Xanthoxyletin Inhibits Proliferation of Human Oral Squamous Carcinoma Cells and Induces Apoptosis, Autophagy, and Cell Cycle Arrest by Modulation of the MEK/ERK Signaling Pathway.

BACKGROUND This study aimed to investigate the effects of xanthoxyletin, a plant-derived coumarin, on human oral squamous cancer cells in vitro and in mouse xenografts in vivo. MATERIAL AND METHODS The study included SCC-1 human oral cancer cells and EBTr normal embryonic bovine tracheal epithelial cells, which were treated with 0 µM, 5 µM, 10 µM, and 20 µM of xanthoxyletin for 24 hours. The MTT assay assessed cell viability, and autophagy was detected by electron microscopy. Cell apoptosis was investigated using 4',6-diamidino-2-phenylindole (DAPI), annexin V, and propidium iodide (PI) fluorescence flow cytometry, which was also used to investigate the cell cycle. Protein expression was measured by Western blot. Mouse xenografts were used for the in vivo evaluation of the effects of xanthoxyletin. RESULTS Xanthoxyletin significantly inhibited the proliferation of oral cancer cells (IC₅₀, 10-30 µM) with lower cytotoxicity for normal cells. Xanthoxyletin treatment was associated with G2/M arrest of the cell cycle and with increased apoptosis and autophagy of SCC-1 cells. Apoptosis and autophagy induced by xanthoxyletin were also associated with changes in expression of the apoptosis-associated proteins, Bax and Bcl-2, and the autophagy-associated proteins, LC3I, LC3II, Beclin 1, p62, and VSp34. Xanthoxyletin inhibited the expression of components of the signaling cascade of the MEK/ERK pathway in the SCC-1 oral cancer cells. The in vivo effects of xanthoxyletin showed inhibition of growth of mouse xenografts. CONCLUSIONS Xanthoxyletin inhibited the proliferation of human oral squamous carcinoma cells and induced apoptosis, autophagy, and cell cycle arrest by modulation of the MEK/ERK signaling pathway.

Background

Oral and pharyngeal squamous cell cancers are the sixth most prevalent cancers worldwide [1]. Although recent advances have been made in cancer research, oral cancer has only a 62% 5-year survival rate, which is very low when compared with breast and prostate cancer, which have a 5-year survival rate of 89% and 99%, respectively [2]. Therefore, more effective strategies for the early detection of oral squamous cell cancer, treatment with more effective chemotherapy agents, and improved post-therapeutic monitoring are needed. Plants and microbes have been sources of therapeutic drugs for human disease for centuries and continue to be sources of drugs that may also treat cancer [3]. Plants produce compounds and secondary metabolites that combat stress, which have been used in the treatment of human disease, including cancer [4]. Taxol and camptothecins are examples of anticancer agents of plant origin [5].

Secondary plant metabolites have been chemically classified into different groups. Coumarins form an important group of secondary plant metabolites with pharmacological potential [6]. Plant coumarins have been shown to have a range of anticancer and antimicrobial activities [7]. Xanthoxyletin is a linear pyranocoumarin that is produced by several species of plants and has anticancer activity [8]. However, its effects on oral squamous cancer cells remain to be studied [9].

The MEK/ERK pathway is involved in cell signal transduction [10,11]. This pathway is involved in the genesis, development, and progression of several cancer types and is considered to be an important therapeutic target [12,13]. To our knowledge, this study is the first to investigate the effects of xanthoxyletin on oral squamous cancer cells. This study aimed to investigate the effects of xanthoxyletin on cell proliferation, apoptosis, autophagy, and the cell cycle in human oral squamous cancer cells in vitro and in mouse xenografts in vivo.

Material and Methods

Culture and maintenance of cell lines

The human oral squamous cancer cell lines CAL-27, FaDu, SCC-4, SCC-9, SCC-1, and SCC-25 and the EBTr normal embryonic bovine tracheal epithelial cells were obtained from American Type Culture Collection (ATCC). Dulbecco’s modified Eagle’s medium (DMEM) (Thermofisher Scientific, Waltham, MA, USA) was used for culture of the cell lines in an incubator at 37°C in an atmosphere of 5% CO2 and a humidity of 98%.

Cell proliferation assay

The antiproliferative effect of xanthoxyletin was assessed on the cell lines by the MTT assay, as previously described [14]. The human oral squamous cancer cells were treated with 0 μM, 5 μM, 10 μM, and 20 μM of xanthoxyletin for 24 hours. The absorbance at 570 nm was measured to determine the rate of cell proliferation.

Electron microscopy

The induction of autophagy in the xanthoxyletin-treated human oral squamous cancer cells was examined by electron microscopy. Following treatment with 0 μM, 5 μM, 10 μM, and 20 μM xanthoxyletin for 24 hours, the cells were collected by trypsinization and subjected washing followed by fixation in glutaraldehyde (2%) in phosphate buffer (0.1 M). The cells were then post-fixed in osmium tetroxide (1%). This was followed by treatment of cells with ethanol and embedding in resin. The thin sections were sectioned with an ultramicrotome and examined by electron microscopy.

Apoptosis assay

The 4′,6-diamidino-2-phenylindole (DAPI) staining method was used to determine whether xanthoxyletin treatment induced cell apoptosis, as described previously [15]. Briefly, the human oral squamous cancer cells were seeded in six-well plates at a density of 0.6×106/well. The plates were incubated for 12 h and then treated with xanthoxyletin (98% pure) (Sigma-Aldrich, St. Louis MO, USA) at 37°C for 24 h. The cell pellets were obtained by centrifugation, and phosphate-buffered saline (PBS) was used for washing the harvested cell pellets. Then, 4′,6-diamidino-2-phenylindole (DAPI), was used to stain the cells. The stained cells were centrifuged and washed with PBS. Fluorescence microscopy was used to analyze the nuclear morphology of stained cells. Annexin V and propidium iodide (PI) staining were used to determine the percentage of the apoptotic cells, as previously described [16].

Analysis of the cell cycle

The technique of flow cytometry was used to investigate the distribution of the phases of the cell cycle of the human oral squamous cancer cells using a PI staining method. Briefly, the cells were grown in six-well plates and treated with xanthoxyletin for 24 h. The cells were then collected and washed in PBS, followed by fixation in ethanol (70%). After overnight incubation at 4°C, the cells underwent PI staining and flow cytometry.

Western blot

After lysis of the human oral squamous cancer cells in RIPA lysis buffer, the estimation of the protein content of each cell lysate was performed using the bicinchoninic acid (BCA) colorimetric assay. The cell lysate samples were onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, which were then transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were incubated with primary antibodies to Bax, Bcl-2, LC3 I, LC3 II, Beclin 1, p62, Vsp34, MEK, p-MEK, ERK, and p-ERK at 4○C for 24 h. The membranes were incubated for 50 min with the secondary antibodies conjugated with horseradish peroxidase (HRP) (1: 1000) at 25°C. The protein bands were visualized using enhanced chemiluminescence (ECL) reagent.

Mouse xenograft in vivo study

The standards of the National Institutes of Health (NIH) and those approved by the Affiliated Cancer Hospital and Institute of Guangzhou Medical University (Approval No. GMU/AE/245A/2018) for the care and use of animals for laboratory purpose were followed. Four-week-old male BALB/c nude mice were used for the tumor xenografts. The animals had free access to a dry pellet diet and free access to water and were housed in ventilated rooms with a controlled 12-hour light and day cycle at a temperature of 28±2°C. The mice were injected with 5×106 SCC-1 cells subcutaneously in the left flank. As the tumors appeared, dimethylsulfoxide (DMSO) (0.1%) was used to dissolved the xanthoxyletin and diluted with 100 μL of normal saline, prepared by serial dilution from a 200 mg/ml stock solution). Mice were injected intraperitoneally at 25 mg/kg body weight on the first day of the experiment. Xanthoxyletin was given three times a week to the mice in the study (N=18). The control mice (N=18) were injected with 0.1% DMSO in normal saline. Isoflurane anesthesia was to euthanize the mice after six weeks, and the tumors were harvested.

Statistical analysis

Each experiment was performed in triplicate. Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism version 7 (GraphPad Software, Inc., La Jolla, CA, USA). A t-test was performed for comparison between two samples. Tukey’s post hoc test was performed for one-way analysis of variance (ANOVA). A P-value <0.05 was considered to be statistically significant.

Results

Xanthoxyletin inhibited the growth of human oral squamous cancer cells

The effects of xanthoxyletin on the proliferation of human oral squamous cancer cells and a normal cell line were studied using the MTT assay (Figure 1A). Xanthoxyletin had an antiproliferative effect on all the human oral squamous cancer cell lines (Table 1). The lowest IC50 of 10 μM was observed for the SCC-1 cell line, which was selected for further studies (Figure 1B). However, the IC50 of xanthoxyletin was comparatively higher than in the EBTr normal embryonic bovine tracheal epithelial cells (IC50, 95 μM) (Figure 1B). The effects of xanthoxyletin on the human oral squamous cancer cells were concentration-dependent.

Figure 1

The chemical structure of xanthoxyletin and its effects on the viability of SCC-1 human oral squamous cancer cells and EBTr normal embryonic bovine tracheal epithelial cells. (A) Chemical structure of xanthoxyletin. (B) Effect of xanthoxyletin on the viability of SCC-1 and EBTr cells determined by the MTT assay. The results are shown as the mean ±SD (*p <0.01). The experiments were performed in triplicate.

Table 1

Anticancer effects of xanthoxyletin on different oral cancer and normal cell line as determined by MTT assay. The experiments were repeated in triplicate and results are expressed as mean ±SD.

S. No.	Cell lines	IC50 (μM)
1	SCC-1	10±0.8
2	SCC-4	15±1.2
3	SCC-9	15±1.0
4	SCC-25	25±2.0
5	CAL-27	15±1.8
6	FaDu	30±2.4
7	EBTr	95±4.0

Xanthoxyletin induced autophagy and apoptosis in SCC-1 human oral squamous cancer cells

The anticancer effects of xanthoxyletin treatment on the SCC-1 oral cancer cells were analyzed by electron microscopy. Autophagosome formation, indicating autophagy, was triggered by xanthoxyletin in the SCC-1 oral cancer cells (Figure 2). Xanthoxyletin also caused shrinkage of the nuclei of SCC-1 cells, indicating apoptosis. Expression analysis of autophagy-associated proteins was performed to confirm the autophagy. Xanthoxyletin treatment resulted in increased levels of Beclin-1 and LC3-II and reduced levels of p62. However, expression of LC3-I and Vps34 was not affected (Figure 3). Cell apoptosis was confirmed by staining with 4′,6-diamidino-2-phenylindole (DAPI), which identified significant changes in the nuclear morphology of the SCC-1 human oral squamous cancer cells (Figure 4). Annexin V and propidium iodide (PI) staining showed that the percentage of apoptotic SCC-1 cells increased from 1.78% in the controls to 43.55% at 20 μM concentration of xanthoxyletin (Figure 5). Apoptosis was confirmed by the increased expression of Bax and reduced expression of the Bcl-2 in SCC-1 cells (Figure 6)

Figure 2

Electron microscopy of the SCC-1 human oral squamous cancer cells treated with xanthoxyletin shows autophagy. A – autophagic vesicles; M – mitochondria; N – nucleus. The experiments were performed in triplicate.

Figure 3

The effect of xanthoxyletin on the expression of autophagy-associated proteins measured by Western blot. The experiments were performed in triplicate.

Figure 4

Flow cytometry images showing induction of apoptosis of the SCC-1 human oral squamous cancer cells treated with xanthoxyletin. Cell apoptosis was investigated using 4′,6-diamidino-2-phenylindole (DAPI), annexin V, and propidium iodide (PI) fluorescence flow cytometry. The experiments were performed in triplicate.

Figure 5

Flow cytometry of the apoptotic cell populations of the SCC-1 human oral squamous cancer cells treated with xanthoxyletin. Cell apoptosis was investigated using annexin V, and propidium iodide (PI) fluorescence flow cytometry. Q1 (Annexin V-negative, PI-positive), necrotic cells; Q2 (Annexin V-positive, PI-positive), middle to late apoptosis; Q3 (Annexin V-negative, PI-negative), live cells; Q4 (Annexin V-positive, PI-negative), early apoptosis. The experiments were performed in triplicate.

Figure 6

Western blot for the expression of Bax and Bcl-2 proteins of the SCC-1 human oral squamous cancer cells treated with xanthoxyletin. The experiments were performed in triplicate.

Xanthoxyletin caused G2/M arrest of SCC-1 human oral squamous cancer cells

Flow cytometry was used to investigate the effects of xanthoxyletin on the distribution of SCC-1 cells in various phases of the cell cycle. Xanthoxyletin treatment significantly increased the percentage of SCC-1 cells in the G2 phase of the cell cycle from 11.62% to 63.15% (Figure 7). These results showed that xanthoxyletin induced G2/M cell cycle arrest of human oral squamous cancer cells.

Figure 7

Flow cytometry of the cell cycle of the SCC-1 human oral squamous cancer cells treated with xanthoxyletin. The experiments were performed in triplicate.

Inhibition of the MEK/ERK signaing pathway by xanthoxyletin

The effects of xanthoxyletin on the MEK/ERK signaling pathway of SCC-1 human oral cancer cells was studied. Xanthoxyletin caused a concentration-dependent inhibition of p-MEK and p-ERK expression, while no apparent effect was observed on the expression of MEK and ERK (Figure 8). These findings suggested that xanthoxyletin inhibited the MEK/ERK signaling pathway in SCC-1 human oral squamous cancer cells.

Figure 8

Western blot for the expression of the MEK/ERK signaling pathway proteins of the SCC-1 human oral squamous cancer cells treated with xanthoxyletin. The experiments were performed in triplicate.

Xanthoxyletin inhibited tumor growth in vivo in the mouse tumor xenografts

Because xanthoxyletin showed anticancer effects on oral cancer cell lines in vitro, the effects in vivo in a xenograft mouse model were studied. Xanthoxyletin at a dose of 25 mg/kg significantly inhibited the growth of xenograft tumors (Figure 9A). Also, xanthoxyletin treatment reduced the weight and volume of the xenograft tumors in a dose-dependent manner (Figure 9B, 9C).

Figure 9

The effect of xanthoxyletin on growth of the mouse xenograft tumors. (A) The appearance of the treated and untreated xenograft tumors. (B) Xenograft tumor volume. (C) Xenograft tumor weight. The results are shown as the mean ±SD (* p<0.01). The experiments were performed in triplicate.

Discussion

Oral squamous cell cancer comprises almost 90% of oral cancers and is classified as a head and neck cancer [17]. Mortality from oral squamous cell carcinoma is mainly due to metastasis [18]. Chemotherapeutic agents used to treat oral squamous cell carcinoma have side effects and can be ineffective [19]. Recently, plant-based anticancer agents have gained interest due to their reduced toxicity, and have been investigated for their anticancer activities [20].

In the present study, the effects of xanthoxyletin at increasing doses were studied in examined against human cancer cell lines. The results indicated that xanthoxyletin could significantly inhibit the proliferation of human oral squamous cancer cells. In a previous study, xanthoxyletin was reported to reduce the growth of gastric adenocarcinoma cells by triggering apoptosis and arrest of the cell cycle [9]. Autophagy, which is a mechanism for the removal of malignant cells is an important physiological and pathological process, which is why it was investigated [21,22].

This study aimed to investigate the effects and possible mechanism of xanthoxyletin on malignant cells. Xanthoxyletin induced autophagy and apoptosis of SCC-1 human oral squamous cancer cells and changes in the expression of autophagy and apoptosis-associated proteins. Xanthoxyletin treatment resulted in the arrest of SCC-1 human oral squamous cell carcinoma cells at the G2/M phase of the cell cycle, which is a mechanism that can halt tumor growth. Previous studies have shown that molecules that have antitumor properties induce autophagy, apoptosis, and cell cycle arrest of cancer cells [23]. Several types of cancer have been shown to progress via the activation of the MEK/ERK pathway, which promotes cell proliferation [10-12]. In the present study, xanthoxyletin inhibited the expression of p-MEK and p-ERK1/2 in SCC-1 cells in a concentration-dependent manner. In this study, because of the in vitro findings, the antiproliferative effects of xanthoxyletin were studied in vivo in a mouse xenograft study. The preliminary findings in the mouse xenograft model supported the in vitro findings, as treatment with xanthoxyletin inhibited the growth of the xenograft tumors. These preliminary findings using oral squamous cell carcinoma cell lines require validation with further in vitro and in vivo studies and also with further studies to investigate the mechanism of action of xanthoxyletin on oral squamous cell carcinoma cells.

Conclusions

The findings from this study showed that xanthoxyletin inhibited the proliferation of human oral squamous carcinoma cells and induced apoptosis, autophagy, and cell cycle arrest by modulation of the MEK/ERK signaling pathway. Based on the findings from this preliminary study, further studies are recommended on the effects of xanthoxyletin on oral squamous carcinoma cells to determine its potential role as a chemotherapeutic agent.