拓蹼酶抑制劑(GL331)對子宮頸癌細胞株HeLa S3細胞毒性機轉之探討

Abstract

GL331是一個半合成podophyllotoxin的衍生物。和其類似物VP-16 (etoposiside)一樣，被視為topoisomerase II (Topo II) poison，具有引發DNA damage的潛在能力。在我們的研究當中，我們深入探討GL331的處理抑制子宮頸癌細胞HeLa S3生長的作用與機轉，GL331透過活化癌症細胞G1/S細胞週期的停滯和細胞凋亡的機轉達到有效抑制癌症細胞。GL331藉由p53的活化促進p21的表現，使得細胞停留在G1/S phase，磷酸化p34cdc2量的增加也表示細胞並未進入M phase。另一方面，GL331的處理啟動細胞中粒線體調控的細胞凋亡途徑(mitochondrial-mediated apoptosis)和接收器調控的細胞凋亡途徑(receptor- mediated apoptosis)，對於GL331所活化的細胞凋亡扮演必要和同等重要的角色。由實驗結果得知，BCL-2在粒線體膜上的表現減少導致細胞中cytochrome c從粒線體釋放到細胞質，隨之caspase 9的活化啟動了粒線體調控的細胞凋亡途徑。GL331也透過增加細胞中FADD的表現和caspase 8的活化啟動了接收器調控的細胞凋亡途徑。因此GL331促進caspase 8和caspase 9的活化進而導致caspase 3的活化和PARP (Poly(ADP-ribose)polymerase)的切割，使得細胞走向死亡。 另外，我們發現mitogen-activated protein kinases (MAPKs)中的extracellular signal-regulated kinase (ERK1/2)明顯的活化，且ERK的活化參與了部份GL331引發的細胞凋亡機轉。為了驗證ERK在GL331處理的細胞中所扮演的角色，我們使用MEK的抑制劑 (U0126)結合GL331的處理，實驗發現U0126具有部分抑制caspase 8和caspase 9活性的能力。我們也首次發現ERK的活化在於FADD protein和mRNA的表現扮演重要的角色，ERK透過活化FADD mRNA的表現促進caspase 8的活化，讓我們重新思考ERK在細胞中的定位，至於ERK是如何促進FADD mRNA的表現將是我們接下研究的主要方向。

GL331 is a semi-synthetic podophyllotoxin-derived compound. Like its congener VP-16 (etoposide), it was originally identified as a potent topoisomerase II (Topo II) poison. In this study, we investigate the anticancer effect of GL331 in human cervical cancer cell line HeLa-S3. GL331 exhibited effective cell growth inhibition by inducing cancer cells to undergo G1/S phase arrest and apoptosis. GL331 induced G1/S arrest by up-regulation of p53-dependant p21CIP1/WAF1 expression. On the other hand, GL331 treatment triggered the mitochondrial - mediated and receptor-mediated apoptotic pathway. Both are essential and equally contributed to GL331-induced apoptosis. GL331 treatment triggered the mitochondrial apoptotic pathway indicated by loss of BCL-2 on the mitochondria membrane , cytochrome c release, and caspase-9 activation. GL331 also induced receptor-mediated apoptosis by increasing the amount of FADD and caspase 8 activation. Take together, activation of both caspase 8 and caspase 9 causes caspase 3 activation and cleavage of PARP (Poly(ADP-ribose)polymerase).The consequential result was the cell apoptosis. Additionally, we found mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK1/2), are significantly activated and partially participate in GL331-induced apoptosis. To demonstrate proapoptotic function of ERK, we combine GL331 treatment with MEK1/2 inhibitor,U0126. U0126 is able to block partially caspase 8 and caspase 9 activity. ERK is a critical mediator in GL331-induced caspase 8 and caspase 9 activation. Surprisingly, we firstly found that ERK activation promotes increasing the amount of FADD protein and mRNA. ERK alternatively induces the expression of FADD and triggers caspase 8 activation in GL331 treatment. And it will be interesting to explore how ERK stimulates the up-regulation of FADD mRNA.