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. The study found that GrA was highly active against pancreatic CSCs. Moreover, GrA showed synergism with gemcitabine in suppressing cancer cell proliferation.
GrA down-regulated the expression levels of CD133, CD44, and CD47 in addition, CD47 re-distribution was observed on cell surface. GrA induced ultrastructural changes, such as the decrease of microvilli-like protrusions on cell surface membrane and the swelling of mitochondria. GrA at 0.1 μM or lower did not cause hemolysis.
Compared on equal concentrations, the efficacy of GrA was stronger than that of Sal. 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. GrA and Sal both inhibited cancer cell proliferation. GrA at 0.05 μM caused tumorspheres disintegration and decrease in number of pancreatic cancer BxPC-3 and MIA PaCa-2 cells. Immunofluorescence staining was performed to observe CD47 re-distribution. Western blot analysis was used to determine relative protein expression levels. Scanning and transmission electron microscopy was used to observe ultrastructural morphological changes on cell membrane surface and mitochondria, respectively. Flow cytometry was performed to detect cell apoptosis and mitochondrial membrane potential. CCK-8 assay was used to detect pancreatic cancer cell proliferation capability. In vitro hemolysis assay was determined to test the borderline concentration of GrA. Tumorsphere formation assay was performed to assess pancreatic CSCs self-renewal potential. This study investigated the effect of GrA on pancreatic CSCs and the mechanism. Whether GrA owns the potential as a therapeutic drug for CSCs still remains unknown. As reported, the ionophore antibiotic salinomycin (Sal) has been proved to kill CSCs effectively. 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. Pancreatic cancer stem cells (CSCs), a special population of cells, renew themselves infinitely and resist to various treatment. Coordinating fate-promoting and -suppressing circuits therefore averts deconstruction of a multifate system into a monopotent system and maintains critical progenitor heterogeneity and functionality. During embryogenesis, the suppressing mechanism dominated in enhancer mutant progenitors, thus yielding progenitors with a predominant monocytic differentiation potential. By increasing GATA2 expression, the enhancer instigates a fate-promoting mechanism while abrogating an innate immunity–linked, fate-suppressing mechanism. Using multiomics and single-cell analyses, we demonstrated that the enhancer orchestrates a balance between pro- and anti-fate circuitry in single cells. Deletion of the murine Gata2−77 enhancer, with a human equivalent that causes leukemia, downregulates the transcription factor GATA2 and blocks progenitor differentiation into erythrocytes, megakaryocytes, basophils, and granulocytes, but not macrophages. Using the hematopoietic system, we analyzed the relative importance of cell fate–promoting mechanisms versus negating fate-suppressing mechanisms to engineer progenitor cells with multilineage differentiation potential. Stem and progenitor cell fate transitions constitute key decision points in organismal development that enable access to a developmental path or actively preclude others. CD47 is the first known gene common to all cancers and is a target for cancer immunotherapy. With these, we discovered that CD47 is an upregulated gene in all human cancers and is a "don't eat me" signal blocking it with antibodies leads to cancer cell phagocytosis. In human acute myelogenous leukemia we showed that all preleukemic mutations occur in HSCs, and determined their order the final mutations occur in a multipotent progenitor derived from the preleukemic HSC clone. Our studies of circulating competing germline stem cells in colonial protochordates led us to document competing HSCs. The induction of immune tolerance with HSCs has led to isolation of other tissue-specific stem cells for regenerative medicine. This led to studies of the thymus as the site of maturation of T cells, which led to the discovery, isolation, and clinical transplantation of purified hematopoietic stem cells (HSCs). I started research in high school, experimenting on immunological tolerance to transplantation antigens.
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