Mice were maintained in accordance with the recommendations, regulations and standards approved by the Federation of European Laboratory Animal Science Association. The mice were used in accordance with institutional guidelines when they were 8-week-old and employed for both tumor maintenance and chemotherapy testing. All the procedures involving animal care and treatment were conducted as stipulated in Italian National Guidelines (D.L. No. 116 G.U., suppl. 40, 18.2.1992, circolare No. 8, G.U. luglio 1994) and in the appropriate European Directives (EEC Council Directive 86/609, 1.12.1987), adhering to the Guide for the Care and Use of Laboratory Animals (United States National Research Council, 1996) and according to an approved protocol reviewed by the National Cancer Research's Institutional Animal Care and Use Committee (Genova, 15 November 2004 n. of reference 149). For ethical reasons, each animal was submitted to euthanasia (in a CO2 atmosphere) when it had lost 20% of its weight as compared with its weight at the time of the tumor graft.
The importance of acetylcholine (ACh) as a neurotransmitter in the nervous system is well-established, but little is yet known of its recently described role as an autocrine/paracrine hormone in non-neuronal cells.1, 2 The numerous signaling pathways mediating by ACh, in these cells, account for the multiple cholinergic effects that cover all vital cell functions during cell life.1, 2 Although the primary functions of non-neuronal AChR have not yet been elucidated, their expression in many different cell types suggests that their role might be fundamental. Consistent with the expression of ACh and AChR in normal lung is their expression in nonsmall cell lung cancer (NSCLC).2–10 Our laboratory's observations suggest that in mesothelioma and NSCLC cells life and death signals are mediated through nicotinic and/or muscarinic receptors.2, 11–13 The nicotinic AChR (nAChR) are ligand-gated ion channels, the binding of ACh allows entry of sodium or calcium into the cell. Nicotine is an exogenous ligand; many investigators, including ourselves, have shown that activation of nAChR by nicotine stimulates lung cancer growth.2, 14–20 This new role for the cholinergic system might suggest a potential new pathway to target tumor growth.
Here, we show that a potent α7-nAChR antagonist, namely α-CbT, decreases NSCLC A549 cell growth both in vitro and in vivo. α-CbT is a long α-neurotoxin (NT) obtained from the venom of Naja kaouthia. A549 cells were chosen since, as reported by different groups, including ourselves, display an active cholinergic system consisting of ACh, choline-acetyltransferase, membrane vesicles that accumulate choline, acetylcholinesterase and functional high affinity nAChR.2, 6, 16, 20–23
Material and methods
Drugs and cells
α-CbT lyophilized powder was resuspended in PBS buffer +0.1% BSA in order to reach the appropriate concentrations. The powder was stable several months at −20°C and the dilution solutions several months at 4°C. The powder obtained from venom is 97% purified by HPLC. Human NSCLC cell line A549 (lung adenocarcinoma) and SK-MES were obtained by our Institutional Cell Repository (Genoa, Italy). Before being utilized in the experiments, cells were analyzed by fingerprinting using 8 different and highly polymorphic STR loci (Powerplex 1.2) by DSMZ (the German National Resource Centre for Biological Material Braunschweig, Germany). The analysis of the DNA pattern revealed that this cell line is genetically identical to the cell line A549 deposited in the DSMZ (ACC 107). NCI-H1650 NSCLC cell line, and AP (mongoose cells) were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in RPMI 1640+10% bovine serum. (Gibco BRL, Grand Island, NY). Natural α-CbT was labeled with the 5-carboxyfluorescein dye according to the following procedure: 100 μg of toxin were dissolved in PBS buffer 0.1 M, pH 7 (90 μl), then 10 μl of a stock solution (1 mg/300 μl) of 5-carboxyfluorescein NHS ester (Fluoprobes, Interchim France) in dimethylsulfoxide/dimethylformamide [1/1] were added and incubated 60 min at 37°C. The reaction was stopped by adding 20 μl of 1 M Tris-HCl, pH 8.0. After incubation at room temperature for an additional 30 min, excess of fluorescein reagent was removed on a PD-10 column (Pharmacia) equilibrated in 10% acetic acid and then labeled toxin was further purified by HPLC. A549 cells were washed twice in PBS, incubated for 1 hr with FITC-conjugated α-CbT and α-CbT as a control at 3 nM concentration and analyzed using a cytofluorimeter CyAn™ (Dako Italy, Milan). Some experiments were performed in the presence of excess (40 μg/ml) of polyclonal α-CbT Antibodies (Santa Cruz, CA). Picture is representative of 3 replicate experiments yielding similar results.
Cell growth decrease was evaluated by Clonogenic and MTT Assay as described.24 24 hr after seeding of 250 cells, drug (α-CbT, mutated-α-CbT or Erabutoxin-a) was added to culture medium. Drugs were administered every 2 days for 15 days. For MTT cells were treated once over 72 hr.
Evidence of apoptosis was identified by cell staining with Annexin V and PI as described.24
Western blotting was performed as described.24 All antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Bands identified in the Western blot assay were analyzed by densitometry (Kodak Digital Science ID Image Analysis Software; Eastman Kodak; Rochester, NY), which measures the surface and intensity of bands. Results were expressed as arbitrary densitometry units. Caspases activation was also detected Using CaspGLOW Fluorescein Caspase-3/9 Staining Kit (Biovision, Mountain View, CA).
Characterization of the basal NF-κB activity was done by means of the Trans-AM NF-κB family transcription factor assay colorimetric kit (active Motif, Carlsbad, CA).
BALB/c nu/nu mice, purchased from Charles-River (Calco, Como, Italy), were housed in specific filter-capped cages, kept in pathogen-free conditions and maintained in the facilities within the Animal Resources Center at the National Cancer Research Institute of Genoa. 5 × 105 A549-luc cells were grafted through the thorax into the left part of the lungs of female mice. The grafts were performed under anaesthesia (Ketamin 70 mg/kg and xylazine 5 mg/kg i.p). Mice were placed in the right lateral decubitus position with all 4 limbs restrained. A 1-cm transverse incision was made in the left lateral skin just below the inferior border of the scapula of the mouse. The muscles were separated from the ribs by sharp dissection, and the intercostal muscles were exposed. The left lung was visible through the intercostal muscles. A 30-gauge needle was inserted ∼5 mm into the lung through the intercostal muscle, and an inoculum of 5 × 105 A549-luc cells was then dispersed into the left lung in a final volume of 10 μl of medium. The procedure required ∼1 min for completion and was performed easily. The skin incision was closed with 3-0 points. Highly reproducible tumor developments (100%) were obtained in each experiment, the median survival of animal with injected A549-luc cells was 50–60 days.
Bioluminescence imaging (BLI) was used to measure luciferase activity with in vivo Imaging System (IVIS) technology (Xenogen, Alameda, CA) optimized for high sensitivity. BLI was done once a week. Mice were anesthetized with Ketamin (70 mg/kg i.p.) and xylazine (5 mg/kg i.p.) followed by the injection of Luciferin (150 mg/kg i.p.; Xenogen) 10 min before imaging. Animals were then put in the light-protected chamber of the IVIS imaging system, and photons emitted were measured over 3 min. Regions of interest were drawn over the area of photon emission and quantified using the “Living Image” software. To avoid at maximum the variability of BLI measures, regions of interest were read 5 times by IVIS and the quantified data represent a mean of these different lectures. As luciferase-bearing cells proliferated in vivo, occasional image saturation was encountered. This was overcome by shortening exposure time to reduce the number of photons collected.
In vivo–in vitro tumor excision cell viability assay
After 4-days drug infusions tumors were excised from mice, weighed, minced with scissors and dissociated using medicons and Medimachine (ConsulTS, Orbassano-Turin). After removal of debris, by passing the tumor suspensions through 70-μm nylon screens, the cells were washed in PBS, counted and stained with PI-quinacrine orange and evaluated using the cytofluorimeter CyAn™ (Dako Italy, Milan).
Statistical analyses and curve fittings were done using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA). Pairs were compared with the Student's t- and Mann-Whitney U-test to show the intergroup differences, a p < 0.05 was considered to be significant.
Results and discussion
When A549 cells were incubated with α-CbT, FITC conjugated, a binding, inhibited by α7-nAChR antibodies, was observed (Fig. 1a). α-CbT significantly lowered the number of A549 cells in a dose-dependent manner; the IC50 value was 3. 2 ± 0.5 nM (Fig. 1b). To check the specificity of this inhibition, we measured the effect on cell number of (i) a mutated α-CbT (CbT-R33E) that has been previously identified for it huge lost of interaction on α7-receptor as compared to wild-type α-CbT25 and (ii) an analogous short-chain toxin (Erabutoxin a) specific of the muscular nAChR subtype. In both cases, no cell growth decrease was observed, confirming the specificity of the α-CbT effect on the α7-neuronal receptor. Furthermore MTT assay was performed on mongoose cells (AP cells). As already reported26 sequences of the α-subunits of the nAChR from mongoose contain several differences in the region between amino acids 183 and 200, consequently this species reveals resistance to the snake α-toxins. In binding experiments no binding of α-CbT FITC-conjugated was observed (data not shown) and no growth decrease was observed after 72 continuous exposures (Fig. 1c).
The concentration of 1.0 μM α-CbT that induced ≈90% of cell decrease (Fig. 1b), was chosen to evaluate death's mechanisms. Annexin V-PI flow cytometric analysis revealed apoptosis that started 18 hr after continuous treatment and raised the maximum after 24 hr (Fig. 1c). To confirm the induction of apoptosis, cell extracts were analyzed for expression of apoptosis biological markers. α-CbT (24 hr treatment) resulted in striking cleavage of procaspase-9, procaspase-3 and poly (ADP-ribose) polymerase (PARP), whereas did not affect procaspase-2 (Fig. 1e and 1g). No cleavage statistical significant was observed for pro-Caspase 2. α-CbT induced release of cytochrome c from mitochondria (Fig. 1h and 1i). Consistent with the absence of caspase-2 activation was the nonactivation of p53 (Fig. 1j). Of interest was the marked reduction of the basal RelA/p65 DNA-binding activity (Fig. 1k). Indeed, A549 cells are considered drug-resistant since display highly activated NF-κB signaling cascade that constitutes one of the major pathways by which tumor cells escape cytotoxic insults.27
The subsequent step was the evaluation of the in vivo α-CbT antitumor activity. To evaluate the efficacy of a drug, as antitumor agent, a good model requires the sacrifice of large cohorts of animals to monitor tumor growth and the effect of an intervention. Hence BALB/c nu/nu mice were orthotopically transplanted with A549 cells modified to stably express luciferase. BLI permits sensitive in vivo detection and quantification of cells specifically engineered to emit visible light. Once a week the same set of animals (8 control and 8 treated) were examined by BLI to measure the effects of α-CbT administration. α-CbT injected i.v., in 50 healthy mice, had an acute (5 days) LD50 of 0.15 mg/kg (LD10 = 0.12; LD90 = 0.20 mg/kg). The effect of α-CbT was tested in well-established xenografts tumors. Mice received the LD10 i.v. once a day, for 5 consecutive days, followed by 2 days of recovery (1 cycle) for a total of 3 cycles (Fig. 2a). An example of serial imaging is shown for both control and treated mouse and showed both the similarity of cell numbers at the beginning of the experiment, the continued increase of BLI with time and the inhibition of BLI by α-CbT. In the entire treated-group, a significant difference with control was first seen by day 7 and persisted for the entire experiment with a 32% of difference at day 21 (Fig. 2b and 2c). Levels of BLI images in vivo corresponded to the frequency and size of cancer lesions in lungs, as subsequently confirmed at necropsy (Fig. 2d and 2e). To gain insight into the mechanism of α-CbT-induced tumor reduction, cells obtained by tumors taken from treated or untreated mice were analyzed. Treated tumors contained 4.6% living and 39% apoptotic cells whereas untreated 31% living and 25% apoptotic cells (Fig. 2f).
The relevance of these results have to be considered taking into account the observations that A549 orthotopic xenografts are resistant to taxol and oxaliplatin, and only weakly sensitive to irinotecan.28
Treatment was well tolerated, no sign of lethality or toxicity such as: body weight loss, serum enzymes (AST, ALT, alkaline phosphatase) or serum chemistry alterations (urea, creatinine), statistically significant (Paired Student's t test p = 0.3488, Mann-Whitney test p > 0.05)], was observed. Moreover, treated and untreated mice, examined for neurological effects assessing autonomic, convulsive, excitability, neuromuscular, sensory-motor and general motor activity domains, did not shown any differences. Evident neurological signs started at the concentration of 0.15 mg/kg.
On the basis of these results we can conclude that α-CbT, a potent antagonist of α7-nAChR, has a significant inhibitory effect on A549 cells growth.
More studies are clearly needed to further understand the complex system α7-nAChR-antagonist in cancer cells and to evaluate α7-nAChR as a potential target in cancer therapy. As a part of an ongoing study we analyzed the ability of α-CbT to decrease the cell growth of 2 additional NSCLC cell lines, namely SK-MES and NCI-H1850 in a clonogenic assay. Under the same experimental conditions used for A549 cells, the IC50 values were 0.04 + 0.001 and 1.0 + 0.5 μM, respectively. Interestingly no effect on cell number decrease (>90.0%) and on apoptosis induction (Fig. 1l) was observed on 2 primary cell cultures of unaffected human bronchoalveolar cells (immunohistologically assessed) exposed to 1.0 μM α-CbT. This last observation is not completely unexpected considering that in α7 knockout (KO) mice the α7 subunit is not essential for normal development or for apparently normal neurological function.29 However, data from α7 KO mice indicates a critical function of the α7 subunit in the normal regulation of systemic inflammatory responses in vivo.30 Finally, in the local environment of tumors, concentrations of ACh at surface receptors may be even higher due to high cell densities in solid masses, proximity of secretion events to receptor location and variations in cholinesterase levels. It has also been reported that levels of circulating cholinesterase is reduced in cancer patients,31 and decreased levels of cholinesterase in NSCLC has been reported,32 with the implication that cholinergic signaling may be further increased in tumors due to decreased cholinesterase activity. If ACh secreted by NSCLC stimulates tumor growth through α7-nicotinic mechanisms, the addition of α7-nAChR antagonists, such as α-CbT, should inhibit NSCLC growth. An important finding of this study is that α-CbT inhibits A549 cell-growth in vivo. As reported, activation of α7-nAChR stimulates growth in multiple tumor types. Thus, if the many tumors that express α7-nAChR also synthesize ACh, as in the case of A549 cells, the cholinergic-autocrine loop may be wide spread in cancers. Different data, including our ongoing study, suggest that a cholinergic loop is expressed in different NSCLC tissues.10
Certainly experiments with α-CbT represent a “working model”. Indeed, α-CbT appears unique among the ligands because of its distinctive binding kinetics and remarkably high affinity and selectivity for α7-nAChR subtypes. It may provide lead compounds for the design of clinically useful drugs. With this knowledge, one can start to imagine how to design smaller inhibitors that act selectively at different nAChR.
Since multiple other cancer types synthesize ACh and express α7-nAChR, antagonists may be an efficacious adjuvant therapy in many different oncologic protocols. In this field we have obtained quite encouraging results inhibiting human mesothelioma cancer cell growth.2, 11
A549 cells that express lentivirally delivered luciferase (A549-luc) were a kid gift of Dr. Jerry W. Shay (Department of Cell Biology and Harold Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas). We thank Ms. Cristina Bruzzo and Mr. Giovanni Battista Oliveri for the excellent technical assistance, and Dr. Angela Alama for helpful discussions. Dr. Alessia Catassi is a fellow of AIRC.