多環芳香烴對人類肺腺癌細胞株纖維母細胞生長因子9之誘導作用

Abstract

近幾年來，肺癌比例在全球有增加的趨勢。在台灣肺癌為女性癌症死亡的主要原因，流病調查資料顯示廚房油煙(cooking oil fumes, COF)、機車廢氣微粒(motorcycle exhaust particulate, MEP)、環境菸草煙(environmental tobacco smoke)皆為肺癌致病之可能環境因子，而這些因子皆具有的多環芳香烴(polycyclic aromatic hydrocarbons, PAHs)為目前已知環境中最大量具致癌性之單一物質；其中benzo[a]pyrene(BaP)具有強致癌性及強突變性，可作為致癌指標。Fibroblast growth factor 9 (FGF9) 為 FGF家族一員，可調控細胞增生、分化及移動。本研究主要目標在探討 FGF9 與這些環境因子交互作用後，增加肺腺癌的轉移與侵襲能力及其機制。首先利用組織免疫染色觀察肺腺癌病人中(65例) FGF9的表現與分布，結果顯示FGF9只表現於肺腺癌細胞之細胞質中，而正常肺泡細胞則無FGF9表現，並且FGF9表現與淋巴轉移程度具有正相關性。以50 μg/ml COF, 100 μg/ml MEP, 10 μg/ml cigarette smoke extract (CSE), 10 μM BaP, 10 μM trans-trans-2,4- decadienal (DDE)與10 μM 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK)處理肺腺癌CL5細胞，分別在6小時後收集RNA及24小時後收集細胞微粒體蛋白觀察FGF9表現。實驗結果顯示，COF、MEP、CSE與BaP可誘導FGF9 mRNA及蛋白表現，但是DDE及NNK則否，可見BaP是環境因子中引起FGF9上升原因之一。而進一步利用mitogen-activated protein kinase抑制劑PD98059與SB202190探討可能誘導機轉，發現BaP及MEP所誘導的FGF9、CYP1A1、IL-1α、IL-6及IL-11可被MEK抑制劑PD98059抑制；而p38 MAP kinase抑制劑SB202190則對被誘導的CYP1A1、CYP1B1、IL-1α、IL-6及IL-11發生抑制作用。在肺腺癌細胞CL5, CL1-0及CL1-5中皆有FGF receptor (FGFR)3IIIc, FGFR2IIIc, FGFR3IIIb及FGFR1IIIc分布。以10 μM BaP處理CL5細胞6小時後則可增加FGFR2IIIc及FGFR3IIIb mRNA的表現。由本研究結果顯示環境汙染物中的PAH類物質BaP是誘發肺腺癌細胞株中FGF9表現的原因之一，而FGF9在肺腺癌中的表現與腫瘤轉移的程度有關，推測環境汙染物可能透由PAHs誘導FGF9而促進肺腺癌侵襲及轉移，藉由本實驗可進一步了解基因與環境因子交互作用在肺癌轉移扮演的可能角色。

Lung cancer is occurring in epidemic proportions worldwide. In recent years, it has been the leading cause of cancer-related mortality in women. Epidemiological studies have indicated that exposures to cooking oil fumes (COF), motorcycle exhaust particulates (MEP), and environmental tobacco smoke (ETS) are environmental factors possibly associated with lung adenocarcinoma in Taiwan. Polycyclic aromatic hydrocarbons (PAHs) are present in these environmental factors. Among these PAHs, benzo[a]pyrene (BaP) is one of the most potent mutagen and carcinogen. Fibroblast growth factor (FGF) 9 belongs to the FGF superfamily which regulates cell differentiation, proliferation and motility. The main hypothesis of this study is that FGF9 gene and environment interaction promotes tumor progression and metastasis in lung adenocarcinoma. The results of immunohistochemistry studies of 65 human lung adenocarcinoma specimens indicated that FGF9 protein expression was moderate to high in lung adenocarcinoma cells but was not detectable in normal lung epithelium and that high FGF9 expression had positive correlation with patients who had advanced lymph node metastasis. Lung adenocarcinoma CL5 cells were treated with 50 μg/ml COF, 100 μg/ml MEP, 10 μg/ml cigarette smoke extract (CSE), 10 μM BaP ,10 μM trans-trans-2,4-decadienal (DDE) and 10 μM 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK). After 6 hr, FGF9 mRNA expression was induced by COF, MEP, CSE and BaP. Induction of FGF9 protein was observed after 24 hr. DDE and NNK had no marked effects on FGF9 mRNA or protein expression. To investigate the induction mechanism of FGF9, mitogen-activated protein kinase signaling pathway inhibitors PD98059 and SB202190 were used. Induction of FGF9, CYP1A1, IL-1α, IL-6 and IL-11 by BaP or MEP was suppressed by PD98059. Induction of CYP1A1, CYP1B1, IL-1α, IL-6 and IL-11 by BaP or MEP was suppressed by SB202190. Fgf receptor (FGFR)3IIIc, FGFR2IIIc, FGFR3IIIb and FGFR1IIIc mRNA were detected in lung adenocarcinoma cells CL5, CL1-0 and CL1-5. Treatment of CL5 cells with 10 μM BaP for 6hr increased FGFR2IIIc and FGFR3IIIb mRNA expression. These studies show that BaP of environmental factors is a chemical responsible for FGF9 induction in lung adenocarcinoma cells. Furthermore, FGF9 expression is correlated with advanced lymph node metastasis in lung adenocarcinoma patients. These results indicate that PAH may possibly mediate female lung adenocarcinoma invasion and metastasis through FGF9 induction and also reveal the possible role of gene and environment interaction in lung cancer metastasis.