探討KMO於犬乳腺腫瘤細胞的功能

Abstract

Kynurenine 3-monooxygenase (KMO)是Tryptophan代謝過程中kynurenine pathway中的一個酵素，位於粒腺體的外膜，可將L-kynurenine降解為3-hydroxy kynurenine，而kynurenine pathway的終產物為quinolinic acid，目前被認為具有活化N-methyl-D-aspartate (NMDA) 受體的能力。在過去的研究中，因為KMO下游產物會造成中樞神經過度興奮而導致神經毒性，故主要被認為與神經退化性疾病有關(例如: Alzhemer’s disease and Huntington’s disease)；然而在我們過去對犬乳腺腫瘤的研究中發現， KMO的表現量分別與腫瘤的惡性程度和病畜生存時間具有相關性，其中越惡性或級數越高的犬乳腺腫瘤其KMO表現量越高，且KMO表現量越高的病畜其存活時間則越短，但KMO在犬乳腺腫瘤細胞中的功能，目前仍不清楚，故本篇研究主要在探討KMO於犬乳腺腫瘤細胞中的功能。首先，我們確認實驗室現有的三株犬乳腺腫瘤細胞株，CMT-1, MPG及CF41.mg皆有表現KMO的mRNA及蛋白質，且在投與專一性的KMO抑制劑(Ro 61-8048)後，KMO蛋白質的表現並不會受到影響。其次，投與該KMO抑制劑，可以抑制犬乳腺腫瘤細胞的增生及移行能力，而在同時投與NMDA受體的致效劑NMDA後，能夠有效的反轉KMO抑制劑造成抑制腫瘤細胞增生能力，但卻不能逆轉被抑制的腫瘤細胞移行能力。另外，在腫瘤細胞訊息傳遞的分析上，我們發現投與KMO抑制劑抑制KMO功能時，降低了腫瘤細胞內ERK, AKT及STAT3等分子的活性，而同時投與NMDA則反轉了ERK及STAT3的活性抑制作用，但不影響AKT的活性抑制作用。顯示KMO可能是藉由ERK及STAT3細胞傳訊路徑影響犬乳腺腫瘤細胞增生能力，而細胞移行能力則可能是藉由AKT細胞傳訊路徑控制。

Kynurenine 3-monooxygenase (KMO), mainly found on the outer membrane of the mitochondria, is one of the enzymes in the kynurenine pathway. KMO catalyzes L-kynurenine to form 3-hydroxy kynurenine and further generates the downstream metabolite quinolinic acid which is an N-methyl-D-aspartate (NMDA) agonist. In the past, KMO was used to associate with neurodegenerative diseases because the downstream metabolites demonstrate excitotoxicity to CNS and act as factors in neurodegenerative diseases. In our previous study has shown that overexpression of KMO is related to tumor malignancy and survival time of cMGT-suffering dogs (in progress). In this study, we investigated the functional characterization of KMO in cMGT cells. First of all, KMO expression of cMGT cells (CMT-1, MPG and CF41.mg) was identified in mRNA and protein level and not changed by application of KMO inhibitor. Second, proliferation and migration of cMGT cells was reduced by KMO inhibitor, and the inhibition of proliferation was restored by NMDA. Third, downregulation of KMO function by KMO inhibitor decreased ERK, AKT and STAT3 activity of cMGTs, and the activity of ERK and STAT3 was restored by NMDA. Our data suggest that KMO involves in the proliferation and migration of cMGT cells. Furthermore, the inhibition of proliferation by KMO inhibitor is mainly through ERK and STAT3 signaling cascades and the signaling pathway may be activated through the NMDA receptor. The inhibition of migration by KMO inhibitor is mainly through AKT signaling cascades.