Abstract 1007: Honokiol inhibits colon cancer stem cell growth and mechanism mediated through Notch signaling pathway
Authors: Subramaniam Dharmalingam , Ponnurangam Sivapriya , Ramalingam Satish , He Zhiyun , Zhang Youcheng , Anant Shrikant 
Published: August 2012

Abstract
Background: Despite therapeutic advances, colon cancer remains the second leading cause of death in the United States. Cancer stem cells are implicated in resistance to radiation and chemotherapy. Honokiol is a biphenolic compound that has been used in the traditional Chinese Medicine for treating various ailments. The current study is designed to determine whether honokiol affected colon cancer stem cells and to identify a mechanism. Methods: Colorectal cancer (CRC) 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, HCT116 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for CD31, stem cell markers and Notch signaling proteins. Results: Honokiol treatment resulted in a significant dose-dependent inhibition of proliferation and colony formation of the two cell lines, but not the normal cells. Treatment also induced CRC cells to undergo apoptosis and a G0/G1 arrest. Furthermore, honokiol reduced the number and size of colospheres, a method used to reflect stem cells in a population. Colon stem cell marker proteins DCLK1, LGR5, CD44 and SOX-9 were also decreased. Further proof was obtained by flow cytometry studies, demonstrating that honokiol reduced the number of DCLK1+ cells. In addition, there were reduced levels of activated Notch-1, its ligand Jagged-1 and the downstream target gene Hes-1. Furthermore, expression of the γ-secretase complex proteins, Presenilin, Nicastrin, Pen2 and APH-1, required for Notch activation were suppressed. On the other hand, ectopic expression of the Notch Intracellular domain partially rescued the cells from honokiol-mediated growth suppression. To determine the effect of honokiol on tumor growth in vivo, nude mice harboring HCT116 tumor xenografts in their flanks were administered the compound intraperitoneally every day for 21 days. Honokiol treatment significantly inhibited tumor xenograft growth, with notably lower tumor volume and weight. Microvessel density, based on CD31 staining was also significantly lower in the tumors following honokiol treatment when compared to controls. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of stem marker and Notch signaling proteins in the honokiol-treated xenograft tissues. Conclusion: Together, these data suggest that honokiol is a potent inhibitor of colon cancer that targets stem cells by inhibiting the γ-secretase complex proteins.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1007. doi:1538-7445.AM2012-1007