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Advances in Brief |
The Departments of Head and Neck Surgery (F. D. H., G. L. C.) and Cancer Biology (G. L. C.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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1% of the mass of typical
antibodies, that meets several criteria for targeted drug
delivery into a solid tumor. First, internalization of HN-1 by human
head and neck squamous cell cancer (HNSCC) cells suggests that HN-1 is
capable of translocating drugs across cell membranes. Second, HN-1
appears to be HNSCC-specific, given its reduced uptake by nonmalignant
human oral keratinocytes and other types of human cells, its
preferential binding to primary HNSCC, and its localization to
HNSCC-derived xenografts. Third, the presence of HN-1 within HNSCC
xenografts suggests that it is capable of penetrating tumor tissues.
Our results establish the utility of tumor-specific peptides for
targeted drug delivery into solid tumors. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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For solid malignancies, which comprise more than 90% of human cancers, antibodies recognizing tumor-specific antigens have provided little utility for drug delivery because the immunoconjugates cannot penetrate tumor tissue (3 , 4) . The development of diverse peptide libraries over the past decade has ushered in the opportunity to identify small peptides that may not be as limited as the larger antibody predecessors.
We believe that promising new therapies for HNSCC will require
tumor-targeted approaches that afford tumor specificity and limited
toxicity. Here we describe the isolation and identification of a novel
peptide, HN-1, a 12-mer peptide with 1% of the mass of
typical antibodies, which may provide the foundation for such an
intervention strategy.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Peptide-Display Library Screening.
Plaque-forming units (2.5 x 1012)
of M13 phage peptide library PhD-12 (New England BioLabs, Beverly, MA)
were incubated with 5 x 106
MDA167Tu cells in growing medium at 37°C in 5%
CO2 for 3 h. Internalized phages were
recovered by lysing with Triton X-100 (1%) detergent and amplified.
Although Triton X-100 could not lyse the nuclei, ionic detergents
capable of disrupting nuclear membrane were avoided because they
inactivate phages. To eliminate phages that became internalized after
interacting with constitutively expressed molecules, the isolated
phages were subtracted using NHFs. Recovered phages were
subjected to five rounds of MDA167Tu-selection successively. These
selections were then followed by three rounds of NHF subtraction in
succession. DNA sequencing of 12 finally selected phages revealed that
they encoded an identical peptide. MDA167Tu cells exhibited 10.3-fold
greater internalization potential for TSPLNIHNGQKL-phage than NHFs.
Basic alignment search tool (BLAST) search revealed no homology with
previously determined sequences.
Gel Electrophoresis.
Cell lysates suspended in Sample buffer [0.12 M Tris (pH 6.8), 2%
SDS, 20% glycerol, and 10% ß-mercaptoethanol were separated
by 17.5% SDS-PAGE and viewed using UV light (5)
. The
images were captured digitally using Kodak Digital Science 1D software.
Peptide Synthesis.
Peptides were synthesized and purified by reverse-phase
high-performance liquid chromatography to >95% purity (Research
Genetics, Huntsville, AL). A fluorescent label was added at the
NH2 terminus, and the COOH-terminus was capped
with an amide group. Mass spectrometry confirmed the predicted mass.
Peptides were further purified by gel electrophoresis, excised,
dialyzed, lyophilized (in the dark), resuspended in PBS, and filter
sterilized.
Fluorescence Microscopy.
Cells were fixed with 3% N-formyl paraformaldehyde, were
mounted using Anti-Fade (Molecular Probe; Eugene, OR), and were viewed
using a Nikon fluorescence microscope Eclipse E400. Images were
captured digitally and analyzed using Metamorph version 3.6a software.
To determine fluorescence intensity, the extent of autofluorescence was
subtracted from the observed intensity.
Competition Assay.
MDA177Tu cells were incubated with FITC-HN-1 in the presence of 200x
molar excess of unlabeled HN-1, a specific competitor, or unlabeled
irrelevant peptide (GIGKFLHSAKKFGKAFVGEIMNS), a nonspecific competitor.
Subcellular Fractionation Study.
Subcellular fractionation was performed as described previously
(6)
. Isolation of nuclear, cytoplasmic, and cell membrane
fractions was confirmed by Western blot analysis using antibodies
specific for human retinoblastoma protein, glutathione transferase, and
GLUT-1 glucose transporter protein, respectively (not shown).
Individual fractions were electrophoresed and viewed. An equivalent
amount of each fraction was loaded.
Protease Protection Assay.
Peptide-incubated cells were rinsed with PBS, scraped, pelleted by
centrifuging at 2000 rpm, and resuspended in 100 µl of PBS. To lyse,
cells were freeze-thawed 10 times using dry ice. After treating with
chymotrypsin (10 units) for 5 min at 25°C, the enzyme was inactivated
with SDS (1%). Samples were suspended in Sample buffer,
electrophoresed, and viewed. No peptide was detected in cell extract
when incubated with chymotrypsin-pretreated peptide (not shown).
Primary Tissue Analysis.
A biopsy of human invasive squamous cell cancer was rapidly frozen in
optimum cutting temperature compound blocks and
4-µm-thick cryostat sections were prepared. No fixative or embedding
material was used to avoid modifying molecules that may interact with
HN-1. As H&E dyes fluoresced under the wavelength used for viewing
fluorescein, an untreated adjacent section was incubated with
FITC-HN-1. After incubating with FITC-HN-1 (2.6 µM) in
PBS-GLY (PBS containing 10 mM glycine and 0.01% BSA) for
12 h at 25°C in a sealed environment, slides were rinsed in
PBS-GLY for 48 h with frequent changes. Samples were mounted and
viewed as described above.
Animal Experiment.
Five week-old female nude mice (Harlan Sprague Dawley) purchased from
Parke-Davis (Morris Plains, NJ) were injected s.c. with 5 x 106 tumor cells suspended in PBS. Mice
harboring tumors (0.5 cm in diameter) were randomized into separate
groups (five per group), and peptides or other indicated agents
(2.6 x 10-8 mole), suspended in
100 µl of PBS, were injected at the tail vein. All of the mice were
maintained under identical conditions. After 48 h, mice were
euthanized, and their tissues were recovered to prepare cryostat
sections. Autofluorescence was suppressed with Eriochrome Black T
(1.3%). For peptide extraction, specimens (harvested after perfusion
with PBS) of equivalent mass were frozen in liquid nitrogen,
pulverized, and resuspended in Lysis buffer [50 mM Tris
(pH 7.4), 250 mM NaCl, 5 mM EDTA, 0.5% NP40,
and protease inhibitors]. After centrifuging to remove nuclei and
other cell debris, the supernatant was electrophoresed and viewed as
described above. For the extract analysis, mice were injected with 2.6
x 10-7 mole of FITC-HN-1. All animal protocols
were reviewed and approved by the institution’s Animal Care and Use
Committee.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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RGE), resulting in abolished binding
properties (7, 8, 9)
.
To mimic drug delivery, synthetic TSPLNIHNGQKL peptide (HN-1) was
conjugated to fluorescein, a complex organic molecule with 44% of
the molecular mass of paclitaxel (Taxol; Ref. 10
). After
incubation in FITC-HN-1 for 48 h, the human HNSCC cell lines that
we examined (MDA138Tu, MDA159Tu, MDA167Tu, MDA686Tu, MDA1986Tu, and
MDA177Tu) exhibited internal fluorescence. In contrast, little
fluorescence was observed with similarly incubated human papilloma
virus-immortalized normal human oral keratinocytes (Fig. 1, a and b)
. The fluorescence intensity was time-
and dose-dependent. The distribution of cells with respect to
fluorescence intensity for each cell line is shown in Fig. 1b
. Internal fluorescence was also observed when the cells
were not fixed, excluding the possibility of the peptide being
artifactually internalized during fixing. The viability of fluorescing
cells was confirmed by trypan blue exclusion (not shown). None of the
cell lines exhibited autofluorescence (see Fig. 1a
for
untreated MDA177Tu cells; others not shown). When the lysate of
FITC-HN-1-incubated MDA177Tu cells was electrophoretically separated
and viewed with UV light, intact peptide was detected (Lane
4 of Fig. 1d
; Lane 3 of Fig. 1e
).
Degradation by an externally applied protease occurred only with prior
cell lysis, confirming the internalization of FITC-HN-1 (Fig. 1d)
. The fact that fluorescein did not get internalized
(Fig. 1a)
, that little dissociation of fluorescein from the
peptide was detected when the medium of FITC-HN-1-incubated cells was
analyzed (not shown), and that little labeling occurred after
incubating with fluorescein and HN-1 separately (Fig. 1a)
suggests that the possibility of the observed fluorescence being
attributable to the uptake of dissociated fluorescein is unlikely.
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and the
subcellular fractionation data (Fig. 1e)
indicate that the
internalized HN-1 was present mainly in the cytoplasm, which is
consistent with the fact that HN-1-displaying phages were isolated from
the cytoplasm during the screening (see "Materials and Methods").
Under higher magnification, a punctate fluorescence pattern was
observed, which suggests that HN-1 may be compartmentalized after entry
into the cell (Fig. 1c)
. The punctate pattern is similar to
that previously observed with internalized epidermal growth factor,
which enters via receptor-mediated endocytosis (11)
.
Internalization of HN-1 was also observed after it was conjugated to
Texas Red (Fig. 1a)
. Because fluorescein (Fig. 1a)
and Texas Red (not shown) are impermeable, the dyes
themselves could not have mediated the internalization of HN-1.
Shifting the relative position of HN-1 with respect to the peptide
[GGGTSPLNIHNGQKLGGGS (HN-2) or GSRRASVTSPLNIHNGQKL (HN-3)] did not
inhibit its internalization (not shown), but jumbling the sequence did
[NQHSKNTLLIGP (HN-J); Fig. 1a
, panel 3], which
suggests that HN-1 internalization is "position-independent" but
"sequence-dependent." The uptake of FITC-conjugated HN-2 or HN-3
excludes the possibility that the ability of HN-1 to enter cells is a
property acquired through its juxtaposition with fluorescein.
To determine whether HN-1 internalization occurs specifically, a
competition assay was performed. Whereas the internalization of
FITC-HN-1 was blocked by unlabeled HN-1 when provided in excess, no
such inhibition occurred with an irrelevant peptide (Fig. 1f)
. This suggests that HN-1 uptake may require a specific
interaction with a heterologous molecule, which may be a
cell-associated molecule or a molecule present in the growth medium.
The latter possibility, however, appears unlikely given that HN-1
internalization also occurred in PBS (not shown).
Intriguingly, DU145 human prostate, SW480 human colon, or U373 MG human
astrocytoma cells displayed little fluorescence (Fig. 1b)
,
even after a prolonged (96–120-h) incubation with FITC-HN-1, which
indicated that HN-1 uptake does not occur ubiquitously. That the lack
of its uptake was not attributable to the degradation of peptide in the
medium was independently confirmed (not shown). The results also
suggest that not all actively dividing cells can internalize HN-1.
An in situ peptide-binding assay performed on cryostat
sections prepared from a biopsy sample of human invasive HNSCC, which
contained invasive malignant cells as well as adjacent nonmalignant
squamous epithelium, showed the preferential binding of FITC-HN-1 to
invasive cancer cells (Fig. 2)
. The inability of fluorescein or FITC-HN-J to bind (Fig. 2)
suggests
that the binding of FITC-HN-1 was mediated by HN-1.
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and 4b)
. As in in vitro, little labeling of the nuclei
was observed in vivo. The untreated tumor cells did not
autofluoresce (Fig. 3a)
. The presence of intact peptide was
confirmed by the electrophoretic analysis of tumor extract (Fig. 3b)
. Inasmuch as the latter was prepared from a
FITC-HN-1-injected mouse harboring MDA167Tu-derived xenograft, it
suggested that HN-1 can be internalized by xenografts derived from
multiple HNSCC cell lines. Little labeling of tumor cells was observed
after injecting equimolar concentrations of fluorescein, FITC-HN-J, or
fluorescein and unlabeled HN-1 separately into mice with size-matched
tumors (Fig. 3a)
. FITC-HN-1 failed to label xenografts
derived from prostate cancer cell line DU145 cells (Fig. 3a)
, which poorly internalizes the peptide in
vitro (Fig. 1b)
. Importantly, brain, heart, lung,
kidney, and liver from FITC-HN-1-injected tumor-bearing mice showed
little labeling (Fig. 3, b and c)
. A similar
result was also obtained with FITC-HN-1-injected tumor-free mice (not
shown), which suggested that the inefficient labeling was not
attributable to peptide depletion by the tumor. The alternate
possibility of its being attributable to rapid peptide degradation in
these tissues or attributable to the inability of the peptide to
recognize the murine homologue of its cognate receptor cannot be
excluded.
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, panel
6, were examined. H&E staining showed that the lower half was
comprised of dispersed tumor cells, whereas the remainder contained
tumor cells compartmentalized (Fig. 4b)
that resembled
well-differentiated human HNSCC (11)
. An adjacent section
from the treated mice showed fluorescing tumor cells (Fig. 4b)
. [The fluorescence was attributable to tumor cells, not
keratins (compare panels 2 and 3 of Fig. 4b
).] Fluorescence appeared ubiquitous, because the tumor
cells that were located centrally as well as peripherally fluoresced
(Fig. 4b)
. Labeling of tumor cells located at the interior
suggests that FITC-HN-1 is capable of penetrating tumor tissues.
Histological sections prepared from other points throughout the tumors
from the treated mice also showed diffuse and homogeneous fluorescing
tumor cells (not shown). |
Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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This study lacks data regarding in vitro internalization and in situ binding assays for squamous cell carcinoma derived from other organ sites including the lung, cervix, esophagus, or skin. The potential of HN-1 to internalize in other squamous malignancies will be further investigated to determine whether this approach may have other far-reaching considerations.
We believe that HN-1 represents a novel peptide and that multiple data suggest that its internalization may be receptor mediated. First, internalization of labeled HN-1 is specific as demonstrated by competition assay. Second, HN-1 entry also occurs in serum free conditions (PBS), which suggests that the interacting molecule of HN-1 is not present in growth medium. Lastly, shifting the position of individual amino acids of HN-1 with respect to the overall peptide did not affect its internalization. All of the above data suggest that HN-1 may require specific interaction with a cognate cellular receptor. The exact sequence that mediates this interaction is currently not known and will require additional analysis.
Previously, it was shown that drugs conjugated to tumor vasculature-specific peptides could eliminate tumors indirectly by destroying endothelial vessels (7) . However, because tumors smaller than 1 mm3 can persist through nutrients obtained from adjacent normal blood vessels, the task of eliminating the remaining tumor still remains (12) . Hopefully, our isolation of HN-1 may allow physicians to provide the necessary dose of a drug to destroy tumors without being restricted by the occurrence of harmful side effects to other cells. The potential of HN-1 as a shuttle is further strengthened by the fact that it is nontoxic (no histological evidence of organotoxicity was observed in HN-1-injected mice), nonimmunogenic in mice (data not shown), stable in vivo, protects its "cargo" during transit, and accumulates efficiently within the tumor in 48 h. As a result, efforts to conjugate paclitaxel, the most potent chemotherapeutic for treating HNSCC, to HN-1 are currently in progress. If the internalized HN-1 is compartmentalized in endosomes, the release of conjugated drugs to cytosol may need to rely on the endosomal degradation of the peptide (13) . Other potential uses of HN-1 may include tumor diagnosis, imaging, or radioablation. It may also provide tumor-specificity to gene transfer approaches (14) ; this is supported by our observation that HN-1 can enhance the transfer of liposome-DNA complexes into HNSCC cells.4
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grant R29 DE11689-01A1 (to
G. L. C.) and an Oral Cancer Research Center of Excellence Award
1P50DE11906-01 (to G. L. C.) from the National Institute of Dental
and Craniofacial Research, Cancer Center Support Grant NIH-NCI-CA
16672, Training of Academic Head and Neck Surgeons Grant T32 CA60374-03
(to G. L. C), the Michael A. O’Bannon Endowment for Cancer Research,
the Betty Berry Cancer Research Fund, and a Tobacco Research grant
appropriated through the State of Texas Legislature. 
2 To whom requests for reprints should be
addressed, at M. D. Anderson Cancer Center, Department of Head and
Neck Surgery, 1515 Holcombe Boulevard, Box 69, Houston, Texas 77005.
Phone: (713) 792-6920; Fax: (713) 794-4662; E-mail: gclayman{at}mdanderson.org
3 The abbreviations used are: HNSCC, head
and neck squamous cell carcinoma; NHF, normal human fibroblasts.
Received 7/21/00. Accepted 10/17/00.
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