檳榔子（areca nut, AN）是一種人類的致癌物，也是促使台灣有很高的口腔癌發生率的原因。奇美醫學中心的口腔腫瘤患者中有超過七成在就醫時仍保有咀嚼檳榔的習慣，故可合理的推論這些患者的口腔腫瘤細胞，包括源自淋巴與上皮的細胞，持續且長期地受到檳榔的刺激。為模擬這種活體（in vivo）的刺激並觀察它對這些細胞的影響，我們以不具有細胞毒性濃度的檳榔子萃取液（AN extract, ANE）或其中經部份純化，分子量介於30-100kDa的部分（簡稱為ANE 30-100K）處理Jurkat白血病 T 細胞與人類口腔鱗狀細胞上皮癌細胞（OECM-1），並分析這些處理對細胞所造成的影響。我們發現，經 ANE 及 ANE 30-100K 處理過的細胞與未經處理的母株細胞相較之下，其低氧的耐受性與對順鉑（cisplatin）及5-氟尿嘧啶（5-fluorouracil, 5-FU）的抗藥性增強。為探討可能的機轉，我們接著尋找可能使這些篩選後的細胞有如此性狀上的改變，且表現量增加的蛋白質。利用免疫轉印法或二維膠體電泳與液相層析質譜儀，我們發現DRAM/LC3-II的表現量與第一型過氧化物還原酶（peroxiredoxin-1, PRDX1）的分泌量在這兩種篩選過的細胞中有增加的現象。此外，免疫組織化學染色法顯示PRDX-1在正常組織中主要位於上皮層，但在腫瘤塊中大部分的細胞卻都有表現，並且在比較四對檳榔使用者及非檳榔使用者的口腔鱗狀細胞癌（oral squamous cell carcinoma, OSCC）組織之後，我們初步的結果顯示PRDX-1在檳榔使用者的樣本中有表現量增高的情形；此外，ANE 30-100K亦以濃度相依的方式在非腫瘤的口腔纖維母細胞CMT-415中刺激PRDX-1的表現。我們現在正試圖以siRNA抑制PRDX-1，藉以分析在兩種篩選過的細胞中，此酵素是否對於低氧與藥物的耐受力有所貢獻。 Areca nut (AN) is a human carcinogen responsible for high oral cancer incidence in Taiwan. More than 70% patients keep on AN chewing when they took medical treatment in Chi Mei Medical Center. It is thus reasonable to speculate that their oral tumor cells, including lymphocytic and epithelial origins, have received a constant and long-term stimulation from AN. To simulate this in vivo stimulation and observe its on these cells, we subjected Jurkat leukemia T and oral epidermoid carcinoma Meng-1 (OECM-1) cells to long-term treatment with noncytotoxic concentrations of AN extract (ANE) or partially purified 30-100 kDa fraction of ANE (ANE 30-100K) and analyze the effects of these treatment on these cells. The results have demonstrated that both ANE- and ANE 30-100K-selected Jurkat T cells exhibited stronger resistance to low oxygen as well as to cisplatin and 5-fluorouracil (5-FU) as compared to non-selected parental cells. To access the possible mechanism, we next searched for upregulated proteins presumed to responsible for such phenotypic changes in these selected cells. By using immunoblotting or two-dimensional gel electrophoresis and liquid chromatography-mass spectrophotometer (LC-MASS), expression level of DRAM/LC3-II and secretion peroxiredoxin-1 (PRDX-1) were identified to be elevated in both selected cells. Immunohistochemistry revealed that PRDX-1 protein is mainly localized at epithelial layer of normal tissues but is expressed in most tumor cells. Moreover, after comparison of 4 pairs of OSCC tissues from AN and non-AN users, our preliminary data indicate on elevated expression level of PRDX1 in the specimens from AN users. In addition, ANE 30-100K also stimulated PRDX-1 expression in non-tumor oral fibroblasts (CMT-415) in a concentration- dependent manner. We are currently try to inhibit PRDX-1 by siRNA to assess whether this enzyme contribute to increase resistance of low oxygen and drugs in both selected cells.
