Abstract 1637: Silibinin causes strong efficacy towards colon cancer stem cells by modulating IL-4 caused CD44 expression

Abstract

Abstract Cancer stem cells (CSC) are now recognized as the main cause for initiation, promotion and progression of most of the epithelial cancers, including colorectal cancer (CRC). Despite this fact, the efficacy of chemopreventive agents towards CSC generation leading to cancer initiation and tumorigenesis has not yet been well-defined. The present study was aimed to provide a new direction to ‘cancer chemoprevention research’ by determining whether silibinin, a natural non-toxic chemopreventive agent, has the potential to target CSC in CRC. In this regard, we determined silibinin effect on the self renewal capacity of the CSC population of CRC cell lines. We isolated different cell populations (CD44+EpCAMhigh, CD44+EpCAMlow, and CD44-EpCAMlow) from the CRC cell lines, SW480, HT-29 and LoVo, and subjected them to sphere cluster formation assays. A limiting dilution approach (cell numbers ranging from 1x104 to 300 cells) was employed to determine the isolated fraction which was enriched in CSC population. Comparative analysis of different populations, in terms of sphere formation, indicated that the CSC enriched fraction was CD44+EpCAMhigh population. Two dilutions (1000 and 3000 cells/well) of the CSC enriched CD44+EpCAMhigh, CD44+EpCAMlow and unsorted populations were then subjected to sphere cluster formation assays, in the absence or presence (a one time treatment) of silibinin (50-100µM). The % of floating spheroids (colonospheres) generated in the sphere cluster assays after 1-2 weeks were determined. The results indicated a dose dependant effect of silibinin on both number and size (volume) of colonospheres, with 100µM silibinin showing a significant reduction in the number and size of generated spheres in all three CRC cell lines. Since formation of spheroids under specific in vitro conditions is a measure of stemness, it is evident that silibinin has the potential to target the self-renewal of CSCs in CRC cell lines. Since, the role of cytokine interleukin (IL)-4 is also implicated in increased resistance of CSC to chemotherapy agents and targeting IL-4 signaling presents a rational approach for the prevention and treatment of CRC, we next determined whether silibinin effect on CSC is mediated via blocking IL-4 signaling in CRC cell lines. In this regard, treatment of all three CRC cell lines with 100µM silibinin was able to significantly reduce IL-4-induced expression of both total CD44 and its variant CD44-v6 (highly expressed in CRC tumors) in a time (12-48h) dependant manner. Subsequent in vitro studies showed this effect to be mediated in part by the inhibitory effect of silibinin on IL-4-induced activation of the transcription factor STAT-3 in terms of its Tyr705 phosphorylation in CRC cells. Together, these results suggest that more studies are needed to establish translational significance of silibinin efficacy on CRC CSCs as this agent is already in clinical-trial in CRC patients. 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 1637. doi:1538-7445.AM2012-1637