sirolimus and perfosfamide

Drug interactions may dictate the failure or success of a treatment. Patients undergoing hematopoietic stem cell transplantation (HSCT) are exposed to various types of drugs, and understanding how these drugs interact is of the utmost importance. The pharmacokinetics of busulfan, melphalan, and cyclophosphamide, drugs commonly used for HSCT, are known to be affected by a variety of other drugs with differing molecular structures. We hypothesized that these structurally unrelated drugs affect the transport of DNA-alkylating agents. To test this hypothesis, we developed a flow cytometry assay that used 5-carboxyfluorescein diacetate acetoxymethyl ester, which is cleaved by nonspecific intracellular esterases to 5-carboxyfluorescein (5-CF), a fluorescent ligand for the drug transporter MRP1. A decreased 5-CF efflux in the presence of a test compound suggests competitive inhibition. We demonstrated that chlorambucil, 4-hydroperoxycyclophosphamide, ketoconazole, ethacrynic acid, everolimus, and sirolimus strongly inhibited 5-CF efflux in lymphoma and leukemia cell lines. The efflux of these drugs partially depends on the glutathione (GSH) level, and their cytotoxicity is synergistic with inhibited GSH synthesis. This is consistent with the hypothesis that their GSH-conjugated products are ligands of a common cellular drug transporter. Our results may explain clinical observations on the effects of various drugs on the pharmacokinetics and pharmacodynamics of alkylating agents, and the assay may be used to deduce interaction mechanisms of drugs transported by a common system.

Topics: Antineoplastic Agents, Alkylating; Biological Transport; Busulfan; Cell Line, Tumor; Cell Survival; Chlorambucil; Cyclophosphamide; Drug Interactions; Ethacrynic Acid; Everolimus; Flow Cytometry; Fluoresceins; Humans; Ketoconazole; Melphalan; Multidrug Resistance-Associated Proteins; Reproducibility of Results; Sirolimus

Bone marrow stromal cell function is a critical influence on hematopoietic reconstitution following progenitor or stem cell transplantation. Stromal cells support hematopoietic cell migration, survival, and proliferation. We have previously reported that stromal cell matrix metalloproteinase-2 (MMP-2) is necessary for optimal support of pro-B-cell chemotaxis through its regulation of stromal cell-derived factor-1 (CXCL12) release. Following exposure to the topoisomerase II inhibitor, etoposide (VP-16), stromal cell MMP-2 protein expression is reduced. The current study investigated the mechanism by which VP-16 may alter translation of MMP-2 in bone marrow stromal cells.. Bone marrow stromal cells were exposed to chemotherapeutic agents etoposide, melphalan, and 4-hydroperoxycyclophosphamide (4HC) and evaluated for MMP-2 expression by enzyme-linked immunosorbent assay and support of pro-B-cell chemotaxis by chemotaxis assay. Western blot analyses were completed to evaluate phosphorylation of stromal cell translational regulatory proteins 4E binding protein-1 (4EBP-1), P70(S6K), and S6 or MMP-2 in the presence of chemotherapy, or the chemical inhibitors rapamycin or LY294002.. Rapid dephosphorylation of 4EBP-1, P70(S6K), and S6 following VP-16 exposure was observed, consistent with blunted translational efficiency. We also observed that inhibition of stromal cell mammalian target of rapamycin with rapamycin, or phosphatidylinositol 3 kinase with LY294002, resulted in inhibition of stromal cell MMP-2 protein. In addition we found that the chemotherapeutic agents melphalan and 4HC disrupt bone marrow stromal cell MMP-2 protein expression and support of chemotaxis.. These data suggest that one mechanism by which chemotherapy may alter stromal cells of the bone marrow microenvironment is through disrupted translation of proteins.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antimetabolites, Antineoplastic; Bone Marrow Cells; Cell Cycle Proteins; Cell Line; Cyclophosphamide; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Etoposide; Humans; Matrix Metalloproteinase 2; Melphalan; Mice; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Stromal Cells; Structure-Activity Relationship; TOR Serine-Threonine Kinases