黑殭菌素B (Destruxin B)對大腸癌細胞HT-29之抑制效果及機制

The Anti-Tumor Effects of Destruxin B on HT-29 Colorectal Cancer

呂佩珊(Pei-Shan Lu)

指導教授 ： 劉正民

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2005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)對人類大腸癌細胞株HT-29細胞造成生長抑制或毒殺作用。由初期的實驗結果得知HT-29在高於1.29uM DB作用下可以有效的抑制癌細胞生長。再利用DNA片段及Annexin V染色的方式更進一步地證實出Destruxin B會引起HT-29細胞死亡是經由細胞凋亡而來，而非壞死。再以西方點墨法的結果證實procaspase 3和PARP蛋白降解有增加的情形，更能證實細胞是由於凋亡而死。HT-29在給於DB 48小時之後，可以由西方點墨的結果得知較上游的caspase 9表現進而使得cytochrome c表現上升;Bcl-2的表現減少，Bax和AIF的增加更可以確定出HT-29經由DB作用後，會經由粒線體的內在路徑而走向細胞凋亡。此外，經由DB刺激下，可誘使p53的表現進而抑制cyclin D1與CDK4的表現而使得細胞週期停滯在G0/G1期。此結果也與流式細胞儀的結果相吻合。CDK2、cdc2和cyclin B些微的減少也導致細胞週期停滯在S和G2/M期。也由螢光顯微鏡的結果顯示出DB會使得HT-29產生一些凋亡核質(apoptotic nuclei)也部份印證出由西方點墨法的結果。 綜合以上結果得知，DB可以抑制人類大腸癌細胞株HT-29的生長是透過內在的細胞死亡路徑及影響細胞週期的G0/G1 期和部份的S和G2/M期所致。

關鍵字

黑殭菌素B ； 細胞凋亡 ； 細胞週期停滯

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 HT-29 human colon adenocarcinoma cell line 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 HT-29 when the doses of DB higher than 1.29uM.Further Annexin V staining and DNA fragmentation experimental results showed that the tumor cells apoptosis is one of the major factor to cause the death of tumor cells.From the Western blot experimental results found that caspase 3 activity and PARP degraded form of protein were increased, it confirmed the previous observations. Caspase 9,one of the upstream caspase which is induced by cytochrome c was increased at 48 hours after DB treatment.The expression of some other signal proteins such as decreasing Bcl-2,increasing Bax and AIF confirmed the effect of DB is followed by the intrinsic pathway of apoptosis which is mediated by mitochondria.In addition,DB may enhance p53 protein expression which corresponded suppression cyclin D1 and CDK4 implied that DB may cause cell cycle arrest at G0/G1 phase.These results are accordant with the result from flow cytometry. The expression of CDK2,cdc2 and cyclin B showed a slightly decreasing at some points may explain that partially interfere the cell cycle at S and G2/M phase. Immuno-fluorescence staining results showed that DB treated HT-29 containing some apoptotic nuclei which may be and indirect evidence to support the results from Western blot. In conclusion,the suppression effects of DB on human HT-29 adenocarcinoma cells are mediated by intrinsic pathway of apoptosis and also interfere cell cycle at G0/G1 phase and partially at S and G2/M phase.

並列關鍵字

Destruxin B ； Apoptosis ； Cell cycle arrest

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