研究滇藏方枝柏粗萃物及合併5-FU對食道癌之協同抑癌作用

食道癌為全球死亡率高的癌症之一，並且較好發於亞洲地區，在台灣男性十大癌症死亡排名第九，在中國男性與女性的發生率分別為第五、八名，死亡率則為第四、第六名，五年存活率約只有19.2%。5-Fluorouracil (5-FU)為臨床上常用的抗癌藥物，可以用來對抗許多癌症，如直腸癌、胃癌、乳腺癌等，而在食道癌中5-FU為主要治療的化療藥物。5-FU的作用機制是抑制胸苷酸合成酶的作用，阻斷DNA複製中胸腺嘧啶的合成，進而抑制癌細胞的增值，但也會產生食欲不振、噁心、嘔吐、口腔炎、掉髮、骨髓抑制等副作用，其中骨髓抑制會導致血球細胞減少，進而降低病人的免疫力。JIn extract為滇藏方枝柏植物萃取液，近年來的研究顯示有抑制癌細胞增生的作用，因此選用JIn extract來測試對食道癌細胞之抑癌作用及合併5-FU之協同作用。首先利用MTT assay觀察到JIn extract會抑制腫瘤及正常細胞生長，但對腫瘤細胞之抑制能力較強，合併5-FU使用也能有效抑制TP53 exon8 R273C突變的細胞株CE81T/VGH，且具有協同作用，而且是透過抑制Akt signaling pathway來達到抑制細胞增生的作用。接著分析藥物對CE81T/VGH細胞株細胞週期之影響，發現JIn extract會使細胞週期停滯在G0/G1期，5-FU則是G0/G1與S期，且藥物合併使用下並不會改變其作用機制，G0/G1和S期的相關蛋白表現量也有下降的現象。此外，藥物處理後Sub-G1 期的百分比也跟著上升，以TUNEL assay證實合併藥物會誘發CE81T/VGH細胞株產生細胞凋亡，進一步利用西方點墨法來確認藥物合併確實會啟動CE81T/VGH細胞株細胞凋亡的內在及外在路徑促進細胞凋亡，並會促使Caspase activity上升，尤其藥物協同的組別上升較快也較強，同時也能抑制食道癌細胞Regrowth。最後，以MTT assay測試後，發現JIn extract在酸性的環境依然下具有抗癌的能力，且在膽酸存在的環境下對食道癌細胞的抑制效果更好，也對腫瘤細胞具有較佳選擇性抑制的能力。總結上述，JIn extract極具研究的潛能，可朝腫瘤治療或預防的方向進行開發。

Esophageal cancer (EC) is one of the highly lethal cancer in the world. The incidence of esophageal cancer is higher in Asia. In Taiwan, mortality of esophageal cancer is the ninth most lethal cancer. In China, the incidence is fifth and eighth in men and women, and the mortality is fourth and sixth, respectively. Five years survival of esophageal cancer is only 19.2% in the world. On the other hand, 5-Florouracil (5-FU) is an anti-cancer drug widely used in clinical, especially for colorectal, stomach, breast cancer. In addition, 5-FU is also a mainly drug for esophageal cancer, which inhibits the function of thymidylate synthase (TS) and blocks the synthesis of thymine in DNA repair. Thus, 5-FU could anti-proliferation in cancer cell. However, 5-FU had side effects such as loss of appetite, nausea, vomiting, mouth sores, alopecia, bone marrow suppression, and so on. Bone marrow suppression might decrease blood cells that reduced patient''s immunity. JIn extract is the plant extract of Juniperus indica, which was proved had anti-proliferation effect in nasopharyngeal carcinoma. Thus, JIn extract was used to estimate the anti-cancer mechanism and synergistic effect combined with 5-FU on esophageal cancer. In MTT assay, JIn extract was found to inhibit cancer and normal cells proliferation, which was higher in esophageal cancer cells. JIn extract combined 5-FU also had synergistic effect by inhibiting Akt signaling pathway in CE81T/VGH cell which has TP53 exon8 R273C mutation. Then, the effects of JIn extract and 5-FU on cell cycle distribution was analyzed by flowcytometry. In CE81T/VGH cell, JIn extract could arrest cell cycle at G0/G1 phase, and 5-FU could arrest cell cycle at both G0/G1 and S phase. Drug combination group didn’t change the mechanism of each drug that evaluated cell cycle regulators proteins expression at G0/G1 and S phase also decreasing. Besides, the percentage of sub-G1 phase increased after treating drugs. Consequently, TUNEL assay was utilized to prove that JIn extract only and combination with 5-FU induced apoptosis in CE81T/VGH cell. Furthermore, combination group could induce apoptosis via intrinsic and extrinsic pathway and increase caspase activity faster and higher. Simultaneously, JIn extract combined 5-FU could inhibit esophageal cancer regrowth. Finally, JIn extract had anti-tumor ability in acid environment and had better anti-proliferation effect on esophageal cancer cell in bile acid containing environment. In conclusion, JIn extract has potential for esophageal cancer treatment or chemoprevention.