黑殭菌素 (Destruxin B)對於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 Effects on Human Acute and Chronic Lymphoma

游雅琪(Ya-Chi Yu)

指導教授 ： 陳建和

共同指導教授 ： 劉正民(Cheng-Ming Liu)

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2007年)

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。

關鍵字

黑殭菌素B(Destruxin B) ； 人類急性淋巴性白血病Molt-4 ； 人類非霍金氏淋巴癌Toledo ； 細胞凋亡 ； 粒線體膜電位

並列摘要

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.

並列關鍵字

Destruxin B ； the Molt-4 human acute lymphoblastic leukemia cell line ； the Toledo human non Hodgkin’s lymphoma cell lines ； Apoptosis ； mitochondrial membrane potential

參考文獻

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