天然物16-hydroxycleroda-3,13-dien-15,16-olide對於癌細胞中 PI3K訊息傳遞與組織蛋白之作用機制

16-hydroxycleroda-3,13-dien-15,16-olide regulates PI3K pathway and histone-modifying enzymes activities involved in cancer cell apoptosis

林逸翔(Yi-Hsiung Lin)

指導教授 ： 吳永昌

高雄醫學大學/藥學院/天然藥物研究所碩士班/博士(2011年)

摘要

在生物細胞中，磷酸肌醇3激酶訊息傳遞鏈(PI3K pathway)，組織蛋白的修飾(histone modification)及其酵素(histone-modifying enzymes)在細胞增生、分化還有血管新生中皆有著舉足輕重的地位，在癌細胞的生長過程中通常會大量表現且具有深遠的影響。本次的實驗主要探討的目標是天然化合物16- hydroxycleroda-3,13- dien-15,16-olide (PL3) 對癌細胞的生長調控作用中，於三者之間的影響，並試著釐清它導致細胞走向細胞凋亡(apoptosis)的機制。我們的結果顯示，PL3在細胞中可作為一個PI3K的抑制劑，並調控組織蛋白修飾酵素的表現，進而影響到細胞的存活率，也影響了其中參予的訊息傳遞及細胞週期的運行。PL3抑制了PI3K及其下游訊息傳遞因子的磷酸化(phosphorylation)，減少組織蛋白修飾酶中的極光激酶(Aurora B)及癌變酵素(EZH2)的表現，並進一步影響其對於下游基因的表現。當Aurora B減少時，會使細胞發生不正當的kinetochore-microtubule 連結，影響紡錘絲校正點(spindle assembly checkpoint，SAC)之功能，而使染色體的分裂及細胞週期產生問題。而EZH2的表現，則直接關係到多種抑癌基因的表現。基因陣列分析也證實，當PRC2複合物受到藥物抑制時，其下所抑制的多個抑癌基因將會重新活化，此結果有可能與PL3所導致的癌細胞細胞凋亡有關。除此之外，PL3與臨床藥物Imatinib(Glivec，基力克)合併使用上也可以將BCR/ABL融合基因T315I突變的BA/F3細胞(Imatinib-resistant)的致死劑量降低百倍以上。總結來說，PL3可以抑制、降低包含PI3K-AKT訊息傳遞鏈及Aurora B、EZH2等組織蛋白修飾酶的活性，而使得癌細胞的基因表現受到干擾，最終走向細胞凋亡。其結果可提供參考應用在癌症化學治療或是標靶藥物的合併療法上使用。

關鍵字

16-hydroxycleroda-3 ； 13-dien-15 ； 16-olide ； 磷酸肌醇3激酶極光激酶B ； PRC2複合物 ； 組織蛋白修飾 ； 細胞凋亡 ； 細胞有絲分裂阻抑

並列摘要

The PI3K-AKT pathway and histone modifications play essential roles in cellular processes including cell survival, differentiation, and angiogenesis, and usually expressed at maximum levels in cancer cell proliferation. Present study investigated the effect of the natural compound, 16-hydroxycleroda-3, 13-dien-15,16-olide (PL3), in regulating the PI3K-AKT pathway, histone modifications and their related enzymes, which may involve in cancer cell apoptosis. PL3 served as a PI3K inhibitor on influencing the cell survival, signaling transduction and cell cycle progression. It was observed that PL3 targeted and induced dephosphorylation of the PI3K pathway, degradation of Aurora B, EZH2 and their down-stream related gene expression, sequentially shut down the cell cycle and led to apoptosis. As Aurora B was down-regulated, the dysfunction of the spindles and destruction of G2/M phase checkpoint resulted in cellar DNA damage and apoptosis. And the deregulation of EZH2 expression was directly related to the repression of lots of tumor suppressor genes activations. Microarray data showed that PL3 could re-activate the genes which were targeted-repressed by PRC2 complex in PL3-treated cells. Moreover, PL3 also resensitized the Imatinib-resistant T315I-mutated Bcr-Abl+ BA/F3 cells. Taken together, PL3 can perturb the PI3K-AKT pathway and Aurora B resulting in gene silencing and cell cycle disturbance. It was demonstrated that PL3 acted like a novel small-molecular PI3K modulator, thereby potentially contributing to cancer chemotherapy and combination medication.

並列關鍵字

16-hydroxycleroda-3 ； 13-dien-15 ； 16-olide ； PI3K ； Aurora B ； PRC2 complex ； histone modification ； apoptosis ； mitotic block

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