Background
Cholangiocarcinoma(CCA), also known as bile duct cancer, is a form of cancer that originates in the epithelial cells of bile ducts, along intrahepatic and extrahepatic biliary tree, that is defined as intrahepatic, peri-hilar and distal CCA [
4,
22]. Due to the high aggressive ability, CCA could easily infiltrate into adjacent organs such like liver, hepatic artery and portal vein [
29]. The infiltration patterns of CCA were distributed in lymph, vascular infiltration site, and lymph node metastases, which is a basic feature of CCA [
6,
12].
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]. Clinical data shows VEGF was significantly increased in the biopsy samples of CCA [
2,
18,
26]. Furthermore, 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].
Apatinib, a tyrosine kinase inhibitor that selectively inhibits the vascular endothelial growth factor receptor-2 (VEGFR2, also known as KDR), could significantly inhibit intracellular VEGF signaling [
28]. Benefiting from the blocking effect of VEGF pathway, apatinib play a prominent role in inhibiting tumor cells anti-apoptosis, cells proliferation in vitro and repressing the growth of xenograft tumor in vivo [
19,
20]. In additon, apatinib reveals inhibition effect on migration and invasion in KIF5B-RET driven tumors therapy [
13]. However, up to now there are currently few studies on the impact of CCA migration and invasion. In this study, we investigated the role of apatinib in CCA migration and invasion via the QBC939 and TFK-1 cell line. Moreover, we also explored the potential mechanism that the inhibition effect of apatinib may via VEGFR2/RAF/MEK/ERK and PI3K/AKT pathways.
Methods
Cell culture and transfection
Human CCA cell lines QBC939 and TFK-1 were purchased from Suer Biological Inc. (Shanghai, China). QBC939 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) and TFK-1 cells were cultured in RPMI-1640 medium (Gibco-BRL, Gaithersburg, MD), both supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and incubated at 37 °C with 5% CO2.
Cells were sub-cultured to 6-well plates until the confluence reached 80%. The final concentration 50 mM siKDR and siControl (labeled with a fluorescent, synthesize by Gene Pharma, Suzhou, China) were diluted in serum-free MEM, and gently mixed with Lipofectamine 2000 (6 μl/well, Sigma-Aldrich, St. Louis, MO, USA) following 5 min stand, respectively. Before added this mixture to cells, another 20 min stand at room temperature is needed. The transfected cells incubated at 37 °C with 5% CO2 for 8 h, and then change the medium into McCoy’s 5A medium containing 10% FBS without antibiotics. 24 h post transfection, the transfection efficiency was checked by fluorescence detection.
RT-qPCR
VEGFR2 mRNA levels from two group cells were tested by RT-qPCR. The first group cells were transfected with siKDR and siControl for 24 h. Cells in the second group was treated with 0, 20, 50, 100, and 200 ng/ml recombinant human VEGF (rhVEGF, PeproTech, 100–20-2) for 2 h. Cells were subsequently homogenized and centrifuged (12,000 x g, 10 min, 4 °C) using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) for total RNA extraction. RNA purity and concentration were determined by Nano-Drop (Thermo Scientific).
1 mg total RNA was reverse transcribed into cDNA using GoScript™ RT system (Promega, Madison, WI, USA). qPCR was performed in triplicate as: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, 58 °C for 10 s and 72 °C for 30 s, subsequently analyze melting curve. GAPDH was used as the reference gene. Primers (forward, reverse) were: VEGFR2 5’-GGACTCTCTCTGCCTACCTCAC-3′, 5’-GGCTCTTTCGCTTACTGTTCTG-3′, GAPDH 5’-AGAAGGCTGGGGCTCATTTG-3′, reverse 5’-AGGGGCCATCCACAGTCTTC-3′. The relative fold change of VEGFR2 was calculated by 2-ΔΔCt method.
MTT assay
After QBC939 cells and TFK-1 cells were cultured to 96-well plates (1 × 105 cells/well) overnight, three conditions of drug treatment were set: (1) cells treated with 0, 10, 100, 1000 and 10,000 nM apatinib (MCE, HY13342) for 24 h; (2) cells treated with 0, 20, 50, 100 and 200 ng/ml rhVEGF for 2 h; (3) cells treated with 100 ng/ml rhVEGF for 2 h following treated with 10, 100, 1000 and 10,000 nM apatinib for 24 h And then cells were cultured for another 24 h, 10 mg/ml MTT was added and incubated for further 4 h. After that, cells were centrifuged at 1,000×g for 5 min at room temperature, removed supernatant, and added 100 μl DMSO to each well for 30 min to dissolve the formazan product. The optical density (OD) was measured at 492 nm by a microplate reader (FLx800; BioTek, Winooski, VT, USA). The relative cell viability was normalized with control group using optical density values.
Wound healing assay
Cells were cultured to 6-well plates (2 × 105 cells/well) until about 100% confluence. 100 nM apatinib or 100 ng/ml rhVEGF + 100 nM apatinib were added into medium and cultured 24 h. 200 μl pipette tip was used to create a wound gap on cell monolayer, and Olympus IX71 microscope (Olympus Corporation, Tokyo, Japan) at 100 times magnification was used for imaging immediately. Migration was then observed 24 h post wound scratched. Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA) was used to calculated the relative migration distant% as: [(The relative distance recorded at 0 h - the relative distance recorded at 24 h)/the relative distance recorded at 0 h] × 100.
Transwell matrix assay
Control and siKDR transfected cells (1 × 104 cells/well) were incubated into the top chamber of matrigel coated polyethylene terephthalate membrane (50 μl/well, Corning, Corning, USA), and 100 nM apatinib or 100 ng/ml rhVEGF were added into the upper chamber,. After culturing for 24 h, cells in the upper chamber were removed gentlyand the invaded cells left at the bottom of chamber were fixed with 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet for 30 min. Following by counting under an optical microscope (Olympus Corporation, Tokyo, Japan) at a magnification of 200.
Western blotting
After cells treated with/without 100 ng/ml rhVEGF, 100 nM apatinib or 100 ng/ml rhVEGF + 100 nM apatinib, cells were lysed using lysis buffer (Cell Signaling Technology, Danvers, USA) to extract total protein. Protein lysates were separated by 10% SDS-PAGE, followed by transfer to nitrocellulose membranes. The membrane was then blocked with 5% milk diluted in PBS at room temperature for 1 h, followed by incubated with 1:1000 VEGFR2 antibody (ab10972, Abcam, Cambridge, MA, USA),1:5000 p-VEGFR2 (ab38473, Abcam), 1:2000 p-MEK (2338, CST), 1:1000 MEK (4694, CST), 1:2000 p-ERK1/2 (4370, CST), 1:1000 ERK (4695, CST), 1:2000 slug (ab51772, Abcam), 1:3000 Snail (ab53519, Abcam), 1:2500 MMP9 (ab38898, Abcam), 1:1500 P-AKT (ab81283, Abcam), 1:1500 AKT (ab179463, Abcam) and 1:5000 GAPDH antibody (ab8245, Abcam) overnight at 4 °C separately. Once primary antibodies were washed, membrane was incubated with goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Sangon Biotech, Shanghai, China). Protein bands were detected by incubating the membrane with Western Bright enhanced chemiluminescence working solution (Advansta, Menlo Park, CA, USA). The film (Kodak XBT-1, Carestream, Xiamen, China) was scanned with Bio-rad Gel Doc XR+ (BIO-RAD, Shanghai, China).
Statistical analyses
Statistical analysis was conducted with the Social Sciences software version 17.0. Quantitative data were presented as mean ± SD. The two-tailed Student’s t test was applied to analyze statistical differences between two groups. For multiple comparisons, the one-way ANOVA was used to analyze the difference. p < 0.05 was considered to be statistically significant. Each test data was repeated at least three times.
Discussion
VEGF exerts its biological effects by combining and activating its receptors, which known as VEGFR2 [
8,
27]. Publications reveal VEGF plays key role in CCA as the high expression level was detected in the patients’ tumor tissues [
1,
15]. Hence, as the antagonist of VEGFR2, apatinib has the potential to become an effective targeted medicine of CCA [
16,
19,
20]. In this study, we firstly confirmed the role of VEGF in QBC939 and TFK-1 cells, and then comfirmed the inhibition functions of apatinib in migration and invasion of these two CCA cell lines. Finally, we analyzed the potential signaling pathways Raf/MEk/ERK and PI3K/AKT that might be influenced by apatinib.
Publication reported VEGF could regulate kinds of cancer cells growth through binding to VEGFR [
10]. Results in our study showed that exogenous rhVEGF activated the VEGFR2 expression and promoted the cell viability both in QBC939 and TFK-1 cells. It was consistent with publications that blocking VEGF/VEGFR2 pathway have inhibition effect on the growth of cancer cells [
7,
14,
32]. Moreover, one paper revealed the role of VEGF in promoting cell growth and inducing the cell apoptosis in CCA [
19,
20]. However, whether VEGF was an necessity for tumor migration or invasion in CCA remains unknown.
As the antagonist of VEGFR2, the biological functions of apatinib towards cell migration and invasion in CCA cell lines were performed in this study, and the results provide a first ever comprehensive elucidation of apatinib in anti-CCA progress. We found that it significantly inhibited cell migration and invasion. Moreover, apatinib significantly decreased the expression of metastatic marker such like Slug, snail, and MMP9 in the CCA cell lines. It was consistent with the previous research in lung adenocarcinomas, which found apatinib inhibits cellular invasion and migration by fusion kinase KIF5B-RET via suppressing RET/Src signaling pathway [
13]. In addition, it was report intracellular autocrine VEGF signaling promotes EBDC cell proliferation, which can be inhibited by apatinib [
19,
20]. And we also found that exogenous rhVEGF significantly promoted migration and invasion in QBC939 and TFK-1 cells, whereas apatinib could reverse these effects. Combine our data and references mentioned above, it exposes the essential role of apatinib in anti-tumour effect, and the apatinib induced inhibition of cell migration and invasion in CCA.
RAF/MEK/ERK pathway has been linked in endothelial cell proliferation [
17] and VEGF mediated cell survival [
3,
11]. To investigate the possible mechanism of how apatinib works on CCA cells. We found exogenous rhVEGF markedly elevated phosphorylation and total of VEGFR2 protein, and the major downstream targets: phosphorylation of RAF, MEK and ERK1/2, but did not affect the levels of total MEK and ERK1/2. On the contrary, apatinibprominently inhibited this promotion. These results disclosed apatinib could efficiently inhibit the activation of VEGFR2/RAF/MEK/ERK1/2 signal transduction which was induced by VEGF, Besides, we also checked the PI3K/AKT pathway, which was identified as a VEGF related signaling pathway [
9,
19,
20,
31]. Same results were gained such like the detection of RAF/MEK/ERK1/2 pathway, which provided another molecular mechanism of apatinib acted on CCA cells. These results are supported by several studies that the anti-apoptosis effect of VEGF closely related to PI3K/AKT/mTOR signaling pathway [
9,
19,
20,
31]. Previous studies has revealed that both VEGF/MEK/ERK and PI3K/AKT pathways play key role in developing CCA [
9,
24,
25,
30]. Here, we found that apatinib could reverse the rhVEGF induced cell migration and invasion by blocking these two pathways. Combined with our data and references we have mentioned above, apatinib has an important role in CCA migration and invasion, and apatinib exerts excellent anti-tumor function in CCA cell lines. However, whether apatinib could perform the same antitumor function of CCA in vivo still needs to be studied. Additionally, since angiogenesis of endothelial cells is an important factor in promoting tumor metastasis, and apatinib might play key role in angiogenesis via VEGFR2 as it expressed in endothelial cells. This would be worth to study in our future work.