Enhancement of the uptake and cytotoxic activity of doxorubicin in cancer cells by novel cRGD-semipeptide-anchoring liposomes.

Novel liposemipeptides hanging cyclic azabicycloalkane-RGD or aminoproline-RGD terminals were synthesized and incorporated into liposomal nanoparticles cAba/cAmpRGD-LNP5 3C/3D. Liposomes with similar composition and lacking semipeptide conjugates were constructed for comparison (LNP, 3A), and physical encapsulation of the anticancer doxorubicin drug in both targeted and untargeted liposomes was accomplished. Microstructural analysis performed by dynamic light scattering (DLS), small-angle neutron scattering (SANS), and electron paramagnetic resonance (EPR) revealed that the conjugated nanoparticles presented an average size of 80 nm and were constituted by 5 nm thick unilamellar liposome bilayer. Flow cytometry and fluorescent microscopy studies showed that 3C-DOXO and 3D-DOXO efficiently delivered the drug into the nuclei of both quiescent and proliferating cells even in a high serum concentration environment. The uptake of doxorubicin when carried by liposomes was faster than that of the free drug, and 30 min incubation was sufficient to load cell nuclei with doxorubicin. Targeted liposomes significantly induced cell death of human breast adenocarcinoma MCF7 cells (IC50 = 144 nM, 3C-DOXO; IC50 = 274 nM, 3D-DOXO), about 2- to 6-fold more potent than free doxorubicin or 3A-DOXO controls (IC50 = 527 and 854 nM, respectively). These results suggest that cAba/cAmpRGD liposomal nanoparticles hold promise for the rapid and efficient delivery of chemotherapeutic agents to αVß3-expressing tumor cells.

Down-regulation of HOXA4, HOXA7, HOXA10, HOXA11 and MEIS1 during monocyte-macrophage differentiation in THP-1 cells.

The translocation t(9;11)(p22;q23) generates the MLL-AF9 oncogene and is commonly associated with monocytic acute myeloid leukemia (AML-M5; FAB-classification). For the oncogenicity of MLL-AF9, the (over)expression of several other genes, including selected HOXA cluster genes as well as MEIS1 (a HOX cofactor), is required. We previously showed that the down-regulation of MLL-AF9 expression is not obligatory for monocyte-macrophage maturation in AML-M5 cells carrying t(9;11)(p22;q23). In this study, we analyzed the expression patterns of HOXA4, 5, 6, 7, 9, 10 and 11 (defined as 'HOXA-code' genes) and MEIS1 by semiquantitative RT-PCR during the monocyte-macrophage differentiation induced by phorbol 12-myristate 13-acetate (PMA) in THP-1 cells carrying t(9;11)(p22;q23) and expressing MLL-AF9. The analyses were performed in THP-1 cells expressing MLL-AF9 even after PMA treatment. The results showed that all the analyzed genes were expressed in untreated THP-1 cells. After the induction of differentiation, we observed a down-regulation of HOXA4, 7, 10, 11 and MEIS1, an up-regulation of HOXA6, and no significant variation in the expression of HOXA5 and 9. These data indicate that the expression of most HOXA-code genes, as well as MEIS1, could be implicated in the differentiation blockage observed in MLL-AF9-related leukemias.