Title: Enoxacin and Bis-enoxacin Alter Regulatory Exosomes Released by Cancer Cells
Demyana Azer (Presenter)
University of Florida College of Dentistry
taylor Vracar, University of Florida College of Dentistry
Jian Zuo, University of Florida College of Dentistry
Christy Mikhael, University of Florida College of Dentistry
Sophia Holliday, University of Florida College of Dentistry
Dontreyl Holsey, University of Florida College of Dentistry
Lindsay VonMoss, University of Florida College of Dentistry
Edward Chan, University of Florida College of Dentistry
Lexie Holliday, University of Florida College of Dentistry
Objectives: Enoxacin and its bisphosphonate ester, bis-enoxacin, have recently emerged as promising therapeutic agents for the treatment of cancer and bone disease. However, very high doses are required to affect cancer growth and survival in vitro. Here, we have sought effects on 4T1 murine breast cancer cells at lower doses that might explain the efficacy of these agents reported in animal models of cancer.
Methods: 4T1 cells were stained with an anti-GW182 antibody, which detects GW bodies, sites associated with microRNA activity. By epifluorescence microscopy, the number of GW bodies per nucleus was determined. 4T1 cells were treated with low dose (50 μM) enoxacin, bis-enoxacin, and vehicle, exosomes were harvested, and tested for their ability to regulate osteoclast and osteoblast formation in calcitriol-stimulated mouse marrow (C-SMM). Quantitative Real Time PCR determined the relative levels of selected microRNAs in cells and in exosomes.
Results: No changes in cell proliferation were detected in the presence of 50 µM enoxacin or bis-enoxacin, but proliferation was reduced at higher concentrations. Both enoxacin and bis-enoxacin (50 µM) triggered increases in the number of GW bodies compared to vehicle (p < 0.0001). Exosomes (5 x 107) from control 4T1 cells stimulated osteoclast and osteoblast differentiation in C-SMM; exosomes from enoxacin and bis-enoxacin treated cells significantly inhibited bone cell maturation. Bis-enoxacin and enoxacin (50 µM) modestly increased cellular levels of miR-146a, let-7b, miR-290, miR-214-3p and miR-689 in 4T1 cells. However, enoxacin and bis-enoxacin significantly reduced levels of miR-146a and let-7b in exosomes released from treated cells. In striking contrast, levels of miR-214-3p in treated exosomes increased 20-30-fold.
Conclusions: Enoxacin and bis-enoxacin cause exosomes released by 4T1 murine breast cancer cells to inhibit osteoclast and osteoblast formation. This could reduce cancer cell’s invasion of bone, which requires bone turnover. Increased levels of miR-214-3p in the exosomes could explain this change.