口腔上皮細胞PARP7表現之功能性探討

Abstract

根據民國95 年行政院衛生署統計，口腔癌已成為台灣男性十大癌症發生率的第五位，且是年增率第一高的癌症死因，而嚼食檳榔已被證實為導致口腔癌發生最主要的危險因子。檳榔鹼 arecoline是檳榔中最主要的興奮成分，在唾液中的濃度可達140 g/ml。過去研究發現它對多種細胞具有細胞毒性以及基因毒性，這些傷害可能造成細胞生長停滯或細胞凋亡，但目前對檳榔是否誘發口腔細胞凋亡及其調控機制瞭解不多。Poly (ADP-ribose) polymerases (PARP) 是存在於多數真核細胞當中一類多功能蛋白質轉譯後修飾酶，由資料庫搜尋，已經找出至少有18種不同的基因產物具有PARP族群的特質。PARP廣泛的參與細胞內多種生理活動, 例如DNA損傷修復與細胞凋亡等。而近期的研究當中，也發現PARP的活性會影響癌症的進程，進而開始研發利用PARP抑制劑作為治療癌症的新方法。在本研究當中，我們利用RT-PCR觀察口腔上皮細胞在經過arecoline處理之後PARPs受到調控的情形。我們首先發現SAS口腔上皮細胞在經過arecoline處理之後,會誘發PARP家族中的一個成員 – PARP7 – 的的表現。我們進一步利用免疫共沉澱的技術發現，PARP7的表現集中在細胞核的位置。接著，經由PARP activity assay，我們發現PARP7確實有poly ADP-ribosylation的活性，但是，與其他PARP家族成員不同的是，他將只會在目標蛋白質上面加上一個或少於六個的ADP-ribose。最後，經由質譜儀的技術我們分析到Nijmegen breakage syndrome 1 (NBS1)，一種參與在DNA雙股斷裂修復 (DNA double strain break) 過程中的蛋白質，會和PARP7有交互作用。總結而言，我們確定了人類新穎基因 – PARP7 – 確實有ADP-ribosylation的活性，另一方面，他可能會經由與NBS1的交互作用，參與在DNA雙股斷裂修復的機制當中。我們希望這些針對口腔癌細胞和PARP7的研究，可以作為將來針對口腔癌治療藥物研發的參考。

There are approximately 2 million habitual chewers in Taiwan. According to various studies, it has been considered that betel quid chewing to be an independent risk factor in the development of oral cancers . As a result, the International Agency for Research on Cancer (IARC) has evaluated Areca nut (AN), the main component in various forms of betel preparations, as a carcinogen to human in 2003. However, the definite pathogenesis and multifactorial mechanisms implicated by AN chewing are still not fully understood. Arecoline is the main alkaloid of AN with a soluble feature in water and alcohol. This compound is mutagenic and increases the risk of chromosome instability. Poly (ADP-ribose) polymerases (PARPs) comprise a protein family involved in a number of cellular processes including DNA repair and programmed cell death. Its functions have been correlated to cancer formation from several aspects and recently the use of PARP inhibitors as anti-cancer therapy becomes longstanding approach from different laboratories in last decades. In this study, we analyzed the expression patterns of PARPs in arecoline induced SAS oral cancer cells. We found that PARP7, as a member of PARPs, is up-regulated in in this condition in both mRNA and protein level. Using immuno-fluorescence staining, we found that exogenous PARP7 locate at the cell nucleus. Furthermore, we demonstrated that the product of PARP7 catalytic domain has ADP-ribosylation activity. Our data reveal that PARP7 is not a conventional PARP, which add long chain of ADP-ribose moieties to acceptor proteins. In contrast, it adds one or less than 6 ADP-ribose to histones. Finally, we analyzed that PARP7 associates with NBS1, one of the key members participating in homologous recombination by protein mass spectrometry. Together, our results indicate that PARP7 may be involved in DNA repair which is induced by arecoline treatment. We anticipate that our results can provide a reference for future drug development for oral cancer therapy.