Oral cancer is a malignant tumor that occurs in the oral and maxillofacial region, and its occurrence has been associated with factors such as chewing betel nuts, smoking, and drinking alcohol. According to statistics, oral cancer accounts for about 3% to 5% of all cancers globally, and the 5-year survival rate for oral malignancies in Taiwan is only approximately 53.9%. Early symptoms of oral cancer are easily neglected or misdiagnosed. As the disease progresses, it may metastasize to the neck lymph nodes, lungs, liver, and other parts of the body through the lymphatic and blood channels, affecting the prognosis and quality of survival.

Cancer metastasis is a major challenge in the treatment of oral cancer, and current research focuses on exploring the molecular mechanisms of metastasis, searching for biomarkers, and developing therapeutic strategies. Pyrocurzerenone is an organic compound from the class of sesquiterpenoids, extracted from Chloranthus sertusra's herbs. However, the effect of pyrocurzerenone and its mechanism of action on oral cancer cells are still unknown.  

Recently, Mu-Kuan Chen's team from Taiwan published an article titled " Pyrocurzerenone suppresses human oral cancer cell metastasis by inhibiting the expression of ERK1/2 and cathepsin S proteins " in the Journal of Cellular and Molecular Medicine. The study examined pyrocurzerenone's antimetastatic effects on oral cancer cells, suggesting that pyrocurzerenone decreased oral cancer cell migration and invasion ability by inhibiting ERK1/2 activation and cathepsin S expression. Therefore, pyrocurzerenone might be a valuable treatment to inhibit human oral cancer cell metastasis.

Materials and Methods

The oral cancer cell lines SCC-9, SAS, and SCC-1 were purchased from the Japanese collection of the Research Bioresource Cell Bank (Shinjuku, Japan). All cell lines were grown in Dulbecco's modified eagle medium (DEME; Life Technologies, Grand Island, NY), supplemented with Ham's F12 nutritional mix, 10% fetal bovine serum (FBS), 1.2 g/L sodium bicarbonate, 15 mM HEPES and 1% penicillin/streptomycin. MTT from MilliporeSigma was used to detect cell viability. The wound-healing assay was used to measure directional cell migration in vitro. The transwell migration and invasion assay was used to measure the capacity of cell motility and invasiveness in vitro. The western blot assay was performed to determine protein expression in pyrocurzerenone-treated oral cancer cells.

Results

Firstly, the study examined the effect of pyrocurzerenone on the viability of oral cancer cells using an MTT assay. SCC-9, SAS, and SCC-1 Cells were treated with pyrocurzerenone at different doses (0, 25, 50, and 100 μM) for 24, 48, and 72 h, and no difference was detected between the vehicle group and the treatment group (Fig. 1B–D). Therefore, pyrocurzerenone had no cytotoxic effect in this cell line.

Fig. 1 Non-cytotoxicity of pyrocurzerenone at a dose less than 100 μM in oral cancer cell lines.

Further study was performed to determine the capacity of SCC-9, SCC-1, and SAS cells to metastasize by assessing cell migration and invasion with wound-healing and transwell assays. It was evident that pyrocurzerenone reduced the motility of SCC-9 cells dose-dependent (Fig. 2A, B), by comparing the 24 h results to those of the control group. At 6 h, identical results were seen in SAS and SCC-1 cell lines (Fig. 2C-F).

Fig. 2 Pyrocurzerenone suppressed cell motility of oral cancer cell lines.

In addition, cell motility was recorded by microphotography at 24 h after treatment with different doses of pyrocurzerenone. It was shown that pyrocurzerenone greatly limited the capacity of all three cell lines to migrate and invade after 24 h treatment in a dose-dependent way (Fig. 3A-D). Collective findings indicate that pyrocurzerenone inhibits cell migration in OSCC cell lines without affecting the cells.

Fig. 3 Pyrocurzerenone suppressed migration and invasion of oral cancer cell lines.

Previous studies have suggested that the ERK1/2 pathway involved oral cancer cell motility. Western blot was used to investigate the expression of proteins ERK1/2, JNK1/2, and p38 linked to the MAPK pathway. The results showed that at 50 and 100 μM, pyrocurzerenone dramatically reduced the amount of ERK phosphorylation in SCC-9 and SCC-1 cells (Fig. 4A–C).

Fig. 4 Pyrocurzerenone decreased ERK protein expression in oral cancer cell lines.

Next, ERK inhibitor U0126 (20 μM) was used with pyrocurzerenone to validate the impact of particular inhibitors on the ERK l/2 pathway in SCC-9 and SCC-1 cells. The results showed that combination treatment increased inhibitory effects on motility (Fig. 5A, B), migration, and invasion (Fig. 5C–E) as compared to the pyrocurzerenone treatment (100 μM) alone.

Fig. 5 Pyrocurzerenone suppressed cell motility and invasion through the downregulation of ERK in oral cancer cells.

Cathepsin S has been shown to participate in several processes, including angiogenesis, invasion, and apoptosis, and it was discovered that cathepsin S inhibitors may be beneficial in preventing or delaying cancer metastasis. To explore the connection between pyrocurzerenone and cathepsin S, the expression level of cathepsin S was detected after pyrocurzerenone therapy. The results demonstrated that the expression of cathepsin S was inhibited by pyrocurzerenone in a concentration-dependent manner (Fig. 6A-D). Further detection revealed that cathepsin S induced migration and invasion in oral cancer cells, while pyrocurzerenone treatment reversed this effect (Fig. 6E–G). These findings suggested that pyrocurzerenone inhibited cathepsin S-induced cell migration and invasion in oral cancer cells.

Fig. 6 Pyrocurzerenone suppressed cell migration and invasion by decreasing cathepsin S.

Collectively, these findings suggest that pyrocurzerenon inhibits oral cancer cell metastasis by reducing the activity of ERK and cathepsin S, implying the potential role of pyrocurzerenon in treating oral cancer.

Conclusion

The study evaluates the antimetastatic capacity of pyrocurzerenone against three different subtypes of oral squamous cell carcinoma cells. It reveals that U0126 and pyrocurzerenone significantly inhibit cell motility and migration in both the SCC-9 and SCC-1 cell lines. To explore the molecular mechanism behind the antimetastatic actions of pyrocurzerenone, the activity and quantity of Cathepsin S and common MAPKs, such as p38, JNK1/2, and ERK1/2, were measured. Results suggest that pyrocurzerenon inhibits metastasis by reducing the activity of ERK and cathepsin S.

In summary, this is the first study to show that pyrocurzerenone reduces the invasiveness of oral cancer by decreasing the production of proteins linked to tumor spread through metastasis. This finding represents a step forward in our understanding of potential therapeutic approaches for oral cancer.

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