多環芳香烴受器於人類肺癌細胞調控IL-6表現與增進抗癌藥物之活性

Aryl Hydrocarbon Receptor Modulated Interleukin-6 Expression and Enhanced Anticancer Drug Activity in Lung Cancer

陳柏宏(Po-Hung Chen)

指導教授 ： 林嬪嬪 ； 蔡菁華

中山醫學大學/醫學院/醫學研究所/博士(2012年)

https://doi.org/10.6834/CSMU.2012.00137

摘要

研究目的：多環芳香烴受器 (Aryl hydrocarbon receptor，AhR) 是一種轉錄因子亦為戴奧辛與多環芳香烴類化合物之受器，當標的細胞暴露於這些污染物質後能使 AhR 活化，進而促進其下游基因表現。我們已於先前研究證實AhR表現在肺腺癌中相較於周邊正常肺組織高，推測AhR在肺腺癌的形成過程中可能扮演重要角色。而許多研究已證實，發炎相關之細胞激素之表現在肺癌形成的過程中亦扮演重要角色。此外AhR已被報導可作為抗癌藥物之作用標的。17-allylamino- 17-demethoxygeldanamycin (17-AAG) 為HSP90之專一性抑制劑，目前已進入多項腫瘤治療之人體臨床試驗。已知未活化之AhR會與HSP90結合並存在於細胞質中，目前有研究指出，17-AAG處理舌部鱗狀上皮癌細胞 MSK-Leuk-1以及人類上皮角質細胞HaCaT細胞，會降低AhR蛋白量及抑制AhR功能。因此本論文研究之目的為：(1) 探討在肺癌中AhR調控細胞激素IL-6表現的分子機轉；(2) 探討在肺腺癌細胞中AhR表現與抗癌藥物17-AAG之藥效之相關性。
研究方法：收集200例非小細胞肺癌患者之肺癌組織，利用組之免疫染色法分析AhR、 RelA與IL-6之相關性。以siRNA、AhR表現質體或是單株細胞方式來調控肺癌細胞中AhR、 RelA與IL-6基因的表現。以聚落形成能力試驗與裸鼠皮下注射試驗來偵測腫瘤生長情形。NFκB活性以reporter gene assay來偵測。17-AAG對細胞之抑癌活性，以計數細胞存活率與細胞週期分佈來分析。利用免疫沉澱法與細胞免疫螢光染色來偵測蛋白於細胞中之存在位置。蛋白與mRNA量分別以西方墨點法與real-time RT-PCR方式進行偵測。
研究結果：於200例非小細胞肺癌檢體之組織免疫化學染色結果顯示，AhR表現與細胞核RelA及IL-6表現有極顯著正向相關性，尤其是在不抽菸的患者。在人類支氣管上皮細胞 BEAS-2B 與人類肺腺癌細胞 H1355中過度表現 AhR時，會增加 IL-6 mRNA 及蛋白的表現量。而經由 reporter gene assay 方法證明調控IL-6之轉錄因子NFκB的活性會隨AhR過度表現而增強，並透過細胞免疫螢光染色法看到 NFκB 有轉移至細胞核之現象，印證增加AhR表現會促進NFκB活化。此外，在高度表現AhR的細胞中，AhR與RelA會形成複合體並進入細胞核與IL-6啟動子上之κB區段鍵結，進而調控IL-6基因表現。17-AAG處理對高度表現AhR之肺腺癌細胞有較佳的抑癌活性。17-AAG能透過降低AhR蛋白量，進而抑制NFκB調控細胞生長之路徑。17-AAG亦能透過降低細胞週期調控蛋白CDK2、CDK4、cyclin D1、cyclin E、phospho-Rb蛋白量，造成G1期停滯而抑制細胞生長；而在AhR表現較高的細胞中，17-AAG處理後上述細胞週期調控蛋白降低的程度更顯著。高度表現AhR之肺腺癌細胞能表現較多的NAD(P)H:quinone oxidoreductase (NQO1)，NQO1能將17-AAG代謝形成更具HSP90鍵結親和力之代謝物，因而17-AAG對高度表現AhR之肺腺癌細胞有較強的抑癌活性。
研究結論：我們的研究結果證明，增加AhR表現能促進NFκB活化並增加 IL-6 表現，此機制可能是AhR促進肺癌發生之重要途徑。而在肺腺癌細胞中，AhR除了是抗癌藥物17-AAG之作用標的外，AhR亦是增加17-AAG之抑癌活性之重要因子。

關鍵字

多環芳香烴受器 ； 肺腺癌

並列摘要

Objective: Lung cancer is the first leading cause of cancer deaths in Taiwan. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is activated by environmental toxicants. Previously, we have demonstrated that AhR is overexpressed in lung adenocarcinoma (AD). Some studies reported that inflammatory cytokines expression, such as IL-6, increased in lung carcinogenesis. AhR usually associates with heat shock protein 90 (Hsp90) in the cytoplasm of cells. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is an Hsp90 inhibitor and evaluated for anticancer activity in clinical trials.17-AAG has been shown to reduce AhR protein levels and functions in tongue squamous cell carcinoma and human epithelial kerationocytes. Therefore, the objectives of this study are: (1) to investigate the relationship between AhR and IL-6 in lung AD; (2) to investigate whether AhR plays a role in 17-AAG-mediated anticancer activity by functioning as a downstream target or by modulating its anticancer efficacy.
Methods: Using immunohistostaining (IHC), AhR, RelA (NF-kappaB subunit) and IL-6 were detected in 200 non-small cell lung cancer (NSCLC) patients. AhR expression in lung AD cells was modulated by RNA interference or overexpression. RelA and IL-6 expressions in lung AD cells was modulated by RNA interference. Tumor growth was determined with colony formation in vitro or in nude mice. NFκB activity was detected by reporter gene assay. Anticancer activity of 17-AAG was determined by measuring cell viability and cell cycle distribution. Proteins localization were examined by immunoprecipitation (IP) and immunofluorescence (IF) staining, respectively. Proteins and mRNA levels were respectively examined by immunoblotting and the real-time reverse transcription-polymerase chain reaction (RT-PCR) method.
Results: With IHC method, we observed that cytosolic and nuclear AhR expression significantly correlated with nuclear RelA and IL-6 in 200 NSCLC patients, especially in never smokers. AhR positively modulated growth of lung AD cells H1355 via RelA-dependent mechanisms. Furthermore, overexpression of AhR increased IL-6 expression in H1355 cells and immortalized human bronchial epithelial cells BEAS-2B. While, AhR overexpression significantly increased NFκB activity, interference of AhR expression significantly reduced IL-6 expression and NFκB activity in H1355 cells. With IP and IF staining, we demonstrated that AhR associated with RelA in the cytosol and nucleus of human lung cells. Furthermore, AhR overexpression enhanced nuclear localization of AhR and RelA, and increased the association of AhR/RelA complex with NFκB response element on the IL-6 promoter. However, neither p50 nor Arnt was involved in AhR/RelA complex formation. Treatment with 17-AAG reduced AhR protein levels and reduced AhR-regulated genes expression in H1355 cells. Interestingly, AhR expression positively increased anticancer activity of 17-AAG. 17-AAG treatment reduced cell viability and increased G1 phase arrest along with reduction in CDK2, CDK4, cyclin E, cyclin D1 and phosphorylated Rb in high AhR lung AD cells. NAD(P)H:quinone oxidoreductase (NQO1), positively regulated by AhR, was reported to metabolically activate 17-AAG and increase its anticancer activity. Knockdown of NQO1 expression attenuated the reduction of CDK2, CDK4, cyclin E, cyclin D1 and phosphorylated Rb by 17-AAG treatment in lung AD cells.
Conclusions: Our results indicate that AhR associated with RelA, positively modulated NFκB activity and then up-regulated IL-6 expression in human lung cells without treatment of AhR ligand. Here we demonstrated a novel mechanism for tumor promotion effect of AhR overexpression in non-smoking lung AD. AhR protein did not only function as a downstream target of 17-AAG, but also enhanced anticancer activity of 17-AAG in lung AD cells.

並列關鍵字

AhR ； RelA ； IL-6 ； 17-AAG ； NQO1

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