8 research outputs found
RPARPAR PAs share the same initial internalization pathway and diverge at later time points.
<p>Confocal micrographs at different time points of chase (pulse: 1 hour) revealed high extent of co-localization (yellow) between PA <b>8</b> (green) and PA <b>4</b> (red) when both PAs were chased for the same time (A,D). When PAs were chased for different time points, co-localization extent was decreased and was dependent on which PA was chased (B,C,E and see text for details). Nuclei stain (blue): Hoechst 33342. Scale Bars: 20 ĀµM.</p
Proposed model for internalization and trafficking of RPARPAR PAs.
<p>Both PAs bind the plasma membrane and are taken up primarily via clathrin-independent pathways (solid line). Both PAs are recycled to the plasma membrane but diC<sub>16</sub> PAs remains anchored to it (dashed line), whereas DSPE-PEG<sub>2000</sub> PAs is washed away (dotted line). The diC<sub>16</sub> tail is directed to lysosomes while DSPE-PEG<sub>2000</sub> is trafficked both to lysosomes and CTb-containing organelles.</p
The non-peptidic part determines PA retention in PPC-1 cells.
<p>(A) Pulse-chase experiments were performed with 10 ĀµM PAs in PPC-1 cells with 1-hour pulse and different chase periods. Cell association of fluorescent PAs was determined and normalized (value of 1 corresponds to no chase). PPC-1-associated levels of PA <b>2</b> remained constant over 3 hours and decreased to half over 24 hours, whereas PA <b>4</b> levels decreased to 25% and 20% at 1 and 3 hours, respectively. PA <b>2</b> fluorescence values/well remained constant during a 24-hour chase (inset). Average values and standard deviations (nā=ā3) are presented. (B) Confocal micrographs of the two PAs at different chase points revealed similar intracellular patterns. Scale bars: 20 ĀµM.</p
diC<sub>16</sub>-Rho-RPARPAR (2) is eventually trafficked to lysosomes while DSPE-PEG<sub>2000</sub>-Rho-RPARPAR (4) is additionally found in CTb-positive vesicles after 24 hour incubation.
<p>Confocal micrographs or RPARPAR PAs following a 1-hour pulse and 24-hour chase in PPC-1 cells treated either with CTb for 1 hour (A, B) or stained with lysotracker (C, D) showed differences in PA localization. PA <b>2</b> co-localized with lysosomes (C) but not CTb (A). PA <b>4</b> on the other hand, co-localized with both CTb (B) and lysosomes (D). Scale bars: 20 Āµm.</p
Internalization of PAs requires cholesterol and is not inhibited by chlorpromazine or amiloride.
<p>(A) PPC-1 cell association of PA <b>2</b> and PA <b>4</b> in presence of MĪ²CD (cholesterol depletion agent) was reduced compared to controls Amiloride did not affect cell-association or internalization of PAs, whereas a low (10%) inhibition of cell-association was noted for PA <b>2</b> in presence of chlorpromazine. Average values and SEM are presented. (B) Qualitatively, the ratio of PAs localized on the plasma mebrane to the PAs found in intracellular vesicles was higher in MĪ²CD treated-cells indicating that internalization was impaired. PA <b>2</b> associated with PPC-1 plasma membrane at 4Ā°C but was not internalized after 1 hour. Scale bars: 20 Āµm.</p
diC16-Rho-RPARPAR (2) co-localizes with CTb following 1-hour incubation in PPC-1 cells and is internalized in a dynamin-2-independent manner.
<p>(AāD) PPC-1 cells incubated with PA <b>2</b> (10 ĀµM) co-localized with CTb (A; yellow indicates co-localization) but not with mitochondria (B; Mitotracker) or lysosomes (C; Lysotracker). A small fraction of intracellular vesicles were positive for both PA <b>2</b> and transferrin (D). PPC-1 cells were transfected with EGFP-coupled dynamin-2 (E) or a dominant negative dynamin-2 mutant (G). 24 hours after transfection, cells were incubated for 1 hour with 10 ĀµM PA <b>2</b>. Absence of co-localization with dynamin-2 (E) and internalization in PPC-1 cells expressing the dominant negative dynamin-2 mutant (G) indicate that PA <b>2</b> enters cells in a dynamin-2-independent manner. Nuclei stain (blue): Hoechst 33342; Scale bars: 20 Āµm.</p
RPARPAR PAs internalize in PPC-1 cells in vitro to a higher extent than the peptide.
<p>(A) Chemical structures of fluorescent peptides, peptide amphiphiles and control amphiphiles used in this study. (B) Quantification of cell-associated peptides, PAs and control amphiphile (concentration: 10 ĀµM) after 1-hour incubation with PPC-1 cells revealed higher association for both types of RPARPAR PAs (<b>2</b>, <b>4</b>) compared to peptide (<b>1</b>). Cell association was similar for RPARPAR PAs with carboxylated (<b>2</b>, <b>4</b>) or amidated C-terminus (<b>3</b>, <b>5</b>). Control amphiphile <b>6</b> and PA <b>7</b> showed lower association compared to RPARPAR PAs (<b>2ā5</b>) Mean values and SEM are presented. (C) Confocal micrographs acquired under the same microscope settings confirmed elevated cellular uptake of PA <b>2</b> compared to peptide (<b>1</b>) and displayed a punctate intracellular fluorescence pattern. Nuclei stain (blue): Hoechst 33342; Scale bars: 40 Āµm.</p
Tumor-Penetrating Nanosystem Strongly Suppresses Breast Tumor Growth
Antiangiogenic
and vascular disrupting compounds have shown promise
in cancer therapy, but tend to be only partially effective. We previously
reported a potent theranostic nanosystem that was highly effective
in glioblastoma and breast cancer mouse models, retarding tumor growth
and producing some cures [Agemy, L. et
al. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 17450ā17455. Agemy, L. et
al. Mol. Ther. 2013, 21, 2195ā2204.]. The nanosystem consists of iron oxide NPs (ānanowormsā)
coated with a composite peptide with tumor-homing and pro-apoptotic
domains. The homing component targets tumor vessels by binding to
p32/gC1qR at the surface or tumor endothelial cells. We sought to
further improve the efficacy nanosystem by searching for an optimally
effective homing peptide that would also incorporate a tumor-penetrating
function. To this effect, we tested a panel of candidate p32 binding
peptides with a sequence motif that conveys tumor-penetrating activity
(CendR motif). We identified a peptide designated as Linear TT1 (Lin
TT1) (sequence: AKRĀGARĀSTA) as most effective in causing
tumor homing and penetration of the nanosystem. This peptide had the
lowest affinity for p32 among the peptides tested. The low affinity
may have moderated the avidity effect from the multivalent presentation
on nanoparticles (NPs), such that the NPs avoid getting trapped by
the so-called ābinding-site barrierā, which can hinder
tissue penetration of compounds with a high affinity for their receptors.
Treatment of breast cancer mice with the LinTT1 nanosystem showed
greatly improved efficacy compared to the original system. These results
identify a promising treatment modality and underscore the value of
tumor penetration effect in improving the efficacy tumor treatment