Background
Stomach cancer is the fifth most common cancer and the third major cause in cancer-related deaths in the word [
1]. Moreover, the mortality of stomach cancer is the second highest among all cancer related deaths in China according to the statistics of 2016 [
2]. It is difficult to detect the early stages of gastric cancer and there are an overwhelming number of patients who are diagnosed late [
3]. Therefore, the study of the mechanisms of cancer cell migration mechanisms during stomach cancer is of great significance for new drug discoveries and for the development of effective treatments for gastric cancer. Currently, the main treatment of gastric cancer consists of surgical treatment, targeted therapy, immunotherapy, radiotherapy and chemotherapy. However, these methods are accompanied by poor patient prognosis with advanced gastric cancer and therefore a novel treatment is urgently needed for people affected by this devastating disease.
Nicotinic acetylcholine receptors (nAChRs) are a type of ligand-gated ion channel proteins, and their expression is not only found on neuronal cells but also in non-neuronal cells including gastric cancer cells [
4,
5]. Alpha7 nicotinic acetylcholine receptor (α7-nAChR) is a member of the family of nAChRs and is widely expressed on stomach epithelial cells [
6,
7]. It has been reported that the activation of α7-nAChR plays an important role in the proliferation and migration of cancer cells. The extracellular signal-regulated kinase (ERK) signaling pathway is involved in a variety of diseases and activated by several stimuli including α7-nAChR [
8].
Epithelial to mesenchymal transition (EMT) is the original biological step required for the invasion of cancer cells and metastasis into tissues. A hallmark of EMT includes the presence of mesenchymal markers such as N-cadherin, Vimentin and the epithelial marker E-cadherin [
9,
10]. EMT is of great significance for tumor migration [11], and a recent study also suggested that alpha 7-nAChR overexpression could enhance EMT to promote proliferation and migration of gastric cancer cells [
11]. Moreover, it was reported that nicotine could promote EMT to induce migration of cancer cells via regulating of the alpha 7-nAChR/MEK/ERK pathway [
12].
Alpha 7-nAChR may be a potential therapeutic key point for the treatment of stomach cancer. Previous studies have also demonstrated that the recombinant avirulent NDV LaSota strain, expressing the rabies virus glycoprotein (rL-RVG), could induce the apoptosis of gastric cancer cells as well as suppress the migration of lung cancer cells by regulating α7-nAChR [
13,
14]. However, it still remains unclear whether rL-RVG could also suppress the migration of gastric cancer and its underlying mechanisms. In this study, we explored if rL-RVG could suppress the migration of gastric cancer cells via regulating α7-nAChR/ERK signaling and EMT.
Methods
Materials
The rL-RVG, NDV, anti-RVG antibody and anti-NDV antibody were stored at − 80 °C supplied by the Harbin Veterinary Research Institute (Harbin, China). The human gastric cancer cell lines SGC7901 and BGC were purchased from the Cancer Cell Repository (Shanghai Cell Bank,2016-11-20) and the American Type Culture Collection (Manassas,VA,USA). The cell line has been authenticated by STR (short tandem repeats, STR) and has been tested for mycoplasma contamination (Additional file
1). Nude mice were purchased from the. Animal Experiment Center of Yangzhou University (Yangzhou, China). Methyllcaconitine citrate hydrate (MLA) (5 mg/mL) which worked as a specific competitive α7-nAChR antagonist was purchased from Santa Cruz (California, USA). Acetylcholine bromide (ACB) (5 mg/mL),an acetylcholine agonist, was obtained from Sigma-Aldrich (St. Louis, MO, USA). In addition, small interfering RNA (si-RNA) for α7-nAChR was purchased from RiboBio (GuangZhou, China). Corynoxenine (10 mM 1 mL in DMSO),the inhibitor of the MEK-ERK pathway, was purchased from MedChemExpress (MCE,USA). Rabbit polyclonal anti-α7 nAChR was purchased from Abcam (London, UK); rabbit polyclonal anti-MEK1/2, anti-P-MEK1/2, anti-ERK1/2, anti-P-ERK1/2 and anti-snail were purchased from Cell Signaling Technology (CST, USA); Mouse monoclonal anti-N-Cadherin, E-Cadherin and Vementin were purchased from Boster (WuHan,China).
Cell culture reagents, viruses and treatment
The human cell lines BGC and SGC were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) in a humidified incubator (37 °C,5%CO2). In addition, when BGC and SGC cell lines reached their logarithmic proliferation phase of up to 80% confluency, they were then sub cultured or used for experiments. The cultured cell lines were randomly divided into rL-RVG, NDV and PBS groups, along with the MLA, ACB, si-RNA of α7-nAChR and corynoxenine pretreatment groups.
CCK-8 assay
The viability of infected BGC and SGC were monitored by performing a CCK-8 assay. BGC and SGC were harvested, pelleted by centrifugation and counted using a blood counting chamber. Cells (6 × 103) were then seeded into 96-well plates and grown in media containing varying dilution titers of rL-RVG or NDV 24 h. The CCK-8 reagent was added into each well incubated for another 4 h. Lastly, the color of the media changed in each well and was measured at 450 nm using a spectrophotometer.
Clonogenic survival assay
BGC and SGC were seeded into 6-well plates (1000 cells per well) and then infected with rL-RVG, NDV at a multiplicity of infection of 10 or incubated with PBS for 24 h. After incubation for 10 days, the cells were fixed with absolute ethyl alcohol for 30 min and stained for 1 h with crystal violet (0.2%) to visualize cell colonies. Each individual experiment was repeated three times.
Wound-healing assay
BGC and SGC were added into 6-well plates and randomly divided into rL-RVG, NDV and PBS treated groups in which cell were infected by rL-RVG,NDV or treated with PBS for 24 h. Groups pretreated with ACB, MLA, si-RNA or corynoxeine for 24 h were also set up. When cells reached 80% confluency, cell monolayers were disrupted with a 10 μL pipette tip after 24 h and their wound healing ability was determined by microscopy.
Migration assay
To carry out migration assays in 24-well transwell plates with polycarbonate membranes (8.0 μm pore size; Costar, MA, USA) were used for in-vitro experiments according to the manufacturer’s protocol. In short, the upper chamber of the filter inserts contained serum free medium with 1 × 105 cells belonging to one of the treatment groups: rL-RVG, NDV, PBS, MLA, ACB, si-RNA of α7-nAChR or corynoxenine. Meanwhile, the lower chamber was filled with 600 μL 10% serum media. After incubation at 37 °C in 5% CO2 for 24 h, the media in the lower chamber were collected and then non migrating cells attached to the membrane of the upper chamber surface were scrubbed. Finally, cells on the bottom wells were stained with 0.1% crystal violet for 20 min,and then washed with PBS for 3 times. Lastly, the migrating cells were counted under the microscope and analyzed by using Image-J software. (National Institutes of Health,USA).
Western blot analysis
After pre-treatment with MLA or ACB or si-RNA of α7-nAChR or corynoxenine for 12 h, cells were infected for 24 h with either rL-RVG,NDV or PBS and then washed with ice-cold PBS for 3 times and lysed by using the lysis buffer RIPA containing 1 mM PMSF for 30 min on ice. Next the lysates were collected and the protein concentrations were quantified using a BCA kit (Thermo Fisher Scientific, USA). Equal quantities of protein were separated by using a 10% SDS-PAGE and the proteins were then transferred to polyvinylidinedifluoride (PVDF) membranes (Bio-Rad Laboratories). The membranes were then blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST, at pH 7.5) for about 2 h at room temperature before washing them with TBST for 15 min for 3 times. Next the membranes were incubated with antibodies at 4 °C overnight with the following antibodies: anti-α7 nAChR, anti-P-MEK, anti-MEK, anti-P-ERK, anti-ERK, anti-E-cadherin, anti-N-cadherin and anti-Vementin. Proteins were detected with HRP-conjugated secondary antibodies for 1 h at room temperature. The protein bands were visualized with a Typhoon 9400 variable mode imager (Amersham Biosciences, UK) using chemiluminescence (ECL Plus Substrate, Thermo Fisher Scientific, USA).
Immunofluorescent assay
BGC and SGC cells were added into 24-well plates and fixed with 4% paraformaldehyde for 2 h at room temperature. Cells were then permeabilized with 0.5% TritonX-100 in PBS for 10 min and blocked with 5% BSA for 1 h. Next the cells were washed 3 times with PBS for 5 min before incubating them with anti-P-ERK at 4 °C overnight. On the next day, the secondary antibody was used to stain cells in order to be detected for immunofluorescence microscopy.
Xenografts
After infection of BGC, SGC cells with rL-RVG, NDV and incubation with PBS for 24 h, the cells were subcutaneously injected into axillary subcutaneous tissues of adult female athymic-nude mice which were randomly selected for the treatment with either rL-RVG,NDV or PBS and housed under specific pathogen free conditions. The size of subcutaneous tumors that developed were measured 2 weeks post treatment using the following calculation: V=W2L0.5 (V is volume, W is width and L is length). In this experiment,we used 2.5% pentobarbital sodium(50 mg/kg) to anaesthetize the athymic-nude mice by intraperitoneal injection. At last,we used 2.5% pentobarbital sodium(200 mg/kg) to overdose the rats with anesthetic to euthanize them.
Statistical analysis
All collected experimental data is presented as mean ± SD. A Student’s t-test or one -way ANOVA with Bonferroni post-test was used to calculate statistical significance using the GraphPad Prism7.0 software (La Jolla,CA, USA). A p-value of P < 0.05 or P < 0.01 was considered statistically significant. Each experiment was conducted independently and repeated at least 3 times.
Discussion
With the development of new drug treatments,the incidence of gastric cancer has declined rapidly over the recent years [
16] but still results in over 1,000,000 new cases and an estimated 783,000 deaths in 2008 (equating to 1 in every 12 deaths globally) [
1]. Early diagnosis and early treatment of gastric cancer is still a major challenge to reduce the mortality of gastric cancer [18]. Tumor metastasis is the main cause of death in patients with gastric cancer. Therefore it is important to understand how to reduce metastasis formation and to find treatment options for curing advanced-stage gastric cancer. In our study, recombinant avirulent NDV LaSota strain expressing the rabies virus glycoprotein rL-RVG did not only suppress migration of gastric cancer cells such as BGC and SGC cells in vitro, but also inhibited growth of subcutaneous tumor in nude mice in vivo.
NDV replicates selectively and destroys tumor cells without damaging healthy cells [
17,
18]. Thus, selective killing of cancer cells is a highly beneficial while side effects are kept to a minimum. The 198–214 amino acid sequence of Rabies virus glycoprotein (RVG) is highly homologous with the 30–56 amino acid sequence in λ-bungarotoxin, which binds nAChRs. Therefore rL-RVG and λ-bungarotoxin have similar inhibitory effects on nAChRs.rL-RVG did not have oncolytic effects but inhibited nAChRs. Propranolol is a nonselective, competitive antagonist of beta adrenergic receptors. In this study,rL-RVG exhibited a similar mechanism of function like to Propranolol and played a role as competitive antagonist of α7-nAChR after infecting gastric cancer cell lines such as BGC and SGC.
nAChR consists of five subunits and assembles into heteromeric or homomeric pentamers. α7-nAChR,another type of nAChR, had a positive effect on cancer cell migration [
19]. Nicotine and NNK act as agonist of α7-nAChR to facilitate the migration of gastric cancer cells [
11,
20] and indirectly activate ERK signaling through promoting the release of epidermal growth factor (EGF) and trans-activation of EGF receptors [
21]. Moreover, the ERK signaling pathway also has influence on EMT that may regulate expression of mesenchymal and epithelial repressor genes [
22]. In our study, we suggest that rL-RVG lowers the phosphorylation levels of ERK signaling and decrease EMT in SGC and BGC cells, indicating that gastric cancer cell migration is associated with the MEK-ERK-EMT signaling pathway.
It is of great significance to activate EMT for invasion and metastasis of gastric cancer cells [
23]. An aberrant EMT activation typically results in the transformation of epithelial cells into mesenchymal cells and leads to phenotypic changes such as the loss of cell-cell adhesion, cell polarity and acquisition of migratory and invasive properties of cells. Cadherin is a significant component of adherent cell junctions. On one hand, aberrant activation of EMT transformed E-cadherin to N-cadherin, which is typically found in mesenchymal cells and promotes the formation of adhesions between cells and the stroma [
24]. On the other hand, Vimentin is a widespread mesenchymal intermediate filament which results in adhesion and migration of activated cells [
25] and is key for the aberrant EMT activation in gastric cancer [
26]. Our previous studies suggest that rL-RVG promotes apoptosis of gastric cancer cells [
13,
27] but the effect of rL-RVG on gastric cancer migration and its underlying mechanism remain unknown. In this study, our results show for the first time that gastric cancer cell migration is suppressed after infection of gastric cancer cells with rL-RVG through competitive inhibition of α7-nAChR. This resulted in the decreased expression of mesenchymal markers including N-cadherin and vimentin and increased levels of E-cadherin. Nicotine promotes the migration of gastric cancer cells via the α7-nAChR pathway [
11]. Thus,we hypothesized that rL-RVG might suppress the migration of cancer cells via α7-nAChR by using ACB, MLA and si-RNA of α7-nAChR to pre-treat SGC cells.
Previous studies suggest that the ERK signaling pathway plays an important role in the proliferation, migration and apoptosis of cells [
28,
29]. Therefore, inhibiting the ERK signaling pathway strengthens the anti-tumor activity of gimatecan in gastric cancer [
30]. Activation of ERK signaling results in the promotion of cervical cancer cell growth and metastasis [
31]. Interestingly, we found that rL-RVG reduces the phosphorylation levels of MEK/ERK and resulted in a decrease of phosphorylation levels of MLA,α7-nAChR and si-RNA pre-treated groups compared to the ACB pre-treated group. Additionally, we also achieved similar results in the change of EMT and migration of SGC cells. Hawsawi O and Henderson V suggested that HMGA2 may induce EMT via ERK signaling pathways [
32]. In our study, after pretreatment with corynoxenine, we found that the expression of N-cadherin and Vimentin was down-regulated and the expression of E-cadherin was upregulated compared to non-pretreated groups. We also found that rL-RVG has a much more inhibitory effect than NDV. Thus, rL-RVG does not only have the same effect as NDV, but also plays a role as competitive antagonist to inhibit α7-nAChR.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.