Abstract 1904: Novel 5-fluorouracil-thymoquinone hybrid kills colon cancer stem cells
2017
Objectives: Recently, it has been shown that a subset population of cancer cells in a tumor, the so-called cancer stem cells (CSCs), majorly contributes to chemoresistance and the ability of a tumor to form recurrences and metastases. Since the response to the standard chemotherapeutic drug 5-fluorouracil (5-FU) in patients with colorectal cancer (CRC) leads only to an inadequate response, we aimed to synthesize new hybrid molecules by combining 5-FU with the plant-derived drug thymoquinone (TQ). Methods: The cytotoxic effect of hybrids, the single drugs and the combination was investigated by crystal violet assays. HCT116, HT29 and 5-FU-resistant colorectal tumor cells were used to assess the spheroid forming capability after treatment with different concentrations of the hybrid and the single drugs by staining with the fluorescent stem cell marker CDy1 and Hoechst 3324 after 21 days of incubation. Western Blot analysis for stem cell surface marker CD133 were performed to verify these results. We have analyzed the anticancer effects of the single drugs TQ and 5-FU and the most effective hybrid on deregulation of cancer signaling pathways utilizing the NANOSTRING cancer pathway panel. In addition, we performed a single cell cloning from spheroids to generate monoclonal cell lines with enriched CSC populations. In vivo effects were studied in the chick chorioallantoic membrane (CAM) assay and in mouse xenografts. Results: The 5FU-TQ hybrid has an increased cytotoxic effect towards CRC cells when compared to single drug treatments. The spheroid formation assay revealed the capability of the hybrid to reduce sphere number and size at fairly low concentrations (10, 20 µM), however at 50 µM there was a complete suppression of spheroid growth. NANOSTRING analysis showed that gene expression of main components of the PI3K/AKT signaling pathway was significantly down-regulated after treatment with all drugs but most prominently with the new hybrid. After data processing we found that AKT3, IGF1, COL11A1, FGF10, GNG7, EPO genes were downregulated in hybrid-treated cells, while CDKN1A, BAX, JUN, MDM2, FAS were upregulated. This was verified via Western Blotting for a panel of markers. Next, we investigated the effectiveness of the hybrid to kill clones having an enriched CSC population. Indeed, CD133 protein levels were significantly decreased in hybrid-treated clones. Histological analysis of in vivo CAM grafts showed that HCT116 control cells developed a well vascularized tumor mass whereas HCT116 cells that were treated with the hybrid showed no more vital tumor cells, but extended areas of necrosis, calcification, and desmoplasia. In vivo mouse xenografts verified the potential of this hybrid as an antitumor agent reducing tumor volume significantly. Conclusion: Our findings strongly suggest that the newly synthesized 5-FU-TQ hybrid might become a promising anticancer drug by directly targeting CSCs. Citation Format: Benardina Ndreshkjana, Aysun Capci Karagoz, Volker Klein, Pithi Chanvorachote, Julienne K. Muenzner, Kerstin Huebner, Chuanpit Ninsontia, Sara Steinmann, Abbas Agaimy, Maamoun Fatfat, Hala Gali-Muhtasib, Svetlana B. Tsogoeva, Regine Schneider-Stock. Novel 5-fluorouracil-thymoquinone hybrid kills 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 1904. doi:10.1158/1538-7445.AM2017-1904
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