Nitric oxide (NO) is a colourless, highly lipophilic and diffusible gas. The molecule is made up of an oxygen atom bound to a nitrogen atom. Most mammalian tissues express different NO synthases (NOS) – a group of enzymes responsible for the synthesis of NO. Three isoforms of NOS enzymes, namely the neuronal NOS (nNOS), the inducible NOS (iNOS) and the endotheial NOS (eNOS) have been detected in a wide range of human cell lines [1]. NO plays an important role in mediating a number of biological processes, such as cell migration [2]. Three different isoforms of NOS (eNOS, nNOS and iNOS) which produce NO, have been found in a wide range of human cell lines. The expression of different NOS isoforms has been reported to increase in breast cancer as well as other cancers such as the cancer of the colon, lung cancer, lymphoma, and melanoma [1,3,4,5,6]. A number of studies have demonstrated a relationship between NO expression and cell migration. For example, in wound healing assays high levels of NO has been found to promote both wound healing as well as the proliferation of human keratinocyte cells [7]. Whereas low levels of NO is conducive to the delaying of wound healing [8].
Endocytosis mediated by caveolae play an important role in NO activity [9]. It is involved in the regulation of a range of important cellular activities such as transmembrane signalling and endocytosis. It has been shown that eNOS directly binds to caveolae and interacts with many signalling factors found within the caveolae coat [10]. A subcellular fractionation of endothelial cells revealed that eNOS was particularly present in the caveolae fraction [11]. NOS mediates S-nitrosylation of proteins which in turn can alter the structure and function of proteins and protein-protein interactions [12,13]. A growing body of evidence suggests that S-nitrosylation is implicated in endocytosis and cell migration [12]. GTP hydrolysis activity of dynamin proteins has been shown to increase following their S-nitrosylation – possibly mediated by eNOS [12,14]. This results in the increase of dynamin GTP hydrolysis activity, which has been shown to accelerate the endocytosis of plasma membrane receptor by enhancing the cleavage of vesicles [12]. Aside from endocytosis, the S-nitrosylation of protein has been implicated in (focal adhesion) FA disassembly in a process that requires the calcium-dependent protease calpain. Calpain 2 also called m-Calpain is a family of calcium dependent proteases that play an important role in cell migration by exerting its proteolytic activity on several FA proteins such as talin, vinculin, paxillin and FAK [15,16]. FA proteins are required to maintain the stability of the actin and integrin assembly [15], and play an important role in cell migration [16]. Although calcium activates calpain, it has also been shown that NO can also regulate the activity of the latter [17]. FA dynamics are considered to be an essential component of cell motility and cell migration [18,19]. Previous studies indicate that NO is involved in the maintenance and attachment of adhesions to ECM in various cell types including endothelial cells, vascular smooth muscle cells and renal meningeal cells [20,21]. In addition, the production of NO was shown to stimulate cell migration of intestinal epithelial cell line through FAK activation [22]. However, little attention has been given to investigate the role of NO in the regulation of FA turnover through affecting endocytic regulation. Since endocytosis is one of the pathways involved in the regulation of FAs dynamics, we aim to determine whether NOS inhibition has any effect on endocytosis. EEA1 positive early endosomes appear to be reduced in number and increased in size upon NOS inhibition and, interestingly, several proteins localised at the level of early endosomes are found to be S-nitrosylated. So, this paper describes some biological effects of the treatment of breast cancer cells with NOS inhibitors, including cell migration, FA turnover and early endosome distribution.
The MDA-MB-231 human invasive breast cancer cell line (ATCC® HTB-26™) was purchased from the American Type Culture Collection cell bank (Manassas, VA). These cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% of foetal bovine serum (Gibco™, A4766801) and 1% v/v penicillin/streptomycin (10,000 units/ml penicillin, 10,000 μg/ml streptomycin, Gibco). For optimal cell growth, these cells were incubated at 37°C in an atmosphere containing 95% air, 5% CO2 and approximately 90% humidity. These cells were tested regularly for mycoplasma with the aid of an EZPCR Mycoplasma Test kit (Geneflow, K1-0210, UK), following the manufacturer’s instruction.
Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) (Abcam, ab120136) and 1400W (Tocris Bioscience, 1415) were used to inhibit all NOS isoforms and iNOS, respectively. PAPA/NO; NOC-15; 1-(3-aminopropyl)-2-hydroxy-3-oxo-1-propyltriazan (PAPA NONOate) (Santa Cruz, 146672-58-4) was used as the NO donor. L-NAME (5 mM), 1400W (2 mM) and PAPA NONOate (50 μM) were diluted in phosphate buffered saline (PBS). Amilorid (50 μM), Pitstop 2 (25 μM) and Dynasore (20 μM) were diluted in dimethyl sulphoxide (DMSO). The vehicle used in control conditions was either PBS or DMSO (0.5%).
MDA-MB-231 breast-cancer cells (1 x 105 cells/ ml) were seeded on a 6-well-plate and were left to incubate overnight under culture conditions mentioned above. These cells were subsequently treated for 24 hours with PBS (vehicle), NO synthase inhibitors such as L-NAME or 1400W or a nitric oxide donor (PAPA NONOate). Live imaging was used to monitor cell migration; an image was taken every 15 minutes for a period of 24 hours. The speed of migration was calculated as a total traveling distance divided by 24 hours.
MDA-MB-231 breast-cancer cells (1 x 105 cells/ ml) were seeded on a 6-well-plate and were left to incubate overnight under the culture condition mentioned above. These cells were subsequently treated for 24 hours with the vehicle, L-NAME (5 mM), 1400W (2 mM) and PAPA NONOate (50 μM). Live-cell imaging was performed to monitor cell migration with a time-lapse microscope (Nikon Eclipse TiE); an image was taken (at 10x magnification) every 15 minutes over a period of 24 hours using the Nikon Eclipse TE200 running NIS elements software and a Nikon DXM1200 camera. The MtrackJ tool of the ImageJ software was used to determine the total distance covered by individual cells. The speed of migration (expressed in μm/hour) was calculated as a total distance covered by individual cells divided by 24 hours.
MDA-MB-231 breast-cancer cells (1 x 105 cells/ ml) were plated onto a 6-well-plate and were grown to 90% confluence. The medium was removed, a 200 μl pipette tip was used to introduce several wound lines by scratching the cell monolayer. Thereafter, cells were washed three times and incubated in DMEM containing either the vehicle, L-NAME, 1400W or PAPA NONOate. The width of the wound was determined immediately after the addition of the DMEM (t = 0 hour) and 24 hours later, using the Nikon Eclipse TiE microscope. Images of the wound lines were taken at 10x magnification. The percentage of covered area of the wound after 24 hours was calculated as (Average initial area, t = 0 hour) – (Average final area, t = 24 hours) x 100.
24-hours prior to transfection, MDA-MB-231 cells were seeded (1 x 104 cells/mm2) onto 35 mm ibidi culture dishes treated with 2 mg/ml collagen (BD Bioscience, 354236). The cells were left to incubate overnight in order to allow their attachment onto the bottom of the plate. These cells were subsequently transfected with 3 μg plasmid DNA consisting of zyxin-mCherry and with the aid of the TurboFect™ transfection reagent (Thermo Scientific, R0532) at a ratio of 1:1 (w/v, DNA: TurboFect). Approximately 16 hours after transfection, the cells were treated with L-NAME, 1400W or the vehicle for 10 minutes. A live-cell imaging was performed at 100x magnification using confocal microscopy (Nikon A1R). Cells were imaged at the low power excitation laser beam at a wavelength of 488 nm and 568 nm. Images were taken every 5 seconds for 10 minutes to monitor the full lifetime of zyxin assembly and disassembly. The average life-time of zyxin was determined with ImageJ.
To differentiate the endocytotic pathways, it is important to perform the transferring and dextran uptake assay. Cells (1 x 105 cells/ml) were seeded in 6 well plates upon which glass coverslips were placed. After an overnight culture, the cell reached approximately 70% confluence, the medium supplemented with foetal bovine serum was replaced with serum free DMEM. These cells were incubated for 3 hours at 37°C in a humidified atmosphere containing 95% air and 5% CO2 and were subsequently treated with the vehicle (PBS or DMSO), L-NAME (5 mM), 1400W (2 mM), PAPA NONOate (50 μM), Amilorid (50 μM), Pitstop 2 (25 μM) and Dynasore (20 μM) for 48 hours. Thereafter, the medium of the cells was replaced with serum free DEMEM containing either transferrin conjugated to Alex Fluor 546 (Thermo Fisher, T13342) (25 μg/ml) or dextran conjugated to fluorescein (Thermo Fisher, D1820) (500 μg/ml) and was left to incubate for 30 minutes. The cells were washed twice with ice-cold PBS then fixed with 4% paraformaldehyde (Sigma Aldrich) diluted in PBS (4°C). The cells were washed 3 times with ice-cold PBS and were subsequently treated with an acidic buffer (1M Glycine, 150 mM NaCl, pH 3). The acidic washing step was performed to strip away transferrin/dextran that are bound to the cell surface, since endocytosis blocked at an intermediate stage will cause transferrin/dextran accumulation on the plasma membrane which in turn will affect the total fluorescence measured in the cell. The cells were permeabilised for 10 minutes with PBS containing 0.5% Triton X-100 and were washed 3 times with ice-cold PBS. Confocal images were taken at different layers of the cell (z-stacks) with a resolution of 1024×1024 pixels in order to capture transferring or dextran uptake. The brightness & contrast were then adjusted using ImageJ, then the fluorescence intensity of transferrin and dextran was measured. Alexa Fluor 546 channels and laser power were adjusted as the following values: HV: 64, Offset: 0 and Alexa Fluor 546 nm: 6.35 for all the experiments. Using ImageJ, the region of interest was selected around the cell and the relative intensity of transferrin or dextran was quantified and subtracted from the average background fluorescence in order to determine the number and the size of endosomes containing dextran or transferrin.
The cellular localisation of iNOS, eNOS and early endosome markers were investigated by immunostaning. Cells were incubated (at 37°C) overnight in 6-well plates containing glass coverslips. Thereafter, the growth medium was discarded and replaced with media containing the inhibitors or the NO donor depending on the experiments. After 24 hours, the cells were washed in PBS and fixed for 15 minutes in 4% paraformaldehyde (PFA) in PBS at room temperature. To permeabilised these fixed cells, the excess PFA was washed away with PBS and the cells were incubated at room temperature for 10 minutes in PBS containing 0.5% Triton X-100. These cells were subsequently treated with PBS containing 10% goat serum for 30 minutes at room temperature. Antibodies raised against EEA1 (Cell Signalling, #2411S), eNOS (Cell Signalling, #9572S) or iNOS (Abcam, ab3523) diluted to 1:100 in PBS containing 2% goat serum were added to the cells and incubated for 60 minutes at room temperature. Unbound antibodies in excess were washed away with PBS and the cells were transferred into a box where they were protected from light. Secondary antibodies conjugated with Alexa Fluor 546 or 488 (Cell signalling, #44125, #4408S) diluted to 1:200 in PBS containing 2% goat serum were added to the cells and incubated for 30–60 minutes at room temperature. These cells were washed with PBS and were visualised using confocal microscopy.
The number and the size of FAs and endosomes were analysed on fixed cells. All images were acquired with a confocal microscopy (Nikon Eclipse Ti Laser-scanner), at 100x magnification and a resolution of 1024 × 1024 pixels using the Nikon confocal system software. Images taken at depths where both endosomes and FA were both present. The acquired images were processed by subtracting the background; the average number and size of both FA and endosomes were determined with ImageJ. To investigated whether the localisations of FA and endosomes were correlated, the Spearman’s rank correlation coefficient was determined within a selected region of interest using ImageJ.
The potential link between early endosomes and NO were investigated using two methods; Biotin switch assay and western blotting to detect whether early endosomes proteins were S-nitrosylated. S-nitrosylated cysteine residues were detected with the S-nitrosylated Protein Detection kit from (Cayman Chemical, 75816-684), following the manufacturer’s instruction. After cells were washed twice with 5 ml ice-cold PBS, they were removed from the flask then centrifuged at 500
GPS-SNO and IntFOLD tools were used to identify buried and exposed cysteines residue in proteins in order to determine what residue which is most likely to be S-nitrosylated. Three dimensional structural models of human early endosomal proteins such as EEA1, APPL1 and Rab5 were generated with the IntFOLD server [23,24]. GPS-SNO analysis was used identify cysteines residues in each protein. Accessibility of cysteine residues within the protein three-dimensional structure was assumed to determine a potential to be S-nitrosylated. The GPS-SNO S-nitrosylation site prediction algorithm was used to predict the specific cysteine residue that is likely to be S-nitrosylated [25]. The prediction algorithm was applied to APPL1, EEA1, Rab5 and other proteins including H2B and ubiquitin.
The data obtained by the imaging software were statistically analysed with GraphPad prism 5 software (GraphPad software, San Diego, CA). One-way analysis of variance (ANOVA) was performed on data resulting from experiment comprising three or more experimental groups. Following a significant difference identified via ANOVA, a Dunnett’s post-hoc test was used for pairwise comparisons in order to compare each treatment to a single control (vehicle). All results were obtained from at least three independent experiments in triplicate (n=3). A value of p-value ≤ 0.05 was considered statistically significant.
To evaluate the effect of NO on cell migration, a wound healing assay and a cell tracking assay was employed on MDA-MB-231 cells treated with the iNOS inhibitor 1400W or the whole NOS inhibitor L-NAME or the NO donor PAPA NONOate. Our results showed that cells treated with L-NAME or 1400W displayed a reduced covering area 24 hours after scratching (
To examine the effect of NOS inhibitors on two endocytotic pathways, namely endocytosis or micropinocytosis, we measured the uptake of transferrin and dextran in MDA-MB-231 cells treated with L-NAME and 1400W. As shown in
To examine whether NO is involved in endocytosis regulation through early endosomal compartments, the impact of NOS inhibition and exogenous NO on the number and size of EEA1-positive endosomes and Rab5-positive vesicles were assessed in MDA-MB-231 cells. As shown in
Given that in our previous results, we demonstrated that the inhibition of NOS regulates the number and the size of EEA1-positive endosomes, we subsequently investigated whether the localisation of EEA1-positive endosomes and that of eNOS or iNOS correlates using immunocytochemistry on fixed MDA-MB-231 cells. As shown in
Following the investigation of the localisation of eNOS and iNOS in relations to EEA1, we assessed the S-nitrosylation of proteins implicated in the formation of early endosomes using the biotin switch assay. The Analyses by western blot revealed that non-S-nitrosylation proteins such as H2B or ubiquitin were not detected by the assay (
Once it was ascertained that early endosome markers were S-nitrosylated, a bioinformatic analysis that uses the GPS-SNO algorithm was performed on APPL1, EEA1, Rab5 and other proteins including H2B and ubiquitin in order to predict cysteine residues with the potential to be subjected to S-nitrosylation. The results showed that APPL1 presented two potential cysteine sites at position 99 and 603 that could be S-nitrosylation; four cysteine sites at position 46, 255, 894 and 1102 were identified in EEA1; a single cysteine site for potential S-nitrosylation was found in Rab5, at position 212. However, both H2B and ubiquitin expectedly showed no potential sites for S-nitrosylation (
Given that NO contributes to cell migration in invasive tumours [1], here we investigated the potential role of NO and protein S-nitrosylation on cell migration and FA turnover, using a pharmacological approach. NOS inhibitors L-NAME and 1400W have been shown to inhibit protein S-nitrosylation [26,27]. Our result showed that the speed of cell migration and the duration of FA turnover were slowed down and reduced, respectively in cells treated with L-NAME or 1400W (
Endocytic processes have been shown to regulate FA turnover [31]. In this study, we investigated the effect of L-NAME and 1400W on transferrin and dextran uptake. NO has been reported to S-nitrosylate dynamin and this in turn promotes cell survival and endocytosis [32]. Furthermore, it has been shown that NO nitrosylates dynamin within its pleckstrin homology (PH) domain on a cystein residue located at position 607 (Cys-607) [12]. The S-nitrosylation of dynamin at Cys-607 induces dynamin oligomerisation and its associated GTPase activity, which in turn leads to increased receptor mediated endocytosis [12,32]. For example, HEK293 cells overex-pressing eNOS, increases β2-adrenergic receptor internalisation [33]. Whilst transferrin and dextran are internalised through micropinocytosis and endocytosis (
A study that examined the role of NO in the regulation membrane fusion by acting through a SNARE complex, demonstrated that NO induced the exocytosis of Weibel-Palade Bodies [34]. These findings suggest that NO regulates vesicle trafficking. As we investigated whether NO might disrupt the trafficking of endosomes, we found that the treatment of MDA-MB-231 cells with L-NAME or 1400W caused a reduction in the number EEA1-positive endosomes while their size was found to increase (
In addition, we showed that the localisation of EEA1 correlates moderately with that of eNOS and iNOS (
In this study, we identified for the first time that NOS may act as positive regulators of cell migration and FA turnover through a process that may rely on early endosomes trafficking in MDA-MB-231 breast cancer cells. Given the limitations of this study, we will need to further investigate the role of NO on early endosomes trafficking. It will be essential to investigate whether the acute effect of NOS inhibitors affect the S-nitrosylation of EEA1 and APPL1. In addition, the effect of NO donors still needs to be established. It would be valuable to investigate whether NO donors reverse the effect of NOS inhibitors. Mutagenesis approaches which would introduce mutations on the dimerisation sites/Rab5 binding domain within EEA1, where the cysteine residues are located should enable prevent their S-nitrosylation in other to further clarify the role of NO in the regulation of endosome trafficking. Similar approaches should be applied to other endosome proteins such as Rab5 and APPL1. The observations on cell migration and FA turnover need to be confirmed with protein knockdown using siRNA or shRNA.
Our work suggests that NO might be involved in the regulation of FA turnover, and this may rely on early endosomal trafficking. This study provides supporting evidence for the claim that NO plays a role in cell migration and FA dynamics, particularly in MDA-MB-231 breast cancer cells – suggesting that these findings may present some therapeutic importance for cancer research.