Pro-opiomelanocortin 抑制黑色素癌之細胞凋亡機轉
指導教授：戴明泓 博士 國立中山大學生物醫學研究所 指導教授：楊文仁 博士 國立高雄大學生物科技研究所 學生：吳健慶 國立高雄大學生物科技研究所 摘要 在我們先前研究顯示 Pro-opiomelanocortin (POMC)基因傳送抑制黑色素瘤的腫 瘤形成過程是經由NFB/COX-2 訊息傳遞路徑，並且降低腫瘤血管新生的能力。 在本篇研究中，我們發現在系統性 Ad-POMC 轉染的腫瘤中，在遠離血管的組織 部位有顯著的細胞凋亡表現，經存活試驗和細胞週期分析指出，在缺氧的情況下 Ad-POMC 感染的細胞會降低細胞增生的能力以及增加細胞週期 pre-G0 時期的數 目。因此，我們的目標是在於研究探討在缺氧情況下 POMC 基因傳送所誘發黑色 素癌細胞走向計畫性細胞凋亡的機制。經由細胞過氧化物和粒線體膜電位測定分 析，我們證明在缺氧的情況下 Ad-POMC 感染的細胞被刺激增加 1.2 倍的過氧化物 產量並且降低粒線體的膜電位。經由免疫螢光染色和細胞質與粒腺體分離的實驗 分析發現，Ad-POMC 感染的細胞中，轉位到粒線體的 Bax 引起一連串的反應包 括 Cytochrome C 和 AIF 從粒線體釋放到細胞質以及活化 caspases cascade。此外Ad-POMC 感染的細胞也增加 1 倍的 caspse-3 活性而在有 ROS 抑制劑 NAC 存在下 則是可以挽救 POMC 所誘導的細胞凋亡以及反轉部分地 caspse-3 活性。接著，我 們評估 POMC 基因衍生的胜肽鏈-MSH, -MSH, ACTH, -MSH 和 -endorphin 在 缺氧情況下 POMC基因傳送所誘發細胞凋亡中扮演的角色，經存活試驗和細胞週 期分析指出，在缺氧狀態下-MSH, -MSH, ACTH 能減低 B16-F10黑色素癌細胞
增生的能力並且增加細胞趨向凋零的數量，而-MSH 和 -endorphin 則不參與在
其中。以上總結，我們證明 POMC基因傳送使黑色素癌細胞對於缺氧的環境更為 敏感並且誘導細胞計畫性凋亡經由增加的過氧化物引發粒線體相關的細胞凋亡路 徑，其中POMC 基因衍生的胜肽鏈-MSH, -MSH, ACTH 參與在 POMC 基因傳 送所誘導的細胞凋亡。
The Apoptotic Mechanism in
Pro-opiomelanocortin-induced Melanoma
Suppression
Advisor: Dr. Ming-Hong Tai
Institute of Biomedical Sciences, National Sun Yat-sen University Advisor: Dr. Wen-Jen Yang
Institute of Biotechnology, Nation University of Kaohsiung Student: Jian-Ching Wu
Institute of Biotechnology, Nation University of Kaohsiung
ABSTRACT
In our previous reports, Pro-opiomelanocortin (POMC) gene delivery could inhibit tumor progression through NFB/COX-2 pathway suppression and attenuate angiogenic processing in melanoma tumors. In this study, in systemic Ad-POMC infected melanoma tumors, a remarkable enhancement of apoptosis was found in the ischemic region away from vessels. Viability assay and cell cycle analysis indicated that Ad-POMC infected of B16-F10 cells reduced proliferation capability and increased the population of pre-G0 phase in hypoxia, respectively. Therefore, our aim was to examine the mechanism of POMC induced apoptosis of melanoma cells in hypoxia. By ROS generation and mitochondrial membrane potential (Ψm) assay analysis, we determined that Ad-POMC infected of melanoma cells could stimulate 1.2-fold of ROS production and decrease mitochondrial membrane potential (Ψm) in hypoxia. Immunofluorescence and cell fractionation identified the translocation of Bax caused sequentially events including release of cytochrome c and AIF from mitochondria to cytoplasm in Ad-POMC infected of melanoma cells. Besides, POMC induced of
melanoma cells could increase 1-fold caspase-3 activity in hypoxia and present of ROS inhibitor NAS could rescue POMC induced of apoptotic cells population and reverse partially caspas-3 activation. Next, we evaluated the role of POMC-derived peptides -MSH, -MSH, ACTH, -MSH and -endorphin participated in apoptotic capacity of POMC-induced in melanoma cells in hypoxia. Viability assay and cell cycle assay elucidated that-MSH, -MSH and ACTH could reduce cell proliferation and enhance the population of apoptotic cells not-MSH and -endorphin in hypoxia. In summary, we demonstrated that POMC gene delivery rendered susceptibility of B16-F10 melanoma cells to hypoxia and induced apoptosis via increase of ROS production trigged mitochondrial associated pathway, including POMC processing of peptides -MSH, -MSH, ACTH participated in POMC induced apoptosis of melanoma cells.
