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Introduction

Lung cancer is the leading cause of cancer-related death in the world, followed by colorectal, liver and stomach cancers (1,2), and non-small cell lung cancer (NSCLC) accounts for ~85% of patients with lung cancer (3,4). Lung squamous cell carcinoma (LSCC) is the second most common histological subtype and is associated with poor prognosis (5). Despite the diagnosis and treatment of NSCLC have been advanced, the main treatment strategy for LSCC remains surgical resection or radiation with chemotherapy (6,7). However, the recurrence rate was high among the patients who suffer postoperative (8). Previously, PD1/PD-L1 immunotherapies have improved the clinical outcome of patients with NSCLC and demonstrated a notable efficacy (9–11), and targeted therapies have improved outcomes for certain subtypes, but patients with LSCC are not likely to benefit from these treatments (12). Thus, it is imperative to search for new drugs and effective therapeutic strategies against LSCC to reduce the mortality rate.

Nowadays, numerous natural medicines have been proved to inhibit or reverse the progression of several cancers and appeared to be promising novel anticancer drugs due to high efficiency and low toxicity. Licochalcone A (LCA) is a natural and useful flavonoid extracted from licorice, which possesses several pharmacological properties such as antibacterial (13), antitumor (14), anti-inflammatory (15,16) and antioxidation properties (17). In recent decades, studies have demonstrated that LCA exhibited potent antitumor effects in several epithelial carcinoma cells by regulating numerous signaling pathways, such as the oxidative stress, the mitogen-activated protein kinase (MAPK) and the apoptosis pathway (18). For example, LCA induced apoptosis of gastric cancer cells by activating the MAPK and PI3K/AKT signaling pathways (17). However, LSCC is a distinct subtype of NSCLC, and the antitumor effects of LCA on LSCC remain unknown.

F-box protein 5 (FBXO5) is involved in the regulation of proliferation, apoptosis, epithelial-mesenchymal transition and drug resistance (19,20). FBXO5 promotes proper mitotic entry by blocking the anaphase-promoting complex/cyclosome activity (21,22). It functions as a cell cycle regulator and plays a crucial role in proliferation and tumorigenesis (23,24). Evidence indicates that FBXO5 is upregulated in various malignant tumors compared with matched normal tissues. Previous research indicated that high FBXO5 expression would cause genomic instability and mitotic catastrophe, contributing to tumorigenesis of breast cancer (25) and esophageal squamous cell carcinoma (26). In addition, existing evidence has suggested that FBXO5 expression level was higher in LSCC compared with normal tissue, and the overexpressed FBXO5 leads to shorter overall survival (OS) in patients with LSCC (27). Therefore, FBXO5 may be a potential oncogene and therapeutic target in LSCC.

The present study aimed to detect the effect of LCA on cell proliferation, cell cycle, apoptosis and gene expression on LSCC in vitro and the antitumor activities in vivo, and further explore the underlying mechanisms.

Materials and methods

Cell culture

The LSCC cells H226 (cat. no. TCHu235), H1703 (cat. no. SCSP-593) and human bronchial epithelial cells (HBE; cat. no. CL-0346) were maintained in RPMI-1640 (cat. no. C11875500BT) media supplemented with 10% fetal bovine serum (cat. no. 10099-141C; Gibco; Thermo Fisher Scientific, Inc.) and cultured in a 37°C incubator set with 5% CO2, humidified environment. H226 and H1703 cells were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. HBE cells were purchased from Procell Life Science & Technology Co., Ltd.

Chemical reagents and antibodies

LCA (Cas. no. 58749-22-7; Fig. 1A; cat. no. S01015) was purchased from Nanjing Dilger Medical Technology Co. Ltd. and dissolved in dimethyl sulfoxide (DMSO; cat. no. D8371; Beijing Solarbio Science & Technology Co., Ltd.). FITC Annexin V Apoptosis Detection kit was acquired from BD (cat. no. 556547, BD Pharmingen; BD Biosciences). CDK2 (1:1,000; cat. no. ab32147; Abcam), CDK4 (1:1,000; cat. no. ab108357; Abcam), cyclin D1 (1:5,000; cat. no. ab134175; Abcam), cyclin E (1:1,000; cat. no. WL01072; Wanleibio Co., Ltd.), cleaved caspase 3 (1:1,000; cat. no. AF7022; Affinity Biosciences, Ltd.), cleaved poly(ADP-ribose)polymerase-1 (PARP1) (1:5,000; cat. no. ab32064; Abcam), ERK (1:1,000; cat. no. ab184699; Abcam), phosphorylated (p-)ERK (1:1,000; cat. no. ab201015; Abcam), p38 MAPK (1:1,000; cat. no. 8690; Cell Signaling Technology, Inc.), p-p38 MAPK (1:1,000; cat. no. 4511; Cell Signaling Technology, Inc.), FBXO5 (1:5,000; cat. no. ab184950; Abcam), GAPDH (1:5,000; cat. no. 60004-1-IG; Proteintech Group, Inc.), HRP-goat anti-mouse (1:5,000; cat. no. EM35110-01; Beijing Emarbio Science & Technology Co., Ltd.) and HRP-goat anti-rabbit antibody (1:5,000; cat. no. EM35111-01; Beijing Emarbio Science & Technology Co., Ltd.).

Figure 1. LCA inhibits lung squamous cell carcinoma cell viability and proliferation. (A) Chemical structure of LCA. (B and C) The viability of H226 and H1703 cells after treatment with LCA was detected by MTT. (D) The viability of HBE cells after treatment with LCA was detected by MTT. (E and F) EdU staining assay was used to detect cell proliferative ability of H226 cells (scale bar, 200 µm). (G and H) EdU staining assay was used to detect cell proliferative ability of H1703 cells (scale bar, 200 µm). The experiments were repeated at least three times (n=3). *P<0.05 and **P<0.01 vs. 0 µM. LCA, licochalcone A; EdU, 5-ethynyl-2′-deoxyuridine.

MTT detection

The cells were seeded in 96-well plates at a density of 5×103 cells per well in a 100-µl medium and incubated overnight. The H226 and H1703 cells were cultured with LCA at a dose of 0, 2, 5, 10, 20 and 40 µM for 24, 48 and 72 h. HBE cells were cultured in the medium with a concentration of 0, 10, 20, 40, 60 and 80 µM LCA for 24 and 48 h. After treatment, 20 µl of 5 mg/ml MTT (Beijing Solarbio Science & Technology Co., Ltd.) was added to the well and incubated for at least 3 h in an incubator set at 37°C, then the medium was removed from each well and 200 µl DMSO was added. The absorbance at the wavelength of 490 nm (OD value) was measured by Infinite M200 Pro NanoQuant (Tecan Group, Ltd.) and then the cell viability and IC50 value were measured according to the OD value. Based on the result of MTT, the IC50 was calculated for H226 and H1703 cells. Subsequently, LCA was selected at the dose of 0, 10, 20 and 40 µM in the following experiments.

5-ethynyl-2′-deoxyuridine (EdU) staining

Cell proliferation was detected using BeyoClick™ EdU-488 Assay kit (cat. no. C0071S; Beyotime Institute of Biotechnology). LSCC cells were plated in six-well plates and incubated overnight, then treated with different doses of LCA for 16 h. EdU solution was prepared and final 10 µM EdU working solution was added to the medium for incubating for 3 h at 37°C. Subsequently, 4% paraformaldehyde was used to fix cells for 15 min and washed off with PBS containing 3% BSA (Beijing Solarbio Science & Technology Co., Ltd.). Next, the cells were permeabilized with enhanced immunostaining permeabilization buffer (cat. no. P0097; Beyotime Institute of Biotechnology) for 15 min and washed. Then, the EdU detection was performed followed the manufacturer's protocols and the nuclei were stained with Hoechst 33342 for 10 min while avoiding light at room temperature. Fluorescence images were obtained by Olympus microscope (Olympus Corporation). The number of EdU-positive cells was counted and calculated in five randomly selected fields. Cell proliferation rate=EdU-positive cells/DAPI-positive cells ×100%. The experiments were performed in triplicate.

Cell cycle assay

The cell cycle distribution was evaluated by FCM using propidium iodide (PI; cat. no. P8080; Beijing Solarbio Science & Technology Co., Ltd.). H226 and H1703 cells were cultured in 6 cm dishes and treated with LCA (0, 10, 20 and 40 µM) for 24 h in a 37°C incubator set with 5% CO2, humidified environment. After treatment, the cells were harvested and resuspended, and subsequently fixed with 70% cold ethyl alcohol overnight at 4°C. The next day, cells were centrifuged at 1,000 × g for 10 min and washed. Following this, the cells were incubated with RNase A and 500 µl PI in a 37°C water bath for 30 min. The cell cycle distribution of LSCC was measured on the Accuri C6 flow cytometer (BD Biosciences) and analyzed with FlowJo 7.6 software (FlowJo LLC).

Cell apoptosis assay

Cellular apoptosis was evaluated by FCM using FITC-Annexin V Apoptosis Detection Kit. H226 and H1703 cells were treated with LCA (0, 10, 20 and 40 µM) for 24 h in a 37°C incubator set with 5% CO2, humidified environment. After treatment, the cells were harvested and assayed according to the manufacturer's instructions. The apoptotic rate was analyzed by the Accuri C6 flow cytometer (BD Biosciences).

4D-data-independent acquisition (4D-DIA) proteomics

H1703 cells were cultured overnight in 100-mm culture dishes until the density reached 40–50%. Subsequently, the cells were treated with or without 40 µM LCA for 24 h in a 37°C incubator set with 5% CO2, humidified environment. The medium was aspirated, and the cells were washed with pre-cooled PBS solution twice. Next, the cells were gently scraped off with cell scraper and transferred to a centrifuge tube, centrifuged at 4°C, 300–500 × g for 5 min, the supernatant was discarded and washed once with PBS. PBS was added to make cell suspensions and 1×107 cells were counted in each sample by a cell counter. The cells were immediately frozen with liquid nitrogen for 5–10 min, then stored in a −80°C refrigerator for proteomics assay. Before the differential protein expression assays, the qualitative and quantitative protein analysis of the 4D mass spectrometry data was performed by DIA-NN (v1.8.1) software. The Pearson's correlation analysis was used to evaluate the correlation among samples. Principal component analysis (PCA) was used to understand the variances between the different sample groups.

Network pharmacological analysis

PubChem (https://pubchem.ncbi.nlm.nih.gov/) was used for searching ‘LCA’ and obtained the SMILES number, the potential targets genes of LCA were predicted by SwissTarget Prediction (http://www.swisstargetprediction.ch/). The potential LSCC therapeutic targets were identified by DisGeNET databases (https://www.disgenet.org/search). The Sangerbox (http://sangerbox.com/) was used to perform the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially expressed genes (DEGs). The DEGs were imported into the Venny 2.1 online tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html) to screen the overlapping genes. Protein-protein interaction (PPI) analysis was performed using the STRINGDB (http://string-db.org/) protein interaction database.

Bioinformatics analyses

The TIMER2.0 database (http://timer.cistrome.org/) and UALCAN database (https://ualcan.path.uab.edu/analysis) were used to study the DEGs between tumor and adjacent normal tissues based on The Cancer Genome Atlas (TCGA) project. In TIMER 2.0 database, the differential expression between tumor and adjacent normal tissues for any gene of interest across all TCGA tumors. Distributions of gene expression levels are displayed using box plots. The statistical significance computed by the Wilcoxon test is annotated by the number of stars (*P<0.05, **P<0.01 and ***P<0.001).

Molecular docking

The canonical 2D structure of Licochalcone A (CAS no. 58749-22-7) was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). ChemOffice software (https://www.downza.cn/) was used to optimize and minimize the structure energy of LCA, then convert the 2D structure to 3D. The ID of FBXO5 (Q9UKT4) was obtained from Uniprot database (https://www.uniprot.org/), and the crystal structure of the FBXO5 (PDB ID: 2M6N) was obtained from PDB database (http://www.rcsb.org/). The mol2 files of LCA and the PDB files of FBXO5 were imported to AutoDocK Tools, converted to PDBQT format and parameters were set to determine the active pocket. Molecular docking was performed with AutoDock 1.5.6 software. The docking image was acquired using PyMol software (https://pymol.org/2/).

Transient transfection of small interfering siRNA

siRNAs against FBXO5 (si-FBXO5) and the negative control siRNA (si-NC) were purchased from CENTRAL BIOL (www.generalbiol.com/). The sequences of si-FBXO5 and si-NC are listed in Table SI. H1703 cells were transfected with si-FBXO5 and si-NC for 24 h at 37°C using Lipofectamine® 3000 (cat no. L3000-015; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The concentration of siRNAs used in the present study was 50 nM. Subsequent experiments were performed 24 h after siRNA transfection.

In vivo xenograft experiment

All operation procedures of this animal study were approved by the Institutional Animal Care and Use Committee of Guilin Medical University (approval no. GLMC-IACUC-2021016; Guilin, China). A total of 20 BALB/c-nu mice (male) at the age of 4 weeks (mean weight, 16±0.5 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd. The mice were housed in pathogen-free environment with controlled temperature (20–25°C), humidity (40–70%), and free access to rodent chow and water. After 5 days of acclimation, equal volumes (200 µl) of 4×106 H1703 cells resuspended in PBS (4×106 cells/mouse) were injected into the right flank of mice. After 7 days, when the tumors were visible, the mice were divided into 4 groups randomly (n=5): i) The control group (saline containing 20% SBE-β-CD), ii) the LCA 7.5 mg/kg group, iii) the LCA 15 mg/kg group and iv) the cisplatin (CDDP) 2 mg/kg group. LCA was dissolved in saline containing 20% SBE-β-CD. The mice were intraperitoneally injected with 200 µl of drugs once a day for 10 days. The body weight, length and width of the tumor were measured every day, and the tumor volume was calculated as follows: length × width2/2. During the process of the animal experiments, none of the mice succumbed. After 10 days of LCA administration, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg) and euthanized by cervical dislocation. The humane endpoints included the tumor volume in the control group (if it reached ~1,500 mm3) and the dietary activities (if they were interfered). The tumors and vital organs were excised and weighted after the absence of a heartbeat and breath, one portion of the tumor was frozen with liquid nitrogen immediately for western blotting, while the other portion was fixed in paraformaldehyde for histopathological experiments. The vital organs (heart, liver, spleen, lung and kidney) were fixed in paraformaldehyde for hematoxylin and eosin (H&E) staining.

Western blot analysis

LSCC cells treated with LCA at the concentration of 0, 10, 20 and 40 µM. After treatment, the cells were harvested and lysed in cold RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) with PMSF and protease inhibitor for 30 min on ice. The supernatant was collected after being centrifuged and the concentration was measured by BCA kit (Beyotime Biotechnology). A total of 20 µg of protein was separated by SDS-PAGE (10–12%) electrophoresis and further transferred onto the nitrocellulose membrane. Subsequently, the membrane was blocked with 5% non-fat milk in TBST buffer at room temperature for 2 h and was then incubated overnight under the primary antibodies at 4°C. After washed for 3 times with TBST, the membrane was incubated with secondary antibody for 1 h at room temperature the next day. The specific bands were visualized by ECL kit (cat. no. P0018S; Beyotime Institute of Biotechnology) and analyzed by ImageJ (v1.48) software (National Institutes of Health).

Histopathology

Tumor tissues and vital organs were fixed in paraformaldehyde (4% PFA) for 24 h at room temperature. A serial alcohol gradient was used to dehydrate the tissues, after dehydration, the tissues were embedded on paraffin wax blocks. The paraffin blocks were cut to 4-µm sections for H&E staining (hematoxylin for 5 min and eosin for 3 min) at room temperature. Images were captured using a light microscope (Olympus Corporation).

Statistical analysis

All data were performed using the GraphPad Prism 5 (Dotmatics) and SPSS software version 23.0 (IBM Corp.). The paired Student's t-test was used on data between two groups after testing for normality and homogeneity of variance, and one-way ANOVA was used for multiple comparisons followed by the post hoc Bonferroni test. The data was expressed as the mean ± standard deviation (SD), and P<0.05 was considered to indicate a statistically significant difference.

Results

LCA inhibits the proliferation in LSCC cells but not in HBE cells in vitro

MTT assay revealed that LCA significantly inhibited the cell viability in H226 (Fig. 1B) and H1703 (Fig. 1C) cells. Furthermore, the inhibitory effect of LCA (5, 10, 20 and 40 µM) on LSCC cells was in a dose and time-dependent manner. The MTT result showed that the cell viability of HBE was 93.31±2.98 and 89.66±2.25% when the concentration of LCA was 60 µM at 24 and 48 h (Fig. 1D). These results indicated that LCA exhibited no significant cytotoxicity against HBE cells in a treatment with up to 60 µM of the drug. EdU staining assay indicated that the proliferation rate was significantly reduced in H226 (Fig. 1E and F) and H1703 cells (Fig. 1G and H) after LCA treatment. These results revealed that LCA suppressed the proliferation capacity of LSCC cells.

LCA induces cell cycle arrest in LSCC cells

To assess the effect of LCA on cell cycle, the distribution of cell cycle in H226 and H1703 cells after treatment with different concentrations of LCA was investigated by FCM. As illustrated in Fig. 2A and B, the proportion of G1 phase at concentrations of 0, 10, 20 and 40 µM in H226 cells were 43.70±0.78, 48.40±2.03, 54.67±2.71 and 61.33±2.19%, respectively, and the ratio of cells at G2 phase was decreased. LCA significantly induced G1 phase arrest in a dose-dependent manner (10, 20 or 40 vs. control: P<0.05, P<0.01 or P<0.01) in H226 cells. Similarly, in H1703 cells, the G1 phase proportion at concentrations of 0, 10, 20 and 40 µM were 52.03±6.09, 61.93±5.92, 73.47±9.07 and 83.40±3.15%, respectively, and the ratio of cells at S phase decreased. LCA at concentrations of 20 and 40 µM could significantly increase the G1 phase proportion (20 or 40 vs. control: P<0.01 or P<0.01) in H1703 cells. These results indicated that LCA treatment significantly increased the distribution of G1 phase in a dose-dependent manner in a certain range of concentrations. According to the results, the expression level of proteins related to G1 phase was further explored. Western blot analysis results (Fig. 2C and D) indicated that LCA decreased the protein levels of cyclin D1, cyclin E, CDK2 and CDK4 in H226 cells and H1703 cells. These results suggested that LCA caused G1 phase arrest by regulating the proteins related to G1 phase.

Figure 2. The effect of LCA on cell cycle in lung squamous cell carcinoma cells. (A and B) Analysis of the cell cycle proportion of H226 and H1703 cells after treated with LCA (0, 10, 20 and 40 µM) by flow cytometry. (C and D) The protein expression of CDK2, CDK4, cyclin D1 and cyclin E in H226 and H1703 cells determined by western blot analysis after treatment with LCA. The intensity of the bands was quantified by ImageJ and GAPDH was used as control. The experiments were repeated at least three times (n=3). *P<0.05 and **P<0.01 vs. 0 µM. LCA, licochalcone A.

LCA induces apoptosis in LSCC cells

The cell apoptosis of LSCC induced by LCA were performed through FCM with the annexin V/PI double staining method. After LCA treated for 24 h, the apoptotic rates of H226 cells at concentrations of 0, 10, 20, and 40 µM were 6.13±1.16, 7.67±1.37, 14.07±1.70 and 28.20±2.47%, respectively. Similarly, in H1703 cells, the apoptotic rates were 5.03±0.64, 8.37±0.95, 14.17±3.65 and 21.93±3.35%, respectively (Fig. 3A and B). At the concentration of 20 and 40 µM, the apoptotic rates were significantly increased in both H226 and H1703 cells compared with control (P<0.01). These results demonstrated that LCA could induce apoptosis in dose-dependent manner within the range of certain concentrations. As demonstrated in Fig. 3C and D, western blot analysis results indicated that LCA significantly increased the expression level of Bax, cleaved PARP1 and cleaved caspase 3 in H226 and H1703 cells, while decreased the level of Bcl-2. Overall, these results indicated that LCA induced cell apoptosis by the mitochondrial-mediated pathway in LSCC cells.

Figure 3. The effect of LCA on cell apoptosis in lung squamous cell carcinoma cells. (A and B) The apoptotic rates of H226 and H1703 cells after treatment with LCA were detected by flow cytometry. (C and D) The level of apoptosis-related proteins of H226 and H1703 cells determined by western blot analysis. The experiments were repeated at least three times (n=3). *P<0.05 and **P<0.01 vs. 0 µM. LCA, licochalcone A; cle-, cleaved; PARP1, poly(ADP-ribose)polymerase-1.

Proteomic analysis

4D-DIA quantitative proteomic technique was used to identify the differential proteins and potential molecular mechanisms after LCA treatment in LSCC cells. Most of the peptides were distributed at 7–20 amino acids and the distribution of peptide length met the quality control requirements (Fig. 4A). Box plots and violin plots (Fig. 4B) indicated favorable consistency within the sample group. Pearson's correlation coefficients indicated high correlation in the expression patterns between the samples (Fig. 4C). PCA revealed satisfactory repeatability in the samples of control and LCA treatment group (Fig. 4D). Histogram (Fig. 4E) and Volcano plots (Fig. 4F) demonstrated a total of 1101 differentially expressed proteins (DEPs) after LCA treated, including 616 upregulated and 485 downregulated DEPs. The heatmap revealed the clustering of DEGs between the control and LCA-treatment group (Fig. 4G). Enrichment analyses of these DEPs based on KEGG databases revealed that PI3K/Akt signaling pathway, AMPK signaling pathway, Hippo signaling pathway, FoxO signaling pathway and MAPK signaling pathway were enriched (Fig. 4H).

Figure 4. Proteomic analysis of control and the LCA-treated LSCC cells. (A) Distribution of peptide length. (B) Distribution of the sample abundance. (C) Pearson's correlation analysis of the protein expression patterns. (D) Principal component analysis. (E and F) The histogram and volcano plots demonstrated the 1101 DEPs including 616 upregulated and 485 downregulated after LCA treatment in LSCC cells. (G) The heatmap revealed the different expression patterns of the differentially expressed genes. (H) Kyoto Encyclopedia of Genes and Genomes pathway analysis. LCA, licochalcone A; LSCC, lung squamous cell carcinoma; ctrl, control; DEPs, differentially expressed proteins.

Network pharmacological analysis

The SwissTarget Prediction database search revealed that 101 relevant targets for LCA were obtained. Disgenet databases identified 671 potential therapeutic targets related to LSCC. Comparative analysis of the targets obtained 17 overlapping genes (Fig. 5A). Moreover, the KEGG Pathway enrichment results detected that the DEPs mainly involved in pathways in cancer, microRNAs in cancer, PI3K/Akt signaling pathway and MAPK signaling pathway (Fig. 5B). These signaling pathways may be closely related to the effects of LCA treatment in LSCC. A PPI network was constructed among the 17 differentially expressed proteins. The interacting proteins were MAPK1 (ERK2), MAPK14 (p38 MAPK), EGFR, PTGS2, PDGFRB, MMP9, MMP14, GLI1, MDM2, HPGDS, ABCB1, RET and THRB (Fig. 5C); and the MAPK1 (ERK2), MAPK14 (p38 MAPK), EGFR, PTGS2, PDGFRB were the core targets. Combined with the results of proteomics, it was hypothesized that LCA inhibits proliferation of LSCC via suppression of the MAPK signaling pathways. Subsequently, western blotting results demonstrated that LCA treatment inhibited the expression of p-p38 MAPK and p-ERK1/2 in a dose-dependent manner while the total p38 MAPK and ERK1/2 were not changed in both H1703 and H226 cells (Fig. 5D-F).

Figure 5. Network pharmacology analysis of the therapeutic target of LCA against LSCC. (A) Venn diagram revealed 17 overlapping genes for LCA target genes and LSCC-related genes. (B) The bubble chart illustrated enriched Kyoto Encyclopedia of Genes and Genomes pathways related to the therapeutic target. (C) Protein-protein interaction analysis revealed interaction of the 17 overlapping genes. (D-F) Western blot analysis results revealed the protein expression of p38 MAPK, p-p38 MAPK, ERK1/2 and p-ERK1/2 after treatment with LCA (0, 10, 20 and 40 µM) in LSCC cells. The experiments were repeated at least three times (n=3). **P<0.01 vs. control. LCA, licochalcone A; LSCC, lung squamous cell carcinoma; p-, phosphorylated.

Bioinformatics analysis

The top 5 upregulated and downregulated DEPs obtained from proteomic analysis were presented in Table I. Bioinformatics analysis of these 10 DEPs through TIMER databases demonstrated that FBXO5 level was higher in 14 types of cancer, such as LIHC, lung adenocarcinoma, LSCC, bladder and breast cancer (Fig. 6A). The FBXO5 expression was significantly elevated in LSCC compared with the corresponding normal tissues through TCGA data (Fig. 6B). Moreover, the expression of FBXO5 was significantly elevated in each pathological stage (Fig. 6C). In addition, the result of western blot analysis demonstrated that FBXO5 expression decreased significantly in a concentration-dependent manner after LCA treatment (Fig. 6D and E). Therefore, it was perceived that FBXO5 may be a potential target of LCA in the LSCC cells. Molecular docking has been widely used in prediction of the interactions between a target protein (enzyme) and drug molecules (ligands) at a molecular level (28). In the present study, the result revealed that LCA is tightly bound to the active site of FBXO5 with a binding energy of-5.58 kcal/mol, in which the hydroxyl group of the LCA molecule forms three hydrogen bonds with the TYR52, CYS40 and GLY 39 (Fig. 6F and G). To identify the effect of FBXO5 in LSCC cells, H1703 cells were transfected with FBXO5 siRNA to knockdown the FBXO5. Si-FBXO5-2 was chosen for its significant inhibition on the expression of FBXO5 in H1703 cells (Fig. 6H). MTT experiment exhibited that the cell viability of H1703 cells treated with FBXO5-2 was lower compared with the negative control group (Fig. S1). Moreover, FBXO5 silencing markedly reduced the expression levels of CDK2, CDK4, cyclin D1 and cyclin E (Fig. 6I). All the aforementioned results indicated that FBXO5 may be a potential target of LCA.

Figure 6. Bioinformatics analysis of FBXO5 based on TCGA. (A) The differential expression of FBXO5 between tumor and adjacent normal tissues across all TCGA tumors. Distributions of gene expression levels are displayed using box plots. The statistical significance computed by the Wilcoxon test is annotated by the number of stars (*P<0.05, **P<0.01 and ***P<0.001). (B) Expression of FBXO5 in LSCC based on sample types across TCGA samples (P<0.001). (C) Expression of FBXO5 in LSCC based on individual cancer stages (P<0.01). (D and E) Western blot analysis results revealed the protein expression of FBXO5 after LCA treatment in LSCC cells. (F and G) Visualization of LCA and FBXO5 molecules. F: The molecular surface form of van der Waals; G: Molecule-protein binding site. (H) Western blot analysis results revealed the protein expression of FBXO5 in control and si-FBXO5-transfected H1703 cells. (I) Western blot analysis results demonstrated the protein expression of CDK2, CDK4, cyclin D1 and cyclin E in control and si-FBXO5-transfected H1703 cells. The experiments were repeated at least three times (n=3). *P<0.05 and **P<0.01 vs. control. FBXO5, F-box protein 5; TCGA, The Cancer Genome Atlas; LSCC, lung squamous cell carcinoma; LCA, licochalcone A; siRNA, small interfering RNA; LUSC, lung squamous cell carcinoma.

Table I. Top 10 differentially expressed proteins (LCA vs. Control).

Table I.

Top 10 differentially expressed proteins (LCA vs. Control).

		LCA treatment vs. Control
Protein	Gene	Fold change	P-value
P01033	TIMP1	12.34	0.0016
Q96PD7	DGAT2	5.71	0.0045
P08138	NGFR	4.5617	0.0001
Q6ZNF0	ACP7	4.3918	0.0095
P00740	F9	4.3059	0.0013
Q6PCB0	VWA1	0.2497	0.0002
P15104	GLUL	0.2529	0.0012
Q6EMK4	VASN	0.2616	0.0376
Q8TDB6	DTX3L	0.2661	0.0011
Q9UKT4	FBXO5	0.2716	0.0146

[i] LCA, licochalcone A.

LCA inhibits the growth of LSCC in vivo

Tumor-transplanted mouse models of H1703 cells were used to evaluate the antitumor effect of LCA in vivo. As revealed in Fig. 7A, images of the mice and tumors of each group were captured. The maximum tumor volume was 1,418.48 mm3 and the weight was 0.9 g in the control group. Compared with control, the weights and volumes of the tumor in LCA treatment groups were significantly decreased (Fig. 7B and C). As shown in Fig. 7D, no significant changes in body weight were detected among groups. The body weight was decreased in the CDDP group compared with control, but the loss was not significant (P>0.05). In toxicological experiments, the changes of organ weight and histological changes are important indexes to evaluate the toxicity of drugs or compounds (29). To detect the possible potential toxicity to organs, the organ weight was measured and histological examination was performed. No significant changes in organ weights were revealed among groups (Fig. 7E). H&E staining results revealed that no significant toxicities were observed in the vital organs of mice (Fig. 7F). Western blot analysis demonstrated that LCA treatment decreased the expression of FBXO5, p-ERK1/2 and p-p38MAPK (Fig. 7G and H) in vivo, which was consistent with the suppression effect of LCA on the MAPK pathway and the expression of FBXO5 in vitro. The aforementioned data indicated that LCA exhibited obvious antitumor activity with low toxicity on LSCC in vivo.

Figure 7. LCA suppresses lung squamous cell carcinoma xenograft tumor growth in vivo (n=5). (A) Representative images of xenograft tumor models and tumor tissues. (B) The wet weight of the mice xenograft tumors. (C) The tumor volumes of the mice xenograft tumors. (D) The body weight of the xenograft tumor mice. (E) The organ weight of the xenograft tumor mice. (F) The hematoxylin and eosin staining representative pictures of vital organs of the xenograft tumors mice (scale bar, 200 µm). (G and H) The levels of FBXO5, p-p38 MAPK and p-ERK1/2 in tumor tissue determined by western blot analysis. The experiments were repeated at least three times (n=3). *P<0.05 and **P<0.01 vs. control. LCA, licochalcone A; FBXO5, F-box protein 5; CDDP, cisplatin; p-, phosphorylated. The body weight of the xenograft tumor mice.

Discussion

Despite advancements in diagnosis and therapeutic techniques, the survival rates of lung cancer remain low in the world. LSCC is a highly heterogeneous malignancy with high tumor mutational burden. The molecular profile of LSCC was significantly different from that of adenocarcinoma (30,31). PD1/PD-L1 immune checkpoint blockade and targeted therapies improved the outcome of patients with lung adenocarcinoma, while these therapies were not available for patients with LSCC. Therefore, it is essential to find novel drugs or therapeutics which are effective and have low side effects. Previous studies demonstrated that Chinese traditional herbal medicine licorice displays anti-inflammatory, antitussive and antitumor activities (32–34). LCA is extracted from licorice and possess antitumor properties in several cancers (35–37).

In the present study, LCA effectively inhibited the proliferation of H226 and H1703 cells, while exhibiting no significant cytotoxicity against HBE cells even at a high concentration. The antitumor effect of LCA was also investigated via subcutaneous xenograft models and it was revealed that LCA suppressed xenograft tumor growth in mice. These results indicated that LCA effectively inhibited cell proliferation in vitro and xenograft tumor growth in vivo. Moreover, H&E staining of vital organs indicated that the application of LCA was safe within a certain concentration range in vivo.

Cell cycle regulation is closely related to proliferation of tumors. Cell cycle is strictly regulated by cyclins and their associated specific CDKs (38), such as cyclin D1, CDK4, cyclin E and CDK2, which are of importance for the transition from G1 to S phase (39). In the present study, it was revealed that LCA increased the cell number in G1 phase in LSCC cells and decreased the protein levels of cyclin D1, cyclin E, CDK2 and CDK4. These findings were consistent with the previous studies that LCA induced G1 phase arrest in MCF-7 (14) and HCT-116 (40) cells. The aforementioned data suggested that LCA induced G1 phase arrest and downregulated the expression level of cell cycle-related protein.

Apoptosis is a programmed cellular process that occurs under either physiological or pathological conditions (41). Apoptosis exerts a vital role in the treatment of cancer, and induction of apoptosis is the eventual aim of numerous cancer therapies (42). The findings of the present study displayed that LCA induced apoptosis in LSCC cells and upregulated the level of cleaved caspase-3, cleaved PARP1 and Bax, meanwhile, LCA downregulated the level of Bcl-2. Therefore, LCA could induce cell apoptosis by the mitochondrial-mediated intrinsic pathway in LSCC cells. In the present study, the observed inhibition of cell viability and proliferation was not only dependent on increased cell apoptosis resulting from LCA treatment, but also in the cell cycle arrest. Apoptotic rates reflect part of proliferation inhibition.

Proteomics is widely used in drug development and cancer research (43). Proteomics combination with bioinformatics can analyze the mechanisms and biochemical processes of numerous complex diseases, such as diabetes and cancers (44). KEGG analyses of proteomic results revealed that the DEPs regulated by LCA were associated with the MAPK signaling pathway. And the network pharmacological analysis indicated that the MAPK1 (ERK2) and MAPK14 (p38 MAPK) were the core targets of LCA against LSCC. MAPK signaling pathways are involved in several biological processes and regulate cell proliferation, apoptosis and immune escape of several cancers (45). The ERK1/2 and p38 MAPK signaling pathways have been found to be activated and overexpressed in lung cancer (46,47). In the present study, LCA significantly suppressed ERK1/2 phosphorylation and p38 MAPK phosphorylation in vivo and in vitro.

Moreover, 4D-DIA proteomics also identified that FBXO5 protein expression was decreased after LCA treatment in LSCC cells. FBXO5 is vital in regulation of cycle proliferation and tumorigenesis. FBXO5 promotes cell proliferation and inhibits DNA re-replication via accumulation of cyclin A2 and geminin (48). FBXO5 was upregulated in various malignant tumors compared with matched normal tissues (25,49). A previous study has demonstrated that targeting FBXO5 could eliminate cutaneous squamous cell carcinoma cells with no effect on normal skin cells (50). Existing evidence has suggested that FBXO5 was upregulated in LSCC compared with normal tissue and associated with shorter OS in patients with LSCC (27). And FBXO5 may function as an oncogene and may be a novel therapeutic target in various malignancies (51). In the present study, compared with the adjacent normal tissue, FBXO5 was highly expressed in LSCC tissues based on the TCGA data, and FBXO5 level was significantly elevated in each pathological stage. The aforementioned results suggested that FBXO5 played an important role in LSCC and may be a therapeutic target for LSCC treatment. Western blot analysis revealed that LCA significantly decreased the FBXO5 protein expression. Furthermore, in order to ascertain the effect of FBXO5, MTT experiments were used to detect the cell viability in FBXO5-silenced H1703 cells. FBXO5 knockdown inhibited the cell viability and decreased the expression levels of CDK2, CDK4, cyclin D1 and cyclin E in H1703 cells. These findings indicated that FBXO5 protein knockdown inhibit the proliferation of H1703 cells.

There are certain limitations to the present study. In order to ascertain whether FBXO5 was the therapeutic target for antitumor effects of LCA, rescue experiments should be performed in the FBXO5-silenced H1703 cells in the future.

In summary, LCA inhibited the proliferation of LSCC cells, induced cell cycle arrest and apoptosis in vitro and significantly inhibited the tumorigenesis with few adverse effects in vivo. In addition, LCA inhibited ERK1/2 phosphorylation and p38 MAPK phosphorylation, and suppressed the expression of FBXO5 in vivo and in vitro. In conclusion, LCA may be a potential therapeutic candidate of LSCC.

Supplementary Material

Supporting Data

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (grant no. 82160615).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

XF, LW, JW and XD designed the experiments. XF and JW performed the experiments and data collection. XF wrote the original draft and revised the manuscript. GG and MJ analyzed the data. LW and JW critically reviewed and edited the manuscript. All authors read and approved the final version of the manuscript. XF and XD confirm the authenticity of all the raw data.

Ethics approval and consent to participate

The present study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University (approval no. GLMC-IACUC-2021016; Guilin, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

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References