Dihydropyrimidines (DPs), one of the six-membered heterocyclic compounds, are well known to have a wide range of pharmacological activities including anticancer. Recently, our previous studies have demonstrated that 6-unsubstituted 2-thioxo-, 2-oxo-, and 2-amino DPs, and 4,4-disubstituted 3,4-dihydropyrimidin-2(1H)-ones and -thiones induced cytotoxicity against human promyelocytic leukemia cell line, HL-60, suggesting that DPs can serve as promising candidates for anticancer treatment. In the current study, the effects of novel 2-methylthio DPs, benzyl 4,4,6-trimethyl-2-methylthio-1,4-dihydropyrimidine-5-carboxylate (DP03) and ethyl 6-methyl-2-methylthio-1,4-dihydropyrimidine-5-carboxylate (DP09) were investigated by focusing on cell viability in human leukemia cell lines, NB4 and HL-60 cells together with other seventeen types of novel 2-methylthio DPs. Our study demonstrated treatment with DP03 at the low concentration for 96 h resulted in a significant decrease in cell viability of NB4 rather than HL-60 cells, whereas treatment with DP09 under the same condition was diametrically opposed to NB4 and HL-60 cells. Whether cell differentiation, cell cycle arrest, and cell death are involved in the DP03-induced cytotoxicity of NB4 cells was further investigated. Instead of necrosis, differentiation, and cell cycle arrest, apoptosis induction was observed in DP03-treated NB4 cells. DP03-triggered apoptosis was accompanied by the activation of caspase-8, -9, and − 3 but not caspase-12, and DP03-induced apoptosis was significantly inhibited by a pan-caspase inhibitor, but not the specific inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, janus kinase/signal transducer and activator of transcription. Their finding suggests that induction of apoptosis associated with the activation of caspase-8, -9, and − 3 contributed to the cytotoxicity of DP03 against NB4 cells.
Research Article
Cytotoxic effects of novel 2-methylthio dihydropyrimidines on human acute myeloid leukemia cell lines
https://doi.org/10.21203/rs.3.rs-2071332/v1
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Dihydropyrimidine
Apoptosis
Caspase signaling pathway
NB4 cells
HL-60 cells
Targeting apoptosis has been recognized as a mainstay and goal of clinical cancer therapy because of a strong link between the dysregulation of apoptosis and cancer development[1, 2]. In addition, differentiation-induction in cancer cells with the irreversible loss of tumor phenotype is currently being applied clinically beyond basic research since the induction of differentiation is generally associated with a loss of proliferative capacity, and cell death regularly accompanies the latter stages of cell maturation[3, 4]. Indeed, differentiation therapy using the combination of retinoic acid (RA) and arsenic results in highly cure against acute promyelocytic leukemia (APL)[3, 5, 6]. Sustention of proliferation signaling, avoidance of growth suppression, and resistance to cell death are the hallmarks of cancer cells[7], and continuous efforts to exploit efficient therapeutic approaches including induction of apoptosis and differentiation selected with a focus on the hallmarks of cancer are ongoing.
Pyridines, pyrimidines, dihydropyrimidinones, dihydropyrimidines (DPs), and their analogs are six-membered heterocyclic compounds and have a wide range of pharmacological activity, such as calcium channel inhibition, anticancer, antiviral, antibacterial, antifungal, antimicrobial, anti-HIV, antimalarial, anti-inflammatory, antihypertensive, and antioxidation activities, ATP-binding cassette transporters inhibition[8–11]. Especially, 5-fluorouracil, a typical pyrimidine compound, is one of the most effective chemotherapeutic agents and leads to DNA damage, S-phase arrest, and apoptosis against cancer cells[12]. Our previous studies have focused on the anticancer activity and safety of natural products including food, traditional herb, kampo, and their phytochemicals[13–17]. Recently, our previous studies also have demonstrated that 6-unsubstituted 2-thioxo-, 2-oxo-, and 2-amino DPs, and 4,4-disubstituted 3,4-dihydropyrimidine-2(1H)-ones and -thiones induced cytotoxicity against a human promyelocytic leukemia cell line, HL-60[18–20], suggesting that DPs might be candidates for anticancer compounds. But the detailed studies of whether 2-methylthio DPs induce cell growth inhibition, cell differentiation, and cell death have not been elucidated.
APL is the uncommon subtype of acute myeloid leukemia (AML) classified according to the French-American-British classification system as AML-M3[6, 21]. A typical APL cell line, NB4, is characterized by harboring the t(15;17) translocation leading to the expression of the fusion protein promyelocytic leukemia-retinoic acid receptor α (PML-RARα), resulting in myeloid differentiation arrest and anti-apoptosis[22, 23]. In addition, it has been considered that mutation of p53 protein in NB4 cells also contributed to anti-apoptosis[24]. Therefore, NB4 cells have been widely used for not only the test of cytotoxicity but also the experiments inducing differentiation and apoptosis along with HL-60 cells[25–28].
In the current study, the effects of novel 2-methylthio DPs, benzyl 4,4,6-trimethyl-2-methylthio-1,4-dihydropyrimidine-5-carboxylate (DP03) and ethyl 6-methyl-2-methylthio-1,4-dihydropyrimidine-5-carboxylate (DP09) (Fig. 1A) were investigated by focusing on cell viability against NB4 and HL-60 cells together with other seventeen types of novel 2-methylthio DPs (supplementary Fig. 1). Furthermore, the induction of cell differentiation, cell cycle arrest, and cell death was also investigated in DP03-treated NB4 cells. To clarify the detail mechanisms in DP03-mediated cytotoxic effects in NB4 cells, the involvement of caspase signaling pathway, mitogen-activated protein kinase (MAP kinase) pathways, phosphatidylinositol 3-kinase (PI3 kinase) pathway, and janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was investigated using each inhibitor.
Materials
5-Methylphenazinium Methyl Sulfate (PMS), Penicillin-Streptomycin Solution (×100) (combination of 10,000 U/ml penicillin and 10,000 μg/ml streptomycin), 0.25w/v% Trypsin-1mmol/L EDTA･4Na Solution (Trypsin-EDTA solution), ATRA, and RPMI-1640 were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Fetal bovine serum (FBS) was obtained from Nichirei Biosciences (Tokyo, Japan). Hoechst 33342 and Cell Counting Kit-8 (WST-8 assay kit) were purchased from Dojindo Laboratories (Kumamoto, Japan). 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), LY 294002 and PD 98059 were purchased from Sigma-Aldrich (MO, USA), SP 600125 and Tyrphostin AG 490 (Tyr AG 490) were obtained from Tokyo Chemical Industry (Tokyo, Japan). SB 203580 (Cat. # 559389) was purchased from Merck. Boc-D-FMK was obtained from ChemScene (NJ, USA). Each Caspases-8, 9, 3, and 12 fluorometric assay kit was purchased from BioVision (Milpitas, CA, USA). Annexin V FITC Assay Kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). PE anti-mouse/human CD-11b and PE Rat IgG2b, κ Isotype Ctrl antibodies were purchased from BioLegend (San Diego, CA, USA).
Synthesis of novel 2-methylthio DPs
Following the literature procedures[29, 30], DP03 and DP09 were prepared. The synthetic procedure for other DPs was shown in Supplementary data.
Culture and treatment
NB4 cells, a human APL cell line with t(15;17), were obtained from Deutsche Sammalung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). HL-60 cells, a human cell line derived from promyelocytic leukemia cells, were obtained from the RIKEN BRC through the National Bio-Resource Project of the MEXT (Resource No. RBRC-RCB3683, Tsukuba, Japan). Their cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% Penicillin-Streptomycin Solution (×100) at 37°C in a humidified atmosphere (5% CO2 in air).
NB4 or HL-60 cells were seeded at a density of 1×105 cells/mL and treated with 0.5% DMSO as a vehicle, various concentrations (2.5, 5.0, 7.5, 10, 25, or 50 μM, respectively) of 19 types of 2-methylthio DPs or 1 μM ATRA for 96 h at the most. Furthermore, NB4 cells were treated with 10 or 20 μM Boc-D-FMK, 1 μM SP 600125, 1 μM PD 98059, 10 μM SB 203580, 1 μM LY 294002, and 5 μM Tyr AG 490, respectively, at the indicated concentrations for 1 h prior to treatment with 10 μM DP03 in the presence or absence of these reagents.
Cell viability assay
Cytotoxicity of 2-methylthio DPs against NB4 or HL-60 cells was measured by XTT assay as described previously[20] or WST-8 assay kit according to the manufacturer’s instructions. Briefly, XTT/PMS mixed solution (1.5 mM XTT and 0.025 mM PMS in PBS) or WST-8 solution of a quarter or a tenth of the medium in well was added followed by incubation at 37 °C for 4 h or 1 h. Next, the plate was mixed on a mechanical plate shaker, and absorbance at 450 nm was measured with a microplate reader, SpectraMax Pro M5e (Molecular Devices, USA). Cell viability was calculated using the following equation:
where A450 sample denotes each analog. The IC50 (half maximal inhibitory concentration) values were calculated using the software, GraphPad Prism Ver 8.4.3 (GraphPad Software, Inc., San Diego, CA, USA).
Differentiation analysis
Differentiated induction was confirmed by the expression of CD11b, a cell surface marker in Myeloid maturation[22]. After each culture supernatant was collected, the adhesion cells were washed twice with PBS. To detach adhesion cells, 3 mL of Trypsin-EDTA solution was added and incubated at 37 °C for 2 min followed by adding 3 mL medium, and the adhesion cells were collected in each culture supernatant. 1×106 cells were washed twice with cell staining buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 14 mM sodium azide, and 2% heat-inactivated FBS), and stained with 5 μg/mL PE anti-mouse/human CD11b antibody or it’s isotype ctrl antibody for 1 h at room temperature in the dark. Cells were then washed twice with cell staining buffer and measured by a flow cytometer (FCM), CytoFLEX S (Beckman Coulter, CA, USA), with an acquisition of 10,000 events using CytExpert Ver 2.4.0.28 software (Beckman Coulter, CA, USA). The data were analyzed by Kaluza 2.1 software (Beckman Coulter, CA, USA).
Cell cycle analysis
Cell cycle analysis was performed using the CytoFLEX S FCM according to the methods previously described with slight modifications [17]. Briefly, cells were washed twice with cold PBS, fixed with 1% paraformaldehyde/PBS on ice for 30 min, washed twice again with cold PBS, permeabilized in 70% (v/v) cold ethanol, and kept at –20°C for at least 4 h. They were then washed twice with cold PBS after centrifugation (430×g for 5 min at 4°C) and cells were resuspended in 1 mL of 4 µg/mL of Hoechst 33342/PBS and incubated for 30 min in the dark at room temperature. Finally, they were measured by CytoFLEX S flow cytometer. A total of 10,000 events were acquired using CytExpert software. Kaluza Analysis 2.1 software was used to calculate the number of cells at G0/G1, S, and G2/M phase fractions, respectively.
Apoptosis/Necrosis analysis
The Annexin V FITC Assay Kit was used for the detection of apoptotic or necrotic cells according to the manufacturer’s instructions. In brief, 5×105 cells were washed with 200 μL of Cell-Based Assay Annexin V Binding Buffer, and 50 μL of the Cell-Base Propidium Iodide and Cell-Base Annexin V FITC combination solution in the Binding Buffer for 10 min at room temperature in the dark. After 150 μL of the Binding Buffer was added, fluorescence intensities of PI and FITC were measured by CytoFLEX S flow cytometer. A total of 10,000 events were acquired using CytExpert software and the data were analyzed by Kaluza Analysis 2.1 software.
Measurement of caspases activity
The activity of caspase-8, -9, -12, and -3 was measured using the caspases fluorometric assay kit according to the method described previously[17, 25]. Briefly, a protein amount of 25 μg/50 μl was plated on a 96-well plate, followed by the addition of 50 μl of 2×reaction buffer containing 10 mM DTT to each sample, and then 5 μl of 1 mM each caspase substrate (final concentration of 50 μM). After incubation at 37°C for 1 h, the fluorescent intensity (Excitation. 400 nm, Emission. 505 nm) was measured using the SpectraMax Pro M5e microplate reader.
Statistical analysis
All experiments were independently carried out at least three times and shown as the means ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.4.3 (GraphPad software, CA, USA) and conducted using one-way ANOVA followed by Dunnett’s or Turkey's post hoc test. A probability level of p < 0.05 was considered to indicate a statistically significant difference.
DP03 was the strongest cytotoxic inducer against NB4 cells
Cytotoxicity of nineteen types of 2-methylthio DPs in NB4 or HL-60 cells was evaluated following treatment with 10, 25, and 50 μM of each compound for 96 h. Very strong cytotoxic effects were observed in both NB4 and HL-60 cells when treated with 25 μM or 50 μM of DP03 and DP09, respectively [Fig. 1B(1)]. By contrast, a strong cytotoxic effect of other seventeen compounds (DP01, DP02, DP04–DP08, DP10, and DP11) was observed in either NB4 or HL-60 cells, but not both cells (supplementary Fig. 2), indicating DP03 and DP09 were more potent cytotoxic inducer at a relatively high concentration among nineteen 2-methylthio DPs in both cells. In addition, only a modest cytocidal effect was confirmed in both cells when exposed to the highest concentrations of 50 μM DP12–DP19 (supplementary Fig. 2). Moreover, treatment with DP03 at concentrations as low as 10 μM significantly caused cytotoxicity in NB4 cells, but not HL-60 cells. In comparison, exposure to 10 μM of DP09 significantly induced cytotoxicity in HL-60 cells, but not NB4 cells[Fig. 1B(1)]. The following experiments were conducted by focusing on the cytotoxicity of DP03 against NB4 cells since NB4 cells are resistant to myeloid differentiation and apoptosis.
To obtain more detailed information regarding the effect of DP03 on NB4 cells, cell viability was further investigated following treatment with 2.5, 5.0, 7.5, 10, and 25 μM for 24, 48, and 96 h, respectively. As shown in Fig. 1B(2),1B(3), and 1B(4), a significant decrease in cell viability was observed in a dose-dependent manner after treatment for 48 and 96 h, although a similar phenomenon was not observed in the short-term treatment (24 h). The IC50 values of DP03 were 13.89 μM and 9.42 μM for 48 h and 96 h treatment, respectively.
DP03 did not induce cell differentiation in NB4 cells
After the exposure of NB4 cells to 1 μM ATRA or 10 μM DP03 for 96 h, the expression of CD11b was measured by FCM. In comparison to control, treatment with ATRA, but not DP03 significantly increased the expression of CD11b in NB4 cells (p < 0.01 vs control) (Fig. 1C).
DP03 did not induce cell cycle arrest in NB4 cells
To evaluate if cell cycle arrest is implicated in the DP03-induced cytotoxicity, cell cycle analysis was performed by FCM. As shown in Fig. 2 and supplementary Fig. 3, almost no significant alterations in G0/G1, S, and G2/M phases of DP03-induced NB4 cells were recognized at 24, 48, 72, and 96 h, respectively.
DP03 induced apoptosis against NB4 cells
After treatment for 24, 48, and 72 h with 10 μM DP03, Annexin V/PI analysis was conducted to explore whether apoptosis and/or necrosis contributed to the cytotoxic effects of DP03. As shown in Fig. 3, a significant increase in apoptotic cells but not necrotic cells was shown in DP03-treated NB4 cells at 24, 48, and 72h, respectively (p < 0.01 vs control, respectively).
DP03-triggered caspase-dependent apoptosis in NB4 cells
To get the more detailed information regarding the DP03-triggered apoptosis in NB4 cells, the activation of caspase-8, -9, -12, and -3 was evaluated in NB4 cells following the treatment for 48 h with 10 μM DP03. As shown in Fig. 4A, treatment with DP03 for 48 h significantly increased the activities of caspase-8, -9, and -3 (p < 0.01 vs control, respectively), but not caspase-12. As shown in Fig. 4B and 4C, the addition of Boc-D-FMK (pan-caspase inhibitor) significantly abrogated DP03-triggered apoptosis of NB4 in a dose-dependent manner (p < 0.01 vs DP03 treatment group). Of note, DP03-induced apoptosis was completely suppressed by the addition of 20 μM Boc-D-FMK at 48 h (Fig. 4C). The involvement of MAP kinase, PI3 kinase, and JAK/STAT pathways was further investigated using their respective inhibitors. DP03-induced apoptosis did not significantly decrease in the presence of SP 600125 (JNK inhibitor) and PD 98059 (MEK1 inhibitor) (1 μM, respectively) (Supplementary Fig. 4, Fig. 5A, and 5B), but significantly enhanced in the presence of SB 203580 (p38 MAP kinase inhibitor), LY 294002 (PI3K inhibitor), and Tyr AG 490 (Stat-3 and JAK2/3 inhibitor) (10 μM, 1 μM, and 5 μM, respectively) (p <0.01, 0.01, 0.05 vs DP03 treatment group, respectively) (supplementary Fig. 4, Fig. 5C, 5D, and 5E) in NB4 cells.
Results from the current study clearly demonstrated that DP03 was the strongest cytotoxic inducer against NB4 cells rather than HL-60 cells, whereas DP09 was the strongest against HL-60 cells rather than NB4 cells [Fig. 1B(1)]. Although at low concentrations, DP01–DP11, and DP19 induce cytotoxic effects against NB4 cells, and DP01–DP11 excluding DP19 also induced cytotoxic effects against HL-60 cells. Furthermore, DP12–DP18 did not induce cytotoxic effects against both cells (supplementary Fig. 2G and 2H). These results suggest that DP03 is the most potent inducer in cytotoxicity against NB4 cells. In addition, it seems that the difference in 6-substituted on 2-methylthio DP affects their induced cytotoxicity against NB4 and HL-60 cells. The structure of DP01–DP11 is introduced 6-methyl or phenyl group on 2-methythio DP, on the other hand, that of P12–DP19 is 6-oxo one. Our previous studies demonstrated that 2-oxo DPs do not induce cytotoxic effects against HL-60 cells[18, 20]. Therefore, all these findings suggest that the introduction of the oxo-group into the structure of DP results in the reduction of cytotoxicity against NB4 and HL-60 cells. Although the exact reason for the difference in 4- and/or 5-substituent in 6-methyl-2-methylthio DPs and 6-phenyl-2-methylthio DP affects cytotoxic induction against both cells is still unclear, it seems to be important for that to be 6-methyl or phenyl group on 2-methythio DP.
The finding of differentiation inducers likely contributes to the development of new differentiation therapy in APL. Indeed, the appearance of arsenic trioxide (ATO) has experienced a dramatic improvement in the cure of relapsed patients with APL[21]. Many research groups including us have demonstrated that ATRA induces cell differentiation against NB4 cells by evaluating NBT reaction and CD11b expression[22, 27, 28, 31]. ATRA-treated NB4 cells were thus used as a positive control in CD11b expression, and the involvement of induction of differentiation in DP03-induced cytotoxicity was investigated against NB4 cells. In addition, cell cycle arrest-inducing activity has been implicated in the antitumor activity in not only basic studies but also clinical cures. Cytarabine, also known as Ara-C and an anti-cancer medicine, induced G0/G1 arrest against NB4 cells[32]. Results from the current study clearly demonstrated that treatment with 10 µM DP03 did not induce the increase of CD11b expression (Fig. 1C) and cell cycle arrest (Fig. 2) in NB4 cells, suggesting that DP03 does not have the potential to induce cell differentiation and cell cycle arrest in NB4 cells. However, 10 µM DP03-treatment induced time-dependent apoptosis in NB4 cells (Fig. 3). Collectively, our study suggests that DP03-triggered apoptosis is the main contributor to its cytotoxicity against NB4 cells.
Caspases are an evolutionarily conserved family of cysteine proteases that are centrally involved in cell death and inflammation responses[33], and caspase-8 and − 9, known as initiators, and caspase-3, known as an effector, are representative apoptotic caspases. The pathway that is mediated by caspase-9 was the intrinsic pathway, while that is mediated by caspase-8 was the extrinsic pathway, and the activation of both caspases triggers the execution phase of apoptosis via the activation of the downstream effector caspase, caspase-3[34]. Caspase-12 phylogenetically clusters with the inflammatory caspase subfamily, and it was suggested that caspase-12 promotes endoplasmic reticulum stress-induced apoptosis in early studies, but the current consensus is that caspase-12 is dispensable for apoptosis[33, 35]. In the current study, a significant increase in caspase-8, -9, and − 3 activities not but caspase-12 was observed in DP03-treated NB4 cells at 48h (Fig. 4A), suggesting that both intrinsic and extrinsic pathways were involved in the DP03-induced apoptosis. As if to prove the involvement of caspases, dose-dependent reduction of apoptotic cells in DP03-treated NB4 cells was further observed in the presence of 10 or 20 µM Boc-D-FMK, a pan-caspase inhibitor, significantly at 48 h (Fig. 4B and 4C). Furthermore, DP03-induced apoptosis was inhibited in the presence of 20 µM Boc-D-FMK completely.
Various pathways are known to evoke the caspase signaling pathway. MAP kinase pathways have been characterized by cascades of three kinases, extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 MAP kinase, and widely known to regulate all aspects of cellular processes, including proliferation, differentiation, apoptosis and stress responses[36–39]. Indeed, our previous studies demonstrated that casticin, a phytochemical derived from Vitex agnus-castus fruits, activates p38 MAP kinase, resulting in the induction of apoptosis against HL-60 cells[17]. In addition, PI3 kinase and JAK/STAT pathways are activated by a wide variety of extracellular stimuli (e.g., growth factors, cytokines, and hormones)[40], and ultimately regulate cell survival, growth, and proliferation[40–42]. Thus, these pathways have been crucial targets in cancer prevention and therapy. Unfortunately, the current study shows that DP03-triggered apoptosis was unaltered in the presence of SP 600125 (JNK inhibitor) and PD 98059 (MEK1 inhibitor)(Fig. 5A and 5B) at 48 h. Furthermore, apoptotic cells in NB4 cells induced by DP03-treatment in the presence of inhibitors, SB 203580 (p38 MAP kinase inhibitor), LY 294002 (PI3K inhibitor), and Tyr AG 490 (Stat-3 and JAK2/3 inhibitor), were increased rather than decreased. Several studies have reported that the combination of apoptosis inducers and inhibitors of p38 MAP kinase, PI3K, or JAK/STAT enhanced the cytotoxicity including apoptosis[43–46]. Thus, the combination of DP03 and the inhibitors of p38 MAP kinase, PI3K, and JAK/STAT signaling pathways possibly be used effectively in combination therapy based on DP03. Collectively, these results suggest that apoptosis induced by DP03 may not involve MAP kinase, PI3 kinase, and JAK/STAT pathways. Therefore, this study could not resolve what kinds of pathway is involved in caspase-8, -9, and − 3 activations, but research is currently underway to determine what factors induce activation of the caspase signaling pathway in DP03-treated NB4 cells.
In conclusion, our results demonstrated that DP03 was the strongest cytotoxic inducer of nineteen types of 2-methylthio DPs against NB4 cells that are resistant to myeloid differentiation and apoptosis. Apoptosis associated with caspases activation, but not differentiation and cell cycle arrest, primarily contributed to DP03-induced cytotoxicity. In addition, the involvement of MAP kinase, PI3 kinase, and JAK/STAT pathways in DP03-triggered apoptosis was little if any observed. Thus further studies on the involvement in the detail pathway are needed. Collectively, these results indicated that DP03 may be a promising candidate as an anticancer therapeutic reagent.
Acknowledgments
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Compliance with Ethical Standards
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Conflict of Interest
Authors declare that they have no conflict of interest.
Funding
YN thanks financial support from the Foundation for Japanese Chemical Research [No. 505(R)].
Author’s Contributions
YT and HK contributed equally to this study. HK conceived and designed the study, and drafted the manuscript together with YT. YN synthesized all of 2-methylthio DPs in this manuscript. YT performed the XTT and WST-8 assays. YT and HK performed flow cytometry analysis and measured caspases activity. HK and BY developed the statistical analysis plan, and YT and RA conducted statistical analyses together with HK. YN, BY, RA, HC, and KS assisted interpretation of the results with HK. All authors read and approved the final manuscript.
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No competing interests reported.