Acetylcholinesterase overexpression mediated by oncolytic adenovirus exhibited potent anti-tumor effect

Background Acetylcholinesterase (AChE) mainly functions as an efficient terminator for acetylcholine signaling transmission. Here, we reported the effect of AChE on gastric cancer therapy. Methods The expression of AChE in gastric cancerous tissues and adjacent non-cancerous tissues was examined by immunohistochemistry. Gastric cancer cells were treated with AChE delivered by replication-deficient adenoviral vector (Ad.AChE) or oncolytic adenoviral vector (ZD55-AChE), respectively, followed by measurement of cell viability and apoptosis by MTT assay and apoptosis detection assays. In vivo, the tumor growth of gastric cancer xenografts in mice treated with Ad.AChE or ZD55-AChE (1 × 109 PFU) were measured. In addition, the cell viability of gastric cancer stem cells treated with Ad.AChE or ZD55-AChE were evaluated by MTT assay. Results A positive correlation was found between higher level of AChE expression in gastric cancer patient samples and longer survival time of the patients. Ad.AChE and ZD55-AChE inhibited gastric cancer cell growth, and low dose of ZD55-AChE induced mitochondrial pathway of apoptosis in cells. ZD55-AChE repressed tumor growth in vivo, and the anti-tumor efficacy is greater than Ad.AChE. Moreover, ZD55-AChE suppressed the growth of gastric cancer stem cells. Conclusion ZD55-AChE represented potential therapeutic effect for human gastric cancer. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-668) contains supplementary material, which is available to authorized users.


Background
Acetylcholinesterase (AChE) terminates cholinergic signaling transmission by hydrolyzing the signaling molecule acetylcholine (ACh), which acts as a stimulus signaling transmitter in neuronal and non-neuronal cholinergic systems. It binds to nicotinic or muscarinic acetylcholine receptors (nAChR or mAChR), and promotes Ca 2 + entry into neuronal and non-neuronal cells, thus stimulating their biological activities [1]. Besides its canonical functions, ACh can also over-activate the nicotinic acetylcholine receptor, which leads to angiogenesis, migration and proliferation of cancer cells [1,2]. Abnormal expression of AChE protein was found in several types of cancer [3,4]. Hepatocellular carcinoma patients with low expression of AChE exhibited poor prognosis [5]. Recently, AChE was reported to be involved in apoptosis [6]. Increased intracellular AChE expression was observed in various cell lines undergoing apoptosis, as well as in animal models of diabetes and Parkinson's disease [6][7][8]. Critical proliferation pathways including PI3K/Akt pathway in cancer cells were inhibited by AChE overexpression [5]. However, there is no report on cancer therapy using AChE protein.
Gastric cancer is one of the leading causes of cancerrelated deaths worldwide. The prevalence of gastric cancer is related to an unhealthy diet, smoking and Helicobacter pylori infection [9,10]. In 2008, about 989,600 patients were diagnosed with gastric cancer and 738,000 patients died [11]. Eastern Asia is one of the regions with a high incidence of gastric cancer. Since early stage gastric cancer may be vague and overlooked, many patients are diagnosed in later life, and the conventional treatments are ineffective for those cases.
In the present study, we detected the expression of AChE in gastric cancerous tissues (CT) and the adjacent non-cancerous tissues (ANCT). Gastric cancer samples presented a low AChE expression compared to the noncancerous samples, and patients with higher AChE levels showed a longer survival. Overexpression of AChE by an oncolytic adenoviral vector (ZD55-AChE) significantly inhibited gastric cancer cell proliferation and reduced growth of gastric tumors in mice. In addition, ZD55-AChE suppressed gastric cancer stem cell growth. Our work demonstrated for the first time that AChE gene mediated by an oncolytic adenovirus is effective for suppressing digestive system cancers.

Patient samples
Ninety-six gastric and seven respective adjacent noncancerous tissues were obtained anonymously from Zhongshan Hospital (Fudan University, Shanghai, China) and Xinhua hospital (Jiaotong Universtiy, Shanghai, China). All the human samples were obtained with informed consent and approval for usage was received from the ethics committee of Zhongshan Hospital and Xinhua Hospital. Studies upon these samples were approved and handled in accordance with the Institutional Review Board of Institute of Biochemistry and Cell Biochemistry, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Immunohistochemistry
Patient samples or tumor xenografts were processed into tissue chips by Shanghai Superchip Biotechnology Corporation (Shanghai, China) and subjected to immunohistochemistry assay according to the standard protocol [12]. Level of AChE expression was set as values range from 0 to 3 according to the intensity, and expression of AChE were calculated as (labelling intensity × percentage of positive cells). Antibody (clone AE1, lot number MAB303) to AChE was purchased from Millipore (Billerica, MA). For primary fibroblast culture, the gastric cancer tissues were washed with Hank's solution (Beyotime, Nantong, China), cut into small pieces and plated into polylysine coated flask. The culture medium is DMEM supplemented with 20% FBS, 100 U/L penicillin-streptomycin, 5 μg/mL amphotericin B (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 50 ng/mL epidermal growth factor (EGF, peprotech, Rocky Hill, NJ) and 50 ng/mL basic fibroblast growth factor (bFGF, peprotech). The fibroblast cells were trypsinized around a month later and moved into a new flask for amplification.

Adenovirus construction
AChE gene was amplified by PCR assay, using AChE forward primer 5'-ATCGAAGCTTATGAGGCCCCCGC AGT-3' and reverse primer 5'-GTACTCTAGATCAC AGGTCTGAGCAGC-3' , with pGS-AChE (T form of AChE) as the template (a gift from Dr. Oksana Lockridge, Eppley Institute, University of Nebraska Medical Center). The PCR fragments were restricted by HindIII and XbaI and inserted into pCA13 shuttle plasmid to form pCA13-AChE. The CMV-AChE-SV40 cassette in pCA13-AChE was restricted by BglII and inserted into pZD55 shuttle plasmid to generate pZD55-AChE. pCA13, pCA13-AChE, pZD55 and pZD55-AChE were co-transfected into HEK293 cells with pBGHE3, respectively. Viral plaques were picked, amplified and purified by gradient CsCl solution centrifugation. The virus titer was measured using Virus titer detection kit (Vector Gene Biotechnology, Beijing, China). The genome of the adenovirus was obtained by using Cell-blood genome extraction kit (Generay, Shanghai, China). AChE gene was identified by PCR assay using the AChE primers mentioned above.

Adenovirus infection
For AChE determination assay, cells were infected with Ad.vector, Ad.AChE, ZD55 or ZD55-AChE at the indicated MOIs. 48 hours post-infection, cell lysis and medium were collected and subjected to AChE determination.
For cell viability assay, cells were infected with Ad.vector, Ad.AChE, ZD55 or ZD55-AChE at different MOIs for 96 hours and then subjected to cell viability detection.

AChE determination
Measurement of AChE activity was performed according to a modified Ellman's assay [13]. Briefly, 1×10 6 cells were washed with cold PBS, incubated with 50 μL lysis buffer (1 M NaCl with 0.5% Tween, 0.05 M K 2 HPO 4 , 0.01 M KH 2 PO 4 ) for 30 min, and then dissociated by ultrasonication. 20 μL cell lysate or culture medium was incubated with 300 μL 0.1 M PB buffer (pH 8.0), 10 μL 0.01 M DTNB and 1.5 μL 0.01 M iso-OMPA for 30 min at room temperature. 20 μL 0.075 M acetylthiocholine was added into the mixture and the absorbance at wavelength 405 nm was measured instantly and then every 5 minutes for a total of 6 times. Experiments were repeated 3 times.

Acetylcholine production level determination
Measurement of human intracellular ACh levels were performed using an ACh determination ELISA Kit (HEA004, Bogoo, shanghai, China) according to the manufacturer's protocol. Cells were collected at a concentration of 10 6 cell/mL and washed by PBS for three times. After repeated freezing and thawing, cells were lysed and the supernatants were applied for measurement in 96-well plates pre-coated with an ACh monoclonal antibody. ACh standard solutions with diluted concentrations were used for standard curve. The results were reflected by absorbance value at a wavelength of 450 nm. Experiments were repeated 3 times.
Hoechst 33258 staining, cell cycle detection and Annexin V/PI staining assay For Hoechst 333258 staining, cells were fixed with 4% paraformaldehyde (PFA) for 15 min, washed with PBS, and incubated with 1 μg/mL Hoechst33258 (Molecular Probe, Grand Island, NY) for 1 min in the dark.
To detect the cell cycle distributions, cells were trypsinized and fixed with 70% ethanol at −20°C overnight. The fixed cells were washed with PBS twice, digested with 20 μg/mL RNase A (Sigma-Aldrich) at 37°C, and incubated with 50 μg/mL propidium iodide (PI, Sigma-Aldrich) for 30 min in the dark.
Apoptotic cells were analyzed using an Annexin V-FITC/PI kit (Biovision, Milpitas, CA) according to the manufacturer's protocol. Cells were trypsinized, washed with binding buffer twice, incubated with 5 μL Annexin V and 10 μL PI for 15 min in the dark, and subjected to flow cytometry analysis.

Mitochondrial membrane potential alteration detection
Mitochondrial membrane potential alteration was detected using a JC-1 staining kit (Beyotime) according to the manufacturer's protocol. Cells were trypsinized, resuspended in 500 μL corresponding culture medium, then mixed with 500 μL JC-1 solution, incubated at 37°C in the dark for 30 min, and analyzed with flow cytometry.

Cell sorting
AGS cells were collected and stained with APC conjugated CD44 antibody (Miltenyi, Bergisch Gladbach, Germany) for 10 min at 4°C in the dark. Cells needed to be continuously shaken during the period of antibody incubation to avoid precipitation. After that, cells were washed and sorted. AGS cells stained with APC conjugated mouse isotype antibody (Miltenyi) or without staining were used as negative controls.

Animal experiments
All the experiments were approved by the Institutional Animal Care and Use Committee of Institute of Biochemistry and Cell Biochemistry, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and manipulated according to the U.S. Public Health Service Policy on Humane Care and the Use of Laboratory Animals.
AGS cells failed to form subcutaneous tumors at 1 × 10 7 cells/mouse. We then mixed AGS cells with matrigel (BD Bioscience, San Jose, CA) at a ratio of 1:1 to enhance the tumor formation ability of the cells. Unfortunately, AGS cells were still unable to form tumors. The experiments to establish AGS xenograft tumors were performed four times without success.
Gastric cancer cell line MGC80-3 cells were utilized for establishing subcutaneous xenograft tumors. Four-week old female nude mice were subcutaneously injected with MGC80-3 cells at 4 × 10 6 cells/mouse. When the size of tumors reached around 90 mm 3 , mice were divided into 5 groups by R software. The mice were intratumorally injected with saline or indicated adenoviruses (1 × 10 9 PFU) every other day. The size of tumors was measured every three days after the treatment, and the volume was calculated as length × width × width/2.

Statistical analysis
Overall survival rate of gastric cancer patients was calculated with Kaplan-Meier. All the statistical data were expressed as mean ± SD. Statistical analysis between two groups were performed with students' t test in R software. P < 0.05 was considered as significant difference.

Results
The expression of AChE is low in gastric cancer patient samples and is associated with survival in patients To detect the expression of AChE in gastric cancerous tissues (CT) and the adjacent non-cancerous tissues (ANCT), we performed immunohistochemistry staining assay in paired tissues. Relatively lower expression level of AChE was observed in gastric cancer tissues compared to the adjacent non-cancerous tissues ( Figure 1A, B). In addition, gastric cancer patients with higher AChE expression levels exhibited longer survival time ( Figure 1C).

Replication-deficient adenovirus carrying AChE gene inhibited cancer cell growth
To determine whether overexpression of AChE could inhibit cell growth, the replication-deficient adenovirus carrying AChE gene (Ad.AChE) was constructed (Additional file 1: Figure S1A, B). Activated AChE protein was expressed in Ad.AChE-infected gastric cancer cell line AGS cells, and can be secreted to culture medium (Figure 2A, B). The AGS cell growth and another gastric cancer cell line NCI-N87 cell growth were significantly inhibited by Ad.AChE at a high MOI level (100 or 200) ( Figure 2C, D). Ad.AChE also decreased cell viability in other digestive system cancers, including liver cancer cell line (Huh-7 and SMMC-7721), colon cancer cell line (SW480) and pancreatic cancer cell line (BxPc-3) (Additional file 1: Figure S1C, D). Ad.AChE did not show cytotoxicity to normal gastric epithelial GES-1 cells or primary fibroblasts ( Figure 2E and Additional file 1: Figure S1E, F). Notably, Ad.AChE was not able to inhibit most cancer cell growth at low MOI level, such as MOI of 1 or 10, compared to the control adenovirus (Ad.vector). In addition, endogenous level of ACh in various cell lines were detected. ACh production level of AGS cells appeared to be the highest one among the gastric epithelia cells (Additional file 2: Figure S2A) and that of pancreatic BxPC-3 cells is relatively high (Additional file 2: Figure S2B).

AChE expressed by oncolytic adenoviral vector presented potential anti-tumor efficiency in vitro and in vivo
Oncolytic adenoviral vector was widely used in cancer therapy with good antitumor effect. To detect the potential cancer therapeutic function of AChE, we constructed an oncolytic adenovirus carrying AChE gene (ZD55-AChE) (Additional file 3: Figure S3A, B). Overexpression of activated AChE protein in AGS cells significantly decreased cell viability as measured by MTT assay (Figure 3A-D). ZD55-AChE suppressed cell growth in gastric cancer NCI-N87 and MGC80-3 cells at a MOI of 10 ( Figure 3E). We also detected the function of ZD55-AChE in the pancreatic cancer cell line BxPc-3, liver cancer cell line Huh-7 and SMMC-7721, and colon cancer cell line SW480 and HCT116, and found that ZD55-AChE inhibited BxPc-3, Huh-7 and HCT116 cell growth at a MOI of 1. However, ZD55-AChE cannot significantly suppress SW480 cell growth at the low MOI level (Additional file 3: Figure S3C). Importantly, ZD55-AChE did not affect normal gastric epithelial GES-1 cells or primary fibroblast cell proliferation ( Figure 3F and Additional file 3: Figure S3D, E).
Next, we sought to explore the anti-tumor effect of ZD55-AChE in vivo. Since AGS cells failed to form subcutaneous xenografts, we evaluated the anti-tumor effect of AChE in the MGC80-3 xenografts. Ad.AChE, ZD55-AChE and the control viruses were intratumoral injected, respectively. The tumor growth was significantly reduced by ZD55-AChE, as compared with the control animals receiving saline. However, Ad.AChE did not inhibit the growth of tumors ( Figure 3G and Additional file 4: Figure S4).

ZD55-AChE induced gastric cancer cell apoptosis through mitochondrial pathway
It was reported that AChE protein participated in apoptosis [6], and we sought to test whether ZD55-AChE induces cancer cell apoptosis. We performed Hoechst333258 staining, and found that ZD55-AChE obviously induced nucleic fragmentation in AGS cells at a MOI of 2 ( Figure 4A). Moreover, the sub-G1 populations and AnnexinV positive AGS cells were increased after ZD55-AChE infection ( Figure 4B, C). We next performed JC-1 staining to investigate the changes in the mitochondrial membrane potential (Δψm). The proportion of AGS cells with loss of mitochondrial membrane potential was increased in the ZD55-AChE infected cells, indicating the involvement of mitochondria in the apoptosis process ( Figure 4D). ZD55-AChE reduced the procaspase 9 protein, which is the essential initiator caspase required for apoptosis signaling through the mitochondrial pathway ( Figure 4E). The expression of procaspase 8, procaspase 3 and the precursor of poly ADP-ribose polymerase (PARP) were also decreased after ZD55-AChE treatment ( Figure 4E). The caspase inhibitor Z-VAD-FMK alleviated the cytotoxicity of ZD55-AChE in AGS cells ( Figure 4F). These data suggested that ZD55-AChE induced AGS cell apoptosis most likely through mitochondrial pathway. Notably, Ad.AChE did not induce AGS cell apoptosis at a MOI of 2 ( Figure 4A-E), which is consistent with our previous data that Ad.AChE inhibited cell growth only at a high MOI level ( Figure 2C, D).

ZD55-AChE inhibited gastric cancer stem cell growth
Increasing researches certified that the quiescent cancer stem cells are critical in cancer initiation, progression, drug resistance and relapse. Since adenovirus can infect quiescent cells, we sought to explore whether ZD55-AChE could inhibit the AGS stem cell growth. AGS cells were sorted based on the cell surface marker CD44 to obtain stem cells [14]. Both CD44 + and CD44 − cells were collected and treated with chemo-therapeutic drugs, etoposide and 5-FU. Compared to CD44 − cells, CD44 + AGS cells afforded resistance to chemo-drugs, which is one of stem cells characteristics [15] (Figure 5A). Ad.AChE decreased CD44 + AGS cell growth at a MOI of 50 ( Figure 5B). Importantly, ZD55-AChE significantly suppressed stem cell growth at a MOI of 0.1 compared to the control virus ( Figure 5C).

Discussion
The well-known function of AChE is to hydrolyze ACh and terminate its neuronal and non-neuronal signaling transmission. Recent studies have disclosed its roles in prolonging survival time in cancer patients and promoting apoptosis [5,16]. To our knowledge, AChE was applied to treat liver cancer delivered by nanoparticles [17,18]. However, no cancer therapy research using viral vector to deliver AChE has been reported. In the present study, two adenoviral vector, including replicationdeficient adenoviral (Ad) and oncolytic adenoviral vector (ZD55), were used to express AChE gene. ZD55-AChE   presented significantly cytotoxicity to gastric cancer cells in vitro and in vivo at a low MOI level (Figure 3C-E, G), and induced mitochondrial apoptosis ( Figure 4).
ACh is not only function as a neurotransmitter in the nervous system, but also involved in some critical signal pathways in cancer cells. ACh stimulates its receptors, nAChR and mAChR, and leads to the activation of the ACh receptor signaling pathway. The precise level of ACh is controlled by the AChE protein. Abnormal expression of AChE will result in the imbalance of ACh degradation and lead to the over-activation of ACh receptors [19]. Aberration of AChE activity in various types of cancers was reported. The lymphocytes from CLL patients showed significantly lower AChE activity than normal [20]. In comparsion of 55 paired tissues of healthy and cancerous gut, 32% decrease of AChE activity was observed in cancerous gut [21]. Increased ACh was found in lung cancer tissues due to the abnormal expression of AChE [22]. Lower level of AChE in cancer tissues and the reverse correlation of AChE expression with survival time were reported in liver cancer patients [5]. In our work, lower expressed AChE protein was found in gastric cancer tissues than adjacent non-cancerous tissues. Patients with high level of AChE protein showed longer survival time (Figure 1). The correlation of AChE expression and prognosis in gastric cancer patients is similar to liver cancer. Therefore, introduction of the AChE protein back may lead to the growth inhibition of cancer cells.
There are three types of AChE mRNA variants, AChE-T, AChE-R and AChE-H [23], which was able to be identified by sendimentation assay [24]. AChE-T, but not AChE-H or AChE-R, is able to anchor to the synaptic membranes or the neuromuscular junctions through ColQ or PRiMA. Only the AChE-T is regarded as truly "synaptic" [25]. Besides, AChE-R promotes cell proliferation and makes cell resistance to genotoxic stress [26,27], however, AChE-T showed the opposite effect [28]. In our work, AChE-T was expressed.
The intracellular level of ACh were measured in various types of cells to get to know whether it related to the response of cancer cells to AChE overexpression. AGS cells expressed high level of ACh (Additional file 2: Figure S2A), and were sensitive to AChE overexpressed by viral vector, suggesting that AChE may inhibit cell growth through hydrolyzing ACh.
Studies indicated that AChE is overexpressed in apoptotic cells [6]. Absence of AChE prohibited stress-induced cells apoptosis [8]. It was also reported that overexpression of AChE renders the cells vulnerable to apoptosis, but cannot initiate apoptosis [29]. A stimulus enabling the translocation of AChE into the nuclei may be required to initiate apoptosis [8,29]. Adenoviral vector was commonly used to express therapeutic genes in cancer therapy. On the other hand, the viral vector can provide the requisite stimulus for initiating apoptosis by itself [30]. We noticed that Ad.AChE inhibited cancer cell growth at a high MOI level ( Figure 2C, D). This illustrated the stimulus effect of the viral vehicles. However, the high MOI request limited Ad.AChE to be further used in cancer therapy.
We found that ZD55-AChE induced the loss of mitochondrial membrane potential and decreased procaspase 9 and procaspase 3 proteins in AGS cells ( Figure 4D, E), indicating the anti-tumor function of ZD55-AChE is associated with induction of mitochondrial apoptosis. Our data partially consistent with previous reports that AChE is required for apoptosome formation [31]. Other researches indicated that ACh stimulated the nicotinic acetylcholine receptor (nAChR) signaling which is pathologically over-activated in tumor [1]. Cholinergic signaling stimulation can promote progression in various types of cancers [32,33]. Nicotine binds to the nAChR, activates Data are shown as fold change relative to that of mock-treated cells. All data shown represent mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.