1.光輝黴素 A經由抑制Eps8造成人類上皮細胞癌細胞株增生及爬行能力降低之機轉探討 2.咖啡酸導致黑色素腫瘤細胞死亡機制之探討

第一部分光輝黴素A (Mithramycin A) 是一種使用已久的抗生素性抗腫瘤藥物，現在已知光輝黴素 A會與SP1轉錄因子競爭結合到DNA上促進子中富含 GC 序列的區域，造成後續mRNA的形成受阻，進而影響蛋白質的形成；而受到光輝黴素 A調控的基因中，除了維持細胞基本功能的管家基因 (housekeeping genes) 之外，還有不少是與腫瘤的生成有相關的原致癌基因 (proto-oncogenes) ，這也是光輝黴素 A能有化學治療功效的機轉之一。但是除了上述這些基因之外，我們發現光輝黴素 A對於Eps8似乎也有調控的效果，因為Eps8的促進子似乎也有富含GC序列的區域。Eps8是表皮生長因子接受器 (EGFR) 下游的受質之一，當它被活化後與有絲分裂、腫瘤的形成有關。 因此，本研究為了證實Eps8是否會被光輝黴素 A調控，利用光輝黴素 A處理癌細胞細胞株，結果顯示光輝黴素 A可以降低Eps8的mRNA和蛋白質表現量，並且細胞生長和爬行的能力也隨著處理光輝黴素 A而明顯降低。另外，由於已知Eps8會受c-Src家族激酶的調控，我們利用已轉殖v-Src的IV5細胞株來當做一個Src會持續活化下游Eps8的模型，當我們使用光輝黴素 A處理IV5細胞株時，發現Eps8的表現和細胞增生與爬行能力依然會被調控而下降，Src並不能改善它，可見光輝黴素 A抑制Eps8所產生的效果與其上游的Src無關；為了更進一步確認Eps8減少和癌細胞生長與爬行能力是相關的，我們以Eps8 siRNA直接抑制癌細胞中的Eps8表現，發現癌細胞的生長和爬行能力果然也有明顯的下降趨勢，所以我們認為Eps8對於調節癌細胞的生長和爬行佔有重要角色，光輝黴素 A也會經由抑制Eps8表現而達到抑癌的效果。第二部分咖啡酸 (Caffeic acid) 是一種自然界植物普遍存在的物質，具有抗氧化、免疫調節、抗發炎與抗癌的效果，黑色素腫瘤則是一種由於皮膚或身體其他部位的黑色素細胞不正常增生所引起，並且是一旦衍生成惡性腫瘤後預後就很差的癌症，因此它的化學預防也就顯得非常重要。有一些研究顯示黑色素腫瘤細胞在受到某些物質或化學治療藥物時，會經由細胞自噬(autophagy)來抑制癌細胞的增生，因此我們利用咖啡酸處理黑色素腫瘤細胞株 (B16-F1 細胞)，發現此成分也能促使B16-F1 細胞走向細胞自噬，造成細胞數目減少、爬行能力降低。而且細胞自噬相關的調控蛋白包括有磷酸化Akt下降、AMPK上升，並且兩者的調控分子FASN (脂肪酸合成酶，一個癌細胞常見相關升高的蛋白) 也有下降的趨勢。此調控機制導致細胞自噬指標如Beclin-1和LC3都有明顯的上升。另外，反向利用Compound C來抑制AMPK的磷酸化之後，也發現之前咖啡酸抑制細胞數目、爬行能力有回復的現象，並且細胞內細胞自噬的相關蛋白都有降低的情形，證實細胞在經過咖啡酸刺激之下，會產生經由AMPK路徑引發的細胞自噬。了解調控黑色素腫瘤細胞死亡的路徑對於其預防是有價值的指標，研究中發現咖啡酸這個食物中常見的抗氧化成分會降低癌細胞活動能力，並經由細胞自噬使細胞走向死亡，其機制與Akt和AMPK的調控有關。此結果顯示了咖啡酸在加強腫瘤化學預防中的相關性角色，在某些條件下也提供一個有效的方法來對抗黑色素腫瘤細胞。

Part1Mithramycin A is an inhibitor of the binding of the Sp family transcription factor to the GC box. Many studies show that Mithramycin A may reduce the expression of many proto-oncogenes by inhibiting the mRNA and protein synthesis and it has been used as an antibiotic chemotherapy drug for a long time. Recently, Eps8 (EGFR pathway substrate 8) has been revealed to be a novel proto-oncogene related to cellular transformation, Rac activation and actin barbed-end-capping activity. Therefore, the aim of this study was to verify whether Eps8 might be regulated by Mithramycin. Results showed that Mithramycin could reduce the mRNA and protein levels of Eps8 in dose- and time-dependent manners in several cancer cell lines. Furthermore, cell growth and migration ability were also reduced significantly by Mithramycin A treatment. Since Src is a well-known Eps8 activity enhancer, a v-Src transfected IV5 cell line was subjected to Mithramycin A treatment and then analyzed to show that Src expression was unable to restore the Mithramycin-induced decrease in Eps8 expression, cell growth, and migration ability. To further confirm the above mentioned results, the expression of Eps8 was eliminated by a transient transfection with siRNA and subsequent analysis showed that silencing of Eps8 might also lead to a reduced growth and migration ability of cancer cells. These findings suggested that Eps8 was involved in the regulation of growth and motility of cancer cells and Mithramycin A might exert its anticancer ability via a pathway involving the downregulation of Eps8.Part2Caffeic acid is an organic compound found in plants which also shows antioxidant, immunomodulatory, antiinflammtory and antitumor activities. Melanoma is tumorigenesis of melanocytes which produce melanin in the skin or other organs. It has a bad prognosis and difficult to treat when tumor become malignant. Studies has showed some chemicals or drugs reduced the proliferation of melanoma cells through inducing autophagy. We treat the melanoma cell line B16-F1 with Caffeic acid and find that cells go autophagy and the cell mobility diminished. The autophagy regulators p-Akt is down-regulated, and p-AMPK is up-regulated. FASN (Fatty acid synthase), a tumor related protein which can be activated by Akt and inhibited by AMPK is decreased. Beclin-1 and LC3, the autophagy regulated proteins, are express increasd. For investigate the roles of AMPK, we block the phosphorylation of AMPK by Compound C. Results show the effects of growth and migration abilities inhibited by Caffeic acid was recovery. Autophagy related proteins Beclin-1 and LC3 was recovery partially. It means that AMPK is one of the key regulator intermediate with Caffeic acid induced autophagy.It is worthy for the prophylaxis of melanoma to comprehend the death mechanisms regulated in cells. Our study shows Caffeic acid, the antioxidant ingredients conclude commonly in the plants, inhibit the cell mobility and decrease the cell growth of B16-F1 cells through autophagy. This mechnism is regulated by Akt and AMPK. Although it need more investigates to realize the factors involved, we still get an active principle for tumor inhibition and it is important for chemoprevention of melanoma.