Abstract 2807: Oral silibinin inhibits tumorigenic potential of colon cancer stem cells
Authors: Kumar Sushil , Kumar Dileep , Raina Komal , Serkova Natalie J. , Agarwal Chapla , Agarwal Rajesh 
Published: August 2015

Recent studies by our group have demonstrated that silibinin, a non-toxic, chemopreventive agent, has an inhibitory effect on the self-renewal capacity and differentiation potential of the cancer stem cell (CSC) population of CRC cell lines. However, these observations were under in vitro conditions. To overcome the limitations of these findings, in the present study, we assessed oral silibinin effect on tumorigenic potential of colon CSC. For this, we employed phenotypic cell isolation strategy to separate CD44+EpCAMhigh (stem cells enriched) HT-29 CRC cells by flow cytometry/magnetic activated sorting. These sorted and unsorted cells were used to generate ectopic xenograft tumors in NOD/SCID mice and silibinin effect on initiation, growth, and metabolism of these tumors was assessed. Briefly, unsorted and sorted population (CD44+EpCAMhigh) from CRC HT29 cells purified using FACS as per defined protocols were injected sub-cutaneously into NOD/SCID mice (6-8 weeks old), and animals were orally fed with vehicle or silibinin (200 mg/ kg body weight), starting 24 h post-injection (n = 14 per group). Unsorted cells were similarly injected as 2 x106 cells/ mouse. Once xenograft started growing, tumor sizes and volumes were calculated. Tumor latency (time from injection to detection of palpable tumor) were also noted. In general, experiment was terminated by 40 days post cell injections or terminated earlier if tumors reach a maximum diameter of 15 mm. To support tumor latency and tumor size results obtained visibly, anatomical proton-density magnetic resonance imaging (MRI) scans (day 2, 7 and 14) were acquired. Functional imaging (performed for 60-80 mm3 tumor and by end of study) was performed to report on tumor cellularity by diffusion-weighted (DW-), vascularity by dynamic contrast-enhanced (DCE-) MRI and glycolytic activity by 18Fluorine-2-Deoxy-Glucose (FDG) positron emission tomography (PET). Tumors harvested at the end of above study were subjected to live cell sorting analysis to determine the percentage of CSC population (CD44+EpCAMhigh cells) in xenografts. To determine oral silibinin effect on CSC self-renewal in serial transplantation studies, these CSC enriched fractions and the unsorted population of tumor cells were transplanted back into NOD/SCID mice and followed for initiation, growth, and tumor phenotype with or without silibinin treatment. Sorted CSC and unsorted tumor cells from both control and silibinin-treated mice were also subjected to in vitro experiments to determine their self-renewal and differentiation capacity. Results show that indeed silibinin feeding has an inhibitory effect on tumorigenic potential of colon CSC; a decrease in latency as well as size/ volume of tumor was observed. This effect was associated with a reduced number of CSC's in silibinin-fed tumors, a decrease in tumor metabolic activity and an inhibition of angiogenic feed to the tumors.

Citation Format: Sushil Kumar, Dileep Kumar, Komal Raina, Natalie J. Serkova, Chapla Agarwal, Rajesh Agarwal. Oral silibinin inhibits tumorigenic potential of colon cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2807. doi:10.1158/1538-7445.AM2015-2807