Down-Regulation of TGF-β Expression Sensitizes the Resistance of Hepatocellular Carcinoma Cells to Sorafenib

Abstract

Purpose: Sorafenib, a multikinase inhibitor, is the standard therapy for patients with advanced-stage hepatocellular carcinoma (HCC). However, resistance develops to the treatment, therefore, we tried to unravel the underlying mechanism in the resistance of HCC cells to sorafenib via the development of more effective therapeutic strategies.

Materials and methods: Various liver cancer cell lines were treated with either sorafenib only or with sorafenib after infection of adenovirus expressing short hairpin RNA (shRNA) against transforming growth factor-β (TGF-β) and p38 activity was examined using western blotting.

Results: p38 MAP kinase activity was inhibited by low concentrations of sorafenib, which could potentially lead to sorafenib resistance in HCC cell lines. Subsequently, we used constitutive form of MKK3/6 (MKK3/6E) to confirm that massive cell death was induced by the activation of p38, and demonstrated the ability to activate p38 without any stimulation. In addition, sorafenib resistance was reduced by the activation of p38. Subsequently, we confirmed that TGF-β shRNA effectively recovered the phosphorylation of p38 inhibited by sorafenib, and increased the sensitivity of HCC cells to sorafenib, thereby inducing cell death and overcoming the resistance of HCC cells to sorafenib.

Conclusion: Our study provides a new therapeutic strategy for HCC that overcomes the resistance of HCC to sorafenib by down-regulation of TGF-β.

Keywords: HCC; TGF-β; adenovirus; p38; resistance; sorafenib.

Fig. 1. Effect of sorafenib on different HCC cell lines. (A) Hep-3B, Huh7, SK-Hep-1, SNU-182, SNU-398, and SNU-449 cells were treated with sorafenib in a dose-dependent manner. After 24 h, cell viability was tested via a MTS viability assay. IC50 of each cell line is indicated in each rectangle. Error bars represent the standard error from three independent experiments. (B) HCC cell lines were treated with sorafenib at IC50 concentrations, respectively. After 24 h, the expressions of p-p38, p38, p-ERK, p-Akt, p-Src, p-p65, and GAPDH were detected by western blot analysis. (C) HCC cell lines were treated with sorafenib in a dose-dependent manner for 24 h. and incubated for additional 14 days for clonogenic assays. (D) HCC cell lines were treated with a low dose of sorafenib (2.5 µM) for 24 h, and changes in the levels of p-Akt p-p65, p-ERK, and p-p38 expression were then detected by western blot analysis. HCC, hepatocellular carcinoma.

Fig. 2. MKK3/6E induced p-p38 activation and massive cell death in HCC cell lines. (A) Hep-3B, Huh7, SNU-398, and SNU-449 cells were transfected with the pCDNA3-MKK3/6E plasmid (1 µg) for 24 h, and treated with sorafenib (2.5 µM) for 24 h. Protein expressions of MKK3, MKK6, p-p38, and GAPDH were estimated via western blot analysis. (B) Hep-3B, Huh7, SNU-398, and SNU-449 cells were transfected with the pCDNA3-MKK3/6E plasmid (1 µg) for 24 h and treated with sorafenib in a dose-dependent manner for 24 h. Cell viability was examined using a MTS viability assay. Error bars represent the standard error from three independent experiments. ^‡p<0.001. HCC, hepatocellular carcinoma. (C) Hep-3B, Huh7, SNU-398, and SNU-449 cells were transfected with the pCDNA3-MKK3/6E plasmid (1 µg) for 24 h, treated with sorafenib in a dose-dependent manner for 24 h, and then incubated for additional 14 days for clonogenic assays. (D) Hep-3B, Huh7, SNU-398, and SNU-449 cells were transfected with the pCDNA3-MKK3/6E plasmid (1 µg) for 24 h, and treated with sorafenib (2.5 µM) for 24 h. Protein expressions of MKK3, MKK6, p-p38, p-Akt, p-p65 and GAPDH were estimated via western blot analysis. HCC, hepatocellular carcinoma.

Fig. 3. Changes in TGF-β expression in response to sorafenib treatment in HCC cell lines. (A) Hep-3B, Huh7, SK-Hep-1, SNU-182, SNU-398, and SNU-449 cells were treated with sorafenib (2.5 µM, IC50) for 24 h, and TGF-β mRNA was estimated by RT-PCR. Error bars represent the standard error from three independent experiments. (B) Hep-3B, Huh7, SK-Hep-1, SNU-398, and SNU-449 cells were treated with sorafenib (2.5 µM) for 24 h and then incubated for additional 14 days for sufficient acquisition of resistance. TGF-β1/2 expression was then detected via ELISA. Error bars represent the standard error from three independent experiments. ^*p<0.05, ^†p<0.01, ^‡p<0.001. TGF-β, transforming growth factor-β; HCC, hepatocellular carcinoma; RT-PCR, real-time polymerase chain reaction; ELISA, enzyme-linked immunosorbent assays; SR, sorafenib resistance. (C) SK-Hep-1, SNU-182, SNU-398, and SNU-449 cells were infected by defective adenoviruses (NC, shT1, and shT2) at 50 MOI. After 2 days, protein expression of p-p38 was detected via western blot analysis. (D) Huh7, SK-Hep-1, SNU-182, SNU-398, and SNU-449 cells were infected by defective adenoviruses (NC, shT1, and shT2) at 50 MOI. After 36 h, cells were treated with low concentration (2.5 µM) of sorafenib for 12 h. Changes in the protein expression of p38 and p-p38 were detected by western blot analysis. (E) SK-Hep-1, SNU-182, SNU-398, and SNU-449 cells were infected by defective adenoviruses (NC, shT1, and shT2) at 50 MOI. After 36 h, cells were treated with low concentration of sorafenib (2.5 µM) for 12 h, and were incubated for additional 14 days for clonogenic assays. TGF-β, transforming growth factor-β; HCC, hepatocellular carcinoma; NC, negative control; MOI, multiplicity of infection.

Fig. 4. Antitumor effects of the combined treatment of sorafenib and an adenovirus co-expressing shTGF-β and shHSP27 in BALB/c nude mice. (A) SNU-449 tumors were grown in male BALB/c nude mice. Tumors were established by subcutaneous injection of 1×10⁷ cells and were allowed to grow to an average size of 60–100 mm³. PBS and adenoviruses were intratumorally injected every other day for a total of 3 injections. Sorafenib (30 mg/kg) was administered via gavage once daily from days 1 to 10. Tumor growth was measured every 2 days for more than 19 days using calipers. (B) Survival rates were calculated every 2 days for more than 19 days. TGF-β, transforming growth factor-β; NC, negative control; PBS, phosphate buffered saline.

Fig. 5. A schematic diagram of sorafenib drug resistance by p38 activation inhibition and TGF-β down-regulation-induced sensitization of the resistance to sorafenib. TGF-β, transforming growth factor-β; VEGFR, vascular endothelial growth factor receptor; PDGFR, platelet-derived growth factor receptor.