Lenvatinib induces anticancer activity in gallbladder cancer by targeting AKT

Abstract

Gallbladder cancer (GBC) is characterized by poor prognosis, early metastasis, and high recurrence rates, which seriously threaten human health. The effect of lenvatinib, a widely used drug in anti-hepatocellular carcinoma in China, on GBC progress, as well as its underlying molecular mechanism, remains unclear. Therefore, the present study investigated the effect of lenvatinib on human GBC GBC-SD and NOZ cells and its underlying mechanisms. A series of experiments, including cell proliferation, clone formation, wound healing, and cell migration and invasion assays, as well as flow cytometry, were performed to investigate the anticancer effect of lenvatinib on GBC. Western blotting was used to detect alterations in protein expression of CKD2, CKD4, cyclin D1, caspase-9, matrix metalloproteinase (MMP)-2, cell migration-inducing protein (CEMIP) and phospho-AKT (p-AKT). In addition, the chemosensitivity of lenvatinib-treated GBC cells to gemcitabine (GEM) and whether the activation of phosphoinositide 3 kinase (PI3K)/AKT contributed to the chemoresistance were determined. Finally, the anticancer effect of lenvatinib in vivo was detected using a xenograft mouse model. These data showed that treatment with lenvatinib inhibited cell proliferation, colony formation ability, migration, induced apoptosis, regulated cell cycle and resulted in decreased resistance to GEM. Treatment with lenvatinib decreased the expression of MMP-2, CEMIP, CDK2, CDK4 and cyclin D1, and increased the expression of cleaved caspase-9, which was mediated by the inactivation of the PI3K/AKT pathway in vitro. In addition, lenvatinib inhibited autophagy in GBC-SD and NOZ cells. Besides, Lenvatinib suppressed GBC cell growth in vivo by targeting p-AKT. In combination, the present data indicated that lenvatinib plays a potential anticancer role in GBC by downregulating the expression of p-AKT.

Keywords: AKT; apoptosis; gallbladder cancer; lenvatinib; migration; proliferation.

Figure 1

Lenvatinib inhibited proliferation and colony formation in GBC-SD and NOZ cells. GBC-SD and NOZ cells were treated with different concentrations of lenvatinib for 24, 48 and 72 h. (A and B) Cell viability was examined by Cell Counting Kit-8 assay. (C) Colony formation. ^*P<0.05 vs. 0 µM. NC, negative control.

Figure 2

Lenvatinib induced apoptosis and regulated cell cycle arrest progression in GBC-SD and NOZ cells. GBC-SD and NOZ cells were treated with 25 or 50 µM lenvatinib for 48 h, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. (A) Quantification of apoptotic rate. (B) Distribution of cell cycle. (C and D) Protein expression of caspase-9, CytoC, Bax, CDK2, CDK4 and cyclin D1 were detected by western blotting. β-actin or GAPDH was used as a loading control. ^*P<0.05 vs. NC. Len, Lenvatinib; FITC, fluorescein isothiocyanate; CytoC, cytochrome c; Bax, Bcl-2-associated X protein; NC, negative control.

Figure 3

Lenvatinib inhibits migration and invasion in GBC-SD and NOZ cells by downregulating MMP-2 and CEMIP. GBC-SD and NOZ cells were treated with 25 or 50 µM lenvatinib for 48 h. (A) Influence of lenvatinib on cell migration by wound closure. (B and C) Influence of lenvatinib on cell migration and invasion by Transwell assays. (D and E) Analysis of MMP-2 and CEMIP following treatment with lenvatinib in GBC‑SD and NOZ cells, as assessed by western blotting. β-actin or GAPDH was used as a loading control. Original magnification, x100. ^*P<0.05 vs. NC. CEMIP, cell migration-inducing protein; Len, lenvatinib. MMP, matrix metalloproteinase; NC, negative control.

Figure 4

Lenvatinib suppressed autophagy in GBC-SD and NOZ cells. GBC-SD and NOZ cells were treated with 25 or 50 µM lenvatinib for 48 h. (A and B) Analysis of LC3II/I ration and p-62 following treatment with lenvatinib in GBC‑SD and NOZ cells, as assessed by western blotting. β-actin or GAPDH was used as a loading control. ^*P<0.05 vs. NC. LC3, microtubules associated proein 1 light chain 3β; NC, negative control.

Figure 5

Lenvatinib suppresses tumor growth in GBC‑SD xenografts. (A) Growth curves are representative of tumor volumes in BALB/c nude mice in the NC or lenvatinib groups. (B) Tumor weight was detected in the NC and lenvatinib groups at the endpoint of the animal experiment. (C) The protein expression of p-AKT in GBC-SD tumor tissues was measured by immunohistochemistry. ^*P<0.05 vs. NC. Len, lenvatinib; NC, negative control; IHC, immunohistochemistry.

Figure 6

Lenvatinib inhibits the expression of p-AKT and enhances GEM-mediated cell growth inhibition in GBC-SD and NOZ cells by inactivating the expression of p-AKT. GBC-SD and NOZ cells were pretreated with lenvatinib for 1 h, then treated with or without lenvatinib for 48 h. (A and B) The protein expression levels of p-AKT and PAPR1 were detected by western blotting with β-actin or GAPDH as a loading control. (C and D) Cell growth proliferation was detected by CCK-8. (E and F) The protein expression levels of p-AKT were measured by western blotting in GBC-SD and NOZ cells following treatment with lenvatinib and GEM. ^*P<0.05 vs. NC. ^**P<0.05 vs. GEM group. NC, negative control; GEM, gemcitabine; PAPR1, poly ADP-ribose polymerase 1; Len, lenvatinib.