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Article

Antitumor Effect of Poplar Propolis on Human Cutaneous Squamous Cell Carcinoma A431 Cells

by
Chuang Zhang
1,†,
Yuanyuan Tian
1,2,†,
Ao Yang
1,
Weihua Tan
3,
Xiaoqing Liu
1 and
Wenchao Yang
1,3,*
1
College of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
College of Juncao Science and Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
3
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(23), 16753; https://doi.org/10.3390/ijms242316753
Submission received: 5 October 2023 / Revised: 22 November 2023 / Accepted: 23 November 2023 / Published: 25 November 2023
(This article belongs to the Special Issue Molecular Research on Skin Disease: From Pathology to Therapy)
Browse Figures
Versions Notes

Abstract

:
Propolis is a gelatinous substance processed by western worker bees from the resin of plant buds and mixed with the secretions of the maxillary glands and beeswax. Propolis has extensive biological activities and antitumor effects. There have been few reports about the antitumor effect of propolis against human cutaneous squamous cell carcinoma (CSCC) A431 cells and its potential mechanism. CCK-8 assays, label-free proteomics, RT–PCR, and a xenograft tumor model were employed to explore this possibility. The results showed that the inhibition rate of A431 cell proliferation by the ethanol extract of propolis (EEP) was dose-dependent, with an IC50 of 39.17 μg/mL. There were 193 differentially expressed proteins in the EEP group compared with the control group (p < 0.05), of which 103 proteins (53.37%) were upregulated, and 90 proteins (46.63%) were downregulated. The main three activated and suppressed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were extracellular matrix (ECM)-receptor interaction, amoebiasis, cell adhesion molecules (CAMs), nonalcoholic fatty liver disease (NAFLD), retrograde endocannabinoid signaling, and Alzheimer’s disease. The tumor volume of the 100 mg/kg EEP group was significantly different from that of the control group (p < 0.05). These results provide a theoretical basis for the potential treatment of human CSCC A431 cell tumors using propolis.

1. Introduction

Cutaneous squamous cell carcinoma (CSCC) is a malignant tumor originating from keratinocytes of the epidermis or appendages and the primary cause of death among nonmelanoma skin tumors [1]. In recent years, approximately 15–35 people in every 100,000 people have been diagnosed with CSCC, with an increase of 2–4% every year, which has become a worldwide threat to public health [2,3]. CSCC is mostly diagnosed in elderly patients, and the ratio of men to women is approximately 2~3:1. Most CSCC occurs in body parts, such as the head and face, exposed to ultraviolet light [4]. Currently, surgery, radiotherapy, and chemotherapy are the main treatment strategies. Surgery is an effective way to remove tumors, but it is not easy to detect cancer at an early stage. Chemotherapy and radiography treatments have side effects, such as damaging normal cells and drug resistance, which reduce the quality of life of patients. The search for alternative treatments is urgently needed.
Propolis (bee glue) is a kind of viscous substance processed by Western honeybee workers that collect mucilage, gums, resins, and lattice from plants, such as Pinus spp., Prunus spp., Acacia spp., Betula pendula, Aesculus hippocastanum, Salix alba, Baccharis dracunculifolia, and Dalbergia ecastaphyllum (L.) Taub., and the collections are mixed with the secretion of worker maxillary glands and beeswax [5,6]. The chemical composition of propolis, mainly including flavonoids, flavonols, flavanones, dihydroflavonoids, and phenylpropane derivatives, varies depending on the plant, geographical origin, and harvest season [7]. There are significant differences in color, active components, and harvesting tools of poplar propolis, red and green propolis [6]. Green Brazilian propolis is derived mainly from the leaf resin of Baccharis dracunculifolia, and red propolis is from the resin of Dalbergia ecastophyllum, while poplar propolis (brown color) is from the resin of Populus nigra L. [6]. The main active components of green propolis were derivatives of phenylpropanoids and diterpenes, chlorophyll, and small amounts of flavonoids, and those of red and poplar propolis were flavonoids [7]. Propolis has a wide range of biological activities, such as antibacterial, antifungal, antiviral, antiparasitic, antioxidant, antitumor, anti-inflammatory, anti-ulcer, and antidiabetic effects [5,8].
The antitumor activity of propolis has attracted much attention. In recent years, the antitumor effects of propolis on cancer and the relevant mechanisms have been reported in in vitro studies on colorectal, lung, breast, melanoma, gastric, lymphoma, tongue, and skin [7,8,9,10,11,12,13,14,15]. Many active ingredients in propolis, such as flavonoids and caffeic acid phenylethyl ester, inhibit tumor activity. Among them, caffeic acid phenylethyl ester has a specific and targeted killing effect on docetaxel-resistant prostate cancer cells [10]. Flavonoids block the cell cycle and then inhibit the proliferation of a human breast cancer cell line (MCF-7) [11]. There are selective antitumor effects of propolis on normal gingival fibroblasts and tongue cancer cells [12]. Red propolis has a cytotoxic effect on the breast cancer MDA MB-231 cell line, which is related to the PI3K/Akt and ERK1/2 pathways [13]. Propolis reduces mitochondrial membrane potential and changes the expressions of specific tumor suppressors (miR-34, miR-15a, and miR-16-5p) and carcinogenic (miR-21) miRNAs by increasing the levels of proapoptotic proteins (p21, Bax, p53, p53 Ser46, and p53 Ser15) in the MCF-7 cell line [14]. Brazilian propolis reduces the proliferation of a head and neck squamous cell carcinoma (HNSCC) cell line [15]. The antitumor effect of poplar propolis on CSCC A431 cells and its potential mechanism are unclear.
In this study, Cell Counting Kit-8 (CCK-8) assays, label-free proteomics, RT–PCR, and a xenograft tumor nude mouse model were employed to determine the effect of propolis on the proliferation, differentially expressed proteins, related pathways in A431 cells, and tumor volume in an animal xenograft tumor model.

2. Results

2.1. Components of Ethanol Extract of Propolis

The total flavonoid content of ethanol extract of propolis (EEP) was 32.04 ± 0.89/100 g. The chromatogram for UPHLC-MS/MS of propolis is shown in Figure 1. The 214 chemical components of EEP dissolved in methanol are presented in Table 1.

2.2. The Antitumor Effect of Ethanol Extract of Propolis (EEP)

There were no significant differences between the viability of A431 cells in the solvent control group and the blank control group. EEP inhibited the proliferation of A431 cells. DMSO (0.05%) had no effect on A431 cells. The inhibition rates are shown in Figure 2. The IC50 of 5-FU and EEP against A431 cells for 48 h was 6.57 and 39.17 µg/mL, respectively.
The morphology of A431 cells after EEP treatment for 48 h is shown in Figure 3. The growth of the cells in the control group was normal, and the treated cells were irregular in shape and distributed in sheets. The number of cells treated with EEP in the microscope field of view decreased, and the cell adhesion ability was weakened with some floating cells.

2.3. The Differentially Expressed Proteins

There were 103 upregulated and 90 downregulated differentially expressed proteins (DEPs) between the EEP group and the control group (screened by FC > 2.0 or FC < 0.5 and p < 0.05). Partial DEPs (p < 0.01) are shown in Table 2.
The volcano plot of proteins in the two groups is shown in Figure 4. The subcellular localization of the DEPs is shown in Figure 5. The DEPs subjected to Gene Ontology (p < 0.05) and Kyoto Encyclopedia of Genes and Genomes (p < 0.05) analyses are shown in Figure 6 and Figure 7, respectively.
These DEPs played roles in different pathways. The significantly enriched pathways (p < 0.05) of upregulated and downregulated proteins were separately analyzed, as shown in Table 3.
All of the DEP interactions are shown in Figure 8. Among the interacting proteins, NADH dehydrogenase [ubiquinone] flavoprotein 2 (mitochondrial), mitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit, NADH dehydrogenase [ubiquinone] flavoprotein 1 (mitochondrial), NADH dehydrogenase (ubiquinone) Fe–S protein 5, 15 kDa (NADH-coenzyme Q reductase), NADH dehydrogenase [ubiquinone] iron–sulfur protein 8 (mitochondrial), and NADH dehydrogenase [ubiquinone] iron–sulfur protein 7 (mitochondrial) had the most protein interactions, with 11 DEPs.

2.4. Relative Gene Expression

The cycle threshold (ct) data of selected genes is shown in Supplementary Table S1. The relative gene expression levels of selected genes encoding differentially expressed proteins are shown in Figure 9. The expression levels of the three genes LAMC1, SDC1, and THBS1 involved in the ECM-receptor interaction pathway were significantly upregulated in the treated group compared with the control group, and the gene expression was consistent with the expression of proteins. The expression levels of three genes, NDUFS1, NDUFV1, and SDHA, involved in the oxidative phosphorylation pathway, were significantly upregulated and inconsistent with the expression of proteins.

2.5. The Effect of EEP on A431 Cell Xenograft Tumors in Nude Mice

The tumor volumes of nude mice in the control group, solvent group, 50 mg/kg propolis group, and 100 mg/kg propolis group after 12 days of gavage are shown in Table 4. There was a significant difference in the 100 mg/kg propolis group compared with the control group (p < 0.05), which indicated that the 100 mg/kg propolis group had in vivo inhibitory effects on A431 cell tumors.
The HE staining results of the tumor tissue of the EEP, solvent control, and control groups are shown in Figure 10. A large number of tumor cells were observed in each group except in the 100 mg/kg EEP group. The morphology and size of cells varied and exhibited atypia, with enlarged nucleoli and unclear cell spacing. There was a small amount of cell necrosis in the control group, solvent control group, and 50 mg/kg group, while a large amount of cell necrosis was observed in the 100 mg/kg EEP group.

3. Discussion

Propolis exhibits antitumor activity against different cell lines. Brazilian red propolis (from Brejo Grande, Brazil) can inhibit the growth of cancer cells, and after 24 h of treatment, the IC50 values against Hep-2 cells and HeLa cells were 63.48 ± 3.30 μg/mL and 81.40 ± 6.40 μg/mL, respectively [16]. EEP (from Ardabil, Iran) has dose-dependent toxic effects on both KB and A431 cancer cells. The IC50 values of EEP in the KB cell line and A431 cell line were 40 ± 8.9 μg/mL and 98 μg/mL, respectively, after 48 h of incubation [17]. The IC50 values of EEPs (from Podlasie, Masovia, and West Pomerania; Poland) against tongue cancer cells treated for 24 h were approximately 88 µg/mL, 110 µg/mL, and 150 µg/mL, respectively [12]. The IC50 values of EEPs range from 26.33 to 143.09 μg/mL against the human colon cancer cell line HCT-16 [18]. The IC50 values of EEP (from Phayao, Chiang Mai, and Nan, Thailand) against A549 cells were 106 ± 0.004 µg/mL, 199 ± 0.009 µg/mL, and 87 ± 0.012 µg/mL, respectively, and for HeLa cells were 81 ± 0.006 µg/mL, 116 ± 0.023 µg, and 54 ± 0.005 µg/mL, respectively [19]. The IC50 of EEP (from Hebei Province, China) against the 5 × l05/mL DLBCL SU-DHL-2 cell line for 24 h was 5.729 μg/mL [9]. In this study, the IC50 of EEP (same as [9]) against A431 cells for 48 h was 39.17 μg/mL (Figure 3). These different median lethal doses against tumor cell lines may be related to the type of cancer cells, concentration of cancer cells, incubation duration, botanical origin of propolis, extraction process of propolis, and storage of propolis.
The cytotoxicity mechanism of propolis against A431 tumor cells was different. Proteins play important roles in the proliferation of cells. Label-free proteomics is commonly used to explore DEPs in cells subjected to different treatments [20,21,22]. In this manuscript, there were 103 upregulated and 90 downregulated DEPs between treatment and control cells. GO enrichment and KEGG enrichment analysis (as shown in Figure 6 and Figure 7) showed that the main upregulated proteins enriched in the ECM-receptor interaction and cell adhesion molecule (CAM) pathways inhibits the ability of A431 cells to metastasize and invade. The downregulated proteins were mainly enriched in adenosine triphosphate (ATP) production by downregulating the main proteins of the retrograde endocannabinoid signaling and oxidative phosphorylation pathways, thereby reducing adenosine triphosphate (ATP) production and inhibiting the proliferation of A431 cells.
The most significantly enriched pathway of the upregulated DEPs was the ECM-receptor interaction pathway. The interaction between tumor cells and extracellular matrix (ECM) components, such as laminin, fibronectin, and collagen, plays a crucial role in tumor invasion and metastasis. The genes LAMC1, SDC1, and THBS1, which are involved in the ECM-receptor interaction pathway, were upregulated. Similar results were also found in gastric cancer, in which the upregulated genes were COL1A2 and COL6A3 [23] or COL6A3, COL3A1, and COL1A1 [24]. There were seven upregulated proteins involved in the ECM-receptor interaction pathway, which is associated with an enhanced migratory/invasive capacity of ovarian cancer cells [25] and non-small cell lung cancer tumors [26,27]. Most of the upregulated DEPs, except Thrombospondin 1, Agrin, and Syndecan-1, involved in the ECM-receptor interaction pathway were also enriched in the amoebiasis pathway. Differentially expressed genes related to cervical cancer were enriched in amoebiasis and other pathways [28,29]. Similar differentially expressed genes were also found in colorectal cancer cells with cetuximab insensitivity [30]. The differentially expressed proteins were involved in amoebiasis pathways of non-small cell lung cancer [26] and gastric cancer tumors of the patients [27].
Another important pathway of the upregulated DEPs was the amoebiasis pathway. The amoebiasis pathway was significantly enriched and identified as one of the important processes or signaling pathways of melanoma metastasis [31,32], the pathogenesis of nasopharyngeal carcinoma [33], carcinogenesis and pathogenesis of cervical cancer [28], breast cancer [34], and gastric cancer [35] by bioinformatics analysis based on the Gene Expression Omnibus database. Our result is in accordance with these previous scientific studies.
The third pathway of the upregulated DEPs was the cell adhesion molecule (CAM) pathway. CAMs, having four main groups including cadherins, integrins, selectins, and immunoglobulins, are primarily glycoproteins on cell surface membranes and can promote homeostasis between cells and between cells and the extracellular matrix. With higher levels of neural cell adhesion molecule expression, neuroblastoma cells have more intense homophilic tumor binding [36]. Knockdown of E-cadherin and cell adhesion molecule 1-related genes decreased cell growth, migration, and cell-to-cell adhesion of BAP1-mutant uveal melanoma cells [37]. High expression of prostaglandin F2 receptor inhibitor (PTGFRN), a type I (single pass) transmembrane Ig superfamily of CAM, could protect cells from apoptosis, thereby promoting growth and migration in glioblastoma cells [38]. It was also shown that the CAM pathway is one of the key processes or signaling pathways of melanoma metastasis and the pathogenesis of nasopharyngeal carcinoma [31,32,33]. CAMs may be the stress response to adverse factors on cancer cells.
The most significantly downregulated pathway was the nonalcoholic fatty liver disease (NAFLD) pathway. NAFLD affected the cell cycle and p53 pathways. SNORA71A knockdown in HT-29 cells led to significant inhibition of cell migration and invasion ability, which targeted LBP to participate in NAFLD in colorectal cancer cells [39]. Corosolic acid inhibited NAFLD-related hepatocellular carcinoma progression by downregulating the NAFLD pathway [40]. Levodopa downregulates oxidative phosphorylation (OXPHO), NAFLD, and Parkinson’s disease-related pathways to inhibit esophageal squamous cell carcinoma cells [41]. Similar results were also obtained in this experiment, in which the OXPHO and Parkinson’s disease-related pathways were also suppressed. OXPHO mainly occurs in the inner mitochondrial membrane of eukaryotic cells or the cytoplasm of prokaryotic organisms [42]. Many tumors require energy from the mitochondria for biosynthesis to synthesize ATP through OXPHO [43]. The average contribution of OXPHOS to ATP generation in normal cells is 80% and 83% in cancer cells [44]. Triple-negative breast cancer (TNBC) cells can be inhibited by a decrease in OXPHOS caused by OXPHOS inhibitors, which causes ATP deficiency that cannot be fully compensated by other mechanisms [45]. Oxidative phosphorylation also acts on other cancer cells, such as liver cancer [46], rectal cancer [47], and pancreatic cancer cells [48]. OXPHO is now widely used as a therapeutic target in cancer [49].
Another suppressed pathway was retrograde endocannabinoid signaling. This pathway was also suppressed in retinoblastoma [50], glioblastoma [51], and glioma patients [52]. The metabolites were also enriched in the retrograde endocannabinoid signaling pathway in breast cancer cells treated with Faecalibacterium prausnitzii [53]. This pathway was also mainly enriched in DEPs and DEGs related to nonfunctional pituitary adenoma [54]. The DEPs between the tumor and adjacent healthy tissue of patients with diffuse gastric cancer and those of patients with advanced gastric cancer were enriched in this pathway [55]. Other suppressed pathways were Alzheimer’s disease-related pathways, Huntington’s disease-related pathways, metabolic pathways, and glycosaminoglycan biosynthesis—keratan sulfate. The proteins involved in these pathways were downregulated and inhibited the growth of A431 cells.
The expression levels of genes subjected to RT–PCR were not completely consistent with the protein expression levels. Some studies have pointed out that transcription levels alone are not sufficient to predict protein levels in many cases [56,57] because the protein concentration is affected by both transcription and translation. Cancer-related genetic changes can affect proteins involved in nearly all levels of transcriptional control [58].
This experiment showed that EEP inhibited tumor growth in nude mice. It was also found that the growth of gastric pyloric tumors and colorectal cancer was inhibited by a diet containing propolis [59,60]. Propolis has immunological enhancement activity [61], which can enhance the immune system of mice and then nonspecificly inhibit tumor growth.
The limitation of this study is that metabonomics, cancer stem cell, molecular docking, or other methods can be employed to explore more accurately the regulation of the EPP antitumor approach against the A431 cells for new drug development. More research about the cell cycle of A431 cells inhibited by EEP and the active components, such as caffeic acid, dihydro cinnamic acid, and p-coumaric acid, or others, who play the main antitumor effect can be determined in the future.

4. Materials and Methods

4.1. Propolis Samples and Its Chemical Components Determination

The crude poplar propolis sample and extraction procedure of ethanol extract of propolis (EEP) were the same as in our previous report [9].
The total flavonoid content determination of EEP was performed using the spectrophotometer method according to the national standards for propolis in China (GB/T 24283-2018)[62]. Rutin was used as a standard substance to determine the absorbance of the samples at 510 nm.
The chemical components of EEP were determined using a UHPLC-MS/MS system (Vanquish UHPLC system (Thermo Fisher Scientific Inc., Germering, Germany) coupled with an Orbitrap Q ExactiveTMHF-X mass spectrometer (ThermoFisher, Germering, Germany)) by Untargeted Metabolomics mothed and performed by Novogene Co., Ltd. (Beijing, China). Simply, The EEP samples (1 mL) were resuspended with prechilled 80% methanol by well vortex and then incubated on ice for 5 min and centrifuged at 15,000× g, 4 °C for 15 min. The supernatant was diluted to 53% methanol final concentration by LC-MS grade water. The solution was transferred to an Eppendorf tube and subsequently centrifuged at 15,000× g, 4 °C for 15 min. The elution conditions and processes were the same as in reference [63]. Q ExactiveTM HF-X mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.5 kV, capillary temperature of 320 °C, sheath gas flow rate of 35 psi, and aux gas flow rate of 10 L/min, S-lens RF level of 60, Aux gas heater temperature of 350 °C.

4.2. Antitumor Bioassay

The human skin squamous cell carcinoma A431 cell line (purchased from Wuhan Purosai Life Sciences Co., Ltd., Wuhan, China) was cultured in a special complete culture medium (Wuhan Purosai Life Sciences Co., Ltd.) in a 5% CO2 humidified incubator at 37 °C (C150, Binder, Tuttlingen, German).
EEP (0.1 g) was dissolved in 0.5 mL DMSO and diluted with a complete culture medium at a concentration of 1 mg/mL. Propolis solution was diluted with a complete medium to 100, 75, 50, and 25 µg/mL. DMSO (0.05%, v/v; equal to DMSO in the 100 µg/mL propolis group) was added to the complete medium as a solvent control.
The A431 cells were irrigated with PBS buffer (pH 7.2–7.4, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) twice and then digested with 1 mL 0.25% trypsin solution (HyClone, Thermo Scientific, Waltham, UT, USA). The digested solution was centrifuged at 137× g for 5 min. The sediment was suspended in 2 mL of complete culture medium. The concentration was determined via 0.4% trypan blue staining (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). At a beginning concentration of 5 × 104 cells/mL, 100 µL cell suspension was added in 96-well plates with 6 repetitions per treatment. After 48 h of cell culture with EEP 100, 75, 50, and 25 µg/mL, and 0.05% DMSO, the viability of cells was determined by a CCK8 kit (DOJINDO, Kumamoto, Japan) at 450 nm using a microplate reader (1510, Thermo Fisher Waltham, MA, USA). The IC50 of EEP on A431 cells for 48 h was calculated using GraphPad Prism 8.4.3 for Windows (GraphPad Software, Inc., La Jolla, CA, USA). 5-Fluorouracil (HPLC grade; purchased from Sigma-Aldrich Co., St. Louis, MO, USA) at 0, 4, 8, 16, 32, and 64 µg/mL were employed to determine the IC50 value against A431 cells.
A431 cells in the logarithmic growth phase were digested with trypsin and seeded in 6-well plates at 1.5 × 105 cells/well. After 24 h of cell culture, the cells were treated with control, and IC50 EEP solution for 48 h, and then the morphological changes of the cells were observed using an inverted microscope (TS-100f, Nikon, Tokyo, Japan).

4.3. Differentially Expressed Proteins in A431 Cells Treated with Propolis

The A431 cells were treated as described for the morphology observation experiment, which was also treated with control and IC50 EEP solution. After these cells were treated with propolis or non-propolis for 48 h, the culture medium was removed. Then, the cells were irrigated twice with precooled PBS buffer, digested, and irrigated twice again. Cells were collected in a centrifuge tube (1.5 mL) after centrifugation. Therefore, these cells were frozen in liquid nitrogen and further stored in a refrigerator at −80 °C (Haier Biomedical, Qingdao, China).
The extraction and concentration determination of total proteins and spectra of proteins were performed as described in our previous report [9].

4.4. Detection of Relative Gene Expression

According to the proteomics results, the genes coded NDUFS1, NDUFV1, and SDHA proteins involved in oxidative phosphorylation and LAMC1, SDC1, and THBS1 ECM-receptor interaction were selected to determine the gene expression levels between the control and IC50 EEP groups using RT–PCR assay. The primers were designed through NCBI’s free online primer design platform, which is given in Supplementary Table S2. The internal reference gene was β-actin.

4.5. Xenograft Tumor Nude Mice

BALB/C male nude mice of SPF grade, 5 weeks old, weighing 18–20 g (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., Shanghai, China) were experimental animals. The A431 cell suspension (1 × 107 cell/mL) (0.1 mL) was injected subcutaneously into the axilla of the right forelimb of nude mice. The small nodules at the inoculation site mean the heterologous tumor model in nude mice was successful. After 1 week, the tumor sizes were 4–7 mm3. Then, 20 nude mice were randomly divided into 4 groups: the control, solvent, 50 mg/kg EEP, and 100 mg/kg EEP groups (according to [64]). They were intragastrically administered 0.2 mL PBS buffer solution, 10% PEG-400 solution, 50 mg/kg, and 100 mg/kg. The tumor volumes of each mouse were measured every 2 days.
These nude mice were sacrificed after the final treatment. The tumor tissue was immediately peeled off with scissors and tweezers, washed with normal saline, and fixed in 4% paraformaldehyde (Biosharp, Labgic Technology Co., Ltd., Beijing, China) for paraffin section preparation and hematoxylin-eosin staining (HE, Wuhan Servicebio Technology Co., Ltd., Wuhan, China).

4.6. Data Analysis

All experiments were performed in triplicate. All these data are expressed as the mean ± standard error. One-way ANOVA was used to analyze the significance of differences (p < 0.01: extremely statistically significant differences between treatment and control groups, p < 0.05: statistically significant differences). The relative gene expression was represented by the ratio of gene expression in propolis-treated cells to that of control cells. Differences in tumor volumes among groups were analyzed using repeated-measures ANOVA using Stat View 5.0.1 (SAS Institute Inc. 1992–1998, Cary, NC, USA).
The spectra obtained from label-free proteomics by LC-MS/MS were analyzed as described in our previous report [9].
The raw data files generated from Untargeted Metabolomics by UHPLC-MS/MS were processed using the Compound Discoverer 3.1 (CD3.1, ThermoFisher) to perform peak alignment, peak picking, and quantitation for each metabolite, whose main parameters were set as follows: retention time tolerance, 0.2 min; actual mass tolerance, 5 ppm; signal intensity tolerance,30%; signal/noise ratio, 3; and minimum intensity.

5. Conclusions

A431 cancer cells can be inhibited by poplar propolis via the main pathways enriched DEPs were ECM-receptor interaction, amoebiasis, cell adhesion molecules (CAMs), nonalcoholic fatty liver disease (NAFLD) related pathway, retrograde endocannabinoid signaling, and other pathways. The inhibition effect was also found in a xenograft tumor for nude mice. Poplar propolis has the potential to be a new treatment strategy for CSCC patients.

Supplementary Materials

The supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms242316753/s1.

Author Contributions

Conceptualization, C.Z. and W.Y.; methodology, W.Y.; data curation, C.Z., Y.T. and X.L.; formal analysis, C.Z., A.Y. and W.T.; writing—original draft preparation, Y.T. and C.Z.; writing—review and editing, W.Y.; supervision, W.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The animal study protocol was approved by the Ethics Committee of Fujian Agriculture and Forestry University (protocol code PZCASFAFU23074).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within this article and supplementary materials.

Acknowledgments

Thanks for the help from Xin Lin during the performance of the protocol.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The chromatogram of ethanol extract of propolis (EEP) for UPHLC-MS/MS: (A) negative polarity mode; (B) positive polarity mode.
Figure 1. The chromatogram of ethanol extract of propolis (EEP) for UPHLC-MS/MS: (A) negative polarity mode; (B) positive polarity mode.
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Figure 2. Inhibitory rates of 5-Fluorouracil (A) and ethanol extract of propolis (EEP) (B) against the proliferation of A431 cells for 48 h (** indicates significant differences compared with the solvent control group. p < 0.01).
Figure 2. Inhibitory rates of 5-Fluorouracil (A) and ethanol extract of propolis (EEP) (B) against the proliferation of A431 cells for 48 h (** indicates significant differences compared with the solvent control group. p < 0.01).
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Figure 3. Effect of ethanol extract of propolis (EEP) on the morphology of A431 cells (100×): (A): Control, (B) IC50 EEP group.
Figure 3. Effect of ethanol extract of propolis (EEP) on the morphology of A431 cells (100×): (A): Control, (B) IC50 EEP group.
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Figure 4. Volcano plot of proteins in A431 cells treated with ethanol extract of propolis (EEP) versus control cells.
Figure 4. Volcano plot of proteins in A431 cells treated with ethanol extract of propolis (EEP) versus control cells.
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Figure 5. The subcellular localization of the differentially expressed proteins.
Figure 5. The subcellular localization of the differentially expressed proteins.
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Figure 6. The differentially expressed proteins enriched in Gene Ontology terms (p < 0.05).
Figure 6. The differentially expressed proteins enriched in Gene Ontology terms (p < 0.05).
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Figure 7. The number of differentially expressed proteins enriched in Kyoto Encyclopedia of Genes and Genomes (p < 0.05).
Figure 7. The number of differentially expressed proteins enriched in Kyoto Encyclopedia of Genes and Genomes (p < 0.05).
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Figure 8. The protein-protein interaction of DEPs.
Figure 8. The protein-protein interaction of DEPs.
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Figure 9. The relative gene expression of selected genes encoding differentially expressed proteins. (A): genes coded proteins enriched in ECM-receptor interaction pathway; (B): genes coded proteins enriched in oxidative phosphorylation pathway. The symbols * and ** indicates significant differences compared with the solvent control group, p < 0.05 or p < 0.01, respectively.
Figure 9. The relative gene expression of selected genes encoding differentially expressed proteins. (A): genes coded proteins enriched in ECM-receptor interaction pathway; (B): genes coded proteins enriched in oxidative phosphorylation pathway. The symbols * and ** indicates significant differences compared with the solvent control group, p < 0.05 or p < 0.01, respectively.
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Figure 10. HE staining of tumors in nude mice of different groups (400×). (A) Control group, (B) Solvent control group, (C) 50 mg/kg EEP group, and (D) 100 mg/kg EEP.
Figure 10. HE staining of tumors in nude mice of different groups (400×). (A) Control group, (B) Solvent control group, (C) 50 mg/kg EEP group, and (D) 100 mg/kg EEP.
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Table 1. Chemical components in ethanol extract of propolis (EEP) (full matched mzCloud).
Table 1. Chemical components in ethanol extract of propolis (EEP) (full matched mzCloud).
IDNameFormulaMolecular WeightRetention Time (min)m/zRelative Quantitative ValuePolarity Mode
11,5,8-Trihydroxy-9-oxo-9H-xanthen-3-yl β-D-glucopyranosideC19H18O11422.083751.31421.0764839,349,735.26negative
2D-ribose 5-phosphateC5H11O8P230.019081.311229.011843,826,332.25negative
3D-Mannose 6-phosphateC6H13O9P260.029921.318259.02264349,455,529.4negative
4Galacturonic acidC6H10O7194.04231.329193.035028,590,476,535negative
5N-Acetyl-α-D-glucosamine 1-phosphateC8H16NO9P301.056281.334300.0489831,064,500.87negative
6D-Saccharic acidC6H10O8210.03721.347209.02992512,353,580.7negative
7Gluconic acidC6H12O7196.057461.358195.050196,478,000,924negative
8UDP-N-acetylglucosamineC17H27N3O17P2607.082041.367606.0747775,450,606.66negative
9D-(−)-FructoseC6H12O6180.062581.375179.055212,010,181,033negative
10N-Acetylneuraminic acidC11H19NO9309.105451.383308.0981867,763,775.54negative
11L-(+)-Tartaric acidC4H6O6150.015711.405149.00844762,277,603.7negative
12Glucuronic acid-3,6-lactoneC6H8O6176.031531.419175.02422433,003,996.6negative
13SucroseC12H22O11342.116161.437341.1087316,004,974,387negative
14D-RaffinoseC18H32O16504.169051.439503.162081,558,022,103negative
15δ-Gluconic acid δ-lactoneC6H10O6178.047041.563177.03976165,652,129.9negative
16Uric acidC5H4N4O3168.027511.886167.020234,413,870,356negative
17D-α-Hydroxyglutaric acidC5H8O5148.036532.092147.02925149,585,529.5negative
18XanthineC5H4N4O2152.032772.181151.02548337,332,340.5negative
19UridineC9H12N2O6244.06972.415243.062681,652,833,848negative
20N-Acetyl-DL-glutamic acidC7H11NO5189.063462.463188.0561853,573,049.69negative
21Ascorbic acidC6H8O6176.031492.814175.02422205,503,907.6negative
222,4-Dihydroxybenzoic acidC7H6O4154.026013.36153.0187492,941,295.62negative
23N-(4-chlorophenyl)-N′-cyclohexylthioureaC13H17ClN2S268.080923.659267.07364271,608,391.5negative
242-Amino-3-(4-hydroxy-3-methoxyphenyl)propanoic acidC10H13NO4211.084174.223210.0769340,438,679.16negative
25XanthosineC10H12N4O6284.076194.747283.0689125,698,102.52negative
26Methylsuccinic acidC5H8O4132.042114.789131.03484147,365,715.6negative
27Gallic acidC7H6O5170.02124.884169.01393542,974,125negative
28ThymidineC10H14N2O5242.090744.895241.0833188,919,605.24negative
293,4,5-trihydroxycyclohex-1-ene-1-carboxylic acidC7H10O5174.05234.907173.0452349,978,794.34negative
301-(2,4-dihydroxyphenyl)-2-(3,5-dimethoxyphenyl)propan-1-oneC17H18O5302.115834.93301.1085543,916,324.75negative
315-Sulfosalicylic acidC7H6O6S217.988484.937216.981233,337,560.45negative
323′,4′-DihydroxyphenylacetoneC9H10O3166.06264.938165.05533255,487,284.2negative
33PorphobilinogenC10H14N2O4226.095434.963225.0881520,599,303.66negative
34D-(−)-Quinic acidC7H12O6192.063085.067191.0558687,769,173.6negative
352,3-Dihydroxybenzoic acidC7H6O4154.026065.072153.018741,430,101,121negative
363-Hydroxy-3-methylglutaric acidC6H10O5162.052195.136161.044921,232,391,129negative
37Vanillyl alcoholC8H10O3154.06245.155153.05511206,029,853negative
38L-Tyrosine methyl esterC10H13NO3195.089265.159194.08197216,011,260.4negative
39Phenylglyoxylic acidC8H6O3150.031115.166149.02383154,372,170.5negative
40EsculinC15H16O9340.080065.172339.0727851,031,924.39negative
412-Isopropylmalic acidC7H12O5176.068075.441175.06079310,134,000.4negative
424-((5-(4-Nitrophenyl)oxazol-2-yl)amino)benzonitrileC16H10N4O3306.074025.445305.06674343,517,763.8negative
43MiquelianinC21H18O13478.074985.464477.0677228,072,478.49negative
44Quercetin-3β-D-glucosideC21H20O12464.096085.483463.08881197,183,482.7negative
453-Coumaric acidC9H8O3164.046925.521163.0396353,776,573,353negative
46HematoxylinC16H14O6302.078325.539301.071043,032,439,830negative
47Gentisic acidC7H6O4154.025985.542153.018691,126,817,012negative
48AgnusideC22H26O11466.147755.545465.1404778,804,038.52negative
49MyricetinC15H10O8318.037645.554317.03036252,777,241.4negative
50Caffeic acidC9H8O4180.041625.562179.0343516,033,774,255negative
513-(4-Hydroxyphenyl)propionic acidC9H10O3166.062275.583165.05499511,602,130.2negative
52Suberic acidC8H14O4174.088785.617173.0815805,556,199.4negative
53trans-Cinnamic acidC9H8O2148.051835.623147.04456299,470,310.6negative
5411-Dehydro thromboxane B2C20H32O6368.220375.64367.212923,259,236,011negative
55CitrininC13H14O5250.084145.709249.07686234,803,880.5negative
56IsorhapontigeninC15H14O4258.088485.722257.0812181,038,331.56negative
57DL-4-Hydroxyphenyllactic acidC9H10O4182.057475.728181.05019365,233,039.2negative
583,8,9-trihydroxy-10-propyl-3,4,5,8,9,10-hexahydro-2H-oxecin-2-oneC12H20O5244.13115.745243.12383213,168,051.7negative
595,7-Dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-oneC16H14O6302.077625.767301.070341,212,404,197negative
60QuercetinC15H10O7302.042625.774301.0353426,894,567,188negative
61LuteolinC15H10O6286.049165.82285.04191,272,812,181negative
62NaringeninC15H12O5272.068835.948271.061552.187 × 1011negative
63β-Estradiol-17β-glucuronideC24H32O8448.210375.95447.20309389,513,176.1negative
64Aflatoxin G2C17H14O7330.072965.965329.06567877,430,305.3negative
65Monobutyl phthalateC12H14O4222.089116.124221.081834,535,327,931negative
66Mycophenolic acidC17H20O6320.125936.188319.1186556,348,359.96negative
67Salvinorin BC21H26O7390.169236.221389.1623235,236,148.01negative
68Dodecanedioic acidC12H22O4230.15196.424229.1446225,219,452.38negative
69AldosteroneC21H28O5360.193966.538359.18668155,818,216.9negative
70Corchorifatty acid FC18H32O5328.225116.568327.217831,609,573,692negative
71N2-(4-iodophenyl)-1,3,5-triazine-2,4-diamineC9H8IN5312.982376.589311.975150,086,503.07negative
722,3-Dinor-8-epi-prostaglandin F2αC18H30O5326.209886.609325.2026174,071,657.03negative
73TroloxC14H18O4250.120826.639499.2344412,254,171,786negative
74GenisteinC15H10O5270.053086.658269.0458153,946,618,740negative
75(±)9(10)-DiHOMEC18H34O4314.245866.658313.238591,801,802,573negative
76[1,1′-biphenyl]-2,2′-dicarboxylic acidC14H10O4242.058046.699241.05077363,776,879.9negative
77Glycocholic acidC26H43NO6465.30986.753464.3025231,784,139.83negative
78GlyciteinC16H12O5284.067956.758283.0606712,479,803,653negative
79Gibberellin A4C19H24O5332.162646.803331.15536935,893,979.2negative
804-(octyloxy)benzoic acidC15H22O3250.157186.852249.14999,593,885.513negative
81(±)-Abscisic acidC15H20O4264.136436.963263.129152,145,442,724negative
82Tetradecanedioic acidC14H26O4258.183397.01257.1761224,368,922.87negative
832-Hydroxymyristic acidC14H28O3244.20347.098243.19612262,979,901.2negative
8413(S)-HOTrEC18H30O3294.219667.48293.21237113,609,783negative
85Pentobarbital-d5C11H13[2]H5N2O3231.162317.484230.155034,925,637.941negative
8615-keto Prostaglandin E1C20H32O5352.225087.857351.217776,575,446.425negative
8716-Hydroxyhexadecanoic acidC16H32O3272.234727.871271.22745609,305,517.6negative
88Protoporphyrin IXC34H34N4O4562.257098.059561.249827,107,790.257negative
8918-β-Glycyrrhetinic acidC30H46O4470.33978.84469.3324311,661,934.32negative
90Arachidic AcidC20H40O2312.303249.988311.2959611,584,703.86negative
91Docosanoic AcidC22H44O2340.3343610.655339.327095,918,139.419negative
9211(Z),14(Z)-Eicosadienoic AcidC20H36O2308.271510.673307.264228,254,456.223negative
93Ursolic acidC30H48O3456.3605510.725455.353277,204,655.792negative
94Lignoceric AcidC24H48O2368.3656810.905367.358420,009,501.76negative
9513Z,16Z-Docosadienoic AcidC22H40O2336.3027811.625335.295541,592,258.77negative
96Stearic acidC18H36O2284.2718911.678283.2646221,499,099.39negative
97N-[2-chloro-6-(trifluoromethoxy)phenyl]-2,2-dimethylpropanamideC12H13ClF3NO2295.058731.268296.06601165,911,372.3positive
98CholineC5H13NO103.099581.274104.106863,351,190,589positive
99Glucose 1-phosphateC6H13O9P260.029591.332261.03687467,937,507.5positive
100Muramic acidC9H17NO7251.100091.337252.107361,505,589,223positive
101BetaineC5H11NO2117.078971.342118.086243,172,931,864positive
1024-Acetamidobutanoic acidC6H11NO3145.07371.379146.08098225,826,130.1positive
103AcetylcholineC7H15NO2145.110221.437146.117493,685,256,761positive
1041-Aminocyclohexanecarboxylic acidC7H13NO2143.094831.438144.10211337,928,527.3positive
105Pipecolic acidC6H11NO2129.079011.495130.08629196,772,335.8positive
106N-AcetylhistamineC7H11N3O153.090261.76154.09753103,842,799.2positive
1074-Guanidinobutyric acidC5H11N3O2145.085271.843146.09254321,810,960.4positive
108Nicotinic acidC6H5NO2123.032251.945124.03953839,395,599.9positive
109NicotinamideC6H6N2O122.04832.071123.05557569,720,151.5positive
1106-Hydroxynicotinic acidC6H5NO3139.027062.112140.034331,036,882,714positive
111Pyrrole-2-carboxylic acidC5H5NO2111.032382.12112.03966479,797,100.7positive
112L-Pyroglutamic acidC5H7NO3129.04282.175130.05005620,786,771.7positive
1133-isopropoxy-4-morpholinocyclobut-3-ene-1,2-dioneC11H15NO4225.10042.395226.1076867,026,578.08positive
114UracilC4H4N2O2112.027522.441113.0348870,107,158.9positive
115AdenineC5H5N5135.054663.527136.06194330,830,026.4positive
116AdenosineC10H13N5O4267.096823.529268.10411,427,095,688positive
117InosineC10H12N4O5268.080943.964269.08823257,548,392.1positive
118HypoxanthineC5H4N4O136.038593.965137.04587887,426,643.9positive
119Diethyl phosphateC4H11O4P154.039593.974155.04686399,273,696.2positive
120DL-StachydrineC7H13NO2143.094844.175144.102111,831,709,107positive
121D(−)-AmygdalinC20H27NO11457.15954.838458.16678209,671,201.6positive
122N-[3-(2-methyl-4-pyrimidinyl)phenyl]-1,3-benzothiazole-2-carboxamideC19H14N4OS346.088244.856347.0955224,561,559.93positive
1231-methyl-2-oxo-1,2-dihydroquinolin-4-yl N,N-dimethylcarbamateC13H14N2O3246.100794.876247.1080637,336,553.33positive
124N-[1-(4-methoxy-2-oxo-2H-pyran-6-yl)-2-methylbutyl]acetamideC13H19NO4253.13154.899254.1387863,994,227.66positive
1252-(tert-butyl)-6,7-dimethoxy-4H-3,1-benzoxazin-4-oneC14H17NO4263.116084.901264.12335185,237,827.6positive
126L-DopaC9H11NO4197.068994.906198.07626140,151,015.2positive
1272-(2-acetyl-3,5-dihydroxyphenyl)acetic acidC10H10O5210.0534.926211.0603298,428,683positive
1281-(3,4-dimethoxyphenyl)ethan-1-one oximeC10H13NO3195.089714.933196.09698614,007,207positive
1294-HydroxybenzaldehydeC7H6O2122.036954.95123.0442432,952,790.1positive
130RetrorsineC18H25NO6351.168354.971352.17566454,873,409.5positive
131N-(5-methylisoxazol-3-yl)-N’-[4-(trifluoromethyl)-3-pyridyl]ureaC11H9F3N4O2286.066884.99287.0741626,800,545.08positive
1322-[(carboxymethyl)(methyl)amino]-5-methoxybenzoic acidC11H13NO5239.079595.014240.08687364,769,903.8positive
1335-[(2-hydroxybenzylidene)amino]-2-(2-methoxyethoxy)benzoic acidC17H17NO5315.110925.047316.1181971,187,584.76positive
1342-(3,4-dihydroxyphenyl)acetamideC8H9NO3167.058415.09168.06569160,810,937.1positive
1353-MethoxybenzaldehydeC8H8O2136.052485.141137.059751,453,566,975positive
136cis,cis-Muconic acidC6H6O4142.026755.227143.03403370,439,328.5positive
137Xanthurenic acidC10H7NO4205.037585.245206.04486142,669,582.9positive
1383-MethylcrotonylglycineC7H11NO3157.0745.255158.081281,115,733,525positive
1393-hydroxy-3,4-bis[(4-hydroxy-3-methoxyphenyl)methyl]oxolan-2-oneC20H22O7374.136465.256375.1437453,726,456.67positive
1405,6-dimethyl-4-oxo-4H-pyran-2-carboxylic acidC8H8O4168.04235.292169.04958265,935,155.1positive
1418-HydroxyquinolineC9H7NO145.052835.294146.0601260,410,244.6positive
142Kynurenic acidC10H7NO3189.042595.32190.049871,905,007,525positive
143SafroleC10H10O2162.068415.32163.07619212,114,032.4positive
144BergaptenC12H8O4216.042335.336217.04961134,452,483.5positive
145Isoferulic acidC10H10O4194.057975.342195.065252,726,827,967positive
1461-(4-butylphenyl)-3-(dimethylamino)propan-1-one hydrochlorideC15H23NO233.178065.354234.1853395,286,786.45positive
1472-PhenylglycineC8H9NO2151.063355.394152.07062248,042,836.8positive
148N1-(2,3-dihydro-1,4-benzodioxin-6-yl)acetamideC10H11NO3193.073935.394194.08121470,764,198.3positive
149Isohomovanillic acidC9H10O4182.057895.476183.06517632,371,956.6positive
150ResveratrolC14H12O3228.07865.495229.08588127,433,383.6positive
151VanillinC8H8O3152.047345.5153.054617,449,671,173positive
1522-(2-hydroxy-3-methylbutanamido)-4-methylpentanoic acidC11H21NO4231.147485.507232.1546516,473,889.02positive
1534-Coumaric acidC9H8O3164.047275.509165.0544910,555,435,607positive
1547-hydroxy-3-phenyl-4H-chromen-4-oneC15H10O3238.062685.518239.0699648,729,023.77positive
155ApocyninC9H10O3166.063055.52167.07033570,626,404.7positive
1564-Phenylbutyric acidC10H12O2164.083815.539165.09109205,439,194.1positive
157Ferulic acidC10H10O4194.057995.545195.0652575,400,334,073positive
1583-benzyl-4-hydroxy-5-(4-hydroxyphenyl)-2,5-dihydrofuran-2-oneC17H14O4282.089075.564283.0963491,049,837.1positive
159TrifolinC21H20O11448.100825.574449.10809207,852,942.9positive
1602-[5-(2-hydroxypropyl)oxolan-2-yl]propanoic acidC10H18O4202.12055.618203.1277889,488,627.25positive
161Isoeugenyl acetateC12H14O3206.094455.678207.10175107,711,395.8positive
162(5E)-7-methylidene-10-oxo-4-(propan-2-yl)undec-5-enoic acidC15H24O3252.172665.712253.179931,402,040,442positive
163(2S)-2-(2-hydroxypropan-2-yl)-2H,3H,7H-furo[3,2-g]chromen-7-oneC14H14O4246.08925.717247.096481,605,550,194positive
1643,4-Dimethoxycinnamic acidC11H12O4208.07365.72209.0808663,552,367,246positive
165Biochanin AC16H12O5284.068535.722285.075815,527,495,198positive
1664-MethoxycinnamaldehydeC10H10O2162.068075.722163.075351,922,721,932positive
167PiceatannolC14H12O4244.073535.735245.08081268,412,733.9positive
168HesperetinC16H14O6302.078875.748303.08615232,819,296.3positive
1697-hydroxy-3-(4-methoxyphenyl)-4H-chromen-4-oneC16H12O4268.073565.759269.080841,376,762,031positive
170GalanginC15H10O5270.052785.766271.0600618,105,451,784positive
171(2R)-2-[(2R,5S)-5-[(2S)-2-hydroxybutyl]oxolan-2-yl]propanoic acidC11H20O4216.136235.791217.14351217,544,557.8positive
172(1E,4Z,6E)-5-hydroxy-1,7-bis(4-hydroxyphenyl)hepta-1,4,6-trien-3-oneC19H16O4308.104725.809309.11246,531,520.91positive
1733-hydroxy-4-methoxy-9H-xanthen-9-oneC14H10O4242.057915.827243.06519295,235,024positive
174DaidzinC21H20O9416.110735.835417.11801638,726,156.6positive
1754,7-dimethoxy-1H-phenalen-1-oneC15H12O3240.078635.844241.085913,312,421,173positive
176NaringeninchalconeC15H12O5272.068315.952273.0755918,351,009,032positive
17712-Oxo phytodienoic acidC18H28O3292.203425.963293.210692,505,657,284positive
178KaempferolC15H10O6286.048235.967287.055519,006,992,981positive
1792,4-DimethylbenzaldehydeC9H10O134.07345.985117.070141,223,537,063positive
1804-Methoxycinnamic acidC10H10O3178.063096.002179.07036,932,438,506positive
181(+)-ar-TurmeroneC15H20O216.151396.016217.158661,027,389,042positive
182ApigeninC15H10O5270.052786.024271.0600614,095,816,138positive
1836-Pentyl-2H-pyran-2-oneC10H14O2166.099396.049167.10667903,441,178.6positive
184Methyl cinnamateC10H10O2162.068086.071163.07535455,777,480.5positive
1854-Phenyl-3-buten-2-oneC10H10O146.073246.076147.08052483,099,106.7positive
1865-(2,5-dihydroxyhexyl)oxolan-2-oneC10H18O4202.120566.094203.1278598,842,367.03positive
187CardamominC16H14O4270.089166.121271.0964417,850,179,874positive
188CitralC10H16O152.120176.184153.127443,164,365,769positive
189FormononetinC16H12O4268.073666.228269.080935,354,174,356positive
190RhamnetinC16H12O7316.057886.28317.065165,100,142,040positive
1913-MethoxyflavoneC16H12O3252.078726.313253.086477,044,431positive
192trans-CinnamaldehydeC9H8O132.057786.316133.064963,462,748,945positive
1935-hydroxy-6,7-dimethoxy-2-phenyl-4H-chromen-4-oneC17H14O5298.084066.338299.09134474,558,537.9positive
1944-HydroxybenzophenoneC13H10O2198.068136.349199.07541858,164,155.1positive
195SakuranetinC16H14O5286.083676.398287.0909410,055,131,651positive
196VeratroleC8H10O2138.068136.414139.075411,285,393,336positive
197PinocembrinC15H12O4256.073566.416257.0808419,143,801,362positive
198NootkatoneC15H22O218.167216.436219.174487,475,410,243positive
1991-oxo-2,3-dihydro-1H-inden-4-yl benzoateC16H12O3252.078726.481253.08686,170,158.6positive
2003,14-dihydro-15-keto-tetranor Prostaglandin E2C16H26O5298.177946.505299.18539363,566,952.2positive
201CarvoneC10H14O150.104566.555151.111831,494,122,211positive
202ChrysinC15H10O4254.057956.577255.0652353,403,620,396positive
203Coenzyme Q2C19H26O4318.182846.717319.19012250,369,688.1positive
204WNKC21H30N6O5446.228386.72447.2356649,336,245.15positive
205WogoninC16H12O5284.068536.735285.0758116,003,176,213positive
2061,7,8-trihydroxy-3-methyl-1,2,3,4,7,12-hexahydrotetraphen-12-oneC19H18O4310.120576.884311.127966,568,647.86positive
2074-methoxy-6-(prop-2-en-1-yl)-2H-1,3-benzodioxoleC11H12O3192.079066.95193.086336,455,924.178positive
2089-Oxo-ODEC18H30O3294.219467295.226728,284,510,598positive
209(2R)-5-hydroxy-7-methoxy-2-phenyl-3,4-dihydro-2H-1-benzopyran-4-oneC16H14O4270.089287.074271.09656638,934,307.1positive
210(−)-Caryophyllene oxideC15H24O220.182887.191221.190164,440,500,049positive
2119-Oxo-10(E),12(E)-octadecadienoic acidC18H30O3294.219487.935295.2268110,127,470,520positive
212DL-DipalmitoylphosphatidylcholineC40H80NO8P733.5640110.387734.57129171,680,834.5positive
213BetulinC30H50O2442.3818110.556443.38919131,107,308.5positive
214Palmitoyl sphingomyelinC39H79N2O6P702.5692611.002703.57654741,892,482.6positive
Table 2. Partial significant (p < 0.01) differentially expressed proteins (DEPs) between the ethanol extract of propolis (EEP) group and the control group (screened by FC > 2.0 or FC < 0.5 and p < 0.05).
Table 2. Partial significant (p < 0.01) differentially expressed proteins (DEPs) between the ethanol extract of propolis (EEP) group and the control group (screened by FC > 2.0 or FC < 0.5 and p < 0.05).
Protein IDNamepRegulated
O75911Short-chain dehydrogenase/reductase 30.000141468down
Q9UPY5Cystine/glutamate transporter0.000659187up
Q6IBA0NADH dehydrogenase (Ubiquinone) Fe-S protein 5, 15 kDa (NADH-coenzyme Q reductase)0.00094246down
A0A024RDX4ATP-dependent (S)-NAD(P)H-hydrate dehydratase0.001028075up
Q9Y4K0Lysyl oxidase homolog 20.001568804up
Q03405Urokinase plasminogen activator surface receptor0.001635932up
Q96JY6PDZ and LIM domain protein 20.001648702up
A0A024R084Stromal cell-derived factor 4, isoform CRA_c0.001660675down
A0A0A6YYF2HCG1811249, isoform CRA_e0.001700981up
B3KN79cDNA FLJ13894 fis, clone THYRO1001671, highly similar to 59 kDa 2-5-oligoadenylate synthetase-like protein0.002052659down
A6NCE7Microtubule-associated proteins 1A/1B light chain 3 beta 20.002064172up
Q9BXY0Protein MAK16 homolog0.003155254down
Q14139Ubiquitin conjugation factor E4 A0.003259227up
A8K878Mesencephalic astrocyte-derived neurotrophic factor0.003402074up
Q9NQC3Reticulon-40.003468251up
Q9Y316Protein MEMO10.003479756down
E7EPT4NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial0.003643833down
P18827Syndecan-10.004028676up
P49821NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial0.004095327down
A8K0B9rRNA adenine N(6)-methyltransferase0.004645847down
A0A3B3ISF9Endothelin-converting enzyme 10.004659358down
Q96PU5E3 ubiquitin-protein ligase NEDD4-like0.004703096down
Q9NX12cDNA FLJ20496 fis, clone KAT087290.005418258up
Q53HG1NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (Fragment)0.005954737down
Q14684Ribosomal RNA processing protein 1 homolog B0.006713584down
Q04828Aldo-keto reductase family 1 member C10.006731072up
Q9GZM7Tubulointerstitial nephritis antigen-like0.007059725up
P22223Cadherin-30.00794582up
O95167NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 30.008545123down
Q496C9D-aminoacyl-tRNA deacylase0.008949922up
A8K8P8Alpha-(1,6)-fucosyltransferase0.00914376down
B4DTK7cDNA FLJ61387, highly similar to Homo sapiens conserved nuclear protein NHN1 (NHN1), Mrna0.009420668up
A0A024R1I7Tuftelin-interacting protein 110.009715326up
Table 3. The significantly enriched pathways (adjusted p < 0.05) of differentially expressed proteins.
Table 3. The significantly enriched pathways (adjusted p < 0.05) of differentially expressed proteins.
Map TitleAdjusted p ValueRegulatedDescription
ECM-receptor interaction5.55 × 10−5upLaminin subunit beta-3, HCG1811249,isoform CRA_e, Laminin subunit gamma-2, Fibronectin 1, isoform CRA_n, Thrombospondin 1, isoform CRA_a, Agrin, Syndecan-1
Amoebiasis0.000108083upLaminin subunit beta-3, HCG1811249, isoform CRA_e, Laminin subunit gamma-2, Fibronectin 1, isoform CRA_n, Ras-related protein Rab-5B, Serpin B6, Leukocyte elastase inhibitor
Cell adhesion molecules (CAMs)0.000267166upCadherin-3, Cadherin-1, Syndecan-1, Programmed cell death 1 ligand 1, Occludin, MHC class I antigen (Fragment), MHC class I antigen (Fragment)
Nonalcoholic fatty liver disease (NAFLD)1.44 × 10−11downSuccinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial,Mitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit, NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial, cDNA FLJ75930, highly similar to Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa (NDUFA9), mRNA, NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial, NDUFA7 protein (Fragment), NADH dehydrogenase (Ubiquinone) Fe-S protein 5, 15 kDa (NADH-coenzyme Q reductase), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2, NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial,NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (Fragment), NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3, Uncharacterized protein (Fragment), Inhibitor of nuclear factor kappa-B kinase subunit beta
Retrograde endocannabinoid signaling1.44 × 10−11downMitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit, NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial, cDNA FLJ75930, highly similar to Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa (NDUFA9), mRNA, NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial, NDUFA7 protein (Fragment), NADH dehydrogenase (Ubiquinone) Fe-S protein 5, 15 kDa (NADH-coenzyme Q reductase), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2, NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial, NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (Fragment), NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3, Uncharacterized protein (Fragment)
Alzheimer’s disease7.51 × 10−10downSuccinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial, Mitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit,NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial,cDNA FLJ75930, highly similar to Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa (NDUFA9), mRNA,NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial,NDUFA7 protein (Fragment),NADH dehydrogenase (Ubiquinone) Fe-S protein 5, 15 kDa (NADH-coenzyme Q reductase),NEDD8-activating enzyme E1 regulatory subunit,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2,NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial,NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (Fragment),NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3,NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3,Uncharacterized protein (Fragment)
Oxidative phosphorylation1.02 × 10−9downSuccinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial,Mitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit,NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial,cDNA FLJ75930, highly similar to Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa (NDUFA9), mRNA,NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial,NDUFA7 protein (Fragment),NADH dehydrogenase (Ubiquinone) Fe-S protein 5, 15 kDa (NADH-coenzyme Q reductase),NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2,NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial,NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (Fragment),NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3,NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3,Uncharacterized protein (Fragment)
Parkinson’s disease1.02 × 10−9downSuccinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial,Mitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit,NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial,cDNA FLJ75930, highly similar to Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa (NDUFA9), mRNA,NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial,NDUFA7 protein (Fragment),NADH dehydrogenase (Ubiquinone) Fe-S protein 5, 15 kDa (NADH-coenzyme Q reductase),NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2,NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial,NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (Fragment),NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3,NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3,Uncharacterized protein (Fragment)
Huntington’s disease3.50 × 10−8downSuccinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial,Mitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit,NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial,cDNA FLJ75930, highly similar to Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa (NDUFA9), mRNA,NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial,NDUFA7 protein (Fragment),NADH dehydrogenase (Ubiquinone) Fe-S protein 5, 15 kDa (NADH-coenzyme Q reductase),NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2,NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial,NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (Fragment),NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3,NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3,Uncharacterized protein (Fragment)
Metabolic pathways5.47 × 10−7downSuccinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial,Mitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit,NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial,cDNA FLJ75930, highly similar to Homo sapiens NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa (NDUFA9), mRNA,NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial,Drug-sensitive protein 1,Alpha-(1,6)-fucosyltransferase,Short-chain dehydrogenase/reductase 3,NDUFA7 protein (Fragment),NADH dehydrogenase (Ubiquinone) Fe-S protein 5, 15 kDa (NADH-coenzyme Q reductase),NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6,Ferrochelatase, mitochondrial,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2,NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial,NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (Fragment),NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial,2-oxoisovalerate dehydrogenase subunit alpha,N-acetylgalactosaminyltransferase 7,Amidophosphoribosyltransferase,NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 3,NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3,Uncharacterized protein (Fragment),DNA-directed RNA polymerase I subunit RPA2,Biliverdin reductase A,Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase,Phosphoribosylformylglycinamidine synthase (FGAR amidotransferase), isoform CRA_b, Dihydropyrimidinase, UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 4
Glycosaminoglycan biosynthesis—keratan sulfate0.025255288downAlpha-(1,6)-fucosyltransferase, UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 4
Table 4. Volume of A431 cell xenograft tumors in nude mice (mm3, n = 5).
Table 4. Volume of A431 cell xenograft tumors in nude mice (mm3, n = 5).
Time (Days)ControlSolvent Control50 mg/kg EEP100 mg/kg EEP
0 106.9 ± 21.64108.06 ± 16.09127.03 ± 31.07153 ± 32.99
3 297.37 ± 43.17223.74 ± 40.86299.84 ± 68.85267.42 ± 25.71
6 472.45 ± 64.62368.35 ± 51.45505.06 ± 112.64424.31 ± 58.3
9 708.31 ± 74.26696.13 ± 145.68765.78 ± 169.3530.67 ± 57.45
12 919.71 ± 56.471118.4 ± 239.051001.22 ± 202.33771.04 ± 79.93 *
* Mean differences compared with the control group (p < 0.05).
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MDPI and ACS Style

Zhang, C.; Tian, Y.; Yang, A.; Tan, W.; Liu, X.; Yang, W. Antitumor Effect of Poplar Propolis on Human Cutaneous Squamous Cell Carcinoma A431 Cells. Int. J. Mol. Sci. 2023, 24, 16753. https://doi.org/10.3390/ijms242316753

AMA Style

Zhang C, Tian Y, Yang A, Tan W, Liu X, Yang W. Antitumor Effect of Poplar Propolis on Human Cutaneous Squamous Cell Carcinoma A431 Cells. International Journal of Molecular Sciences. 2023; 24(23):16753. https://doi.org/10.3390/ijms242316753

Chicago/Turabian Style

Zhang, Chuang, Yuanyuan Tian, Ao Yang, Weihua Tan, Xiaoqing Liu, and Wenchao Yang. 2023. "Antitumor Effect of Poplar Propolis on Human Cutaneous Squamous Cell Carcinoma A431 Cells" International Journal of Molecular Sciences 24, no. 23: 16753. https://doi.org/10.3390/ijms242316753

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