缺氧相關分子腎上腺髓質素在急性骨髓性白血病中角色之研究

Abstract

根據文獻，低氧 (hypoxia) 狀態會促進固態腫瘤細胞進行侵犯 (invasion) 或轉移 (metastasis) 到正常的組織；骨髓內的環境即呈現低氧狀態，但於白血病 (leukemia) 的研究並不多，而急性骨髓性白血病 (AML) 是在一般成人中最常被診斷的白血病，因此我們想探討缺氧狀態在 AML 中所扮演的角色。首先我們利用微陣列分析培養於氧氣濃度 21 % (正常氧) 與 1 % (低氧) 環境下 8 小時的白血病細胞株 (OCI/AML3)，發現在低氧狀態下，腎上腺髓質素 (adrenomedullin，AM) mRNA 表現量約為正常氧的 9 倍。同時，我們利用 Oncomine 網路資料庫發現在 AML 病人與正常人中有不同程度的 AM mRNA 表現，但正常人與 AML 病人之間或不同 AML 亞型之間無明顯差異；我們也分析不同白血病細胞株 (U937、K562、HL-60、OCI/AML3) 及隨機取樣的 AML 病人檢體其 AM mRNA 表現量在不同氧分壓下的變化。結果顯示在低氧環境下，AM mRNA 於各血癌細胞株及 AML 病人檢體的初代培養 (primary culture) 中，表現量有增加的趨勢 (增加倍率有2 - 150倍的差異)。為了實際反應出骨髓內 AM 的角色，我們進一步利用免疫組織化學法分析 AML 病人及骨髓正常之病患的骨髓切片，結果顯示， 在 22 位 AML 病患中，有三位病患具 AM 蛋白的表現，而在 11 例骨髓正常之病患中則有 4 位。已有文獻指出在固態腫瘤中，AM 能透過活化 PI3K/Akt 路徑或 Ras/Raf 路徑，促使腫瘤細胞增生，因此我們欲探討 AM 對白血病細胞的影響。結果顯示，在正常氧環境下，給予 U937 細胞株 AM (1 uM) 48 小時後，會促使 U937 細胞數目增加 20 %；反之，若給予拮抗劑 (1 uM) 48 小時，細胞數目則會降低 20 %；但於其他細胞株無明顯差異。為了釐清 AM 影響 U937 細胞數目的機制，我們分析細胞週期、細胞凋亡比例及觀察促凋亡蛋白 (Bax) 和抗凋亡蛋白 (Bcl-2、Bcl-xL) 表現量之變化。結果顯示，不論是細胞週期、細胞凋亡比例、促凋亡蛋白或抗凋亡蛋白表現量，U937 細胞於加藥前後皆無差異。本研究指出在不同血癌細胞株和部分臨床 AML 病人檢體中可觀察到有 AM mRNA 及蛋白表現，雖然目前結果未能釐清其對血癌細胞的影響與作用，但仍有許多方向可供探討。

Privious studies showed that hypoxia condition can promote cancer cells invasion and metastasis in solid tumors. In fact, the bone marrow microenvironment is in hypoxia condition. However, the involvement of hypoxia in leukemia has not been clearly documented. Because AML is the most common in leukemia, we investigated the roles of hypoxia in AML. First, we analyzed the microarray data of leukemia cell line (OCI/AML3) under normoxia or hypoxia condition. We found that adrenomedullin (AM) mRNA expression under hypoxia is 9-fold higher than normoxia. Meanwhile, we found that AM mRNA were detected in AML patients by Oncomine website database. In addition, we analyzed AM mRNA expression in other leukemia cell lines and clinical AML samples by RT-PCR and real-time RT-PCR. The results also showed that AM mRNA expression under hypoxia increased compared to normoxia. Furthermore, the AM signals were detected in three of twenty- two AML bone marrow samples by immunohistochemstry. However, there were also detected in four of eleven non-AML bone marrow samples. To understand the effects of AM in leukemia, we observed the effects of cell viability in leukemia cell lines. The results revealed in normoxia condition, 1 uM AM treament increased cell numbers of U937 cells. In contrast, when treated with AM antagonist, AMA (1 uM), reduced the cell numbers of U937 cells. To understand the mechanism of AM, which increased cell numbers of U937 cells, we analyzed cell cycle distribution, sub-G1 DNA contents and protein expression of apoptotic-related molecules. However, there were no significant differences in either one of these experiments between drug-treated and untreated cells. The study indicated that there were AM mRNA and protein expression in leukemia cells and clinical AML patient samples. However, the effects and mechanisms of AM in AML are still not clear.