Effects of mTOR inhibitors and cytoskeletal-directed agents alone and in combination against normal and neoplastic hematopoietic cells in vitro

The mechanistic target of rapamycin (mTOR) controls cell growth and enlargement and has been found to be aberrant in a wide variety of malignancies. Although mTOR is already an attractive antineoplastic target, overexpression or aberrant expression of mTOR may also provide an opportunity to further increase the size differential between malignant and normal cells, providing an opportunity to amplify and exploit cell size differences between neoplastic cells and their normal counterparts using physiochemical treatment modalities. Therefore, this study sought to quantify the concentration response and time course effects of rapamycin on cell cycle entry, cell enlargement, and cell proliferation in U937 human monocytic leukemia and human hematopoietic stem cells (hHSCs). In addition, the effects of combination treatment with mTOR inhibitors (rapamycin, everolimus, and temsirolimus) and cytoskeletal-directed agents (cytochalasin B and vincristine) in leukemic cells (U937, THP1, K562, Molt-4, and L1210) were assessed for potential drug synergy. While both U937 cells and hHSCs exhibited a marked reduction in cell volume, U937 cells were able to proliferate in the presence of rapamycin ranging from 0.5 nM to 10 μM (10,000 nM), whereas hHSCs were able to proliferate only at lower concentrations, and were completely inhibited from proliferation by 8 nM rapamycin. These effects were observed with as little as 0.5 nM rapamycin, demonstrating the profound affinity the compound has for FK-binding protein 12 (FKBP12), which subsequently forms the FKBP12/rapamycin complex to inhibit mTOR. Rapamycin continued to exert effects on cell size and proliferation even at 10 μM, without producing marked cytotoxicity. Although cytochalasin B and vincristine were unable to substantially enlarge rapamycin-treated leukemia cells, it appears that rapamycin and its associated analogs everolimus and temsirolimus have notable synergistic potential with microfilament-disrupting cytochalasin B and microtubule-disrupting vincristine as assessed by comparative effects on cell growth, annexin V staining, IC(30) isobolograms, and Chou-Talalay statistics. These observations indicate a potentially novel therapeutic rationale for hematological malignancies and for other cancers to elicit the preferential destruction of neoplastic cells that aberrantly express mTOR.