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研究生:郭餘芬
研究生(外文):Yu-Fen Kuo
論文名稱:POMC過度表現刺激B16-F10黑色素癌細胞之MITF/HIF-1α路徑
論文名稱(外文):POMC Overexpression Stimulates MITF/HIF-1α Survival Pathway in B16-F10 Melanoma Cells
指導教授:戴明泓
指導教授(外文):Ming-Hong Tai
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:57
中文關鍵詞:黑色素癌細胞
外文關鍵詞:Heme oxygenase-1AdenovirusProopiomelanocortinmelanoma
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黑色素癌是由黑色素細胞所產生的癌症,是皮膚癌中最嚴重的一類型,在細胞無限制生長的情況下形成癌症,它會破壞組織的融合、打斷並重新排列正常的組織與改變其功能。正常的黑色素細胞是存在於皮膚的外層並產生棕色的色素,稱為黑色素,而它主要負責皮膚色素的產生。當黑色素細胞發展成癌症,增生且侵入其他組織時,就會形成黑色素癌。POMC是一個帶有241胺基酸的多胜肽荷爾蒙,其中包含了ACTH、α-MSH與β-EP。近年來,我們的研究結果證實在黑色素癌細胞大量表現POMC,不論在體內或體外的實驗上的確會抑制腫瘤生長與轉移的能力。然而,僅管能夠抑制腫瘤的增生與血管新生,B16-F10黑色素癌仍然會在POMC基因治療後存活,其存活機制目前仍不明確。MITF是一個具有螺旋-環狀-螺旋結構的轉錄因子。它不僅與黑色素的生成有關,同時在黑色素細胞的發展與存活上扮演了重要的角色。此外,MITF會結合在HIF-1α的啟動子上並刺激、活化其轉錄作用。在此研究中,我們探討在B16-F10黑色素癌細胞中,POMC基因傳送對MITF/HIF-1α生存路徑的影響。於即時定量PCR與西方墨點分析中顯示了在黑色素癌細胞中,POMC基因傳送會增加MITF mRNA與蛋白質的量;根據報告基因分析來看,發現在黑色素癌細胞中,POMC基因傳送會顯著增加HIF-1α冷光的活性。由轉殖技術與puromycin的篩選,我們得到了具有穩定表現short hairpin RNA抑制MITF特性的MITF-knockdown B16-F10 黑色素癌細胞。而其細胞的生長、侵入與群聚形式相似於vector控制組。經由組織學分析可知以MITF-knockdown細胞殖入處理過的腫瘤會顯著性的減少CD31-positive血管,並且伴隨下降Ki-67-positive增生的細胞數目和增加TUNEL-positive凋亡細胞數目。然而,MITF-KD細胞的殖入會導致黑色素癌腫瘤尺寸顯著減少。ASA是一種被廣泛使用的解熱陣痛劑。而它作用在生物上的影響很廣泛,包括抗熱休克、中風及預防一些癌症的發展;而在我們的研究發現,ASA是可以增加細胞增生的。然而,在侵入試驗中,ASA對於細胞的移動是沒有影響的。此外,經由POMC正向調節的MITF與HIF-1α在MITF-knockdown B16-F10細胞中同樣會明顯的減少。POMC基因傳送會增加HO-1 mRNA與蛋白質的量。HO-1是HIF-1α路徑下游的一個受動器,同時也是一種酵素,它會催化血色素轉換成鐵、一氧化碳與膽綠素。抑制HO-1的活性會增加POMC基因傳送後抑制黑色素癌細胞的增生、移動與細胞生長的能力。而這些研究顯示出MITF/HIF-1α確實會在POMC基因傳送後有助於黑色素癌的生存。
Melanoma is a cancer of the pigment producing cells, melanocytes, and is the most serious type of skin cancer. Cancer is a condition in which one type of cell grows without limit in a disorganized fashion, disrupting and replacing normal tissues and their functions. Normal melanocytes reside in the outer layer of the skin and produce a brown pigment called melanin, which is responsible for skin color. Melanoma occurs when melanocytes become cancerous, grow, and invade other tissues. Pro-opiomelanocortin (POMC) is a precursor polypeptide of 241 amino acids and the prohormone of various neuropeptide, including corticotropin (ACTH),
α-melanocyte-stimulating hormone (α-MSH), and β-endorphin (β-EP). Recently, we demonstrated that systemic POMC overexpression potently suppresses the growth and metastasis of B16-F10 melanoma in vitro and in vivo. However, despite potent inhibition of tumor proliferation and angiogenesis, B16-F10 melanoma still managed to survive after POMC gene therapy. The underlying survival mechanism of B16-F10 melanoma remains unclear. Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix transcription factor that plays a key role not only in melanin synthesis, but also in melanocyte development and survival. Besides, MITF binds to
the hypoxia-inducible factor-1α (HIF-1α) promoter to stimulate its transcriptional activity. In this study, we investigate the influence of POMC gene delivery on the
pro-survival MITF/HIF-1α pathway in B16-F10 melanoma cells. Quantitative RT-PCR and western blot analysis revealed that POMC gene delivery increased the MITF mRNA and protein level in B16-F10 melanoma cells. Besides, POMC gene delivery significantly enhanced the HIF-1α-driven luciferase activities in melanoma cells. By transfection and puromycin selection, we generated and characterized a MITF-knockdown B16-F10 melanoma cells (MITF KD) stably expressing short hairpin RNA against MITF. The growth, invasion, and colonies formation of MITF-KD were similar to those of vector control. However, implantation of MITF-KD cells led to melanoma with significantly reduced tumor size compared with those in mice implanted with vector control cells. Histological analysis revealed a significant reduction of CD31-positive blood vessels in implantation of MITF-KD cells-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, POMC-mediated upregulation of MITF and HIF-1 α was significantly attenuated in MITF KD-B16-F10 cells. Acetylsalicylic acid (aspirin; ASA) is widely used as an
analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. In our study, we found ASA enhanced cell proliferation. However, in invasion test, ASA had no effect on cell migration. POMC gene delivery elevated the mRNA and protein level of hemeoxygenase-1 (HO-1), a downstream effector of HIF-1α pathway and an enzyme catalyzing the converting reaction of heme to carbon monoxide, ion and biliverdine. Inhibition of HO-1 activities augmented the inhibitory effect of POMC gene delivery on proliferation, migration and anchorage-independent
growth of B16-F10 melanoma cells. These studies indicated that activation of MITF/HIF-1α/HO-1 indeed contributes to melanoma survival after POMC gene
delivery.
Abbreviations 3
Abstract in Chinese 4
Abstract in English 6
Introduction 8
Materials and Methods 13
Results 19
Discussion 24
References 27
Figures and Legends 32
Appendix 54
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