Creation of a novel peptide with enhanced nuclear localization in prostate and pancreatic cancer cell lines.

BACKGROUND: For improved uptake of oligonucleotide-based therapy, the oligonucleotides often are coupled to peptides that facilitate entry into cells. To this end, novel cell-penetrating peptides (CPPs) were designed for mediating intracellular uptake of oligonucleotide-based therapeutics. The novel peptides were based on taking advantage of the nuclear localization properties of transcription factors in combination with a peptide that would bind putatively to cell surfaces. It was observed that adding a glutamate peptide to the N-terminus of the nuclear localization signal (NLS) of the Oct6 transcription factor resulted in a novel CPP with better uptake and better nuclear colocalization than any other peptide tested. RESULTS: Uptake of the novel peptide Glu-Oct6 by cancer cell lines was rapid (in less than 1 hr, more than 60% of DU-145 cells were positive for FITC), complete (by 4 hr, 99% of cells were positive for FITC), concentration-dependent, temperature-dependent, and inhibited by sodium azide (NaN3). Substitution of Phe, Tyr, or Asn moieties for the glutamate portion of the novel peptide resulted in abrogation of novel CPP uptake; however none of the substituted peptides inhibited uptake of the novel CPP when coincubated with cells. Live-cell imaging and analysis by imaging flow cytometry revealed that the novel CPP accumulated in nuclei. Finally, the novel CPP was coupled to a carboxyfluorescein-labeled synthetic oligonucleotide, to see if the peptide could ferry a therapeutic payload into cells. CONCLUSIONS: These studies document the creation of a novel CPP consisting of a glutamate peptide coupled to the N-terminus of the Oct6 NLS; the novel CPP exhibited nuclear colocalization as well as uptake by prostate and pancreatic cancer cell lines.

HIF-1 mediates pathogenic inflammatory responses to intestinal ischemia-reperfusion injury.

Acute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) are major causes of death in trauma patients. Gut inflammation and loss of gut barrier function as a consequence of splanchnic ischemia-reperfusion (I/R) have been implicated as the initial triggering events that contribute to the development of the systemic inflammatory response, ALI, and MODS. Since hypoxia-inducible factor (HIF-1) is a key regulator of the physiological and pathophysiological response to hypoxia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Utilizing partially HIF-1α-deficient mice in a global trauma hemorrhagic shock (T/HS) model, we found that HIF-1 activation was necessary for the development of gut injury and that the prevention of gut injury was associated with an abrogation of lung injury. Specifically, in vivo studies demonstrated that partial HIF-1α deficiency ameliorated T/HS-induced increases in intestinal permeability, bacterial translocation, and caspase-3 activation. Lastly, partial HIF-1α deficiency reduced TNF-α, IL-1ß, cyclooxygenase-2, and inducible nitric oxide synthase levels in the ileal mucosa after T/HS whereas IL-1ß mRNA levels were reduced in the lung after T/HS. This study indicates that prolonged intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. Consequently, these results provide unique information on the initiating events in trauma-hemorrhagic shock-induced ALI and MODS as well as potential therapeutic insights.

Intravenous injection of trauma-hemorrhagic shock mesenteric lymph causes lung injury that is dependent upon activation of the inducible nitric oxide synthase pathway.

OBJECTIVE: To test the hypothesis that gut-derived factors carried in trauma-hemorrhagic shock (T/HS) lymph is sufficient to induce lung injury. Additionally, because our previous studies showed that T/HS-induced nitric oxide production was associated with lung injury, we examined whether T/HS lymph-induced lung injury occurs via an inducible nitric oxide synthase (iNOS)-dependent pathway. BACKGROUND: We have previously shown that T/HS-induced lung injury is mediated by gut-derived humoral factors carried in the mesenteric lymph. However, it remains unclear whether T/HS lymph itself is sufficient to induce lung injury, or requires the activation of other factors during the T/HS period to exert its effect. METHODS: Mesenteric lymph collected from T/HS or trauma-sham shock (T/SS) animals was injected intravenously into male rats at a rate of 1 mL/h for 3 hours. At the end of infusion, lung injury was assessed by lung permeability and lung histology. The effect of iNOS inhibition on T/HS lymph-induced lung injury was studied and this was further confirmed in iNOS knockout mice. Finally, iNOS immunohistochemistry was performed to identify the cells of origin of iNOS. RESULTS: The injection of T/HS lymph, but not sham shock lymph, caused lung injury. This was associated with increased plasma nitrite/nitrate levels as well as induction of iNOS protein in the lung, liver, and gut. Treatment with the selective iNOS inhibitor aminoguanidine prevented T/HS lymph-induced lung injury. iNOS knockout mice, but not their wild-type controls, were resistant to T/HS lymph-induced lung injury. By immunohistochemistry, neutrophils and macrophages, rather than parenchymal cells, were the source of T/HS lymph-induced lung iNOS. CONCLUSIONS: These results indicate that T/HS lymph is sufficient to induce acute lung injury and that lymph-induced lung injury occurs via an iNOS-dependent pathway.