Ⅰ. INTRODUCTION
Apoptosis, a highly regulated and systematic multi-step process in living organisms, plays a vital role in processes such as human development and immune responses under normal conditions (1). However, disruptions in the regulatory mechanisms of apoptosis can result in various diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions (2). Consequently, inducing apoptosis in cancer cells has become a key and widely employed strategy in cancer treatment (3).
Thuja orientalis, a herbaceous perennial plant native to Korea, Japan, and Southeast Asia, contains bioactive compounds such as sauchinone and neoglignans. The methanol extract of Thuja orientalis (METO) has demonstrated anticancer activity in several cancer cell lines, including stom- ach and lung cancers, though its effects on oral squamous cell carcinoma (OSCC) cells remain largely unexplored.
While anticancer agents are designed to target rapidly dividing cancer cells, they often lack selectivity, affecting normal cells that also divide rapidly. This lack of specificity can result in side effects such as bone marrow depression, lymphoreticular toxicity, gastrointestinal toxicity (e.g., diarrhea and mucositis), and chemotherapy-induced alopecia (4). To minimize these side effects and enhance treatment efficacy, researchers are actively exploring alternative anticancer agents, particularly plant extracts and plant-derived natural compounds, which have emerged as promising candidates. Many of these natural substances exhibit anticancer effects primarily through mechanisms like DNA damage induction and the promotion of apoptosis in cancer cells.
SOCS3, a crucial member of the SOCS family, functions as a negative regulatory protein in cytokine- and growth factor- related signaling pathways. By acting as a negative regulator of Janus Kinase (JAK) and Signal Transducers and Activators of Transcription (STAT) signaling, SOCS3 plays a vital role in preventing malignant transformation and promoting apoptosis in tumor cells. When SOCS3 expression is suppressed, persistent activation of STAT3 can lead to excessive cell proliferation and inhibition of apoptosis (5, 6).
The MAPK pathway is classified into four subgroups: extracellular signal-regulated kinase (ERK) 1/2, JNK, p38, and ERK5 (7). ERK1/2 regulates crucial cellular processes such as proliferation, differentiation, and apoptosis (8), and its activation has been linked to pro-apoptotic signaling, underscoring its role in apoptosis regulation (9). Similarly, c-Jun N-terminal kinase (JNK), another member of the MAPK family, translates external stimuli into diverse cellular responses, including proliferation, differentiation, survival, migration, invasion, and apoptosis (10). Upon activation, JNK triggers a signaling cascade involving the sequential activation of MAP kinase kinase kinase (MAP3Ks), MAP kinase kinase (MAP2Ks), and MAP kinase, which regulates downstream cellular processes (11).
The present study focuses on identifying natural substances with anticancer potential, specifically investigating the anticancer effects of METO on OSCC cells. Additionally, the study aims to elucidate the molecular mechanisms through which METO exerts its effects on OSCC cells.
Ⅱ. MATERIALS AND METHODS
1. METO Chemicals
The METO used in this experiment was purchased from the Korea Plant Extract Bank (Cheongwon-gu, Chungbuk, Korea), dissolved in dimethyl sulfoxide (DMSO), and stored at –20°C in a refrigerator prior to use.
2. HSC4 cells and culture conditions
The oral squamous cell carcinoma cell (OSCC) line HSC4, purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB) in Tokyo, Japan, was used in this experiment. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (GIBCO, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin- streptomycin antibiotics. After being passaged in vitro for more than two days in a 37°C incubator with 5% CO2, the experiment was initiated when the HSC4 cells reached 70–80% confluency in a 60 cm² culture flask.
3. Reagents and antibodies
The Cell Counting Kit-8 (CCK-8) assay kit was obtained from Dojindo Laboratories, Kumamoto, Japan) For the tumor spheroid culture process, a Costar® 24-well Ultra-Low Attachment Surface plate (Corning, 3473) was used as the low-attachment plate. The culture medium was prepared by mixing serum-free DMEM/F12 with 1X B27 (Invitrogen), 20 ng/ml EGF (PeproTech), 20 ng/ml bFGF, and 4 μg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA). The Annexin V FITC Apoptosis Detection Kit I was obtained from BD Bioscience (Heidelberg, Germany). Antibodies targeting Bcl-2, Bax, p53, survivin, cytochrome C, SOCS3, ERK, phosphorylated ERK, phosphorylated JNK, and JNK were procured from Abcam (Cambridge, MA). Additionally, beta-actin, anti-rabbit IgG antibody, and rabbit anti-mouse IgG antibody were sourced from Santa Cruz Biotechnology (Santa Cruz, CA).
4. Analysis of cell viability
In this experiment, the CCK-8 assay was conducted to measure cell viability, following these steps: 1.5 × 10⁴ HSC4 cells were seeded into each well of a 96-well plate and stabilized overnight to assess the effect of METO on cell viability. After stabilization, METO solution was added at concentrations of 35, 50, 70, and 100 μg/mL, and the cells were incubated for 24 and 48 hours at 37°C. Following the incubation, 10 μL of CCK-8 solution was added to each well, and the plate was incubated for approximately 1 hour. Finally, absorbance was measured at a wavelength of 450 nm using a microplate reader, and the average of three repeated experiments was calculated as the estimated value.
5. Production of 3-Dimensional (3-D) tumor spheres
To generate 3-D tumor spheres, 1 × 10⁴ HSC4 cells were seeded into each well of a 6-well plate, with each well containing 2 mL of a serum-free DMEM/F12 solution supplemented with 1X B27, 20 ng/mL of EGF, 20 ng/mL of bFGF, and 4 μg/mL of heparin. The cells were incubated at 37°C for 7 days. Pipetting was performed gently every 2–3 days to mix the cells, and to prevent disruption or aspiration of the developing tumor spheres, only half of the medium was cautiously replaced during each media change.
6. SOCS3 siRNA transfection
The HSC4 cells were prepared and cultured specifically for SOCS3 siRNA transfection. For this process, the following SOCS3 siRNA sequence (Bioneer, Daejeon, Korea) was used:
SOCS3 siRNA (100 ng/mL) was diluted in Opti-MEM medium along with Lipofectamine 3000 (5 μL/mL) following the manufacturer’s instructions. The diluted siRNA and transfection reagent were incubated together at room temperature for 1 hour to allow the formation of siRNA-transfection reagent complexes. Subsequently, the cells were exposed to these siRNA complexes and incubated for 48 hours in a 6-well plate. After transfection, the efficiency of SOCS3 knockdown was evaluated using Western blotting.
7. ANNEXIN V/PI staining
To identify the type of cell death induced by METO treatment in HSC4 cells, ANNEXIN V/PI staining was conducted. HSC4 cells treated with METO solution at predetermined concentrations were harvested and stained using the reagents provided in the ANNEXIN V/PI kit, following the manufacturer’s protocol. The stained cells were then analyzed for staining patterns using FACS FACScan flow cytometry.
8. Western Blotting
To verify the types of cell death induced by METO and its mechanism of inhibiting cell proliferation, Western blot analysis was conducted. Proteins were extracted from METO-treated HSC4 cells, and their concentrations were measured using the Bradford assay. Approximately 40 μg of protein was subjected to electrophoresis on a 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and incubated overnight at 4°C. The membrane was then probed with a primary antibody, washed with phosphate-buffered saline (PBS), and treated with a horseradish peroxidase (HRP)-tagged secondary antibody at room temperature for 2 hours. Protein expression levels were subsequently analyzed using a chemiluminescence detection system.
9. Statistical analysis
Statistical analysis of the experimental data was performed using SPSS 23.0 software, with a p-value of less than 0.05 (p < 0.05) considered statistically significant.
Ⅲ. RESULTS
1. Analysis of changes in cell viability and the number of 3-D tumor spheres in METO-treated HSC4 cells
To assess the inhibitory effect of METO on cell proliferation, HSC4 cells were treated with METO at concen- trations of 0, 35, 50, 70, and 100 μg/mL for 24 and 48 hours. Cell viability was analyzed using a CCK-8 assay, revealing a statistically significant reduction in cell viability in HSC4 cells treated with 100 μg/mL of METO, compared to the control group (Fig. 1A, 1B). Furthermore, when 3-D tumor spheres were cultured in the presence of METO, the number of tumor spheres also decreased significantly.
2. Analysis of the cell cycle for HSC4 cells
To evaluate the effect of METO treatment on inhibiting cell growth in the HSC4 cell line, FITC Annexin-V/PI double staining was conducted. Following 24 hours of METO treatment, the inhibitory effect on cell proliferation was assessed by summing the number of cells in the early and late apoptosis stages and comparing these counts between the control and experimental groups. HSC4 cells treated with METO at a concentration of 100 μg/mL exhibited a significant increase in apoptosis in a dose- and time-dependent manner compared to the control group (Fig. 2).
3. Analysis of the expression of apoptosis and related pathway
To confirm that METO's mechanism of inhibiting cell proliferation is apoptosis and to investigate the related pathways, we analyzed the expression of Bcl-2, Bax, p53, survivin, cytochrome C, and SOCS3 using Western blotting, with beta-actin serving as the internal control. In METO-treated cells, the Bax/Bcl-2 ratio was significantly increased compared to the control group, and p53 expression was also elevated, supporting that METO inhibits cell proliferation through apoptosis (Fig. 3A). Moreover, the expression levels of survivin, cytochrome C, and SOCS3 were also notably increased (Fig. 3A, 3B).
To further elucidate METO's mechanism of action, cells were co-treated with METO and the proteasome inhibitor MG132. This co-treatment revealed that METO-induced sur- vivin upregulation was reversed by MG132 (Fig. 3C), suggesting that METO promotes apoptosis via the proteasome degradation pathway.
Additionally, transfecting SOCS3 siRNA into HSC4 cells followed by METO treatment partially restored cell proliferation, which had been inhibited by METO. This indicates that METO's inhibitory effect on cell proliferation is mediated, at least in part, through SOCS3 expression (Fig. 3D).
4. Activation of ERK and JNK pathways in METO-treated HSC4 cells
To investigate the mechanism underlying the inhibitory effect of METO on cell proliferation, the authors analyzed the expression levels of ERK and JNK proteins in HSC4 cells treated with METO. Using western blotting, they examined the levels of phosphorylated ERK (p-ERK), ERK, phosphorylated JNK (p-JNK), and JNK 24 hours after METO treatment. The expression of p-ERK protein showed a dose-dependent increase compared to the control group following METO treatment (Fig. 4A). In contrast, the expression of p-JNK protein increased at a METO concentration of 35 μg/ml but gradually decreased at higher concentrations of 50, 70, and 100 μg/ml (Fig. 4A). Notably, co-treatment with MG132 and METO resulted in an increase in both p-ERK and p-JNK levels. These findings suggest that METO induces apoptosis in HSC4 cells through the stress-related JNK pathway (Fig. 4B).
Ⅳ. DISCUSSION
The purpose of this experiment was to investigate the inhibitory effects of METO on cell proliferation in the OSCC cell line and to explore its underlying mechanisms. METO treatment of the HSC4 cell line and tumor spheroids derived from it demonstrated a significant concentration-dependent inhibition of cell proliferation and tumor spheroid reduction. To evaluate this effect, HSC4 cells were treated with METO at concentrations of 0, 35, 40, 70, and 100 μg/ml for 24 and 48 hours, followed by a CCK-8 assay. The results showed a significant, time-dependent decrease in viable cell numbers in METO-treated groups compared to the control group, along with a marked reduction in tumor spheroids. These findings suggest that METO inhibits cell proliferation in OSCC cells, likely through apoptosis mediated by the proteasome pathway and influenced by SOCS3.
To better understand the mechanism underlying METO-induced inhibition of cell proliferation, an investigation into the downstream pathways of METO-induced apoptosis revealed that apoptosis is likely mediated through the activation of the ERK and JNK pathways. MJ Hsieh et al. reported that treatment with Dioscin in lung cancer cell lines induced both apoptosis and autophagy, which were associated with the phosphorylation of the ERK and JNK pathways (12). Similarly, Chin-Chuan Su et al. demonstrated that treating the human tongue SCC cell line (SAS cells) with Docetaxel led to concurrent activation of the ERK1/2 and JNK pathways, with the activation of the ERK1/2 pathway promoting pro-apoptotic signaling and inducing apoptosis (9). Additionally, when cisplatin was administered to human ovarian cells, ERK1/2 activation and increased p53 protein levels were observed in a concentration-dependent manner (13).
The observed increase in ERK1/2 protein levels is thought to result from the activation and accumulation of p53, while activated JNK proteins are believed to further contribute to p53 activation (13). In this study, phosphorylation of the ERK and JNK proteins, along with an increase in p53, was observed, suggesting that METO treatment may activate the MAPK pathway in HSC4 cells through p53 upregulation. Although the ERK pathway has traditionally been associated with survival signaling related to cell proliferation and survival, recent studies have highlighted its pro-apoptotic role in neuronal cells, indicating that the function of the MAPK pathway varies depending on the type of stimulus and cell model (14, 15).
SOCS3, a gene with tumor suppressor properties, is known to inhibit cancer cell proliferation (16). Consequently, Abnormal expression or dysfunction of SOCS3 has been linked to tumor development and progression (17). In mammary differentiated cells, SOCS3 expression has been reported to promote apoptosis (18). In cancer cells, Ehlting et al. observed that LPS-induced SOCS3 activates the ERK1/2 and STAT3 pathways (19). Furthermore, evidence indicates that OSM, an IL-6-type cytokine, induces SOCS3 expression and is linked to the activation of the ERK1/2 pathway. Consistently, this study observed an increase in SOCS3 protein and ERK phosphorylation following METO treatment, aligning with previous findings.
Natural products derived from various plants have been extensively studied for their anticancer potential for more than half a century. With a growing need for alternative therapies that can enhance patient survival and quality of life without compromising efficacy, plants have emerged as a rich source of bioactive compounds capable of targeting diverse pathways. Among these, bioactive compounds such as alkaloids, polyphenols, and phytochemicals have demonstrated significant effects against various types of cancer and are considered promising candidates for chemotherapeutic agents. These substances can be employed in diverse ways for cancer treatment and are particularly relevant for addressing altered protein homeostasis in cancer (20). Natural products are generally known to induce apoptosis and autophagy by activating their underlying mechanisms.
This study demonstrated that METO inhibits HSC4 cell proliferation primarily through apoptosis, mediated by altered ERK and JNK signaling and regulation via p53 and SOCS3. These findings suggest that METO holds potential as an anticancer drug for OSCC treatment. However, further studies are required to confirm its efficacy in various cancer cell lines and in vivo models, as well as to clarify its precise mechanisms of action.
