Abstract 2579: Silibinin inhibits tumor associated macrophages-induced lung cancer cell growth and invasiveness by down-regulating macrophage migration inhibitory factor .
Authors: Ramasamy Kumaraguruparan , Deep Gagan , Tyagi Alpna , Agarwal Chapla , Agarwal Rajesh 
Published: August 2014

Lung cancer is the leading cause of cancer related deaths worldwide. According to ACS, 226,160 new lung cancer cases and 160,340 associated deaths are estimated in the United States for 2012. The alarming statistics demand additional preventive and therapeutic strategies to reduce lung cancer burden. One of the novel and rationalized approaches to control lung cancer is to target tumor microenvironment (TM) along with cancer cells. Tumor associated macrophages (TAMs) are important component of TM, and play a pivotal role in the growth and progression of lung cancer. Clinical studies have shown a positive correlation between TAMs and poor prognosis in lung cancer, confirming TAMs as an important drug target. Earlier, we have reported that natural flavonolignan silibinin feeding inhibits urethane-induced lung tumorigenesis by targeting TAMs in the TM. Here, we developed cell culture models to study inhibitory effects and mechanism of silibinin action against macrophages-induced growth and invasiveness in lung cancer cells. We exposed U937 and THP-1 monocytes to phorbol 12-myristate 13-acetate (PMA) to generate M2-type macrophages that closely resemble TAMs. Next, PMA-induced U937 and THP-1 macrophages were treated with DMSO (control) or silibinin (25-100 μM) for 24-72 h; then fresh serum free media was added and at definite time-points control conditioned media (CCM) and silibinin-treated conditioned media (SBCM) were collected. Thereafter, lung cancer A549 cells were exposed to CCM or SBCM and cell proliferation was studied. Results showed that A549 cells proliferation was increased by CCM (from both U937 and THP-1 macrophages) but inhibited by SBCM. Exogenous addition of silibinin (25-100 μM) in CCM also inhibited the growth of A549 cells. Also, A549 cells co-cultured with macrophages showed a ∼3 fold increase in invasiveness, while silibinin (100 μM) treatment of U937 and THP-1 macrophages inhibited the A549 cells invasiveness by 50% (p&lt;0.001) and 55% (p&lt;0.001), respectively. Cytokine array analyses showed that silibinin decreased the level of 15 cytokines (MIF, MCP1, VEGF, HGF, TGFβ, and TNFα etc) and increased the level of 3 cytokines (ENA78, IL12 and MIP-1β) in U937 macrophages. Among the several silibinin-modulated cytokines, we focused on MIF (macrophage migration inhibitory factor) as this cytokine is overexpressed in lung tumors and implicated in all stages of the neoplasia. We confirmed cytokine array results by ELISA that showed 62% and 50% decrease in MIF in SBCM from U937 and THP-1 macrophages, respectively. Importantly, supplementation of recombinant MIF (70 pg) in SBCM reversed SBCM-caused growth inhibition of A549 cells. Similar results were also observed in lung cancer cell line H322. Taken together, these results suggest that silibinin could effectively target TAMs in the TM and inhibit lung tumor growth and invasiveness.

Citation Format: Kumaraguruparan Ramasamy, Gagan Deep, Alpna Tyagi, Chapla Agarwal, Rajesh Agarwal. Silibinin inhibits tumor associated macrophages-induced lung cancer cell growth and invasiveness by down-regulating macrophage migration inhibitory factor . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2579. doi:10.1158/1538-7445.AM2013-2579