β-lapachone抑制cisplatin對纖維母細胞損傷之機制

Abstract

DNA損傷藥劑在臨床上被用來做為腫瘤的化學治療藥物已有很長的歷史，cisplatin (CDDP)屬於此類藥物且經常被用來當作第一線治療藥物。Cisplatin結構中含有一個鉑、兩個氨離子以及兩個氯離子，在進入細胞後，cisplatin的氯離子易解離而使得其中的鉑與DNA鹼基發生配位，形成配位結構後，會影響複製叉的形成而導致細胞週期異常。除了傷害細胞核的DNA外，研究中也指出cisplatin會傷害粒線體DNA，導致粒線體無法正常合成電子傳遞鏈上蛋白質，造成細胞內ROS堆積而使細胞走向死亡。基於這樣的機轉，cisplatin在癌細胞的治療上具有很大的潛力；然而，這些損傷的效應不是只有發生在目標癌細胞上，在其他正常組織也會受到影響而導致副作用的產生，因此，降低cisplatin副作用在臨床上是個非常重要的議題。 先前研究顯示，β-lapachone (β-lap)有促進傷口癒合與細胞增生等功效。β-lap在細胞中的活性主要是受到NAD(P)H:quinone dehydrogenase 1 (NQO1) 酵素活性影響，在先前研究中NQO1 酵素被指出可能穩定p53蛋白質，而p53又與核酸穩定有關，因此我們推測NQO1酵素在維持細胞中基因完整性扮演了重要角色。近年來研究指出β-lap可有效改善cisplatin在小鼠上造成的腎臟損傷以及聽力功能受損，基於上述的研究結果，我們推測當正常細胞被給予cisplatin前，利用β-lap預處理可能藉由降低cisplatin對於細胞核DNA及粒線體DNA的損害效應而對細胞具有保護的功效。除了上述機制外，近年來文獻指出，細胞自噬(autophagy)雖然被認為是細胞的保護機制，且可能參與cisplatin的抗藥性，但此現象可能也扮演了促死亡的角色。因此在本篇研究中，我們使用NIH3T3纖維母細胞來進行實驗，檢測β-lap對cisplatin所引致損傷效應之抑制機制，也探討細胞自噬在其中所扮演之角色。 本篇研究結果發現，β-lap有利於挽救cisplatin所造成之細胞死亡並改善cisplatin造成的DNA損傷，也改善cisplatin導致之NAD+/NADH比例下降現象，並具有保護粒線體的功能。除此之外，我們還發現給予細胞cisplatin，與細胞自噬相關蛋白質 LC3BII的表現量及自噬溶小體(autolysosme)染色有明顯上升，而預處理β-lap可抑制LC3BII及自噬溶小體的表現，我們推測細胞自噬可能也和β-lap的保護機制有關，因此，β-lap具有很大的潛力可作為cisplatin治療之保護劑。

DNA-damaging agents can be used as chemotherapeutic drugs,which have a long history in clinical use. Cisplatin (CDDP) is categorized as the most popular and the first line chemotherapeutic drug. It has a platinum core with two chloride leaving groups and two amine non-leaving groups. When cisplatin enters cells, the aquation of the leaving groups allows the platinum core to bind to DNA bases. Thus, adducts are formed on the DNA. These structures block the progression of the replication fork and causing cell cycle abnormality. In addition, studies have shown that cisplatin may also form DNA adduct on mitochondria DNA. These structures interfere with the production of the proteins involved in the electron transport chain, therefore, causing mitochondria dysfunction and ROS production. Based on the fact, cisplatin has a promising potential on treating solid tumors. However, the damaging effects not only affect the targeted cancer cells, but also the non-targeted normal tissues. It is important for clinic to discover the solution to this problem. Previous studies have shown that β-lapachone (β-lap) is effective in proliferation of many types of cells in vitro and promoting wound healing in vivo. The effect of β-lap in cells is tightly relied on the activity of NAD(P)H:quinone dehydrogenase 1 (NQO1), which is shown to involve in the maintenance of chromosomal integrity through p53 stabilization. Moreover, recent studies have reported that β-lap pretreatment ameliorates cisplatin-induced acute kidney injury and hearing impairment. Those evidences imply that it is possible for non-cancer cells to be protected by β-lap, while treating with cisplatin, with blocking the DNA damaging effect of cisplatin on nuclear DNA and mitochondria DNA. In addition to the above-mentioned mechanism, recent studies have shown that despite the protecting role of autophagy in the occurrences of cisplatin-resistance, it may play a pro-death role under certain conditions. In this study, NIH3T3 fibroblast was used as a cell model to investigate the inhibitory mechanisms of β-lap on the damages caused by cisplatin and to explore the role of autophagy. We found that β-lap was effective in rescuing the cisplatin-induced cell death, NAD+/NADH ratio decline and protecting cells from cisplatin-induced DNA damage. Besides, the pretreatment of β-lapachone protected cells from cisplatin-mediated mitochondrial dysfunction. We also discovered a phenomenon that the levels of autophagy markers, LC3BII and MDC fluorescences, were increased by cisplatin treatment, and the increase can be blocked by β-lap. Therefore, autophagy may also play a role in the protecting mechanisms of β-lap. According to the above results, β-lap may be a potential chemoprotective agent in the treatment of cancer.