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Transducible Peptide Therapy for Uveal Melanoma and Retinoblastoma
J. William Harbour, MD;
Lori Worley, BS;
Duanduan Ma, PhD;
Michael Cohen, BS
Arch Ophthalmol. 2002;120:1341-1346.
ABSTRACT
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Objective To determine whether transducible peptides that inhibit the oncoproteins
HDM2 and Bcl-2 may selectively kill uveal melanoma and retinoblastoma cells.
Methods Peptides were tested by viability assay, flow cytometry, TUNEL (terminal
deoxynucleotidyl transferase–mediated fluorescein-dUTP nick-end labeling)
assay, Western blot analysis, and reverse transcription–polymerase chain
reaction in cultured eye tumor cells and normal cells. Preclinical studies
were performed in a rabbit xenograft model of retinoblastoma.
Main Outcome Measures Cell survival, apoptosis, gene expression, and tumor regression.
Results The anti–Bcl-2 peptide induced apoptosis in tumor cells, but it
also caused apoptosis in normal cells in culture and induced retinal damage
after intravitreal injection. In contrast, the anti-HDM2 peptide induced rapid
accumulation of p53, activation of apoptotic genes, preferential killing of
tumor cells, and minimal retinal damage after intravitreal injection. The
anti-HDM2 peptide also induced regression of human retinoblastoma cells in
rabbit eyes.
Conclusions Peptide transduction is a promising new approach to molecular eye cancer
therapy. Inhibition of HDM2 can selectively activate p53 in transformed cells
and may be an effective strategy for inducing apoptosis in eye cancer cells
with minimal damage to normal ocular tissues.
Clinical Relevance Molecular characteristics of uveal melanoma and retinoblastoma may be
used to design novel therapeutic agents that have greater specificity and
fewer adverse effects than current therapies.
INTRODUCTION
UVEAL MELANOMA and retinoblastoma are the most common primary intraocular
cancers in adults and children, respectively. Current treatments for eye cancer,
such as radiation and chemotherapy, are effective in many patients. However,
the mechanisms of action of these modalities are nonspecific, leading to substantial
injury to normal tissues and consequent dose-limiting complications.1 Molecular therapy may reduce the complications of
conventional therapies by antagonizing specific molecules in cancer cells
and selectively killing tumor cells with less damage to normal cells.
Several oncoproteins have been identified as potential targets for therapeutic
inhibition, including HDM2 and Bcl-2. HDM2 inhibits p53 and maintains it at
very low levels in normal cells.2 However,
some cancers, such as uveal melanoma, seem to functionally inhibit p53 by
overexpressing HDM2.3-4 Because
p53 is rarely mutated in uveal melanoma,3, 5 inhibition
of HDM2 may trigger apoptosis in uveal melanoma cells by releasing the endogenous,
wild-type p53 from its negative interaction with HDM2.6 Bcl-2
blocks apoptosis by inhibiting pro-apoptotic proteins such as Bax.7 Bcl-2 is overexpressed in most uveal melanomas and
is the most consistently observed molecular abnormality in this eye cancer.3, 8-9 Thus, inhibition of
Bcl-2 may liberate pro-apoptotic proteins and allow them to trigger apoptosis
in uveal melanoma cells.10 Although HDM2 and
Bcl-2 have not been adequately studied in retinoblastoma, inhibition of these
proteins may also be effective in this pediatric eye cancer, which usually
expresses wild-type p53 and is susceptible to apoptosis.11-12
Transducible peptides represent a promising new technology for efficiently
delivering designer therapeutic molecules into cells.10, 13-14 Transducible
peptides contain a therapeutic domain linked to a transduction domain that
allows the chimeric peptide directly to traverse, or "transduce," cell membranes
independent of cell surface receptors or other transport molecules.15 Eye cancers provide an attractive model for testing
transducible peptides because therapeutic agents can be delivered locally
to the tumor using standard ophthalmic techniques. In this article, we present
preliminary studies to examine transducible peptide inhibitors of HDM2 and
Bcl-2 in cultured eye tumor cells and in a rabbit model of retinoblastoma.
METHODS
CULTURED CELLS
Primary uveal melanoma cells (MM-23, MM-24, and MM-26) were obtained
from 3 patients and established in short-term culture (cells used after  3
passages). Cultures of normal choroidal fibroblasts and melanocytes (MM-23N
and MM-26N) were obtained from 2 of these patients. Y79 and WERI-RB1 retinoblastoma
cells, U2OS osteosarcoma cells, and C33A cervical carcinoma cells were obtained
from American Type Culture Collection (Manassas, Va). Adult human diploid
fibroblasts and mouse embryonic fibroblasts were obtained from BioWhittaker
Inc (Walkersville, Md). Cells were maintained in Dulbecco Modified Eagle Medium
with 0.1% or 10% fetal calf serum (as indicated) at 50% to 75% confluence
by splitting 1 to 3 every 4 to 7 days. These experiments conformed to the
human studies committee protocol of Washington University.
IMMUNOHISTOCHEMICAL ANALYSIS
Immunohistochemical analysis was performed as previously described.3 Briefly, we used the streptavidin-biotin method with
the Vector ABC Elite kit (Vector Laboratories Inc, Burlingame, Calif) and
nuclear fast red for counterstain. Four-micron sections of paraffin-embedded
primary tumor tissue (MM-23, MM-24, and MM-26) or cytospins of 105 cultured
cells (U2OS, Y79, and WERI) were deparaffinized, rehydrated with alcohol,
and treated with 0.3% hydrogen peroxide. Heat-induced antigen retrieval was
performed. Antibodies against HDM2 (NCL-MDM2; Novocastra Laboratories Ltd,
Newcastle, England), 1:30 dilution; p53 (clone 1801; Biogenics, Napa, Calif),
1:80 dilution; and Bcl-2 (Dako, Glostrup, Denmark), 1:500 dilution, were applied
at 4°C overnight. The percentage of positive cells for each antibody was
estimated by counting at least 200 cells in at least 8 fields with magnification
x40 for each specimen.
PEPTIDES
Transducible peptides were generated by linking the Tat transduction
sequence YGRKKRRQRRRG to the amino-terminus of the following peptides: Tat- HDM2
(a 12–amino acid peptide derived from the p53 sequence that mediates
binding to HDM2), Tat-HDM2-ala (a 12–amino acid control mutant
of the HDM2 binding peptide in which the residues required for binding to
HDM2, corresponding to Phe-19 and Trp-23 in p53, are converted to alanine),
Tat-Bcl2 (a 19–amino acid peptide derived from the BH3 domain
of the protein Bad, which binds and inhibits the anti-apoptotic proteins Bcl-2
and Bcl-XL), Tat-scrambled (an 8–amino acid control peptide
with a randomly scrambled sequence), Bcl2 (the Bcl-2 binding peptide
without Tat), and Tat alone. Peptides were synthesized (John Gorka, PhD, Biomolecules
Midwest, Inc, St Louis, Mo) using an ABI 431 synthesizer and eluted with 0.1N
acetic acid. Amino acid content and purity greater than 90% were confirmed
by mass spectrometry. Peptides were solubilized in sterile water and analyzed
at a range of 50 to 300µM. The optimal ratio of tumor cell–normal
cell killing was observed at 200µM, so most subsequent studies were
performed at this concentration.
CELL STUDIES
Cell survival was measured by MTS assay (Promega Inc, Madison, Wis).
Cells were seeded onto 96-well plates at 104 cells/well in Dulbecco
Modified Eagle Medium with 10% fetal calf serum. Peptides were added to the
media the next day, and spectrophotometric assay was performed 24 hours later.
TUNEL (terminal deoxynucleotidyl transferase–mediated fluorescein-dUTP
nick-end labeling) assay was performed 8 hours after addition of the indicated
peptides using the Apoptag kit (Intergen Inc, Purchase, NY). The broad-spectrum
caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone (BIOMOL
Research Laboratories Inc, Plymouth Meeting, Pa) was added to MTS plates as
indicated. For Western blot analysis and reverse transcription–polymerase
chain reaction (RT-PCR), cells were obtained at various points after the addition
of 200µM Tat-HDM2, lysates were obtained, and protein concentration
was normalized. Western blot analyses were performed using a polyclonal p53
antibody (sc-6243; Santa Cruz Biotechnology Inc, Santa Cruz, Calif), dilution
1:500, and a polyclonal p73 antibody (sc-7957; Santa Cruz Biotechnology),
1:500 dilution. For semiquantitative RT-PCR, total RNA was obtained using
the RNEasy kit (Qiagen, Valencia, Calif). Reverse transcription was performed
in 20 µL of RNase-free water containing 5mM magnesium chloride, 10mM
Tris-hydrochloride (pH 8.8), 50mM potassium chloride, 0.1% Triton X-100, 1mM
dNTP, 20 U of rRNase inhibitor, 15 U of AMV reverse transcriptase (Promega
Inc), and 0.5 µg oligo(dT)15 nucleotide primers at 42°C for 60 minutes,
99°C for 5 minutes, and then 5°C for 5 minutes. For PCR, 5 µL
of the reverse transcription product was added to a 25-µL mixture containing
primers for p21, Bax, Pig3, or GAPDH, and 2.5 U of Taq polymerase in standard
PCR buffer (GIBCO BRL, Grand Island, NY). Reaction cycles (n = 20-25) and
temperatures varied for each gene (details and primer sequences are available
from the authors on request).
ANIMAL EXPERIMENTS
New Zealand White rabbits (2-4 kg) were used. For toxicity studies,
peptides were injected into the vitreous cavity, and eyes were obtained 48
hours later for histopathologic examination. To create intraocular tumors,
107 WERI cells were injected into the right anterior chambers of
animals immunosuppressed with cyclosporine (15 mg/kg per day). Tumors were
observed daily by visual inspection of the anterior chamber, and they were
observed to implant and begin growing within 1 to 2 weeks. Peptides were dissolved
in sterile water and injected into the anterior chambers at an estimated intraocular
concentration of 200µM. Two injections were administered over 3 days,
and animals were humanely killed 48 hours later. Eyes were embedded in paraffin,
stained with hematoxylin-eosin, or analyzed for apoptosis using the Apoptag
TUNEL kit. Tumor destruction was quantified by projecting histopathologic
sections and scoring areas of viable and apoptotic tumor. Studies conformed
to the animal studies committee protocol of Washington University.
RESULTS
Amino acid sequences for peptides used in this study are listed in Table 1. The immunohistochemical expression
of p53, HDM2, and Bcl-2 for each tumor sample is summarized in Table 2. Transducible peptides tagged with fluorescein were shown
to enter approximately 100% of cells and cell nuclei within 1 to 2 minutes
of addition to culture media (data not shown), as previously described.15
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Table 1. Peptides Used in This Study
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Table 2. Immunohistochemical Expression Profile of Tumors in This Study
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INHIBITION OF Bcl-2
Tat-Bcl2 efficiently induced cell death in MM-23, MM-24, MM-26,
Y79, WERI, and U2OS cells (Figure 1 and Figure 2A). However, Tat-Bcl2 also
induced variable levels of cell death in normal primary choroidal cells (MM-23N
and MM-26N), mouse embryonic fibroblasts, and normal human diploid fibroblasts
(Figure 2A). Intravitreal injection
of Tat-Bcl2 in normal rabbit eyes (approximate intraocular concentration,
100µM) caused mild to moderate disruption of retinal cells, especially
in the inner nuclear layer (Figure 2B). Approximately 75% of the apoptosis induced by Tat-Bcl2 was blocked
by the caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone
(Figure 3A).
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Figure 1. Representative 24-hour survival
curves measured by MTS assay (Promega Inc, Madison, Wis) (in triplicate) in
MM-23 uveal melanoma cells, WERI retinoblastoma cells, U2OS osteosarcoma cells,
and C33A cervical carcinoma cells after treatment with the indicated peptides.
Error bars represent SEMs.
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Figure 2. Effect of Tat-Bcl2 and
Tat-HDM2 on tumor cells vs normal cells. A, Cell death (measured by
24-hour MTS assay [Promega Inc, Madison, Wis]) after treatment of cultured
cells with 200µM Tat-Bcl2 or 200µM Tat-HDM2. Cells
include uveal melanoma cells (MM-23, MM-24, and MM-26), retinoblastoma cells
(Y79 and WERI), normal primary choroidal cells (MM-23N and MM-26N), normal
mouse embryo fibroblasts (MEFs), and normal human diploid fibroblasts (NHDFs).
B, Histologic sections of rabbit retina obtained 24 hours after intravitreal
injection of Tat-Bcl2 or Tat-HDM2 and stained with hematoxylin-eosin
(original magnification x20). Error bars represent SEMs.
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Figure 3. A, Cell death (measured by MTS
assay [Promega Inc, Madison, Wis]) in U2OS cells treated with the indicated
peptides and the caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone
(Z-VAD). Error bars represent SEMs. B, TUNEL (terminal deoxynucleotidyl transferase–mediated
fluorescein-dUTP nick-end labeling) staining of U2OS cells that were untreated
(left) or treated for 8 hours with 200µM Tat-HDM2 (right). Dark
brown nuclear staining indicates a positive TUNEL reaction (original magnification
x40). Note the nuclear blebbing and DNA margination consistent with
apoptosis. C, Western blot analysis using anti-p53 or anti-p73 antibodies
and semiquantitative reverse transcription–polymerase chain reaction
using primers for p21, Pig3, Bax, and GAPDH (control).
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INHIBITION OF HDM2
Tat-HDM2 efficiently killed MM-23, MM-24, MM-26, Y79, WERI, U2OS,
and C33A cells (Figure 1 and Figure 2A). Tat-HDM2 induced positive
TUNEL staining in tumor cells consistent with apoptosis (Figure 3B). At concentrations that induced apoptosis in tumor cells,
Tat-HDM2 had little effect on normal cells (Figure 2A and Figure 4).
Intravitreal injection of Tat-HDM2 in normal rabbit eyes (approximate
intraocular concentration, 200µM) induced minimal histologic damage
to the retina and other ocular tissues (Figure
2B). Treatment of cells with Z-Val-Ala-Asp(OCH3)-fluoromethylketone
blocked approximately 65% of the apoptosis induced by Tat-HDM2 (Figure 3A). In U2OS cells, which express
low levels of wild-type p53 due to overexpression of HDM2, Tat-HDM2
caused a rapid increase in p53 protein levels, which remained high for 6 hours
after treatment. Tat-HDM2 also caused an increase in p73 protein levels
(Figure 3C). By semiquantitative
RT-PCR, Tat-HDM2 activated the p53 target genes p21, Pig3, and Bax,
which reached peak protein levels 4 to 6 hours after treatment (Figure 3C).
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Figure 4. Cultured normal primary choroidal
cells (MM-23N) (A) and low-passage uveal melanoma cells (MM-23) (B) from the
same patient. Treatment of cells with 200µM Tat-HDM2 for 8 hours
resulted in minimal change in the normal cells (C) and extensive death of
melanoma cells, with nuclear blebbing and fragmentation consistent with apoptosis
(arrow) (D) (hematoxylin-eosin, original magnification x40).
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An animal model of intraocular retinoblastoma was created by injecting
107 WERI human retinoblastoma cells into the anterior chambers
of rabbit eyes and allowing the tumors to implant and grow (Figure 5A). After injection of Tat-HDM2 (approximate intraocular
concentration, 200µM), tumors began to dissolve into a fine cloud within
24 hours. In contrast, tumors treated with the Tat-scrambled control peptide
remained unchanged from their pretreatment appearance. Peptides were injected
again 24 hours later, and the eyes were obtained. By histopathologic examination,
Tat-HDM2 caused extensive tumor destruction, with a 76% reduction in
viable tumor mass compared with tumors treated with the control peptide (Figure 5B-E). A much larger proportion of
tumor cells were positive for TUNEL staining in eyes treated with Tat-HDM2
compared with eyes treated with the control peptide (Figure 5F and G). No histologic damage to the cornea, lens, retina,
or other ocular tissues was detected.
COMMENT
In this study, we tested transducible peptides that inhibit HDM2 and
Bcl-2 for their ability to induce tumor-specific apoptosis in uveal melanoma
and retinoblastoma cells. An anti–Bcl-2 peptide induced apoptosis in
tumor cells but also caused variable levels of toxicity in normal cells and
tissues. In contrast, an anti-HDM2 peptide induced apoptosis in tumor cells,
with little effect on normal cells in a therapeutic dose range. This peptide
also caused regression of retinoblastoma in rabbit eyes, with minimal damage
to normal ocular tissues. We conclude that inhibition of HDM2 may be a promising
strategy for the treatment of uveal melanoma and retinoblastoma and that transducible
peptides may be an effective technology for local delivery of anticancer therapy
to the eye.
Since Bcl-2 is frequently overexpressed in uveal melanomas and is thought
to protect tumor cells from cell death,3, 8-9 we
tested whether inhibition of Bcl-2 might lead to apoptosis in uveal melanoma
cells. For these experiments, we generated the peptide Tat-Bcl2, which
is derived from the BH3 domain of the pro-apoptotic protein Bad, which has
been shown to bind and inhibit the anti-apoptotic proteins Bcl-2 and Bcl-XL.10 Tat-Bcl2 efficiently induced
apoptosis in each of the uveal melanoma cell lines, which were all derived
from tumors that overexpressed Bcl-2 (Table
2). This peptide induced apoptosis that was largely caspase dependent,
consistent with activation of the Bcl-2 mitochondrial apoptotic pathway. Tat-Bcl2
also killed U2OS osteosarcoma cells and Y79 and WERI retinoblastoma cells,
although Bcl-2 was not overexpressed in these cells (Table 2). In addition, the peptide had a moderate cytotoxic effect
on normal cells, suggesting that inhibition of Bcl-2 may trigger apoptosis
in a broad spectrum of cells by destabilizing the delicate balance between
pro-apoptotic and anti-apoptotic proteins in the Bcl-2 pathway. Activation
of this pathway, which is downstream of the p53 tumor suppressor checkpoint,
may then lead to generalized activation of the apoptotic program. Therefore,
the anti–Bcl-2 peptide did not demonstrate a significant therapeutic
window and was not studied further.
HDM2 binds and inhibits p53, maintaining it at low concentrations in
normal cells. In cells that sustain oncogenic mutations, the HDM2-p53 interaction
is disrupted, allowing p53 to accumulate, become activated, and induce growth
arrest or apoptosis.16 Therefore, most tumors
develop strategies for circumventing this tumor suppressor checkpoint, such
as by mutating p53 or overexpressing HDM2. Because HDM2 is strongly expressed
in most uveal melanomas and may serve to functionally inactivate p53,3-4 we became interested in HDM2 as a potential
therapeutic target. We hypothesized that a peptide that blocks HDM2 may liberate
p53, which may then become activated in response to the oncogenic changes
in cancer cells but may remain inactive in normal cells. For these experiments,
we developed a novel peptide, Tat-HDM2, which contains the Tat transduction
domain linked to a sequence from p53 that contains the binding site for HDM2.17 A similar peptide has been shown to competitively
inhibit the binding of HDM2 to p53 and to trigger the accumulation of activated
p53.6, 18 For some of the initial
experiments, we used U2OS osteosarcoma cells because they overexpress HDM2
and express wild-type p53 (similar to uveal melanomas), and they are more
practical to work with in culture.19 As expected,
Tat-HDM2 caused a rapid increase in p53 protein levels and activation
of p53 target genes involved in cell cycle arrest and apoptosis (eg, p21,
Bax, and Pig3). These molecular changes were accompanied by apoptosis in U2OS
cells and in all 3 uveal melanomas that were tested. Tat-HDM2 was also
effective against Y79 and WERI retinoblastoma cells, which express wild-type
p5311 but do not overexpress HDM2 (Table 2), suggesting that HDM2 overexpression
is not a requirement for this peptide to be effective. Tat-HDM2 also
killed C33A cells, which express a mutant form of p53, suggesting that expression
of wild-type p53 may also not be a requirement for this peptide to be effective.
This observation may be because Tat-HDM2 also induces the accumulation
of p73, a member of the p53 family that binds HDM2 and can mediate p53-independent
apoptosis.20 Tat-HDM2 may also stabilize
mutant p53, as has been observed with other peptide fragments of p53.14 These findings, along with the observation that Tat-HDM2
triggers caspase-dependent and caspase-independent apoptotic mechanisms (Figure 4A), suggest that Tat-HDM2
may have multiple mechanisms of action. Tat-HDM2 caused minimal damage
to normal cells, and injection of the peptide into the vitreous cavity of
rabbit eyes did not result in histologic damage to the retina or other ocular
tissues. The selective killing of tumor cells by p53 may be due to the unique
ability of p53 to become activated in tumor cells to a greater extent than
in normal cells.21
Because Tat-HDM2 showed the most specificity for tumor cells
in cultured cells, we further tested this peptide in a rabbit xenograft model
of retinoblastoma. Tat-HDM2 caused tumor dissolution and apoptosis
of tumor cells without significant damage to normal ocular tissues. In these
preliminary studies, the peptide was delivered directly into the anterior
chamber, but we would not anticipate intraocular injection in patients with
eye cancer owing to concern about tumor dissemination. Instead, it may be
possible to deliver transducible peptides by topical, subconjunctival, retrobulbar,
or systemic routes because these peptides have been shown to rapidly cross
cell membranes, diffuse through tissues, and cross the blood-brain barrier.22 Because transducible peptides function independently
of transport proteins, they may also be helpful in overcoming multidrug resistance
in retinoblastoma resulting from overexpression of P-glycoprotein.23 These studies provide an initial framework for future
studies to determine appropriate molecular targets, optimal routes of administration,
and indications for and limitations of transducible peptide therapy for eye
cancers.
AUTHOR INFORMATION
Submitted for publication October 16, 2001; final revision received
March 28, 2002; accepted June 13, 2002.
This work was supported by grants K08 EYOO382-01 and RO1 EY13169-01
(Dr Harbour) and a departmental core grant from the National Eye Institute,
Bethesda, Md, and by an unrestricted departmental grant from Research to Prevent
Blindness Inc, New York, NY.
Corresponding author and reprints: J. William Harbour, MD, Department
of Ophthalmology and Visual Sciences, Washington University School of Medicine,
Campus Box 8069, 660 S Euclid Ave, St Louis, MO 63110 (e-mail: harbour{at}vision.wustl.edu).
From the Department of Ophthalmology and Visual Sciences and the Division
of Molecular Oncology, Washington University, St Louis, Mo.
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