探討DNA Damager MNNG誘發細胞壞死訊息傳遞路徑之研究

Abstract

N-Methyl-N’-nitro-N’-nitrosoguanidine (MNNG) 是一種烴化藥物，會使細胞內大量的去氧核醣核酸斷裂，造成負責修補核酸斷裂的核蛋白PARP-1過度活化進而造成細胞壞死。然而，目前對於活性氧化物 (ROS) 的產生，鈣離子的增加，ATP的大量排空，JNK的活化等等各種已知細胞壞死相關現象，和PARP-1活化之間的調控機轉，尚未被完全研究清楚。於是我們利用MNNG處理小鼠纖維母細胞來研究因PARP-1活化而導致細胞壞死的訊息傳遞路徑。我們發現，MNNG會造成propidium iodide uptake的增加，也就表示MNNG會造成細胞壞死，而且非選擇性細胞凋亡蛋白酶(caspase) 抑制劑 ZVAD對於MNNG造成的細胞壞死並沒有保護的效果。在MNNG造成的細胞死亡中，我們發現的現象有：PARP-1的快速活化，JNK的活化，兩階段性的細胞內活性氧化物產生，細胞內鈣離子的增加，以及持續的ATP排空現象。從實驗結果中發現，在藥物處理後1分鐘，就有細胞內活性氧化物的產生，而在5到15分鐘內達到巔峰；因為我們使用DPI可以抑制這個現象，所以推測這個早期細胞內活性氧化物的產生可能是來自於NADPH oxidase。而發生於30分鐘並隨著時間逐漸增加、持續至6小時的後期活性氧化物，可能是由粒線體所產生的。當我們給予抗氧化劑NAC，MNNG所造成的各種現象都會消失。我們也利用鈣離子螯合劑BAPTA/AM，去釐清鈣離子在訊息傳遞中的角色。實驗結果中發現，MNNG造成鈣離子的快速上升，是屬於早期活性氧化物累積的下游反應，而且鈣離子也參與了PARP-1和JNK的活化。此外，PARP-1抑制劑 (3AB)可以抑制MNNG導致PARP-1活化、ATP排空、後期活性氧化物累積、鈣離子增加以及細胞死亡，但是並不影響JNK的快速活化。另一方面，使用JNK抑制劑SP600125所作的相關研究，顯示JNK參與在後期活性氧化物累積的過程中。比較MNNG處理WT和RIP1-/-小鼠纖維母細胞的結果，顯示RIP1參與了後期活性氧化物產生、後期鈣離子釋放，以及ATP排空的訊息路徑中。總而言之，我們釐清在MNNG所導致的細胞壞死過程中，PARP-1、活性氧化物、鈣離子、JNK以及RIP1的訊息傳遞路徑。這些結果提供了在細胞壞死調控過程中的新觀點，以及在細胞壞死相關疾病的臨床研究上一些新方向。

Alkylating agent N-methyl-N’-nitro-N’-nitrosoguanidine (MNNG) can cause excess DNA strands break then leads to PARP-1 overactivation and necrotic cell death. However, the regulatory mechanism of PARP-1 activation in detail relating to identified necrotic features, such as reactive oxygen species (ROS) production, calcium elevation, ATP depletion and JNK activation, have not been fully understood. In this study, we used MNNG-treated mouse embryonic fibroblasts (MEFs) to study the signaling pathways of PARP-1-mediated necrosis. We found that MNNG treatment can induce cell necrosis as assessed by the increased uptake of propidium iodide, and no protective ability of pan-caspase inhibitor ZVAD. MNNG induced cell death is featured by the rapid PARP-1 activation, JNK activation, biphasic ROS production, intracellular calcium increase and sustained ATP depletion. Results indicate that the early ROS production which occurred at 1 min and peaked at 5-15 min after MNNG treatment is resulting from NADPH oxidase, as DPI is able to inhibit such response. In contrast, the late phase of ROS production which occurred after 30 min and time-dependently increased up to 6 hr after MNNG treatment was generated by mitochondria. When treating cells with antioxidant NAC, all the phenomena caused by MNNG were abrogated. Studies with calcium chelator BAPTA/AM suggest that the rapid calcium rise, an event downstream of early ROS production, is involved in PARP-1 and JNK activation. Moreover, PARP-1 inhibitor (3AB) is able to reduce MNNG-induced PARP-1 activation, ATP depletion, late phase ROS production, calcium elevation, and cell death, but cannot affect the rapid JNK activation. On the other hands, studies with JNK inhibitor SP600125 indicate that JNK is involved in the late phase of ROS production. When comparing MNNG induced events in WT and RIP1-/- MEFs, our results indicate the necessity of RIP1 for mediating the sustained ROS production, calcium increase, as well as ATP depletion. Taken together, we have clarified the signaling crosstalk between PARP-1, ROS, calcium, JNK and RIP1 in MNNG elicited necrosis of MEFs. These results provide not only new insight into necrotic regulating mechanisms, but also into clinical benefits to patients in a variety of pathophysiological conditions associated with tissue necrosis.