Tumor stem cell targeted therapy with mTOR inhibitor RAD001 and hedgehog signalling inhibitor Cyclopamine reverts chemoresistance towards 5-Fluorouracil in human pancreatic carcinoma cells

Abstract

Background: In the present study, we aimed to identify and to eliminate putative cancer stem cells (CSC) within different human pancreatic cancer cell lines as well as within 5-Fluorouracil-resistant pancreatic cancer cell lines established in our laboratory. Methods: Pancreatic CSC were identified and characterized by flow cytometry, using Hoechst 33342 dye staining. CSC from 5-FU-resistant cell lines were isolated by high-speed flow cell sorting (MoFlo) and the CSC self-renewal pathways were further analyzed by RT-PCR and Western blotting. Isolated CSC within 5-FU-resistant cell lines were exposed to the cancer stem cell targeted therapeutics (RAD001, Cyclopamine) and stained for Propidium iodide and BrdU. Furthermore, isolated CSC as well as non-tumorigenic cancer cells within 5-FU-resistant cell lines were orthotopically xenografted in nude mice and the efficacy of stem-cell-targeted therapy in combination with 5-FU was investigated. Results: Flow cytometry analysis revealed a significantly high amount of CSC in all chemotherapy-resistant cell lines. Combined 5-Fluorouracil IC50 and cancer stem cell targeted therapy resulted in the reduction of tumor cell proliferation independently on 5-FU-chemoresistance status, as detected by Propidium iodide and BrdU staining. In vivo the combination of 5-FU with cancer stem cell targeted therapeutics RAD001 or Cyclopamine significantly decreased the tumorigenic potential of CSC derived from 5-FU-resistant cell lines and dramatically reduced primary pancreatic tumor volumes and weight. Conclusions: Our results demonstrate that chemotherapy-resistant cancer cells contain the increased CSC population (as compared to their parental sensitive cells) that is highly resistant to standard chemotherapy but not towards CSC-targeted therapy in vitro and in vivo. The further characterization of such cells might therefore lead to the development of new molecular and pharmaceutical therapeutics and better anti-cancer strategies.