The ionophore antibiotic gramicidin A inhibits pancreatic cancer stem cells associated with CD47 down-regulation.

ABSTRACT

BACKGROUND: Pancreatic cancer stem cells (CSCs), a special population of cells, renew themselves infinitely and resist to various treatment. Gramicidin A (GrA), an ionophore antibiotic derived from microorganism, can form channels across the cell membrane and disrupt cellular ionic homeostasis, leading to cell dysfunction and death. As reported, the ionophore antibiotic salinomycin (Sal) has been proved to kill CSCs effectively. Whether GrA owns the potential as a therapeutic drug for CSCs still remains unknown. This study investigated the effect of GrA on pancreatic CSCs and the mechanism. METHODS: Tumorsphere formation assay was performed to assess pancreatic CSCs self-renewal potential. In vitro hemolysis assay was determined to test the borderline concentration of GrA. CCK-8 assay was used to detect pancreatic cancer cell proliferation capability. Flow cytometry was performed to detect cell apoptosis and mitochondrial membrane potential. Scanning and transmission electron microscopy was used to observe ultrastructural morphological changes on cell membrane surface and mitochondria, respectively. Western blot analysis was used to determine relative protein expression levels. Immunofluorescence staining was performed to observe CD47 re-distribution. RESULTS: GrA at 0.05 µM caused tumorspheres disintegration and decrease in number of pancreatic cancer BxPC-3 and MIA PaCa-2 cells. GrA and Sal both inhibited cancer cell proliferation. The IC50 values of GrA and Sal for BxPC-3 cells were 0.025 µM and 0.363 µM; while for MIA PaCa-2 cells were 0.032 µM and 0.163 µM, respectively. Compared on equal concentrations, the efficacy of GrA was stronger than that of Sal. GrA at 0.1 µM or lower did not cause hemolysis. GrA induced ultrastructural changes, such as the decrease of microvilli-like protrusions on cell surface membrane and the swelling of mitochondria. GrA down-regulated the expression levels of CD133, CD44, and CD47; in addition, CD47 re-distribution was observed on cell surface. Moreover, GrA showed synergism with gemcitabine in suppressing cancer cell proliferation. CONCLUSIONS: The study found that GrA was highly active against pancreatic CSCs. It indicates that GrA exerts inhibitory effects against pancreatic CSCs associated with CD47 down-regulation, implying that GrA might play a positive role in modulating the interaction between macrophages and tumor cells.