探討Pyrvinium Pamoate在Molm13血癌細胞株中的抗癌機制

Abstract

急性骨髓性白血病(AML)是由於骨髓系造血前驅細胞異常增生且無法分化所導致的血液惡性腫瘤，當不成熟的骨髓系造血細胞堆積在骨髓和周邊血中，會干擾白血球、紅血球及血小板的成熟與功能。大約有三分之一的AML病人帶有FLT3基因突變，其中又以FLT3內部串聯重複(FLT3-ITD)最常見，並且具有FLT3-ITD突變的病人通常預後不佳。FLT3是一種酪胺酸激酶受體(receptor tyrosine kinase, RTK)，FLT3基因的變異會導致FLT3受體異常活化，進而引起下游與細胞生長、存活與增生有關之訊息傳遞路徑不斷地被活化，因此FLT3被認為有潛力作為治療AML的標的。雖然目前有許多FLT3標靶藥物進入臨床試驗的階段，但是這些藥物只能讓FLT3突變的病人得到暫時緩解，仍然有很大機率會再復發。對於產生抗藥性的原因至今仍不清楚，因此發展一個新的治療策略是必要的。 在實驗室之前的研究中發現，FLT3標靶藥物cabozantinib (XL184)能夠選擇性的抑制FLT3-ITD突變的AML細胞株Molm13之生長，在動物實驗中也發現cabozantinib能有效抑制Molm13腫瘤的生長。為探討cabozantinib的抗藥性，實驗室先前也建立了一株對於cabozantinib具有抗藥性的細胞株Molm13-XR。 Pyrvinium pamoate (簡稱PP)是經由FDA核准的蟯蟲驅蟲劑，最近有許多研究指出PP對於不同的癌細胞都有抑制的作用，例如乳癌、胰臟癌和大腸直腸癌等等。目前有許多研究探討PP對於不同癌細胞的作用機制，但是PP對於AML的影響尚不明瞭。因此本篇想要探討PP對於AML細胞株Molm13的作用機制，另外藉由實驗室先前建立的Molm13-XR細胞株測試PP是否對於具有FLT3標靶藥物抗藥性的細胞也有相同的作用機制。 本篇研究發現，PP在低濃度下即能有效抑制Molm13-P細胞的存活，其IC50為18.43 ± 6.45 nM，而且存活率的降低是因為PP引起了細胞凋亡，而不是因為抑制細胞生長。為找出PP造成細胞凋亡的可能原因，參考過去文獻找出一些PP可能的作用機制。結果發現在Molm13-P細胞中，PP些微減少FLT3下游的STAT5、WNT/β-catenin和STAT3訊息傳遞的活化，但是細胞核內β-catenin和p-STAT3蛋白量以及其下游標的基因轉錄卻都不受影響。另外，PP雖然會阻斷Molm13-P細胞的細胞自噬機制，但與細胞自噬相關基因的轉錄無關。如同過去文獻的發現，在Molm13-P細胞中，PP也會轉移到粒線體上，因而抑制粒線體經由氧化磷酸化合成ATP的能力，而粒線體質量減少且ROS增加的現象也說明了在PP作用之下粒線體的功能會受到損害。 接著探討PP在Molm13-XR細胞中是否也是相似的作用。結果顯示，PP在低濃度時即會促進細胞凋亡而抑制Molm13-XR的存活，其IC50為6.63 ± 4.32 nM。以低濃度PP處理Molm13-XR細胞即出現FLT3、WNT/β-catenin和STAT3訊息傳遞路徑分子的活化被抑制的情形，但同樣不影響其細胞核內β-catenin和 p-STAT3的含量，以及下游標的基因的轉錄。另外，PP同樣會阻斷細胞自噬機制，而且也不是經由抑制細胞自噬相關基因的轉錄所導致。如同過去文獻以及Molm13-P細胞的結果，PP同樣會轉移到Molm13-XR細胞的粒線體上，並且影響其粒線體的功能，包括粒線體氧化磷酸化的抑制、粒線體質量的減少以及ROS增加。最後，不論是Molm13-P或是Molm13-XR細胞中，PP與cabozantinib的抑殺效果都有協同作用。 整體來說，不論Molm13-P細胞或是有抗藥性的Molm13-XR細胞，PP皆能有相當好的毒殺效果，並且皆會抑制粒線體的功能以及細胞自噬機制。本篇研究也發現Molm13-XR內過度活化的訊息傳遞路徑會受PP所抑制，可能因此造成Molm13-XR的IC50略低於Molm13-P的結果。PP雖然改變了Molm13-XR內訊息傳遞分子的活化卻不會影響下游基因的轉錄，或許暗示了PP的作用是經由影響訊息傳遞路徑與粒線體之間的交互關係，而不是經由影響訊息分子進入細胞核內調控轉錄的功能。

Acute myeloid leukemia (AML) is a hematological malignancy characterized by the aberrant proliferation and differentiation block of precursor myeloid cells. Approximately one third of AML patients harbor FLT3 mutations. Internal tandem duplications mutations in FLT3 (FLT3-ITD) is the most common form and usually correlated with poor prognosis. FLT3 has been considered as a promising therapeutic target for AML, therefore, several FLT3 inhibitors have been used in clinical trials, including cabozantinib (XL184). Our previous studies demonstrated that cabozantinib selectively inhibited the viability of AML cell lines with FLT3-ITD mutations, including Molm13 cell line (also known as Molm13-P), both in vitro and in vivo. Since drug resistance of FLT3 inhibitors has emerged in clinical, our lab established cabozantinib resistant Molm13 cell line (also known as Molm13-XR) to investigate the mechanisms underlying the drug resistance. Recent studies indicated that pyrvinium pamoate (PP), an FDA-approved anthelmintic drug, had a potent anticancer activity against various cancer cells. The mechanisms of PP are currently under extensive investigation. However, the anticancer effects and mechanisms of PP toward AML cells still remain unknown. We revealed that PP caused cytotoxicity toward Molm13-P by inducing cell apoptosis. The IC50 for Molm13-P was 18.43 ± 6.45 nM. Our studies demonstrated that PP slightly inhibited STAT5, WNT/β-catenin and STAT3 signaling pathway in Molm13-P. However, the protein levels of β-catenin and p-STAT3 in nucleus were not decreased, consistent with the unaffected downstream target genes transcription. In addition, PP blocked autophagy without inhibiting autophagy-associated gene transcription. We also found that PP localized on mitochondria and inhibit mitochondrial oxidative phosphorylation, resulting in reduced ATP production in Molm13-P. As a result of impaired mitochondrial function, our studies showed that the mitochondria mass decreased and ROS level increased after PP treatment. In turn, we also revealed that PP also caused cytotoxicity toward Molm13-XR by inducing cell apoptosis. The IC50 for Molm13-XR was 6.63 ± 4.32 nM. Our studies indicated that PP inhibited STAT5, AKT and ERK activation, and also suppressed WNT/β-catenin and STAT3 signaling pathway in Molm13-XR. However, the protein levels of β-catenin and p-STAT3 in nucleus, as well as the downstream target genes transcription, were not decreased by PP treatment in Molm13-XR,. In addition, PP blocked autophagy without disturbing autophagy-associated gene transcription. PP also localized on mitochondria and inhibited mitochondria oxidative phosphorylation, resulting in decreased ATP production in Molm13-XR. Decreased mitochondria mass and increased ROS level in PP-treated Molm13-XR also implied the mitochondria dysfunction caused by PP. Moreover, we showed that PP and cabozantinib have synergistic effect on Molm13-P and Molm13-XR. In conclusion, this study supports that PP potently inhibited the viability of Molm13 cell lines with or without cabozantinib resistance through cell apoptosis. Our results demonstrated that PP inhibited mitochondrial function and autophagy both in Molm13-P and Molm13-XR. Although some signaling molecules inactivated by PP, the target gene transcription was not subsequently inhibited. These evidences implied that the effects of PP might primarily change the interaction between signaling pathway and mitochondria, instead of altering the nuclear function.