U.S. patent application number 12/055164 was filed with the patent office on 2009-04-30 for selective inhibitors of nuclear factor kappab activation and uses thereof.
Invention is credited to Bharat B. Aggarwal, Sujay Singh.
Application Number | 20090111754 12/055164 |
Document ID | / |
Family ID | 34590199 |
Filed Date | 2009-04-30 |
United States Patent
Application |
20090111754 |
Kind Code |
A1 |
Aggarwal; Bharat B. ; et
al. |
April 30, 2009 |
Selective Inhibitors of Nuclear Factor KappaB Activation and Uses
Thereof
Abstract
The present invention provides cell permeable NF-.kappa.B
inhibitors consist of a polypeptide derived from the p65 subunit of
NF-.kappa.B and a protein transduction domain derived from
antennapedia third helix sequence. The inhibitor suppressed
NF-.kappa.B activation induced by TNF, LPS, IL-1, okadaic acid,
PMA, H.sub.2O.sub.2 and cigarette smoke condensate.
NF-.kappa.B-regulated reporter gene expression induced by TNF,
TNFR1, TRADD, TRAF2, NIK, IKK and p65 was suppressed by the
inhibitor. The inhibitor enhanced TNF- and chemotherapeutic
agent-induced apoptosis. Overall these results demonstrate a
NF-.kappa.B inhibitor that can selectively inhibit NF-.kappa.B
activation induced by various inflammatory stimuli, down-regulate
NF-.kappa.B mediated gene expression and upregulate apoptosis.
Inventors: |
Aggarwal; Bharat B.;
(Houston, TX) ; Singh; Sujay; (San Diego,
CA) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, L.L.P.
600 CONGRESS AVENUE, SUITE 2400
AUSTIN
TX
78701
US
|
Family ID: |
34590199 |
Appl. No.: |
12/055164 |
Filed: |
March 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10981082 |
Nov 4, 2004 |
7368430 |
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12055164 |
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60517852 |
Nov 6, 2003 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
C07K 14/4702 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
514/14 |
International
Class: |
A61K 38/10 20060101
A61K038/10 |
Goverment Interests
FEDERAL FUNDING LEGEND
[0002] This invention was produced in part using funds obtained
through a Department of Defense US Army Breast Cancer Research
Program grant (BC010610), a PO1 grant (CA91844) from the National
Institutes of Health and a P50 Head and Neck SPORE grant from the
National Institutes of Health. Consequently, the federal government
has certain rights in this invention.
Claims
1-14. (canceled)
15. A method of treating cancer in an individual, comprising the
step of administering to the individual a composition comprising:
(a) a polypeptide comprising (i) a sequence selected from the group
consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; and (ii)
a transduction sequence selected from the group consisting of SEQ
ID NO:3, a herpes virus structural protein transduction sequence,
and HIV tat protein; and (b) a pharmaceutically acceptable
carrier.
16. The method of claim 15, further comprising the step of
administering to said individual a chemotherapeutic agent.
17. The method of claim 16, wherein the chemotherapeutic agent is
doxorubicin or cisplatin.
18. The method of claim 15, wherein the cancer is metastatic
cancer.
19. The method of claim 15, wherein the composition is administered
intraperitoneally, intramuscularly, subcutaneously, intravenously,
or orally.
20. The method of claim 15, wherein the polypeptide comprises SEQ
ID NO:5.
21. The method of claim 15, wherein the polypeptide comprises SEQ
ID NO:6.
22. The method of claim 15, wherein the polypeptide comprises SEQ
ID NO:7.
23. The method of claim 15, wherein the polypeptide comprises SEQ
ID NO:8.
24. The method of claim 15, wherein the polypeptide comprises SEQ
ID NO:9.
25. The method of claim 15, wherein the polypeptide comprises SEQ
ID NO:11.
26. The method of claim 15, wherein the polypeptide comprises SEQ
ID NO:12.
27. The method of claim 15, wherein the polypeptide comprises SEQ
ID NO:13.
28. The method of claim 15, wherein the transduction sequence is
SEQ ID NO:3.
29. The method of claim 15, wherein the transduction sequence is a
herpes virus structural protein transduction sequence.
30. The method of claim 15, wherein the transduction sequence is
HIV tat protein.
31. The method of claim 15, wherein the polypeptide consists of (i)
a sequence selected from the group consisting of SEQ ID NO:5, SEQ
ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ
ID NO:12 and SEQ ID NO:13; and (ii) a transduction sequence
selected from the group consisting of SEQ ID NO:3, a herpes virus
structural protein transduction sequence, and HIV tat protein.
32. The method of claim 31, wherein the transduction sequence is
SEQ ID NO:3.
33. The method of claim 31, wherein the transduction sequence is a
herpes virus structural protein transduction sequence.
34. The method of claim 31, wherein the transduction sequence is
HIV tat protein.
35. A method of treating cancer in an individual, comprising the
step of administering to the individual a composition comprising:
(a) a polypeptide comprising SEQ ID NO:4 or SEQ ID NO:10; and (b) a
pharmaceutically acceptable carrier.
36. The method of claim 35, wherein the polypeptide comprises SEQ
ID NO:4.
37. The method of claim 35, wherein the polypeptide comprises SEQ
ID NO:10.
38. The method of claim 35, wherein the polypeptide consists of SEQ
ID NO:4.
39. The method of claim 35, wherein the polypeptide consists of SEQ
ID NO:10.
40. The method of claim 35, further comprising the step of
administering to said individual a chemotherapeutic agent.
41. The method of claim 40, wherein the chemotherapeutic agent is
doxorubicin or cisplatin.
42. The method of claim 35, wherein the cancer is metastatic
cancer.
43. The method of claim 35, wherein the composition is administered
intraperitoneally, intramuscularly, subcutaneously, intravenously,
or orally.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This patent application claims benefit of priority of
provisional patent application U.S. Ser. No. 60/517,852, filed Nov.
6, 2003, now abandoned.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the molecular
biology of nuclear factor-kappa B (NF-.kappa.B). More specifically,
the present invention relates to polypeptides that can selectively
inhibit NF-.kappa.B activation, downregulate NF-.kappa.B mediated
gene expression and enhance apoptosis induced by TNF and other
apoptotic stimuli.
[0005] 2. Description of the Related Art
[0006] Nuclear Factor-KB (NF-.kappa.B) represents a group of five
proteins, namely c-Rel, Rel A (p65), Rel B, NF-kB1 (p50 and p105),
and NF-.kappa.B2 (p52). NF-.kappa.B is regulated by a family of
inhibitors called I.kappa.B. In an inactive state, NF-.kappa.B is
present in the cytoplasm as a heterotrimer consisting of p50, p65,
and I.kappa.B.alpha. subunits. In response to an activation signal,
the I.kappa.B.alpha. subunit is phosphorylated at serine residues
32 and 36, ubiquitinated at lysine residues 21 and 22, and degraded
through the proteosomal pathway, thus exposing the nuclear
localization signals on the p50-p65 heterodimer. The p65 is then
phosphorylated, leading to nuclear translocation and binding to
specific DNA sequence, which in turns results in transcription of
various genes including cyclin D1, cyclooxyenase (COX) 2 and matrix
metalloproteinase (MMP) 9.
[0007] The p65 subunit of NF-.kappa.B, which contains at least two
strong transactivation domains (TAD) within the C terminus (TA1 30
amino acid; TA2 90 amino acid), has been shown to undergo
phosphorylation upon activation. The sites of phosphorylation and
the kinase responsible for p65 phosphorylation remain
controversial. For instance, phosphorylation at Ser 276 by protein
kinase A, at Ser 529 by casein kinase II, at Ser 536 by IKK-.beta.,
and at serine 471 by PKC-.epsilon. have been demonstrated. In
addition, phosphorylation of p65-TAD by glycogen synthase
kinase-3.beta. and by Ca.sup.2+/calmodulin-dependent protein kinase
IV have been demonstrated.
[0008] NF-.kappa.B has been shown to regulate the expression of a
number of genes whose products are involved in inflammation, viral
replication, carcinogenesis, anti-apoptosis, invasion and
metastasis. These include anti-apoptosis genes, adhesion molecules,
chemokines, inflammatory cytokines, and cell cycle regulatory
genes. Thus agents that can suppress NF-.kappa.B activation have
the potential to treat a variety of diseases that involves
inflammation, apoptosis and carcinogenesis.
[0009] Most proteins enter the cell through their specific cell
surface receptors. Recent studies, however, indicate that certain
short protein sequences can enter the cells without any receptors
and such proteins have been described as protein transduction
domain (PTD) peptides (Lindgren et al., 2000; Schwarze and Dowdy,
2000). Most of the protein transduction domain peptides are
arginine-rich peptides (Futaki et al., 2003). Importantly,
conjugation of proteins, peptides and antisense oligonucleotides to
these protein transduction domain peptides has been shown to
deliver these cargos effectively, allowing observation of
biological action in several cell and animal models (Lindgren et
al., 2000; Schwarze and Dowdy, 2000). Peptides derived from third
helix of the antennapedia homeodomain, herpes virus structural
protein, and HIV tat protein have been used to deliver both small
and large peptides of interest to the cells through an energy- and
receptor-independent mechanism (Derossi et al., 1994; Elliott and
O'Hare, 1997; Fawell et al., 1994).
[0010] Using these protein transduction domain peptides, several
peptides based on protein-protein interaction domains have been
delivered to the cells to suppress cell signaling. These include
Grb2 binding peptide, mitogen-activated protein kinase, STAT3,
NEMO-IKK interacting peptide, and peptides carrying nuclear
localization sequences. Besides peptides, protein transduction
domain peptides have also been used to deliver larger full length
polypeptides, including I.kappa.B.alpha., cyclin-dependent kinase
inhibitory protein p27, anti-apoptotic proteins Bcl-xl, and
proapoptotic proteins.
[0011] The prior art is deficient in providing a cell permeable
inhibitor specific for NF-.kappa.B. The present invention fulfills
this long-standing need and desire in the art by disclosing the
construction of a cell permeable NF-.kappa.B-specific inhibitor
comprising a NF-.kappa.B polypeptide linked to an
antennapedia-derived protein transduction domain. This inhibitor
can suppress NF-.kappa.B activation, suppress NF-.kappa.B-mediated
gene transcription and enhance apoptosis induced by TNF and other
apoptotic stimuli.
SUMMARY OF THE INVENTION
[0012] The present invention is directed to a cell permeable NF-KB
inhibitor comprising (i) a polypeptide of SEQ ID NO. 5 or 11, or
homologues or derivatives thereof, and (ii) a protein transduction
domain which is able to transport the polypeptide across cell
membrane. In general, the protein transduction domain is derived
from the third helix of the antennapedia homeodomain, herpes virus
structural protein, or HIV tat protein. In one embodiment of the
present invention, the protein transduction domain derived from the
third helix of the antennapedia homeodomain has the sequence of SEQ
ID NO. 3, and the cell permeable. NF-.kappa.B inhibitor has the
sequences of SEQ ID NO. 4 or 10.
[0013] In another aspect, the present invention provides methods of
using the NF-.kappa.B inhibitor to inhibit DNA binding activity of
NF-.kappa.B or enhance apoptosis in a cell. In general, DNA binding
activity of NF-.kappa.B induced by TNF, LPS, IL-1, okadaic acid,
PMA, H.sub.2O.sub.2, cigarette smoke condensate, TNF receptor 1
(TNFR1), TNF receptor-associated death domain (TRADD), TNF
receptor-associated factor 2 (TRAF2), NF-.kappa.B-inducing kinase
(NIK), or I.kappa.B.alpha. kinase (IKK) could be inhibited by the
inhibitor, whereas apoptosis induced by TNF, or chemotherapeutic
agent such as doxorubicin or cisplatin could be enhanced by the
inhibitor.
[0014] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention. These
embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-1B show the structure of the p65 subunit of
NF-.kappa.B. FIG. 1A show the p65 consists of a DNA-binding and
dimerization domain (RHD), nuclear localization domain (NLS), and
transactivation domain (TD). The p65 phosphorylation sites are
indicated.
[0016] FIG. 1B shows the sequence of cell-permeable peptide and the
p65 peptides. The protein transduction domain (PTD) sequence
derived from antennapedia was conjugated with p65-P1 or p65-P6 for
in vivo study. Other p65 peptides (p65-Pt, -P8) without
antennapedia segment were used for in vitro study.
[0017] FIGS. 2A-2B shows the effect of various peptides containing
the phosphorylation site of p65 on NF-.kappa.B activation. FIG. 2A
shows KBM-5 cells were treated with 0.1 nM TNF for 30 min, and the
nuclear extracts were prepared, incubated for 30 min with various
peptides, and then assayed for NF-.kappa.B activation by
electrophoretic mobility shift assay (EMSA).
[0018] FIG. 2B shows dose-dependent effect of p65-P1 peptide on
NF-.kappa.B binding to DNA in vitro. Nuclear extracts were prepared
from TNF-treated cells, incubated for 30 min with various
concentrations of peptides, and then assayed for NF-.kappa.B
activation by EMSA.
[0019] FIGS. 3A-3D shows PTD-p65-P1 polypeptide inhibits
TNF-induced NF-.kappa.B activation. FIG. 3A shows KBM-5 cells were
incubated with 150 .mu.M peptides for 1 h and treated with 0.1 nM
TNF for the indicated times. Nuclear extracts were prepared, and
then NF-.kappa.B activation was analyzed by EMSA.
[0020] FIG. 3B shows dose dependent effect. KBM-5 cells were
incubated with various concentrations of peptides for 1 hour and
treated with 0.1 nM TNF for 30 minutes. Nuclear extracts were
prepared, and then NF-.kappa.B activation was analyzed by EMSA.
[0021] FIG. 3C shows PTD-p65-P6 peptide inhibits TNF-induced
NF-.kappa.B activation. KBM-5 cells were incubated with various
concentrations of peptides for 1 h and treated with 0.1 nM TNF for
30 min. Nuclear extracts were prepared, and then NF-.kappa.B
activation was analyzed by EMSA.
[0022] FIG. 3D shows PTD-p65 peptides specifically inhibits
TNF-induced NF-.kappa.B activation. KBM-5 cells were treated with
0.1 nM TNF for 30 min. Nuclear extracts were prepared, incubated
for 30 min with different antibodies, preimmune serum (PIS),
unlabeled NF-.kappa.B oligo probes (Competitor), or mutant
NF-.kappa.B oligo probe, and then assayed for NF-.kappa.B
activation by EMSA.
[0023] FIGS. 4A-4B shows the effect of PTD-p65-P1 on TNF-induced
AP-1 activity. FIG. 4A shows KBM-5 cells were incubated with
various concentrations of peptides for 1 hour and treated with 0.1
nM TNF for 30 min. Nuclear extracts were prepared, and then
NF-.kappa.B activation was analyzed by EMSA.
[0024] FIG. 4B shows PTD-p65-P1 peptide inhibits NF-.kappa.B
activation induced by different activators. KBM-5 cells were
incubated with 150 .mu.M peptide for 1 h, treated with 0.1 nM TNF,
1 .mu.g/ml LPS, 100 ng/ml IL-1, 500 nM okadaic acid (OA), 10 ng/ml
PMA, 500 .mu.M H.sub.2O.sub.2, or 1 .mu.g/ml cigarette smoke
condensate (CSC), and then analyzed for NF-.kappa.B by EMSA.
[0025] FIGS. 5A-5C shows PTD-p65-P1 has no effect on the
TNF-induced I.kappa.B.alpha.phosphorylation or degradation, but
inhibits p65 phosphorylation and nuclear translocation. Cells were
incubated with 150 .mu.M PTD-p65-P1 for 1 h and treated with 0.1 nM
TNF for the indicated times. Nuclear and cytoplasmic extracts were
prepared and then fractionated on 10% SDS-PAGE. Western blot
analysis was performed using with phospho-specific
anti-I.kappa.B.alpha., anti-I.kappa.B.alpha., phospho-specific
anti-p65, anti-p65, and .beta.-actin.
[0026] FIG. 5B shows PTD-p65-P1 peptide has no effect on the
TNF-induced IKK activation. Cells were incubated with 150 .mu.M
PTD-p65-P1 for 1 h and treated with 0.1 nM TNF for the indicated
times. Whole-cell extracts were prepared, incubated with
anti-IKK-.alpha. antibody, and then immunoprecipitated using
protein A/G-Sepharose beads. Immunocomplex kinase reaction was
performed as described below. Whole-cell extracts were fractionated
on 7.5% SDS-PAGE and immunoblotted using anti-IKK-.alpha. and
anti-IKK-.beta. antibodies.
[0027] FIG. 5C shows IKK phosphorylates p65 peptides in cell-free
system. Whole-cell extracts were prepared from TNF-treated cells
and immunoprecipitated with antibody against IKK-.alpha..
Thereafter immunocomplex kinase assay was performed in the presence
of the peptides as a substrate.
[0028] FIGS. 6A-6C shows PTD-p65-P1 inhibits TNF-induced expression
of NF-.kappa.B-dependent gene. A293 cells were transiently
transfected with NF-.kappa.B-containing plasmid linked to the SEAP
gene and incubated with 150 .mu.M PTD-p65-P1. Cells were treated
with 1 nM TNF, and supernatants were collected and assayed for
SEAP.
[0029] FIG. 6B shows A293 cells were transiently transfected with a
NF-.kappa.B-containing plasmid along with indicated plasmids and
then incubated with 150 .mu.M PTD-p65-P1. Cells were exposed to
TNF, and supernatants of the culture medium were assayed for SEAP.
Results are expressed as fold activity of the vector control.
[0030] FIG. 6C shows KBM-5 cells were incubated with 150 .mu.M
PTD-p65-P1 for 1 h and treated with 0.1 nM TNF for the indicated
times. Nuclear and cytoplasmic extracts were prepared and then
fractionated on 10% SDS-PAGE.
[0031] FIGS. 7A-7D shows protein transduction domain-p65-P1
enhances TNF-induced cytotoxicity. Five thousand KBM-5 cells were
seeded in triplicate in 96-well plates. Cells were pretreated with
100 .mu.M PTD-p65-P1, and then incubated with the indicated
concentrations of TNF for 72 hour. Thereafter, cell viability was
analyzed by MTT assay.
[0032] FIG. 7B shows 1.times.10.sup.5 cells were pretreated with
100 .mu.M protein transduction domain-p65-P1, and then incubated
with 1 nM TNF for 16 hour. Cells were stained with Live/Dead assay
reagent for 30 min, and then analyzed under a fluorescence
microscope.
[0033] FIG. 7C shows 1.times.10.sup.5 cells were pretreated with
100 .mu.M PTD-p65-P1, and then incubated with 1 nM TNF for 16 h.
Cells were fixed, stained with TUNEL assay reagent, and then
analyzed under a fluorescence microscope.
[0034] FIG. 7D shows protein transduction domain-p65-P1 enhances
chemotherapy-induced cytotoxicity. Five thousand cells were seeded
in triplicate in 96-well plates. Cells were pretreated with 100
.mu.M protein transduction domain-p65-P1 and then incubated with
indicated concentrations of TNF or doxorubicin or cisplatin for 72
hr. Thereafter, cell viability was analyzed by the MTT method.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The following abbreviations are used herein: TNFR 1, TNF
receptor 1; PIS, preimmune serum; I.kappa.B, inhibitory subunit of
NF-.kappa.B; IKK, I.kappa.B.alpha. kinase; NIK,
NF-.kappa.B-inducing kinase; TRAF 2, TNF receptor-associated
factor-2; TRADD, TNF receptor-associated death domain; EMSA,
electrophoretic mobility shift assay; SEAP, secretory alkaline
phosphatase; IL-1, interleukin-1; PMA, phorbol myristate acetate;
SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel
electrophoresis; ALLN, N-acetyl-leucyl-leucyl-norleucinal; PTD,
protein transduction domain.
[0036] The nuclear transcription factor NF-.kappa.B has been shown
to mediate inflammation, viral replication, carcinogenesis,
anti-apoptosis, invasion and metastasis. Thus, specific inhibitors
of this factor have therapeutic potential.
[0037] The present invention identifies NF-.kappa.B inhibitors that
can suppress TNF-induced NF-.kappa.B activation in vivo. The
NF-.kappa.B inhibitor was generated by linking a cell-delivery
peptide to a polypeptide derived from the p65 subunit of
NF-.kappa.B. More specifically, polypeptides which contain
phosphorylation sites from the p65 subunit of NF-kB (e.g. SEQ ID
NO. 5 or 11) were linked to the protein transduction domain
peptides (SEQ ID NO. 3) derived from antennapedia third helix
sequence. The resulting NF-.kappa.B inhibitors include protein
transduction domain-p65-P1 (SEQ ID NO. 4, containing amino acid
271-282 of the p65 subunit) and protein transduction domain-p65-P6
(SEQ ID NO. 10, containing amino acid 525-537 of the p65
subunit).
[0038] Deletion of amino acids either from the C-terminus or the
N-terminus of the p65-derived peptides abolished the NF-.kappa.B
suppressive activity. Substitution of serine with alanine residue
also abolished the inhibitory activity of the inhibitor. A
concentration of 150 .mu.M peptide is required to suppress
NF-.kappa.B activation. Protein transduction domain-p65-P1 and
protein transduction domain-p65-P6 inhibited TNF-induced
NF-.kappa.B activation in vivo only when linked to the protein
transduction domain peptide. Linkage to cell-permeable peptide was
not required to suppress the binding of p50-65 to DNA in vitro.
[0039] Inhibitor PTD-p65-P1 had no effect on TNF-induced AP-1
activation, did not affect IkB.alpha. kinase (IKK) activation,
IkB.alpha. phosphorylation or degradation, but did suppress p65
phosphorylation and nuclear translocation. Whether p65
phosphorylation is needed for nuclear translocation is not fully
understood. It was also found that the polypeptide itself (p65-P1
and p65-P6) undergoes phosphorylation upon treatment with IKK.
These results are consistent with previous reports which
demonstrated IKK can induce phosphorylation of p65.
[0040] PTD-p65-P1 suppressed NF-.kappa.B activation induced by TNF,
LPS, IL-1, okadaic acid, PMA, H.sub.2O.sub.2 or cigarette smoke
condensate. These results indicate that the peptide inhibitor
affects a common step in NF-kB activation.
[0041] PTD-p65-P1 also suppressed NF-kB regulated reporter gene
expression induced by TNF, TNF receptor 1 (TNFR1), TNF
receptor-associated death domain (TRADD), TNF receptor-associated
factor 2 (TRAF2), NF-kB-inducing kinase (NIK), IKK and p65, and
enhanced apoptosis induced by TNF and chemotherapeutic agents There
are numerous reports which suggest that NF-.kappa.B mediates
suppression of apoptosis. Several genes that are involved in
suppression of apoptosis are regulated by NF-.kappa.B. These
include cIAP, TRAF1, TRAF2, cFLIP, survivn, bcl.sub.xl, and XIAP.
It is possible that the polypeptide inhibitors of the present
invention suppress the expression of these genes and thus
potentiate apoptosis. Several genes that are involved in
tumorigenesis, metastasis, angiogenesis and inflammation are also
regulated by NF-kB. Thus, the polypeptide inhibitors reported
herein have a potential in suppressing the synthesis of all these
gene products and may have potential for therapeutic
applications.
[0042] The present invention is directed to a cell permeable NF-KB
inhibitor comprising (i) a peptide fragment of the p65 subunit of
NF-.kappa.B, or homologues or derivatives thereof, and (ii) a
protein transduction domain which is able to transport said peptide
fragment across cell membrane. Preferably, the peptide fragment of
the p65 subunit comprises phosphorylation site(s) of the p65
subunit of NF-.kappa.B. Examples of the p65 peptide fragments
include peptides with the sequence of SEQ ID NO. 5 or 11. In
general, the protein transduction domain is derived from the third
helix of the antennapedia homeodomain, herpes virus structural
protein, or HIV tat protein. In one embodiment of the present
invention, the protein transduction domain derived from the third
helix of the antennapedia homeodomain has the sequence of SEQ ID
NO. 3. Representative examples of the NF-.kappa.B inhibitors
include peptides with the sequence of SEQ ID NO. 4 or 10.
[0043] In another embodiment, the present invention provides
methods of using the NF-.kappa.B inhibitor of the present invention
to inhibit DNA binding activity of NF-.kappa.B or enhance apoptosis
in a cell. In general, DNA binding activity of NF-.kappa.B induced
by TNF, LPS, IL-1, okadaic acid, PMA, H.sub.2O.sub.2, cigarette
smoke condensate, TNFR1, TRADD, TRAF2, NIK, or IKK could be
inhibited by the inhibitor, whereas apoptosis induced by TNF, or
chemotherapeutic agent such as doxorubicin or cisplatin could be
enhanced by the inhibitor.
[0044] In yet another embodiment, the NF-.kappa.B inhibitor of the
present invention can be used to treat cancer in an individual. It
is well known in the art that NF-.kappa.B activation plays an
important role in cancer development, and inhibition of NF-.kappa.B
activities is generally believed to be beneficial in cancer
treatment. Moreover, the NF-.kappa.B inhibitor disclosed herein can
be used in combination with chemotherapeutic agent in the treatment
of cancer.
[0045] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion. The present
examples, along with the methods, procedures, treatments,
molecules, and specific compounds described herein are presently
representative of preferred embodiments. One skilled in the art
will appreciate readily that the present invention is well adapted
to carry out the objects and obtain the ends and advantages
mentioned, as well as those objects, ends and advantages inherent
herein. Changes therein and other uses which are encompassed within
the spirit of the invention as defined by the scope of the claims
will occur to those skilled in the art.
EXAMPLE 1
Reagents and Cell Lines
[0046] Bacteria-derived human recombinant TNF, purified to
homogeneity with a specific activity of 5.times.10.sup.7 U/mg, was
kindly provided by Genentech (South San Francisco, Calif.).
Penicillin, streptomycin, Iscove's modified Dulbecco's medium, and
FBS were obtained from Invitrogen (Carlsbad, Calif.).
Lipopolysaccharide, PMA, okadaic acid, H.sub.2O.sub.2 and
anti-.beta.-actin antibody were obtained from Sigma Chemical (St.
Louis, Mo.). The cigarette smoke condensate was provided by Dr. C.
Gary Gariola (Univ. of Kentucky, Lexington, Ky.).
[0047] Polyclonal anti-p65, anti-p50, anti-I.kappa.B.alpha.,
anti-cyclin D1 and anti-MMP-9 antibodies were obtained from Santa
Cruz Biotechnology (Santa Cruz, Calif.). Phospho-specific
anti-I.kappa.B.alpha. (Ser32) antibody was purchased from Cell
Signaling (Beverly, Mass.). Phospho-specific anti-p65 antibody was
kindly provided by Rockland Laboratory. Anti-IKK-.alpha. and
anti-IKK-.beta. antibodies were kindly provided by Imgenex (San
Diego, Calif.). Anti-COX2 antibody was obtained from BD Biosciences
Pharmingen (San Diego, Calif.).
[0048] All peptides (see FIG. 1) were synthesized using an
automated peptide synthesizer (Symphony Multiplex, Rainin
Instruments, MA). The peptides were purified to more than 90%
purity using HPLC.
[0049] Leukemic cell line KBM-5 is phenotypically myeloid with
monocytic differentiation. Cells were cultured in Iscove's modified
Dulbecco's medium supplemented with 15% FBS, 100 U/ml penicillin,
and 100 mg/ml streptomycin. A293 embryonic kidney cells were
maintained in minimum essential medium supplemented with 10% FBS,
with 100 U/ml penicillin, and 100 .mu.g/ml streptomycin.
EXAMPLE 2
Electrophoretic Mobility Shift Assays
[0050] NF-.kappa.B activation was examined by electrophoretic
mobility shift assays as described (Chaturvedi et al., 1994; Takada
and Aggarwal). Briefly, nuclear extracts prepared from TNF-treated
cells (1.times.10.sup.6/ml) were incubated with
.sup.32P-end-labeled 45-mer double-stranded NF-.kappa.B
oligonucleotide (10 ug of protein with 16 fmol of DNA) from the
human immunodeficiency virus long terminal repeat,
5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTC CAGGGAGGCGTGG-3' (SEQ ID NO. 1,
boldface indicates NF-.kappa.B binding sites) for 30 min at
37.degree. C., and the DNA-protein complex formed was separated
from free oligonucleotide on 6.6% native polyacrylamide gels. A
double-stranded mutated oligonucleotide,
5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3' (SEQ ID NO. 2)
was used to examine the specificity of binding of NF-.kappa.B to
the DNA. The specificity of binding was also examined by
competition with unlabeled oligonucleotide.
[0051] For supershift assays, nuclear extracts prepared from
TNF-treated cells were incubated with antibodies against either the
p50 or p65 subunits of NF-.kappa.B for 30 min at 37.degree. C. and
then the complex was analyzed by electrophoretic mobility shift
assays. Antibodies against cyclin D1 and preimmune serum (PIS) were
included as negative controls. The dried gels were visualized, and
radioactive bands quantitated by a PhosphorImager (Molecular
Dynamics, Sunnyvale, Calif.) using Imagequant software.
EXAMPLE 3
I.kappa.B.alpha. Kinase (IKK) Assay
[0052] The IKK assay was performed by a method described previously
(Manna et al., 2000a). Briefly, IKK complex from whole-cell extract
was precipitated with antibody against IKK-A, followed by treatment
with protein A/G-Sepharose beads (Pierce, Rockford, Ill.). After a
2-h incubation, the beads were washed with lysis buffer and then
assayed in kinase assay mixture containing 50 mM HEPES (pH 7.4), 20
mM MgCl.sub.2, 2 mM dithiothreitol (DTT), 20 mCi [.gamma.-.sup.32P]
ATP, 10 mM unlabeled ATP, and 2 .mu.g of substrate
GST-I.kappa.B.alpha. (1-54). After incubation at 30.degree. C. for
30 min, the reaction was terminated by boiling with SDS sample
buffer for 5 min. Finally, the protein was resolved on 10%
SDS-PAGE, the gel was dried, and the radioactive bands were
visualized by PhosphorImager.
[0053] To determine the total amounts of IKK-.alpha. and IKK-.beta.
in each sample, 30 mg of the whole-cell protein was resolved on
7.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and
then blotted with either anti-IKK-.alpha. or anti-IKK-.beta.
antibodies. Cell-free phosphorylation of peptide by IKK was also
determined using 10 .mu.g of peptides as a substrate in the kinase
reaction mixture described above, and then fractionated on 20%
SDS-PAGE in 2.times.SDS electrophoresis buffer.
EXAMPLE 4
NF-.kappa.B-Dependent Reporter Secretory Alkaline Phosphatase
(SEAP) Expression Assay
[0054] The effect of the inhibitory peptides on TNF-, TNFR-,
TRADD-, TRAF 2-, NIK-, IKK, and p65-induced NF-.kappa.B-dependent
reporter gene transcription was analyzed by SEAP assay as
previously described (Manna et al., 2000b). Briefly, A293 cells
(5.times.10.sup.5 cells/well) were plated in 6-well plates and
transiently transfected by calcium phosphate method with
pNF-kB-SEAP (0.5 .mu.g). To examine TNF-induced reporter gene
expression, the cells were transfected with 0.5 .mu.g of SEAP
expression plasmid and 2 .mu.g of control plasmid pCMVFLAG1 DNA for
24 hours. Thereafter the cells were treated for 24 hours with 150
.mu.M peptides, and then stimulated with 1 nM TNF for 24 hours. The
cell culture medium was then harvested and analyzed for alkaline
phosphatase (SEAP) activity according to the protocol essentially
as described by the manufacturer (Clontech, Palo Alto, Calif.)
using a 96-well fluorescence plate reader (Fluoroscan II,
Labsystems, Chicago, Ill.) with excitation set at 360 nm and
emission at 460 nm.
EXAMPLE 5
Cytotoxicity Assay (MTT Assay)
[0055] The cytotoxic effects of LPS were determined by the MTT
uptake method as described (Manna et al., 2000a). Briefly, 5000
cells were incubated with synthetic peptides for 1 hour in
triplicate in 96-well plates, and then treated with various
concentration of TNF for 72 hours at 37.degree. C. Thereafter, MTT
solution was added to each well. After a 2-hr incubation at
37.degree. C., extraction buffer (20% SDS, 50% dimethylformamide)
was added, the cells were incubated overnight at 37.degree. C., and
then the OD was measured at 570 nm using a 96-well multiscanner
(Dynex Technologies, MRX Revelation; Chantilly, Va.).
[0056] The cytotoxic effects of TNF were determined by the
Live/Dead assay. Briefly, 1.times.10.sup.5 cells were incubated
with 100 .mu.M PTD-p65-P1 for 1 h, and then treated with 1 nM TNF
for 16 hr at 37.degree. C. Cells were stained with Live/Dead
reagent (5 .mu.M ethidium homodimer, 5 .mu.M calcein-AM), and then
incubated at 37.degree. C. for 30 min. Cells were analyzed under a
fluorescence microscope (Labophot-2, Nikon, Tokyo, Japan).
EXAMPLE 6
TUNEL Assay
[0057] The TNF-induced apoptosis was determined by TUNEL assay
using In Situ Cell Death Detection reagent (Roche Applied Science).
Briefly, 1.times.10.sup.5 cells were incubated with PTD-p65-P1 for
1 h, and then treated with 1 nM TNF for 16 hr at 37.degree. C.
Thereafter, cells were plated on a poly 1-lysine-coated glass slide
by centrifugation using a cytospin 4 (Thermoshendon, Pittsburgh,
Pa.), air-dried, fixed with 4% paraformaldehyde, and permeabilized
with 0.1% of Triton-X 100 in 0.1% sodium citrate. After washing,
the cells were incubated with reaction mixture for 60 min at
37.degree. C. Stained cells were mounted with mounting medium
purchased from Sigma Chemicals and analyzed under a fluorescence
microscope (Labophot-2).
EXAMPLE 7
Cell Permeable Peptides Derived from p65 Subunit of NF-.kappa.B
Inhibits TNF-Induced NF-.kappa.B Activation
[0058] The p65 subunit of NF-.kappa.B was targeted to design
polypeptides that suppress NF-.kappa.B activation. The p65 consists
of a DNA-binding and dimerization domain (RHD), a nuclear
localization domain (NLS), and a transactivation domain (TD). The
phosphorylation residue Ser 276 present in the DNA-binding and
dimerization domain, and residues Ser 529 and Ser 536 in the
transactivation domain were targeted (see FIG. 1A). Polypeptides
derived from the p65 subunit were linked to the protein
transduction domain (PTD) derived from the third helix of the
antennapedia homeodomain (FIG. 1B). These polypeptides were then
tested for its ability to suppress NF-.kappa.B activation induced
by various proinflammatory stimuli.
[0059] To determine the effects of peptide containing Ser 276
(PTD-p65-P1), KBM-5 cells was preincubated with the peptides for 1
h, and then treated with 0.1 nM TNF for the indicated times.
Nuclear extracts were prepared, and NF-.kappa.B activation was
analyzed by electrophoretic mobility shift assays (EMSA).
TNF-induced NF-.kappa.B activation in a time dependent manner and
pretreatment with PTD-p65-P1 completely abolished the TNF-induced
NF-.kappa.B activation (FIG. 3A). Neither a protein transduction
domain nor p65-P1 alone had any effect on TNF-induced NF-.kappa.B
activation, indicating that p65-P1 must be attached to a protein
transduction domain for it to enter the cells. Minimum dose of
PTD-p65-P1 required to suppress NF-.kappa.B activation was also
investigated. PTD-p65-P1 suppressed TNF-induced NF-.kappa.B
activation by 25% at 100 .mu.M and completely at 150 .mu.M (FIG.
3B).
[0060] To determine the effects of peptide containing Ser 529 and
536 (PTD-p65-P6), KBM-5 cells were preincubated with various
concentrations of peptides for 1 h and then treated with 0.1 nM TNF
for 30 min. PTD-p65-P6 inhibited TNF-induced NF-.kappa.B activation
in a dose dependent manner. Neither a protein transduction domain
nor p65-P6 alone had any effect on TNF-induced NF-.kappa.B
activation (FIG. 3C).
EXAMPLE 8
Specific Amino Acid Sequence Required for Suppression of
NF-.kappa.B Activation
[0061] The above in vitro assay was used to determine amino acid
sequence required for NF-.kappa.B inhibition (FIG. 2A). Peptide in
which Ser 276 was mutated (p65-P2) did not inhibit NF-.kappa.B
binding to the DNA. Peptides in which five amino acid residues were
deleted from the C-terminus (p65-P4) or three amino acid residues
were deleted from the N-terminus (p65-P5) were also inactive. The
minimum peptide required for suppression of NF-.kappa.B activation
was QLRRPSDRELSE (p65-P1, SEQ ID NO. 5).
[0062] Whether p65-P6 can suppress p50-p65 binding to DNA was
examined. Peptide in which Ser 529 was mutated (p65-P7) or Ser 536
was mutated (p65-p8) did not inhibit NF-.kappa.B binding to DNA
(FIG. 2A). These results suggest that the inhibition of TNF-induced
NF-.kappa.B activation by p65-P6 requires the presence of both
phosphorylation sites, Ser529 and 536. In contrast, p65-P1 contains
single phosphorylation site and it is needed to inhibit NF-.kappa.B
activity. All subsequent studies were performed with protein
transduction domain-p65-P1.
[0063] The dose-dependent effect of protein transduction
domain-p65-P1 and p65-P1 on p50-p65 binding to DNA was
investigated. Nuclear extracts from TNF-treated cells were
incubated with different concentrations of the peptide and then
examined for DNA binding. p65-P1 inhibited NF-.kappa.B binding in a
dose-dependent manner, and maximum inhibition occurred at 50 .mu.M
(FIG. 2B). PTD-p65-P1 also inhibited NF-.kappa.B binding at the
same concentration. The protein transduction domain alone had no
effect.
EXAMPLE 9
Specificity of NF-.kappa.B Inhibition by PTD-p65-P1
[0064] Since NF-.kappa.B is a complex of proteins, various
combinations of Rel/NF-.kappa.B protein can constitute an active
NF-.kappa.B heterodimer that binds to a specific sequence in the
DNA. To show that the retarded band visualized by EMSA in
TNF-treated cells was indeed NF-.kappa.B, nuclear extracts from
TNF-stimulated cells were incubated with antibodies to either the
p50 (NF-.kappa.B1) or the p65 (RelA) subunit of NF-.kappa.B. Both
shifted the band to a higher molecular mass (FIG. 3D), suggesting
that the TNF-activated complex consisted of p50 and p65 subunits.
Neither preimmune serum (PIS) nor the irrelevant antibody
anti-cyclin D1 had any effect. Excess unlabeled NF-.kappa.B
(100-fold; competitor) caused complete disappearance of the band,
but not by mutant oligonucleotide (Mutant oligo).
EXAMPLE 10
TNF-Induced AP-1 Activation is not Inhibited by PTD-p65-P1
[0065] Like NF-.kappa.B, TNF is also a potent activator of AP-1.
Whether PTD-p65-P1 affects TNF-induced AP-1 activation was
therefore investigated. To determine this, cells were treated with
0.1 nM TNF for the indicated times, nuclear extracts were prepared
and assayed for AP-1 activation by EMSA (FIG. 4A). TNF activated
AP-1, but protein transduction domain-p65-P1 had no effect on the
activation of AP-1.
EXAMPLE 11
PTD-p65-P1 Inhibits NF-.kappa.B Activation Induced by Different
Activators
[0066] Lipopolysaccharide, IL-1, okadaic acid, PMA, H.sub.2O.sub.2,
and cigarette smoke condensate are potent activators of
NF-.kappa.B, but the mechanisms differ. The inventors examined
whether PTD-p65-P1 could suppress NF-.kappa.B activated by these
agents. Cells were preincubated with 150 .mu.M protein transduction
domain-p65-P1 for 1 h, treated with 0.1 nM TNF, 1 .mu.g/ml LPS, 100
ng/ml IL-1, 500 nM okadaic acid, 10 ng/ml PMA, 500 .mu.M
H.sub.2O.sub.2, or 1 .mu.g/ml cigarette smoke condensate and then
analyzed for NF-.kappa.B activation by EMSA. PTD-p65-P1 suppressed
the activation of NF-.kappa.B induced by all these agents (FIG.
4B), suggesting that the PTD-p65-P1 acts at a step common to all
these agents.
EXAMPLE 12
PTD-p65-P1 has No Effect on I.kappa.B.alpha. Phosphorylation or
Degradation
[0067] The translocation of NF-.kappa.B to the nucleus is preceded
by the phosphorylation, ubiquitination, and proteolytic degradation
of I.kappa.B.alpha.. To determine whether PTD-p65-P1 inhibits
TNF-induced NF-.kappa.B activation by inhibition of
I.kappa.B.alpha.degradation and phosphorylation, cells were
pretreated with the polypeptide for 1 h, and then exposed to 0.1 nM
TNF for the indicated times. I.kappa.B.alpha.status in the
cytoplasm was examined by Western blot analysis. As shown in FIG.
5A, pretreatment of cells with protein transduction domain-p65-P1
had no effect on either TNF-induced phosphorylation or degradation
of I.kappa.B.alpha..
EXAMPLE 13
PTD-p65-P1 Inhibits p65 Phosphorylation and Nuclear
Translocation
[0068] The effect of PTD-p65-P1 on TNF-induced phosphorylation and
nuclear translocation of p65 was also analyzed. Western blot
analysis showed that TNF induced nuclear translocation of p65 in a
time-dependent manner. As early as 5 min after TNF stimulation, p65
was translocated to the nucleus, and remained constant till 30 min
(FIG. 5A, middle panel). The results also show that TNF induced
phosphorylation of p65 in a time-dependent manner, whereas protein
transduction domain-p65-P1 suppressed it almost completely (FIG.
5A, bottom). These results suggest that protein transduction
domain-p65-P1 suppressed TNF-induced NF-.kappa.B activation by
inhibiting phosphorylation and nuclear translocation of p65.
EXAMPLE 14
PTD-p65-P1 has No Effect on TNF-Induced IKK Activation
[0069] Since IKK is required for TNF-induced NF-.kappa.B
activation, the effect of protein transduction domain-p65-P1 on
TNF-induced IKK activation was determined next. Immune complex
kinase assays showed that TNF activated IKK as early as 5 min after
TNF treatment, and protein transduction domain-p65-P1 had no effect
on this activation (FIG. 5B).
EXAMPLE 15
IKK Phosphorylates p65-Peptides in Cell-Free System
[0070] p65-P1 and p65-p6 have one or two serine residues
respectively. Whether these serine residues can be phosphorylated
by IKK was investigated. Whole-cell extracts from TNF-treated cells
were immunoprecipitated with antibody against IKK and then
immunocomplex kinase assay was performed using p65-peptides as
substrates. After reaction, samples were fractionated on 20%
SDS-PAGE with 2-fold electrophoresis buffer. FIG. 5C shows that
precipitated IKK complex can phosphorylate p65-P1 and p65-p6,
indicating that synthetic peptides from the p65 subunit of
NF-.kappa.B can be phosphorylated by IKK complex. The results also
showed that p65-peptides in which Ser 276, 529 or 536 were mutated
into alanine did not undergo phosphorylation by the IKK complex,
indicating that Ser 276, 529 and 536 are necessary for p65 to be
phosphorylated by IKK.
EXAMPLE 16
PTD-p65-P1 Inhibits TNF-Induced NF-.kappa.B-Dependent Reporter Gene
Expression
[0071] Although electrophoretic mobility shift assays show that
protein transduction domain-p65-P1 blocks NF-.kappa.B activation,
DNA binding does not always correlate with NF-.kappa.B-dependent
gene transcription, suggesting there are additional regulatory
steps. To determine the effect of protein transduction
domain-p65-P1 on TNF-induced NF-.kappa.B-dependent reporter gene
expression, cells were transiently transfected with the
NF-.kappa.B-regulated SEAP reporter construct. The cells were
incubated with the polypeptide, and then stimulated with TNF.
[0072] An almost 4-fold increase in SEAP activity over vector
control was observed upon stimulation with TNF. Polypeptide protein
transduction domain-p65-P1 completely suppressed the TNF-induced
stimulation, but protein transduction domain or p65P1 alone failed
to suppress it (FIG. 6A). These results demonstrate that PTD-p65-P1
also represses NF-.kappa.B-dependent reporter gene expression
induced by TNF.
[0073] TNF-induced NF-.kappa.B activation is mediated through
sequential interaction of the TNF receptor with TRADD, TRAF 2, NIK,
and IKK, resulting in phosphorylation of I.kappa.B.alpha.. To
delineate the site of action of PTD-p65P1 in the TNF-signaling
pathway leading to NF-.kappa.B activation, cells were transfected
with TNFR 1-, TRADD-, TRAF 2-, NIK-, IKK-, and p65-expressing
plasmids and then monitored for NF-KB-dependent SEAP expression. As
shown in FIG. 6B, all of the plasmid transfected cells induced
NF-.kappa.B-SEAP gene expression, and protein transduction
domain-p65P1 suppressed NF-.kappa.B reporter gene expression
induced by all. These results suggest that protein transduction
domain-p65 P1 affects NF-.kappa.B activation at a terminal
step.
EXAMPLE 17
PTD-p65-P1 Inhibits TNF-Induced NF-.kappa.B-Dependent Cyclin D1,
COX2 and MMP-9 Gene Expression
[0074] TNF-treatment induces expression of cyclin D1, COX-2 and
MMP-9 which have NF-.kappa.B binding sites in their promoters. The
investigators next examined whether protein transduction
domain-p65-P1 inhibits TNF-induced cyclin D1, COX-2 and MMP-9.
Cells were pretreated with PTD-p65-P1 for 1 h, then treated with
TNF for the indicated times, and whole-cell extracts were prepared
and analyzed by Western blot analysis for the expression of cyclin
D1, COX-2 and MMP-9.
[0075] As shown in FIG. 6C, TNF induced Cyclin D1, COX-2 and MMP-9
expressions in a time-dependent manner. PTD-p65-P1 blocked
TNF-induced expression of these gene products.
EXAMPLE 18
PTD-p65-P1 Enhances TNF-Induced Cytotoxicity
[0076] Activation of NF-.kappa.B has been shown to inhibit
TNF-induced apoptosis, whereas suppression of NF-.kappa.B
stimulates TNF-induced apoptosis. Whether suppression of
NF-.kappa.B by protein transduction domain-p65P1 affects
TNF-induced cytotoxicity was investigated by MTT assay.
[0077] As shown in FIG. 7A, TNF was cytotoxic to KBM-5 cells and
protein transduction domain-p65P1 enhanced TNF-induced
cytotoxicity. PTD or p65 P1 by alone had no effect on TNF-induced
cytotoxicity.
[0078] Whether suppression of NF-.kappa.B by protein transduction
domain-p65-P1 affects TNF-induced apoptosis was also investigated
by live and dead assay (FIG. 7B) and annexin V staining (FIG. 7C).
These results show that TNF induced apoptosis in KBM-5 cells and
PTD-p65 P1 enhanced TNF-induced apoptosis from 4% to 45% (see red
staining in FIG. 7B).
EXAMPLE 9
PTD-p65P1 Potentiates Chemotherapy-Induced Cytotoxicity
[0079] Chemotherapeutic agents are known to activate NF-.kappa.B
and mediate chemoresistance. Whether suppression of NF-.kappa.B by
PTD-p 65-P1 affects chemotherapy-induced cytotoxicity was
investigated by the MTT assay.
[0080] As shown in FIG. 7D, cytotoxicity induced by doxorubicin
(top panel) and cisplatin (bottom panel) was potentiated by protein
transduction domain-p65P1. These results suggest that protein
transduction domain-p65-P1 has a therapeutic potential in combining
with chemotherapy.
[0081] The following references were cited herein: [0082]
Chaturvedi et al., J Biol Chem. 269:14575-14583 (1994). [0083]
Derossi et al., J Biol Chem. 269:10444-10450 (1994). [0084] Elliott
and O'Hare, Cell 88:223-233 (1997). [0085] Fawell et al., Proc Natl
Acad Sci USA. 91:664-668 (1994). [0086] Futaki et al., Curr Protein
Pept Sci. 4:87-96 (2003). [0087] Lindgren et al., Trends Pharmacol.
Sci. 21:99-103 (2000). [0088] Manna et al., J. Immunol. 165:4927-34
(2000a). [0089] Manna et al., Cancer Res. 60:3838-47 (2000b).
[0090] Schwarze and Dowdy, Trends Pharmacol Sci. 21:45-48 (2000).
[0091] Takada and Aggarwal, J Biol Chem. 278:23390-23397.
[0092] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these patents and publications are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
Sequence CWU 1
1
13145DNAArtificial SequenceSynthetic primer 1ttgttacaag ggactttccg
ctggggactt tccagggagg cgtgg 45245DNAArtificial SequenceSynthetic
primer 2ttgttacaac tcactttccg ctgctcactt tccagggagg cgtgg
45318PRTArtificial SequenceSynthetic peptide 3Asp Arg Gln Ile Lys
Ile Trp Phe Gln Asn Asn Arg Arg Met Lys Trp1 5 10 15Lys
Lys430PRTArtificial SequenceSynthetic peptide 4Asp Arg Gln Ile Lys
Ile Trp Phe Gln Asn Asn Arg Arg Met Lys Trp1 5 10 15Lys Lys Gln Leu
Arg Arg Pro Ser Asp Arg Glu Leu Ser Glu 20 25 30512PRTArtificial
SequenceSynthetic peptide 5Gln Leu Arg Arg Pro Ser Asp Arg Glu Leu
Ser Glu1 5 10612PRTArtificial SequenceSynthetic peptide 6Gln Leu
Arg Arg Pro Ala Asp Arg Glu Leu Ser Glu1 5 10712PRTArtificial
SequenceSynthetic peptide 7Gln Leu Arg Arg Pro Ala Asp Arg Glu Leu
Ala Glu1 5 1087PRTArtificial SequenceSynthetic peptide 8Gln Leu Arg
Arg Pro Ser Asp1 599PRTArtificial SequenceSynthetic peptide 9Arg
Pro Ser Asp Arg Glu Leu Ser Glu1 51030PRTArtificial
SequenceSynthetic peptide 10Asp Arg Gln Ile Lys Ile Trp Phe Gln Asn
Asn Arg Arg Met Lys Trp1 5 10 15Lys Lys Asn Gly Leu Leu Ser Gly Asp
Glu Asp Phe Ser Ser 20 25 301112PRTArtificial SequenceSynthetic
peptide 11Asn Gly Leu Leu Ser Gly Asp Glu Asp Phe Ser Ser1 5
101212PRTArtificial SequenceSynthetic peptide 12Asn Gly Leu Leu Ala
Gly Asp Glu Asp Phe Ser Ser1 5 101312PRTSynthetic SequenceSynthetic
peptide 13Asn Gly Leu Leu Ser Gly Asp Glu Asp Phe Ser Ala1 5 10
* * * * *