Silver nanoparticles of Albizia adianthifolia: the induction of apoptosis in human lung carcinoma cell line

Background Silver nanoparticles (AgNP), the most popular nano-compounds, possess unique properties. Albizia adianthifolia (AA) is a plant of the Fabaceae family that is rich in saponins. The biological properties of a novel AgNP, synthesized from an aqueous leaf extract of AA (AAAgNP), were investigated on A549 lung cells. Cell viability was determined by the MTT assay. Cellular oxidative status (lipid peroxidation and glutathione (GSH) levels), ATP concentration, caspase-3/-7, -8 and −9 activities were determined. Apoptosis, mitochondrial (mt) membrane depolarization (flow cytometry) and DNA fragmentation (comet assay) were assessed. The expression of CD95 receptors, p53, bax, PARP-1 and smac/DIABLO was evaluated by flow cytometry and/or western blotting. Results Silver nanoparticles of AA caused a dose-dependent decrease in cell viability with a significant increase in lipid peroxidation (5-fold vs. control; p = 0.0098) and decreased intracellular GSH (p = 0.1184). A significant 2.5-fold decrease in cellular ATP was observed upon AAAgNP exposure (p = 0.0040) with a highly significant elevation in mt depolarization (3.3-fold vs. control; p < 0.0001). Apoptosis was also significantly higher (1.5-fold) in AAAgNP treated cells (p < 0.0001) with a significant decline in expression of CD95 receptors (p = 0.0416). Silver nanoparticles of AA caused a significant 2.5-fold reduction in caspase-8 activity (p = 0.0024) with contrasting increases in caspase-3/-7 (1.7-fold vs. control; p = 0.0180) and −9 activity (1.4-fold vs. control; p = 0.0117). Western blots showed increased expression of smac/DIABLO (4.1-fold) in treated cells (p = 0.0033). Furthermore, AAAgNP significantly increased the expression of p53, bax and PARP-1 (1.2-fold; p = 0.0498, 1.6-fold; p = 0.0083 and 1.1-fold; p = 0.0359 respectively). Conclusion Data suggests that AAAgNP induces cell death in the A549 lung cells via the mt mediated intrinsic apoptotic program. Further investigation is required to potentiate the use of this novel compound in cancer therapy trials.


Introduction
Cancer is a leading cause of global morbidity and mortality [1]. In 2006 there were 4525 deaths due to lung cancer in South Africa [1]. As much as 80-90% of lung cancer cases are attributed to smoking, with the smaller proportion (10-20%) as a result of occupational exposure to heavy metals [2,3]. Much recently, an association has been found between the acquired immunodeficiency syndrome (AIDS) and the development of lung cancer [4]. This is of concern considering the crisis of AIDS in South Africa. The financial strain of anti-retroviral treatment and cancer therapy necessitates the need for alternate means of cancer treatment that is cost effective, easily accessible and safe.
Nanoparticles (NPs) are small sized (1-100 nm) compounds that are able to function as whole units. These compounds are becoming widespread for their use in consumer products and medical applications; with potential for utilization as therapeutic compounds, transfection vectors, anti-microbial agents and fluorescent labels [5]. Silver NPs are the most commercialized and prominent group of nano-compounds, attributed to their diverse applications in the health sector. Silver (Ag) possesses unique and unusual chemical, physical as well as biological properties [6]. Silver, in a colloidal form, is used for the treatment of bacterial infections in open wounds, and preparation of ointments, bandages and wound dressings [7]. Additionally, nanosilver has been used as a contraceptive, and marketed as a water disinfectant [8,9].
Silver NPs are now being exploited for the treatment of various diseases such as retinal neurovascularization [10,11] and acquired immunodeficiency syndrome as a result of human immunodeficiency virus [12]. Additionally, AgNPs are well known for their anti-microbial properties and are used as antiviral agents against hepatitis B, herpes simplex virus type 1, monkey pox virus and respiratory syncytial virus [13,14].
Concerns on environmental exposure to AgNPs have initiated toxicity studies. Silver NP-hydrogel induced DNA damage and the production of reactive oxygen species (ROS) in cultured HeLa cells [15]. A study using human lymphocytes revealed that AgNPs caused DNA damage and cell death [16]. Additionally, AgNPs induced oxidative stress and caused impairment of nuclear DNA in Swiss albino mice [16].
Recently, the use of AgNPs as anti-cancer agents has proved promising [6]. Various attempts to incorporate AgNPs into cancer treatments have been made, with positive outcomes [17]. Although the induction of oxidative stress by AgNP induced mt damage has been observed as the general mode of AgNP toxicity, mechanistic pathways remain unclear [18].
Albizia adianthifolia, a plant member of the Fabaceae family found abundantly on the east coast of South Africa, contains saponins such as prosapogenins and triterpene saponins [19,20]. Saponins are plant glycosides that were found to induce cell cycle arrest in a human breast cancer cell line and initiation of apoptosis in a leukemia cell line [21]. Additionally, certain classes of saponins can sequester serum cholesterol and modulate the immune response [22]. The individual properties of Ag and AA were considered for the synthesis of a novel AgNP using aqueous leaf extracts of the plant.
The A549 cell line (human lung carcinoma) is well characterized and extensively used in in vitro nanotoxocity studies [23]. A recent study postulated that the induction of ROS and alterations in mt membrane permeability were possible mechanisms by which AgNP exerted its toxic effects in A549 cells [24]. The aim of this study was to investigate the effects of AA AgNP on lung cancer cells. It was hypothesized that AA AgNP induced cell death by apoptosis as a result of AA AgNP mediated generation of ROS. We report on a possible mechanism by which AA AgNP induced apoptosis in the A549 cells.

Cell viability assay
Toxicity of AA AgNP to A549 cells was determined using the MTT assay. A dose-dependent decline in cell viability was observed using AA AgNP concentrations in the range 0 to 75 μg/ml for 6h (95% Cl = 31.96 to 57.72) (Figure 1). An IC 50 value of 43 μg/ml was obtained and used in all subsequent assays.

Analysis of caspases
AA AgNP significantly increased the activities of caspase-3/-7 (1.7-fold, 95% Cl = −1,000,000 to −260,000; p = 0.0180)  Figure 1 A dose-dependent decline in A549 cell viability after AA AgNP treatment. The MTT assay was used to determine effect of AA AgNP on A549 cell viability. Cells were exposed to AA AgNP in the range 0-75 μg/ml for 6h, after which the formazan product was quantified spectrophotometrically. A distinct dose dependent effect was observed where AA AgNP at lower concentrations induced minimal cell death. A gradual decrease in cell viability occurred with increasing AA AgNP concentration (95% Cl = 31.96 to 57.72). An IC 50 value of 43 μg/ml was obtained and used for subsequent assays. and −9 (1.4-fold, 95% Cl = −610,000 to −220,000; p = 0.0117) compared to the control. The activity of caspase-8 however was significantly decreased by AA AgNP compared to untreated cells (2.5-fold, 95% Cl = 550,000 to 840,000; p = 0.0024) ( Table 1).

Discussion
Several pathological syndromes such as liver failure, stroke or heart attack are associated with abrupt death of tissue or organs as a result of apoptotic dysregulation. Conversely, the survival of abnormal cells, due to aberrant  apoptosis, may lead to tumorigenesis [25]. Apoptosis is commonly altered in cancerous cells, which have the ability to evade the apoptotic cascade. Silver nanoparticles of AA significantly increased PS externalization, a transmembrane glycoprotein, in the treated A549 cells ( Figure 4A).
The signal transduction of apoptosis involves a cascade of initiator and executioner caspases [26]. Executioner caspases-3/-7 cleave specific substrates leading to alteration changes linked with apoptosis and ultimately cell death [27]. Initiator caspses-8 and −9 are responsible for the activation of executioner caspases. Silver nanoparticles of AA significantly up regulated the activities of caspases-3/-7 and −9 (Table 1). Furthermore AA AgNP increased DNA fragmentation ( Figure 5)-an end stage characteristic of apoptosis. In response to this DNA damage, the nuclear enzyme PARP-1 catalyzes the transfer of NAD + to a specific set of nuclear substrates [28]. During apoptosis, PARP-1 is cleaved, by executioner caspases-3/-7, to a 24kDa DNA binding domain and an 89kDa fragment containing catalytic activity. Silver nanoparticles of AA was responsible for the cleavage of PARP-1 as evidenced by the significantly increased expression of 24kDa fragment compared to untreated control cells ( Figure 6B).
Mitochondria play an important role in apoptosis, via the intrinsic apoptotic program. An initial step for activation of the intrinsic apoptotic pathway is the depolarization of the mt membrane. Depolarized mt is as a result of the formation of mt permeability transition (PT) pores [29]. Mitochondrial PT has been associated with various metabolic consequences such as halted functioning of the electron transport chain with associated elevation in ROS and decreased production of ATP [30]. Bax, a pro-apoptotic protein of the Bcl-2 family, translocates from the cytosol to the outer mt membrane during apoptosis where it interacts with lipids and induces mt PT pores. A significant increase in mt depolarization was observed after AA AgNP treatment ( Figure 4B), with an accompanying decrease in ATP concentration ( Figure 2). The high levels of bax expression ( Figure 6C), high mt depolarization and decreased ATP suggest that AA AgNP induces cellular apoptosis in cancerous lung cells via the intrinsic apoptotic pathway.
Silver has a high affinity for thiol (−SH) groups [31]. In this study, the levels of cysteine-rich GSH were decreased whilst lipid peroxidation was significantly elevated by AA AgNP (Figure 3). This oxidant/anti-oxidant imbalance has previously been documented as an apoptotic mechanism by AgNP mediated cytotoxicity [32,33].
The extrinsic apoptotic pathway is mediated by CD95 death receptor, which recruits Fas-associated protein with death domain (FADD) adapter protein. The adapter protein FADD binds to and activates caspase-8 via the formation of a death-inducing signaling complex [25]. The results of this study show that CD95 expression ( Table 2) and caspase-8 activity (Table 1) was significantly decreased by AA AgNP . The extracts of AA are rich in saponins, which will promote rapid entry of the AA AgNP into the cells resulting in mt mediated intrinsic apoptotic pathway. A well characterized biological action of saponins is their ability to induce cell membrane permeabilization [34]. Decreased ATP concentrations and increased MDA as a result of ROS may be due to disruptions in the mt respiratory chain. Nanoparticles preferentially localize in mt and cause oxidative stress as Figure 4 A) Percentages of apoptotic and necrotic cells and B) Mt depolarization after treatment with AA AgNP . Flow cytometry was used to examine PS translocation (annexin-V-Fluos assay (Roche)) and mt membrane integrity (BD™ MitoScreen kit (BD Biosciences). A significant increase in apoptosis (57 ± 0.59% vs. control: 10 ± 0.84%, 95% Cl = −50 to −43; p < 0.0001) and necrosis (17 ± 0.79% vs. control: 4.6 ± 0.70%, 95% Cl = −16 to −9.2; p < 0.0001) following treatment was seen. Furthermore, mt membrane depolarization was significantly higher in AA AgNP treated cells (77 ± 0.88% vs. control: 23 ± 1.8%, 95% Cl = 44 to 57; p < 0.0001). well as potentiate structural damage [35,36]. Various studies have associated AgNP toxicity with mt damage [37,38]. Several pro-apoptotic molecules are released from the mt during apoptosis. In the presence of ATP, mt released cytochrome c associates with apoptotic protease-activating factor (Apaf)-1 in the cytosol inducing its oligomerization. An apoptosome is then formed with the oligomeric Apaf-1 complex and procaspase-9, inducing the activation of caspase-9, which in turn activates effector caspases-3 and −7 [26]. An interesting finding in this study was that although ATP levels were reduced post AA AgNP treatment, the activity of caspase-9 was still elevated (Table 1).
A class of molecules involved in the regulation of apoptosis is the inhibitor of apoptosis (IAP) proteins. These proteins avert cell death by suppressing the activity of caspases. X-chromosome-linked inhibitor of apoptosis (XIAP) is the most well characterized member of IAPs [25]. The ability of IAPs to act as endogenous suppressors of procaspase activation is attributed to the presence of domains referred to as baculoviral IAP repeats (BIR).
In particular, BIR3 and a region adjacent to BIR2 are responsible for the inhibition of caspases-9, and −3 and −7 respectively. Smac/DIABLO, a mt protein, is able to abolish the inhibitory effects of XIAP [26]. Both smac/ DIABLO and caspases-3,-7 and −9 contain IAP-binding motifs that fit into the BIR domains of XIAP. Thus, smac/ DIABLO is able to relieve inhibition by replacing and releasing caspases-3, -7 and −9 from the XIAP inhibitory complex [25,26]. We postulated that AA AgNP released smac/DIABLO from the mt. The increased intracellular staining ( Table 2) and expression of smac/DIABLO by western blotting (Figure 6D) confirmed that AA AgNP induced its release from the mt.
The p53 protein mediates a range of anti-proliferative processes in response to different stress stimuli by directly activating apoptosis and promoting the release of bax [39] and inducing executioner caspase activity [40] Also, p53 interferes with mt integrity and function leading to the release of pro-apoptotic molecules and the generation of ROS [41]. This study clearly shows the increased expression of p53 after addition of AA AgNP to cells ( Figure 6A).

Conclusion
In conclusion, this novel AA AgNP possesses potent proapoptotic potential. We have shown, mechanistically, that AA AgNP activates the intrinsic apoptotic pathway in A549 lung carcinoma cells. The findings of this study suggest the potential for AA AgNP in drug development against cancer. None the less, further studies need to be conducted to ascertain if the effects of AA AgNP are consistent in other cancerous cell lines and also non-toxic to healthy systems.

Materials and methods
Materials A549 cells were purchased from Highveld Biologicals (Johannesburg, South Africa). Cell culture reagents were purchased from Whitehead Scientific (Johannesburg, South Africa). LumiGLO W chemiluminescent substrate kit was purchased from Gaithersburg (USA) and western blot reagents were purchased from Bio-Rad (USA). All other reagents were purchased from Merck (South Africa).

Synthesis of AA AgNP
Synthesis and characterization of AA AgNP , described by Gengan et al. 2013, was conducted at the Steve Biko campus, Durban University of Technology, Durban, South Africa. A one-pot green synthesis technique was used [42]. Briefly, fresh leaves of AA were extracted with deionized water. The crude extract was filtered and the supernatant was allowed to react with silver nitrate solution at room temperature (RT). Silver nanoparticles of AA solution (pH 7) were then characterized using UV spectrometry. Particle size was determined by transmission electron microscopy. To assess the interaction between nanosilver  and compounds of the aqueous extracts of AA leaves, Fourier transform infrared spectrophotometry was employed [42]. Ethical approval from the Biomedical Research Ethics Administration Office of the University of KwaZulu-Natal (Reference number: BE050/08) was obtained.
Cell culture and exposure protocol The A549 cells were cultured in Eagle's minimum essential medium supplemented with 1% L-glutamine, 1% penstrepfungizone and 10% fetal bovine serum. Cultures were maintained at 37°C with 5% CO 2 . For the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, cells were seeded into a 96-well microtitre plate, allowed to attach overnight and treated with AA AgNP solution (0-75 μg/ml). For flow cytometry assays, caspase, ATP and lipid peroxidation assays, cells were cultured to 90% confluency in 25 cm 2 tissue flasks and treated with AA AgNP . For the GSH assay, cells were plated in 96-well microtitre plates and allowed to attach overnight, followed by treatment with AA AgNP . For western blot analysis and the comet assay, cells were grown to 90% confluency in 6-well culture plates and treated with AA AgNP .

Cell viability assay
Cell viability was determined using the MTT assay. Approximately 20,000 cells (in six replicates) were used for exposure to AA AgNP concentrations in the treatment range. After incubation with AA AgNP for 6 h, cells were washed twice with 0.1M phosphate buffer saline (PBS) and incubated with MTT salt solution (5 mg/ml in 0.1M PBS) and complete culture medium (37°C, 4 h). Thereafter, 100 μl of dimethyl sulfoxide was added to each well and incubated (37°C, 1 h). Optical density of the formazan product was measured using a spectrophotometer (Bio-tek μQuant) at 570/690 nm. The results were expressed as percentage cell viability vs. concentration of AA AgNP , from which the half maximal inhibitory concentration (IC 50 ) was determined.

ATP assay
Cells (20,000/well in six replicates) were aliquoted in an opaque 96-well microtitre plate to which the ATP CellTitre Glo (Promega, Madison, USA) reagent (50 μl) was added and allowed to react in the dark (30 min, RT). After incubation, the luminescent signal proportional to the cellular ATP content was detected with a Modulus ™ microplate reader (Turner Biosystems, Sunnyvale, USA).
The results were expressed as mean relative light units (RLU).

Glutathione assay
The GSH-Glo ™ Glutatione assay (Promega, Madison, USA) was utilized to quantify intracellular GSH levels. Subsequent to treatment of cells (10,000 cells/well in six replicates) in an opaque 96-well microtitre plate, culture medium was removed and 25 μl of 1X GSH-Glo ™ reagent (prepared according to manufacturer's guidelines) was added to each well. Glutathione standards (0-5M) were serially diluted (two-fold) from a 5 mM stock in deionized water. After brief mixing on a shaker and 30 min incubation at RT, 100 μl of Luciferin detection reagent was added to the wells (15 min, RT). Luminescence was detected on a Modulus ™ microplate luminometer (Turner Biosystems, Sunnyvale, USA). A calibration curve was constructed and sample GSH concentrations (μM) were calculated.

Lipid peroxidation
To investigate the AA AgNP -mediated generation of reactive oxygen species (ROS), malondialdehyde (MDA-a product of lipid peroxidation) levels were measured using the thiobarbituric acid reactive substances (TBARS) assay. All tubes were incubated in a water bath (100°C, 15 min) and after cooling; butanol (1.5 ml) was added to each tube, vortexed for 10 seconds and allowed to separate into two distinct phases. Approximately 800 μl of the upper butanol phase was then transferred to 1.5 ml tubes and centrifuged (840 x g, 24°C; 6 min). To a 96-well micotitre plate, 100 μl of supernatant was transferred in six replicates and the absorbance read using a spectrophotometer (Bio-tek μQuant) at 532/600 nm. The mean absorbance was divided by the extinction co-efficient (156 mM -1 ) and results were expressed as μM concentrations.

JC-1 MitoScreen assay
Mitochondrial membrane potential (Δ Ψ) was assayed with the BD ™ MitoScreen kit (BD Biosciences). JC-1, a cationic dye, is sensitive to Δ Ψ and accumulates in mt with polarized membranes. JC-1 working solution was prepared and 100 μl added to each flow cytometry tube, followed by 100 μl of cell suspension. Tubes were incubated at 37°C with 5% CO 2 for 15 min, after which 100 μl of JC-1 wash buffer was added. Approximately 50,000 events were analyzed for mt depolarization. A FACS Calibur flow cytometer was used and data were analyzed using CellQuest PRO v4.02 software. Cells were gated to exclude cellular debris using FlowJo v7.1 software. The results were expressed as percentage of the total events.

Comet assay
The comet assay was used to determine DNA fragmentation in the AA AgNP treated lung cells. Briefly, three slides per sample were prepared with a first layer containing 400 μl of 1% low melting point agarose (LMPA, 37°C), a second layer of 25 μl of cells from each sample with 175 μl 0.5% LMPA (37°C) and a third layer containing 200 μl of 1% LMPA (37°C). Cover slips were removed and slides were subjected to lysis (4°C, 1 hr, protected from light) by being submerged in cells lysis buffer (2.5M NaCl, 100 mM EDTA, 1% Triton X-100, 10 mM Tris (pH 10) and 10% DMSO

Statistical evaluation
Statistical analyses were performed using GraphPad Prism version 5.00 software package (GraphPad PRISM W ). Data are expressed as mean ± standard error of the mean (sem). Comparisons were made using unpaired t tests. Statistical significance was set at 0.05.