Creation of a novel peptide with enhanced nuclear localization in prostate and pancreatic cancer cell lines
- H Dan Lewis†1,
- Ali Husain†1,
- Robert J Donnelly2,
- Dimitrios Barlos1,
- Sheraz Riaz1,
- Kalyani Ginjupalli1, 5,
- Adetola Shodeinde3 and
- Beverly E Barton1, 4Email author
© Lewis et al; licensee BioMed Central Ltd. 2010
Received: 11 February 2010
Accepted: 28 October 2010
Published: 28 October 2010
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.
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.
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.
Experimental therapeutic approaches using oligonucleotides for prostate and pancreatic cancer are actively investigated in many laboratories, including ours [1, 2]. 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 (CPP) to a therapeutic payload, such as a peptide nucleic acid (PNA). 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 (NLS). 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 [5, 6], 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 p16 successfully inhibited cell growth , and the insulin-like growth factor loop 1 peptide IGF1 is being tried for imaging of early pancreatic tumors .
Our laboratory has been involved in STAT3 inhibition for cancer therapy for a number of years. Previously, we designed oligonucleotides that inhibited STAT3 expression with concomitant abrogation of STAT3 target gene expression [1, 2]. It had long been our intention to use PNAs as therapeutic entities for STAT3, 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(s) 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 STAT3, 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 (PNAs) found optimal efficacy under serum-free conditions at 5 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 (EEEAA) to the N-terminus of the Oct6 transcription factor NLS, a novel CPP was created that entered prostatic and pancreatic cancer cell lines readily. As little as 30 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 Oct6 NLS to the glutamate-rich peptide enhanced uptake 100-fold. Combining the glutamate peptide to the Oct6 NLS created a novel peptide with better nuclear localization properties than either the glutamate-rich peptide or the Oct6 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-145 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-145 cells
Peptides and Peptide-PNAs Included in Studies
Addition of glutamate-rich peptide EEEAA to the Oct6 NLS enhanced its uptake
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 (peptides Phe-Oct6-FITC, Phe-Oct6-Dansyl, and Phe-Oct6), Asn residues (peptide Asn-Oct6-FITC), or Tyr residues (peptides Tyr-Oct6-FITC and Tyr-Oct6). The resulting peptides were incubated at 0 to 1000 nM with cells for 1 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 300 nM (geometric MFI increased from approximately 40 to 100; the increase was not significant by paired ANOVA). 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 (Figure 2D). Because labeling the Phe-Oct6 peptide with either FITC or dansyl resulted in peptides that had cytotoxicity at high concentrations (300 and 1000 nM; data not shown), the experiment was performed again using unlabeled Phe-Oct6 peptide in the presence of FITC-labeled Glu-Oct6-FITC, to see if Phe-Oct6 could compete with Glu-Oct6-FITC for binding and uptake. The results shown in Figure 2E reveal that incubating cells with Phe-Oct6 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.
Cell Viability after Incubation with Peptides Glu-Oct6 and Phe-Oct6
Uptake of all peptides was temperature-dependent, but uptake of peptides containing the Oct6 NLS only was inhibited by NaN3
Sodium Azide Inhibited Uptake of Peptides with Oct6 NLS
Avg Geometric MFI
Peptide Glu-Oct6-FITC colocalized to the nucleus
Peptide Glu-Oct6 facilitated transport of a synthetic oligonucleotide into prostatic and pancreatic cancer cell lines
Glu-Oct6 Facilitated Nuclear Colocalization of PNA 13778a
mean % nuclear FITC
mean nuclear FITC intensity
mean % cyto. FITC
mean cyto. FITC intensity
Peptide Glu-Oct6 facilitated transport of an anti-STAT3 PNA into cancer cell lines, resulting in apoptosis
PNA Glu-Oct6-13410a Induced Apoptosis in DU-145 and PANC-1 Cells
The use of NLS peptides was explored because of their potential to ferry therapeutic cargoes efficiently. Previously, it was observed that the Oct6 NLS peptide accumulated in the endosomal compartments of cells . However, it was observed that addition of peptide EEEAA to the N-terminus of the Oct6 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-Oct6 facilitated entry of a PNA into DU-145 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-Oct6 ( and Figures). It is entirely possible that Glu-Oct6 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-Oct6 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 (4 to 12°C), 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 (Figure 3) was observed in DU-145 and PANC-1 cell lines; furthermore Glu-Oct6-FITC partitioned across nuclei and cytoplasm (Figure 4A and 4B), whereas neither peptides Glu-FITC nor Oct6-FITC exhibited appreciable nuclear localization (Figure 4C and 4D). The Oct6 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 Oct6 NLS and others ; accumulation across cytoplasm and nucleus by Glu-Oct6-FITC indicates that addition of the Glu-rich peptide to the Oct6 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 V79 and PC12 cell lines . Furthermore, the uptake of NLS peptides by the MCF-7 breast cancer cell line was found to be temperature-dependent but unaffected by the presence of NaN3 .
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-145 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-7 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 [27, 28]. PSMA we believe is not involved because DU-145 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-Oct6 binds to on DU-145, LNCaP, and PANC-1 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 Oct6 NLS peptide as a CPP was explored. Peptide Glu-Oct6-FITC was shown to gain entry into DU-145, PANC-1, 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 Oct6 NLS peptide alone and lacked the N-terminal glutamate-rich peptide, did not localize to the nucleus. The ability of Glu-Oct6 to function as a CPP was lost when Phe, Tyr, or Asn were substituted for the Glu residues. Peptide Glu-Oct6 facilitated entry of a carboxylysyl-fluorescein PNA into DU-145 and PANC-1 cells. Finally, peptide Glu-Oct6 facilitated transport of an anti-STAT3 PNA into DU-145 and PANC-1 cells, resulting in significant apoptosis. Therefore, Glu-Oct6 may be a peptide useful for therapeutic applications.
Synthesis of Peptides and Peptide-PNA
Peptides used are listed in Table 1 and are referred to by their synthesis numbers for convenience. The carboxyfluorescein and carboxydansyl amino acids were purchased from Bachem. All peptides used had a molar ratio of FITC or dansyl to peptide of 1. All peptides were synthesized by the Molecular Resources Facility at the University of Medicine and Dentistry, New Jersey Medical School (Newark, NJ) on an Applied Biosystems model 433 peptide synthesizer using FMoc chemistry. After cleavage and deprotection, the peptides were purified by high-performance liquid chromatography (HPLC) and analyzed by both HPLC and sequencing on an Applied Biosystems Procise 494C sequenator. The FITC-labeled peptide-PNAs FITC-PNA-EEE, FITC-TAT-PNA, and Glu-Oct6-PNA-FITC were synthesized by BioSynthesis (Lewisville, TX). They were purified by HPLC and their structures verified by MALDI-TOF. Because of the requirements of PNA chemistry, FITC was added to the C-terminus of Glu-Oct6-PNA as lysyl FITC.
DU-145 and LNCaP cells were the gift of Dr. James Turkson (University of Central Florida, Orlando, FL). DU-145 cells were grown in DMEM/Ham's F12 (Invitrogen, Carlsbad, CA) plus 10% newborn bovine serum (Hyclone, Logan, UT). LNCaP cells were maintained in RPMI-1640 (Invitrogen) plus fetal bovine serum (Hyclone). PANC-1 cells were the gift of Dr. James Freeman, University of Texas Health Sciences Center, San Antonio TX. They were grown in the same medium as the DU-145 cells. Cell viabilities were determined using fluorescein diacetate (Sigma Chemical Co., St. Louis, MO) and a Universal RIII fluorescence microscope (Zeiss, Jena, Germany).
Uptake/Fluorescence Quantification and Nuclear Colocalization Studies
Peptides were added to subconfluent cultures of cells at times, temperatures, and concentrations indicated in experiments. Concentrations ranging from 0 to 1000 nM were assayed. Fluorescence was normalized using calibration beads (Becton-Dickinson; BD). Cells were analyzed in the presence of 5 mM 7-AAD (eBioscience, San Digeo CA) to gate on live events. Fluorescence was quantified, after cells were harvested, using a BD FACScan flow cytometer. At least 10,000 events were acquired using CellQuest Pro software and an Apple Macintosh G4 dual coprocessor computer running OS X 10.3.9. Fluorescence detectors on the instrument were standardized prior to each acquisition run, so that fluorescence intensities from different days could be compared. Because the FACScan employs logarithmic amplifiers on the fluorescence detectors, the more accurate parameter with which to compare fluorescence intensities for different samples is the geometric mean fluorescence intensity (geometric MFI). For the studies examining uptake of carboxyfluorescein peptides plus carboxydansyl peptides, fluorescence was quantified on a BD LSR II flow cytometer. In nuclear colocalization studies, cells were stained with the nuclear stain DRAQ5 (Axxora, San Diego, CA) at 5 mM final concentration following uptake of peptides. Fixed cells (4% paraformaldehyde/DPBS) were then analyzed on an Amnis ImageStream 200 imaging cytometer using IDEAS 3.0 software. Four to five thousand events were collected for analysis.
Live Cell Imaging Studies
A Zeiss Axiovert 200 inverted phase-contrast microscope outfitted with epifluorescence was used for live imaging studies. Subconfluent cultures of cells in 12-well plates were incubated with 500 nM peptides as indicated. DRAQ5 (Axxora; 10 mM final concentration) was added for the last hour of incubation, then the cells were washed twice with warm phenol red-free buffer. Cells were examined in phenol red-free buffer plus 10% serum. Images were acquired and analyzed using Zeiss Axiovision software.
DU-145 and PANC-1 cells were incubated with PNAs Glu-Oct6-13410a or 13410a (no CPP attached) at 0, 300, 600, or 1000 nM for 48 hr. Cells were harvested, then stained with FITC-annexin V (Abcam; Cambridge, MA) and counterstained with propidium iodide (Sigma, St.Louis, MO). Cells were analyzed on a BD FACScan for fluorescence in the FL1 and FL3 channels; CellQuest Pro software was used to quantify fluorescence and determine the extent of apoptosis.
The graphing program Kaleidagraph 4.2 (Synergy Software, Reading, PA) and the statistical program InStat3 (GraphPad Software, San Diego, CA) were used for data analyses unless otherwise indicated.
The authors acknowledge Dr. Sukhwinder Singh for acquiring data on the Amnis ImageStream and Mr. Richard A. DeMarco of Amnis Corporation for help analyzing data files. This work was supported by NIH grant CA 121782 (BEB) and a Research & Development Merit Award from the Department of Veterans Affairs (BEB).
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