The metastasis of cancer cells is considered as a major cause of human death and mortality in any type of cancer. Treatment is difficult if cancer cells spread beyond the primary site of the tumor. Therefore, innovative strategies are required to be developed for the prevention of the invasive potential of cancer cells. In this study we found that head and neck cutaneous SCC cells are much more aggressive in terms of their invasion potential than other human skin cancer cells, such as A431 cells, which are well known human epidermoid carcinoma cells. Milliri et al  reported that the invasion potential of SCC-derived cells is dependent upon EGF stimulation, and this response to EGF does not occur in benign epidermal cells. Also, this response does not occur in A431 cells because these cells have sustained expression of the c-Jun deletion mutant, TAM67, which inhibits EGF-induced cytoskeletal rearrangements necessary for lamellipodia formation and cell rounding and ultimately cell motility and invasion.
The significant findings in the present study are that the treatment of head and neck cutaneous SCC cells with GSPs inhibits invasive potential of cells in a dose-dependent manner, and that is associated with the down-regulation of EGFR expression in cells. The head and neck cutaneous SCC13 cells over-express EGFR, and the inhibition of EGFR by GSPs contributes to the inhibition of cell invasion of these cells. This concept is supported by the evidence that treatment of the SCC13 cells with gefitinib or erlotinib, which are potent inhibitor of EGFR, resulted in a reduction of cell invasion. Similar effects were also noted when the SCC13 cells were transfected with EGFR-siRNA. Treatment of cells with EGF stimulates EGFR, and we observed that treatment of SCC13 cells with EGF enhances cell invasion ability, and that this EGF-induced cell invasion was blocked by the treatment of cells with GSPs. These observations support the evidence that inhibition of head and neck cutaneous squamous cell carcinoma cell invasion by GSPs is mediated through their inhibitory effects on EGFR expression. It has been reported that inhibitors of EGFR can prevent the growth and progression of HNSCC; however, long term use may also induce some form of toxicity [19, 20]. This possibility is not expected with the use of GSPs as these are dietary components and toxicity has not been observed in animal models [11, 12].
Proteins of MAPK family are a downstream target of EGFR, and have been shown to play a crucial role in cancer cell invasion. Our results show that inhibition of invasion of SCC13 cells by GSPs is associated with the inhibition of ERK1/2 phosphorylation. The inhibition of MEK with UO126, a MEK inhibitor, blocked the invasion capacity of SCC13 cells which is similar to the action of GSPs. These observations suggest a possible involvement of ERK1/2/MAPK pathway in inhibition of the invasion of cutaneous HNSCC cells. Activation of the proteins of MAPK family leads to the activation NF-κB which play an important role in multiple biological processes, including inflammation, cell proliferation and angiogenesis [21–23]. Importantly, NF-κB has been identified as an important regulator of EMT in several cancer cell types [21–24]. EMT has been implicated in invasion and metastasis of epithelial tumors. EMT can render tumor cells migratory and invasive through the involvement of all stages: invasion, intravasation and extravasation [13, 14]. During the process of EMT, cells can change from an epithelial to a mesenchymal state. They lose their characteristic epithelial traits and instead gain properties of mesenchymal cells. This process is primarily coordinated by the disappearance or loss of epithelial biomarkers such as E-cadherin with the concomitant appearance or gain of mesenchymal markers such as vimentin, fibronectin and N-cadherin, etc. In the present study, GSPs treatment of SCC13 cells showed the suppression of mesenchymal biomarkers, such as vimentin, fibronectin and N-cadherin while restored the levels of epithelial biomarker such as, E-cadherin, in human cutaneous head and neck SCC cells which suggest that GSPs have the ability to reverse the EMT process in HNSCC cells. These information suggest that reversal of EMT in SCC13 cells by GSPs may also be one of the possible mechanisms through which GSPs reduce the invasiveness of cutaneous head and neck squamous cell carcinoma cells and that lead to inhibition of invasion of SCC13 cells in our system. A recent study showed that GSPs inhibit invasion of melanoma cancer cells and this inhibitory effect of GSPs on melanoma cell invasion was associated with their inhibitory effect on COX-2 overexpression and successive down-regulation of NF-κB and reversal of EMT process . Similar to GSPs, other phytochemicals, such as berberine, have also been shown to inhibit the invasion potential of cancer cells. Berberine inhibits the invasion of melanoma cancer cells through its inhibitory effect on endogenous COX-2 overexpression and successive down-regulation of prostaglandin E2 and prostaglandin E2 receptors .