Abstract LB-179: Effect of silibinin on the growth kinetics of 3D tumor spheroids: Colorectal cancer cells enriched for cancer stem cell phenotype
Authors: Sushil Kumar, Komal Raina, Chapla Agarwal, Rajesh Agarwal
Journal: Cancer Research (2012)

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in US; statistical estimates for 2012 indicate ∼103,170 new CRC cases and 51,690 associated deaths. Several studies have reported the chemopreventive potential of a wide range of natural agents against CRC; however, their efficacy towards cancer stem cell (CSC) generation leading to cancer initiation and tumorigenesis has not yet been well-defined. In this regard, recent studies by our group have demonstrated that silibinin, a non-toxic, chemopreventive agent, strongly decreases the percentage of sphere formation of CRC cells (a stem cell characteristic). The present study was aimed to provide an understanding of the mechanisms involved in the inhibitory effect of silibinin on the CSC population in CRC. Specifically, we carried out studies on how silibinin modulates the dynamics of CSC spheroids under in vitro conditions, by examining its effect on the kinetics of CSC spheroids generated from three CRC cell lines, SW480, HT-29 and LoVo, and subjected them to sphere cluster formation assays. Both single and multi-treatment approaches with silibinin were employed to determine the effect, for 1-2 weeks, on the cycling properties of CSC which were enriched in the individual spheroids. Comparative analysis of different cell lines in terms of sphere formation kinetics indicated that the CSC enriched spheroids varied significantly in their growth kinetics, measured as a function of time and related to the individual diameter/ area of the spheroids and the nutrient uptake. Effect of silibinin on the growth kinetics and cycling properties of these CSC enriched spheres indicated a significant but differential modulatory effect of silibinin on these properties across cell lines. Further, 2D and 3D differentiation assays of these spheroids carried under serum conditions in the presence and absence of silibinin, indicated towards the formation of more differentiated clones by treatment. Subsequent in vitro studies, employing real time RT-PCR, showed silibinin effect to be mediated in part by significant down regulation of the expression of the CSC marker CD44 and Notch mediated signaling in these cell lines. Further studies showed that silibinin also decreased protein levels of cleaved Notch and its transcriptional target Hes-1. The implications of such an effect are tremendous, since the disruption of homeostasis (regulated in part by Notch signaling) among stem cells and progenitor cells in intestinal region results in the expansion of CSC pool together with an increase in proliferative cell populations. These are extremely important findings as they clearly show that silibinin interferes with kinetics of CSC pool expansion via targeting various regulatory signals associated with the survival and multiplication of colon CSC pool leading to effective CRC prevention. 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 LB-179. doi:1538-7445.AM2012-LB-179