Abstract
BackgroundCholangiocarcinoma (CCA) is a form of cancer that easily aggress to contiguous structures. Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) are increased in majority species of cancers and suppress tumor progression by blocking VEGF/VEGFR2. Apatinib is a highly selective VEGFR2 antagonist which has inhibitive effect on antiapoptotic and cell growth in CCA. While, the effect of apatinib cell migration and invasion in CCA is still unknown.MethodsCCA cell lines QBC939 and TFK-1 were transfected with siKDR to establish the KDR function loss cell model, and recombined human VEGF (rhVEGF) protein was added into the culture medium to enhance the VEGF expression. RT-qPCR and western bloting were used to detect the mRNA and protein expression levels of VEGFR2 to investigate whether it was effectively repressed or activated with rhVEGF or apatinib treatment. Then, MTT, wound healing assay, and transwell matrix assay were applied to measure the effect of apatinib and rhVEGF on cell viability, migration and invasion, respectively.ResultsThe mRNA and protein expressions of VEGFR2 were significantly reduced with KDR RNAi in both QBC939 and TFK-1 cells, and rhVEGF treatment increased these expression levels (p < 0.05). Apatinib dramatically suppressed VEGF-mediated cell migration and invasion at the concentration of 100 nM treatment and significantly decreased the expression of metastasis-associated protein such as Slug, snail and MMP9. Moreover, all of these inhibiting effects of apatinib depended on the VEGFR2 existence. In addition, VEGFR2/RAF/MEK/ERK and PI3K/AKT signal pathways were enhanced by the introduction of rhVEGF, but were dramatically suppressed after the apatinib treatment.ConclusionApatinib inhibit VEGF-mediated cell migration and invasion in CCA cell lines via inhibiting the VEGFR2/RAF/MEK/ERK and PI3K/AKT pathways. It will be a potentially effective targeted drug for CCA.
Highlights
Cholangiocarcinoma (CCA) is a form of cancer that aggress to contiguous structures
VEGF receptor 2 (VEGFR2) mRNA level reduced significantly, showed five and two times lower in Small interference KDR (siKDR) group compared to siControl group
Apatinib inhibites the migration and invasion of QBC939 and TFK-1 cells There were no changes of relative cell viability on both QBC939 and TFK-1 cells with 10 and 100 nM apatinib treatment, but 1,000 and 10,000 nM apatinib caused a greatly reduction of relative cell viability compared to control group, suggested 1,000 nM and higher concentration of apatinib could cause cytotoxicity on CCA cells (Fig. 3a)
Summary
Cholangiocarcinoma (CCA) is a form of cancer that aggress to contiguous structures. Vascular endothelial growth factor (VEGF), originally known as vascular permeability factor (VPF), is a signal protein produced by epithelial cells [23]. It has been identified as a key player in neovascularization and cell proliferation in a variety of cancers, including the fatal biliary CCA [5, 21]. There is evidences that blocking VEGF/VEGFR2 pathway can effectively inhibit the proliferation, migration, invasion, survival and adhesion ability of hepatocellular carcinoma, hyperplastic cholangiocyte and non-small cell lung cancer [14, 32]. A tyrosine kinase inhibitor that selectively inhibits the vascular endothelial growth factor receptor-2 (VEGFR2, known as KDR), could significantly inhibit intracellular VEGF signaling [28]. We explored the potential mechanism that the inhibition effect of apatinib may via VEGFR2/RAF/MEK/ERK and PI3K/AKT pathways
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