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

• Main Authors: Po-Hung, 陳柏宏
• Other Authors: Pinpin Lin
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/86316077224741790117

博士 === 中山醫學大學 === 醫學研究所 === 100 === 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.