Head and neck squamous cell carcinoma (HNSCC) are a widespread and increasingly prevalent neoplastic disease worldwide. More than 60% of patients with HNSCC are diagnosed at stage III or IV tumors and approximately 10% of patients have distant metastasis [1]. Currently, HNSCC is available a comprehensive treatment combining chemotherapy, radiation, and surgery [2]. Nevertheless, the 5-year survival rate in patients with HNSCC remains unsatisfactory for a variety of reasons including late stage detection, recurrence potential, serious adverse events, and drug resistance [3]. Clinical treatment for head and neck cancer can cause side effects related to language functional changes, swallowing, or authoring ability [4]. Therefore, much attention is paid to the development of natural anticancer drugs that can minimize side effects while maintaining the effectiveness of anticancer drugs [5].

Apoptosis a regulatory process for removing unnecessary and potentially harmful cells from an organism [3]. Dysfunctional cell death contributes to many diseases and the development of immune system abnormalities. Analysis of the process of apoptosis is invaluable and helps us understand the etiology of the disease caused by dysfunctional apoptosis [3]. In cancer, apoptosis may occur via the death receptor-dependent exogenous pathway or the mitochondrial-dependent endogenous pathway induced by chemotherapy [6].

Betulinic acid (3-beta-hydroxy-lup20[29]-en-28-oic acid) has gained significant interest due to its various biological and pharmacological activities attributed to this compound, including anti-inflammatory, antimicrobial, antiviral, antidiabetic, antimalarial, anti-human immunodeficiency virus, and anti-tumor effects [7]. Previous studies have shown that betulinic acid may help improve breast, pancreatic, and ovarian cancer [8]. However, whether betulinic acid is effective in treating oral cancer is not yet known. In this study, we investigated the effect of betulinic acid on cell growth and the mechanism of apoptosis induced by betulinic acid in FaDu human pharyngeal carcinoma cells.

Materials

Betulinic acid (Fig. 1) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Thermo Fisher Scientific, Waltham, MA, 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.

Fig. 1. Chemical structure of betulinic acid.

Cell line and cell cultures

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

Cell viability assay (MTT assay)

The FaDu cells were seeded at a concentration of 5×10³ cells/well in 48-well plates. After 24 hr of growth, the cells were treated with betulinic acid at various concentrations for 24 hr. The cell viability assay was evaluated using an MTT assay. At least four separate experiments were performed on each concentration combination.

DAPI staining

FaDu cells (5×10³ cells/well) were incubated on 8-well chamber slide and allowed to adhere to the bottom of the chamber slide overnight. After 24 hr growth, cells were treated with 0, 10, or 20 µM betulinic acid 24 hr. DAPI staining was performed according to the method described above [6]. The stained cells were observed by fluorescent inverted microscopy (Eclipse TE2000; Nikon Instruments, Melville, NY, USA).

LIVE/DEAD cell assay

The FaDu cells (5×10³ cells/well) were cultured on 8-well chamber slide, and allowed to adhere to the bottom of the chamber slide overnight. Thereafter, FaDu cells were treated with 0, 10, or 20 µM betulinic acid for 24 hr at 37℃ and stained using the LIVE/DEAD assay kit. FaDu cells were imaged using a fluorescence microscope (Eclipse TE2000). The LIVE/DEAD assay kit uses green calcein AM to stain the live cells (green fluorescence) and ethidium homodimer 1 to stain the dead cells (red fluorescence).

Immunoblotting

FaDu cells were treated with 0, 10, or 20 µM betulinic acid for 24 hr. Immunoblotting was performed with minor modification according to previously described method [6]. 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 antibody was used as the primary antibody.

Data analysis

All experiments were conducted at least four times. Results were presented as mean±standard error of the mean. Statistical significance was analyzed using Student’s t-test for two groups and one-way analysis of variance for multiple group comparisons. All statistical analyses were performed using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA) [9]. A p-value <0.05 was considered statistically significant.

Betulinic acid is cytotoxic to FaDu human pharyngeal cells

To analyze the effect of betulinic acid on the viability of FaDu cells, cells were treated with various concentrations of betulinic acid for 24 hr. Treatment with betulinic acid at concentrations ranging from 12.5 to 100 µM decreased the viability of FaDu cells in a dose-dependent manner compared to the control group (Fig. 2). These results suggest that betulinic acid induces FaDu cell death in a dose-dependent manner.

Fig. 2. Effect of betulinic acid on cell viability in FaDu pharyngeal carcinoma cells. The effect of betulinic acid on the viability of FaDu cells was assessed by MTT assay. The percentage of cell viability was calculated as the ratio of A570 nms of betulinic acid treated 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 (control cells measured in the absence of betulinic acid).

Betulinic acid induces apoptosis in FaDu pharyngeal carcinoma cells

To investigate the underlying mechanism of betulinic acid-induced FaDu cell self-destruction, we treated FaDu cells with 10 µM and 20 µM betulinic acid for 24 hr before performing DAPI staining and determining chromatin condensation. The number of FaDu carcinoma cells with condensed nuclei significantly increased after betulinic acid treatment (Fig. 3A). To determine the cytotoxicity of betulinic acid in FaDu cells, live and dead cells stained with calcein AM (green fluorescence) and ethidium homodimer 1 (red fluorescence) were visualized under a microscope, respectively. As shown in Fig. 3B, the concentration-dependent number of FaDu cells treated with betulinic acid was increased by ethidium bromide homodimer 1. These results suggest that betulinic acid induces FaDu cell apoptosis a dose-dependent manner.

Fig. 3. Betulinic acid-induced apoptosis in FaDu pharyngeal carcinoma cells. (A) Changes in nuclear morphology by betulinic acid. Cells were treated with 0, 10, and 20 μM betulinic acid for 24 hr. 4',6-diamidino-2-phenylindole staining showed that betulinic acid increased the number of FaDu cells with nuclear condensation. (B) FaDu cells emitting green fluorescence are live cells stained with green calcein AM, whereas cells emitting red fluorescence are dead cells stained with ethidium homodimer 1. Images were observed under a fluorescence microscope (Eclipse TE2000; Nikon Instruments, Melville, NY, USA).

Betulinic acid induces extrinsic apoptosis mediated by death receptor

Western blot analysis was performed to determine whether death receptors induce FaDu apoptosis by betulinic acid. Fas, an apoptosis ligand that induces the death receptor-dependent extrinsic apoptosis pathway in cancer cells, was induced by betulinic acid in FaDu cells (Fig. 4). As shown in Fig. 4, the expression level of cleaved caspase-8, a downstream target of the pro-apoptotic factor Fas, was subsequently increased by betulinic acid. These data suggest that the extrinsic death receptor-mediated apoptosis pathway is involved in FaDu cell death induced by betulinic acid.

Fig. 4. Expression levels of exogenous-apoptosis-related proteins in FaDu pharyngeal carcinoma cells treated with betulinic acid. FaDu cells were treated with 0, 10, and 20 μM of betulinic acid for 24 hr. (A) Expression of Fas cleaved caspase-8, an apoptosis-related protein. (B) Quantitative data of (A) were analyzed using Image J. Data are expressed as mean standard deviations from three independent experiments. **p<0.01, ***p<0.001 as compared to controls.

Betulinic acid induces apoptosis in FaDu pharyngeal carcinoma cells

To analyze whether mitochondria-dependent intrinsic apoptosis is involved in betulinic acid-induced FaDu cell death, western blotting was performed. To investigate the expression levels of anti-apoptotic factors Bcl-xL and Bcl-2, betulinic acid down-regulated factors involved in the intrinsic mitochondria-dependent apoptotic pathway in FaDu cells. In contrast, the expression levels of mitochondria-dependent apoptotic factors such as Bax and Bad were up-regulated by betulinic acid in FaDu cells (Fig. 5). In addition, when FaDu cells were treated with betulinic acid, the expression level of cleaved caspase-9 in FaDu cells increased (Fig. 5). These data suggest that betulinic acid-induced FaDu cell death is associated with the intrinsic mitochondrial-dependent apoptotic pathway.

Fig. 5. Expression levels of unique apoptosis-related proteins in FaDu treated with betulinic acid. FaDu cells were treated with 0, 10, and 20 μM betulinic acid for 24 hr. (A) Expression of apoptosis-related proteins, Bcl-2, Bcl-xl, Bax, Bad, and cleaved caspase-9. (B) Quantitative data in (A) were analyzed using Image J. Data are expressed as the mean standard deviation of three independent experiments compared to the control group. *p<0.05, **p<0.01, ***p<0.001.

Betulinic acid induces FaDu pharyngeal carcinoma cell death via extrinsic and intrinsic apoptotic mechanisms

After betulinic acid treatment, cleaved caspase-3, caspase-8, and caspase-9 after PARP expression both acted on the extrinsic death receptor-mediated and intrinsic mitochondria-dependent apoptotic pathways in FaDu cells (Fig. 6). These results suggest that betulinic acid induces FaDu cell death, which is mediated through cell death receptor-mediated extrinsic and mitochondria-dependent intrinsic apoptosis via activation of the caspase cascade in FaDu pharyngeal carcinoma cells.

Fig. 6. Betulinic acid induces FaDu carcinoma cell death through extrinsic and intrinsic apoptosis mechanisms. (A) Expression of apoptosis-related proteins, cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP). (B) Quantitative data from (A) were analyzed using Image J. Data are expressed as the mean standard deviation of three independent experiments, **p<0.01, ***p<0.001 compared to the control group.

This study, we analyzed the cell growth inhibitory effect and mechanism of betulinic acid on pharyngeal carcinoma cell FaDu has been used as an anti-inflammatory and anti-cancer drug, and has been used as a treatment for several cancer cells [10]. However, the efficacy of betulinic acid against oral cancer cells is not yet known. The study analyzed the inhibitory effects and mechanisms of human-derived oral squamous cell FaDu in order to study the anticancer activity of betulinic acid in oral cancer. Through this study, we aim to increase the value of betulinic acid as a treatment for oral cancer cells and suggest its potential as a functional material for future development of anticancer drugs [11].

The biggest problem with currently used anticancer drugs is that they affect the growth of both normal cells and cancer cells, causing side effects such as gastrointestinal disorders, kidney function, decreased immunity, and decreased bone marrow function [12]. Therefore, the discovery of a substance that targets only cancer cells is expected to have very high clinical application value as it can reduce side effects and expect anticancer effects [13].

Betulinic acid significantly inhibited the proliferation of FaDu, a major oral cancer cell (Fig. 2). These results are consistent with the growth inhibitory effects of betulinic acid on other cancer cell lines. Güttler et al. [8] reported that breast cancer cell line MDA-MB-231 was effective in inhibiting growth by approximately 50% when treated with 10 µM of betulinic acid for 24 hr. In our cell viability (Fig. 2), the cell viability of FaDu pharyngeal carcinoma cells was gradually decreased by the betulinic acid in a dose-dependent manner. Treatment with 12.5 µM and 25 µM betulinic acid resulted in a 66% and 40% reduction in FaDu cell viability compared to the untreated control group, respectively. Inducing the apoptosis of cancer cells is considered the best strategy to suppress unlimited proliferation.

Most anticancer drugs on the market are based on this mechanism [14]. In this study, we confirmed that the proliferation inhibitory effect of oral cancer cells by betulinic acid is mediated by apoptosis, and analyzed caspase signaling and mitochondrial dependence mechanisms by mechanism. Changes in caspase activity are also closely related to the exogenous pathway through ligand binding of death receptors and the endogenous pathway by mitochondrial outer membrane. It is also known to induce apoptosis due to the deficiency of elements required for cell survival [15]. Caspase-8 induces apoptosis signaling through the death receptor, and hydrolyzed caspase-8 activates the mitochondria-dependent apoptosis signaling pathway through hydrolysis of mitochondrial tBID [16]. Betulinic acid induced the hydrolysis of caspase-8, caspase-9, and caspase-3 through death receptors. Ultimately, it cleaved PARP, which performs DNA repair function (Fig. 4-6). In addition, the expression of Bcl-2 and Bcl-xl, which function as anti-apoptotic factors in the mitochondrial outer membrane, was reduced. In addition, as shown in the results in Fig. 5, the expression of Bax and Bad, which are apoptosis promoters, increased. These results are consistent with other studies on the anticancer efficacy and mechanism of betulinic acid. Betulinic acid induces cell cycle arrest in breast cancer cell lines (MDA-MB-231), and the Güttler et al. [8] reported that betulinic acid activates autophagy to cause cell death. Our findings confirmed that betulinic acid activates caspase-3 to induce the death of FaDu cells.

In this study, we identified the value of betulinic acid 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 (2024).

The authors declare that they have no competing interests.