Betanodavirus B2 protein triggers apoptosis and necroptosis in lung cancer cells that suppresses autophagy

The betanodavirus B2 protein targets the mitochondria and acts as a “death factor”, but its effect on lung cancer cells is unknown. We examined the effect of the B2 protein on triggering apoptosis or necroptosis via P53-dependent and P53-independent pathways and increased in suppression of autophagy. The B2 protein targets the mitochondria of A549 (P53+/+) and H1299 (P53—/—) lung cancer cells due to a specific signal sequence (41RTFVISAHAA50). This triggers generation of reactive oxygen species within the mitochondria, and a minor stress response in A549 cells, but a strong stress response in H1299 cells. We examined the molecular mechanism of this cell death pathway, and found that B2 protein induces the P53/Bax-mediated apoptotic pathway in A549 cells, and that a P53 specific inhibitor (pifithrin-α) switches this response to RIP3-mediated necroptosis. On the other hand, B2 induces RIP3-mediated necroptosis pathway in H1299 cells, and a necroptosis inhibitor (necrostatin-1) switches this response to the apoptotic pathway. Both types of cell death signals inhibited autophagy via a tightly increased balance of beclin-1 and Bcl-2. Thus, B2 protein triggers P53-dependent apoptosis in A549 cells and ROS/RIP3-mediated necroptosis in H1299 cells, and crosstalk of these pathways limits initiation of autophagy. These findings provide new insights into the possible control and treatment of lung cancer.


INTRODUCTION
Betanodaviruses are the causative agents of viral nervous necrosis (VNN) in fish, an infectious neuropathological condition characterized by necrosis of the central nervous system, including the brain and retina [1]. Clinical signs include abnormal swimming behavior and darkening of the fish [2]. VNN can cause massive dying off of the larval and juvenile populations of several marine teleost species [3]. Little is known about the molecular pathogenesis of VNN.
The nodavirus genome comprises two singlestranded molecules of positive polarity, RNA1 and RNA2 that are approximately 3.1 and 1.4 kb in length, respectively. RNA1 encodes a nonstructural protein of approximately 110 kDa, designated RNA-dependent RNA polymerase or protein A that is vital for replication of the viral genome. RNA2 encodes a 42-kDa capsid protein [4,5] that may induce post-apoptotic necrotic cell death through a pathway mediated by cytochrome c release [6]. In RNA replication, betanodaviruses synthesize a sub-genomic RNA3 from the 3' terminus of RNA1 that encodes two proteins, B1 and B2 [1,7,8]. In RGNNV, B1 plays anti-necrosis functions [9]. B2 acts as a host siRNA silencing suppressor in alpha- [10][11][12] and beta-nodavirus [7]. Recently, the B2 protein can induce oxidative stressmediated cell death via mitochondrial targeting in vitro and in vivo [13].
The tumor suppressor protein P53 plays a major role in the cellular response to DNA damage and in protecting the genome from mutations. Activation of p53 can promote cell death or survival [14]. The P53 protein mediates cellular stress responses, in that it can initiate DNA repair, cell-cycle arrest, and senescence [15][16][17][18]. Importantly, P53 also regulates apoptosis, necroptosis, and autophagy [19]. When DNA repair fails, p53 initiates apoptosis by transactivating pro-/anti -apoptotic proteins that have roles in the signal transduction of apoptosis and necroptosis [20].
Apoptosis occurs normally during development and aging, and functions as a homeostatic mechanism to maintain cell populations in tissues. Apoptosis also functions as a defense mechanism, as in immune reactions or responses to cell damage from diseases or harmful agents. There are two major apoptotic pathways: the extrinsic (or death receptor) pathway and the intrinsic (or mitochondrial) pathway [21]. The extrinsic pathway is characterized by transmembrane receptor-mediated interactions, in which death receptors (members of the tumor necrosis factor [TNF] receptor gene superfamily) have a role [22]. The intrinsic pathway has a diverse array of non-receptor-mediated stimuli that produce intracellular signals, which act directly on targets within the cell, and are mitochondria-initiated events.
Recent studies indicate that necrosis is not just a series of unregulated processes, but is a series of programmed events, termed necroptosis [23]. In fact, TNFα, FasL, and TRAIL, the same ligands that activate apoptosis, can also stimulate necroptosis. Receptor interacting protein (RIP) kinases are also crucial regulators of cell survival and death [24]. There are seven proteins in the RIP family, each of which has a kinase domain (KD). Importantly, activation of RIP1 kinase regulates the necroptotic death pathway [25].
Autophagy is a highly conserved catabolic process, in which there is degradation of proteins and organelles that promote survival or death, depending on the physiological and pathological conditions [26]. A key part of autophagy is the sequestration of proteins and organelles within double-membrane structures, termed autophagosomes. Lysosomes target the autophagosomes, which degrade them to autophagic vacuoles or autophagolysosomes. Induction of several autophagyrelated genes, including LC3, phosphatidylinositide 3-kinase, and Beclin 1 (which is regulated by Bcl-2 and Bcl-xL proteins) [27][28][29], initiates the formation of an autophagosome.
We previously studied the effect of B2 protein on ATP depletion-induced cell death in vitro and in vivo [13,30,31] in a line of fish cells and a zebra fish model system. However, the effect of the B2 protein on the cell death pathways in lung cancer cells is still unclear. In this study, we used the novel viral B2 protein to induce different cell death pathways in A549 lung cancer cells, which express P53 (P53 +/+ ), and H1299 lung cancer cells, which do not express P53 (P53 -/-), and also examined relationship of activation of these different pathways with suppression of autophagy. These data may provide new insight into the control and treatment of lung cancer.

B2 protein targets lung cancer cell mitochondria
The betanodavirus B2 protein targets mitochondria via a specific signal peptide ( 41 RTFVISAHAA 50 ) [13]. We determined if the B2 protein can also target the mitochondria of human lung cancer cell lines A549 (P53 +/+ ) and H1299 (P53 -/-). Thus, we used fulllength EYFP-B2 and EYFP-ΔB2, which has a deleted targeting region ( Figure 1B and 1C) that further have predicted the 3D-structure of full length (1-75 aa) and B2 mitochondria targeting domain (36 aa) as a major alpha helix confirmation, and measured localization of B2 protein using MitoTracker and measurement of green fluorescence. The results show green fluorescence in the mitochondria of cells transfected with the full-length EYFP-B2 ( Figure 1A and 1Ag-1Ai: A549 cells; p-r: H1299 cells). In contrast, cells of the EYFP group ( Figure  1A Figure 1D). These results confirm that B2 targets the mitochondria in A549 and H1299 cells that were transfected with the fulllength EYFP-B2, but not in cells of the other groups.

B2 protein induces stronger ROS production in H1299 (P53 -/-) cells than A549 (P53 +/+ ) cells
Previous studies of fish indicated that B2 protein targeting of mitochondria correlates with ROS production [30]. Thus, we measured B2-induced generation of ROS at 48 h post-transfection in both lines of cancer cells. We also determined P53 expression in A549 cells (Figure 2A Figure 2B, 2Bg and 2Bh in A549 cells). Relative to cells treated with NAC, there was a 4-fold increase of ROS generation in A549 cells, and a 6-fold increase in H1299 cells ( Figure 2D). We also found that B2-induced ROS production upregulates P53 by ~1.3-fold in A549 cells ( Figure 2E, lane 2 and 2F). Moreover, B2-induced ROS significantly increased P53 phosphorylation on serine residue 15 (for DNA damage) and serine residue 46 (for apoptosis), but not serine residue 392 (for tumor induction) [16,17].

B2-induces apoptosis in A549 (P53 +/+ ) cells and necroptosis in H1299 (P53 -/-) cells
Next, we determined the mechanism(s) by which the B2 protein induces cell death in both lines of lung cancer cells by use of flow cytometric analysis with Annexin-V-FITV and PI staining ( Figure 3). The results show that B2 protein induces apoptosis in 13% of A549 cells ( Figure 3A and 3B), but induces necroptosis in 10% of H1299 cells ( Figure 3A and 3B).
We also found that B2 increased the expression of the pro-apoptotic gene Bax by ~2.5-fold ( Figure 3C, lane 2) in A549 cells, but not in H1299 cells ( Figure 3C, lane 4). On the other hand, B2 increased the expression of Bcl-2 in H1299 cells by ~4.5-fold, but only by ~20% in A1299 cells ( Figure 3D). In summary, B2 protein induces Bax-mediated apoptosis in A549 cells, but induces RIP3mediated necroptosis in H1299 cells.

A specific P53 inhibitor (pifithrin-α) leads to B2 protein-induction of necroptosis in A549 cells
Next, we determined the role of P53 on apoptosis and necroptosis by double staining A549 cells with PI and annexin V-FITC, and treatment with a specific inhibitor of P53 (Pifithrin-α, 30 μM) ( Figure 4). The results show that Pifithrin-α blocked P53 activity ( Figure 4A   confirmed these differences were significant (p < 0.05 for all comparisons).
We also analyzed markers of apoptosis and necroptosis in A549 cells ( Figure 4C and 4D). The results show that B2 induces a ~1.3-fold increase in the proapoptotic gene Bax ( Figure 4C, lane 2) and that Pifithrin-α treatment blocked this effect. Blockage of P53 activity was also associated with a ~23% increase in RIP3 expression ( Figure 4C, lane 4 and 4D). These differences were statistically significant (p < 0.01 for all comparisons).

A specific inhibitor of necroptosis (necrostatin-1) leads to B2 protein-induction of apoptosis in H1299 cells
We also used a specific inhibitor of necroptosis (necrostatin-1, 40 μM) to determine the effect of blocking necroptosis in H1299 cells ( Figure 5). Necrostatin-1 inhibits RIPK1 by blocking its association with RIP3.
Based on the PI/Annexin V double staining assay, we found that treatment of H1299 cells with necrostatin-1 inhibited necroptosis, and increased apoptosis ( Figure  5A, 5Ad-5Af and 5Aj-5Al). Quantification of green fluorescence (Annexin-V-FITC) with red fluorescence (PI) confirmed that this difference was significant (p < 0.01).
As previously, we also examined markers of apoptosis and necroptosis in H1299 cells ( Figure 5C and 5D). The results show that B2 protein upregulated the necrosis gene RIP3 by ~1.35-fold and Bcl-2 by ~1.5-fold ( Figure 5C

B2 protein regulates cell death, but limits induction of autophagy by maintaining a balance of beclin-1 and Bcl-2 in lung cancer cells
The interaction of B2 protein-induced cell death with autophagy is unknown. We examined the effect of B2 protein-induced cell death on autophagy by treatment of cells with the P53 inhibitor Pifithrin-α or the necrotsis inhibitor necrostatin-1.
Thus, at 48 h post-transfection, B2 expression inhibited autophagy initiation in A549 cells ( Figure  6A) and H1299 cells ( Figure 6B) based on the LC3-II/ LC3-I ratio, a marker of autophagy. There was also downregulation of the autophagy regulation genes, beclin 1 and Bcl-2, in A549 cells, although there was upregulation of Bcl-2 and downregulation of beclin-1 in H1299 cells. Blockage of P53 in A549 cells increased the LC3-II/LC3-I ratio ( Figure 6A, lane 4), and this correlated with increased expression of beclin1 and decreased expression of Bcl-2. In contrast, blockage of necrosis in H1299 cells is inhibted autophagy ( Figure 6B, lane 4) on LC3-II/LC3-I ratio ( Figure 6B, lane 4), and increased minor upregulation of Bcl-2 and beclin 1. Quantification of these results indicated the differences were significant (p < 0.01 for all comparisons) ( Figure 6C and 6D). In summary, these experiments indicate the P53 gene has a role in supression of autophagy in A549 cells, but not in H1299 cells.  Figure 3C. Error bars represent the SEM of 3 independent experiments. All data were analyzed using a paired or unpaired Student's t-test, as appropriate. * P < 0.01 significantly different from the control. www.impactjournals.com/oncotarget DISCUSSION B2 protein expression induces necroptosis and breakdown of mitochondria in aquatic fish cells, and this correlates with B2 targeting of mitochondria, ROS generation, and ATP depletion from complex-V (F 0 F 1 -ATP synthase). Furthermore, B2 protein induces cell death in zebrafish during the early embryonic stage (within 12 h post-infection) [13,30,31]. These results suggest that transfection experiments in which B2 protein expression is induced in other types of cells, such as lung cancer cells, may help to elucidate the mechanisms of cell death.
The present study examined the effect of the nonstructural protein B2 as a novel "death factor" that targets mitochondria and regulates apoptosis and necroptosis in lung cancer cells, depending on the presence of P53. We found that B2 protein targets the mitochondria of lung cancer cells, and that targeting increases to ROS production and apoptosis in A549 cells (P53 +/+ ) and necroptosis in H1299 cells (P53 -/-). Our results also suggest that the presence of pathways for two types of cell death -apoptosis and necroptosis --limits the extent of autophagy in these cells.

B2 protein targets mitochondria and triggers stress signals in lung cancer cells
Our results indicate that the B2 signal region was between amino acids 41-50 ( 41 RTFVISAHAA 50 ), and includes 10 amino acids [13] that target the mitochondria of A549 and H1299 cells ( Figure 1B). This signal region is different from the signal regions of other proteins that target mitochondria (HSP60 and TOM5; Figure 1C) that protein B2 targeting domain was shown the alpha helix structure. Our results also show that removal of this signal peptide blocked the ability of B2 protein to target mitochondria ( Figure 1A). Furthermore, we found that B2 mitochondrial targeting increases to ROS production in both cell types, although with a there was a 4-fold increase of ROS in A549 cells and an 8-fold increase of ROS in H1299 cells (Figure 2). Thus, P53 appears to have a more important role in directly regulation of oxidative stress A549 cells, but additional factors may also regulate stress in H1299 cells.
Furthermore, we found that B2 protein expression has different effects on cells with different genetic backgrounds (i.e. A549 cells (P53 +/+ ) and H1299 cells [P53 -/-]). B2 protein triggers apoptosis through P53/ Bax signaling in A549 cells (Figure 4) [30,31]. However, blockage of P53 function in A549 cells by a specific inhibitor switched the mechanism to necroptosis, as indicated by downregulation of Bax and upregulation of RIP3 ( Figure 4C). By contrast, we found that B2 protein triggers necroptosis via the ROS/RIP3-pathway in H1299 cells ( Figure 5), and that treatment of these cells with a specific inhibitor of necroptosis switched to mechanism to apoptosis ( Figure 5C), as indicated by downregulation of Bcl-2 expression.

Induction of two cell death pathways by B2 protein inhibits initiation of autophagy
In normal physiological situations, autophagy is always occurring at a basal level, and it functions as an intracellular quality-control system that maintains homeostasis by removal of superfluous and/or damaged proteins [34][35][36][37][38][39][40][41][42][43][44][45][46]. Autophagy and apoptosis both occur when cells are under stress [46]. Normally, autophagy precedes apoptosis and maintains cell homeostasis, with a tight crosstalk between these pathways. Some factors function in apoptosis and autophagy, such as Beclin-1 and Bcl-2, that protein interaction on control to apoptosis or autophagy was required [47].
We found that B2 triggered P53/Bax-mediated cell death via apoptosis in A549 lung cancer cells, and that crosstalk with the autophagy pathway occurs through downregulation of Beclin-1 and Bcl-2 ( Figure 6A) [46,48]. Furthermore, inhibition of P53 by Pif-α switched the cells from apoptosis to autophagy via minor Beclin-1 upregulation and minor Bcl-2 downregulation. This indicates that P53-mediated apoptosis can directly regulate autophagy. On the other hand, B2 induces ROS/RIP3mediated cell death via necroptosis in H1299 cells, and also limits autophagy via minor Beclin-1 downregulation and strong Bcl-2 upregulation ( Figure 6B). Then, further

Figure 6: Crosstalk of apoptosis and necroptosis pathways limits initiation of autophagy in human lung cancer cells.
Immunoblotting at 48 h post-transfection using monoclonal antibodies against autophagy-related proteins (LC3-I, LC3-II, Beclin-1, and Bcl-2) in A549 cells, with and without a P53 inhibitor (A) and in H1299 cells, with and without a necrosis inhibitor (B). ß-actin was used as a loading control. (C and D) Quantification of the results in A and B, respectively. Error bars represent the SEM of 3 independent experiments. All data were analyzed using a paired or unpaired Student's t-test, as appropriate. * p < 0.01 indicates statistical significance. necroptotic process inhibited by necroptosis inhibitor Nec-1 was found that minor double upregulated the Beclin-1 and Bcl-2 expression, which necroptotic death signals is involved in autophagy regulation.
In summary, we found that the B2 protein triggers death of lung cancer cells via promotion of P53/ Bax-mediated apoptosis (A549 cells) and by ROS/ RIP3-mediated necroptosis (H1299 cells) (Figure 7). Moreover, ROS generation and metabolism has roles in the P53-dependent and P53-independent pathways [49]. Our findings also suggest that crosstalk of the apoptosis and necroptosis pathways can reduce autophagy by altering the balance of beclin-1 and Bcl-2.
If P53 activity is blocked, then Rif-α can switch from apoptosis to necroptosis, with only minor promotion of autophagy in A549 and H1299 cells. On the other hand, we found that blockage of necroptosis switches cells to apoptosis, but with no apparent initiation of autophagy in H1299 lung cancer cells, in contrast to A549 cells. Our findings indicate that B2 protein induces two cell death pathways -a P53-dependent pathway and a P53independent pathway. Thus, the genetic background of a cell determines which pathway is triggered, a death signals to regulate autophagy initiation between beclin-1/ Bcl-2 interaction, and this provides new insight into cancer control and therapy.

Cell transfection
Polyethylenimine (PEI; Sigma Aldrich, 408727) was used as the transfection agent [50,51]. For cell transfection, 4×10 5 cells were seeded in 6-well culture plates. On the following day, 3.2 μg of recombinant plasmid was mixed with 3.2 μg of PEI, and the transfection procedure was carried out according to the manufacturer's instructions.

Preparation of mitochondria from B2transfected cells
A549 and H1299 cells were seeded in 60-mm diameter culture dishes with 4 mL of medium (10 5 cells/ mL) for 24 h. These cells were then transfected with EYFP or EYFP-B2 for 48 h. At each change of the culture medium, 1 mL of medium was removed. Mitochondria were isolated by modification of a previously described protocol [30]. Briefly, cells (2×10 6 ) were washed with PBS and homogenized in 0.3 mL of mitochondria isolation buffer (0.35 M mannitol, 10 mM HEPES, pH 7.2, 0.1% bovine serum albumin) using a glass homogenizer. Unbroken cells and nuclei were pelleted by centrifugation (600 g for 5 min at 4°C). Then, the mitochondrial pellet was isolated by centrifugation (10,000 g for 10 min at 4°C) and the supernatant was collected and mixed with 25 μL of 10×SDS sample buffer. Samples (50 μL) were boiled and subjected to western blot analysis as previously described [13].

Mitochondrial staining assay
To track changes in mitochondrial morphology, cells were transfected as described above. After culture for 48 h, cells were stained with MitoTracker Red CM-H 2 XRos (Invitrogen) in accordance with the manufacturer's instructions. Then, cells were analyzed by fluorescence microscopy, with excitation at 488 nm green fluorescence measured with a 515-nm long-pass filter, and with 510 nm excitation and red fluorescence measured with a 590-nm long-pass filter, as previously described [30].

Intracellular ROS content of lung cancer cells
The generation of ROS was evaluated by a fluorescent-cytometry assay based on intracellular oxidation of H 2 DCFDA (Life Technologies, Carlsbad, CA, USA) [55]. Cells in the logarithmic growth phase were incubated in a 6-well plate overnight. Then, the medium was replaced with B2 transfection medium for 48 h. Cells were then washed with phosphate-buffered saline (PBS), resuspended at a concentration of 1 × 10 6 cells/mL, and stained for 30 min at 37°C. Cells were observed by fluorescence microscopy, with excitation at 488 nm and measurement of green fluorescence using a 515-nm longpass filter [30].

Assays for apoptosis and necrosis
The Annexin V-FITC/Propidium iodide (PI) flow cytometric assay was used to measure early and late apoptosis, according to the manufacturer's instructions (Annexin V-FITC/PI, Rocha). Briefly, A549 and H1299 cells were transfected with FLAG or FLAG-B2 plasmids for 48 h at 37°C, then washed twice with cold PBS, and centrifuged at 1000 rpm for 5 min. The harvested cells were resuspended in 200 μL binding buffer that contained 10 μL Annexin V-FITC. After 15 min, the cells were washed twice and resuspended in 300 μL binding buffer, and 10 μL of PI was added. Then, the cells were immediately analyzed by flow cytometry using a FACS Vantage cell sorter (Becton-Dickinson, San Jose, CA, USA). PI red fluorescence was measured using a 650-nm long-pass filter. Apoptotic and necroptotic cells have higher PI fluorescence (PI + ) than intact cells (PI − ). Each analysis examined at least 10,000 cells in the gated region, based on light scattering properties. Fluorescence data are displayed on one or two major scales, as previously described [13].

3D-structure prediction
SWISS-MODEL Repository (SMR) and Phyre2 web portal are a database of annotated 3D protein structure models generated by the automated SWISS-MODEL homology modeling pipeline [56,57] and Phyre2 web portal system [58]. In the 3D-strcuture prediction, the full length RGNNV B2 (1-75 aa) and B2 mitochondrial targeting domain (36-61 aa) alone sequence for comparing from either SWISS-MODEL Repository system or Phyre2 web portal system. Two systems we have found that received very similar results. Then further confirmed the 3D-structure of protein B2 to published alpha-nodavirus protein B2 in dimer form structure [59] that still received the consistent result, have shown the alpha helix structure.

Statistical analysis
All western blot images are representative of at least three independent experiments. The level of ROS production (H 2 DCFDA assay) and percentage of Annexin-V and PI-fluorescein-positive cells was determined by counting 200 cells per sample. Each result is expressed as the mean ± SEM. Data were analyzed using the paired or unpaired Student's t-test, as appropriate. For comparison of group means, a P value less than 0.05 was considered statistically significant.