모자반류 유래 천연물에 의한 구강암세포의 세포사멸 효과

Abstract

The main cause of oral squamous cell carcinoma with smoking, drinking, eating spicy food that irritates the tongue lining. Treatment options for oral squamous cell carcinoma today are surgery, radiotherapy, and chemotherapy. Among seaweeds such as the Sargassum spp., it was reported that concentration-dependent proliferation was suppressed for human liver cancer, colon cancer, cervical cancer, and gastric cancer cells. The Sargassum spp. used in this study were Sargassum confusum (S. confusum), Sargassum macrocarpum (S. macrocarpum), Sargassum siliquastrum (S. siliquastrum), Sargassum thunbergii (S. thunbergii), Sargassum horneri (S. horneri) and Sargassum fulvellum (S. fulvellum) and oral cancer cell YD-9 and normal oral cell PDL were used as cell lines. 6 spp. of Sargassum were extracted with methanol, and as a result of analyzing the yield, S. fulvellum was the highest at 9.8%. In addition, the total polyphenol content was high in S. macrocarpum (250.39 mg GAE/mL) and S. siliquastrum (223.982 mg GAE/mL), and the total flavonoid content was also high in S. macrocarpum (27.449 mg QE/mL) and S. siliquastrum (165.366 mg QE/mL) was high. Oral cancer cell (YD-9) and normal cell (PDL) were treated with the extracts from 6 spp. of Sargassum. As a result of analyzing cytotoxicity and cell viability, YD-9 cells were inhibited by 80% at a concentration of 400 ug/mL, and normal cell viability was over 90% in all concentration. Based on the above experimental results, an additional experiment was performed by finally selecting S. siliquastrum from 6 spp. of Sargassum was finally selected. The apoptosis mechanism of S. siliquastrum extract (SSE) against oral cancer cells was verified by MTT assay, live and dead assay, DAPI staining, H&E staining, colony formation, wound healing assay and immunoblotting. As a result of analyzing the cytotoxic effect of SSE through Live & Dead assay, significant results were confirmed at concentrations of 200 and 400 ug/mL. The result of analyzing the cell morphological changes of oral cancer cells by H & E staining was confirmed that the cytoplasm was condensed in the concentration range of 100 to 400 ug/mL. In addtion, colony formation and cell proliferation of YD-9 cell were decreased at the concentration of SSE of 50 and 100 ug/mL. As a result of analyzing cancer cell metastasis potential through wound-healing assay, YD-9 cells lost wound-healing activity at 50 and 100 ug/mL of SSE, and it was confirmed that this was mediated through inhibition of MMP-2 and MMP-9 activity. In addition, as the concentration of SSE increased in YD-9 cells, many nuclear changes such as cytoplasmic condensation and nuclear fragmentation were observed, indicating that apoptosis was induced. The SSE affected the activation of PARP following protein division of caspase-3 against YD-9 cells. SSE increased the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 decreased. Thereafter, decreased MMP-2 and MMP-9 through phosphorylation of PI3K/Akt. As a result, it was confirmed that the S. siliquastrum extract inhibits cell growth and induces cell death in YD-9, an oral squamous cell carcinoma cell, through an endogenous apoptosis pathway. The above research results suggest that it is necessary to separate active substances from S. siliquastrum extract through additional research, and that S. siliquastrum can be used as a natural material to inhibit the proliferation of oral cancer cells.