Anticancer effects of 6-shogaol via the AKT signaling pathway in oral squamous cell carcinoma

Abstract Objective Oral squamous cell carcinoma (OSCC) is one of the common type of cancer that leads to death; and is becoming a global concern. Due to the lack of efficient chemotherapeutic agents for patients with oral cancer, the prognosis remains poor. 6-shogaol, a bioactive compound of ginger, has a broad spectrum of bioactivities and has been widely used to relieve many diseases. However, its effects on human oral cancer have not yet been fully evaluated. In our study, we investigated the anticancer effects of 6-shogaol on the proliferation, migration, invasion, apoptosis, and underlying mechanisms within human OSCC cell lines. Methodology We investigated the effect of 6-shogaol on the growth of OSCC cells by cell viability and soft agar colony formation assay. Migration and invasion assays were conducted to confirm the effect 6-shogaol on OSCC cell metastasis. Apoptosis was detected by flow cytometry and the underlying mechanism on the antigrowth effect of 6-shogaol in OSCC cells was assessed using western blotting. Results In our results, 6-shogaol not only suppressed proliferation and anchorage-independent cell growth in OSCC cells, but also induced apoptosis by regulating the apoptosis-associated factors such as p53, Bax, Bcl-2, and cleaved caspase-3. Migration and invasion of OSCC cells were inhibited following the regulation of E-cadherin and N-cadherin by 6-shogaol. Additionally, 6-shogaol treatment significantly inhibited the PI3K/AKT signaling pathway. Conclusion Therefore, our results may provide critical evidence that 6-shogaol can be a potential new therapeutic candidate for oral cancer.


Introduction
Cancer within the oral cavity and pharynx is one of the most common types worldwide, occurring more often in men than women. 1 Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers and is caused by various factors, including tobacco, alcohol, and heredity. 2 Since most oral cancers are diagnosed at a late stage, the 5-year survival rate for OSCC patients was reported to be approximately of 55%. 3 Surgical resection is the primary treatment for most oral cancers, followed by either radiation or chemotherapy, or a combination thereof. 4 Despite advances in anticancer treatment techniques, patients with oral cancer develop esthetic and functional impairments due to the surgical defect at the head and neck region. 5 As a result, the patient's quality of life deteriorates. Therefore, it is important to develop nontoxic and effective new drugs for the prevention and treatment of oral cancer in patients.
Compared to traditional cytotoxic agents, recent oncology has focused on molecules regulating signal transduction pathways, which are key factors in cancer development. Of these pathways, AKT signaling is a central mechanism for carcinogenic activation.
The AKT-modified expression has been implicated in various cancers, including esophageal and colon cancer, and is also associated with cell growth, apoptosis or epithelial-mesenchymal transition (EMT) during the process of carcinogenesis. 6 Figure   3C). To confirm the mechanism of apoptosis which was induced by 6-shogaol, the level of apoptosisrelated proteins was also investigated using western blotting. 6-shogaol treatment significantly increased the expression of p53, Bax, and cleaved caspase-3 in a dose-dependent manner, but decreased the antiapoptotic protein Bcl-2 ( Figure 3C). These results

6-shogaol blocks the PI3K/AKT signaling pathway in OSCC cells
To elucidate the regulatory mechanism underlying the antigrowth effect of 6-shogaol in OSCC cells, we assessed the expression of critical PI3K/AKT pathway proteins. We conducted an ex vivo pull-down assay with OSCC cell lysates to establish whether AKT is a target of 6-shogaol. We identified that 6-shogaol did indeed bind with AKT directly ( Figure 4A). Significant proteins involved in the PI3K/AKT/mTOR signaling pathway were subsequently analyzed using western blotting. Figure 4B shows that 6-shogaol significantly decreased the expression of p-PI3K, p-AKT, and p-mTOR in a dose-dependent manner in both YD-10B and Ca9-22 cells. However, no significant change in the total protein expression of PI3K, AKT, and mTOR was observed. Additionally, glycogen synthase kinase 3beta (GSK3β) was also inhibited following 6-shogaol treatment. These results indicate that 6-shogaol may increase apoptosis of OSCC cells through the PI3K/ AKT/mTOR signaling pathway.

Discussion
Oral cancer is a common fatal cancer and remains a major threat to global public health. Local recurrence and lymph node metastasis are considered the mainly responsible for the low survival rate in patients with OSCC. 18,19 Therefore, although current therapeutic methods, such as surgical resection and radiotherapy,   28 It has been shown that targeting the TLR4 signaling pathway with 6-shogaol plays an important role in cancer therapy. 29 Back to our concern, our findings suggest that 6-shogaol may affect oral cancer cell growth by inhibiting AKT pathway. Notably, 6-shogaol is a multi-targeted drug.
Our study showed that 6- The results also indicate that 6-shogaol inhibits the EMT process and triggered apoptosis by activating the PI3K/AKT/mTOR pathway in OSCC cells. Our findings support the development of natural product-derived compound as a new therapeutic candidate for patients with oral cancer. However, since these are cellular-level results, in vivo studies should be further evaluated for the potential application of 6-shogaol in the prevention and treatment of oral cancer.

Conflicts of interest
The authors declare no conflicts of interest.