Abstract 3227: Novel Marmelin analog DBQ targets Notch signaling pathway in colon cancer stem cells
Authors: Subramaniam Dharmalingam , Ponnurangam Sivapriya , Dandawate Prasad R. , Kaushik Gaurav , Tawfik Ossama W. , Jensen Roy A. , Santra Santimukul , Padhye Subhash B. , Weir Scott J. , Anant Shrikant 
Published: July 2017

Abstract
Background: Colon cancer is the second leading cause of death in the United States. Previously, we have reported that the identification of a novel compound Marmelin from Aegle marmelos and potent anti-colon cancer activity. We have developed novel Marmelin analogue THB from this structure showed potent anti-cancer activity and its inhibitory constant value was 10 µM. From this, we developed a second series of analogs, of which DBQ is even more potent than THB. The current study is designed to determine whether DBQ affects colon cancer stem cells and identify a mechanism.
Method: Colon cancer cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we performed studies in HCT116 tumor xenografts. Immunohistochemistry was determined for stem cell markers and Notch signaling proteins.
Results: DBQ treatment induced significant dose-dependent inhibition of proliferation and colony formation of HCT116 and SW480 cells, but not that of the normal FHC colon epithelial cells. DBQ also significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, DBQ reduced the levels of colon stem cell marker proteins DCLK1, LGR5, and CD44. We obtained further confirmation by flow cytometry, where DBQ treatment reduced the number of DCLK1+ cells. We next determined whether DBQ affects the Notch signaling, a pathway that is important in maintaining CSC population. Notch receptor and its ligands are up-regulated in human colon cancer tissues. DBQ treatment significantly downregulated the expression of all four Notch isoforms, its ligands Jagged 1, 2 and DLL1, 3, 4 and downstream target protein Hes1. Notch activation requires cleavage by the γ-secretase complex. DBQ treatment inhibits the expression of γ-secretase complex proteins. To confirm that DBQ effect is thorough downregulating Notch activation, we ectopically expressed the Notch Intracellular domain. DBQ effect was significantly mitigated in this condition. To determine the effect of DBQ on tumor growth in vivo, we administered DBQ intraperitoneally (5mg/kg bw) every day for 21 days in mice carrying HCT116 tumor xenografts. DBQ treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition of CSC marker proteins DCLK1, LGR5 and CD44 and also the Notch signaling proteins in the DBQ-treated xenograft tissues.
Conclusion: Together, these data suggest that DBQ treatment suppresses colon cancer growth that targets stem cells in part by inhibiting Notch signaling pathway.
Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Prasad R. Dandawate, Gaurav Kaushik, Ossama W. Tawfik, Roy A. Jensen, Santimukul Santra, Subhash B. Padhye, Scott J. Weir, Shrikant Anant. Novel Marmelin analog DBQ targets Notch signaling pathway in colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3227. doi:10.1158/1538-7445.AM2017-3227