黑將菌素 (Destruxin B)對人類急性和慢性淋巴癌之抗癌機制探討
Study the Mechanisms of Apoptosis of Destruxin B Anti-tumor Effect on Human Acute and Chronic Lymphoma
中文摘要
黑殭菌素(Destruxin)是由昆蟲寄生性真菌(entomogenous fungi)之一 (Matarhizium anisopliae)所分泌出的毒素，在已分離出的三十餘種中，本研究中採用的是由此 一菌種分泌的二次代謝物 Destruxin B (DB)作為抗腫瘤製劑。DB 對昆蟲具有毒性 且運用在生物農藥已有數年之久。在之前的研究中發現到在體外試驗或是活體試 驗 DB 對於 DBA/2 老鼠 L5178Y 淋巴癌細胞有抑制的效果。本論文將探討由黑 殭菌素 B (Destruxin B)對人類急性淋巴性白血病 Molt-4 和人類非霍金氏淋巴癌 Toledo 細胞造成生長抑制或毒殺作用。由初期的實驗結果得知 Molt-4 細胞在高 於 5.05 uM DB 作用下可以有效的抑制癌細胞生長；而 Toledo 細胞則在 1.26 uM DB 作用下明顯的抑制癌細胞生長。利用 Annexin V 染色及 JC-1 染色的方式更進 一步地證實出 Destruxin B 會引起 Molt-4、Toledo 細胞死亡是經由粒線體膜電位 改變引起細胞凋亡，而非壞死。Destruxin B 作用在 Molt-4 和 Toledo 細胞 48 小 時後，以西方點墨法的結果證實，AIF 和 Bax 蛋白表現有增加的情形，Bid 和 Bcl-2 蛋白表現量遞減，更能證實細胞是經由粒線體膜電位改變引起細胞凋亡而死；並 且也觀察到 FADD、caspase 8、caspase 3 的活化，發現 Destruxin B 可導致細胞 經由死亡接受器途徑而凋亡。綜合以上初步結果得知，Destruxin B 可以抑制人 類急性淋巴性白血病 Molt-4 和人類非霍金氏淋巴癌 Toledo 細胞的生長，其機制 是經由死亡接受器途徑和粒線體途徑而使細胞走向凋亡。
英文摘要
Destruxins are second metabolic products form an entomogenous fungus,
Mararhuzuim anisopliae. These substances are toxic to insect and have been used as an insecticide for decades. Over thirty analogues of destruxins have been isolated by different laboratories, the one used in this study is Destruxin B (DB). The previous study in our laboratory revealed that DB has potent anti-tumor activity in mouse L5178Y lymphoma cells in DBA/2 mice both in vitro and in vivo experiments. In this report, the Molt-4 human acute lymphoblastic leukemia cell line and the Toledo human non Hodgkin’s lymphoma cell lines was used as a subject to evaluate its anti-tumor activity and study the mechanism of the effect. The initial experimental results showed that DB possesses a potent growth suppression effect on Molt-4 when the doses of DB higher than 5.05 uM and Toledo obviously had suppression cell growth at 1.26 uM DB. Further Annexin V and JC-1 staining experimental results showed that Destruxin B caused Molt-4, Toledo cell death by mitochondrial
membrane potential changed to induce apoptosis. From the Western blot experimental results found that the expression of AIF and Bax was increased and that decreased in Bid and Bcl-2 expression in Molt-4 and Toledo cells at 48 hours after DB treatment, thus confirming cell death via mitochondrial membrane potential changed to induce apoptosis. We also found the activation of FADD, caspase 8, caspase 3, thus
confirming cell death may via death receptor pathway to induce apoptosis also. In conclusion, the mechanisms of cell apoptosis by DB treatment may be due to change mitochondrial membrane potential and the death receptor pathway.
