cabozantinib 誘導K562細胞株進行紅血球系列分化之機制的探討

Study on the molecular mechanism of cabozantinib-induced erythroid differentiation of K562 erythroleukemia cells

楊宜茹(Yi-Ju Yang)

指導教授 ： 林亮音

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.03188

摘要

大部分慢性骨髓性白血病(chronic myeloid leukemia, CML)的病人體內會存在第 9 對及第 22 對染色體轉位所產生的 BCR-ABL 融合蛋白，此融合蛋白會不斷啟動下游訊息傳遞蛋白造成細胞不斷增生、並且阻止細胞分化。因此，利用酪胺酸激酶抑制劑抑制BCR-ABL融合蛋白酪胺酸激酶的活性為治療辦法之一。Cabozantinib(XL184,N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-N-(4-fluorophenyl) cyclopropane-1, 1-dicarboxamide)為一多重酪酸胺激酶抑制劑，此藥物經美國FDA核准用於治療腎上腺髓質癌，建議每日用量與每日最大用量分別為140毫克與175毫克。根據藥物動力學研究顯示此藥物在每日用量175毫克時，人體血漿濃度可達2.8uM。本實驗室先前利用cabozantinib 進行實驗時，發現cabozantinib處理 K562 細胞放置數天後，細胞會變成紅色，故推論 cabozantinib 具有誘導K562細胞分化走向紅血球系列之能力，因此，我們主要目的是探討其中的機制。
首先，我們證實在小於血漿可達濃度之1uM cabozantinib處理K562細胞4天後，細胞會走向紅血球系列分化而非血小板系列。我們發現經1uM cabozantinib處理之K562細胞利用二胺基聯苯胺鹽酸(benzidine dihydrochloride)試驗與流式細胞儀，偵測細胞內血色素與細胞表面抗原CD71以及CD235a的表現量，證實經由cabozantinib處理之K562細胞，其紅血球系列分化相關之蛋白表現上升。而qRT-PCR 分析cabozantinib處理之K562細胞與紅血球分化相關之基因表現，例如：r-globin、transferrin receptor(TfR、CD71)、glycoforin A(GPA、CD235a)等基因，也呈現顯著上升的趨勢。我們發現cabozantinib可抑制K562細胞生長速率，而利用流式細胞儀分析細胞週期發現有細胞周期停滯的現象。進一步探討cabozantinib調控K562細胞分化之機制，我們發現cabozantinib處理K562細胞6小時後，細胞中BCR-ABL及其下游蛋白ERK, STAT5,AKT磷酸化之情形會隨著藥物濃度上升而下降。實驗結果亦顯示cabozantinib處理K562細胞48小時後，可造成c-myc蛋白表現下降，伴隨著p27蛋白表現上升。根據以上結果我們認為cabozantinib可藉由負調控AKT磷酸化使得p27蛋白表現增加，進而刺激K562細胞往紅血球系列分化。另外，我們也發現cabozantinib處理K562細胞6小時後，細胞中JNK及其下游分子磷酸化情形上升，並且利用JNK抑制劑可有效中和cabozantinib所刺激之分化現象，故我們認為cabozantinib亦可能透過JNK路徑調控K562細胞進行分化。

關鍵字

carbozantinib ； 紅血球系列分化 ； BCR-ABL融合蛋白

並列摘要

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic stem cell disease which arises from the bcr-abl oncogene. The Bcr-Abl oncoprotein, as a constitutively activated tyrosine kinase, could activate multiple signaling pathways for the malignant transformation. Therefore, specific inhibitors of tyrosine kinases are attractive therapeutic agents. Cabozantinib (XL184, N-(4-((6, 7-Dimethoxyquinolin-4-yl) oxy) phenyl)-N-(4-fluorophenyl) cyclopropane-1, 1-dicarboxamide), a multiple tyrosine kinase inhibitor, has recently been approved by US FDA for the treatment of medullary thyroid cancer (MTC). The recommended daily dose and the maximum-tolerated dose (MTD) of cabozantinib are 140mg and 175 mg, respectively. Pharmacokinetics study revealed that the steady-state plasma levels were 2.8μM when administrating MTC patients with 175mg daily. In our preliminary results revealed that treating K562 cells harboring bcr-abl oncogene with 1μM cabozantinib after a few days demonstrated an erythroid differentiation phenotype.
Treatment with plasma-reachable dose (1μM) of cabozantinib in K562 leukemia cells for 96 hrs induced erythroid differentiation but not megakaryocytic maturation. We observed a significant population of cells with cytological features of early erythroid differentiation and significantly increased benzidine-positive cells (up to 50%) accompanying with significant accumulation of γ-globin. Expression of transferrin receptor (TfR, CD71) and glycophorin A (GPA, CD235a), both well documented erythroid-specific gene, were induced 3 to 4-fold relative to untreated control cells. We found that cabozantinib could inhibit cell growth of K562 cells, and cell cycle analysis revealed that cabozantinib could also result in G0/G1 cell cycle arrest in K562 cells. Signaling pathway analysis revealed that cabozantinib could decrease phosphorylation of BCR-ABL, ERK, STAT5 and AKT at 6 hours in a dose-dependent manner. Further analysis revealed that cabozantinib treatment could decrease c-myc with a concomitant induction of p27 at 48 hours in a concentration- dependent manner. Taken together, we proposed that cabozantinib could induce K562 cell differentiation toward erythroid lineage through inhibiting PI3K/AKT pathway, of which, p27 was involved. In addition, we found that cabozantinib treatment could up-regulate phosphorylation of JNK and its downstream protein and JNK inhibitor could reduce the cabozantinib-induced erythroid differention on K562 cells. We considered that cabozantinib could also induce K562 cell differentiation toward erythroid lineage through JNK pathway.

並列關鍵字

cabozantinib ； erythroid differentiation ； BCR-ABL fushion protein

參考文獻

1. Baccarani M, Dreyling M, Group EGW. Chronic myeloid leukaemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2010;21 Suppl 5:v165-167.

2. Sawyers CL. CHRONIC MYELOID LEUKEMIA. N Engl J Med. 1999;340:1330-1340.

3. Jabbour E, Kantarjian H. Chronic myeloid leukemia: 2014 update on diagnosis, monitoring, and management. Am J Hematol. 2014;89(5):547-556.

4. Bizzozero OJ, Jr., Johnson KG, Ciocco A. Radiation-related leukemia in Hiroshima and Nagasaki, 1946-1964. I. Distribution, incidence and appearance time. N Engl J Med. 1966;274(20):1095-1101.

5. Ren R. Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat Rev Cancer. 2005;5(3):172-183.