Study on the inhibitory mechanism of mitoxantrone on HIF-1α pathway

實質固態瘤（solid tumor）大都處在一個缺氧的微環境中，而參與缺氧調節的核心因子－缺氧誘導因子-1α（hypoxia-inducible factor-1α，HIF-1α）在腫瘤的發生及進程以及腫瘤細胞對化療及放射線治療的耐受性上扮演著極為重要的角色。因此，HIF-1α是很有潛力的腫瘤治療標的物。在本論文中，我們發現一個新型HIF-1α抑制劑並深入探討該藥物抑制HIF-1α的分子機制。我們分析了臨床藥物拓撲異構酶Ⅱ（topoisomerase II，TOP2）抑制劑對於HIF-1α表現的抑制效果，並以mitoxantrone（MX）為重點藥物做進一步的研究。出乎意料的是，利用藥理性抑制、RNA干擾技術以及TOP2突變型細胞株，我們發現MX抑制HIF-1α的機制並不透過TOP2。MX也不影響HIF-1α的蛋白穩定度及RNA轉錄（transcription）程度，但其抑制卻與cycloheximide的作用機制類似。這些實驗間接說明了MX抑制了HIF-1α的轉譯（translation）。利用體外轉譯（in vitro translation）HIF-1α mRNA的方法也進一步證實MX能夠抑制HIF-1α的轉譯。有趣的是，細胞在處理MX後，多核醣體（polysome）上的VEGF-A mRNA如預期的減少但是HIF-1α mRNA卻有些許地增加。因此，我們猜測MX有可能抑制了HIF-1α mRNA在轉譯延長（translation elongation）的步驟。綜合上述的實驗結果，我們認為MX除了能夠抑制TOP2還另外具有抑制HIF-1α的能力，開廣了MX在癌症治療上的應用範圍及效力。Solid tumors encounter a growth-limiting hypoxic microenvironment as they develop. Hypoxia-inducible factors (HIF) play important roles in hypoxia-associated tumor development and therapeutic resistance. Targeting the HIF pathway (especially HIF-1α) represents a promising cancer treatment strategy. In this dissertation, a novel class of HIF-1α inhibitors and the possible molecular basis of inhibition were studied. We analyzed the inhibitory effects of clinically used topoisomerase II (TOP2)-targeting drugs on HIF-1α expression with a primary focus on mitoxantrone (MX), which showed the most potent inhibitory effect. The potential role of TOP2 in MX-inhibited HIF-1α expression was studied using pharmacological inhibition, a knockdown approach and TOP2 mutant cells. Moreover, involvement of MX in proteasome-mediated degradation, transcription and translation of HIF-1α was examined. The TOP2-targeting MX, but neither doxorubicin nor etoposide, strongly inhibited HIF-1α expression under hypoxic conditions in a dose- and time-dependent manner. Surprisingly, the MX-mediated inhibition of HIF-1α expression was largely independent of two TOP2 isozymes, proteasomal degradation and transcription. Furthermore, MX inhibited HIF-1α expression and function in a similar fashion as cycloheximide, suggesting that MX might inhibit HIF-1α via a blockage at its translation step. In vitro translation experiments using HIF-1α mRNA further confirmed inhibition of HIF-1α translation by MX. Interestingly, levels of the polysome-bound HIF-1α and VEGF-A mRNA were elevated and decreased after MX treatment, respectively. In conclusions, we have identified the TOP2-targeting compound, MX, as a HIF-1α inhibitor possibly through a translation inhibition mechanism, suggesting the possibility of an additional anticancer activity for MX