Silibinin inhibits cytokine-induced signaling cascades and down-regulates inducible nitric oxide synthase in human lung carcinoma A549 cells Manesh Chittezhath, 1 Gagan Deep, 1 Rana P. Singh, 1,3 Chapla Agarwal, 1,2 and Rajesh Agarwal 1,2 1 Department of Pharmaceutical Sciences, School of Pharmacy, and 2 University of Colorado Cancer Center, University of Colorado Denver, Denver, Colorado; and 3 Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Abstract Recently, we reported that silibinin inhibits primary lung tumor growth and progression in mice and down-regulates induciblenitricoxidesynthase(iNOS)expressionintumors; however, the mechanisms of silibinin action are largely not understood. Also, the activation of signaling pathways inducing various transcription factors are associated with lung carcinogenesis and their inhibition could be an effective strategy to prevent and/or treat lung cancer. Herein, we used human lung epithelial carcinoma A549 cells to explore the potential mechanisms and observed strong iNOS expression by cytokine mixture (containing 100 units/mL IFN-; + 0.5 ng/mL interleukin-1B + 10 ng/mL tumor necrosis factor-A). We also examined the cytokine mixture–activated signaling cascades, which could potentially up-regulate iNOS expression, and then examined the effect of silibinin (50-200 Mmol/L) on these signaling cascades. Silibinin treatment inhibited, albeit to different extent, the cytokine mixture–induced activation of signal transducer and activator of transcription 1 (Tyr 701 ), signal transducer and activator of transcription 3 (Tyr 705 ), activator protein-1 family of transcription factors, and nuclear factor-KB. The results for activator protein-1 were correlated with the decreased nuclear levels of phosphorylated c-Jun, c-Jun, JunB, JunD, phosphorylated c-Fos, and c-Fos. Further, silibinin also strongly decreased cytokine mixture–induced phosphorylation of extracellular signal-regulated kinase 1/2 but only marginally affected JNK1/2phosphorylation.Silibinintreatmentalsodecreased constitutive p38 phosphorylation in the presence or absence of cytokine mixture. Downstream of these path- ways, silibinin strongly decreased cytokine mixture– induced expression of hypoxia-inducible factor-1A without any considerable effect on Akt activation. Cytokine mixture–induced iNOS expression was completely inhibited by silibinin. Overall, these results suggest that silibinin could target multiple cytokine-induced signaling pathways to down-regulate iNOS expression in lung cancer cells and that could contribute to its overall cancer preventive efficacy against lung tumorigenesis. [Mol Cancer Ther 2008;7(7):1817–26] Introduction Lung cancer is the leading cause of cancer burden worldwide with >3 million incidences and 1 million deaths annually. In United States, it is the second leading cause of cancer-related incidences and is the leading cause of cancer-related deaths (1). It is estimated that, among men, lung cancer alone will kill (31% of total cancer deaths) more than the next three cancers combined [prostate (9%), colon and rectum (9%), and pancreas (6%; ref. 1)]. Despite extensive research, the overall 5-year survival rate in lung cancer patients is only 8% to 14% and has improved only marginally during the past 25 years (1, 2). These alarming statistics suggest the need for effective preventive measures to lower the burden of this malignancy. Chemoprevention is a potentially important approach to reduce the large number of lung cancer–related deaths. Numerous studies have found that chemoprevention using phytochemicals, especially flavonoids, can prevent variety of cancers including lung cancer (3–5). In this regard, a large clinical study has suggested the presence of inverse association between flavonoid intake and subsequent lung cancer incidences (6). Silibinin, a flavonoid, is the major bioactive constituent present in silymarin, which is isolated from milk thistle (Silybum marianum), and widely used as a hepatoprotective agent and has been marketed as a dietary supplement. In vitro and in vivo studies have revealed pleiotropic capabilities of silibinin against various epithelial cancers including lung cancer (3, 7–12). Silibinin treatment induces a significant growth inhibition, a moderate cell cycle arrest, and a strong apoptotic death in SHP-77 cells (small cell lung carcinoma cells) and A-549 cells (non-small cell lung carcinoma cells; ref. 13). Dietary silibinin (up to 1%, w/w) strongly retards the urethane-induced lung tumor growth and progression via inhibition of proliferation and angio- genesis (3). Further, we observed that silibinin suppresses the expression of proliferating cell nuclear antigen, cyclin Received 3/15/08; revised 5/1/08; accepted 5/17/08. Grant support: National Cancer Institute grant RO1 CA113876. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Rajesh Agarwal, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Box C238, East 4200 9th Street, Denver, CO 80262. Phone: 303-315-1381; Fax: 303-315-6281. E-mail: Rajesh.Agarwal@uchsc.edu Copyright C 2008 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-08-0256 1817 Mol Cancer Ther 2008;7(7). July 2008 Downloaded from http://aacrjournals.org/mct/article-pdf/7/7/1817/1879807/1817.pdf by guest on 10 June 2022