Enoxacin and its bone-seeking bisphosphonate derivative, bis-enoxacin, have recently captured attention as potential therapeutic agents for the treatment of cancer and bone disease. No differences in growth or survival of 4T1 murine breast cancer cells were detected at a concentration of 50 µM of enoxacin or bis-enoxacin. Growth was perturbed at higher concentrations. Both 50 µM enoxacin and bis-enoxacin stimulated increases in the number of GW/Processing bodies, but there were minimal changes in microRNA levels. Extracellular vesicles (EVs) released from 4T1 cells treated with 50 µM enoxacin or 50 µM bis-enoxacin stimulated proliferation of RAW 264.7 cells, and both significantly inhibited osteoclastogenesis in calcitriol-stimulated mouse marrow. EVs from 4T1 cells treated with enoxacin and bis-enoxacin displayed small reductions in the amount of microRNA (miR)-146a-5p and let-7b-5p. In marked contrast, miR-214-3p, which has been shown to regulate bone remodeling, was increased 22-fold and 30-fold respectively. We conclude that enoxacin and bis-enoxacin trigger the release of EVs from 4T1 cancer cells that inhibit osteoclastogenesis.

The (pro)renin receptor (PRR) is a multifunctional integral membrane protein that serves as a component of the vacuolar H+-ATPase (V-ATPase) and also activates (pro)renin. We recently showed that full-length PRR, found as part of a V-ATPase sub-complex, is abundant in extracellular vesicles shed by osteoclasts. Here, we tested whether these extracellular vesicles stimulate (pro)renin. Extracellular vesicles isolated from the conditioned media of RAW 264.7 osteoclast-like cells or primary osteoclasts were characterized and counted by nanoparticle tracking. Immunoblotting confirmed that full-length PRR was present. Extracellular vesicles from osteoclasts dose-dependently stimulated (pro)renin activity, while extracellular vesicles from 4T1 cancer cells, in which we did not detect PRR, did not activate (pro)renin. To confirm that the ability of extracellular vesicles from osteoclasts to stimulate (pro)renin activity was due to the PRR, the “handle region peptide” from the PRR, a competitive inhibitor of PRR activity, was tested. It dose-dependently blocked the ability of extracellular vesicles to stimulate the enzymatic activity of (pro)renin. In summary, the PRR, an abundant component of extracellular vesicles shed by osteoclasts, stimulates (pro)renin activity. This represents a novel mechanism by which extracellular vesicles can function in intercellular regulation, with direct implications for bone biology.

Extracellular vesicles (EVs) are 30-150 nm in diameter and are released by cells into the extracellular environment. They facilitate intercellular communication and may be involved in cell survival. Although serum-dependent cells are not obviously affected by depletion of EVs from the serum, we previously reported that cells in serum that contain EVs were more resistant to the drugs, enoxacin and bis-enoxacin, compared to cells in serum without EVs. This change in resistance led us to examine whether EVs are sufficient to maintain serum dependent cells in vitro. Here, we tested the effect of EVs on the survival RAW 264.7 cells and on 4T1 murine breast cancer cells. EVs were isolated by differential centrifugation. EVs were counted over various time periods. Cells were grown in alpha or Dulbecco’s minimal essential media supplemented with FBS, EVs from various sources, or with no supplements. Cell growth was determined by cell counts. In media without supplementation, the RAW 264.7 cells and 4T1 cells died by three days. Media supplemented with EVs from FBS allowed the cells to survive in the absence of FBS for one week or more.  Being able to perform experiments on cells supported by EVs will simplify the interpretation of experiments. Efforts are underway to determine their mechanism by which EVs support cell survival.