Head and neck squamous carcinoma cells (HNSCCs) occur in the mucosal epithelium of the oral cavity, pharynx, and larynx and are the most common malignant tumor of the head and neck [1]. The incidence of HNSCC is generally associated with tobacco and alcohol abuse, and its incidence has been increased by human papillomavirus infection [2]. The treatment method for head and neck cancer consists of a combination of chemotherapy and radiation for oral cancer. Radiation therapy is primarily used for cancer of the pharynx and larynx [3,4]. Clinical treatment of head and neck cancer can cause side effects related to functional changes in speech, swallowing, or chewing ability [5]. Therefore, a lot of attention is being paid to developing of natural anticancer drugs that can minimize side effects while maintaining the effectiveness of anticancer drugs [6].

Hyperoside (quercetin 3-o-β-d-galactopyranoside) is a flavonoid glycosides with anti-depressant, anti-inflammatory, and anti-cancer effects [7]. The biological functions of peroxides primarily include antioxidant, hypoglycemic, anticancer, anti-inflammatory and cardiovascular effects [8]. Previous studies have shown that peroxide may help improve lung, pancreatic, prostate, and colorectal cancer [9,10]. However, whether peroxides are effective in treating oral cancer is not yet known. In this study, we investigated the effect of hyperoside on cell growth and the mechanism of apoptosis induced by hyperoside in FaDu human pharyngeal carcinoma cells. Our results showed that hyperoside can inhibit cell survival and induce cell death in a dose-dependent manner in FaDu human pharyngeal carcinoma cells.

Materials

Hyperoside (Fig. 1) and 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA). Anti-cleaved caspase-3, -8, -9, anti-Fas, anti-cleaved poly (ADP-ribose) polymerase (PARP), anti-Bcl-2, anti-Bcl-xL, anti-Bax, anti-Bad, and anti-β-actin antibodies were supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA). The live/dead cell viability assay kit was purchased from ThermoFisher Scientific Inc. (Waltham, MA, USA). The 4′,6-diamidino-2-phenylindole (DAPI) stain was purchased from ThermoFisher Scientific, Inc. The hematoxylin and eosin (H&E) staining kit was purchased from Abcam (Cambridge, UK).

Fig. 1. Chemical structure of hyperoside.

Cell line and cell cultures

The FaDu human pharyngeal carcinoma cells obtained from the American Type Culture Collection (Rockville, MD, USA). FaDu cells were grown in minimum essential medium (WelGene, Daegu, Korea) containing 10% fetal bovine serum (WelGene) at 37°C in an atmosphere containing 5% CO₂.

Cell viability test (MTT assay)

The FaDu cells were seeded at a concentration of 1×10⁴ cells/well in 48-well plates. After 24 hr of growth, the cells were treated with hyperoside at various concentrations for 24 hr. The cell viability test was evaluated using an MTT assay. At least four separate experiments were performed on each concentration combination.

DAPI staining

FaDu cells (1×10⁴ cells/well) were cultured in an 8-well chamber slide and allowed to attach to the bottom of the chamber slide overnight. After 24 hr growth, the cells were treated with 0, 100, or 200 μM hyperoside 24 hr. DAPI staining was done according to the previously described method [11]. The stained cells were examined by fluorescent inverted microscopy (Eclipse TE2000; Nikon Instruments, Melville, NY, USA).

Live/dead cell assay

The FaDu cells (1×10⁴ cells/well) were cultured in an 8-well chamber slide, and allowed to attach to the bottom of the chamber slide overnight. Thereafter, the cells were treated with 0, 100, or 200 µM hyperoside for 24 hr at 37°C and stained using the live/dead cell viability assay kit. The cells were imaged using a fluorescence microscope (Eclipse TE2000). The live/dead cell viability assay kit uses green calcein-AM to stain the live cells (green fluorescence) and ethidium homodimer1 to stain the dead cells (red fluorescence).

H&E staining

Morphological alteration as cell shrinkage is a representative characteristic of apoptosis. Hence to observe the morphological alteration, FaDu cells (1×10⁴ cells/mL) were cultured in 8-well chamber slides and allowed to adhere for 24 hr. The cultured FaDu cells were then treated with 0, 100, or 200 μM hyperoside for 24 hr at 37°C. After cultivation, the cells were rinsed with phosphate buffered saline and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 minutes at 4°C. The morphology of FaDu cells incubated with hyperoside was observed by H&E staining. Images of these cells were captured by a microscope (Leica DM750; Leica Microsystems, Heerbrugg, Switzerland). A relative percentage of total cells was counted by cells with both altered and intact morphologies.

Immunoblotting

The FaDu cells were treated with 0, 100, or 200 μM hyperoside for 24 hr. Immunoblotting was done according to the previously described method with minor modifications [11]. The anti-cleaved caspase-3, -8, -9, anti-Fas, anti-cleaved PARP, anti-Bcl-2, anti-Bcl-xL, anti-Bax, anti-Bad, or anti-β-actin antibodies were purchased from Cell Signaling Technology, Inc.

Data analysis

All experiments were performed at least four times. The results were presented as mean±standard error of the mean. The statistical significance was analyzed using Student’s t-test for two groups and a one-way analysis of variance for multi-group comparisons. All statistical analyses were performed using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA). A p-value <0.05 was considered statistically significant.

Hyperoside is specifically cytotoxic to FaDu human pharyngeal cells

To analyze the effect of hyperoside on the survival rate of L-929 cells, different concentrations of hyperoside were treated on the cells for 24 hr. The 25 to 200 μM hyperoside treatment observed no significant changes compared to the control group (Fig. 2A). On the other hand, different concentrations of hyperoside were treated on the cells for 24 hr to analyze the effect of hyperoside on the survival rate of FaDu cells. The 25 to 200 μM hyperoside treatment reduced the survival rate of FaDu cells dose-dependent compared to the control group (Fig. 2B). Based on this result, the IC₅₀ value of hyperoside in FaDu cancer cells was estimated to be approximately 197.82 μM. These results suggest that hyperoside specifically induces FaDu cell apoptosis dose-dependent.

Fig. 2. Effect of hyperoside on cell viability in FaDu pharyngeal carcinoma cells. The effect of hyperoside on the viability of L-929 cells (A) and FaDu cells (B) was accessed by the MTT assay. The percentage of cell viability was calculated as a ratio of A570_nms of hyperoside treated with cells and untreated control cells. Each data point represents the mean±standard error of the mean of four experiments. **p<0.01 vs. control and ***p<0.001 vs. control (the control cells measured in the absence of hyperoside).

Hyperoside induces apoptotic cell death in FaDu pharyngeal carcinoma cells

To investigate the underlying mechanism of FaDu cell self-destruction by hyperoside, DAPI staining was performed and treated with 100 μM and 200 μM hyperoside for 24 hr before determining chromatin condensation in FaDu cells. After hyperoside treatment, the number of FaDu carcinoma cells in which the nucleus was condensed was significantly increased (Fig. 3A). To confirm cytotoxicity by hyperoside in FaDu cells, living and dead cells stained with calcein-AM (green fluorescence) and ethidium homodimer 1 (red fluorescence) were visualized using a microscope, respectively. As shown in Fig. 3B, the concentration-dependent number of FaDu cells treated with hyperoside was increased by Ethidium bromide homodimer 1. In addition, as shown in Fig. 3C, it was confirmed that FaDu cells decreased in a concentration-dependent manner in H&E stain.

Fig. 3. Apoptosis-inducing effect of hyperoside on FaDu pharyngeal carcinoma cells. (A) Changes in nuclear morphology by hyperoside. The cells were treated with 0, 100, and 200 μM hyperoside for 24 hr. 4′,6-diamidino-2-phenylindole staining revealed that hyperoside increased the number of FaDu cells with nuclear condensation. (B) FaDu cells emitting green fluorescence are live cells stained by green calcein-AM, whereas cells emitting red fluorescence are dead cells stained by ethidium homodimer-1. Images were observed by fluorescence microscopy (Eclipse TE 2000; Nikon Instruments, Melville, NY, USA). (C) H&E staining revealed that hyperoside increased the number of FaDu cells with nuclear condensation. Images of these cells were captured by a microscope (Leica DM750; Leica Microsystems, Heerbrugg, Switzerland).

Hyperoside induces extrinsic apoptosis by death receptor

Immunoblotting analysis was performed to confirm whether the death receptor induced the apoptosis of FaDu by hyperoside. Fas, an apoptosis ligand that induces death receptor-dependent exogenous apoptosis pathways in cancer cells, was induced by hyperoside in FaDu cells (Fig. 4). As shown in Fig. 4, the expression level of severed caspase-8 as a downstream target of the pro-apoptotic factor Fas was subsequently increased by hyperoside. These data suggest the involvement of extrinsic death receptor-mediated apoptosis pathways in FaDu cell apoptosis induced by hyperoside.

Fig. 4. The expression levels of extrinsic-apoptosis-related protein in hyperoside treated FaDu pharyngeal carcinoma cells. FaDu cells were treated with 0, 100, and 200 μM of hyperoside for 24 hr. (A) The expression of apoptosis-related protein, Fas cleaved-caspase-8. (B) Quantitative data of (A) were analyzed using Image J. Data are expressed as means standard deviation of three independent experiments. **p<0.01, ***p<0.001 compared with the control group.

Hyperoside induces apoptotic cell death in FaDu pharyngeal carcinoma cells

Immunoblotting was performed to analyze whether mitochondrial-dependent endogenous apoptosis was involved in FaDu cell apoptosis by hyperoside. To investigate the expression levels of Bcl-2 and Bcl-xL, anti-apoptotic factors associated with intrinsic mitochondrial-dependent apoptosis pathways, were downregulated by hyperoside in FaDu cells. On the other hand, expression levels of mitochondrial-dependent pro-apoptotic factors such as Bax and Bad were upregulated by hyperoside in FaDu cells (Fig. 5). In addition, when treated with hyperoside in FaDu cells, the expression level of caspase-9 cut in FaDu cells was increased (Fig. 5). These data show that FaDu apoptosis induced by hyperoside involves an intrinsic mitochondrial-dependent apoptosis pathway.

Fig. 5. The expression levels of intrinsic-apoptosis-related protein in hyperoside-treated FaDu. FaDu cells were treated with 0, 100, and 200 μM of hyperoside for 24 hr. (A) The apoptosis-rerlated protein expression, Bcl-2, Bcl-xl, Bax, Bad, and cleaved caspase-9. (B) Quantitative data of (A) were analyzed using Image J. Data are expressed as means standard deviation of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared with the control group.

Hyperoside induces FaDu cancer cell apoptosis through exogenous and endogenous-cell apoptosis mechanisms.

Both cleaved caspase-8 and caspase-9 acted on exogenous death receptor-mediated and intrinsic mitochondrial-dependent apoptosis pathways in FaDu cells after hyperoside treatment following expression of cleaved caspase-3 and PARP (Fig. 6). These results suggest that hyperoside induces FaDu cell death, which is mediated by death receptor-mediated exogenous and mitochondrial-dependent intrinsic apoptosis through activation of caspase cascade in FaDu pharyngeal cancer cells.

Fig. 6. Hyperoside induces FaDu cancer cell apoptosis through exogenous and endogenous cell apoptosis mechanisms. (A) The expression of apoptosis-related protein, cleaved caspase-3 and poly (ADP-ribose) polymerase. (B) Quantitative data of (A) were analyzed using Image J. Data are expressed as means standard deviation of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared with the control group.

This study, we analyzed the cell growth inhibitory effect and mechanism of hyperoside on oral cancer cell FaDu. Hyperoside has been used as an anti-inflammatory and anti-cancer drug, and has been used as a treatment for several cancer cells [6]. However, the efficacy of hyperoside on oral cancer cells has not yet been identified. This study analyzed the inhibitory effect and mechanism of human-derived oral squamous epithelial cell FaDu to study the anticancer activity of hyperoside in oral cancer. Through this study, we would like to increase the value of hyperoside as a treatment for oral cancer cells and present the potential as a functional natural material for the development of anticancer drugs in the future [7].

The biggest side effects of anticancer drugs currently in use affect the growth of normal cells and cancer cells, resulting in side effects such as gastrointestinal disorders, kidney function, decreased immunity, and decreased bone marrow function [12]. Therefore, the discovery of substances that only target cancer cells without affecting the growth of normal cells can reduce side effects and expect anticancer effects, so the clinical application value is expected to be very high [13]. Hyperoside did not affect the growth of L-929 cells used as normal cells. The proliferation of FaDu, the main oral cancer cell, was significantly suppressed (Fig. 2). These results are consistent with the growth inhibitory effect of hyperoside on other cancer cell lines. Qui’s research team [14] reported that 100 μM of hyperoside, a breast cancer cell line, is effective in suppressing growth by about 50% when treated 24 hr. In our cell survival rate (Fig. 3), the cell survival rate of FaDu pharyngeal carcinoma cells was gradually reduced by hyperoside in a dose-dependent manner. Additionally, similar to the DAPI staining results, H&E staining also showed that upon hyperoside treatment, not only did the number of FaDu cells decrease in a dose-dependent manner, but the number of cells with altered morphology increased (Fig. 3C). Taken together, these data consistently suggested that hyperoside-induced cell death is involved in apoptosis of FaDu cells. Treatment with 100 μM and 200 μM Arctigenin resulted in 75% and 50% reduction in FaDu cell survival compared to untreated controls, respectively.

Inducing apoptosis of cancer cells is considered the most effective strategy to suppress unlimited proliferation, and most anticancer drugs on the market are based on this mechanism [15]. This study, it was confirmed that the effect of inhibiting the proliferation of oral cancer cells by hyperoside was mediated by apoptosis, and caspase signaling and mitochondrial-dependent mechanisms were analyzed as mechanisms. The activity of caspases is also closely related to the exogenous pathway through ligand binding of death-receptors and the endogenous pathway by mitochondrial outer membranes, and is known to induce cell death by lacking elements necessary for cell survival [16]. Caspase-8 induces apoptosis signaling through death-receptor, and the hydrolyzed caspase-8 activates the mitochondrial-dependent apoptosis signaling pathway through the hydrolysis of the mitochondrial tBID [16]. Hyperoside induced hydrolysis of caspase-8, caspase-9, and caspase-3 through a death-receptor, and eventually the PARP, which performs DNA recovery function, was cut (Fig. 4-6). In addition, the expression of Bcl-2 and Bcl-xl, which function as anti-cell death factors, was reduced in the mitochondrial outer membrane. The expression of Bax and Bad, which are cell death promoters, increased (Fig. 5). These results are consistent with other studies on hyperoside’s anti-cancer efficacy and mechanism. Hyperoside induces cell cycle arrest in breast cancer cell lines (MCF-7 and 4T1), and the Qui’s research team [14] reported that hyperoside activates caspase-3 to cause cell death. In this study, we identified the value of hyperoside as a potential treatment for human oral cancer through effective anti-cancer activity analysis for oral cancer cells.

This study was supported by research fund from Chosun University (2023).

The authors declare that they have no competing interests.