Regorafenib inhibits tumor progression through suppression of ERK/NF-κB activation in hepatocellular carcinoma bearing mice

Abstract

Regorafenib has been demonstrated in our previous study to trigger apoptosis through suppression of extracellular signal-regulated kinase (ERK)/nuclear factor-κB (NF-κB) activation in hepatocellular carcinoma (HCC) SK-Hep1 cells in vitro However, the effect of regorafenib on NF-κB-modulated tumor progression in HCC in vivo is ambiguous. The aim of the present study is to investigate the effect of regorafenib on NF-κB-modulated tumor progression in HCC bearing mouse model. pGL4.50 luciferase reporter vector transfected SK-Hep1 (SK-Hep1/luc2) and Hep3B 2.1-7 tumor bearing mice were established and used for the present study. Mice were treated with vehicle or regorafenib (20 mg/kg/day by gavage) for 14 days. Effects of regorafenib on tumor growth and protein expression together with toxicity of regorafenib were evaluated with digital caliper and bioluminescence imaging (BLI), ex vivo Western blotting immunohistochemistry (IHC) staining, and measurement of body weight and pathological examination of liver tissue, respectively, in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice. The results indicated regorafenib significantly reduced tumor growth and expression of phosphorylated ERK, NF-κB p65 (Ser536), phosphorylated AKT, and tumor progression-associated proteins. In addition, we found regorafenib induced both extrinsic and intrinsic apoptotic pathways. Body weight and liver morphology were not affected by regorafenib treatment. Our findings present the mechanism of tumor progression inhibition by regorafenib is linked to suppression of ERK/NF-κB signaling in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice.

Keywords: Bioluminescence imaging; Regorafenib; apoptosis; hepatocellular carcinoma; nuclear factor kappaB.

Figure 1. Schematic depiction of experimental protocol

Please refers to “Material and methods” section in context for detail; HE, hematoxylin and eosin; IHC, immunohistochemistry.

Figure 2. Effect of regorafenib on tumor growth in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice

Mice were treated with vehicle (140 μl of PBS plus 10 μl of DMSO per day) or regorafenib (20 mg/kg/day) by gavage for 14 days. Tumor growth was evaluated with digital caliper and BLI. (A) SK-Hep1/luc2 tumor volume was measured by digital caliper on day 0, 2, 4, 6, 8, 10, 12, and 14. (B) Hep3B 2.1-7 tumor volume was measured by digital caliper on day 0, 2, 4, 6, 8, 10, 12, and 14. (C) SK-Hep1/luc2 tumor growth was monitored with BLI on day 0, 7, and 14; ^aP<0.01 and ^bP<0.05 as compared with vehicle group. BLI, bioluminescence imaging; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered solution.

Figure 3. Effect of regorafenib on expression of P-ERK, AKT (Ser473), NF-κB p65 (Ser473), and NF-κB-modulated downstream effector proteins in SK-Hep1/luc2 tumor and Hep3B 2.1-7 bearing mice

Mice were killed on day 14 after treatments and protein levels in tumor tissues were evaluated with IHC staining. (A) Protein levels of MMP-9, VEGF, MCL-1, XIAP, C-FLIP, Cyclin-D1, and P-ERK, AKT (Ser473), NF-κB p65 (Ser473) on SK-Hep1/luc2 tumor by IHC. (B) IHC staining of Hep3B 2.1-7 tumor. (C) Phosphorylation oncogenes and apoptosis-related cleavage proteins expression which validated by Western blotting on SK-Hep1/luc2 tumor. (D) Western blotting of Hep3B 2.1-7 tumor. (E) Expression of antiapoptotic proteins (active Capase-9, -8, and -3) on SK-Hep1/luc2 tumor by IHC. (F) IHC staining of Hep3B 2.1-7 tumor; ^aP<0.01 as compared with vehicle group; C-FLIP, cellular FADD-like IL-1β-converting enzyme-inhibitory protein; IHC, immunohistochemistry; MCL, myeloid leukemia cell differentiation protein; MMP, matrix metallopeptidase; NF-κB, nuclear factor-κB; P-ERK, phosphorylated extracellular signal-regulated kinase; VEGF, vascular endothelial growth factor; XIAP, X-linked inhibitor of apoptosis protein.

Figure 3. Effect of regorafenib on expression of P-ERK, AKT (Ser473), NF-κB p65 (Ser473), and NF-κB-modulated downstream effector proteins in SK-Hep1/luc2 tumor and Hep3B 2.1-7 bearing mice

Mice were killed on day 14 after treatments and protein levels in tumor tissues were evaluated with IHC staining. (A) Protein levels of MMP-9, VEGF, MCL-1, XIAP, C-FLIP, Cyclin-D1, and P-ERK, AKT (Ser473), NF-κB p65 (Ser473) on SK-Hep1/luc2 tumor by IHC. (B) IHC staining of Hep3B 2.1-7 tumor. (C) Phosphorylation oncogenes and apoptosis-related cleavage proteins expression which validated by Western blotting on SK-Hep1/luc2 tumor. (D) Western blotting of Hep3B 2.1-7 tumor. (E) Expression of antiapoptotic proteins (active Capase-9, -8, and -3) on SK-Hep1/luc2 tumor by IHC. (F) IHC staining of Hep3B 2.1-7 tumor; ^aP<0.01 as compared with vehicle group; C-FLIP, cellular FADD-like IL-1β-converting enzyme-inhibitory protein; IHC, immunohistochemistry; MCL, myeloid leukemia cell differentiation protein; MMP, matrix metallopeptidase; NF-κB, nuclear factor-κB; P-ERK, phosphorylated extracellular signal-regulated kinase; VEGF, vascular endothelial growth factor; XIAP, X-linked inhibitor of apoptosis protein.

Figure 4. Toxicity investigation of regorafenib in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice

Toxicity of regorafenib was evaluated with body weight measurement and pathological examination of mice livers. (A) Body weight of both HCC bearing mouse were measured on day 0, 2, 4, 6, 8, 10, 12, and 14. (B) Mice were killed on day 14 after treatments and pathological examination of livers was evaluated with H&E staining; HE, hematoxylin and eosin.