Histone Potentiates Inositol Hexakisphosphate in Inducing Apoptosis of HONE-1 Nasopharyngeal Cancer Cells

Abstract

Inositol hexakisphosphate (InsP₆) requires relatively high concentrations to induce apoptosis of cancer cells, which can possibly cause apoptosis of normal cells. Anticancer ability of InsP₆ could be preserved by combining with histone, so InsP₆ can be used at low concentrations. The effect of InsP₆ and histone combination has not been investigated on nasopharyngeal cancer cells. The current study elucidated the effect of InsP₆ and its combination with histone on the apoptosis of HONE-1 cells. The most effective concentration and the cellular mechanisms by which this combination exerts its anticancer effects were also investigated. HONE-1 and NIH3T3 cells (as normal control cells) were treated with InsP₆ and/or histone in different concentrations. Apoptosis percentages of the treated cells were measured with sub-G1 assay. Nuclear fragmentation and mitochondrial membrane potential (∆Ψm) reduction in the treated HONE-1 cells were confirmed with 4',6-diamidino-2-phenylindole (DAPI) staining and ∆Ψm assay, respectively. The combination of 10 μM InsP₆ and 10 μg/mL histone had the optimal ability to induce the apoptosis of HONE-1 cells. This combination did not induce apoptosis of NIH3T3 cells. The apoptosis-inducing ability of this combination was higher than that of 10 μM InsP₆ merely. The ability of InsP₆ to induce apoptosis of HONE-1 cells could be enhanced by histone application. Combination of 10 μM InsP₆ and 10 μg/mL histone might be the optimal concentrations for inducing apoptosis in HONE-1 cells.

Keywords:
inositol hexakisphosphate; histone; apoptosis; HONE-1 cells.

HIGHLIGHTS

InsP₆ has the ability to induce apoptosis in HONE-1 nasopharyngeal cancer cells.

Apoptosis-inducing ability of InsP₆ can be enhanced by histone application.

Combination of InsP₆ and histone decreases ∆Ψm in HONE-1 cells.

Combination of InsP₆ and histone induces nuclear fragmentation in HONE-1 cells.

INTRODUCTION

Natural products have recently received a great deal of attention as potential anticancer supplements due to the safety and efficacy of their use compared to chemical-based drugs. One of the natural products that has attracted attention is inositol hexakisphosphate (InsP₆). InsP₆ can be easily found in cereals and legumes as well as mammalian cells together with lower inositol phosphates (InsP_1-5) and inositol [¹]. Extracellular application of InsP₆ has been reported to induce apoptosis of several types of cancer cells, both in vitro [²,³] and in vivo [⁴,⁵].

When entering the target cells, InsP₆ is internalized via endocytosis and further dephosphorylated to InsP_1-5 in lysosomes [⁶]. InsP_1-5 have been known to be involved in signal transduction pathways and directly affect crucial cellular processes in cancer cells [¹]. Despite having potent anticancer activity, InsP₆ itself requires relatively high concentrations (≥75 μM) to exert its effect on some cancer cells [⁷]. High concentration of InsP₆ can possibly cause apoptosis in normal cells as well.

InsP₆ can be used at low concentrations without reducing its effectiveness by neutralizing the negative charges possessed by this substance to enhance its intracellular uptake [⁸,⁹]. This could be achieved by several approaches, including delivery with synthetic cationic oligoproline [⁸] and synthesis of hydrophobic inositol phosphates derivate [⁹]. Other natural delivery systems, such as histone, are also preferred in attempt to mask the negative charges [¹⁰]. By combining with histone, the effective concentration of InsP₆ to induce apoptosis in cervical cancer cells can be reduced ~10 fold [²].

Anticancer substances-induced apoptosis might involve the reduction of mitochondrial membrane potential (∆Ψm) and could result in DNA fragmentation [¹¹]. Alterations in specific apoptotic mechanisms might cause cancer resistance toward the current standard treatments [¹²]. Thus, there is a need to target the altered mechanisms for achieving successful cancer treatment.

InsP₆ potencies have been reported in several cancers, including cervical [²] and colon [³] cancer cells. However, to date none has been reported towards nasopharyngeal cancer (NPC), one of the most common head and neck cancers. NPC is still a burden in certain regions and countries, especially in South-Eastern Asia and China [¹³]. Therefore, the potency of InsP₆ was investigated by using NPC cell model, HONE-1 cell line. NPC cell lines, including HONE-1, could be induced to undergo cell death through apoptosis [¹⁴]. Apoptosis of NPC cells could possibly also be induced by InsP₆ treatment. Up to now, the effect of InsP₆ and histone combination has not been investigated on NPC cells. The current study elucidated the effect of InsP₆ and its combination with histone on the apoptosis of HONE-1 cells. The most effective concentration and the cellular mechanisms by which this combination exerts its anticancer effects were also investigated.

MATERIAL AND METHODS

Cell Culture

NIH3T3 and HONE-1 cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Sigma, St. Louis, MO, USA) and RPMI 1640 medium (Sigma), respectively. These media were added with 10% fetal bovine serum (Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin (Sigma). NIH3T3 and HONE-1 cells were maintained in a humidified, 37°C, 5% CO₂ incubator. Upon reaching 80% confluency, cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Sigma) and sub-cultured.

Sub-G1 Assay

NIH3T3 and HONE-1 cells (5×10⁴ cells) were seeded into 48-well plates and starved for 12 hours. After that, the cells were treated with medium merely, 0.037% H₂O₂, 10 ng/mL TNF-related apoptosis-inducing ligand (TRAIL) (Sigma), InsP₆ (Sigma) and/or histone (Merck, Darmstatdt, Germany) at various concentrations for 48 hours. The percentage of apoptotic cells was evaluated by propidium iodide staining as described previously [¹⁵] using a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ) at 400 events/second.

4',6-diamidino-2-phenylindole (DAPI) Staining

HONE-1 cells (5×10⁴ cells) were distributed onto coverslips for seeding, starved for 12 hours, and treated for 24 hours. Treated HONE-1 cells were washed with phosphate-buffered saline (PBS) and then treated with ice-cold trichloroacetic acid and gradient ethanol for fixation. Fixed HONE-1 cells were permeabilized with a solution containing 0.1% Triton X-100 and 0.1% bovine serum albumin, stained with 1 μg/mL DAPI (Merck, Darmstadt, Germany), washed with PBS, mounted onto glass slides using glycerol, and examined under a fluorescence microscope.

∆Ψm Assay

∆Ψm assay was performed as described previously [¹¹]. HONE-1 cells were starved for 12 hours and treated for 6 hours. The treated cells were subsequently collected, suspended in 20 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC₆) in PBS, and left to incubate for 15 minutes. FACSCanto II flow cytometer was utilized to measure ∆Ψm. Flow cytometry histogram from the untreated group was then merged with the ones from the experimental groups. The shifted histogram area was quantified with ImageJ (National Institute of Health, Bethesda, MD, USA).

Statistical Analyses

Statistical analyses were performed for sub-G1 assay and ∆Ψm assay results. Data was analyzed using SPSS Statistics Version 20.0 (IBM, Armonk, NY, USA). Kruskal-Wallis test followed by Mann-Whitney post-hoc or Welch analysis of variance (ANOVA) followed by Games-Howell post-hoc was conducted to point out the difference of each group. p<0.05 was considered as statistically significant.

RESULTS

Combination of InsP₆ and Histone Did Not Induce Apoptosis in NIH3T3 Cells

As the positive control, treatment with H₂O₂ significantly induced apoptotic NIH3T3 cells than the negative control (untreated) (p<0.05, Mann-Whitney test). When treated with 1-10 μM InsP₆, the percentages of apoptotic NIH3T3 cells were not significantly different as compared with the one untreated. When treated with 100 μM InsP₆, the percentage of apoptotic NIH3T3 cells was significantly higher than the one untreated (p<0.05, Mann-Whitney test) (Figure 1, Table 1). In addition, the apoptosis percentage of 500 μM InsP₆-treated NIH3T3 cells was significantly higher when compared with that of 100 μM InsP₆-treated cells (p<0.05, Mann-Whitney test) (Table 1). Although 100 μM InsP₆ induced the apoptosis of NIH3T3 cells, which is a non-malignant cell, the concentration of 100 μM InsP₆ was still further investigated in cancer cell model, but higher InsP₆ concentration (500 μM InsP₆) was not further investigated due to its high cytotoxicity in normal cells.

Table 1
Mann-Whitney post-hoc statistical analyses of the InsP₆-histone-treated apoptotic NIH3T3 cells.

Treatment	Untreated	H2O2	InsP6 (μM)					Histone (μg/mL)					10 μM InsP6 + Histone (μg/mL)					100 μM InsP6 + Histone (μg/mL)
			1	10	100	500		1	10	100	500		1	10	100	500		1	10	100	500
Untreated	-	*	NS	NS	*	*		NS	NS	NS	NS		NS	NS	NS	NS		*	*	*	*
H2O2	*	-	*	*	*	*		*	*	*	*		*	*	*	*		*	*	*	*
1 μM InsP6	NS	*	-	NS	*	*		NS	NS	NS	NS		NS	NS	NS	NS		*	*	*	*
10 μM InsP6	NS	*	NS	-	*	*		NS	NS	NS	NS		NS	NS	NS	NS		*	*	*	*
100 μM InsP6	*	*	*	*	-	*		*	*	*	*		*	*	*	*		NS	*	*	*
500 μM InsP6	*	*	*	*	*	-		*	*	*	*		*	*	*	*		*	*	NS	NS
1 μg/mL histone	NS	*	NS	NS	*	*		-	NS	NS	NS		NS	NS	NS	NS		*	*	*	*
10 μg/mL histone	NS	*	NS	NS	*	*		NS	-	NS	NS		NS	NS	NS	NS		*	*	*	*
100 μg/mL histone	NS	*	NS	NS	*	*		NS	NS	-	NS		NS	NS	NS	NS		*	*	*	*
500 μg/mL histone	NS	*	NS	NS	*	*		NS	NS	NS	-		NS	NS	NS	NS		*	*	*	*
10 μM InsP6 + 1 μg/mL histone	NS	*	NS	NS	*	*		NS	NS	NS	NS		-	NS	NS	NS		*	*	*	*
10 μM InsP6 + 10 μg/mL histone	NS	*	NS	NS	*	*		NS	NS	NS	NS		NS	-	NS	NS		*	*	*	*
10 μM InsP6 + 100 μg/mL histone	NS	*	NS	NS	*	*		NS	NS	NS	NS		NS	NS	-	NS		*	*	*	*
10 μM InsP6 + 500 μg/mL histone	NS	*	NS	NS	*	*		NS	NS	NS	NS		NS	NS	NS	-		*	*	*	*
100 μM InsP6 + 1 μg/mL histone	*	*	*	*	NS	*		*	*	*	*		*	*	*	*		-	*	*	*
100 μM InsP6 + 10 μg/mL histone	*	*	*	*	*	*		*	*	*	*		*	*	*	*		*	-	*	*
100 μM InsP6 + 100 μg/mL histone	*	*	*	*	*	NS		*	*	*	*		*	*	*	*		*	*	-	NS
100 μM InsP6 + 500 μg/mL histone	*	*	*	*	*	NS		*	*	*	*		*	*	*	*		*	*	NS	-

• NS = not significant;
• ^*
  significant with p<0.05.Histone did not induce apoptosis of NIH3T3 cells at concentration of 1-500 μg/mL, as indicated by the apoptosis percentages that were not significantly different when compared with that of untreated NIH3T3 cells. The combination of 1-500 μg/mL histone + 10 μM InsP₆ also did not induce apoptosis of NIH3T3 cells (Figure 1, Table 1). However, when 1-500 μg/mL histone was combined with 100 InsP₆, apoptosis percentages of NIH3T3 cells were significantly higher as compared with combination of 1-500 μg/mL histone + 10 μM InsP₆ (p<0.05, Mann-Whitney test) (Table 1).

Figure 1
Percentage of InsP₆-histone-treated apoptotic NIH3T3 cells. NIH3T3 cells were treated with InsP₆ and/or histone in different concentrations and analyzed with sub-G1 assay according to the Material and Methods. Experiments were performed in sixtuplicate. *p<0.05 vs. untreated cells; ^#p<0.05 vs. 100 μM InsP₆-treated cells, Mann-Whitney test.

The apoptosis-inducing ability of 100 μM InsP₆ increased upon histone addition, as demonstrated by higher apoptosis percentages of NIH3T3 cells compared with that of 100 μM InsP₆ merely. As histone concentrations increased, the apoptosis percentages of 100 μM InsP₆-treated NIH3T3 cells were also increased. The combination of 100 μM InsP₆ and histone reached its optimal ability at histone concentration of 100 μg/mL (Figure 1). No further increase of apoptosis percentage was observed after the addition of 500 μg/mL histone (Figure 1), as indicated by the apoptosis percentage of 100 μg/mL histone + 100 μM InsP₆-treated NIH3T3 cells that were not significantly different as compared with that of 500 μg/mL histone + 100 μM InsP₆-treated NIH3T3 cells (Table 1). These data suggested that InsP₆ did not induce apoptosis in normal cells, so that it could be used at low concentration, particularly at <100 μM.

Combination of InsP₆ and Histone Induced Apoptosis in HONE-1 Cells

As for HONE-1 cells, apoptosis percentage of TRAIL-treated cells (positive control) was significantly higher than that of the untreated (negative control) (p<0.05, Mann-Whitney test) (Figure 2, Table 2). InsP₆ treatment induced apoptosis in a concentration-dependent manner (Figure 2). A significant increase of apoptosis percentage was observed starting from concentration of InsP₆ as low as 10 μM (p<0.05, Mann-Whitney test). Meanwhile, histone treatment did not induce apoptosis of HONE-1 cells at all (Figure 2, Table 2).

Table 2
Mann-Whitney post-hoc statistical analyses of the InsP₆-histone-treated apoptotic HONE-1 cells.

Treatment	Untreated	TRAIL	InsP6 (μM)				Histone (μg/mL)					10 μM InsP6 + Histone (μg/mL)					100 μM InsP6 + Histone (μg/mL)
			1	10	100		1	10	100	500		1	10	100	500		1	10
Untreated	-	*	NS	*	*		NS	NS	NS	NS		*	*	*	*		*	*
TRAIL	*	-	*	*	*		*	*	*	*		*	*	*	*		*	*
1 μM InsP6	NS	*	-	*	*		NS	NS	NS	NS		*	*	*	*		*	*
10 μM InsP6	*	*	*	-	*		*	*	*	*		NS	*	*	*		*	*
100 μM InsP6	*	*	*	*	-		*	*	*	*		*	*	*	*		NS	*
1 μg/mL histone	NS	*	NS	*	*		-	NS	NS	NS		*	*	*	*		*	*
10 μg/mL histone	NS	*	NS	*	*		NS	-	NS	NS		*	*	*	*		*	*
100 μg/mL histone	NS	*	NS	*	*		NS	NS	-	NS		*	*	*	*		*	*
500 μg/mL histone	NS	*	NS	*	*		NS	NS	NS	-		*	*	*	*		*	*
10 μM InsP6 + 1 μg/mL histone	*	*	*	NS	*		*	*	*	*		-	*	*	*		*	*
10 μM InsP6 + 10 μg/mL histone	*	*	*	*	*		*	*	*	*		*	-	NS	NS		*	NS
10 μM InsP6 + 100 μg/mL histone	*	*	*	*	*		*	*	*	*		*	NS	-	NS		*	NS
10 μM InsP6 + 500 μg/mL histone	*	*	*	*	*		*	*	*	*		*	NS	NS	-		*	NS
100 μM InsP6 + 1 μg/mL histone	*	*	*	*	NS		*	*	*	*		*	*	*	*		-	*
100 μM InsP6 + 10 μg/mL histone	*	*	*	*	*		*	*	*	*		*	NS	NS	NS		*	-

• NS = not significant;
• ^*
  significant with p<0.05.

Figure 2
Percentage of InsP₆-histone-treated apoptotic HONE-1 cells. HONE-1 cells were treated with InsP₆ and/or histone in different concentrations and analyzed with sub-G1 assay according to the Material and Methods. Experiments were performed in sixtuplicate. *p<0.05 vs. untreated cells; ^$p<0.05 vs. 10 μM InsP₆-treated cells; ^#p<0.05 vs. 100 μM InsP₆-treated cells, Mann-Whitney test.

The apoptosis-inducing ability of 10 μM InsP₆ + 1 μg/mL histone was not significantly different as compared with that of 10 μM InsP₆ merely. By the addition of 10-500 μg/mL histone, the apoptosis-inducing ability of 10 μM InsP₆ was significantly higher compared with that of 10 μM InsP₆ merely (p<0.05, Mann-Whitney test). The combination of 10 μM InsP₆ and histone reached its optimal ability at histone concentration of 10 μg/mL. No further increase of apoptosis percentage was observed after the addition of 100-500 μg/mL histone (Figure 2, Table 2).

The apoptosis-inducing ability of 100 μM InsP₆ + 1 μg/mL histone was not significantly different as compared with that of 100 μM InsP₆ merely. However, by the addition of 10 μg/mL histone, the apoptosis-inducing ability of 100 μM InsP₆ was significantly higher compared with that of 100 μM InsP₆ merely (p<0.05, Mann-Whitney test) (Figure 2, Table 2).

Combination of InsP₆ and Histone Induced Nuclear Fragmentation in HONE-1 Cells

To confirm the results of sub-G1 assay, DAPI staining of HONE-1 cells was performed accordingly. Treatment of HONE-1 cells with 10 μM InsP₆ caused nuclear fragmentation, an indication of apoptosis. Meanwhile, treatment with 10-100 μg/mL histone did not cause nuclear fragmentation. The combination of 10 μM InsP₆ + 10 μg/mL histone appeared to increase the number of HONE-1 cells that underwent nuclear fragmentation as compared with the one of 10 μM InsP₆ merely. A higher number of cells with fragmented nuclei was observed in 10 μM InsP₆ + 100 μg/mL histone-treated HONE-1 cells when compared with that in 10 μM InsP₆ + 10 μg/mL histone-treated HONE-1 cells (Figure 3).

Figure 3
DAPI staining of InsP₆-histone-treated apoptotic HONE-1 cells. HONE-1 cells were treated with InsP₆ and/or histone in different concentrations and analyzed with DAPI staining according to the Material and Methods. Experiments were performed in sixtuplicate. White arrowhead: Fragmented nucleus. White bar: 50 μm.

Combination of InsP₆ and Histone Reduced ∆Ψm in HONE-1 Cells

Upon 10 μM InsP₆ treatment, ∆Ψm of HONE-1 cells slightly shifted to the left side of the histogram, indicating slight ∆Ψm reduction. Meanwhile, no ∆Ψm alterations were observed in histone-treated cells. The reduction of ∆Ψm in 10 μM InsP₆ + 10 μg/mL histone-treated cells were significantly higher than that of 10 μM InsP₆-treated cells (p<0.05, Games-Howell test). Moreover, a significant higher ∆Ψm reduction was observed in 10 μM InsP₆ + 100 μg/mL histone-treated cells when compared with that of 10 μM InsP₆ + 10 μg/mL histone-treated cells (p<0.05, Games-Howell test) (Figure 4).

Figure 4
∆Ψm of InsP₆-histone-treated apoptotic HONE-1 cells. HONE-1 cells were treated with InsP₆ and/or histone in different concentrations and analyzed with ∆Ψm assay according to the Material and Methods. (A) Merged results of flow cytometry histogram; (B) Shifted area of flow cytometry histogram for each experimental group. Experiments were performed in sixtuplicate. The measurement of the shifted area was performed in triplicate. *p<0.05 vs. 10 μM InsP₆-treated cells; ^#p<0.05 vs. 10 μM InsP₆ + 10 μg/mL histone-treated cells, Games-Howell test.

DISCUSSION

The results of the current study showed that InsP₆ induced apoptosis of HONE-1 cells in a concentration-dependent manner. InsP₆ could also induce apoptosis in several types of cancer, including cervical [²] and colorectal cancers [³,¹⁶], as well as hepatocellular carcinoma [¹⁷]. In the current study, a significant increase in HONE-1 cell apoptosis was observed starting from a concentration of 10 μM InsP₆. Furthermore, no cytotoxicity was observed on 10 μM InsP₆-treated NIH3T3 normal cells. InsP₆ has been reported to induce apoptosis in different types of cancer at different minimum concentrations. Minimum InsP₆ concentration as high as 4 mM induces apoptosis in oral squamous cell carcinoma [¹⁸]. Additionally, InsP₆ induces apoptosis in liver cancer cells at concentrations as minimal as 0.25 mM [¹⁹]. Meanwhile, the range of minimum InsP₆ concentration required for inducing apoptosis in melanoma cells is 0.1-0.3 mM [²⁰,²¹].

The current results also showed that the ability of 10 μM InsP₆ to induce apoptosis of HONE-1 cells could be enhanced by combining with histone. The increase in HONE-1 cell apoptosis was evident after the addition of 10 μg/mL histone. However, higher histone concentrations (100-500 μg/mL) did not further increase the apoptosis percentage of HONE-1 cells. In addition, histone, alone or in combination with 10 μM InsP₆, did not induce apoptosis of normal cells. These results suggested that 10 μM InsP₆ + 10 μg/mL histone might be the best combination for inducing apoptosis of HONE-1 cells. Application of histone enhances intracellular uptake of InsP₆ by masking negative charges of phosphate groups of this substance [¹⁰]. An earlier study reported that histone lowered the effective concentration to induce apoptosis of cervical cancer cells [²].

Upon endocytosis, internalized InsP₆ does not directly exert its anticancer effects, but first enters the lysosomes, where it is subsequently dephosphorylated to InsP_1-5 and inositol [⁶]. These dephosphorylation products might mediate the activation of apoptotic cascades [⁷,²²] that trigger ∆Ψm reduction and the subsequent DNA fragmentation [²³]. In the current study, DAPI staining results showed that InsP₆ triggered nuclear fragmentation in HONE-1 cells. InsP₆ could also induce DNA fragmentation in hepatocellular carcinoma [¹⁷], prostate cancer [²⁴], and glioblastoma [²⁵]. Furthermore, histone increased InsP₆-induced nuclear fragmentation in HONE-1 cells in a concentration-dependent manner. Aligned with DAPI staining results, InsP₆ merely induced slight ∆Ψm reduction in HONE-1 cells and the addition of histone increased InsP₆-induced ∆Ψm reduction in a concentration-dependent manner. Reduction of ∆Ψm is one of the crucial steps in the intrinsic apoptotic pathway. This phenomenon leads to the increase of mitochondrial membrane permeabilization, which in turn causes the release of apoptogenic factors and the activation of downstream effector caspases [²³].

After 6-24 hours, nuclear fragmentation and ∆Ψm reduction were higher in 10 μM InsP₆ + 100 μg/mL histone-treated HONE-1 cells compared with those treated with 10 μM InsP₆ + 10 μg/mL histone. However, after 48 hours, the apoptotic percentages between 10 μM InsP₆ + 10 μg/mL histoneand 10 μM InsP₆ + 100 μg/mL histone-treated HONE-1 cells were similar. These discrepancies might be caused by the differences in treatment period between each examination and may relate to the sequence of events that occur during apoptosis process. The combination of 10 μM InsP₆ + 100 μg/mL histone might be more effective to induce events preceding to the cell death, including ∆Ψm reduction and initiation of nuclear fragmentation, than the one of 10 μM InsP₆ + 10 μg/mL histone. However, these combinations might have a similar effectiveness to induce HONE-1 cell death, the results of those events.

The findings of the current study provide novel understanding into the mechanisms of InsP₆ and histone combination in exerting its anticancer properties toward HONE-1 cells. The signaling cascade by which InsP₆ and histone combination causes ∆Ψm reduction and nuclear fragmentation needs to be further investigated.

CONCLUSION

InsP₆ has the ability to induce apoptosis in HONE-1 cells through ∆Ψm reduction, and this ability could be enhanced by histone application. Combination of 10 μM InsP₆ (low concentration) and 10 μg/mL histone might be the optimal concentrations for inducing apoptosis in HONE-1 cells. The combination of 10 μM InsP₆ and 10 μg/mL histone did not induce apoptosis in NIH3T3 cells, hence it might be safe for normal cell.

• Funding:
  This research received no external funding.

Acknowledgments:

The authors would like to thank to Prodia StemCell Indonesia Laboratory for facility and technical support.

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• Editor-in-Chief: Paulo Vitor Farago

• Associate Editor: Fábio André dos Santos

Publication Dates

• Publication in this collection
  31 Mar 2025
• Date of issue
  2025

History

• Received
  01 Feb 2024
• Accepted
  02 Nov 2024