根據統計，西方國家的睪丸癌發生率有逐年上升的趨勢，從0.0037%上升到0.0059%，而惡性睪丸癌的治療方式多採取為手術結合化學藥物，因此尋找有效的抗癌藥物是非常重要的。導眠靜（midazolam）和異丙酚（propofol）都是具有麻醉和鎮定效果的藥物，主要透過調控中樞神經系內的GABA 受體，而達到鎮定效果。從之前的研究中，我們已經發現導眠靜能透過活化硫胱氨酸蛋白酶（caspase）路徑，進而誘發MA-10 小鼠萊氏腫瘤細胞走向細胞凋亡。在其他研究中，也發現到異丙酚能誘發內質網壓力進而造成肺癌細胞凋亡現象產生。然而導眠靜和異丙酚對於萊氏腫瘤細胞的抗癌能力和調控細胞凋亡的詳細機制尚未清楚。因此在本研究中會進一步探討。我們使用不同濃度的導眠靜和異丙酚處理MA-10 和TM3（小鼠萊氏細胞）細胞株。結果發現MA-10 和TM3 細胞分別30~150 或150~300 μM 導眠靜作用12 小時以後，其細胞存活率都有顯著的下降。而此兩種細胞經由300~600 μM 異丙酚作用24 小時以後，其細胞存活率也都有顯著的下降。利用流式細胞儀分析後，可觀察到subG1 期的顯著上升；透過細胞雙染試驗，更加驗證麻醉劑（導眠靜和異丙酚）確實會誘導這兩種細胞株走向細胞凋亡。此外，此兩種藥物也會促使硫胱氨酸蛋白酶（caspase-8, -9 and -3）的活化以及聚（腺苷二磷酸-核糖）多聚酶（Poly ADP ribose polymerase, PARP）的裂解。除此之外，在MA-10 細胞中，導眠靜造成Bax 移位和cytochrome C 的釋放進而誘發細胞凋亡。絲分裂活化蛋白質激酶（Mitogen-activated protein kinase,MAPK）訊息傳遞路徑的活化，包括ERK1/2, JNK, p38 蛋白，也參與在導眠靜對於TM3 細胞或異丙酚對於此兩種細胞所誘導的細胞凋亡當中。然而，在這兩種細胞中，異丙酚會近一步降低Akt 的磷酸化。另外，導眠靜透過誘發內質網壓力，進一步活化EIF2α 磷酸化、 ATF4、 ATF3 以及CHOP 蛋白表現，讓MA-10細胞產生凋亡。除此之外，導眠靜在MA-10 和TM3 細胞中，會透過調控p53 進而抑制cyclin A、cyclin B 和CDK1 的表現，而調控細胞週期。根據以上實驗結果發現，麻醉劑（導眠靜和異丙酚）在MA-10 和TM3 細胞中，透過活化硫胱氨酸蛋白酶和MAPK，進一步抑制Akt 訊息傳遞路徑，而誘發細胞凋亡。另外，導眠靜在MA-10 細胞中，能造成內質網壓力和調控細胞週期，進而誘發細胞凋亡。此外實驗結果也顯示出對於小鼠萊氏腫瘤來說，導眠靜比異丙酚更適合做為抗癌藥物。

The incident rate of the testicular cancer is increasing in western world, and the overall incidence rate of testicular cancer rose over time, from 3.7 (per 100,000) in 1975 to 5.9 (per 100,000) in 2007. The treatment upon malignant testicular cancer is usually combined surgery with chemical drugs. Midazolam and propofol are widely used as sedative and anesthetic induction agents by modulating the different GABA receptors in the central nervous system. In previous study, we have found that midazolam could induce MA-10 mouse Leydig tumor cell apoptosis by activating caspase cascade. In addition, study has shown that propofol could induce apoptosis in lung cancer through endoplasmic reticulum (ER) stress. However, the detail mechanisms how midazolam and propofol regulating caspase and/or ER stress pathways in Leydig tumor cells remain elusive. In the present study, MA-10 cells and TM3 cells (a mouse Leydig normal cell line) were used with the treatments of midazolam or propofol. Results showed that cell viability significantly decreased by midazolam from 30 to 150 µM in MA-10 cells and from 150 to 300 µM in TM3 cells for 12 hr, respectively, in a dose-dependent manner (p<0.05). Cell viability of MA-10 and TM3 cells also significantly decreased as the dosage of propofol increased (300 to 600 μM) for 24 hr, respectively (p<0.05). In flow cytometry analysis, both midazolam and propofol significantly increased the amounts of subG1 phase cell numbers in both cell lines (p<0.05). AnnexinV/PI double staining further confirmed that two sedative agents induced apoptosis in both cell lines. Moreover, cleaved caspase-8, -9, -3 and/or PARP were significantly activated after treatment of midazolam and propofol in both cell lines. Besides, Bax translocation and cytochrome C release were also involved in midazolam-induced MA-10 cell apoptosis. In TM3 cell line, the phosphorylation of JNK, ERK1/2 and p38 were elevated by midazolam treatment. On the other hand, the expression of p-JNK, p-ERK1/2 and p-p38 were activated by propofol treatment in both cell lines, which indicated that two sedative agents could induce apoptosis through MAPK pathway. In addition, propofol diminished the phosphorylation of Akt to induce apoptosis in both cell lines. Furthermore, the expression of p-EIF2α, ATF4, ATF3 and CHOP could be induced by midazolam in both cell lines, which appeared that midazolam could induce apoptosis probably through endoplasmic reticulum (ER) stress. The expressions of cyclin A, cyclin B and CDK1 could be inhibited by midazolam through the regulation of p53 in MA-10 and TM3 cells, suggesting midazolam could regulate cell cycle to induce apoptosis in mouse Leydig cell lines. In conclusion, midazolam and propofol could induce cell apoptosis by activating caspases and MAPKs pathways, and inhibiting Akt pathway in mouse Leydig cell lines. In addition, midazolam could induce cell apoptosis through activation of ER stress and regulation of cell cycle, and midazolam might be a better anticancer therapy than propofol in Leydig cancer.

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