An in vitro study on anti-carcinogenic effect of remdesivir in human ovarian cancer cells via generation of reactive oxygen species

Abstract

Background

Remdesivir is an anti-viral drug that inhibits RNA polymerase. In 2020, remdesivir was recognized as the most promising therapeutic agents against coronavirus disease 2019 (COVID-19). However, the effects of remdesivir on cancers have hardly been studied.

Purpose

Here, we reported that the anti-carcinogenic effect of remdesivir on SKOV3 cells, one of human ovarian cancer cell lines.

Research design

We anlalyzed the anti-carcarcinogenic effect of remdesivir in SKOV3 cells by performing in vitro cell assay and western blotting.

Results

WST-1 showed that remdesivir decreased cell viability in SKOV3 cells. Experiments conducted by Muse Cell Analyzer showed that remdesivir-induced apoptosis in SKOV3 cells. We found that the expression level of FOXO3, Bax, and Bim increased, whereas Bcl-2, caspase-3, and caspase-7 decreased by remdesivir in SKOV3 cells. Furthermore, we observed that intracellular reactive oxygen species (ROS) level increased after treatment of remdesivir in SKOV3 cells. Interestingly, cytotoxicity of remdesivir decreased after treatment of N-Acetylcysteine.

Conclusion

Taken together, our results demonstrated that remdesivir has an anti-carcinogenic effect on SKOV3 cells vis up-regulation of reactive oxygen species, which suggests that remdesivir could be a promising reagent for treatment of ovarian cancer.

Keywords

Remdesivir apoptosis reactive oxygen species ovarian cancer

Introduction

A recent statistic reported that ovarian cancer accounted for 2.5% of all cancers among women but 5% of deaths in women due to low survival rates.¹ Ovarian cancers are divided into more than 10 histologic subtypes and ovarian epithelial tumors account for approximately 90%.² The five major subtypes of ovarian epithelial tumors are considered different diseases because each type has different risk factors.³ Furthermore, histological subtypes show the different responses to therapeutic methods such as chemotherapy, probably due to genetic differences.^4–6 For instance, TP53 is mutated in 90% of patients in high-grade serous ovarian cancer which is the most common subtype.⁷ Thus, we can think about another tumor suppressive transcriptional factor such as FOXO3 as the possible target for treating ovarian cancer, which can substitute the role of the mutated p53. Even though therapeutic technologies in surgery have been improved, its prognosis is still poor.⁸ Therefore, it is necessary to find an alternative method for ovarian cancer treatment.

Due to the rapid development of molecular and cellular biology, mechanism of cancer development and survival have been investigated well. Recently, many drugs targeting specific mediator in cells have been developed based on these knowledges.^9–11 Typical example is celecoxib showing the anti-inflammatory activity vis regulation of COX-2.¹² These days, many researchers and scientists have been trying to find the effective reagents to inhibit cancer cells because target therapy is less harm to normal cells and has fewer side effects.^13–15 Recent studies have shown the effective anti-carcinogenic effects of anti-viral reagents in cancers. It was reported that anti-viral reagent acyclovir had the suppressive effect on breast cancer MCF7 cells.¹⁶ A recent study has revealed that carotenoids produced by haloalkaliphilic archaeon Natrialba sp. M6 exerted not only anti-viral effect on hepatitis C and hepatitis B, but also anti-cancer effect on various cancer cell lines such as MCF-7, HepG-2, and Caco-2 cells.¹⁷ It is crucial to study the effect of anti-viral reagents on cancers or the other way around because it could broaden therapeutic applications by taking advantage of the same or common working mechanism of the specific medicine in the different disease.

Inhibition of RNA polymerase has been recognized as an important target for cancer treatment because dysregulation of ribosomal RNA genes (rDNA) transcription is one of common features in human cancers.^18,19 Several studies showed the inhibitory effect of RNA polymerase inhibitor on cancers.²⁰ It was reported that tamoxifen which is an antagonist of the estrogen receptor inhibited breast cancer via inhibition of RNA polymerase III gene transcription.²¹ CX-5461, selective rDNA transcription inhibitor, showed anti-tumor activity on patients with advanced hematologic cancers.²² In this study, we evaluate the anti-tumor activity of remdesivir that has been known as viral RNA-dependent polymerase inhibitor on human ovarian cancer SKOV3 cells. Remdesivir is a nucleoside analogue prodrug that inhibits viral RNA polymerase.²³ This chemical was famous because it showed inhibitory effects on coronavirus 2019 (COVID-19).²⁴ Because of outbreak of COVID-19, the inhibitory effects of remdesivir on COVID-19 has been investigated well, however, anti-carcinogenic effect of remdesivir on cancers has hardly been understood. In this study, we tried to investigate the basic mechanisms of apoptosis and involvement of reactive oxygen species (ROS) in remdesivir-induced apoptosis in SKOV3 cells.

Materials and methods

Reagents

Remdesivir was obtained from Selleckchem (Houston, TX, USA). Muse cell analyzer was purchased from Millipore Corporation (Hayward, CA, USA). FlowJo software was from FlowJo LCC (Ashland, OR, USA). Antibodies against PARP-1 and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against FOXO3, Bax, Bcl-2, Bim, caspase-3, and caspase-7 were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against rabbit and mouse were purchased from Santa Cruz Biotechnology.

Cells and cell culture

SKOV3 cells were from Korean Cell Line Bank (Seoul, South Korea). Cells were maintained in RPMI-1640 medium (Thermo fisher scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, Sigma, St. Lois, MO, USA) and 1% streptomycin/penicillin antibiotics (Thermo fisher scientific) in a humidified incubator (5% CO₂ and 37°C).

WST-1 assay

SKOV3 cells were seeded in 96-well plate (3 × 10³ cells/well) and incubated for 18 h. Then, cells were treated with remdesivir (DMSO, 5, 10, and 20 μM). After incubation for 72 h, the media was replaced with and fresh media containing 10% EZ-cytox (DogenBio, Seoul, South Korea) and incubated for 1 h in the dark. The absorbance (450 nm) was measured by plate-reader (Molecular Devices, Mountain View, CA, USA).

Ki67 assay by Muse cell analyzer

SKOV3 cells were seeded in 6-well plate (2 × 10⁵ cells/well) and incubated for 18 h. Then, cells were treated with remdesivir (DMSO, 5, 10, and 20 μM). After incubation for 72 h, the population of Ki67 positive cell was measured by Muse cell analyzer using Muse Ki67 Proliferation Kit (Luminex Corporation, Austin, TX, USA, Part number: MCH100114) according to manufacturer’s instructions.

Viability assay by Muse cell analyzer

SKOV3 cells were seeded in 6-well plate (2 × 10⁵ cells/well) and incubated for 18 h. Then, cells were treated with DMSO or 10 μM of remdesivir in the presence or absence of 10 mM of N-Acetylcysteine (NAC, Sigma). After incubation for 72 h, cell viability was measured by Muse cell analyzer using Muse Count and Viability Kit (Luminex corporation, Austin, TX, USA, Part number: MCH100102) according to manufacturer’s instructions.

Cell counting assay

SKOV3 cells were seeded in 6-well plate (2 × 10⁵ cells/well) and incubated for 18 h. Then, cells were treated with remdesivir (DMSO, 5, 10, and 20 μM). For evaluating the effect of ROS induced by remdesivir on cell viability, cells were treated with DMSO or 10 μM of remdesivir in the presence or absence of 10 mM of NAC. After incubation for 72 h, the live cell was counted with hemocytometer (Thermo fisher scientific).

Colony formation assay

SKOV3 cells were seeded in 60 mm dishes (5 × 10² cells/dish) and incubated for 18 h. Then, cells were treated with remdesivir (DMSO, 5, 10, and 20 μM). After incubation for 72 h, the cell culture media was changed every 2 day for 11 days. After then, cells were fixed with 4% formaldehyde for 25 min in room temperature. After PBS washing two times, the colony was stained with 1% crystal violet solution (Sigma) and the number of colonies was counted with microscope (Nikon Eclipse TE 2000-U, Tokyo, Japan).

Apoptosis assays by Muse cell analyzer

SKOV3 cells were seeded in 6-well plate (2 × 10⁵ cells/well) and incubated for 18 h. Two plates were prepared for two experiments. For first plate, cells were treated with remdesivir (DMSO, 5, 10, and 20 μM). For second plate, cells were treated with DMSO or 10 μM of remdesivir in the presence or absence of 10 mM of NAC. After incubation for 72 h, the percentage of apoptotic cells was analyzed by Muse cell analyzer using Muse Annexin V and Dead Cell Kit (Luminex Corporation, Part number: MCH100105), Muse Mito-Potential Kit (Luminex Corporation, Part number: MCH100110), Muse Caspase-3/7 Kit (Luminex Corporation, Part number: MCH100108), and Muse Multi-Caspase Kit (Luminex Corporation, Part number: MCH100109) according to manufacturer’s instructions.

Cell cycle analysis by Muse cell analyzer

SKOV3 cells were seeded in 6-well plate (2 × 10⁵ cells/well) and incubated for 18 h. Then, cells were treated with remdesivir (DMSO, 5, 10, and 20 μM). After incubation for 72 h, cell cycle was analyzed by Muse cell analyzer using Muse Cell Cycle Kit (Luminex Corporation, Part number: MCH100106) according to manufacturer’s instructions.

Oxidative stress analysis by Muse cell analyzer

SKOV3 cells were seeded in 6-well plate (2 × 10⁵ cells/well) and incubated for 18 h. Two plates were prepared for two experiments. For first plate, cells were treated remdesivir (DMSO, 5, 10, and 20 μM). For second plate, cells were treated with DMSO or 10 μM of remdesivir in the presence or absence of 10 mM of NAC. After incubation for 72 h, reactive oxygen species (ROS) level in SKOV3 cells induced by remdesivir was measured by Muse cell analyzer using Muse Oxidative Stress Kit (Luminex Corporation, Part number: MCH100111) according to manufacturer’s instructions.

ATM and H2AX phosphorylation analysis by Muse cell analyzer

SKOV3 cells were seeded in 6-well plate (2 × 10⁵ cells/well) and incubated for 18 h. Then, cells were treated with remdesivir (DMSO, 5, 10, and 20 μM). After incubation for 72 h, phosphorylation level of ATM and H2AX in SKOV3 cells induced by remdesivir was measured by Muse cell analyzer using Muse Multi-Color DNA Damage Kit (Luminex Corporation, Part number: MCH200107) according to manufacturer’s instructions.

Mitochondria potential analysis by Muse cell analyzer

SKOV3 cells were seeded in 6-well plate (2 × 10⁵ cells/well) and incubated for 18 h. Then, cells were treated with remdesivir (DMSO, 5, 10, and 20 μM). After incubation for 72 h, mitochondria potential in SKOV3 cells induced by remdesivir was measured by Muse cell analyzer using Muse Mito-Potential Kit (Luminex Corporation, Part number: MCH100110 according to manufacturer’s instructions.

Western blotting analysis

SKOV3 cells were seeded in 60 mm dishes (5 × 10⁵ cells/dish) and incubated for 18 h. Then, cells were treated with DMSO or remdesivir (5 or 10 μM). After incubation for 72 h, cells were washed with cold PBS and suspended with RIPA buffer (Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma). After sonicating cell suspension in ice, the lysates were centrifuged (10,000 r/min, 10 min, 4°C). Then the supernatant was mixed with 4 × sample buffer (Bio-Rad, Hercules, CA, USA) containing 10% β-mercaptoethanol (Sigma) and boiled for 5 min (100°C). The same amount of protein lysates was loaded and resolved in 8, 10, or 12% SDS-PAGE (100 V, 2 h). Separated proteins were transferred onto 45 μm PVDF Immobilon-P membrane (Merck Millipore, Burlington, MA, USA). The membrane was blocked with 3% bovine serum albumin (BSA, Sigma) for 1 h on the shaking incubator at room temperature. The membranes were incubated with primary antibodies (1:1,000 dilution in 3% BSA) overnight at 4°C. After washing membranes with TBS-T three times, secondary antibodies (1:5000 dilution in TBS-T) for 1 h at room temperature. After washing membranes with TBS-T three times, bands were detected by Chemi-Doc system (Bio-Rad) using enhanced chemiluminescence reagent (ECL, Bio-Rad).

Statistical analysis

Data were presented as mean ± standard deviation (SD). Statistical significance between DMSO-treated group and remdesivir-treated group was analyzed using ANOVA test and p < .05 was considered as statistically significant.

Results

Remdesivir inhibited the proliferation of SKOV3 cells

The cytotoxicity and anti-proliferative activity of remdesivir on human ovarian cancer SKOV3 cells was determined by WST-1, cell counting, colony formation, and Ki67 staining assay. The cells were treated with remdesivir (DMSO, 5, 10, and 20 μM) for 72 h. Our results showed that cell viability decreased after treatment of remdesivir with a dose-dependent way (Figure 1(a)). Additionally, we found that the number of living cells decreased with a dose-dependent way (Figure 1(b)). As shown in Figure 1(c), the number of colonies was decreased with a dose-dependent way. Furthermore, we found that Ki67 positive cells decreased after treatment of remdesivir (Figure 1(d)). Taken together, our results demonstrated that remdesivir suppressed cell proliferation in SKOV3 cells.

Figure 1.

Anti-proliferative effect of remdesivir on human ovarian cancer SKOV3 cells. Cells were treated with remdesivir (DMSO, 5, 10, and 20 μM) for 72 h. (a) Cell viability was measured by WST-1 assay after treatment of remdesivir for 72 h. (b) The number of living cells was counted by hemocytometer after treatment of remdesivir for 72 h. (c) The number of colonies was counted after treatment of remdesivir for 72 h and culture for 11 days. (d) The population of Ki67-positive cells was measured by Muse cell analyzer and FlowJo program after treatment of remdesivir for 72 h. Experiments were repeated at least 3 times independently and statistically analyzed (*p < .05).

Remdesivir-induced the apoptosis in SKOV3 cells

To determine the apoptosis inducing capability of remdesivir in SKOV3 cells, analysis for Annexin V staining was performed using Muse cell analyzer. Cells were treated with remdesivir (DMSO, 5, 10, and 20 μM) for 72 h. As shown in Figure 2(a), our results showed that the population of total apoptotic cells (early + late apoptotic cells) increased after treatment of remdesivir in SKOV3 cells with a dose-dependent manner. Additionally, we measured the population of mitochondria-depolarized cells in SKOV3 cells treated with remdesivir for 72 h. We found that the percentage of mitochondria-depolarized cells increased after treatment of remdesivir (Figure 2(b)) with a dose-dependent manner. Furthermore, we analyzed cell cycle after treatment of remdesivir for 72 h using Muse cell analyzer in SKOV3 cells. Interestingly, the population of SubG1 cells increased after treatment of remdesivir with a dose-dependent manner (Figure 2(c)). Additionally, activities of caspase-3, caspase-7, and multi-caspase were measured using Muse cell analyzer. As shown in Figure 2(d) and (e), caspases activity increased after treatment of remdesivir for 72 h with a dose-dependent manner. The expression level of proteins related to apoptotic signaling pathway was analyzed by western blotting (Figure 2(f) and (g)). Our results showed that FOXO3, Bax, and Bim increased, whereas Bcl-2, caspase-3, and caspase-7 decreased after treatment of remdesivir for 72 h with a dose-dependent manner. Interestingly, PARP-1 increased in 5 μM treated group but decreased in 10 μM treated group. Taken together, our results demonstrated that remdesivir induced the apoptosis in SKOV3 cells.

Figure 2.

Induction of apoptosis by remdesivir in human ovarian cancer SKOV3 cells. Cells were treated with remdesivir (DMSO, 5, 10, and 20 μM) for 72 h. (a) The population of apoptotic (early + late apoptosis) cells, (b) the population of cells with the depolarized mitochondria, (c) SubG1 population of cell, (d) the activity of multi-caspase, and (e) the activity of caspase-3 and caspase-7 were measured by Muse cell analyzer and FlowJo program after treatment of remdesivir for 72 h. (f) The expression level of PARP-1, FOXO3, Bax, Bcl-2, Bim, caspase-3, and caspase-7 was evaluated by western blotting. (g) Quantification of western blot data. β-actin was used as a loading control. Experiments were repeated at least 3 times independently and statistically analyzed (*p < .05).

Remdesivir increased the intracellular oxidative stress in SKOV3 cells

To measure the intracellular ROS induced by remdesivir in SKOV3 cells, we treated the cells with different concentrations of remdesivir (DMSO, 5, 10, and 20 μM) for 72 h and analyzed the population of ROS positive cell using Muse cell analyzer (Figure 3(a)). We found that intracellular ROS level increased with a dose-dependent manner. Additionally, we found that the expression level of the phosphorylated ATM and γH2AX (two of DNA damage marker) increased after treatment of remdesivir (Figure 3(b)). To further investigate the intracellular ROS induced by remdesivir, we treated the cells with 10 μM remdesivir or/and 10 mM N-acetyl-l-cysteine (NAC, antioxidant reagent) for 72 h and analyzed cells by Muse cell analyzer. As shown in Figure 4(a), intracellular ROS level of 10 μM remdesivir-treated group was higher than 10 μM remdesivir and 10 mM NAC-treated group. The number of living cells after treatment of remdesivir or/and NAC was counted (Figure 4(b)). Our results showed that the number of living cells in remdesivir-treated group was lower than remdesivir and NAC-treated group. Additionally, we found that cell viability in remdesivir-treated group were lower than remdesivir and NAC-treated group (Figure 4(c)). Total apoptotic cells in remdesivir-treated group were higher than remdesivir and NAC-treated group. Taken together, these results suggested that ROS induced by remdesivir could be an important mediator of apoptotic processes in SKOV3 cells.

Figure 3.

Induction of ROS generation and DNA damage by remdesivir in human ovarian cancer SKOV3 cells. Cells were treated with remdesivir (DMSO, 5, 10, and 20 μM) for 72 h. (a) Intracellular ROS level (b) the population of DNA damage (level of the phosphorylated ATM and γH2AX) was measured by Muse cell analyzer and FlowJo program after treatment of remdesivir for 72 h. Experiments were repeated at least 3 times independently and statistically analyzed (*p < .05).

Figure 4.

Apoptosis depended on the intracellular ROS induced by remdesivir in SKOV3 cells. Cells were treated with DMSO or 10 μM remdesivir in the presence or absence of 10 mM NAC for 72 h. (a) Intracellular ROS level was evaluated by Muse cell analyzer and FlowJo program after treatment of remdesivir for 72 h. (b) The number of living cells was counted by hemocytometer after treatment of remdesivir for 72 h. (c) The population of dead cells and (d) the population of total apoptotic cells (early + late apoptosis) was measured by Muse cell analyzer and FlowJo program after treatment of remdesivir for 72 h. Experiments were repeated at least 3 times independently and statistically analyzed (*p < .05).

Discussion

Remdesivir is a viral RNA-dependent RNA polymerase inhibitor that has shown inhibitory activity against COVID-19 in vitro and in vivo.^25–27 Interestingly, even though anti-viral effect of remdesivir against COVID-19 has been studied well, anti-cancer effect of remdesivir has hardly been investigated. Anti-cancer effects of remdesivir on various cancer cell lines might be studied to verify the efficacy of remdesivir on cancers. Here, we focused on the effect of remdesivir on human ovarian cancer SKOV3 cells. To evaluate the inhibitory effect of remdesivir on SKOV3 cells, we first performed WST-1 and viability assays (Figure 1). We found that cell viability decreased significantly after remdesivir treatment, which suggested that remdesivir has a suppressive effect on proliferation of SKOV3 cells. Basically, it was reported that remdesivir showed no significant effect on human RNA polymerase II and mitochondrial RNA polymerase,²⁸ which is why it was recognized as promising reagent for treatment of COVID-19. In the next study, we might need to examine whether the remdesivir has the inhibitory effect on human RNA polymerases or not and determine the possible side effect of remdesivir in human body.

To determine the apoptotic effect of remdesivir on SKOV3 cells, we performed Annexin V, mito-potential, cell cycle arrest assays, caspase activity assay, and western blotting analysis (Figure 2). Annexin V assay is the most typical experiment evaluating apoptotic effect. Loss of mitochondrial membrane potential is one of characteristics that is shown in apoptotic cells.²⁹ We found that mitochondrial membrane potential decreased after treatment of remdesivir in SKOV3 cells. DNA damage is another characteristic of apoptotic cells, which can be evaluated by measurement of SubG1 by cell cycle assay.³⁰ We found that the population of SubG1 increased after treatment of remdesivir in SKOV3 cells. Many studies have shown that intracellular ROS induced apoptosis in cancer cells.^31–33 Our results indicated that remdesivir increased intracellular ROS level in SKOV3 cells, which could damage DNA in cancer cells. Additionally, we performed western blotting to evaluate the expression level of apoptosis-related proteins in SKOV3 cells treated with remdesivir. We found that the expression of the pro-apoptotic proteins such as FOXO3, Bax, and Bim increased, whereas the expression of Bcl-2, caspase-3, and caspase-7 decreased. Interestingly, PARP-1 expression increased in 5 μM remdesivir-treated group but decreased in 10 μM remdesivir-treated group. PARP-1 is a first responder to DNA damage and facilitates DNA repair.³⁴ In apoptotic cells, cleavage of PARP-1 was frequently detected.^35,36 We speculated that PARP-1 was increased to compensate for DNA damage induced by 5 μM of remdesivir but decreased in 10 μM of remdesivir group due to the apoptotic effect of remdesivir.

It has been reported that intracellular ROS played crucial roles in cancer cell survival.^37–39 To verify the effect of ROS induced by remdesivir, we treated the cells with remdesivir or/and NAC which is well-known ROS scavenger in SKOV3 cells. We uncovered that intracellular ROS induced by remdesivir decreased was reduced by NAC. We additionally evaluated the cytotoxic effect of ROS induced by remdesivir in SKOV3 cells. As shown in Figure 4, cytotoxic effect of remdesivir was attenuated by the treatment of NAC, which suggested that ROS induced by remdesivir could be an important mediator of apoptotic process in SKOV3 cells. In the next study, we are planning to determine the level of lipid peroxidation products such as 4-hydroxynonenal (4-HNE) in the cell culture media as an indicator of ROS generation in cells by remdesivir.

There are several points we need to discuss in this paper. Basically, even though cell proliferation assays and apoptotic assays showed the same tendency, the effects of remdesivir are a little different outcome depending on assay types. For example, in Figure 1(a)–(c), 20 µM of remdesivir showed the dramatic effect on cell viability, which is why we decided to use this range of concentration and treatment time. However, when we performed apoptosis assays, it turned out the effect of remdesivir wasn’t so strong as in cell viability assays. Interestingly, as shown in Figure 2(f), there were dramatic expression level changes in apoptotic proteins such as FOXO3 and Bax, which suggested that specific apoptotic signaling pathways were significantly activated. Based on various kinds of assays related to apoptosis, our results strongly supported that apoptosis was induced by remdesivir in SKOV3 cells. We also think that it will be promising if remdesivir can be used as anti-cancer drug through combination with the previously approved and clinically used promising anti-cancer drug for the synergic effect.

In this study, we tried to investigate the basic mechanisms of apoptosis and involvement of ROS in remdesivir-induced apoptosis in SKOV3 cells. However, there are limitations of our study. First, specific and detailed mechanisms of apoptotic process and the effect of remdesivir on various cell lines should be verified. For example, we need to investigate the involvement of FOXO3 in the apoptosis process by remdesivir on ovarian cancer cells using FOXO3 knocked down or knocked out cell line model. Second, the effect of remdesivir in animal ovarian cancer xenograft or orthotic model should be evaluated. In conclusion, our study demonstrated that remdesivir has anti-carcinogenic effect on human ovarian cancer SKOV3 cells. We found that remdesivir decreased cell viability, induced apoptosis, and increased intracellular ROS level in SKOV3 cells. Interestingly, we found that intracellular ROS induced by remdesivir had a cytotoxic effect on SKOV3 cells. Taken together, our results suggested that remdesivir has a potential to be a therapeutic reagent for ovarian cancer treatment.

Footnotes

Authors contributions

CL, M-AK, and JB conceived the presented idea, carried out experiments, and wrote manuscript KP and Y-HY analyzed the data and revised the manuscript.

JL and KJ analyzed the data, revised the manuscript, and supervised all of processes S-HP analyzed the data, revised manuscripts, supervised all of processes, and acquisition of funds.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the National Research Foundation of Korea (NRF, NRF2014R1A6A3A04054307 and NRF2017R1A5A2015061) funded by the Ministry of Science and ICT (MSIP).

ORCID iD

See-Hyoung Park

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