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Pentoxifylline Enhances the Apoptotic Effect of Carboplatin in Y79 Retinoblastoma Cells

Background/Aim: Retinoblastoma (RB) is the most common primary intraocular malignancy. Carboplatin (CPt) is a DNA damage-inducing agent that is widely used for the treatment of RB. Unfortunately, this drug also activates the transcription factor nuclear factor-kappa B (NF-ĸB), leading to promotion of tumor survival. Pentoxifylline (PTX) is a drug that inhibits the phosphorylation of I kappa B-alpha (IĸBα) in serines 32 and 36, and this disrupts NF-ĸB activity that promotes tumor survival. The goal of this study was to evaluate the effect of the PTX on the antitumor activity of CPt. Materials and Methods: Y79 RB cells were treated with CPt, PTX, or both. Cell viability, apoptosis, loss of mitochondrial membrane potential, the activity of caspase-9, -8, and -3, cytochrome c release, cell-cycle progression, p53, and phosphorylation of IĸBα, and pro- and anti-apoptotic genes were evaluated. Results: Both drugs significantly affected the viability of the Y79 RB cells in a time- and dose-dependent manner. The PTX+CPt combination exhibited the highest rate of apoptosis, a decrease in cell viability and significant caspase activation, as well as loss of mitochondrial membrane potential, release of cytochrome c, and increased p53 protein levels. Cells treated with PTX alone displayed decreased I kappa B-alpha phosphorylation, compared to the CPt treated group. In addition, the PTX+CPt combination treatment induced up-regulation of the proapoptotic genes Bax, Bad, Bak, and caspases- 3, -8, and -9, compared to the CPt and PTX individual treated groups. Conclusion: PTX induces apoptosis per se and increases the CPt-induced apoptosis, augmenting its antitumor effectiveness.

Cell viability. Y79 cells were seeded in 96-well plates (2×10 4 cells/well) and were treated with RPMI-S, PTX, CPt or PTX+CPt for 24 h. Cell viability was assessed using the cell proliferation reagent WST-1 (Commercial Kit, Sigma Aldrich), according to the manufacturer's instructions. After incubation in WST-1, an electron coupling reagent (ECS) was added, and the Y79 cells were incubated for another 3 h. Absorbance was measured in a microtiter plate reader (Synergy™ HT Multi-Mode Microplate Reader; Biotek, Winooski, VT, USA) at 450 nm with a reading reference wavelength at 690 nm. Data are reported as a percentage of cell viability compared to the respective percentage in UCG cells considered as 100%.
Assessment of Annexin-V, mitochondrial membrane potential loss (ΔΨm), and DNA fragmentation. Y79 cells seeded in 6-well plates were treated with the appropriate drug, drug combination, or medium (control) for 24 h; apoptosis was evaluated by different methods.
Early detection of apoptosis was performed using the Annexin-V-FLUOS staining Kit (Sigma Aldrich, St Louis, MO, USA) according to the manufacturer's protocol. Briefly, 1×10 6 cells were collected and resuspended in 500 μl 1x Annexin-V binding buffer. Afterward, cells were incubated with FITC-conjugated Annexin-V FLUOS for 15 min and were analyzed by flow cytometry. For mitochondrial membrane potential assays, 1×10 6 cells/ml were collected and stained for 20min with MitoCapture™ staining solution (MitoCapture™ Mitochondrial Apoptosis Detection Kit, BioVision Research, Mountain View, CA, USA) followed by two washes with in vivo 33: 401-412 (2019) PBS prior to analysis by flow cytometry. As an internal positive control for the ΔΨm loss, cells were treated for 4 h with 150 μM of protonophore Carbonyl Cyanide m-ChloroPhenylhydrazone (CCCP, Sigma Aldrich), which induces mitochondrial depolarization (26). The percentage of cells with ΔΨm loss was analyzed by flow cytometry using an Attune™ flow cytometer (Life Technologies, Carlsbad, CA, USA); at least 20,000 events were acquired for each sample and were analyzed using Attune software version 2.1 (Life Technologies). Results are represented as the percentage of Annexin-V and ΔΨm loss.
Apoptotic DNA fragmentation is a crucial feature of apoptosis (27); for this reason, internucleosomal DNA fragmentation was quantitatively assayed by antibody-mediated capture and detection of cytoplasmic mononucleosome-and-oligonucleosome-associated histone-DNA complexes (Cell Death Detection ELISA PLUS Kit; Sigma Aldrich). Briefly, Y79 cells were cultured in 96-well plates at 2×10 4 cells/well and treated with CPt 30 μg/ml, PTX 4 mM, or combined PTX 4 mM+CPt 30 μg/ml, for 24 h. The cell culture supernatants were removed, then the cells were resuspended in 200 μl of lysis buffer™ and lysed directly in the well, centrifugated (1,200 rpm, 10 min), and 20 μl of the cytoplasmic fraction was used to determinate DNA fragmentation according to the manufacturer's standard protocol. Subsequently, absorbance was measured in a microplate reader (Synergy™ HT Multi-Mode Microplate Reader; Biotek, Winooski, VT, USA) at 405 nm. In the DNA fragmentation test, the rate of apoptosis is reflected by the enrichment (fold increase) of mono-and oligonucleosomes accumulated in the cytoplasm and was calculated according to the following formula:

Rate of Apoptosis=Absorbance of Sample cells/Absorbance of Control cells.
Pan-caspase activity by flow cytometry and colorimetric assay for determining the specific caspase -9, -8, and-3 activity. Determination of activated caspases was performed using the Generic Caspase Activity Assay FITC staining kit for use in flow cytometry (Abcam, Cambridge, UK). This assay can detect activated caspases-1, -3, -4, -5, -6, -7, -8, and -9 in cells undergoing apoptosis. After seeded in 6-well plated and treated with the appropriate drug, drug combination or medium (control) for 24 h, cells were incubated with 1 μl/ml of 500× TF2-VAD-FMK for 1 h at 37˚C in 5% CO 2 . Finally, cells (1×10 6 cells) were harvested, washed twice with PBS, and were resuspended in PBS before being analyzed by flow cytometry. Flow cytometry experiments were carried out as before.
In a second step, caspase-9, -8, and -3 activities were determined using colorimetric kits (Abcam, Cambridge, UK). Y79 cells (10×10 6 cells) were treated for 24 h with the set of drugs mentioned previously. Subsequently, the cells were washed twice with PBS, were resuspended in 300 μl of cell lysis buffer™ containing protease inhibitor (cOmplete™, Mini, EDTA-Free, Roche-Sigma Aldrich) and were incubated on ice for 10 min. Crude lysates were centrifuged for 10 min at 12,000 rpm. Protein concentrations were determined by the Bradford assay using the Dc Protein Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's instructions. Finally, absorbance was measured in a microplate reader (Synergy™ HT Multi-Mode Microplate Reader; Biotek, Winooski, VT, USA) at 405 nm. Results are represented as a percentage of pancaspase activity, and the mean±standard deviation of the optical density (OD) values obtained in each group. Protein extraction for Cytochrome C level measurement. Cytochrome C (CytC) was determined using the Cytochrome C Profiling ELISA kit (Abcam, Cambridge, UK). Y79 cells (10×10 6 cells) were treated with CPt, PTX, or both drugs for 24 h. The cells were then harvested, washed twice with PBS, and were resuspended in lysis buffer (Standard Cell Fractionation buffer, Abcam, Cambridge, UK) containing a cocktail of protein inhibitors (cOmplete™, Mini, EDTA-Free, Roche-Sigma Aldrich), according to the manufacturer's instructions, so as to obtain the cytosolic and mitochondrial fractions. Protein concentrations were determined by the Bradford assay as before. For CytC profiling of subcellular fractions, equal amount of protein (60 μg for cytosolic or mitochondrial fractions) from each sample was added and was analyzed according to the manufacturer's instructions. The absorbance of cytosolic fractions and mitochondrial were measured at 660 nm using a plate, and the values for each standard concentration were plotted on the vertical (Y) axis versus the corresponding CytC concentration on the horizontal (X) axis, using the Gen5™ software (BioTek Instruments, Inc., Winooski, VT, USA). The results are expressed in μg/ml.

Cell-cycle distribution analysis.
For the cell-cycle analysis, the Y79 cells were initially synchronized. In brief, cells were cultured in RPMI-1640 containing 5% FBS for 12 h. Subsequently, the cells were washed and cultured in RPMI-1640 containing 1% FBS. After incubation in this medium overnight, the cells were washed with PBS and were incubated in serum-free medium for 18 h. Finally, the cells were split and were released into cell cycle by the addition of 15% FBS in RPMI-1640 culture medium. A total of 2×10 6 cells were treated with each drug alone or with both drugs for 24 h. The BD Cycletest™ Plus DNA Reagent kit was used according to the manufacturer's instructions (BD Biosciences, San Jose, CA, USA). DNA quality control (QC) Particles (BD Biosciences) were used for verification of the instrument performance and quality control of the Attune™ flow cytometer used for DNA analysis. For each sample, at least 30,000 events were acquired, and data were processed with Kaluza v2.0 software (Beckman Coulter) (18).
Detection of total-IĸBa and IĸBα (pS32/36) proteins by ELISA. Y79 cells (10×10 6 ) were treated with CPt, PTX, and PTX+CPt for 24 h. After treatment, cells were harvested, and cell protein extracts were prepared by lysing the cells using buffer lysis™ provided in the Cell Extraction/Enhancer Buffer PTR kit (Abcam, Cambridge, UK), according to the manufacturer's instructions. Protein concentrations were determined by the Bradford assay as before. Total and phosphorylated I-kappa B-alpha (IĸBα) protein levels from the cell extracts were measured using the IĸBα (pS32/36)+Total IĸBα SimpleStep ELISA kit (Abcam, Cambridge, UK), according to the manufacturer's protocol. Equal amounts of protein (50 μg) were used in each experiment, and the absorbance (OD) was measured at 450 nm with a microtiter plate reader. The values of absorbance at 450 nm minus the values for each standard concentration were plotted on the vertical (Y) axis versus the corresponding IĸBα (pS32/36) and Total IĸBα concentrations on the horizontal (X) axis using the Gen5™ software. The results are expressed in μg/ml.

RNA extraction and quantitative real time-PCR.
Total RNA of Y79 cells (10×10 6 cell) was obtained after 3 h of incubating the cells with the different treatments, using the column-based RNA purification kit (GeneJET™ RNA purification kit, Thermo Scientific Waltham, MA, USA), following the manufacturer' s instructions, The cDNA was synthesized starting from 5 μg of total RNA using the Transcript First Strand cDNA (Roche). Real-Time PCR was carried out with the System Light Cycler ® 2.0 apparatus (Roche Applied Science, Mannheim, Germany), using DNA Master plus SYBR Green I (Roche Applied Science). The final concentration of primers in a real time-PCR reaction was 5 pg. The PCR program consisted of an initial 10 min step at 95˚C, and 40 cycles of 15-sec at 95˚C, 5-sec at 60˚C, and 15-sec cycles at 72˚C. Analysis of PCR products was performed using LightCycler ® software (Roche Applied Science). Data are expressed as relative normalized quantities employing L32 Ribosomal Protein (RPL32) gene expression to verify the specificity of the amplified reaction, which was nearly 100%. Each sample was processed in triplicates to verify the specificity of the amplification reaction. The oligonucleotides (Invitrogen Corporation, Carlsbad, CA, USA) used to amplify human Bad, Bak, Bax, caspase-3, -8, and -9, Bcl-XL, Bcl-2, and RPL32 are shown in Table I. The oligonucleotides were designed using Oligo software (version 6). Gene sequences were obtained from the GenBank Nucleotide Database of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) (19).
Statistical analysis. All experimental procedures were performed in triplicates and were repeated three times. The values represent the mean±standard deviation from the actual values obtained. Statistical analysis was performed using the non-parametric Mann-Whitney U test to compare two groups. Considering values of p<0.05 as significant. In some experiments, we calculated the Δ%, which represents the percentage of increase or decrease in relation to the corresponding UCG. For the determination of significant variation in gene expression levels, this was set at a fold-change≥1.3 between the experimental and control groups.  the CPt or PTX alone groups, and a significant cytotoxic effect was observed after 24 h of incubation with 30 μg/ml of CPt or PTX 4 mM (45% inhibition of cell viability, p<0.05) in comparison to UCG ( Figure 1A and B). Cell viability diminished progressively after 48 h. These results show that the CPt and PTX alone exhibit important antitumoral activity. We then performed experiments to verify whether the combination of the two drugs at doses close to IC 50 may be more efficient than the CPt alone. Figure 1C illustrates that the combined treatment with PTX (4 mM)+CPt ( activities of caspase-9, -8, and -3 in the different experimental groups were further evaluated ( Figure 2B). Analysis revealed that the combination treatment with PTX+CPt significantly increased caspase-9, and -3 activity (Δ%=42.1% and Δ%=30.9%, respectively; p<0.001 compared to the UCG). Caspase-8 activity also exhibited significant changes following treatment with PTX alone and CPt alone (Δ%=48.1%, Δ%=54.3%, respectively; p<0.007, compared to UCG), while the highest change was observed in the PTX+CPt-treated group (Δ%=79.0%, p<0.007) compared to the UCG. Taken together, these results suggest that in treated groups, caspase-9, -3 and -8 play an important role in the apoptosis observed in Y79 cells.

Dose-and time-dependent effects of CPt and PTX treatments
PTX+CPt increased CytC release. The release of CytC in cells treated with PTX, CPt or with both drugs combined was determined, as this comprises a central event in apoptosis induced by the mitochondrial/caspase-9 pathway. As shown in Figure 3A, the cells treated with PTX or the PTX+CPt displayed decreased levels of mitochondrial CytC release (p<0.05) in comparison with the levels in UCG (Δ%=-23.7% and Δ%=-25.4% respectively). Therefore, the PTX and the PTX+CPt groups demonstrated important increase of cytosolic CytC with a Δ%=1,250% and Δ%=2,350%, respectively (p<0.001) ( Figure 3B), compared to the UCG. No significant changes were observed in the CPt-treated group. These results reveal the role of CytC in the induction of cell death by PTX alone or when combined with CPt and suggest that mitochondria play an important role in the CytC-induced apoptosis.   Effect of PTX on Y79 cell-cycle progression. We were next interested in elucidating whether the aforementioned treatments modulate the cell cycle progression in Y79 cancer cells. The cells were, as previously, treated with PTX or CPt alone or their combination for 24 h, and subsequently, the cell cycle was determined by flow cytometry. As shown in Figure 4, 61.8% of UCG cells were in G 1 phase, while the cells in the PTX and PTX+CPt treated groups exhibited similar G 1 levels, with a variation at Δ%=4.6% and Δ%=-2.2% respectively. Interestingly, 54.5%, of cells   Determination of total-IĸBα and phosphorylated IĸBα (pIĸBα) protein by ELISA. At 24 h post-treatment with the aftermentioned drugs, the total-IĸBα was quantified in Y79 cells.

. Pentoxifylline (PTX) alone and combined with carboplatin (CPt) decrease mitochondrial and increase cytosolic levels of cytochrome C (CytC) in Y79 cells. Mitochondrial (A) and cytosolic (B) CytC levels in Y79 cells treated with PTX, CPt or PTX and
In Figure 5A, an important increase of total Iĸβα was observed in the groups treated with PTX and PTX+CPt (105.0±1.0 μg/ml and 90.1±2.1 μg/ml, respectively). These values represent an increment of Δ%=184.86% and Δ%=144.49%, respectively in comparison with the UCG (p<0.001) and CPttreated group (p<0.001). In contrast, the pIĸBα increased after the CPt-treatment Δ%=221.02% (p<0.05) compared to the untreated group ( Figure 5B). In the case of PTX or PTX+CPt groups, a reduction of the pIĸBα was observed; Δ%=-56.45% and Δ%=-45.16%, respectively (p<0.05) compared to CPt treated group. These experiments strongly suggest that the PTX treatment decrease phosphorylation of the IĸBα induced by CPt, and confirm precedents observations (19).
PTX+CPt increase p53 protein in Y79 cells. Western blotting analyses allowed us to assess the effects of CPt, PTX, and PTX+CPt on the expression of the p53 protein. Figure 6 shows that untreated Y79 cells express the p53 protein constitutively. However, treatment with CPt increased the level of p53 expression in a time-dependent response after 24 h by 1.70-fold and at 48 h by 2.5-fold (p<0.05) compared to the PTX group and UCG. On the contrary, PTX treatment of Y79 cells resulted in slight increased the p53 levels at 24 h by 1.15-fold as compared to the UCG. When the two drugs were combined (PTX+CPt), the levels of p53 after 24 h increased by 1.5-fold, compared to the UCG (p<0.05). These results further support the role of these two drugs in increasing the levels of apoptosis in Y79 cells.
Changes in the expression of proapoptotic and anti-apoptoticrelated genes. Real-time PCR was employed to determine the relative changes in the expression of apoptosis-related genes mRNA (Figure 7). Upregulation or downregulation of gene expression was considered when fold-change in mRNA levels compared to the UCG was ≥1.3. PCR assay revealed that CPt treatment did not cause any significant changes in the expression levels of the examined genes. On the contrary, when Y79 RB cells were treated with PTX, mRNA levels of Bak, Bax, Bad, caspase-9, caspase-8, and caspase-3 were significantly increased; however, fold-change was <1.3. When Y79 RB cells were treated with the combination of PTX+CPt, the proapoptotic genes Bax, Bad, and caspase 8 had significantly increased mRNA levels (p<0.05; fold change <1.3), while the highest increase was observed in Bak, caspase 9, and caspase 3 expression (3.0-, 2.5-, and 1.8-fold change, respectively; p<0.05). In general, the data obtained suggested that only the PTX+CPt treatment of Y79 RB cells favored the activation of genes with proapoptotic activity.

Discussion
Our study has focused on assessing the ability of PTX, CPt, and their combination to induce cell death in Y79 retinoblastoma cells. The Y79 RB cell line represents one of   the most useful in vitro model systems for investigating human retinoblastoma (28). The potential of PTX as an antitumor agent is great, and successful combinations of PTX with antitumor treatments, including Adriamycin, Cisplatin, MG132, Perillyl alcohol, and radiotherapy produce a synergistic more robust antitumoral response (16,17,19,29). In this study, we observed that the PTX and the CPt treatments alone exhibited an important antitumoral activity per se in the Y79 RB cells; this effect was stronger when the drugs were combined, suggesting a possible synergistic effect. This was supported by the evaluation of cell viability and apoptosis, and DNA fragmentation assays.
In response to DNA damage, cell cycle arrest in G 1 phase allows cells to undergo apoptosis instead of progressing to S phase (30). Interestingly, in our study, the used treatments caused significant changes in the control of Y79 cell cycle. The highest amount of cells in S phase was under the effect of CPt, whereas slightly fewer cells were in S phase after treatment with PTX+CPt. It has been previously reported that CPt induces cell-cycle arrest in S-phase and directs tumor cells towards to senescence and drug resistance (31). These results are in agreement with our previously reported evidence that treatment of U937 cells with PTX alone or in combination with the proteasome inhibitor MG132 induces cell arrest in G 1 phase (18). Moreover, it has been demonstrated that PTX induces cell cycle arrest at G 0 /G 1 phase and apoptosis in HepG2 cells (32). Therefore, it is possible that the increment in the percentages of Y79 cells in G 1 phase may be induced by PTX. These observations suggest different mechanisms action of the two drugs in the cell cycle progression.
By combining different assays, we observed that the disruption of ΔΨm by PTX or PTX+CPt appears to precede the activation of caspase-3, -8 and -9. Following the apoptosis-induced permeabilization of the mitochondrial membrane and CytC release, other proapoptotic proteins are released into the cytosol. Stronger caspase-8 activation was observed when Y79 cells were treated with PTX and CPt alone or with the combination of the two drugs. A similar report has shown that the use of PTX alone or in combination with other chemotherapeutic agents induces the activation of caspase-8 in cervical tumor cells (19). CPt exhibited a different behavior from that of PTX, as it did not induce a ΔΨm loss or CytC release. These results provide evidence that CPt has a different function from PTX.
In RB cells, it has been observed that involvement of the p53 can mediate cell death (33,34). In the present study, the magnitude and kinetics of the changes in p53 caused by CPt alone were quite different from that caused by PTX alone or by the combined actions of PTX and CPt. The accumulation of p53 in cells incubated with CPt for 24-48 h was considerably increased as compared to the PTX, PTX+CPt treated groups and UCG. It has been well documented that cisplatin and CPt increase p53 levels and facilitate the apoptotic response in these cells (35). Contrary to the CPt, treatment with PTX alone did not change p53 levels in Y79 RB cells. In agreement with this finding, other investigators have reported that PTX sensitizes and enhanced antitumor effect of cisplatin in tumor cells independently of p53 status (19,36). In addition, we previously showed that PTX sensitizes p53-null cells to perillyl alcohol-induced apoptosis (17). Taken together, these data suggest that PTX and CPt induce apoptosis in retinoblastoma Y79 cells via different mechanisms.
On the other hand, NF-ĸB is constitutively activated in many types of cancer and can prevent the apoptosis induced by antitumor drugs (37). It is well documented that CPt can induce NF-ĸB activation in cervical and ovarian cancer (38). In addition, NF-ĸB plays a fundamental role in the survival of tumor cells and occasionally confers significant resistance of cancer cells to the different antitumoral therapies (39). We found a great increase in the levels of IĸBα phosphorylation in Y79 cells treated with CPt compared to UCG (p<0.05), which, in turn, translates to an increased activity of NF-ĸB. At the same time, PTX prevented the activation of NF-ĸB by avoiding the breakdown of its inhibitory molecule, IĸBα. When PTX plus CPt was used, the IĸBα phosphorylation was decreased compared to the CPt-alone treatment. This suggested that diminution of phosphorylation of IĸBα keeps NF-ĸB inactive in the cytoplasm, thus inhibiting tumor resistance and survival of these cancer cells. Our findings are in agreement with observations from other groups, who have reported that inhibition of the translocation of NF-ĸB to the nucleus significantly reduces retinoblastoma-cell invasion, migration, and viability, and decreases the resistance of tumor cells to different chemotherapy drugs (37).
Tumor cells develop resistance to apoptosis through multiple mechanisms, including the expression of antiapoptotic genes (10). NF-ĸB activation induces the modulation of several genes related to inhibition of apoptosis. Our results demonstrate that the Y79 cells exposed to both PTX and CPt show an up-regulation of proapoptotic genes (Bak, Bax, Bad, caspases-9 -8 and -3). Among the proapoptotic genes analyzed, we found that the highest upregulation occurred in the Bak and caspase-9 genes (18). In contraposition, PTX alone induced the overexpression of Bak, caspase-3 and -9 genes. The latter suggests that there is a gene balance that favors apoptosis in the PTX-treated cells, and differences in the expression of these genes strongly suggest alternative mechanisms of gene regulation. Taken together, our results point to a general mechanism of apoptosis in retinoblastoma cells induced by PTX or PTX and CPt combined, that involves parallel or overlapping cascades of events that lead to the disruption of NF-ĸB, cellcycle arrest, and caspase activation with consequent apoptotic cell death. All these data demonstrate that PTX in combination with CPt can favor a proapoptotic machinery in in vivo 33: 401-412 (2019) Y79 retinoblastoma cells, further supporting previous observations from our group (7). Importantly, PTX is a strong inhibitor of PDE activity with PDE1 and PDE4 expression predominating in Y79 cells. It may be possible that PTX may exhibit its proapoptotic activity in these cells through inhibition of PDE 4, as it has been reported to be the case in other types of cells (40).
Even though PTX is experimentally used to sensitize tumor cells to chemotherapy and can exhibit similar or, in some cases, better efficacy against tumor cell survival, it is not classified as an antitumor drug. In our study, we generated a considerable amount of in vitro evidence of the antitumoral role of PTX, similar to other studies performed in lymphoblastic-leukemia and cervical cancer cells (20,23,24). The importance of this lies in the necessity to find chemotherapeutic agents with fewer adverse effects and robust antitumoral activity. With more than 30 years of clinical use and fewer adverse effects, PTX (41,42) can provide protection against the adverse symptoms from chemotherapy (43,44). In addition, PTX is not as expensive as other antitumor drugs, and it displays excellent tolerance even in children with cancer (20,21). Due to these characteristics, PTX may represent a new alternative for the treatment of retinoblastoma. Thus, it would be interesting to include more specific studies on the function of PTX and evaluate it as an antitumoral drug and not only as an adjuvant agent.

Conclusion
PTX and CPt showed antitumor activity in the in vitro model with human Y79 RB cells, while the two drugs combined had an additive effect on apoptosis. These findings suggest that the combination of PTX and CPt may comprise a promising strategy for the treatment of retinoblastoma tumors.

Conflicts of Interests
The Authors declare that they have no conflicting interests.