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前言：中医药副作用低具治疗特定疾病的潜力，过去研究发现紫草萃取成份紫草素对於癌症有治疗的效果，但对於口腔癌研究仍不足，口腔癌一旦转为恶性肿瘤将严重威胁患者性命，本研究深入探索紫草素应用於口腔癌细胞治疗的治疗价值。
研究方法：本研究使用紫草素 Shikonin 和人类口腔癌的细胞株（SCC-4 & SAS ），分别进行细胞生长和毒性分析（CCK-8 assay）；另外使用 In Situ Cell Apoptosis 检测套组应用 terminal deoxynucleotidyl transferase(TdT)-mediated deoxyuridine triphosphate-biotin nick end-labelling（TUNEL）原理侦测肿瘤检体中细胞凋亡；在肿瘤动物实验中将细胞培养液200μl皮下注射至每一只老鼠的下腹部，待肿瘤长至100 mm3 ，即开始给药，接着观察每一只老鼠的肿瘤变化情形及体重共28天；所有实验数据以平均值±标准差（mean ± S.E.M）表示，两组之间的比较以 Student’s t-test 分析，p < 0.05 视为有统计上的意义。
结果：实验结果发现外给予紫草素能专一的对口腔癌细胞产生作用而非一般细胞，并呈现药物浓度梯度抑制其生长，利用非致死剂量也发现紫草素能有效藉由抑制细胞中细胞金属螯合酶的产生，进而抑制癌细胞的爬行与愈合能力，同时也会降低肿瘤恶化因子，如癌细胞血管新生与淋巴管新生前期所释放的 VEGF-A与VEGF-G，同时随着剂量加剧诱发细胞自嗜作用，活化ATG-5、LC-3B 、Beclin-1等蛋白质表达量，达到致死剂量後进一步造成粒线体模电位的下降，增加钙离子的释放诱发细胞凋亡的途径，而给予钙离子螯合剂（BAPTA-AM）可以抑制紫草素产生的细胞凋亡，由上述结果显示紫草素会诱发口腔癌细胞的自嗜作用与活化凋亡途径，在小鼠模式上也同样观察到紫草素显着抑制口腔癌生长及造成凋亡的作用。
结论：综述以上发现紫草素能有效抑制口腔癌细胞的生长、转移以及降低癌症恶化的相关蛋白质，考量口腔癌的患处可直接投药，未来临床应用深具价值，更盼能改善患者的愈後与提升治愈率。
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Chien-Min Chen1 Min-Huan Wu2,* You-Jen Tang3
1 Traditional Chinese medicine department, Singying Hospital, Ministry of Health and welfare
2 Tunghai University Bachelor of Science in Senior Wellness and Sports Science, Taichung, Taiwan
3 Traditional Chinese medicine department, Chiayi Branch, Taichung Veterans General Hospital
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Introduction: Traditional Chinese medicine has few side effects, and can be used to treat specific diseases. Past studies have found that Shikonin has a therapeutic effect on cancer, but there is still insufficient research on the effect of Shikonin on oral cancer. Once oral cancer turns into a malignant tumor, it will seriously threaten the lives of patients. This study therefore seeks to explore application value of Shikonin treatment on oral cancer cells.
Methods: The shikonin and human oral cancer cell lines (SCC-4 & SAS) were used in this study. The intensity of CCK-8 reagent is proportional to the number of viable cells, allowing direct analysis of cell growth and toxicity. Additionally, The In Situ Cell Apoptosis Detection Kit was used to detect cell apoptosis in tumor samples based on the principle of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL). In animal experiment, a 200 μl cell suspension was then subcutaneously injected into the lower abdomen of each rat. Drug administration began when the tumor reached 100 mm3, and each rat’s tumor size and body weight were observed until day 28. All experimental data are presented as mean ± standard error of the mean (mean ± S.E.M). Pairwise comparisons were performed using Student’s t-test, and p < 0.05 was considered statistically significant.
Results: Our results found that Shikonin can specifically target oral cancer cells and present a doses dependent to inhibit cell growth. Using non-lethal doses to observe the migration and wound healing ability of Shikonin on cancer cells, we also found that Shikonin can effectively inhibit cells through decrease Matrix metalloproteinases levels, which in turn inhibits the ability of cancer cells to migrate and promote wound healing, while reduce the oncogene, such as VEGF-A and VEGF-G released in the early stage of cancer cell angiogenesis and lymphangiogenesis. Meanwhile, as the dose intensifies, it induces cell autophagy and activates the expression of ATG-5, LC-3B, Beclin-1 and related autophagy proteins. Once reaching a lethal dose, it further causes a decrease in the mitochondrial phantom potential and release of calcium ions to induce cell apoptosis.
Conclusion: The above results show that Shikonin can induce autophagy and activate the apoptosis pathway of oral cancer cells. In the mouse model, Shikonin was also observed to significantly inhibit the growth of oral cancer and cause apoptosis. A review of the research results found that Shikonin can effectively inhibit the growth and metastasis of oral cancer cells and reduce the related proteins of cancer progression, and because oral cancer is easier to administer to the affected area, it is of great value in clinical development in the future, and it is hoped that it can improve patients’ healing and improving the cure rate.
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【Keywords】Shikonin, oral cancer, apoptosis, autophagy, migration, wound healing
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263.3 紫草素抑制口腔癌细胞爬行与诱发自嗜与凋亡作用的分子机制研究
