Baicalein induces apoptosis by inhibiting the glutamine-mTOR metabolic pathway in lung cancer

Jingyang Li; Di Zhang; Shaohui Wang; Peng Yu; Jiayi Sun; Yi Zhang; Xianli Meng; Juan Li; Li Xiang

doi:10.1016/j.jare.2024.02.023

. 2024 Mar 2;68:341–357. doi: 10.1016/j.jare.2024.02.023

Baicalein induces apoptosis by inhibiting the glutamine-mTOR metabolic pathway in lung cancer

Jingyang Li ^a, Di Zhang ^a, Shaohui Wang ^a, Peng Yu ^a, Jiayi Sun ^a, Yi Zhang ^a, Xianli Meng ^a,^⁎, Juan Li ^b,^⁎, Li Xiang ^a,^⁎

PMCID: PMC11785570 PMID: 38432394

Graphical abstract

Keywords: Baicalein, Non-small-cell lung cancer, Apoptosis, Metabonomics, Glutamine-mTOR metabolic pathway

Highlights

•
Baicalein exhibits inhibitory effects on lung cancer both in vitro and in vivo.
•
Baicalein significantly inhibits glutamine metabolism in NSCLC cells.
•
The mTOR inhibition of baicalein were achieved by regulating glutamine metabolism.
•
Baicalein induces apoptosis by inhibiting the glutamine-mTOR metabolic pathway.
•
Baicalein exhibits potential as a pro-apoptotic agent in lung cancer therapy.

Abstract

Introduction

Baicalein, a bioactive component of Scutellaria baicalensis Georgi, has been shown to promote apoptosis in non-small cell lung cancer cells. However, previous studies have not determined if baicalein exerts proapoptotic effects by modulating the metabolic pathways.

Objective

To investigate if baicalein induces apoptosis in lung cancer cells by modulating the glutamine-mTOR metabolic pathway.

Methods

The in vivo anti-lung cancer activity of baicalein (50, 100, and 200 mg/kg) was evaluated using a xenograft model. In vitro experiments were used to assess the efficacy of baicalein (for H1299: 12.5, 25, and 50 μM; for A549: 10, 20, and 40 μM) on lung cancer cell proliferation, colony formation, and apoptosis. Metabolomics analysis was performed using liquid chromatography-mass spectrometry. The binding of baicalein to glutamine transporters and glutaminase was examined using molecular docking. The overexpression of glutamine transporters was validated using qRT–PCR and western blot analyses. The levels of ASCT2, LAT1, GLS1, p-mTOR, mTOR, and apoptosis-related proteins were evaluated using western blot analysis.

Results

Baicalein inhibited lung cancer xenograft tumor growth in vivo and suppressed proliferation and promoted apoptosis in lung cancer cells in vitro. Additionally, baicalein altered amino acid metabolites, especially glutamine metabolites, in H1299 and A549 cells. Mechanistically, baicalein interacted with glutamine transporters as well as glutaminase and inhibited their activation. The expression of mTOR, an apoptosis-related protein and downstream target of glutamine metabolism, was also inhibited by baicalein treatment. Importantly, we next demonstrated the suppression of mTOR signaling and the induction of apoptosis by baicalein were achieved by regulating glutamine metabolism.

Conclusion

Baicalein inhibited the mTOR signaling pathway and induced apoptosis by downregulating glutamine metabolism. The potential of baicalein to induce apoptosis in lung cancer cells by selectively targeting the glutamine-mTOR pathway suggests an encouraging approach for treating lung cancer.

Introduction

The occurrence and mortality rates of lung cancer, especially in patients with non-small cell lung cancer (NSCLC), which accounts for more than 85 % of all cases, are notably increasing worldwide [1]. Despite notable advancements in surgical techniques, novel chemotherapy regimens, and radiation therapies, the prognosis for lung cancer patients is still lacking. The 5-year comparative survival rate of patients with NSCLC is only 23 % [2]. This can be attributed to the increased probability of reoccurrence and the drug resistance in NSCLC patients after undergoing chemotherapy, surgery, or radiation therapy [3]. Therefore, it is imperative to dedicate efforts toward the discovery of novel therapeutics and targets to prevent and manage NSCLC.

The metabolic profile is markedly altered in patients with NSCLC [4]. Compared with nonmalignant cells, lung cancer cells exhibit a regulatory response toward changes in nutrient availability, leading to the modulation of the anabolic and catabolic pathways to facilitate rapid cell proliferation [5]. The rapid proliferation of lung cancer cells requires increased energy consumption, which contributes to the generation of various metabolites [6]. Currently, the tumor metabolome can be analyzed to effectively assess treatment efficacy. The metabolomic profile of NSCLC provides valuable insights into disease impact and metabolic aberrations in NSCLC cells, contributing to advances in cancer research. Therefore, leveraging the global advantages of metabolomics is crucial for investigating metabolic alterations in drug-treated NSCLC cells.

Natural products have a critical role in drug development as they serve as a prolific source of novel drugs. Extensive research has been conducted to explore the antitumor activity of these compounds [7]. Baicalein, a characteristic component of Scutellaria baicalensis Georgi, has applications in the prevention and treatment of various diseases, including lung cancer [8], [9]. Recent studies have demonstrated that baicalein exerts potent antitumor effects through various mechanisms, including the induction of cellular apoptosis, the suppression of lung cancer cell proliferation, and the impediment of tumor invasion and metastasis [10], [11], [12]. However, the metabolic regulatory and anti-NSCLC mechanisms of baicalein have not been elucidated. This study demonstrated that baicalein exerted growth-inhibitory effects against NSCLC by effectively suppressing proliferation and inducing apoptosis both in vitro and in vivo. Furthermore, baicalein suppressed the mTOR pathway and induced apoptosis by regulating glutamine metabolism. Our findings suggest that targeting the glutamine-mTOR pathway using baicalein to induce apoptosis in cancer cells is a prospective therapeutic approach for lung cancer.

Materials and methods

Regents and drugs

Baicalein (purity: 99.64 %, No. 491-67-8) was obtained from Lemeitian Biological Technology Co., Ltd. (Chengdu, China). Cisplatin (CDDP, P4394-25 MG) was obtained from Sigma Biological Technology Co., Ltd. (Sigma, USA). Elabscience Biological Technology Co., Ltd., provided the CCK-8 reagent (Elabscience, Wuhan, China). Apoptosis and cell cycle detection kits were procured from Yeasen BioTech Co., Ltd. (Yeasen, Shanghai, China).

Cell culture

H1299 and A549 NSCLC cells, and Lewis lung cancer cells were procured from Biotechnology Co., Ltd. of Zhong Qiaoxinzhou (Shanghai, China). Human healthy lung epithelial cells (BEAS-2B) were acquired from Hunan Fenghui Biotechnology Co., Ltd. H1299 and A549 cells were cultivated in RPMI-1640 medium, while BEAS-2B and Lewis lung cancer cell lines were cultured in DMEM. The cell cultures were kept in an environment at a temperature of 37 °C and a CO₂ concentration of 5 %.

Lung cancer xenograft mouse model

The animal experiments carried out in this study received ethical approval from the Animal Ethical and Welfare Committee at Chengdu University of Traditional Chinese Medicine [No. SCXK (Chuan) 2020–124]. The experiments were conducted in strict accordance with both the “Guide for Laboratory Animal Care and Use” and the American Research Institute (ARRIVE) guidelines. C57BL/6 mice (n = 40) aged between 6 and 8 weeks were obtained from GemPharmatech Co., Ltd. (Nanjing, China). Lewis lung cancer cells (1 × 10⁶) were introduced into the right flanks of the mice through subcutaneous injection utilizing a suspension of 0.2 mL basic DMEM. When the tumor volumes varied between 50 and 100 mm³, mice (n = 40) were allocated randomly into five groups (8 mice/group). The groups receiving baicalein treatment were administered doses of 50, 100, and 200 mg/kg [9]. The CDDP was administered at a dose of 3 mg/kg through intraperitoneal injection every three days for a duration of ten days [13]. 0.5 % CMC-Na was used as a control. The weights and sizes of the tumors were recorded at three-day intervals. The tumor volume was evaluated by applying the formula V = (width)² × length/2. At the end of the experiment, the mice were euthanized. The tumor tissues were immediately separated and weighed. Additionally, the liver, heart, spleen, lung, and kidney tissues were extracted and weighed.

CCK-8 assay

Cellular proliferation was assessed using the CCK-8 method. A 96-well plate was used, in which 1 × 10⁵ cells were seeded in each well and exposed to different concentrations of baicalein (0, 10, 20, 30, 50, 100, 200 and 500 μM). After a culture period of 24 h, 10 μL CCK-8 reagent was added to each well. The cells were incubated r for one and a half hours. Subsequently, a microplate reader (SpectarMaxiD5; USA) was used to measure the optical density (OD) at a wavelength of 450 nm.

Colony formation assay

In each well of a 6-well plate, a total of 500 cells were seeded and subjected to treatment with CDDP (10 μM) or various concentrations of baicalein for a period of 14 days. The visible colonies were immobilized using methanol and subjected to staining with a solution comprising 0.5 % crystal violet (Solarbio, Beijing, China). The counts of cell colonies and images were captured.

Wound-healing assay

H1299 and A549 cells were seeded in six-well plates until they reached 90 % confluence. A vertical incision was made in the center of each well using a pipette tip with a volume of 20 μL. Later, the plates were washed three times to eliminate the scraped cells. After the addition of CDDP (10 μM) or various concentrations of baicalein, the cells were cultured. The effects of CDDP and baicalein on H1299 and A549 cell migration were observed under a microscope for 24 h and 48 h by measuring changes in scratch width. The scratch healing rate (%) was determined by subtracting the scratch width at a specified time from the initial scratch width, dividing it by the initial scratch width, and multiplying it by 100 %.

Cell cycle analysis

The cells were exposed to baicalein for 24 h. After that, the cells were fixed overnight at a temperature of 4 °C using frozen ethanol. Next, the cells were suspended in a solution containing RNase A (100 μL). The suspension was immersed in a 37 °C water bath for 30 min. The PI dye solution (400 μL) was then gently added to the mixture, which was subsequently cultivated in the dark for 30 min. To analyze the cell cycle distribution, flow cytometry was performed using a FACSCalibur instrument (BD Biosciences, USA), followed by analysis using FlowJo software version 10.8.1.

Apoptosis analysis

An apoptosis detection kit was used to assess the impact of baicalein on H1299 and A549 cells in terms of apoptosis. After baicalein administration, the cells were collected in 100 μL of 1 × Annexin V binding solution. The cell suspension was gently mixed with 2.5 μL of Annexin V-FITC solution and 2.5 μL of PI staining solution, followed by incubation on ice for 20 min in the dark. The sample was then mixed gently with an additional 400 μL of 1 × Annexin V binding solution before fluorescence analysis using both flow cytometry and fluorescence microscopy.

LC-MS metabolomic analysis

Sample preparation: The cell samples were incubated with a mixture of 100 mg of glass beads and 1000 µL of a solution containing acetonitrile, methanol, and water (2:2:1, V/V/V) at 4 °C. Subsequently, the samples were snap-frozen in liquid nitrogen for 5 min. After thawing, the samples were homogenized using a tissue grinder at a frequency of 55 Hz for 2 min. The supernatant, which was obtained using centrifugation, was concentrated and freeze-dried. Next, the samples were dissolved in 300 µL of a solution containing acetonitrile-2-amino-3-(2-chlorobenzene)-propionic acid (4 ppm) in the ratio of 1:9 (v/v) with formic acid (0.1 %) and stored at 4 °C. Further, the samples were passed through a 0.22 μm membrane, and the filtrate was prepared for LC-MS analysis.

LC conditions: LC analysis was performed using a Vanquish UHPLC System (Thermo Fisher Scientific, USA) under the following conditions: column, ACQUITY UPLC® HSS T3 column (150 × 2.1 mm, 1.8 µm) (Waters, Milford, MA, USA); column temperature, 40 °C; injection volume, 2 μL; flow rate, 0.25 mL/min. The specific chromatographic parameters are listed in Table S1.

Mass spectrometry was performed on an Orbitrap Exploris 120 (Thermo Fisher Scientific, USA) with an electrospray ionization source to detect metabolites. To obtain relevant data, a combined approach of simultaneously acquiring MS1 and MS/MS spectra was employed. The specific chromatographic parameters are shown in Table S2.

The identification of metabolites was based on precise mass measurements (within 30 ppm) and comparisons of their MS/MS data with the information available in the HMDB (https://www.hmdb.ca) [14], massbank (https://www.massbank.jp/) [15], LipidMaps (https://www.lipidmaps.org) [16], mzclound (https://www.mzcloud.org) [17], and KEGG (https://www.genome.jp/kegg/) [18]. The spectra of standard substrates were used to validate the identified discriminant metabolites.

Glutamine uptake assay

The cells were treated with baicalein for 24 h. The culture supernatant and cell pellet were collected for quantification of glutamine content using the glutamine assay kit (Abcam, AB197011, USA) following the manufacturer's instructions.

Molecular docking

The structures of ASCT2, LAT1, and GLS1 proteins were obtained from the RCSB PDB database (https://www.pdbus.org/) in the PDB format [19]. The 3D structure of baicalein was acquired from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) as a MOL2 file [20]. Maestro Version 11.5 software was used to dehydrate the hydrogenates and minimize the amount of energy needed for the protein structures. Molecular docking was performed using the Glide module. The binding energy was used to assess the stability of the receptor–ligand matching.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from the cells using a Foregene RNA isolation Kit (Chengdu, China). The density of the RNA was determined using a Nanodrop 2000 (Thermo Scientific). A reverse transcription kit for qPCR (ABclonal, Wuhan, China) was used to obtain cDNA. Fast qPCR mix (ABclonal, Wuhan, China) was used for qRT-PCR. The expression levels of target genes were normalized to those of GAPDH. The primers used in this study were as follows: ASCT2 (SLC1A5), forward: 5′-CAGTCCTTGGACTTCGTAAAGA-3′; reverse: 5′-CCAGGATCAAGGAGATATGGTC. LAT1 (SLC7A5), forward: 5′-ACCCACCACAACAAGCAAGT-3′; reverse: 5′-CATTACTCGGCAGAGGGTGT-3′. GAPDH, forward: 5′-CCTGGTATGACAACGAATTTG-3′; reverse: 5′- CAGTGAGGGTCTCTCTCTTCC-3′.

Transfection

E. coli strains harboring plasmid DNA overexpressing ASCT2 (SLC1A5) or LAT1 (SLC7A5) were generated via MIAOLING biology (Wuhan, China). Plasmid DNA extraction was performed utilizing a TIANpure Midi Plasmid Kit (TIANGEN, Beijing, China). The DNA concentration was measured with a Nanodrop 2000 (Thermo Scientific). pLV3-NC, pLV3-ASCT2, and pLV3-LAT1 were transfected into H1299 and A549 cells using Lipo8000TM (Beyotime, Shanghai, China). The evaluation of transfection efficacy was performed by visualizing the presence of green fluorescent protein (GFP) under a fluorescence microscope. ASCT2 and LAT1 overexpression was measured via qRT–PCR and western blot analysis.

Western blot and antibodies

Total protein was extracted using RIPA lysis buffer. The protein extracts were then separated through electrophoresis on SDS-PAGE gels with different percentages of gels (10 % and 12 %). The resolved proteins were transferred onto PVDF membranes. Subsequently, specific antibodies were incubated with the PVDF membrane at a temperature of 4 °C. Proteins were detected using enhanced chemiluminescence reagent (UVP ChemStudio, Analytik Jena, Germany). The primary antibodies used in this study were as follows: anti-ASCT2 (Proteintech, 20350–1-AP), anti-LAT1 (Proteintech, 11326–1-AP), anti-GLS1 (HUAabio, ET1611-5), anti-mTOR (HUAbio, ET1608-5), anti-p-mTOR (HUAbio, HA600094), anti-Bax (HUAbio, ET1603-34), anti-Bcl-2 (HUAbio, ET1702-53), anti-cleaved caspase 9 (HUAbio, ET1610-95), anti-cleaved caspase 3 (Annoron, YC0006), anti-GAPDH (Servicebio, GB15004-100) and anti-β-actin (Servicebio, GB15003-100).

Statistical analysis

All statistical analyses were performed using SPSS 20.0 and GraphPad Prism 7, employing one-way ANOVA and nonparametric testing. The data from at least three independent experiments are presented as standard deviations (SD). Differences were considered significant at P < 0.05 (*P < 0.05, **P < 0.01, and ***P < 0.001).

Results

Baicalein inhibits tumor growth in the lung cancer xenograft mouse model

The growth-inhibitory effect of baicalein (whose structure is shown in Fig. 1A) against lung tumor xenografts were examined. The protocols for establishing the xenograft model and administration drugs were shown in Fig. 1B. The tumor size in the baicalein-treated and CDDP-treated groups was lower than that in the control group (Fig. 1C). Additionally, the tumors subjected to baicalein treatment were significantly smaller than those in the control group (Fig. 1D). The average tumor weight in the group treated with baicalein and CDDP was significantly lower than that in the control group (Fig. 1E). Notably, CDDP administration markedly decreased the bodyweight of mice. However, the bodyweight was not significantly different between the control and the baicalein-treated groups (Fig. 1G). Additionally, baicalein treatment did not result in damage to the mouse organs, including the liver, heart, spleen, lung, or kidney (Fig. 1F). In conclusion, our findings suggest that baicalein exerts minimal in vivo toxic effects and effectively suppress xenograft tumor growth.

Baicalein inhibits proliferation and induces apoptosis in NSCLC cells

Next, the in vivo effect of baicalein on NSCLC cells was examined. The effects of different doses of baicalein (0, 10, 20, 30, 40, 50, 100, 200, and 500 μM) on the proliferation of H1299 and A549 cells were examined using CCK-8 method. Baicalein significantly inhibited the proliferation of H1299 (IC₅₀ value: 25 μM) and A549 cells (IC₅₀ value: 20 μM) (Fig. 2A). The IC₅₀ value was considered the intermediate dose with half the concentration of IC₅₀ used as the low dose and twice the concentration of IC₅₀ value used as the high dose for subsequent experiments. Therefore, the experimental concentrations of baicalein for H1299 cells were 0 (control), 12.5, 25, and 50 μM, while those for A549 cells were 10, 20, and 40 μM. This study also examined the toxic effects of baicalein on BEAS-2B cells to ensure its safety profile. As shown in Fig. 2A, baicalein (0–50 μM) did not exert toxic effects on BEAS-2B cells, indicating that the dosage of baicalein used in this study was within the safe range. Consistently, the colony formation assays results revealed that baicalein significantly suppressed the proliferation of H1299 and A549 cells (Fig. 2B). Almost no clones were observed in baicalein-treated groups. Furthermore, we performed cell scratch experiments to examine the potential suppressive impact of baicalein on the migration of H1299 and A549 cells. The findings revealed that baicalein had a notable dose- and time-dependent effect on suppressing the migration of both H1299 and A549 cells (Fig. 2C).

Fig. 2 — **Proliferation and migration were inhibited by baicalein in NSCLC cells.** (A) Baicalein was administered to H1299, A549, and BEAS-2B cells at various concentrations for 24 h, after which cell viability was measured using the CCK-8 assay. (B) Colony formation assays were performed after treating H1299 and A549 cells with baicalein for 12 days to assess the impact of baicalein on cell proliferation. (C) The impact of baicalein on cell migration was assessed through cell scratch assays. **p <* 0.05, ***p <* 0.01, ****p <* 0.001 compared to the control.

Next, a dual staining method using Annexin V-FITC and PI was used to assess the apoptosis in H1299 and A549 cells. Baicalein dose-dependently increased the apoptosis rate in both H1299 and A549 cells (Fig. 3A and Fig. S1). The apoptotic rate in H1299 and A549 cells aligns with the results of the cell viability assay. Additionally, baicalein dose-dependently suppressed the protein levels of Bax, cleaved caspase 9, and cleaved caspase 3 in H1299 and A549 cells. Furthermore, baicalein upregulated the levels of Bcl-2 (Fig. 3B). Flow cytometry analysis revealed that at high treatment concentrations, baicalein resulted in cell cycle arrest in A549 cells, specifically in the G0/G1 phase. However, baicalein did not arrest the cell cycle of H1299 cells (Fig. S2). These findings indicate that baicalein inhibits proliferation and induces the apoptosis in NSCLC cells.

Fig. 3 — **Baicalein induces apoptosis in NSCLC cells** (A) The impact of baicalein on cellular apoptosis was assessed using flow cytometry analyses. (B) Densitometric quantification of the Bax/GAPDH, Bcl-2/GAPDH, cleaved caspase 9/GAPDH, and cleaved caspase 3/GAPDH ratios. Statistical analysis revealed significant differences (**p < 0.01 and ***p < 0.001) compared to those in the control group.

Baicalein altered amino acid metabolism, especially glutamine metabolism

The samples of the control and baicalein-treated groups were subjected to metabolomic analyses. MS data were used for the identification of metabolites in these groups using principal component analysis (PCA). The PCA score plot revealed that the metabolites of the control and baicalein-treated groups exhibited distinct clustering. OPLS-DA was further conducted to elucidate the baicalein-induced metabolic alterations in H1299 and A549 cells (Fig. 4A). The volcano plot illustrated that the upregulated metabolites in the baicalein-treated group relative to the control group were located on the right side of the valley, whereas those with decreased expression are positioned on the left side (Fig. 4B). Different levels of metabolite abundance are represented by varying shades ranging from red to blue (Fig. 4C). Additionally, Z-score conversion and numerical analysis revealed the correlation between multiple metabolites, especially amino acid metabolism-related metabolites (Fig. 5A). The impact of baicalein on the levels of differently abundant amino acid metabolites in H1299 and A549 cells is shown in Table 1. The findings demonstrated that baicalein primarily modulates amino acid metabolism, especially glutamine metabolism.

Fig. 4 — **Metabolomic profile alterations in baicalein-treated NSCLC cells.** (A) PCA score plots and OPLS-DA score plots were generated for H1299 and A549 cell samples obtained from both the control group and the baicalein-treated group. (B) Volcano plot analysis of differentially abundant metabolites in H1299 and A549 cells after treatment with baicalein. Metabolites showed significant upregulation are denoted by red squares, while downregulated metabolites are represented by blue dots. (C) A hierarchical clustering heatmap was generated to visualize the differentially abundant metabolites selected using t-tests/ANOVA. The degree of change is indicated by red (upregulated) and blue (downregulated). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 — **Glutamine metabolism is altered in baicalein-treated NSCLC cells.** (A) Z score plots depicting differential amino acid metabolites in H1299 and A549 cells. The y-axis represents the metabolite, while the dot color indicates different groups. The x-axis denotes the relative content of the metabolite in each group, obtained through Z score conversion. Moving toward the right signifies a greater number of metabolites within the group. (B) Significantly altered glutamine metabolites between the control and baicalein-treated groups. (C) Pathway analysis overview depicting altered metabolic pathways in H1299 and A549 cells from the control and baicalein-treated groups. (D) Changes in glutamine levels in baicalein-treated NSCLC cells.

Table 1.

Analysis of metabolic variations among amino acids.

Cell types	Metabolite name	*m/z*^a	Rt(min)^b	Expression
H1299	4-Acetamidobenzoic acid	180.065	452.6	↑
	5-Aminopentanoic acid	116.927	30.8	↑
	Aminoadipic acid	162.021	95.1	↑
	L-Threonine	118.051	85.2	↑
	L-Aspartic acid	134.044	83.1	↓
	L-Arginine	175.118	79.8	↑
	Pyrrolidonecarboxylic acid	128.9598	83.7	↑
	O-Acetylserine	148.0606	114.9	↑
	L-Histidine	154.0625	93.9	↑
	cis-4-Hydroxy-D-proline	129.9762	84.6	↑
	L-Phenylalanine	164.0719	312.6	↑
	N-Alpha-acetyllysine	188.0707	388.1	↑
	L-Lysine	197.8079	379.7	↑
	L-Glutamic acid	146.0455	77.8	↓
	N-methyl-L-glutamic Acid	162.0758	116.8	↓
	L-Glutamine	145.0622	95.8	↓
A549	cis-4-Hydroxy-D-proline	129.9762	84.6	↑
	D-Aspartic acid	114.0197	71.9	↑
	L-Phenylalanine	164.0719	312.6	↓
	Glutathione	378.0975	125.6	↓
	S-Formylglutathione	318.067	157.6	↓
	Pyroglutamic acid	129.0547	87.4	↓

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Measurement of the mass associated with the specific ion characteristic of the metabolite.

Time of retention.

Next, the effect of baicalein on glutamine metabolism was examined. Baicalein altered glutamine metabolism. Compared with those in control H1299 cells, the levels of L-glutamic acid and N-methyl-L-glutamic acid were downregulated in baicalein-treated H1299 cells. Meanwhile, the levels of glutathione (GSH), S-formylglutathione, and pyroglutamic acid in baicalein-treated A549 cells were downregulated when compared to that in control group (Fig. 5B). To screen and identify distinct metabolites, we performed an enrichment analysis of the KEGG pathways. The top 5 enriched pathways were analyzed, revealing the significant impacts of baicalein administration on phenylalanine metabolism, histidine metabolism, and phenylalanine metabolism in H1299 cells. Additionally, notable effects were observed for histidine metabolism; nicotinate and nicotinamide metabolism; D-glutamine metabolism; and alanine, aspartate and glutamate metabolism in A549 cells (Fig. 5C). These findings indicate that baicalein significantly alters the amino acid metabolite profile, especially glutamine metabolism and pathways associated with glutamine-glutamate metabolism.

The glutamine content was analyzed in the supernatant and pellet of baicalein-treated H1299 and A549 cells. Baicalein dose-dependently downregulated the glutamine levels in the cell pellet and dose-dependently increased the glutamine concentration in the supernatant (Fig. 5D). These findings indicate that baicalein inhibits cellular glutamine uptake, which is consistent with the findings of metabolomics studies.

Baicalein downregulates glutamine metabolism-related proteins and suppresses mTOR activation downstream of glutamine metabolism

Molecular docking was performed to predict the interactions between baicalein and glutamine metabolism-associated proteins, including ASCT2, LAT1, and GLS1 (Fig. 6A and Fig. S3). The docking score of baicalein and LAT1 exhibited remarkable superiority, as they formed hydrogen bonds with GLY67, SER66, and ILE63. Moreover, baicalein established hydrogen bonds with ASP (D: 327) and ARG (A: 317) while also participating in π-π stacking interactions with the amino acids PHE (D: 322) and TYR (D: 394) at the active sites of GLS1. For ASCT2, the amino acids that interact with baicalein involve ALA113 via hydrogen bonds. The molecular docking data suggested a potential 3D interaction between baicalein and glutamine metabolism-related proteins.

Fig. 6 — **Baicalein interacts with ASCT2, LAT1, and GLS1 and downregulates the glutamine-mTOR metabolic pathway** (A) Molecular docking was used to simulate the interaction between baicalein and glutamine transporters (ASCT2 and LAT1) and glutaminase (GLS1). (B) Densitometric quantification of the ASCT2/GAPDH, LAT1/GAPDH, and GLS1/GAPDH ratios. (C) Densitometric quantification of the p-mTOR/mTOR ratio. Statistical analysis indicated notable differences (**p < 0.01 and ***p < 0.001) in comparison to those in the control group.

Based on the evidence that baicalein binds to glutamine metabolism-related proteins, we investigated the influence of baicalein on the expression of proteins associated with glutamine metabolism. Our findings demonstrated that baicalein dose-dependently inhibited the expression of ASCT2, LAT1, and GLS1 (Fig. 6B). Additionally, baicalein effectively inhibited the mTOR signaling pathway, which is associated with apoptosis downstream of glutamine metabolism, by downregulating the protein levels of mTOR and p-mTOR (Fig. 6C). This impedes the glutamine-mTOR metabolic pathway, promoting apoptosis in lung cancer cells.

Baicalein suppresses mTOR activation through the inhibition of glutamine metabolism

To further investigate the ability of baicalin to target the glutamine-mTOR metabolic pathway, ASCT2 and LAT1 were overexpressed by transfecting cells with pLV3-ASCT2 and pLV3-LAT1, respectively. Baicalein treatment effectively reduced the upregulation of ASCT2 and LAT1 in H1299 and A549 cells, as observed through fluorescence microscopy (Fig. S4). The ASCT2 and LAT1 mRNA and protein expression in H1299 and A549 cells was significantly upregulated by pLV3-ASCT2 and pLV3-LAT1 compared to the pLV3-NC. The administration of baicalein resulted in a significant reduction in both the mRNA and protein levels of these genes (Fig. 7A-B). Next, the effect of baicalein on the mTOR metabolic pathway was examined in ASCT2-overexpressing and LAT1-overespressing H1299 and A549 cells. Baicalein did not markedly affect the expression levels of p-mTOR or mTOR. This indicated that the upregulation of ASCT2 and LAT1 can offset the effects of baicalein on H1299 and A549 cells. Thus, the targeted inhibition of glutamine metabolism using baicalein suppresses the mTOR metabolic pathways.

Fig. 7 — **Baicalein inhibits the glutamine-mTOR metabolic pathway** (A) qRT–PCR was conducted on H1299 and A549 cells, in which ASCT2 and LAT1 were transfected with the negative controls pLV3-ASCT2 or pLV3-LAT1. Baicalein treatment was administered during the experiment. GAPDH was utilized as a reference gene for normalization purposes. (B) Protein levels of ASCT2, LAT1, p-mTOR, and mTOR in H1299 and A549 cells transfected with pLV3-NC, pLV3-ASCT2 or pLV3-LAT1 and treated with baicalein were detected. β-Actin was used as a reference for normalizing protein loading. **p <* 0.05, ***p <* 0.01, ****p <* 0.001 versus pLV3-NC; ^#*p <* 0.05, ^##*p <* 0.01 versus pLV3-ASCT2 or pLV3-LAT1.

Baicalein induces apoptosis by targeting the glutamine-mTOR metabolic pathway

Next, the effect of baicalein on apoptosis-related proteins was examined in ASCT2-overexpressing and LAT1-overexpressing H1299 and A549 cells. Baicalein significantly downregulated the protein expression of Bax, cleaved caspase 9, and cleaved caspase 3 and upregulated the levels of Bcl-2 in the pLV3-NC-transfected group. However, the expression levels of apoptosis-related proteins in cells overexpressing ASCT2 and LAT1 were not significantly affected by baicalein (Fig. 8). These results are in accordance with the findings regarding the impact of baicalein on the mTOR metabolic pathway in cells overexpressing ASCT2 and LAT1. Thus, baicalein does not significantly affect the mTOR pathway or apoptotic proteins downstream of glutamine metabolism upon overexpression of glutamine transporters. Additionally, baicalein promotes apoptosis in lung cancer cells by specifically targeting the glutamine-mTOR metabolic pathway.

Fig. 8 — **Baicalein targets the glutamine-mTOR metabolic pathway to induce apoptosis.** The protein levels of Bax, Bcl-2, cleaved caspase 9, and cleaved caspase 3 were assessed in H1299 and A549 cells treated with baicalein after transfection with pLV3-NC, pLV3-ASCT2 or pLV3-LAT1. β-Actin was used as a reference for normalizing protein loading. **p <* 0.05, ***p <* 0.01, ****p <* 0.001 versus the pLV3-NC group.

Discussion

Given the critical treatment landscape of lung cancer, there is an escalating demand for novel anticancer therapies with reduced adverse effects, thereby stimulating research on novel natural sources of pharmacologically active compounds against lung cancer [21]. Baicalein, an active constituent derived from Scutellaria baicalensis Georgi, has been extensively investigated in the field of cancer research. Multiple studies have demonstrated the therapeutic efficacy of baicalein against NSCLC, and baicalein has been shown to exhibit multifaceted tumor inhibitory effects [22], [23], [24], [25]. Our previous studies have also reviewed the notable anti-lung cancer effects of baicalein, which potentially enhances apoptosis in lung cancer cells, induces cell cycle arrest, and suppresses metastasis in lung cancer [26]. Baicalein has potential in the treatment of NSCLC, and elucidating its mechanism of action has profound implications.

This study demonstrated that baicalein exerts growth-inhibitory effects on lung cancer xenograft models in vivo. In contrast to that between the CDDP and control groups, the bodyweight was not significant different between the baicalin low-, medium- or high-dose and control group. Therefore, baicalein has several advantages when compared with CDDP, including decreased nephrotoxicity, enhanced in vivo safety profile, and limited adverse reactions. However, further verification is required to confirm these results. Furthermore, the CCK-8 and colony formation assays results revealed that baicalein dose-dependently inhibited H1299 and A549 cell proliferation. Baicalein downregulated Bcl-2 expression and upregulated Bax expression, contributing to the upregulation of cleaved caspase 9 and cleaved caspase 3. Moreover, the results obtained from flow cytometry and fluorescence microscopy demonstrated a positive correlation between baicalein concentration and the percentage of apoptotic H1299 and A549 cells, which was consistent with the observed changes in apoptotic protein expression. These findings demonstrate the anti-lung cancer efficacy of baicalein, which is consistent with previously reported results.

Metabolic dysregulation is a critical factor driving the pathogenesis of NSCLC. NSCLC has an intricate network regulatory system characterized by alterations in gene expression, protein levels, and metabolite profiles [27], [28], [29]. Therefore, this study examined if baicalein promotes NSCLC cell apoptosis by regulating lung cancer metabolism. Baicalein significantly altered the metabolic profiles of H1299 and A549 cells, especially the amino acid metabolites. The differential amino acid metabolites between H1299 and A549 cells exhibited distinct patterns following baicalein treatment. The metabolic alterations in H1299 cells were associated with the benzoic acid, threonine, aspartic acid, arginine, histidine, proline, phenylalanine, lysine, glutamine-glutamate metabolism pathways, among others. Meanwhile, the metabolic alterations in A549 cells involved the proline, aspartic acid, phenylalanine and glutamate metabolism pathways. Although the two baicalein-treated cell lines exhibited differential metabolite profiles, several metabolic pathways were shared. The glutamine metabolism pathway has a critical role in lung cancer pathogenesis and progression. Hence, this study focused on the glutamine-glutamate metabolic pathway, which was significantly altered in baicalein-treated cells. The effect of baicalein on glutamine levels was examined using the glutamine assay kit. Baicalein downregulated the glutamine levels, which was consistent with the findings of the metabolomics analysis. These findings suggest that baicalein exerts therapeutic effects on NSCLC by modulating the glutamine metabolic pathways in lung cancer cells. Additionally, baicalein differentially affected the phenylalanine content in H1299 and A549 cells. This indicates that baicalein differentially affected the phenylalanine catabolism pathways in H1299 and A549 cells or the uptake or export of phenylalanine in a cell type-dependent manner. A comprehensive literature review revealed that the level of phenylalanine in lung cancer varied between different studies. Lu et al. reported that the phenylalanine level in lung cancer tissues was lower than that in normal paracancerous tissues [30]. In contrast, Cao et al. and Li et al. demonstrated that higher levels of phenylalanine in both lung cancer tissues and serum samples from lung cancer patients [31], [32]. However, studies on lung cancer treatment have indicated the upregulation of phenylalanine metabolism after treatment [33]. Therefore, further studies are needed to explore the expression level of phenylalanine within the lung cancer microenvironment and its impact on patient prognosis during treatment. The findings of our study align with those reported in previous studies, which reported differential phenylalanine levels between H1299 and A549 cells after baicalein treatment. However, further studies are needed to elucidate the underlying reasons behind these observations—a topic that will be addressed in our future work.

Glutamine, a vital amino acid present at high concentrations in the bloodstream, is involved in various biochemical processes, such as the production of proteins and purines and in the TCA cycle [34], [35]. Additionally, glutamine is involved in the regulation of intracellular signaling pathways, modulating malignant tumor development [36]. Recent studies have revealed the upregulation of glutamine levels in lung cancer tissues [37] and cells [38], suggesting that lung cancer cells are strongly reliant on glutamine metabolism. These results highlight the targeting glutamine metabolism is a viable approach for lung cancer. This study demonstrated that baicalein downregulates glutamine metabolism-related metabolites in H1299 and A549 cells. In particular, baicalein effectively downregulated the expression levels of L-glutamic acid and N-methyl-glutamic acid in H1299 cells, as well as suppressed the expression levels of pyroglutamic acid, S-formylglutathione, and GSH in A549 cells. Moreover, KEGG enrichment analysis of the differentially abundant metabolites revealed that baicalein exerts regulatory effects on glutamine–glutamate metabolic pathway in H1299 and A549 cells. These findings align with the reported research direction, ensuring the scientific rigor of this study. Thus, baicalein alters the metabolism of H1299 and A549 cells, regulating amino acid metabolism through the modulation of glutamine metabolism-related metabolites.

Further studies are needed to elucidate the mechanisms underlying the inhibitory effects of baicalein on the levels of the intracellular glutamine metabolite GSH. Baicalein may induce oxidative stress in cancer cells, leading to the utilization of GSH, the main endogenous antioxidant, to mitigate damage caused by reactive oxygen species (ROS) [39]. The downregulation of GSH can also be attributed to the baicalein-induced disruption of the availability of precursor amino acids, such as glutamate (required for GSH synthesis) by inhibiting GSH uptake or modulating metabolic pathways [40]. This prevents lung cancer cells from effectively utilizing GSH to counteract the high levels of ROS, leading to apoptosis induction. Furthermore, the ionic environment significantly regulates cellular homeostasis and glutamine metabolism. For example, zinc is reported to promote oxidative stress by augmenting ROS production and inhibiting GSH reductase activity [41]. However, glutamate exerts protective effects against zinc-induced toxicity through various mechanisms, such as the chelation of zinc ions and serves as a precursor for GSH synthesis or is converted into intermediates in the TCA cycle [42]. As zinc regulates cellular homeostasis and baicalein modulates the GSH levels, this study aimed to further investigate the underlying mechanism regulating zinc and glutamine metabolism during cancer cell apoptosis. Baicalein promoted apoptosis in lung cancer cells by regulating glutamine metabolism in H1299 and A549 cells. This provides a foundation for future studies on the anti-lung cancer mechanism of baicalein.

The two major transporters involved in glutamine uptake are ASCT2 and LAT1. ASCT2 belongs to the solute carrier 1 family and functions as a sodium-dependent transporter, facilitating the movement of neutral amino acids across cell membranes in peripheral tissues [43]. Its functional significance lies in its crucial role in mediating more than 50 % of the transmembrane influx of glutamine [44]. LAT1 facilitates the transport of glutamine from the cells to the extracellular space and simultaneously allows leucine to enter cells [45]. Thus, LAT1 maintains cellular glutamine homeostasis. Glutaminase catalyzes the conversion of intracellular glutamine to glutamate, which subsequently enters the TCA cycle and participates in mitochondrial energy metabolism [46]. The expression levels of both the glutamine transporters and glutaminase have been extensively demonstrated to be upregulated in lung cancer, promoting the uptake and metabolism of glutamine by lung cancer cells [47]. Thus, the inhibition of ASCT2, LAT1, and GLS can effectively suppress glutamine metabolism and impair the proliferation of lung cancer cells, offering a novel therapeutic approach for lung cancer. Furthermore, mutations in the mTOR signaling pathway-related proteins constitute a prevalent subset of activating mutations in lung cancer. Glutamine is reported to be indispensable for the activation of the mTOR pathway, a pivotal signaling hub regulating protein translation, cellular proliferation, and apoptosis. The activation of the mTOR pathway is facilitated by LAT1 through its interaction with glutamine and transportation of essential amino acids, such as leucine into the cellular environment. ASCT2 plays a vital role in regulating intracellular amino acid levels and facilitating LAT1 function, thereby exerting a substantial impact on the mTOR pathway [48]. The combined effect of ASCT2 and LAT1 enhances the activation of the mTOR signaling pathway, facilitating an increased energy supply for the proliferation of lung cancer cells and simultaneously suppressing apoptosis [49]. Rajasinghe et al. demonstrated that glutamine facilitates the transport of crucial amino acids in rapidly dividing cells, promoting mTOR activation in A549 and H1299 lung cancer cells [50]. Activated mTOR stimulates the production of nucleotides and proteins by activating its downstream effector 4EBP1, facilitating cancer cell proliferation and suppressing apoptosis[51], [52]. Thus, targeting the glutamine-mTOR metabolic pathway is a potential therapeutic strategy for lung cancer. The signaling pathway is depicted in Fig. 9.

Fig. 9 — **Baicalein induces NSCLC cell apoptosis by blocking the glutamine-mTOR metabolic pathway** Lung cancer cells are dependent on glutamine and exhibit upregulated glutamine metabolism. Glutamine is transported into the cell through specific transporters (ASCT2 and LAT1), while glutamate is generated by glutaminase (GLS1) and subsequently enters the TCA cycle. Baicalein effectively suppressed the protein expression levels of ASCT2, LAT1, and GLS1, exerting inhibitory effects on glutamine metabolism. Additionally, baicalein inhibited the mTOR pathway downstream of glutamine, which plays a crucial role in apoptosis induction. Furthermore, baicalein effectively suppressed proliferation, migration and nucleotide and protein biosynthesis in H1299 and A549 cells, promoting apoptosis in lung cancer cells.

This study demonstrated that baicalein stably binds to ASCT2 and LAT1 through hydrogen bonding, as well as to GLS through both hydrogen bonding and π-π stacking, highlighting the potential molecular interactions between baicalein, ASCT2, LAT1, and GLS. However, the specific amino acid binding sites of baicalein were not investigated in this study. Molecular docking identified key residues that potentially interacted with baicalein (ASP D: 327, ARG A: 317, PHE D: 322, and TYR D: 394 and ALA113). Based on the factors, such as the ionic environment, steric hindrance, and nuclear charge in the lung tumor microenvironment, the effect of mutating the residues predicted to interact with baicalein on resistance to baicalein treatment must be examined, which will be addressed in future studies.

Western blot analysis in this study revealed that baicalein downregulates the protein expression of ASCT2, LAT1, and GLS1 in H1299 and A549 cells, which are involved in glutamine transport and metabolism. As ASCT2, LAT1, and GLS1 are involved in glutamine metabolism, their interaction with baicalein was hypothesized to yield consistent outcomes. The consistent outcomes can be due to the multifaceted effect of baicalein on the glutamine metabolic pathway. Therefore, the inhibition of GLS1 may upregulate the intracellular glutamine levels provided that glutamine transport is unaffected. However, the simultaneous downregulation of ASCT2 and LAT1 may downregulate the influx of glutamine, potentially counterbalancing the effect of GLS1 inhibition. Thus, the overall intracellular concentration of glutamine may remain low, not because of its conversion to glutamate but due to reduced import. This can explain the reason for the baicalein-induced downregulation of ASCT2, LAT1, and GLS resulting in a uniform inhibitory effect on the glutamine pathway. Additionally, the downregulation of intracellular glutamine can also trigger cellular stress responses that further inhibit glutamine utilization pathways. Moreover, the downregulation of multiple proteins in a metabolic pathway can exert an amplifying effect, especially when both transporters and enzymes in the same pathway are targeted by a therapeutic compound. To address this complexity and resolve the apparent paradox, this study quantified the glutamine levels in baicalein-treated cells to directly measure the intracellular glutamine levels. ASCT2, LAT1, and GLS1 collectively play crucial roles in the glutamine metabolic pathway, facilitating the proliferation of lung cancer cells. However, the regulatory mechanisms underlying the effect of baicalein on ASCT2 and LAT1 transcription or posttranscriptional modification were not elucidated in this study. This study hypothesized that baicalein downregulates ASCT2 and LAT1 by inhibiting their transcriptional activity. Further studies are needed to validate this hypothesis.

Additionally, the mechanism through which baicalein inhibits glutaminase activity will be examined in future studies. Yang et al. demonstrated that baicalein effectively suppresses energy metabolism in lung cancer cells by downregulating the glutamine/glutamate/α-KG metabolic axis [35]. Consequently, the inhibition of glutaminase activity can impede glutamine utilization in the TCA cycle, leading to the downregulation of α-KG levels and ATP synthesis and the suppression of lung cancer cell proliferation. Thus, glutaminase activity inhibition is a potential therapeutic strategy for lung cancer. Furthermore, glutamine metabolism is regulated via a feedback mechanism[53]. This study hypothesized that the glutaminase inhibition-induced upregulation of intracellular glutamine levels promotes negative feedback regulation of glutamine uptake by transporters. Simultaneously, the entry of glutamine into the TCA cycle is suppressed, impairing the energy supply for the proliferation of lung cancer cells and consequently inhibiting their growth. However, further studies are needed to validate this hypothesis.

Finally, this study examined the effect of baicalein on the mTOR signaling pathway. Baicalein inhibited the mTOR metabolic pathway downstream of glutamine metabolism, which is involved in inducing apoptosis, and downregulated the levels of the mTOR and p-mTOR in H1299 and A549 cells. These findings suggest that baicalein may exert proapoptotic effects on lung cancer cells through the inhibition of the glutamine-mTOR metabolic pathway. Furthermore, the regulatory effect of overexpressing the glutamine transporters ASCT2 and LAT1 on glutamine metabolism and the mTOR signaling pathway was analyzed. Baicalein did not significantly affect the mTOR metabolic pathway or apoptosis-related proteins in ASCT2-overexpressing and LAT1-overexpressing cells. Therefore, baicalein exerted targeted inhibitory effects on ASCT2 and LAT1, inducing apoptosis in lung cancer cells by specifically targeting the glutamine-mTOR metabolic pathway. This validated the findings of this study. However, gene knockout experiments will further validate the findings of this study.

Conclusion

This study demonstrated that baicalein induces cell apoptosis by suppressing the mTOR pathway through the downregulation of glutamine metabolism. Although this study has some limitations, the findings of this study demonstrated that baicalein is a novel glutamine-targeting therapeutic agent that induces apoptosis by inhibiting the glutamine-mTOR metabolic pathway in lung cancer. Thus, baicalein is a potential therapeutic agent for NSCLC.

CRediT authorship contribution statement

Jingyang Li: Conceptualization, Methodology, Validation, Writing – original draft. Di Zhang: Methodology. Shaohui Wang: Writing – review & editing. Peng Yu: Formal analysis, Methodology. Jiayi Sun: Methodology. Yi Zhang: Writing – review & editing. Xianli Meng: Project administration, Software, Supervision. Juan Li: Project administration, Software, Supervision. Li Xiang: Conceptualization, Validation, Visualization, Funding acquisition, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by Sichuan Science and Technology Program (No. 2023NSFSC0684), and the Xinglin Scholar Research Premotion Project of Chengdu University of TCM (No. QJRC2022053).

Footnotes

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jare.2024.02.023.

Contributor Information

Xianli Meng, Email: mxl999@cdutcm.edu.cn.

Juan Li, Email: lijuan74@scszlyy.org.cn.

Li Xiang, Email: xianglydr@cdutcm.edu.cn.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1

mmc1.docx^{(1.2MB, docx)}

Supplementary data 2

mmc2.docx^{(17.9KB, docx)}

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Associated Data

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Supplementary Materials

Supplementary data 1

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Supplementary data 2

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PERMALINK

Baicalein induces apoptosis by inhibiting the glutamine-mTOR metabolic pathway in lung cancer

Jingyang Li

Di Zhang

Shaohui Wang

Peng Yu

Jiayi Sun

Yi Zhang

Xianli Meng

Juan Li

Li Xiang

Graphical abstract

Highlights

Abstract

Introduction

Objective

Methods

Results

Conclusion

Introduction

Materials and methods

Regents and drugs

Cell culture

Lung cancer xenograft mouse model

CCK-8 assay

Colony formation assay

Wound-healing assay

Cell cycle analysis

Apoptosis analysis

LC-MS metabolomic analysis

Glutamine uptake assay

Molecular docking

RNA extraction and quantitative real-time PCR (qRT-PCR)

Transfection

Western blot and antibodies

Statistical analysis

Results

Baicalein inhibits tumor growth in the lung cancer xenograft mouse model

Fig. 1.

Baicalein inhibits proliferation and induces apoptosis in NSCLC cells

Fig. 2.

Fig. 3.

Baicalein altered amino acid metabolism, especially glutamine metabolism

Fig. 4.

Fig. 5.

Table 1.

Baicalein downregulates glutamine metabolism-related proteins and suppresses mTOR activation downstream of glutamine metabolism

Fig. 6.

Baicalein suppresses mTOR activation through the inhibition of glutamine metabolism

Fig. 7.

Baicalein induces apoptosis by targeting the glutamine-mTOR metabolic pathway

Fig. 8.

Discussion

Fig. 9.

Conclusion

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgements

Footnotes

Contributor Information

Appendix A. Supplementary data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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