Inhibition of angiogenesis- and inflammation-inducing factors in human colon cancer cells in vitro and in ovo by free and nanoparticle-encapsulated redox dye, DCPIP

<p>Abstract</p> <p>Background</p> <p>The redox dye, DCPIP, has recently shown to exhibit anti-melanoma activity <it>in vitro </it>and <it>in vivo</it>. On the other hand, there is increasing evidence that synthetic nanoparticles can serve as highly efficient carriers of drugs and vaccines for treatment of various diseases. These nanoparticles have shown to serve as potent tools that can increase the bioavailability of the drug/vaccine by facilitating absorption or conferring sustained and improved release. Here, we describe results on the effects of free- and nanoparticle-enclosed DCPIP as anti-angiogenesis and anti-inflammation agents in a human colon cancer HCT116 cell line <it>in vitro</it>, and in induced angiogenesis <it>in ovo</it>.</p> <p>Results</p> <p>The studies described in this report indicate that (a) DCPIP inhibits proliferation of HCT116 cells <it>in vitro</it>; (b) DCPIP can selectively downregulate expression of the pro-angiogenesis growth factor, VEGF; (c) DCPIP inhibits activation of the transcriptional nuclear factor, NF-κB; (d) DCPIP can attenuate or completely inhibit VEGF-induced angiogenesis in the chick chorioallantoic membrane; (e) DCPIP at concentrations higher than 6 μg/ml induces apoptosis in HCT116 cells as confirmed by detection of caspase-3 and PARP degradation; and (f) DCPIP encapsulated in nanoparticles is equally or more effective than free DCPIP in exhibiting the aforementioned properties (a-e) in addition to reducing the expression of COX-2, and pro-inflammatory proteins IL-6 and IL-8.</p> <p>Conclusions</p> <p>We propose that, DCPIP may serve as a potent tool to prevent or disrupt the processes of cell proliferation, tissue angiogenesis and inflammation by directly or indirectly targeting expression of specific cellular factors. We also propose that the activities of DCPIP may be long-lasting and/or enhanced if it is delivered enclosed in specific nanoparticles.</p

Nanoparticle-based delivery of siDCAMKL-1 increases microRNA-144 and inhibits colorectal cancer tumor growth via a Notch-1 dependent mechanism

<p>Abstract</p> <p>Background</p> <p>The development of effective drug delivery systems capable of transporting small interfering RNA (siRNA) has been elusive. We have previously reported that colorectal cancer tumor xenograft growth was arrested following treatment with liposomal preparation of siDCAMKL-1. In this report, we have utilized Nanoparticle (NP) technology to deliver DCAMKL-1 specific siRNA to knockdown potential key cancer regulators. In this study, mRNA/miRNA were analyzed using real-time RT-PCR and protein by western blot/immunohistochemistry. siDCAMKL-1 was encapsulated in Poly(lactide-<it>co</it>-glycolide)-based NPs (NP-siDCAMKL-1); Tumor xenografts were generated in nude mice, treated with NP-siDCAMKL-1 and DAPT (γ-secretase inhibitor) alone and in combination. To measure <it>let-7a </it>and <it>miR-144 </it>expression <it>in vitro</it>, HCT116 cells were transfected with plasmids encoding the firefly luciferase gene with <it>let-7a </it>and <it>miR-144 </it>miRNA binding sites in the 3'UTR.</p> <p>Results</p> <p>Administration of NP-siDCAMKL-1 into HCT116 xenografts resulted in tumor growth arrest, downregulation of proto-oncogene c-Myc and Notch-1 via <it>let-7a </it>and <it>miR-144 </it>miRNA-dependent mechanisms, respectively. A corresponding reduction in <it>let-7a </it>and <it>miR-144 </it>specific luciferase activity was observed <it>in vitro</it>. Moreover, an upregulation of EMT inhibitor <it>miR-200a </it>and downregulation of the EMT-associated transcription factors ZEB1, ZEB2, Snail and Slug were observed <it>in vivo</it>. Lastly, DAPT-mediated inhibition of Notch-1 resulted in HCT116 tumor growth arrest and down regulation of Notch-1 via a <it>miR-144 </it>dependent mechanism.</p> <p>Conclusions</p> <p>These findings demonstrate that nanoparticle-based delivery of siRNAs directed at critical targets such as DCAMKL-1 may provide a novel approach to treat cancer through the regulation of endogenous miRNAs.</p

A schematic of two-state energy level diagram depicting the thermodynamic and mechanical stabilization of M-crystallin upon Ca²⁺ binding.

<p>Ligand binding not only stabilizes the native state (N) by ΔG∼11.4 kcal/mol but also reduces the unfolding potential width (Δx_u) from 0.55 nm to 0.38 nm. Estimates of unfolding transition state (TS) energy barriers (ΔG^‡) are also indicated. See text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094513#pone-0094513-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094513#pone-0094513-t002" target="_blank">2</a> for more details.</p

Structure and 2D topology diagram of two β-sandwich proteins with Greek key motifs used in this study.

<p>The pulling direction used in the single-molecule force spectroscopy (SMFS) experiments is shown by arrows. (<i>A</i>) NMR structure of I27 (PDB ID: 1TIT). Terminal β-strands A′ and G are directly connected by H-bonding, shearing this “mechanical-clamp” results in the mechanical unfolding of the protein. The rupture of H-bonds between A and B strands constitutes the less stable mechanical intermediate. (<i>B</i>) 2D topology diagram of I27. The five-stranded (BCDEF) ‘double’ Greek key (3,2)₃ formed by overlapping (3,1)_N and (2,2)_C Greek keys (as defined by Hutchinson and Thornton <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094513#pone.0094513-Hutchinson1" target="_blank">[53]</a>). (<i>C</i>) NMR structure of M-crystallin (PDB ID: 2K1W) bound to two Ca²⁺ ions (shown as black spheres). The terminal β-strands A and H are not directly bonded and they need to be “peeled” away from each other to unfold the protein. (<i>D</i>) 2D topology diagram of M-crystallin showing the two (3,1)_C Greek keys formed by ABCD and EFGH. In both cases, the backbone H-bonding around the terminal strands is shown.</p

Mechanical unfolding of (M-crystallin)₈ using SMFS.

<p>(<i>A</i>) A pair of typical single-molecule force extension (FX) traces of apo protein (black). A series of equidistant force peaks in FX traces indicating the sequential unfolding of individual M-crystallin units in the octamer during the mechanical stretching (pulling speed is 1000 nm/s). The unfolding force peaks in sawtooth pattern are fitted with WLC model (grey). The contour length change is ∼29 nm and the unfolding force is ∼90 pN. Histograms of contour length change fitted to Gaussian distribution (<i>B</i>) and unfolding force (<i>C</i>) are shown.</p

Mechanical unfolding of Ca²⁺ bound (M-crystallin)₈.

<p>(<i>A</i>) A pair of FX traces obtained in the presence of 10 mM Ca²⁺. The contour length change upon unfolding is ∼29 nm and the unfolding force is ∼125 pN in the sawtooth curves (black). WLC fits are also shown (grey). (<i>B</i>) The unfolding force histograms of (M-crystallin)₈ in holo (filled) and apo (unfilled) show that Ca²⁺ stabilizes the protein mechanically by ∼35 pN.</p

[Ca²⁺] dependent unfolding forces of (M-crystallin)₈.

<p>The unfolding force histograms at various Ca²⁺ concentrations are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094513#pone.0094513.s001" target="_blank">Figure S7 in File S1</a>. The increase in unfolding force in two phases is consistent with two Ca²⁺ binding sites (see text for more details).</p

Thermodynamic parameters of two Ca²⁺ binding sites in M-crystallin monomer and octamer.

<p>Thermodynamic parameters of two Ca²⁺ binding sites in M-crystallin monomer and octamer.</p