BACKGROUND
experimental therapeutic approaches using oligonucleotides for prostate and pancreatic cancer are actively investigated in many laboratories, including ours  <cit> .
such inhibitors are attractive in theory but lack a practical method for delivery in the clinical setting.
one possible approach to overcome this roadblock is to use peptide-mediated transport, thereby coupling a cell-penetrating peptide  to a therapeutic payload, such as a peptide nucleic acid .
an inherent advantage of using cpps is the ability to design cell specificity in the sequence, as well as target organelle specificity through inclusion of nuclear localization signals .
cpp-mediated can be quite efficient, allowing for rapid and complete uptake and delivery of a pna payload for the treatment of hiv  <cit> .
cpps for delivery of therapeutic oligonucleotides have gained attention in recent years; an excellent review describing the major categories of cpps was published earlier this year  <cit> .
cpps for prostate cancer have been examined in conjunction with delivery of methotrexate-loaded liposomes  <cit> , double-stranded decoys  <cit> , and radioactive gadolinium complexes targeted to c-myc  <cit> .
as for pancreatic cancer, the antennepedia protein antp when coupled to the tumor suppressor p <dig> successfully inhibited cell growth  <cit> , and the insulin-like growth factor loop  <dig> peptide igf <dig> is being tried for imaging of early pancreatic tumors  <cit> .
our laboratory has been involved in stat <dig> inhibition for cancer therapy for a number of years.
previously, we designed oligonucleotides that inhibited stat <dig> expression with concomitant abrogation of stat <dig> target gene expression  <cit> .
it had long been our intention to use pnas as therapeutic entities for stat <dig>  given the superior properties of pnas compared to oligonucleotides for this purpose.
pnas bind strongly to rna or dna, more strongly than antisense or rnai, thereby inhibiting transcription of gene through the creation of triple helices.
the structure of pnas makes them highly resistant to nucleases and proteases  <cit> .
finally, pnas form triple helices with duplex dna, making them ideal candidate molecules for inhibiting transcription factors  <cit> .
however, pnas need suitable cpps for transport into cells.
and in the case of inhibiting a transcription factor such as stat <dig>  nuclear colocalization is highy desirable since the nucleus is the main seat of transcription factor activity.
one strategy for cpp design recently examined is to use the nls peptides of transcription factors themselves as cpps.
the nls of several transcription factors have been compared in various tumor types with varying degrees of efficacy with regard to uptake and nuclear localization, however sequestration in endosomes was observed for many of the peptides tested  <cit> .
as for delivery of an oligonucleotide or pna payload, one study using cpps consisting of cell surface ligands linked to nls and conjugated to peptide nucleic acids  found optimal efficacy under serum-free conditions at  <dig> mm, a concentration that is not commercially feasible due to prohibitive costs  <cit> .
clearly, more studies on designing and optimizing cpps for delivery of therapeutic oligonucleotide or pna payloads are needed in order to bring new therapeutic entities to the clinic.
in this paper, the creation of a novel cpp combining the nls of a transcription factor with another peptide that was observed to enhance cellular uptake and nuclear localization.
by adding a glutamate-rich peptide  to the n-terminus of the oct <dig> transcription factor nls, a novel cpp was created that entered prostatic and pancreatic cancer cell lines readily.
as little as  <dig> nm of the fitc-labeled glutamate-rich peptide was sufficient to stain 60-70% of the cells.
addition of a lysyl peptide, kkk, to the glutamate-rich peptide enhanced uptake 10-fold, and addition of the oct <dig> nls to the glutamate-rich peptide enhanced uptake 100-fold.
combining the glutamate peptide to the oct <dig> nls created a novel peptide with better nuclear localization properties than either the glutamate-rich peptide or the oct <dig> nls alone.
substituting other amino acids for the glutamate residues abrogated uptake, but coincubation of substituted peptides with the novel cpp did not inhibit uptake, meaning that uptake of the novel cpp was sequence-specific.
finally, when the novel peptide was coupled to a carboxyfluorescein-labeled pna, uptake and nuclear localization by du- <dig> cells was observed.
the rational design of cpps using the nls of transcription factors for enhanced cancer cell uptake is worthy of study for delivery of therapeutic payloads into targeted cells.
RESULTS
neither a pmsa-targeted peptide nor tat resulted in efficient pna uptake in du- <dig> cells
because pmsa  is overexpressed on most prostate cancer cells, it is an attractive target for therapeutic delivery.
being a carboxyglutamate peptidase, it cleaves c-terminal glu residues.
c-terminal peptides such as eee have been used previously to deliver methotrexate-loaded liposomes to prostate cancer cell lines  <cit> ; whether a c-terminal glu peptide could deliver a pna to prostate cancer cells had not been investigated.
tat peptide, a 13-amino acid peptide comprised of residues  <dig> to  <dig> of the human immunodeficiency virus- <dig> tat protein, has been shown to be an efficient cpp for delivery of anti-viral oligonucleotides and pnas  <cit> ; as is the case for eee, it had not been fully evaluated in most cancer lines for delivery of pnas.
therefore, fitc-labeled pnas bearing either a carboxy-terminal peptide  or an n-terminal tat peptide were synthesized and incubated with du- <dig> cells .
surprisingly, only about 30-50% of the du- <dig> cells were fluorescent  <dig> hr after start of incubation  and the proportion of fluorescent cells markedly decreased over time in the case of the eee-labeled pna .
based on these results, it was decided to try other strategies for design of suitable cpps to deliver pnas into cancer cell lines.
carboxyfluorescein and carboxydansyl peptides were synthesized by fmoc chemistry at the molecular resources facility, umdnj.
they were purified by hplc and sequence verified by the same technique.
carboxydansyl peptides are synthesized using lysyl dansyl moieties.
the peptide-pnas were synthesized by biosynthesis; they were purified by hplc and verified by maldi-tof.
the letters in bold indicate the pna portions of the molecules.
the fitc moiety was coupled to glu-oct6-pna as a lysyl-fitc, due to the requirements of pna chemistry.
addition of glutamate-rich peptide eeeaa to the oct <dig> nls enhanced its uptake
uptake of peptides by live cells was compared by flow cytometry, using either 7-amino actinomycin d  or propidium iodide  to exclude dead cells from analyses.
because logarithmic amplification of fluorescence detectors were employed, the geometric mean fluorescence intensities  were compared among samples.
in comparing the geometric mfis among the peptides, it was noted that peptides glu-fitc and glu-ala-fitc  exhibited the lowest values, whereas addition of either kkk or the oct <dig> nls  significantly increased the geometric mfis after  <dig> hrs incubation.
these data suggested the possibility that inclusion of even a minimal nls such as kkk could profoundly influence cellular uptake.
the influence of the oct <dig> nls peptide on uptake was even more dramatic; however, uptake of the oct <dig> nls alone  was not nearly as great as that of glu-oct6-fitc.
therefore it is possible that both parts of the glu-oct <dig> peptide, eeeaa and the oct <dig> nls, contributed to the enhanced property of glu-oct6-fitc to function as a cpp.
the kinetics of uptake for glu-oct6-fitc were found to be rapid, although saturation was not reached.
figure 2b shows that in both du- <dig> and lncap cells, the geometric mfi of  <dig> nm at  <dig> hr extrapolated from the graph is nearly the same as that measured at  <dig> hr .
in the presence of  <dig> mm, the geometric mfis of the two cell lines was  <dig> to  <dig>  and uptake was still linear.
inclusion of higher concentrations of peptide was not feasible, due to the limited amount of peptide.
substitution of phe, asn, or tyr for glu abrogated peptide uptake
in order to determine if uptake of peptide glu-oct6-fitc was sequence-specific, glu residues were substituted by phe residues , asn residues , or tyr residues .
the resulting peptides were incubated at  <dig> to  <dig> nm with cells for  <dig> hr.
cells were then harvested and processed for analysis by flow cytometry.
figure 2c shows that there was very little uptake of peptides phe-oct6-fitc, asn-oct6-fitc, or tyr-oct6-fitc up to  <dig> nm .
coincubation of glu-oct6-fitc with either carboxydansyl peptides phe-oct-dansyl or asn-oct-dansyl resulted in nearly unaltered uptake of peptide glu-oct6-fitc without concomitant uptake of either phe-oct6-dansyl or asn-oct6-dansyl .
because labeling the phe-oct <dig> peptide with either fitc or dansyl resulted in peptides that had cytotoxicity at high concentrations , the experiment was performed again using unlabeled phe-oct <dig> peptide in the presence of fitc-labeled glu-oct6-fitc, to see if phe-oct <dig> could compete with glu-oct6-fitc for binding and uptake.
the results shown in figure 2e reveal that incubating cells with phe-oct <dig> in the presence of glu-oct6-fitc did not affect uptake of glu-oct6-fitc.
all these data taken together demonstrate that substituting asn, phe, or tyr for glu did not interfere with uptake of glu-oct6-fitc, indicating that uptake of glu-oct6-fitc had some specificity requirement for glu on the peptide.
to check for cytotoxicity in the unlabeled phe-oct <dig> and glu-oct <dig> peptides, these peptides were incubated with panc- <dig> and du- <dig> cells for  <dig> and  <dig> hrs.
at the appropriate times, cells were harvested and stained with fitc-labeled annexin v plus pi and analyzed by flow cytometry, in order to assess induction of apoptosis.
cell line viabilities at the start of the experiments was 98% ; after treatment with phe-oct <dig> or glu-oct <dig> peptides, cell viabilities were observed to be  <dig> to 100% , indicating that the cytotoxicities seen with the fitc- or dansyl-labeled phe-oct <dig> peptides were not due to the peptide part of those molecules, but rather due to the combination of the peptide plus the fluorochromes .
the conclusion from this series of experiments is that glu-oct <dig> and phe-oct <dig> peptides in and of themselves are not cytotoxic and do not induce apoptosis.
the results of the experiments presented in this subsection are evidence that uptake is specific to the sequence of the glu-oct <dig> peptide.
du- <dig> and panc- <dig> cells were incubated with  <dig> -  <dig> nm peptides for  <dig> hr, then harvested, stained with fitc-annexin v/pi, washed and analyzed by flow cytometry to determine the cell viability using a facscan flow cytometer and cellquest pro software.
the average of  <dig> replicate experiments is shown.
uptake of all peptides was temperature-dependent, but uptake of peptides containing the oct <dig> nls only was inhibited by nan3
in order to determine if peptides entered cells by diffusion or by an atp-dependent process, the following experiments were performed.
first, uptake of  <dig> nm peptides glu-fitc, glu-ala-fitc, glu-lys-fitc, glu-oct6-fitc, and oct6-fitc was compared at  <dig>   <dig>  and 37°c for  <dig> hr in du- <dig> cells.
as shown in figure  <dig>  uptake of peptides tested was temperature-dependent, with best uptake observed at 37°c.
even at 4°c, peptides glu-oct6-fitc and oct6-fitc exhibited greatly enhanced uptake, compared with the other peptides .
in the case of panc- <dig> cells, a similar pattern of temperature dependence of uptake was observed.
at 4°c, the average geometric mfis for all peptides were found to be  <dig> to  <dig> ; at 25°c the average geometric mfis increased again increased slightly for glu-fitc  and glu-ala-fitc .
however, the average geometric mfi of glu-lys-fitc increased to about  <dig>  that of glu-oct <dig> to about  <dig>  and the average geometric mfi of oct6-fitc was about  <dig>  at 37°c, the average geometric mfis of the glu-fitc and glu-ala-fitc peptides were still less than  <dig> , whereas the average geometric mfi of the peptides containing the nls either remained constant , increased to over  <dig> , or decreased .
these data confirm the temperature-dependent uptake of oct <dig> nls-containing peptides in panc- <dig> cells.
usually, marked temperature dependence such as observed for these peptides is indicative of uptake by endoctytosis  <cit> .
if this is the case, then uptake would be expected to be inhibited by depletion of atp.
to investigate this, experiments were performed for  <dig> hr at 37°c in the presence of nan <dig>  table  <dig> shows that the addition of up to 1% nan <dig> to du- <dig> cells had little effect on the uptake of peptides glu-fitc, glu-ala-fitc, or glu-lys-fitc at  <dig> nm .
however, the geometric mfi of  <dig> nm peptide oct6-fitc was reduced from nearly  <dig> to  <dig> in the presence of 1% nan <dig>  while the geometric mfi of peptide glu-oct6-fitc was decreased from  <dig> to  <dig> in the presence of 1% nan <dig>  the addition of as little as  <dig> % nan <dig> inhibited the average geometric mfi for oct6-fitc, reducing it from  <dig> to  <dig> .
these data indicate that uptake of the smaller peptides was atp-independent but that uptake of the larger peptides containing the oct <dig> nls was atp-dependent.
uptake of peptides lacking the oct <dig> nls was observed to be temperature-dependent but uptake was unaffected by up to 1% nan3; on the other hand uptake of peptides glu-oct6-fitc and oct6-fitc, both of which contain the oct <dig> nls, was inhibited by 1% nan <dig> by over 50% .
these preliminary studies demonstrated that uptake of peptides lacking the oct <dig> nls is likely through a non-endocytic mechanism and is not receptor-mediated whereas uptake of peptide containing the oct <dig> nls proceed through a different mechanism, but more studies have to be conducted to confirm if this observation is generalizable to other nls peptides.
du- <dig> cells were incubated with  <dig> nm peptides for  <dig> hr in the presence or absence of nan <dig>  cells were washed and analyzed by flow cytometry to determine the geometric mean fluorescence intensities using a facscan flow cytometer and cellquest pro software.
the average of  <dig> replicate experiments is shown.
peptide glu-oct6-fitc colocalized to the nucleus
since flow cytometry only gives data regarding total shifts in fluorescence of a population, imaging flow cytometry and live imaging microscopy studies were performed to determine if the nls augmented nuclear accumulation of the peptides.
imaging flow cytometry was chosen to provide data on intracellular distribution of fitc-labeled peptides.
inverted epifluorescence microscopy was chosen to eliminate inadvertant artifacts that may arise from fixation, such as nuclear translocation  <cit> .
for these studies, the cell-permeable fluorochrome draq <dig> was used because it readily enters and stains nuclei of viable cells  <cit> .
imaging flow cytometry revealed that peptide glu-oct6-fitc accumulated in the nucleus of du- <dig> and lncap cells much better than did peptide glu-ala-fitc; at  <dig> nm glu-oct6-fitc was distributed approximately 50% in the nucleus and 50% in the cytoplasm.
it was not retained in the plasma membrane .
in contrast, peptide glu-ala-fitc showed much less accumulation in the cells when the plasma membrane was excluded from the analysis.
to be certain these results were not due to artifactual translocation due to fixation, live cell imaging studies were performed.
we observed that  <dig> nm of peptide glu-oct6-fitc, but not peptides glu-fitc, glu-ala-fitc, or oct6-fitc, accumulated in the nuclei of du- <dig> and lncap cells after  <dig> hrs incubation .
the translocation of peptides to nuclei was complete in the sense that fitc and draq <dig> fluorescence colocalized in all viable cells, although cytoplasmic fluorescence was visible as well.
the fitc distribution was somewhat punctate in the cytoplasmic portion of cells, leading us to conclude that the peptide may be accumulating within endosomes.
these studies show that the presence of the glutamate-rich peptide eeeaa on the n-terminus of the oct <dig> nls of peptide glu-oct6-fitc enhanced nuclear colocalization as well as uptake.
peptide glu-oct <dig> facilitated transport of a synthetic oligonucleotide into prostatic and pancreatic cancer cell lines
because the ultimate goal of these studies is to use peptide glu-oct <dig> to ferry in a therapeutic cargo, the hybrid molecule glu-oct6-pna was synthesized, which consisted of glu-oct <dig> at the n terminus and the carboxyfluorescein peptide nucleic acid  having the inert sequence tatgatctcctccgt-lysyl-fitc at the c terminus .
glu-oct6-pna-fitc was incubated with du- <dig> and panc- <dig> cells at  <dig>   <dig>  and  <dig> nm for  <dig> hr, at which time cells were harvested and processed for flow cytometry.
figure  <dig> shows that increasing the concentration of glu-oct6-pna-fitc increased the mfi of the cells, and the percent of cells positive for fitc fluorescence.
analysis of panc- <dig> cells by imaging flow cytometry, which can quantify colocalization of fluorescent dyes to subcellular organelles  <cit>  revealed that glu-oct6-pna-fitc colocalized to a great extent with the nuclear stain draq <dig> .
ideas software analysis of the  <dig> to  <dig> thousand cellular events acquired revealed that the extent of colocalization was approximately 70% nucleus and 20% cytoplasmic across all events examined .
the fluorescent intensities for pna-fitc in nuclei of both cell lines was much greater than those measured in the cytoplasmic portions , indicating that the pna accumulated in the nuclei to a much greater extent than in the cytoplasm.
the percents do not add up to 100% because they exclude the portion that localized to the cell membrane asa well as whatever amount of pna clung to cellular debris.
in addition, a small but highly fluorescent population of cells  was apparent, even as low as  <dig> nm .
furthermore, panc- <dig> cells increased in proportion of fitc positive with increasing concentration of glu-oct6-pna-fitc .
therefore, conjugating glu-oct <dig> peptide to a pna facilitated entry and nuclear colocalization of the pna into both du- <dig> and panc- <dig> cell lines.
cells were incubated with  <dig> nm glu-oct6-pna-fitc for  <dig> hr, at which time cells were fixed with 4% paraformaldehyde, stained with  <dig> μm draq <dig> for nuclear localization, and analyzed on an amnis imagestream imaging cytometer.
four to  <dig> thousand events were collected and analyzed using ideas software .
peptide glu-oct <dig> facilitated transport of an anti-stat <dig> pna into cancer cell lines, resulting in apoptosis
to determine if the creation of an anti-stat <dig> pna that is transported into cells by the novel cpp glu-oct <dig> is feasible, a pna was synthesized bearing glu-oct <dig> on the n terminal portion, plus an anti-stat <dig> sequence, 13410a  <cit> .
a control pna consisted of the 13410a sequence without a peptide coupled to it.
these were incubated with du- <dig> or panc- <dig> cells for48hr, at which times cells were harvested, stained, and analyzed by flow cytometry to quantify apoptosis.
table  <dig> shows that significant apoptosis was induced in both du- <dig> and panc- <dig> cells by as little as  <dig> nm glu-oct6-13410a , whereas addition of pna 13410a, lacking any cpp, induced very little apoptosis at  <dig> nm .
these data are encouraging for the future development of experimental therapeutics using glu peptides bound to transcription factor nls peptides for aiding the translocation of active payloads into cells.
further work in this area of peptide design is planned.
cells were incubated with pnas glu-oct6-13410a or 13410a for  <dig> hr, at which time they were harvested and stained with fitc-annexin v plus pi, then apoptosis was quantified by flow cytometry.
pna 13410a was added at  <dig> nm.
the percents apoptosis are the means of  <dig> experiments; sd = standard deviation.
* p <  <dig>  by repeated anova
discussion
the use of nls peptides was explored because of their potential to ferry therapeutic cargoes efficiently.
previously, it was observed that the oct <dig> nls peptide accumulated in the endosomal compartments of cells  <cit> .
however, it was observed that addition of peptide eeeaa to the n-terminus of the oct <dig> nls enhanced cellular uptake and also enhanced nuclear localization.
the aa residues were added so that the glu peptide would be spatially separated from the nls portion, to maximize binding of each.
ala-rich linker peptides have been described before to link functional domains of various proteins  <cit> .
it was further observed that although glu-oct <dig> facilitated entry of a pna into du- <dig> cells, higher concentrations and longer incubation times were required for the pna than for the peptide glu-oct6-fitc.
it is conceivable that there is a large energy barrier to overcome for efficient transport of pna into cells, despite their neutral charge and despite the apparently enhanced cell uptake properties of glu-oct <dig> .
it is entirely possible that glu-oct <dig> would function as a more efficient cpp if the form of the therapeutic payload were changed from a pna to a different entity, such as a locked nucleic acid.
notwithstanding, glu-oct <dig> deserves further study as a potential probe for studying nuclear localization events, and as a cpp for ferrying other forms of therapeutic payloads, such as peptides or liposomes.
temperature-dependence of peptide uptake has been found to correlate as well with segregation to intracellular compartments.
fretz and coworkers observed that at lower temperatures , l- and d-octa-arginine peptides partitioned across nuclear and cytoplasmic compartments equally, moving to the endosomes of cd34+ leukemia cells when ambient temperature rose to 30°c and higher  <cit> .
they further observed that raising concentration affected which intracellular compartments were labeled by peptides  <cit> .
similarly, temperature-dependent uptake of peptide glu-oct6-fitc  was observed in du- <dig> and panc- <dig> cell lines; furthermore glu-oct6-fitc partitioned across nuclei and cytoplasm , whereas neither peptides glu-fitc nor oct6-fitc exhibited appreciable nuclear localization .
the oct <dig> transcription factor is known to shuttle between cytoplasm and nucleus  <cit> ; this would be expected of transcription factors.
however, the nls peptide of a transcription would not be expected to shuttle, since it is not activated.
rather, one might expect that an nls peptide accumulates in one or more select organelles.
indeed, ragin and coworkers observed such accumulation with regard to the oct <dig> nls and others  <cit> ; accumulation across cytoplasm and nucleus by glu-oct6-fitc indicates that addition of the glu-rich peptide to the oct <dig> nls prevents the endoplasmic sequestration by a mechanism that has yet to be explored.
several mechanisms of peptide uptake are known; atp-dependent and atp-independent peptide uptake are two major differentiating features of peptide uptake but by no means the only ones.
even within the same cell line, investigators have observed multiple modes of peptide uptake; endocytic and non-endocytic modes of uptake were noted in the v <dig> and pc <dig> cell lines  <cit> .
furthermore, the uptake of nls peptides by the mcf- <dig> breast cancer cell line was found to be temperature-dependent but unaffected by the presence of nan <dig>  <cit> .
pnas have been a focus of cancer researchers for over a decade.
early work on pnas for cancer therapy showed that anti-sense pnas directed against the androgen receptor and tata-binding protein genes worked by hybridization with cag triplet repeats in lncap and du- <dig> cell lines; no binding to c-myc, which lacks the cag repeats was observed  <cit> .
although marked effects on the transcriptosomes of the androgen receptor and tata-binding proteins genes were observed, the pnas were not designed to be therapeutically active.
elegant studies on t47d and mcf- <dig> breast cancer cell lines with pna-peptide conjugates targeting human progesterone receptor gene isoforms a and b revealed the extent to which expression of progesterone receptor protein was attenuated  <cit> .
more recently, investigators showed therapeutic efficacy in a mouse model of burkitt's lymphoma using an anti-sense pna targeted to the em enhancer region of the h chain locus, which in burkitt's lymphoma is transposed near the c-myc locus  <cit> .
glutamate receptors are known to be overexpressed by cancer cells.
in prostate cancer, the best known is psma, which binds carboxy glutamates  <cit> .
psma we believe is not involved because du- <dig> cells are psma-negative  <cit> , and because the glutamate is on n-termini of the peptides.
metabotropic glutamate receptors are usually found on neuronal cells but are found to be aberrantly expressed by malignant cells.
these glutamate receptors mediated 5-fluorouracil resistance in human colon cancer cells  <cit> .
glutamate receptors are implicated in transformation to malignancy; it's hypothesized that glutamate receptors overexpression may be a common feature of tumor pathogenesis.
thus glutamate receptors may be typical of what glu-oct <dig> binds to on du- <dig>  lncap, and panc- <dig> cells.
the activity of normal glutamate receptors in ectopic cellular environments may involve signaling pathways, which dysregulate cell growth, ultimately leading to tumorigenesis.
thus, dysregulated and aberrantly-expressed glutamate receptors may function as oncogenes  <cit> .
malignant prostatic neuroendocrine cells proliferate more when glutamate receptors are stimulated; they use glutamate as a substrate for nadh biosynthesis, producing increased levels of free fatty acids.
these activities correlate with the aggressive nature of these tumors  <cit> .
glutamate receptors have been understudied and certainly have not yet been widely used for cancer-specific targeting.
since glutamate receptors are overexpressed on a variety of solid tumors  <cit> , they should lend themselves well to cancer cell targeting by a variety of strategies, including cpp design.
this should be the focus of future work.
recent papers on peptide uptake indicates that nuclear colocalization, even by transcription factor nls peptides, is not easily achieved  <cit> .
here we describe the enhancement of uptake and nuclear localization of a nls through addition of a peptide.
whether addition of the peptide enhances uptake and nuclear localization when coupled to the nls of other transcription factors and whether the enhancement is sequence-specific is under investigation.
CONCLUSIONS
the use of the oct <dig> nls peptide as a cpp was explored.
peptide glu-oct6-fitc was shown to gain entry into du- <dig>  panc- <dig>  and lncap cells quickly and efficiently, and localized to the nucleus.
its ability to function as a cpp was concentration- and temperature-dependent, and abrogated in the presence of azide.
the homologous peptide oct6-fitc, which consisted of the oct <dig> nls peptide alone and lacked the n-terminal glutamate-rich peptide, did not localize to the nucleus.
the ability of glu-oct <dig> to function as a cpp was lost when phe, tyr, or asn were substituted for the glu residues.
peptide glu-oct <dig> facilitated entry of a carboxylysyl-fluorescein pna into du- <dig> and panc- <dig> cells.
finally, peptide glu-oct <dig> facilitated transport of an anti-stat <dig> pna into du- <dig> and panc- <dig> cells, resulting in significant apoptosis.
therefore, glu-oct <dig> may be a peptide useful for therapeutic applications.

 experimental therapeutic approaches using oligonucleotides for prostate and pancreatic cancer are actively investigated in many laboratories, including ours. such inhibitors are attractive in theory but lack a practical method for delivery in the clinical setting. one possible approach to overcome this roadblock is to use peptide-mediated transport, thereby coupling a cell-penetrating peptide to a therapeutic payload, such as a peptide nucleic acid. an inherent advantage of using cpps is the ability to design cell specificity in the sequence, as well as target organelle specificity through inclusion of nuclear localization signals. cpp-mediated can be quite efficient, allowing for rapid and complete uptake and delivery of a pna payload for the treatment of hiv. cpps for delivery of therapeutic oligonucleotides have gained attention in recent years; an excellent review describing the major categories of cpps was published earlier this year. cpps for prostate cancer have been examined in conjunction with delivery of methotrexate-loaded liposomes, double-stranded decoys, and radioactive gadolinium complexes targeted to c-myc. as for pancreatic cancer, the antennepedia protein antp when coupled to the tumor suppressor p successfully inhibited cell growth, and the insulin-like growth factor loop peptide igf is being tried for imaging of early pancreatic tumors. our laboratory has been involved in stat inhibition for cancer therapy for a number of years. previously, we designed oligonucleotides that inhibited stat expression with concomitant abrogation of stat target gene expression. it had long been our intention to use pnas as therapeutic entities for stat given the superior properties of pnas compared to oligonucleotides for this purpose. pnas bind strongly to rna or dna, more strongly than antisense or rnai, thereby inhibiting transcription of gene through the creation of triple helices. the structure of pnas makes them highly resistant to nucleases and proteases. finally, pnas form triple helices with duplex dna, making them ideal candidate molecules for inhibiting transcription factors. however, pnas need suitable cpps for transport into cells. and in the case of inhibiting a transcription factor such as stat nuclear colocalization is highy desirable since the nucleus is the main seat of transcription factor activity. one strategy for cpp design recently examined is to use the nls peptides of transcription factors themselves as cpps. the nls of several transcription factors have been compared in various tumor types with varying degrees of efficacy with regard to uptake and nuclear localization, however sequestration in endosomes was observed for many of the peptides tested. as for delivery of an oligonucleotide or pna payload, one study using cpps consisting of cell surface ligands linked to nls and conjugated to peptide nucleic acids found optimal efficacy under serum-free conditions at mm, a concentration that is not commercially feasible due to prohibitive costs. clearly, more studies on designing and optimizing cpps for delivery of therapeutic oligonucleotide or pna payloads are needed in order to bring new therapeutic entities to the clinic. in this paper, the creation of a novel cpp combining the nls of a transcription factor with another peptide that was observed to enhance cellular uptake and nuclear localization. by adding a glutamate-rich peptide to the n-terminus of the oct transcription factor nls, a novel cpp was created that entered prostatic and pancreatic cancer cell lines readily. as little as nm of the fitc-labeled glutamate-rich peptide was sufficient to stain 60-70% of the cells. addition of a lysyl peptide, kkk, to the glutamate-rich peptide enhanced uptake 10-fold, and addition of the oct nls to the glutamate-rich peptide enhanced uptake 100-fold. combining the glutamate peptide to the oct nls created a novel peptide with better nuclear localization properties than either the glutamate-rich peptide or the oct nls alone. substituting other amino acids for the glutamate residues abrogated uptake, but coincubation of substituted peptides with the novel cpp did not inhibit uptake, meaning that uptake of the novel cpp was sequence-specific. finally, when the novel peptide was coupled to a carboxyfluorescein-labeled pna, uptake and nuclear localization by du- cells was observed. the rational design of cpps using the nls of transcription factors for enhanced cancer cell uptake is worthy of study for delivery of therapeutic payloads into targeted cells.  neither a pmsa-targeted peptide nor tat resulted in efficient pna uptake in du- cells because pmsa is overexpressed on most prostate cancer cells, it is an attractive target for therapeutic delivery. being a carboxyglutamate peptidase, it cleaves c-terminal glu residues. c-terminal peptides such as eee have been used previously to deliver methotrexate-loaded liposomes to prostate cancer cell lines; whether a c-terminal glu peptide could deliver a pna to prostate cancer cells had not been investigated. tat peptide, a 13-amino acid peptide comprised of residues to of the human immunodeficiency virus- tat protein, has been shown to be an efficient cpp for delivery of anti-viral oligonucleotides and pnas; as is the case for eee, it had not been fully evaluated in most cancer lines for delivery of pnas. therefore, fitc-labeled pnas bearing either a carboxy-terminal peptide or an n-terminal tat peptide were synthesized and incubated with du- cells. surprisingly, only about 30-50% of the du- cells were fluorescent hr after start of incubation and the proportion of fluorescent cells markedly decreased over time in the case of the eee-labeled pna. based on these results, it was decided to try other strategies for design of suitable cpps to deliver pnas into cancer cell lines. carboxyfluorescein and carboxydansyl peptides were synthesized by fmoc chemistry at the molecular resources facility, umdnj. they were purified by hplc and sequence verified by the same technique. carboxydansyl peptides are synthesized using lysyl dansyl moieties. the peptide-pnas were synthesized by biosynthesis; they were purified by hplc and verified by maldi-tof. the letters in bold indicate the pna portions of the molecules. the fitc moiety was coupled to glu-oct6-pna as a lysyl-fitc, due to the requirements of pna chemistry. addition of glutamate-rich peptide eeeaa to the oct nls enhanced its uptake uptake of peptides by live cells was compared by flow cytometry, using either 7-amino actinomycin d or propidium iodide to exclude dead cells from analyses. because logarithmic amplification of fluorescence detectors were employed, the geometric mean fluorescence intensities were compared among samples. in comparing the geometric mfis among the peptides, it was noted that peptides glu-fitc and glu-ala-fitc exhibited the lowest values, whereas addition of either kkk or the oct nls significantly increased the geometric mfis after hrs incubation. these data suggested the possibility that inclusion of even a minimal nls such as kkk could profoundly influence cellular uptake. the influence of the oct nls peptide on uptake was even more dramatic; however, uptake of the oct nls alone was not nearly as great as that of glu-oct6-fitc. therefore it is possible that both parts of the glu-oct peptide, eeeaa and the oct nls, contributed to the enhanced property of glu-oct6-fitc to function as a cpp. the kinetics of uptake for glu-oct6-fitc were found to be rapid, although saturation was not reached. figure 2b shows that in both du- and lncap cells, the geometric mfi of nm at hr extrapolated from the graph is nearly the same as that measured at hr. in the presence of mm, the geometric mfis of the two cell lines was to and uptake was still linear. inclusion of higher concentrations of peptide was not feasible, due to the limited amount of peptide. substitution of phe, asn, or tyr for glu abrogated peptide uptake in order to determine if uptake of peptide glu-oct6-fitc was sequence-specific, glu residues were substituted by phe residues, asn residues, or tyr residues. the resulting peptides were incubated at to nm with cells for hr. cells were then harvested and processed for analysis by flow cytometry. figure 2c shows that there was very little uptake of peptides phe-oct6-fitc, asn-oct6-fitc, or tyr-oct6-fitc up to nm. coincubation of glu-oct6-fitc with either carboxydansyl peptides phe-oct-dansyl or asn-oct-dansyl resulted in nearly unaltered uptake of peptide glu-oct6-fitc without concomitant uptake of either phe-oct6-dansyl or asn-oct6-dansyl. because labeling the phe-oct peptide with either fitc or dansyl resulted in peptides that had cytotoxicity at high concentrations, the experiment was performed again using unlabeled phe-oct peptide in the presence of fitc-labeled glu-oct6-fitc, to see if phe-oct could compete with glu-oct6-fitc for binding and uptake. the results shown in figure 2e reveal that incubating cells with phe-oct in the presence of glu-oct6-fitc did not affect uptake of glu-oct6-fitc. all these data taken together demonstrate that substituting asn, phe, or tyr for glu did not interfere with uptake of glu-oct6-fitc, indicating that uptake of glu-oct6-fitc had some specificity requirement for glu on the peptide. to check for cytotoxicity in the unlabeled phe-oct and glu-oct peptides, these peptides were incubated with panc- and du- cells for and hrs. at the appropriate times, cells were harvested and stained with fitc-labeled annexin v plus pi and analyzed by flow cytometry, in order to assess induction of apoptosis. cell line viabilities at the start of the experiments was 98%; after treatment with phe-oct or glu-oct peptides, cell viabilities were observed to be to 100%, indicating that the cytotoxicities seen with the fitc- or dansyl-labeled phe-oct peptides were not due to the peptide part of those molecules, but rather due to the combination of the peptide plus the fluorochromes. the conclusion from this series of experiments is that glu-oct and phe-oct peptides in and of themselves are not cytotoxic and do not induce apoptosis. the results of the experiments presented in this subsection are evidence that uptake is specific to the sequence of the glu-oct peptide. du- and panc- cells were incubated with - nm peptides for hr, then harvested, stained with fitc-annexin v/pi, washed and analyzed by flow cytometry to determine the cell viability using a facscan flow cytometer and cellquest pro software. the average of replicate experiments is shown. uptake of all peptides was temperature-dependent, but uptake of peptides containing the oct nls only was inhibited by nan3 in order to determine if peptides entered cells by diffusion or by an atp-dependent process, the following experiments were performed. first, uptake of nm peptides glu-fitc, glu-ala-fitc, glu-lys-fitc, glu-oct6-fitc, and oct6-fitc was compared at and 37°c for hr in du- cells. as shown in figure uptake of peptides tested was temperature-dependent, with best uptake observed at 37°c. even at 4°c, peptides glu-oct6-fitc and oct6-fitc exhibited greatly enhanced uptake, compared with the other peptides. in the case of panc- cells, a similar pattern of temperature dependence of uptake was observed. at 4°c, the average geometric mfis for all peptides were found to be to; at 25°c the average geometric mfis increased again increased slightly for glu-fitc and glu-ala-fitc. however, the average geometric mfi of glu-lys-fitc increased to about that of glu-oct to about and the average geometric mfi of oct6-fitc was about at 37°c, the average geometric mfis of the glu-fitc and glu-ala-fitc peptides were still less than, whereas the average geometric mfi of the peptides containing the nls either remained constant, increased to over, or decreased. these data confirm the temperature-dependent uptake of oct nls-containing peptides in panc- cells. usually, marked temperature dependence such as observed for these peptides is indicative of uptake by endoctytosis. if this is the case, then uptake would be expected to be inhibited by depletion of atp. to investigate this, experiments were performed for hr at 37°c in the presence of nan table shows that the addition of up to 1% nan to du- cells had little effect on the uptake of peptides glu-fitc, glu-ala-fitc, or glu-lys-fitc at nm. however, the geometric mfi of nm peptide oct6-fitc was reduced from nearly to in the presence of 1% nan while the geometric mfi of peptide glu-oct6-fitc was decreased from to in the presence of 1% nan the addition of as little as % nan inhibited the average geometric mfi for oct6-fitc, reducing it from to. these data indicate that uptake of the smaller peptides was atp-independent but that uptake of the larger peptides containing the oct nls was atp-dependent. uptake of peptides lacking the oct nls was observed to be temperature-dependent but uptake was unaffected by up to 1% nan3; on the other hand uptake of peptides glu-oct6-fitc and oct6-fitc, both of which contain the oct nls, was inhibited by 1% nan by over 50%. these preliminary studies demonstrated that uptake of peptides lacking the oct nls is likely through a non-endocytic mechanism and is not receptor-mediated whereas uptake of peptide containing the oct nls proceed through a different mechanism, but more studies have to be conducted to confirm if this observation is generalizable to other nls peptides. du- cells were incubated with nm peptides for hr in the presence or absence of nan cells were washed and analyzed by flow cytometry to determine the geometric mean fluorescence intensities using a facscan flow cytometer and cellquest pro software. the average of replicate experiments is shown. peptide glu-oct6-fitc colocalized to the nucleus since flow cytometry only gives data regarding total shifts in fluorescence of a population, imaging flow cytometry and live imaging microscopy studies were performed to determine if the nls augmented nuclear accumulation of the peptides. imaging flow cytometry was chosen to provide data on intracellular distribution of fitc-labeled peptides. inverted epifluorescence microscopy was chosen to eliminate inadvertant artifacts that may arise from fixation, such as nuclear translocation. for these studies, the cell-permeable fluorochrome draq was used because it readily enters and stains nuclei of viable cells. imaging flow cytometry revealed that peptide glu-oct6-fitc accumulated in the nucleus of du- and lncap cells much better than did peptide glu-ala-fitc; at nm glu-oct6-fitc was distributed approximately 50% in the nucleus and 50% in the cytoplasm. it was not retained in the plasma membrane. in contrast, peptide glu-ala-fitc showed much less accumulation in the cells when the plasma membrane was excluded from the analysis. to be certain these results were not due to artifactual translocation due to fixation, live cell imaging studies were performed. we observed that nm of peptide glu-oct6-fitc, but not peptides glu-fitc, glu-ala-fitc, or oct6-fitc, accumulated in the nuclei of du- and lncap cells after hrs incubation. the translocation of peptides to nuclei was complete in the sense that fitc and draq fluorescence colocalized in all viable cells, although cytoplasmic fluorescence was visible as well. the fitc distribution was somewhat punctate in the cytoplasmic portion of cells, leading us to conclude that the peptide may be accumulating within endosomes. these studies show that the presence of the glutamate-rich peptide eeeaa on the n-terminus of the oct nls of peptide glu-oct6-fitc enhanced nuclear colocalization as well as uptake. peptide glu-oct facilitated transport of a synthetic oligonucleotide into prostatic and pancreatic cancer cell lines because the ultimate goal of these studies is to use peptide glu-oct to ferry in a therapeutic cargo, the hybrid molecule glu-oct6-pna was synthesized, which consisted of glu-oct at the n terminus and the carboxyfluorescein peptide nucleic acid having the inert sequence tatgatctcctccgt-lysyl-fitc at the c terminus. glu-oct6-pna-fitc was incubated with du- and panc- cells at and nm for hr, at which time cells were harvested and processed for flow cytometry. figure shows that increasing the concentration of glu-oct6-pna-fitc increased the mfi of the cells, and the percent of cells positive for fitc fluorescence. analysis of panc- cells by imaging flow cytometry, which can quantify colocalization of fluorescent dyes to subcellular organelles revealed that glu-oct6-pna-fitc colocalized to a great extent with the nuclear stain draq. ideas software analysis of the to thousand cellular events acquired revealed that the extent of colocalization was approximately 70% nucleus and 20% cytoplasmic across all events examined. the fluorescent intensities for pna-fitc in nuclei of both cell lines was much greater than those measured in the cytoplasmic portions, indicating that the pna accumulated in the nuclei to a much greater extent than in the cytoplasm. the percents do not add up to 100% because they exclude the portion that localized to the cell membrane asa well as whatever amount of pna clung to cellular debris. in addition, a small but highly fluorescent population of cells was apparent, even as low as nm. furthermore, panc- cells increased in proportion of fitc positive with increasing concentration of glu-oct6-pna-fitc. therefore, conjugating glu-oct peptide to a pna facilitated entry and nuclear colocalization of the pna into both du- and panc- cell lines. cells were incubated with nm glu-oct6-pna-fitc for hr, at which time cells were fixed with 4% paraformaldehyde, stained with μm draq for nuclear localization, and analyzed on an amnis imagestream imaging cytometer. four to thousand events were collected and analyzed using ideas software. peptide glu-oct facilitated transport of an anti-stat pna into cancer cell lines, resulting in apoptosis to determine if the creation of an anti-stat pna that is transported into cells by the novel cpp glu-oct is feasible, a pna was synthesized bearing glu-oct on the n terminal portion, plus an anti-stat sequence, 13410a. a control pna consisted of the 13410a sequence without a peptide coupled to it. these were incubated with du- or panc- cells for48hr, at which times cells were harvested, stained, and analyzed by flow cytometry to quantify apoptosis. table shows that significant apoptosis was induced in both du- and panc- cells by as little as nm glu-oct6-13410a, whereas addition of pna 13410a, lacking any cpp, induced very little apoptosis at nm. these data are encouraging for the future development of experimental therapeutics using glu peptides bound to transcription factor nls peptides for aiding the translocation of active payloads into cells. further work in this area of peptide design is planned. cells were incubated with pnas glu-oct6-13410a or 13410a for hr, at which time they were harvested and stained with fitc-annexin v plus pi, then apoptosis was quantified by flow cytometry. pna 13410a was added at nm. the percents apoptosis are the means of experiments; sd = standard deviation. * p < by repeated anova discussion the use of nls peptides was explored because of their potential to ferry therapeutic cargoes efficiently. previously, it was observed that the oct nls peptide accumulated in the endosomal compartments of cells. however, it was observed that addition of peptide eeeaa to the n-terminus of the oct nls enhanced cellular uptake and also enhanced nuclear localization. the aa residues were added so that the glu peptide would be spatially separated from the nls portion, to maximize binding of each. ala-rich linker peptides have been described before to link functional domains of various proteins. it was further observed that although glu-oct facilitated entry of a pna into du- cells, higher concentrations and longer incubation times were required for the pna than for the peptide glu-oct6-fitc. it is conceivable that there is a large energy barrier to overcome for efficient transport of pna into cells, despite their neutral charge and despite the apparently enhanced cell uptake properties of glu-oct. it is entirely possible that glu-oct would function as a more efficient cpp if the form of the therapeutic payload were changed from a pna to a different entity, such as a locked nucleic acid. notwithstanding, glu-oct deserves further study as a potential probe for studying nuclear localization events, and as a cpp for ferrying other forms of therapeutic payloads, such as peptides or liposomes. temperature-dependence of peptide uptake has been found to correlate as well with segregation to intracellular compartments. fretz and coworkers observed that at lower temperatures, l- and d-octa-arginine peptides partitioned across nuclear and cytoplasmic compartments equally, moving to the endosomes of cd34+ leukemia cells when ambient temperature rose to 30°c and higher. they further observed that raising concentration affected which intracellular compartments were labeled by peptides. similarly, temperature-dependent uptake of peptide glu-oct6-fitc was observed in du- and panc- cell lines; furthermore glu-oct6-fitc partitioned across nuclei and cytoplasm, whereas neither peptides glu-fitc nor oct6-fitc exhibited appreciable nuclear localization. the oct transcription factor is known to shuttle between cytoplasm and nucleus; this would be expected of transcription factors. however, the nls peptide of a transcription would not be expected to shuttle, since it is not activated. rather, one might expect that an nls peptide accumulates in one or more select organelles. indeed, ragin and coworkers observed such accumulation with regard to the oct nls and others; accumulation across cytoplasm and nucleus by glu-oct6-fitc indicates that addition of the glu-rich peptide to the oct nls prevents the endoplasmic sequestration by a mechanism that has yet to be explored. several mechanisms of peptide uptake are known; atp-dependent and atp-independent peptide uptake are two major differentiating features of peptide uptake but by no means the only ones. even within the same cell line, investigators have observed multiple modes of peptide uptake; endocytic and non-endocytic modes of uptake were noted in the v and pc cell lines. furthermore, the uptake of nls peptides by the mcf- breast cancer cell line was found to be temperature-dependent but unaffected by the presence of nan. pnas have been a focus of cancer researchers for over a decade. early work on pnas for cancer therapy showed that anti-sense pnas directed against the androgen receptor and tata-binding protein genes worked by hybridization with cag triplet repeats in lncap and du- cell lines; no binding to c-myc, which lacks the cag repeats was observed. although marked effects on the transcriptosomes of the androgen receptor and tata-binding proteins genes were observed, the pnas were not designed to be therapeutically active. elegant studies on t47d and mcf- breast cancer cell lines with pna-peptide conjugates targeting human progesterone receptor gene isoforms a and b revealed the extent to which expression of progesterone receptor protein was attenuated. more recently, investigators showed therapeutic efficacy in a mouse model of burkitt's lymphoma using an anti-sense pna targeted to the em enhancer region of the h chain locus, which in burkitt's lymphoma is transposed near the c-myc locus. glutamate receptors are known to be overexpressed by cancer cells. in prostate cancer, the best known is psma, which binds carboxy glutamates. psma we believe is not involved because du- cells are psma-negative, and because the glutamate is on n-termini of the peptides. metabotropic glutamate receptors are usually found on neuronal cells but are found to be aberrantly expressed by malignant cells. these glutamate receptors mediated 5-fluorouracil resistance in human colon cancer cells. glutamate receptors are implicated in transformation to malignancy; it's hypothesized that glutamate receptors overexpression may be a common feature of tumor pathogenesis. thus glutamate receptors may be typical of what glu-oct binds to on du- lncap, and panc- cells. the activity of normal glutamate receptors in ectopic cellular environments may involve signaling pathways, which dysregulate cell growth, ultimately leading to tumorigenesis. thus, dysregulated and aberrantly-expressed glutamate receptors may function as oncogenes. malignant prostatic neuroendocrine cells proliferate more when glutamate receptors are stimulated; they use glutamate as a substrate for nadh biosynthesis, producing increased levels of free fatty acids. these activities correlate with the aggressive nature of these tumors. glutamate receptors have been understudied and certainly have not yet been widely used for cancer-specific targeting. since glutamate receptors are overexpressed on a variety of solid tumors, they should lend themselves well to cancer cell targeting by a variety of strategies, including cpp design. this should be the focus of future work. recent papers on peptide uptake indicates that nuclear colocalization, even by transcription factor nls peptides, is not easily achieved. here we describe the enhancement of uptake and nuclear localization of a nls through addition of a peptide. whether addition of the peptide enhances uptake and nuclear localization when coupled to the nls of other transcription factors and whether the enhancement is sequence-specific is under investigation.  the use of the oct nls peptide as a cpp was explored. peptide glu-oct6-fitc was shown to gain entry into du- panc- and lncap cells quickly and efficiently, and localized to the nucleus. its ability to function as a cpp was concentration- and temperature-dependent, and abrogated in the presence of azide. the homologous peptide oct6-fitc, which consisted of the oct nls peptide alone and lacked the n-terminal glutamate-rich peptide, did not localize to the nucleus. the ability of glu-oct to function as a cpp was lost when phe, tyr, or asn were substituted for the glu residues. peptide glu-oct facilitated entry of a carboxylysyl-fluorescein pna into du- and panc- cells. finally, peptide glu-oct facilitated transport of an anti-stat pna into du- and panc- cells, resulting in significant apoptosis. therefore, glu-oct may be a peptide useful for therapeutic applications. 
these studies document the creation of a novel cpp consisting of a glutamate peptide coupled to the n-terminus of the oct nls; the novel cpp exhibited nuclear colocalization as well as uptake by prostate and pancreatic cancer cell lines. live-cell imaging and analysis by imaging flow cytometry revealed that the novel cpp accumulated in nuclei. uptake of the novel peptide glu-oct by cancer cell lines was rapid, complete, concentration-dependent, temperature-dependent, and inhibited by sodium azide. finally, the novel cpp was coupled to a carboxyfluorescein-labeled synthetic oligonucleotide, to see if the peptide could ferry a therapeutic payload into cells. it was observed that adding a glutamate peptide to the n-terminus of the nuclear localization signal of the oct transcription factor resulted in a novel cpp with better uptake and better nuclear colocalization than any other peptide tested. 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. 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.
 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 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 of the oct transcription factor resulted in a novel cpp with better uptake and better nuclear colocalization than any other peptide tested.  uptake of the novel peptide glu-oct by cancer cell lines was rapid, complete, concentration-dependent, temperature-dependent, and inhibited by sodium azide. 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.  these studies document the creation of a novel cpp consisting of a glutamate peptide coupled to the n-terminus of the oct nls; the novel cpp exhibited nuclear colocalization as well as uptake by prostate and pancreatic cancer cell lines. 