Studies on the Mechanism of Minocycline on the Hypoxia-inducible Factor-1α Expression in Cancer Cells

缺氧誘導性因子-1α(HIF-1α) 為一種對於氧分壓敏感的轉錄因子，在正常氧分壓下HIF-1α很快就被prolyl hydroxylase氫氧化進入26S蛋白酶體降解，當細胞處理缺氧環境時HIF-1α會累積並與HIF-1β形成複合物進行轉錄活化的作用；HIF-1α所調控的基因包括影響能量供給、細胞生長、血管新生等，被認為在腫瘤發育過程扮演促進者的角色。 米諾黴素(Minocycline)為一種廣效性四環黴素類的抗生素，藉由與原核生物之30S核醣體結合干擾蛋白質合成進而抑制細菌的生長；近年來許多研究指出米諾黴素除了抗菌外，對於真核細胞的發炎反應、細胞凋亡及血管新生皆有所影響，且能抑制腫瘤的生長與微血管生成；本研究希望能夠透過進一步實驗，觀察處於缺氧微環境的腫瘤細胞處理米諾黴素時，對於缺氧適應反應調控者、血管新生促進者－HIF-1α是否會造成影響及可能牽涉的相關訊息機轉。本研究採用非小細胞肺癌細胞株A549和H1299做為材料，以西方墨點法分析，發現在正常氧分壓下單獨處理米諾黴素，並不影響HIF-1α表現；但若使用氯化亞鈷(II)或0.5%氧分壓的缺氧環境下使HIF-1α大量表現時，前處理米諾黴素則可抑制HIF-1α累積的現象，以MTT試驗觀察米諾黴素並未對細胞產生毒性；米諾黴素不會影響HIF-1αmRNA的表現，而利用蛋白轉譯抑制劑cycloheximide觀察HIF-1α降解情況，顯示米諾黴素能加速HIF-1α蛋白的降解速率，的確米諾黴素造成缺氧誘導因子-1α累積表現減少的現象會隨著26S蛋白酶體抑制劑MG132的處理而消失；此外米諾黴素的處理不改變細胞中pVHL蛋白(E3 ubiquitin ligase)表現量但增加pVHL與HIF-1α間的鍵結程度；訊息傳遞路徑方面，米諾黴素能夠抑制由缺氧所活化之AKT、ERK磷酸化現象；HIF-1α下游基因：VEGF、MMP-2的表現也會受到米諾黴素的影響，並能藉由抑制HIF-1α的表現達到抑制內皮細胞管狀構造的形成。總結以上結果，米諾黴素直接或間接影響增加AKT、ERK訊息傳遞，促進pVHL蛋白與HIF-1α間的鍵結加速HIF-1α降解速率，並降低其下游基因表現及抑制血管新生體外模式－HUVECs管狀形成。HIF-1α is an oxygen-regulated protein and is recognized as a positive factor to tumorigenesis. During nomoxia, HIF-1α is rapidly hydroxylated by prolyl hydroxylase then goes into 26S proteasome degradation pathway. During hypoxia, HIF-1α accumulates and dimerizes with HIF-1β to transactivate hypoxia-responsive gene expression. There are more than 100 HIF-1 downstream genes identified with varying functions, including glucose metabolism, angiogenesis, cell proliferation/apoptosis. Minocycline is a second-generation tetracycline, and remains widely used clinically. Several studies have demonstrated that it also affects many cellular functions relevant to human vascular diseases including inflammation, matrix remodeling and angiogenesis. Moreover, minocycline also can repress tumor growth and angiogenesis. In this study, we found that minocycline will not affect on HIF-1α expression during nomoxia ,but significantly inhibit HIF-1α accumulation during hypoxia and CoCl2 treatment without affecting cell viability. By using the translation inhibitor cycloheximide, we found that minocycline reduced HIF-1α protein expression via accelerating HIF-1α degradation rate, but had no significant effect on HIF-1α mRNA expression. Minocycline caused a decrease in the level of HIF-1α could be rescued by MG132. The pVHL(E3 ubiquitin ligase) protein levels were unchanged by minocycline, but pVHL binding to HIF-1α was increased. Minocycline also inhibited hypoxia-induced activation of Akt and ERK1/2. In addition, minocycline inhibited the mRNA level of hypoxia-responsive gene such as VEGF and MMP-2. Furthermore, Minocycline inhibited hypoxia-induced tubule formation in HUVECs. In conclusion, Minocycline can enhance pVHL binding to HIF-1α via Akt、ERK signaling pathway and thereby accelerate degradation of HIF-1α.This result leads to decrease VEGF and MMP-2 expression and inhibite hypoxia-induced endothelial cell tubule formation.口試委員會審定書..……………………………………………….…………………….i誌….……………………………………………….………………………………….ii寫表(Abbreviations)..……………………………………………….………………iii表目錄(Figures and Tables)…………………………...……………………………iv文摘要……………………………………………………………………………..... vi文摘要………………………………………………………………..…………...... vii一章 緒論(Introduction).1腫瘤發育與缺氧…………………………………………………..……...………….2.2缺氧狀態的調節者－缺氧誘導因子-1(Hypoxia inducible factor-1；HIF-1)……….2.3缺氧誘導因子-1α(HIF-1α)的表現與調控……………………..……...……………4.4米諾黴素 (Minocycline)之背景、藥物動力學、毒理介紹..……...………………9.5米諾黴素今日之新應用…………………………………………..……...………10.6研究動機……………...……………………………………………...……………..13二章 實驗材料與方法 (Materials and Methods) .1實驗材料………………………………………………..……...……………..…….15.1.1藥品……………………………………………….…..……...……………..…….15.1.2抗體、酵素與試劑……………………………….…..……...……………..…….15.2實驗方法 .2.1細胞培養和缺氧系統培養箱(Cell culture and Hypoxia treatment ) ……..…….16.2.2細胞毒性測試(Cell viability test/MTT assay) …….…..……...……………..…..17.2.3細胞總蛋白質液收集(Cell lysate collection) …….…..……...….…………..…..17.2.4西方墨點法(Western blot analysis) …….…..……...…………….…………..…..17.2.5免疫沉澱法(Immunoprecipitation)….…..……......…………….…………..…..18.2.6萃取細胞RNA(RNA extraction) …….…..……......…………….…………..…..18.2.7 反轉錄鏈鎖聚合酶反應(Reverse transcription polymerase chain reaction).…..19.2.8酵素免疫法測試VEGF蛋白表現(VEGF ELISA assay) ……….…………..…..20.2.9管狀形成實驗(In vitro matrigel capillaries formation) ……….…………..……..20.2.10統計分析(Statistic analysis) ……….…………..……………………………….20三章 實驗結果(Results).1 在正常氧分壓下單獨處理米諾黴素不影響缺氧誘導因子1α表現…………….22.2 米諾黴素能抑制由氯化亞鈷(II)處理或處於缺氧狀態造成的缺氧誘導性因1α累積之現象……….…………..…………………………………………………...22.3 米諾黴素抑制缺氧誘導因子-1α累積之現象並非是由於細胞死亡所造成…....23.4 米諾黴素的處理會影響缺氧誘導因子1α的穩定性，加速其降解速率…...........24.5 米諾黴素抑制缺氧誘導因子1α累積之現象是藉由26S蛋白酶體路徑..............26.6 米諾黴素能增加pVHL蛋白與缺氧誘導因子-1α之間的鍵結但不影響細胞中pVHL蛋白的表現量…………..………………………………………………….27.7 米諾黴素能抑制由缺氧狀態刺激所引起的AKT與ERK磷酸………….……...28.8 米諾黴素能抑制由缺氧狀態所誘發的血管內皮生長因子表現量增加的現象..29.9 米諾黴素能抑制缺氧狀態誘導的人類臍靜脈內皮細胞管狀構造形成作用….31四章 討論(Discussion) .1 米諾黴素抑制由缺氧狀態所誘發的缺氧誘導因子-1α累積現象之可能機轉…35.2 米諾黴素抑制由缺氧狀態活化之Akt和ERK磷酸化…………………………38.3 米諾黴素對於缺氧狀態下血管新生相關機制的影響…….…………………….39.4 米諾黴素與缺氧狀態對A549與H1299細胞MMP-2 mRNA表現的影響…….40五章 結論(Conclusion) ……………………………………...……………………43考文獻(References) …………………………………………………………………45表集(Figures and Tables) ….………………………………………………………..6