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前言：中醫藥副作用低具治療特定疾病的潛力，過去研究發現紫草萃取成份紫草素對於癌症有治療的效果，但對於口腔癌研究仍不足，口腔癌一旦轉為惡性腫瘤將嚴重威脅患者性命，本研究深入探索紫草素應用於口腔癌細胞治療的治療價值。
研究方法：本研究使用紫草素 Shikonin 和人類口腔癌的細胞株（SCC-4 & SAS ），分別進行細胞生長和毒性分析（CCK-8 assay）；另外使用 In Situ Cell Apoptosis 檢測套組應用 terminal deoxynucleotidyl transferase(TdT)-mediated deoxyuridine triphosphate-biotin nick end-labelling（TUNEL）原理偵測腫瘤檢體中細胞凋亡；在腫瘤動物實驗中將細胞培養液200μl皮下注射至每一隻老鼠的下腹部，待腫瘤長至100 mm3 ，即開始給藥，接著觀察每一隻老鼠的腫瘤變化情形及體重共28天；所有實驗數據以平均值±標準差（mean ± S.E.M）表示，兩組之間的比較以 Student’s t-test 分析，p < 0.05 視為有統計上的意義。
結果：實驗結果發現外給予紫草素能專一的對口腔癌細胞產生作用而非一般細胞，並呈現藥物濃度梯度抑制其生長，利用非致死劑量也發現紫草素能有效藉由抑制細胞中細胞金屬螯合酶的產生，進而抑制癌細胞的爬行與癒合能力，同時也會降低腫瘤惡化因子，如癌細胞血管新生與淋巴管新生前期所釋放的 VEGF-A與VEGF-G，同時隨著劑量加劇誘發細胞自嗜作用，活化ATG-5、LC-3B 、Beclin-1等蛋白質表達量，達到致死劑量後進一步造成粒線體模電位的下降，增加鈣離子的釋放誘發細胞凋亡的途徑，而給予鈣離子螯合劑（BAPTA-AM）可以抑制紫草素產生的細胞凋亡，由上述結果顯示紫草素會誘發口腔癌細胞的自嗜作用與活化凋亡途徑，在小鼠模式上也同樣觀察到紫草素顯著抑制口腔癌生長及造成凋亡的作用。
結論：綜述以上發現紫草素能有效抑制口腔癌細胞的生長、轉移以及降低癌症惡化的相關蛋白質，考量口腔癌的患處可直接投藥，未來臨床應用深具價值，更盼能改善患者的癒後與提升治癒率。
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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 紫草素抑制口腔癌細胞爬行與誘發自嗜與凋亡作用的分子機制研究
