- 學位論文
Denbinobin, Moscatilin 及 Cucurbitacin B 在人類結腸直腸癌 引起細胞凋亡作用機轉之探討
The Apoptotic Mechanisms of Denbinobin, Moscatilin and Cucurbitacin B in Human Colorectal Cancer Cell Lines
摘要
許多抗癌藥物是由天然藥物所萃取或是再經過化學結構的修飾而來的，近年來公佈將近有一半的小分子全新化學物質新藥 (NCE)，是經由天然物和其衍生物所衍生而來的，所以運用天然藥物作為新藥開發，在全球是非常具有潛力與發展力的。本篇論文中，我們發現石斛的萃取活性成分 denbinobin 與 moscatilin 可以對人類結腸直腸癌細胞株 HCT-116 在 in vitro 有引起細胞凋亡的作用，在 in vivo 有抑制腫瘤生長的效果。另外，南投秋海棠的萃取活性成分 cucurbitacin B 對人類結腸直腸癌細胞株 HT-29 則可誘發細胞凋亡作用。本篇論文探討這三個藥物是否具有開發為抗結腸直腸癌臨床用藥的潛力。 論文的第一部分為 denbinobin，是從大爪石斛 (Ephemerantha lonchophylla) 的莖所萃取出來的純化物，我們發現此一成分可以引起人類結腸直腸癌細胞株 HCT-116 細胞凋亡。研究結果發現 denbinobin 所造成的細胞凋亡機轉不是經由活化 caspase 途徑而來，denbinobin 主要是引起粒線體釋放出 apoptosis-inducing factor (AIF)，AIF 會再進入細胞核引起細胞凋亡作用。另外，我們也發現 denbinobin 會引起 DNA damage，活化 p53 與其下游調控的蛋白。在活體動物實驗中，denbinobin 也能有效地抑制 HCT-116 腫瘤的生長，且對小鼠的毒性低。綜合以上實驗發現，denbinobin能經由粒線體釋放 AIF 以及引起 DNA damage，造成人類結腸直腸癌細胞株HCT-116 的細胞凋亡，加上動物實驗也證實能有效抑制 HCT-116 腫瘤細胞生長，所以我們認為 denbinobin 可以開發為新的抗癌藥物。 論文的第二部分為 moscatilin，是由環草石斛 (Dendrobrium loddigesii) 的莖所萃取出的純化物，我們發現此一成分可以引起人類結腸直腸癌細胞 HCT-116 細胞凋亡。Moscatilin 使細胞週期停止在 G2/M，並伴隨著 subG1 期的細胞(凋亡的細胞)數量增加。我們也發現 moscatilin 可以抑制微管蛋白的聚合作用，所以推測 moscatilin 會與微管蛋白結合。Moscatilin 也可以活化 JNK 及其下游的蛋白 c-Jun，進而活化 caspase 途徑而引起 HCT-116 細胞凋亡。另外，我們也發現 moscatilin 會引起 DNA damage，活化下游的 p53 蛋白與 p21WAF1/CIP1 蛋白。在活體動物實驗中，moscatilin 也證實呈現無毒性且能有效抑制 HCT-116 腫瘤細胞的生長。綜合以上實驗結果，我們認為 moscatilin 經由抑制微管蛋白聚合以及產生 DNA damage，經由活化 JNK 而啟動內生性途徑─ caspase-9 和 caspase-3，造成人類結腸直腸癌細胞株 HCT-116 的細胞凋亡作用。由於在動物實驗也有效的抑制 HCT-116 腫瘤生長，所以我們認為 moscatilin 可以開發為新的抗癌藥物。 論文的第三部分為 cucurbitacin B，從南投秋海棠 (Begonia nantoensis)所萃取出的三萜成分，可以在低濃度引起人類結腸直腸癌細胞 HT-29 細胞凋亡。Cucurbitacin B 會造成粒線體膜電位降低，並引起 Bcl-xL 蛋白磷酸化，Mcl-1 蛋白表現減少。Cucurbitacin B 也會造成 caspase-8、caspase-9、caspase-3 和 caspase-6 的活化，並造成下游受質 PARP 的分解，這表示 cucurbitacin B 可以引起內在途徑與外在途徑的 caspases 活化，進而造成細胞凋亡。Cucurbitacin B 會引起 HT-29 細胞週期停止在 G1 期；並增加 p21WAF1/CIP1 及 p27KIP1 的蛋白表現，抑制 CDK2、CDK4 及 cyclin E 的蛋白表現。Cucurbitacin B 也會造成 ERK、p38、JNK 及 Akt 的活化和抑制 STAT3 的磷酸化。總而言之，cucurbitacin B 會引起人類結腸直腸癌細胞 HT-29 細胞週期停止在 G1 期並且引起細胞凋亡。
並列摘要
Natural products and their bio-active chemical derivatives have historically been invaluable as a source of novel cancer therapeutic agents. In recent decades, almost half of small-molecule New Chemical Entities (NCEs) were originally from natural products and their derivatives. To use natural products as a rich source of new drug development is a potential trend in the worldwide. In this thesis, we found denbinobin and moscatilin, isolated form traditional Chinese medicine - Herba dendrobii, were effective against the human colorectal cancer cells in vitro and in vivo. Cucurbitacin B, isolated from Begonia nantoensis, can induce apoptosis in human colorectal HT-29 cells. In this thesis, we study if these natural components can be new drugs against human colorectal cancer. Denbinobin, a phenanthraquinone derivative isolated from the stems of Ephemerantha lonchophylla, induces apoptosis in human colon cancer HCT-116 cells. Interestingly, denbinobin induces apoptosis via caspase-independent pathway in HCT-116 cells. Denbinobin triggered the translocation of apoptosis-inducing factor (AIF) from the mitochondria into the nucleus. Denbinobin treatment also caused DNA damage, activation of the p53 tumor suppressor gene, and upregulation of numerous downstream effectors. A HCT-116 xenograft model demonstrated the in vivo efficacy and low toxicity of denbinobin. Taken together, our findings suggest that denbinobin induces apoptosis of human colorectal cancer HCT-116 cells via DNA damage and an AIF-mediated pathway. These results indicate that denbinobin has potential as a novel anti-cancer agent. Moscatilin, purified from the stem of the orchid (Dendrobrium loddigesii), induces apoptosis of human colorectal cancer HCT-116 cells. Moscatilin induced arrest of the cell-cycle at G2/M, with an associated decrease in cells at the G1 and an increase of cells at subG1. In addition, Moscatilin inhibited tubulin polymerization, suggesting that that it might bind to tubulins. Moscatilin also induced the phosphorylation of JNK1/2 and c-Jun, and this may contribute to caspase-dependent apoptosis signaling. Moreover, moscatilin induces DNA damage, activation of p53, and activation of the downstream effector p21WAF1/CIP1. A HCT-116 xenograft model demonstrated the in vivo efficacy and low toxicity of moscatilin. In summary, our results suggest that moscatilin induces apoptosis of colorectal HCT-116 cells via tubulin depolymerization and DNA damage stress and that this leads to the activation of JNK and mitochondria-involved intrinsic apoptosis pathway. These results indicate that moscatilin has potential as a novel anti-cancer agent. Cucurbitacin B, a triterpenoid isolated from Begonia nantoensis, induces apoptosis of human colorectal cancer HT-29 cells at low concentration. Cucurbitacin B reduced mitochondrial membrane potential, induced Bcl-xL phosphorylation, and reduced expression of Mcl-1. Cucurbitacin B-induced apoptosis of HT-29 cells was dependent on activation of the extrinsic and intrinsic apoptosis pathways, as indicated by its activation and processing of procaspase-8, -9, -3, -6 and PARP. Cucurbitacin B blocked HT-29 cells at the G1 phase, elevated the levels of p21WAF1/CIP1 and p27KIP1, and suppressed expression of CDK2, CDK4, and cyclin E. Cucurbitacin B significantly activated MAPKs (ERK, p38, JNK) and Akt, but inhibited STAT3 phosphorylation. Taken together, the results of our studies of HT-29 cells provide insight to the cucurbitacin B-induced arrest of cells at the G1 phase and the apoptosis of these cells.
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