The destruction of cells using the mechanical activation of magnetic nanoparticles with low-frequency magnetic fields constitutes a recent and interesting approach in cancer therapy. Here, we showed that superparamagnetic iron oxide nanoparticles as small as 6 nm were able to induce the death of pancreatic cancer-associated fibroblasts, chosen as a model. An exhaustive screening of the amplitude, frequency, and type (alternating vs. rotating) of magnetic field demonstrated that the best efficacy was obtained for a rotating low-amplitude low-frequency magnetic field (1 Hz and 40 mT), reaching a 34% ratio in cell death induction; interestingly, the cell death was not maximized for the largest amplitudes of the magnetic field. State-of-the-art kinetic Monte-Carlo simulations able to calculate the torque undergone by assemblies of magnetic nanoparticles explained these features and were in agreement with cell death experiments. Simulations showed that the force generated by the nanoparticles once internalized inside the lysosome was around 3 pN, which is in principle not large enough to induce direct membrane disruption. Other biological mechanisms were explored to explain cell death: the mechanical activation of magnetic nanoparticles induced lysosome membrane permeabilization and the release of the lysosome content and cell death was mediated through a lysosomal pathway depending on cathepsin-B activity. Finally, we showed that repeated rotating magnetic field exposure halted drastically the cell proliferation. This study established a proof-of-concept that ultra-small nanoparticles can disrupt the tumor microenvironment through mechanical forces generated by mechanical activation of magnetic nanoparticles upon low-frequency rotating magnetic field exposure, opening new opportunities for cancer therapy.

A region between residues 38 and 42 of the human cholecystokinin-A (CCK-A) receptor was shown to be involved in the binding of CCK but not in that of JMV 179 and JMV 180, two peptides closely related to CCK (Kennedy, K., Escrieut, C., Dufresne, M., Clerc, P., Vaysse, N., and Fourmy, D. (1995) Biochem. Biophys. Res. Commun. 213, 845-852). In the present study, we have identified the residues of both the receptor and the ligand responsible for this differential binding. Residues Trp-39 and Gln-40 of the receptor were crucial for binding of the C-terminal nonapeptide of CCK as W39F and Q40N mutants demonstrated parallel decreases in both affinity and potency to induce accumulation of inositol phosphates (12.9- and 20.9-fold). The W39F and Q40N mutant receptors bound CCK analogues modified at their C-terminal end, including JMV 179 and JMV 180, as well as the C-terminal amidated heptapeptide of CCK, with identical affinities to the wild-type receptor. In contrast, W39F and Q40N mutants bound CCK octapeptide with the same decreased affinity as the CCK nonapeptide. The modeling of the CCK-A receptor and the docking of the peptide agonists [Thr,Nle]CCK9 and CCK-8 indicated that their N terminus was connected to the receptor through a strong bond network involving Trp-39 and Gln-40 thus confirming experimental data. These first molecular data identifying the agonist binding site of the human CCK-A receptor represent an important step toward the complete delineation of the agonist binding site and the understanding of the molecular mechanisms that govern differential activation of this receptor by CCK-related peptides.

Despite decades of effort in understanding pancreatic ductal adenocarcinoma (PDAC), there is still a lack of innovative targeted therapies for this devastating disease. Herein, we report the expression of apelin and its receptor, APJ, in human pancreatic adenocarcinoma and its protumoral function. Apelin and APJ protein expression in tumor tissues from patients with PDAC and their spatiotemporal pattern of expression in engineered mouse models of PDAC were investigated by immunohistochemistry. Apelin signaling function in tumor cells was characterized in pancreatic tumor cell lines by Western blot as well as proliferation, migration assays and in murine orthotopic xenograft experiments. In premalignant lesions, apelin was expressed in epithelial lesions whereas APJ was found in isolated cells tightly attached to premalignant lesions. However, in the invasive stage, apelin and APJ were co-expressed by tumor cells. In human tumor cells, apelin induced a long-lasting activation of PI3K/Akt, upregulated β-catenin and the oncogenes c-myc and cyclin D1 and promoted proliferation, migration and glucose uptake. Apelin receptor blockades reduced cancer cell proliferation along with a reduction in pancreatic tumor burden. These findings identify the apelin signaling pathway as a new actor for PDAC development and a novel therapeutic target for this incurable disease.