活化p53調控人類非小型肺癌細胞凋亡的藥物與機制

Abstract

Part I 本論文發現三環化合物 (Teroxirone) 在低濃度下能抑制人類非小型肺癌細胞 (NSCLC cells) 的增生，不論在體外實驗或體內實驗都能具有顯著的效果。藥物所誘發的p53依賴型的細胞凋亡 (p53-dependent apoptosis) 是經由破壞癌細胞內的DNA結構所引發的，讓癌細胞p53上升，進而活化下游caspase-3，導致最終的細胞凋亡。而caspase-3的抑制劑 (DEVD-CHO) 或是利用 si-RNA p53抑制細胞內的p53，皆能抑制藥物所引發的細胞凋亡現象。另外，此藥物在缺失p53的細胞H1299 cells中，也能引起些微的細胞毒性。在體內實驗中，此藥物對於裸鼠身上的癌細胞也具有顯著抑制生長的功效。進一步發現，此藥物會引發癌細胞內的氧化壓力、產生活性氧化物 (ROS)、讓細胞內的粒腺體外膜崩壞，進而產生p53依賴型的內生性細胞凋亡 (intrinsic apoptosis pathway)。而利用ROS的抑制劑 (NAC) 進行處理後，也會抑制藥物所引發的細胞凋亡現象。由結果顯示，此藥物在低濃度時就能引發癌細胞的細胞凋亡並會顯著抑制癌細胞增生的結果，在未來可以是一個具有潛力治療人類非小型肺癌細胞的藥物。 Part II 玫瑰樹鹼 (Ellipticine) 是一種DNA拓樸異構酶II抑制劑，能夠有效的抑制人類非小型肺癌細胞 (NSCLC cells) 的增生。之前研究指出，此藥物能讓p53及Akt活化且共同轉移至細胞核內，並可藉由磷酸化AktS473去誘導癌細胞產生細胞自噬 (autophagy)，進而引發細胞死亡。另外，此藥物也可抑制能穩定表現p53功能的細胞株 (利用轉殖wild-type p53質體進入原本缺失p53的細胞株 (H1299 cells) 內，並讓其穩定表現 p53功能的細胞株) 的生長。在本研究中，我們進一步發現到，將AktS473的磷酸化位點突變成alanine以及利用shRNA p53去knockdown p53後，皆能抑制玫瑰樹鹼對於p53和Akt的活化以及轉移至細胞核內的程度，也能同時減少細胞凋亡的產生。因此，p53能夠讓此誘導細胞凋亡，並且能協助磷酸化AktS473進入細胞核內，進而引發細胞自噬作用，導致細胞死亡。本研究指出，玫瑰樹鹼能活化p53及磷酸化AktS473，並藉由這兩者間的協同作用去抑制癌細胞的生長。對於原本只有DNA拓樸異構酶II抑制功能的藥物，能提供另一個治療癌症抑制的新思維。

Part I In this study, we demonstrated that the growth of human non-small cell lung cancer (NSCLC) cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells. The results indicated that low concentrations of teroxirone suppressed the growth of human non-small cell lung cancer cells. The induced apoptotic cell death can be reverted by caspase-3 inhibitor, DEVD-CHO. The reduced cell viability is closely related to p53-activated apoptosis. Furthermore, we also found that teroxirone-induced p53-dependent apoptosis was through regulating intrinsic pathway via ROS generation and mitochondria dysfunction, which can reverted by antioxidant NAC. Teroxirone provides a good candidate for lung cancer treatment by suppressing cellular proliferation. Part II Topoisomerase II inhibitor ellipticine effectively suppressed the growth of human non-small cell lung cancer (NSCLC) epithelial cells. Previously, we reported the drug activity was consummated through parallel nucleus migration of p53 and Akt in A549 cells. While inducing cell death, the drug activity was proved related to autophagy through phosphorylated Akt at S473. In addition, ellipticine induced cytotoxicity in p53-null H1299 cells with stable expression of ectopic p53. In this work, we further demonstrated that dominant-negative AktS473A or p53 shRNA inhibited ellipticine-mediated translocalization of p53 and Akt and attenuated apoptotic cell death in A549 cells. The presence of p53 predates ellipticine-mediated apoptotic cell death, assists in nucleus translocation of phosphorylated Akt and activation of autophagy pathway. Growth inhibition through collaborating p53 and phosphorylated Akt473 in lung epithelial cancer cells provided a new perspective of the topoisomerase inhibitor as an effective cancer therapy agent.