transcriptional level of IL-6 signaling-related genes in a dose-dependent manner (Single*, double** and triple*** marked represent statistical significance in p < 0.05, p < 0.01 and p < 0.001 in comparison with their control samples, respectively). (b) sgp130 inhibits HepG2 cell growth in a dose-dependent manner as measured with MTT. (c) Apoptosis of HepG2 cells analyzed with FACS after IL-6 or sgp130 treatment for 24 h. (d) Observed with the PI and Annexin-FITC stained HepG2 cells before FACS analysis (red image observed with UV laser; green image observed with blue light).

Figure 2. Sgp130 suppresses IL-6 signaling pathway in HepG2 cells.

(a) Lysates of HepG2 cells treated with IL-6 and sgp130 for 48 h were analyzed for global tyrosine phosphorylation at position 705 of STAT3. (b) sgp130 inhibits IL-6 induced transcriptional activation of inflammatory factors in HepG2 cells treated with sgp130 or IL-6 for 48 h (1: control; 2: positive sample; 3: sgp130 treated sample). Relative expression levels of cytokine mRNAs isolated from cells and analyzed by real-time PCR. Data are mean ± SD. n = 3 (Single*, double** and triple*** marked represent statistical significance in p < 0.05, p < 0.01 and p < 0.001, respectively). (c) sgp130 decreases IL-6-induced increase of Ki67 in IL-6 and sgp130 co-treated HepG2 cells for 48 h. The cells were stained with a specific antibody against Ki67 and the treatment followed with HRP-coupled secondary antibody (upper panel); sgp130 suppresses IL-6 activated growth of HepG2 cells which was analyzed by soft agar assay (lower panel). (1 × 10³ cells/well).

Figure 3. Sgp130 reduces DEN-induced inflammatory responses in mouse liver tissue at 16 weeks.

(a) Schematic diagrams of sgp130 in inhibiting IL-6 signaling. (b) Expression of p-STAT3, IL-6, IL-6R and gp130 in mouse liver tissue (400×). (A: Control; B: model group; C: sgp130 treatment group). (c) sgp130 changes the DEN-induced high level of liver related factors in mouse sera. (d) B-ultrasonography of liver for detecting DEN-induced hepatic fibrosis (Arrowheads point to hepatic fibrosis bright spots or stripes; (A: Control; B: model group; C: sgp130 treatment group)). (e) type IV collagen expressed in tissue of mouse livers, detected with immunohistochemical staining (400×) (A: Control; B: model group; C: sgp130 treatment group).

Figure 4. Sgp130 effects tumor initiation and development in mouse liver.

(a) The changes of α-AFP levels in mouse sera detected at 16 weeks and 36 weeks respectively. (b) Expression of liver cancer marker genes in mouse liver tissue analyzed with immunohistochemical staining at Week 16 (100×). (c) Tumors on the surface of mouse livers after treatment for 36 weeks. (A: Control; B: model group; C: sgp130 treatment group).

Figure 5. Sgp130 suppresses tumor growth in nude mice.

(a) The xenograft tumors and their curve on nude mice receiving a subcutaneous injection of 5 × 10⁶/mL HepG2 cells. Data are mean ± SD, n = 6 (Single*, double** and triple*** marked represent statistical significance in p < 0.05, p < 0.01 and p < 0.001, respectively). Each experiment was repeated three times, and data shown are representative one of three independent experiments. (b) Immunohistochemical analysis of IL-6, IL-6R, gp130, Ki67 and PCNA expression in xenograft tumors (100×) (A: Control; B: sgp130 treatment group). (c) Detection of HepG2 cell metastasis in nude mouse lungs through analyzing expression of CD44 and Ki67 lung tissues (100X) (A: Negative control; B: Control; C: sgp130 treatment group).