PVM/MA-shelled selol nanocapsules promote cell cycle arrest in A549 lung adenocarcinoma cells
- Ludmilla Regina de Souza1,
- Luis Alexandre Muehlmann2,
- Mayara Simonelly Costa dos Santos1,
- Rayane Ganassin2,
- Rosana Simón-Vázquez3,
- Graziella Anselmo Joanitti2,
- Ewa Mosiniewicz-Szablewska4,
- Piotr Suchocki5, 6,
- Paulo César Morais7, 8,
- África González-Fernández3,
- Ricardo Bentes Azevedo2 and
- Sônia Nair Báo2Email author
© de Souza et al.; licensee BioMed Central Ltd. 2014
Received: 24 June 2014
Accepted: 12 August 2014
Published: 23 August 2014
Selol is an oily mixture of selenitetriacylglycerides that was obtained as a semi-synthetic compound containing selenite. Selol is effective against cancerous cells and less toxic to normal cells compared with inorganic forms of selenite. However, Selol’s hydrophobicity hinders its administration in vivo. Therefore, the present study aimed to produce a formulation of Selol nanocapsules (SPN) and to test its effectiveness against pulmonary adenocarcinoma cells (A549).
Nanocapsules were produced through an interfacial nanoprecipitation method. The polymer shell was composed of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer. The obtained nanocapsules were monodisperse and stable. Both free Selol (S) and SPN reduced the viability of A549 cells, whereas S induced a greater reduction in non-tumor cell viability than SPN. The suppressor effect of SPN was primarily associated to the G2/M arrest of the cell cycle, as was corroborated by the down-regulations of the CCNB1 and CDC25C genes. Apoptosis and necrosis were induced by Selol in a discrete percentage of A549 cells. SPN also increased the production of reactive oxygen species, leading to oxidative cellular damage and to the overexpression of the GPX1, CYP1A1, BAX and BCL2 genes.
This study presents a stable formulation of PVM/MA-shelled Selol nanocapsules and provides the first demonstration that Selol promotes G2/M arrest in cancerous cells.
Low therapeutic efficacy and drug resistance are the most common problems related to the currently available chemotherapeutic agents used in tumoral clinical application. In the search for new chemotherapeutic drugs, several selenium (Se) compounds shown anticancer and anticarcinogenic activities [1,2]. In particular, those containing Se at its 4+ oxidation state, namely selenite, present the highest antioxidant and anticancer activities . However, Se(IV)-containing compounds generally present high systemic toxicity, limiting their clinical application. In this context, a selenite-containing compound named Selol, which was first obtained at Warsaw Medical University, Poland , has shown antitumor activity and low systemic toxicity [4,5]. Selol is a mixture of different selenitetriacylglycerides and appears to act primarily through the induction of oxidative stress in cancer cells . Interestingly, Selol was shown to sensitize leukemia cells to the cytotoxicity of vincristine and doxorubicin, insomuch that it was suggested that Selol could be used in combination with other drugs in chemotherapeutic protocols .
The potential synergism of Selol with classical anticancer drugs can be exploited to treat tumors, such as non-small cell lung cancer (NSCLC) . NSCLC, whose most frequently observed histological subtype is adenocarcinoma, is particularly aggressive and the leading cause of cancer death worldwide [6,7]. It is estimated that more than 75% of patients with NSCLC present locally advanced or metastatic disease, severely limiting the success of treatments [8,9]. Platinum-based treatment, which is the most recommended first-line therapy, reaches response rates of only 20-40% and mean survivals between 7 and 12 months [8,10,11]. Multidrug protocols and a treatment break with non-platinum-based drugs after a fixed course of initial chemotherapy have been shown to prolong the survival of NSCLC patients [12,13]. Thus, Selol may be a potential candidate for combined anti-NSCLC strategies .
Despite the therapeutic potential presented by Selol, its hydrophobicity is a major obstacle to its biological application. For instance, high hydrophobicity often hinders intravenous (iv) administration and may thus confer an undesirable pharmacokinetic profile . This problem can be circumvented through the nanoencapsulation of Selol in an aqueous vehicle to form a nanocapsule-based drug delivery system. The nanoencapsulation of Selol with a polymer presenting highly reactive chemical groups allowing surface modification could bring up new possibilities for delivering this anticancer agent. In this context, the copolymer poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) has been reported to be a biocompatible and biodegradable material useful for preparing drug delivery systems . Additionally, the copolymer PVM/MA presents a surfactant effect and anhydride groups, which readily react with a series of molecules. On that ground, we report the development and the first in vitro efficacy tests of a PVM/MA-shelled Selol nanocapsule formulation intended for the treatment of lung adenocarcinomas.
Results and discussion
The interfacial precipitation method of preformed polymer through solvent displacement yields nanosized Selol capsules only within a certain range of solute and solvent concentrations. Thus, to identify the best formulation parameters, different concentrations of each component were tested.
Next, different concentrations of Selol plus PVM/MA were tested. The concentrations of acetone, ethanol and water were set to 20, 40 and 40% (v:v), respectively. As expected, smaller nanocapsules were obtained at the lowest concentrations of Selol plus PVM/MA (Figure 1(b)). The PDI was not significantly affected by this variable, remaining close to 0.1. The concentration of Selol plus PVM/MA was set to 0.8% (w:v) in further experiments because it provided good colloidal characteristics in addition to a good yield of nanocapsules.
Then, different concentrations of acetone and ethanol were tested in the process of encapsulation with 0.8% Selol plus PVM/MA and a ratio of 1.0 Selol-to-PVM/MA. Different volumes of acetone were used for dissolving a fixed amount of Selol and PVM/MA, and the final volume reached 100% with ethanol:water (1:1, v:v). As shown in Figure 1(c), a major change in the nanocapsule HD was observed with 40% acetone, but the PDI remained below 0.1. When the concentration of acetone was set to 20% and varying volumes of ethanol were added, it was observed that the HD of the nanocapsules decreased with higher concentrations of ethanol (Figure 1(d)). The highest HD and PDI values were obtained with 20% ethanol.
Given the results described above, the protocol of Selol nanoencapsulation was established as follows: 1) 100 mg of PVM/MA and 100 mg of Selol were dissolved in 5 mL of acetone; 2) 10 mL of ethanol and 10 mL of water were added; and 3) the purification steps were then performed.
The method of nanoprecipitation by solvent displacement yielded monodisperse nanocapsules at almost all of the conditions tested and also allowed modulation of the nanocapsule diameter. Noteworthy, by varying the concentrations of acetone and ethanol, nanocapsules of different HDs were obtained, likely due to differences in solvent diffusion, as previously suggested . Even for a concentration of nanocapsule components (Selol plus PVM/MA) near the upper critical limit of 2%, as noted by Aubry et al. (2009)  for this method, stable and monodisperse nanocapsules were obtained. As expected, at higher concentrations of Selol, larger capsules were obtained, which can be attributed to the nucleation-and-growth phenomenon [19,20].
Characterization of Selol nanocapsules
The Selol nanocapsules (SPN) formulation presented a single population of nanocapsules with an HD of 344.4 ± 4.8 nm, a PDI of 0.061 ± 0.005 and a zeta potential (ζ po tential) of −29.3 mV ± 1.5. The transmission electron microscope (TEM) image revealed a population of nanocapsules with an average diameter of 207.9 ± 80.9 nm (Figure 1(e)). These nanometric structures presented a spherical shape and slightly rough surface, as observed with a scanning electron microscope (SEM) (Figure 1(f)). A spherical equilibrium shape is expected with this method due to the three-dimensional primordial droplet nuclei growth conferred by the interfacial tension between the droplets and the dispersant [20,21]. Furthermore, according to the TEM images, the Selol nanocapsules appear to be deformable. Both the deformability and the spherical shape of the nanocapsules are interesting for their administration through parenteral routes .
Thermodynamic stability and dispersibility studies of Selol-PVM/MA nanocapsules
Hydrodynamic diameter ± S.D. (nm)
Zeta potential (mV)
344.4 ± 4.8a
349.7 ± 8.2a
338.9 ± 6.4a
344.3 ± 2.0a
336.6 ± 4.6a
344.4 ± 4.8a
390.2 ± 1.0b
391.3 ± 7.5b
373.9 ± 15.0b
381.6 ± 8.9b
380.6 ± 7.1b
344.4 ± 4.8a
414.2 ± 7.7c
408.2 ± 10.7c
471.0 ± 1.3d
615.2 ± 13.3e
566.3 ± 14.8f
Freezing (−20°C) and thawing cycle
344.4 ± 4.8
414.2 ± 7.74
414.1 ± 10.0
406.7 ± 4.5
410.2 ± 4.7
416.5 ± 4.3
405.5 ± 6.0
425.8 ± 0.9
467.3 ± 9.9
486.5 ± 17.1*
495.1 ± 1.0*
509.3 ± 6.6*
566.2 ± 3.5*
865.1 ± 50.4*
989.1 ± 26.6*
1027.5 ± 60.7*
Selol nanocapsules affect cell viability in a concentration- and time-dependent manner
SPN reduces cell proliferation and promotes cell cycle arrest
As expected, both untreated normal and A549 cells proliferate in culture, as evidenced by the time-dependent increase in the number of cells (p < 0.001 for both 24 vs. 48 h and 48 vs. 72 h for A549; p < 0.01 for 24 vs. 48 h, and p < 0.05 for 48 vs. 72 h for normal cells) (Figure 3 (g and h)). SPN treatment inhibited the proliferation of both cell types, but the intensity of this effect was cell line-dependent. The number of both normal and tumor cells in the SPN-treated groups did not vary over the treatment time (p > 0.05). Reductions of 40.4 ± 2.7% and 64.7 ± 1.7% in the number of SPN-treated A549 cells were evidenced at 48 h (p < 0.001) and 72 h (p < 0.001), respectively, relative to the control A549 cells. However, normal cells were affected to a lesser extent because a significant reduction in their number was observed only at 72 h (24.8 ± 6.7%, p < 0.01) compared with the control normal cells.
Effect of Selol-PVM/MA nanocapsule (SPN) treatment on the cell cycle distribution of A549 cells
Control 48 h
69.6 ± 2.9
18.4 ± 2.3
12.2 ± 1.3
SPN 48 h
56.6 ± 3.8
26.5 ± 0.9
16.9 ± 3.7*
Control 72 h
70.7 ± 3.9
18.8 ± 2.9
10.6 ± 1.7
SPN 72 h
56.2 ± 1.3
25.0 ± 1.9
18.7 ± 2.7*
In relation to normal cells, A549 cells were more susceptible to Selol, both free and encapsulated, which may be partially due to enhanced endocytic activity . Another possible explanation that is supported by the results of the present study is that tumor cells may be more sensitive to Selol due to their higher proliferation rates because Selol appears to primarily act as an inhibitor of cell proliferation. SPN significantly increased the percentage of A549 cells arrested in the G2/M phase of cell cycle and consequently reduced the number of living cells. In the G2 phase, the wee1 protein inactivates the mitosis promoting factor (cyclin B1/CDK1), and cdc25C is a positive regulator of this complex. As shown in previous studies, G2/M arrest may require activation of wee1 in addition to inactivation of cdc25C [28,29].
Additionally, the decrease in cell proliferation induced by Selol was not associated with a decreased energy metabolism coordinated by the main mitochondrial deacetylase sirtuin 3 , as observed by the unchanged transcript levels of the SIRT3 gene (FC < 2.0) (Figure 7(a)). Moreover, subtle changes in the expression of the β-actin gene (ACTB) in SPN-treated cells compared with untreated cells showed that this gene is not suitable as an endogenous control for SPN treatment (Figure 7(a)), likely due to association of the β-actin protein with cytoskeletal components and consequent cell division events .
Phase contrast microscopy revealed that neither SPN nor free Selol induced morphological changes in A549 cells. SPN treatment did not induce any morphological alterations in normal cells because most of them were shown to be spindle-shaped with cytoplasmic projections, as expected. In contrast, free Selol induced visible cytoplasmic retraction (arrows) in normal cells (Figure 6). Comparatively, pronounced morphological changes were induced by SS in both normal and A549 cells.
Cell death signaling
DNA fragmentation increased after 48 h (19.8 ± 1.1% vs. 3.6 ± 1.0% for control, p < 0.001) and 72 h (24.0 ± 2.5% vs. 4.6 ± 1.0% for control, p < 0.001) of SPN treatment (Figure 11(c)). As shown in Figure 11c, this treatment resulted in an increase in the sub-G1 cell population, a clear reduction in the percentage of cells in the G1 phase and an increase in the percentage of cells in the G2/M phase (Figure 11(d)).
The levels of transcripts related to apoptosis, namely BAX (BCL2-associated X protein) and BCL-2 (B-cell lymphoma 2), increased after 48 h (2-fold and 3-fold for BAX and BCL-2, respectively) and 72 h (2-fold for both BAX and BCL-2) (Figure 7(a)). These results suggest that Selol triggers damages that are able to activate the apoptosis mechanisms. However, the BAX/BCL2 ratio, which is recognized as an initiator of the caspases activation pathway, was slightly increased only at 72 h. The results presented suggest that the mechanism of action of Selol in A549 cells is not crucially dependent on the direct induction of apoptosis or necrosis. Suchocki et al. (2007)  demonstrated mitochondrial changes and DNA fragmentation in a leukemia cell line, and Estevanato et al. (2012)  and Wilczynska et al. (2011)  observed the translocation of phosphatidylserine in breast and cervix cancer cell lines. In the present study, the exposition of phosphatidylserine, changes in the mitochondrial membrane potential, DNA fragmentation, and/or alterations in the plasma membrane permeability were observed in low percentages of A549 cells after treatment with free or encapsulated Selol. These findings, however, do not exclude the possibility that Selol may induce extensive A549 cell death at the highest concentrations and/or after prolonged treatment times. Prolonged cell cycle arrest may elicit apoptosis insomuch that cell death may not only be due to primary damage but also result from accumulation of damages arising from the cell cycle arrest itself .
Oxidative stress in A549 cells
The production of reactive oxygen species (ROS) was evaluated in A549 cells compared with HL60 promyelocytic cells. The HL60 cell line is a cell model that is frequently used to access the oxidant or antioxidant potential of different compounds [34,35]. In the current study, Selol surprisingly did not induce an oxidative burst on HL60 cells (Figure 7(b)). Otherwise, ROS accumulation was observed in A549 cells at higher SPN exposure times (24, 48 and 72 h) (Figure 7(b)). These findings suggest that the SPN-induced oxidative burst is variable according to the cell type.
Additionally, increased expression of GPX1 (glutathione peroxidase 1) (5-fold and 7-fold at 48 h and 72 h, respectively) was evidenced on A549 cells (Figure 7(a)). Increased levels of ROS coincident with the up-regulation of GPX1 indicate that A549 cells responded in response to exposure to SPN by activation of the antioxidant defense systems. Suchocki et al. (2010)  showed the involvement of ROS and the inhibition of CYP1A1 (cytochrome P450) induced by Selol in cervix cancer cell lines. Conversely, in this study, the expression of CYP1A1 (4-fold on both 48 h and 72 h) on A549 cells was enhanced by SPN treatment (Figure 7(a)). The metabolism and detoxification of xenobiotics performed by cytochrome P450 may be associated with SPN metabolism and ROS generation . Therefore, the present study suggests that ROS production on A549 cells may be associated with cell death and inhibition of proliferation, as evidenced by Chen et al. (2013) .
Despite the induction of oxidative stress observed on A549 cells, SPN had no effect on the gene expression of catalase at the concentration and times evaluated (Figure 7(b)), corroborating the results of a study on prostate cancer cells treated with Selol for 24 and 48 h .
This study demonstrates that Selol can act as a cytostatic agent and corroborates previous reports indicating that it is effective against cancerous cells and safer for clinical applications than sodium selenite. Furthermore, the present study includes the development of a stable and monodisperse aqueous vehicle for Selol delivery, namely PVM/MA-shelled Selol nanocapsules, which are able to maintain the activity of free Selol against pulmonary adenocarcinoma cells, exhibit reduced toxicity to non-tumor cells in vitro and are thus potentially suitable for the treatment of some types of lung cancer. Further in vivo studies should be performed to evaluate the potential of this formulation for human therapy.
Selol composed of 5% selenium (w:w) was provided by Warsaw Medical University (Poland). PVM/MA (Gantrez AN 119) was kindly gifted by ISP Corp. (Brazil). Human lung adenocarcinoma (A549) and promyelocytic leukemia (HL60) cell lines were purchased from American Type Culture Collection (USA). Sodium selenite, trifluoroacetic acid, dimethyl sulfoxide and rhodamine 123 were purchased from Sigma (USA). Dulbecco’s modified Eagle’s medium, F12 medium, Roswell Park Memorial Institute medium, 3,4,5-dimethylthiazol-2,5 biphenyl tetrazolium bromide (MTT), annexin V-FITC and propidium iodide (PI) were provided by Invitrogen (USA). Fetal bovine serum, penicillin and streptomycin were purchased from Gibco (USA). RNase A was obtained from Promega (USA). Primers and 2’ ,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) were acquired from Life Technology (USA). Phorbol 12-myristate 13-acetate (PMA) was obtained from Abcam (England). GeneJet RNA purification and cDNA Maxima kits were provided by Life Science (USA). DNase I, free-RNase kit was purchased from Thermo Scientific (USA). The TaqMan gene expression assay was obtained from AB Applied Biosystems (USA). Grids and supports of copper and osmium tetroxide were obtained from Electron Microscopy Sciences (USA). Dichloromethane was purchased from Vetec (Brazil). Ethanol and acetone were purchased from J. T. Baker (USA). Phosphate buffer saline was obtained from Laborclin (Brazil).
Preparation of nanocapsules
Nanocapsules were prepared through an interfacial nanoprecipitation method. Briefly, Selol (oil phase) and PVM/MA (surfactant) were dissolved in acetone at room temperature (RT). Next, ethanol and distilled water were sequentially added to the acetone solution, under mild stirring, to form a yellowish, opaque suspension. The organic solvents were removed by distillation at 45°C under reduced pressure (80 mbar) in a rotavapor apparatus (Rotavapor RII®, Buchi Switzerland). Next, the resulting oily nanocapsules were centrifuged at 22,000 × g for 30 min, the transparent aqueous supernatant was removed, and the pellet was resuspended in distilled water. This preparation was immediately characterized and/or stored at 4°C until usage.
The nanocapsules were dispersed in phosphate buffer saline (PBS) (pH 7.4) at a concentration of Selol equivalent to 100 μg/mL. Then, the hydrodynamic diameter (HD) and polydispersity index (PDI) and the zeta potential (ζ potential) were measured at 25°C by dynamic light scattering (DLS) and electrophoretic laser Doppler anemometry (ZetaSizer Nano ZS®, Malvern Instruments), respectively.
Surface morphology and structure
The shape and surface morphology of the capsules were investigated using a field emission scanning electron microscope (SEM) (JEOL JSM 7001-F®, Japan). Before analysis, the composition was diluted with ultrapure water to 5% (v:v), and 20 μL was deposited onto copper supports. Next, the sample was fixed with 1% osmium tetroxide vapor (w:v) for 1 h, left to dry at RT and coated with gold using a Blazers SCD 050® sputter coater (Blazers Union AG, Liechtenstein). The images were digitized using an UltraScan® camera connected to the Digital Micrograph® 3.6.5 computer software (Gatan, USA).
The diameter values of 300 nanocapsules were measured with the Image Pro-Plus® 5.1 software from images captured with a transmission electron microscope (TEM) (JEOL JEM 1011®, Japan). Before this analysis, the sample was diluted with ultrapure water to 3% (v:v) and deposited onto a copper grid. The dried sample was fixed and contrasted with 1% osmium tetroxide vapor (w:v) for 20 min. The images were digitized using an UltraScan® camera connected to the Digital Micrograph 3.6.5® computer software (Gatan, USA).
Efficiency of Selol encapsulation
The Selol concentration in the nanocapsules was estimated according to Suchocki et al. (2003) . Briefly, 300 μL of the nanocapsules dispersion was centrifuged at 22,000 × g and 4°C for 30 min. The pellet was left to dry at RT for two days. Next, Selol was extracted from the pellet with 800 μL of dichloromethane and oxidized with 200 μL of trifluoroacetic acid. The selenium absorbance was measured at a wavelength of 380 nm in a quartz cuvette. The efficiency of Selol encapsulation was calculated considering the ratio of the encapsulated mass to the mass of Selol that was initially used.
Thermodynamic stability studies
Storage at room temperature, 4°C and −20°C. Aliquots of the nanocapsule dispersion were stored at RT, 4°C or −20°C, and their colloidal characteristics (HD, PDI and ζ potential) were evaluated every 15 days.
Freezing and thawing cycles. Fifteen cycles of freezing (−20°C) and thawing (25°C) were applied to a nanocapsule aliquot. After each cycle, the HD, PDI and ζ potential were evaluated.
The A549 human lung adenocarcinoma cell line was cultured with a 1:1 (v:v) mixture of Dulbecco’s modified Eagle’s medium (DMEM) and F12 medium supplemented with 10% (v:v) fetal bovine serum (FBS) and 1% (v:v) antibiotic solution (100 units/mL penicillin and 100 mg/mL streptomycin). Human connective tissue cells harvested from the dental pulp of normal teeth were maintained in primary culture and used as non-tumor control cells, namely normal cells . These cells were grown in DMEM supplemented with 10% FBS and 1% antibiotic solution, as described above. Both cells were maintained at 37°C in a 5% CO2 and 80% humidity environment.
The cells were allowed to adhere to culture microplates for 24 h and were then treated as follows: (1) Selol-PVM/MA nanocapsules (SPN), (2) free Selol (S), (3) blank polymeric nanoparticles without Selol (Bl), and (4) sodium selenite (SS). Cells treated with culture medium, culture medium/acetone and culture medium/PBS corresponded to the control groups of SPN, S and SS, respectively. For the free Selol treatments, Selol was dissolved in acetone and then added to the culture medium, as described by Suchocki et al. (2007) . Before the tests, a viability study was performed to ensure that the volumes of acetone required for each Selol concentration would not be cytotoxic themselves. Bl nanoparticles were prepared using the same method with the same concentrations of the components used for SPN but without Selol. Each treatment was performed in triplicate with different Se concentrations (50, 100 and 150 μg/mL) and times of exposure (24, 48 and 72 h). The concentration of PVM/MA in the treatments with Bl was equivalent to that used in the SPN treatment.
To verify whether the biological activity of SPN was maintained during storage at 4°C, the cell viability was also evaluated on days 1st and 60th after preparation of the SPN.
The cells were seeded and treated as described above. Next, the cells were incubated with 0.5 mg/mL MTT for 2.5 h. The MTT solution was removed, and formazan was extracted from the cells with dimethyl sulfoxide . The absorbance at a wavelength of 595 nm was measured using a spectrophotometer (Spectramax M2, USA) and was used as an index of cell viability. The results were expressed as percentages relative to the control groups after 24 h of treatment.
A549 and normal cells were treated with SPN (100 μg/mL) for 24, 48 and 72 h. Next, the cells were harvested and quantified using a Scepter™ Cell Counter (Millipore, USA).
Morphology and confluence of cells
The morphology and confluence of the cells were analyzed using a phase contrast microscope (Zeiss, Germany) and the AxioVision® software (Zeiss, Germany). For ultrastructural analysis, the cells were fixed, contrasted and dehydrated in agreement with the method described by Carneiro et al. (2011) . Ultrathin sections were observed through TEM, and the images were digitized.
Real-time cell index
Real-time cell analysis was performed with a RTCA instrument (xCelligence, Roche, Switzerland) . Briefly, A549 cells were seeded for 24 h on plates containing microelectronic sensor arrays and later incubated with medium or treated with different concentrations of SPN (50, 100, and 150 μg/mL). The cells were automatically monitored every 15 min for up to 100 h. Two independent experiments were performed.
Reactive oxygen species
The intracellular production of reactive oxygen species (ROS) was measured using 2’ ,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) as an oxidation-sensitive fluorescent probe. The ROS production was evaluated in A549 and HL60 cells [34,35]. The HL60 cells were cultured in RPMI (Roswell Park Memorial Institute medium) supplemented with 10% FBS and 1% antibiotic solution and were maintained at 37°C, 5% CO2 and 80% humidity. Then, the cells treated or not treated with SPN (100 μg/mL) for different times (1, 4, 8, 24, 48 and 72 h) were stained with 2.5 μM DCFH-DA for 30 min at 37°C. HL60 cells treated with 10 mM phorbol 12-myristate 13-acetate (PMA) were used as a positive control. A total of 10,000 events per sample were analyzed using a FC500® cytometer (Beckman Coulter, USA) and the FlowJo 7.6.3 software.
Annexin V-FITC/propidium iodide staining
The externalization of phosphatidylserine and the loss of plasma membrane integrity, which are signs of apoptosis and necrosis, respectively, were assessed with a double-staining kit consisting of FITC-labeled annexin V and propidium iodide (PI). The cells were incubated with 100 μL of binding buffer containing 10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl and 2.5 mM CaCl2. Next, 5 μL of annexin V-FITC and 10 μL of PI (50 μg/mL) were added, and the cells were incubated for 15 min in the dark at RT. The cells were analyzed with a CyFlow® space cytometer (Partec, Germany), and 10,000 events were counted per sample. Cells not incubated with Annexin V-FITC and PI were used as the negative control. Cells incubated with 100 μg/mL SS for 24, 48 and 72 h were used as annexin V staining-positive cells. Cells killed by heating at 60°C for 5 min were used as PI staining-positive cells. All of the cytometry results reported in the CyFlow® space were analyzed using the Windows™ Flow Max® and FlowJo 7.6.3 software programs.
DNA fragmentation and cell cycle analysis
The cell cycle was evaluated by the quantification of total DNA. A549 cells were fixed with 70% ethanol for 2 h at 4°C, rinsed with PBS, incubated with 50 μg/mL RNase A for 30 min at 37°C and stained with 50 μg/mL PI for 30 min at RT . A total of 10,000 events per sample were counted with a CyFlow® cytometer, and the percentage of cells in different phases of the cell cycle was determined. Only those cells presenting DNA content in the range of 2n-4n were considered in the cell cycle analysis. Fragmented DNA was identified in the sub-G1 (DNA content < 2n) population and calculated considering the totality of events.
Mitochondrial membrane potential
The fluorescent cationic substrate rhodamine 123 (Rho123) was used to assess the mitochondrial membrane potential (∆Ψm) in A549 cells. The cells were incubated with 5 μg/mL Rho123 for 15 min at RT and washed twice with PBS . A total of 10,000 events were analyzed per sample using a CyFlow® cytometer.
Quantitative RT-PCR (qRT-PCR)
A549 cells were harvested after SPN treatment (100 μg/mL) for 24, 48 and 72 h. The total RNA was extracted using a GeneJet RNA purification kit and was then treated with DNase I, free-RNase kit. cDNA was synthesized from mRNA using cDNA Maxima reverse transcription reagents. qRT-PCR was performed using a TaqMan gene expression assay, and the amplification reactions were performed using a Fast Real-time System 7900HT (Applied BioSystems, USA). The following primers (and their specifications) were used: ACTB (Hs99999903_m1), BAX (Hs00180269_m1), BCL2 (Hs00608023_m1), CAT (Hs00156308_m1), CDC25C (Hs00156411_m1), CCNB1 (Hs01030099_m1), CCND1 (Hs00765553_m1), CCNE1 (Hs01026536_m1), CYP1A1 (Hs00153120_m1), GAPDH (Hs02758991_g1), GPX1 (Hs00829989_gH), SIRT3 (Hs00953477_m1), and WEE1 (Hs00268721_m1). All of the kits were used according to the manufacturer’s instructions.
The cDNA dilutions were defined based on threshold cycles (CT) (17 – 20) derived from the amplification of the constitutive gene GAPDH (R2 ≥ 0.9974). Each sample was normalized based on the mRNA expression level of GAPDH. The gene expression values were obtained using the 2-∆∆CT equation. A gene was considered to be differentially expressed when the transcript rate (FC, fold change) was at changed by at least twofold compared with the untreated control sample .
All of the experiments were performed in triplicate and repeated three times. The results are represented as the means ± standard deviation. Significant differences were assessed by one- or two-way analyses of variance followed by Tukey or Bonferroni’s post-tests (α = 0.05) using the GraphPad Prism 5.0 software.
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), the Financiadora de Estudos e Projetos (FINEP), the Fundação de Empreendimentos Científicos e Tecnológicos (FINATEC), the Instituto Nacional de Ciência e Tecnologia (INCT), the European Union Seventh Framework Programme [FP7/REGPOT-2012-2013.1] under grant agreement BIOCAPS-316265 and Xunta de Galicia (INBIOMED 2012/273, DXPCTSUG-FEDER), and the National Science Centre in Poland (Grants N N405 360639 and N N202 166440).
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