U.S. patent application number 16/945382 was filed with the patent office on 2020-11-19 for composition for autophagy inhibiting in cell, and pharmaceutical composition for preventing or treating neoplastic disease or inhibiting anti-cancer agents resistance containing the same.
This patent application is currently assigned to L-BASE CO., LTD. The applicant listed for this patent is L-BASE CO., LTD. Invention is credited to Do Yong JEON, Ji Eun JUNG, Chang Hoon MOON.
Application Number | 20200361989 16/945382 |
Document ID | / |
Family ID | 1000004989979 |
Filed Date | 2020-11-19 |
United States Patent
Application |
20200361989 |
Kind Code |
A1 |
JEON; Do Yong ; et
al. |
November 19, 2020 |
COMPOSITION FOR AUTOPHAGY INHIBITING IN CELL, AND PHARMACEUTICAL
COMPOSITION FOR PREVENTING OR TREATING NEOPLASTIC DISEASE OR
INHIBITING ANTI-CANCER AGENTS RESISTANCE CONTAINING THE SAME
Abstract
Provided are a polypeptide having the activity of inhibiting
autophagy in a cell, a composition for inhibiting autophagy in a
cell, and particularly in a tumor cell, containing the polypeptide
as an active ingredient, and a pharmaceutical composition
containing the polypeptide, and more specifically, a composition
for inhibiting autophagy in a cell containing a polypeptide
inhibiting the control mechanism of autophagy as an active
ingredient, and a pharmaceutical composition for preventing or
treating a neoplastic disease or for inhibiting resistance to
anticancer agents, containing the polypeptide.
Inventors: |
JEON; Do Yong; (Seoul,
KR) ; MOON; Chang Hoon; (Seoul, KR) ; JUNG; Ji
Eun; (Goyang-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
L-BASE CO., LTD |
Seoul |
|
KR |
|
|
Assignee: |
L-BASE CO., LTD
Seoul
KR
|
Family ID: |
1000004989979 |
Appl. No.: |
16/945382 |
Filed: |
July 31, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15780347 |
Dec 5, 2018 |
10766926 |
|
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PCT/KR2018/006023 |
May 28, 2018 |
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16945382 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 38/00 20130101; C07K 7/06 20130101 |
International
Class: |
C07K 7/06 20060101
C07K007/06; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 23, 2018 |
KR |
10-2018-0046935 |
Claims
1. A polypeptide having the activity of inhibiting autophagy in a
cell, represented by the General Formula of a sequence below:
X3-X1-T-X1-K-X2 [General Formula] wherein, in General Formula of a
sequence above, T is threonine, K is lysine; X1 is at least one
amino acid selected from the group consisting of alanine, glycine,
isoleucine, leucine, methionine, proline, and valine; X2 is at
least one amino acid selected from the group consisting of alanine,
threonine, cysteine, asparagine, proline, glutamine, and serine;
and X3 is at least one amino acid selected from the group
consisting of alanine, glutamine, threonine, serine, asparagine,
and glycine, or is absent.
2. The polypeptide of claim 1, wherein X1 and X2 are alanine.
3. The polypeptide of claim 1, wherein X1 is proline; and X2 is
threonine.
4. The polypeptide of claim 1, wherein X1 is isoleucine; and X2 is
threonine.
5. The polypeptide of claim 1, wherein X3 is glutamine or
threonine.
6. The polypeptide of claim 1, wherein X3 is serine.
7. The polypeptide of claim 1, wherein X3 is glycine, asparagine,
or threonine.
8. The polypeptide of claim 1, wherein the polypeptide inhibits
autophagy in a cell by reducing the expression of at least one
protein selected from the group consisting of p-AMPK, ATG5-ATG12,
Beclin1, p-Beclin1, LC3-I, and LC3-II.
9. The polypeptide of claim 1, wherein the polypeptide inhibits
autophagy in a cell by increasing the expression of p-mTOR or p62
protein.
10. The polypeptide of claim 1, wherein the polypeptide is labeled
with any one labeling material selected from the group consisting
of chromogenic enzymes, radioisotopes, chromophores, luminescent
materials, fluorescers, magnetic resonance imaging (MRI) materials,
super paramagnetic particles, and ultrasuper paramagnetic
particles.
11. The polypeptide of claim 1, wherein the cell is a tumor
cell.
12. A composition for inhibiting autophagy in a cell comprising the
polypeptide of claim 1 as an active ingredient.
13. A pharmaceutical composition for preventing or treating a
neoplastic disease comprising the polypeptide of claim 1 as an
active ingredient.
14. The pharmaceutical composition of claim 13, wherein the
neoplastic disease is selected from the group consisting of lung
cancer, liver cancer, colon cancer, pancreatic cancer, stomach
cancer, breast cancer, ovarian cancer, kidney cancer, thyroid
cancer parathyroid cancer, esophageal cancer, prostate cancer,
brain cancer, skin cancer, osteosarcoma, soft tissue sarcoma,
glioma, lymphoma, nasopharyngeal cancer, larynx cancer, adrenal
gland cancer, colon carcinoma, ureteral cancer, gallbladder cancer,
bladder cancer, testis cancer, uterine cervical cancer, endometrial
cancer, choriocarcinoma, head and neck cancer, malignant melanoma,
leukemia, multiple myeloma, chronic myeloid leukemia,
neuroblastoma, and aplastic anemia.
15. A pharmaceutical composition for inhibiting resistance to
anticancer agents, comprising the polypeptide of claim 1 as an
active ingredient.
16. The pharmaceutical composition of claim 15, wherein the
composition has resistance to at least one anticancer agent
selected from the group consisting of erlotinib, celastrol,
cisplatin, docetaxel, osimertinib, taxol, pemetrexed, and
tamoxifen.
Description
BACKGROUND OF THE INVENTION
Field of the invention
[0001] The present invention relates to a polypeptide having the
activity of inhibiting autophagy in a cell, a composition for
inhibiting autophagy containing the polypeptide as an active
ingredient, and a pharmaceutical composition containing the
polypeptide, and more specifically, a composition for inhibiting
autophagy in a cell, and particularly in a tumor cell, containing a
polypeptide inhibiting the control mechanism of autophagy as an
active ingredient, and a pharmaceutical composition for preventing
or treating a neoplastic disease or inhibiting resistance to
anticancer agents, containing the polypeptide.
Related Art
[0002] Autophagy is a process for removing unnecessary or impaired
organelles and proteins from cells and it helps to maintain cell
homeostasis and is a mechanism for cell survival.
[0003] Autophagy is known to play many roles especially in various
diseases such as cancer, inflammatory diseases, degenerative
neurological diseases, immunological diseases, etc. In particular,
many studies have revealed that autophagy has complex roles
according to the progression, type, genotype, etc. of cancer and is
thus closely associated with cancer.
[0004] When cancer is formed by a rapid increase of cells, the
cells become starved due to inappropriate supply of nutrients from
the surrounding environment. At this time, the cells are provided
with nutrients through autophagy by recycling harmful protein
deposits and damaged organelles, and the energy supply by autophagy
in an environment unfavorable for cancer growth promotes the
survival of cancer cells.
[0005] The protein known to be involved in cell survival under
these circumstances is AMP-activated protein kinase (AMPK). It is
well known that AMP plays an important role in inducing metabolic
action by being activated under the conditions of energy depletion,
release of extracellular matrix, increase of reactive oxygen
species, and hypoxia. Additionally, it is known that the activated
AMPK inhibits the action of mammalian target of rapamycin (mTOR) in
a signaling system of autophagy.
[0006] The mTOR is a serine/threonine protein kinase that belongs
to PI3K-related kinase family and is known to regulate cell growth
and proliferation, survival, migration, protein synthesis, and
transcription, and inhibit autophagy. Many studies that have been
published report that the mTOR mechanism is not only involved in
cancer, but also in various diseases such as metabolic diseases,
degenerative neurological diseases, cardiovascular diseases,
etc.
[0007] Beclin1 is a protein encoded by the BECN1 gene and is an
autophagy inducer. Beclin1 plays an important role in tumor
suppression and this tumor suppressive function is known to be
associated with programmed cell death of autophagy.
[0008] Recently, there is a growing interest in study results that
autophagy plays an important role in the formation, growth, and
treatment of tumors. The role of autophagy in cancer is so complex
that it is known to have two opposite functions as a tumor
suppressor or tumor promoter. Therefore, the differentiation and
proper regulation of the role of autophagy in various conditions of
cancer has become a new strategic method of cancer therapy, and
study results have shown that the inhibition of autophagy reduces
the resistance to anticancer agents, which is a major problem in
cancer therapy, due to various chemotherapies (2017, Nature Reviews
Cancer 17, 528-542).
[0009] Under these circumstances, the present inventors have made
an effort to develop a polypeptide capable of controlling autophagy
which plays an important role in cancer. As a result, they have
confirmed that a composition for inhibiting autophagy in a cell,
and particularly in a tumor cell, containing a polypeptide
inhibiting the control mechanism of autophagy as an active
ingredient, and a pharmaceutical composition for preventing or
treating a neoplastic disease or inhibiting resistance to
anticancer agents, containing the polypeptide, have the effects of
inhibiting the promotion of tumor growth caused by autophagy and
reducing resistance to anticancer agents, by activating mTOR that
inhibits autophagy and inhibiting autophagy through the
inactivation of AMPK and Beclin1 that induce autophagy, thereby
completing the present invention.
Prior Art References
[0010] (Patent Document 1) KR10-1645359 B
[0011] (Patent Document 2) KR10-2018-0007307 A
SUMMARY OF THE INVENTION
[0012] An object of the present invention is to provide a
polypeptide, which has an activity of inhibiting the promotion of
tumor growth caused by autophagy and reducing resistance to
anticancer agents in a cell, and particularly in a tumor cell, by
activating mTOR that inhibits autophagy and inhibiting autophagy
through the inactivation of AMPK and Beclin1 that induce
autophagy.
[0013] Another object of the present invention is to provide a
composition for inhibiting autophagy in a cell, and particularly in
a tumor cell, using the polypeptide.
[0014] Still another object of the present invention is to provide
a pharmaceutical composition for preventing or treating a
neoplastic disease using the polypeptide.
[0015] Still another object of the present invention is to provide
a pharmaceutical composition for inhibiting resistance to
anticancer agents using the composition for inhibiting
autophagy.
[0016] According to an aspect of the present invention, it provides
a polypeptide having the activity of inhibiting autophagy in a
cell, represented by General Formula of a sequence below:
X3-X1-T-X1-K-X2 [General Formula]
[0017] in which, in General Formula of a sequence above,
[0018] T is threonine,
[0019] K is lysine;
[0020] X1 is at least one amino acid selected from the group
consisting of alanine, glycine, isoleucine, leucine, methionine,
proline, and valine;
[0021] X2 is at least one amino acid selected from the group
consisting of alanine, threonine, cysteine, asparagine, proline,
glutamine, and serine; and
[0022] X3 is at least one amino acid selected from the group
consisting of alanine, glutamine, threonine, serine, asparagine,
and glycine, or is absent.
[0023] Autophagy is induced in a cell that is under stress by a
cell survival mechanism. Although autophagy inhibits tumor before
the onset of cancer or during the development of cancer, it
contributes to the survival of tumor cells once cancer is
developed. Therefore, it is thought that autophagy can reduce the
activity of tumor cells by inhibiting the mechanism of autophagy in
tumor cells where cancer has developed.
[0024] In this regard, the present inventors have invented a
polypeptide for inhibiting autophagy in cells where cancer has
developed, and particularly in tumor cells, and have confirmed that
the polypeptide represented by General Formula can inhibit the
promotion of tumor growth caused by autophagy and reducing
resistance to anticancer agents, by inhibiting autophagy, thereby
completing the present invention.
[0025] In the polypeptide having the activity of inhibiting
autophagy of the present invention, the cell may be a tumor
cell.
[0026] As used herein, the term "in a tumor cell" may refer to a
tumor cell after cancer development, and may refer to solid cancer.
Additionally, the term "tumor cell" may refer to a tumor cell
having resistance to anticancer agents.
[0027] In the polypeptide having the activity of inhibiting
autophagy of the present invention, X1 may be an amino acid which
is non-polar and has an aliphatic R group, and preferably X1 may be
alanine, and X2 may be an amino acid which is non-polar and has an
uncharged R group, and preferably X2 may be alanine.
[0028] Additionally, in the polypeptide having the activity of
inhibiting autophagy of the present invention, X1 may be an amino
acid which is non-polar and has an aliphatic R group, and
preferably proline, and X2 may be an amino acid which is polar and
has an uncharged R group, and preferably threonine.
[0029] Additionally, in the polypeptide having the activity of
inhibiting autophagy of the present invention, X1 may be an amino
acid which is non-polar and has an aliphatic R group, and
preferably isoleucine, and X2 may be an amino acid which is polar
and has an uncharged R group, and preferably threonine.
[0030] In the polypeptide having the activity of inhibiting
autophagy of the present invention, X3 may be glutamine or
threonine.
[0031] Additionally, in the polypeptide having the activity of
inhibiting autophagy of the present invention, X3 may be
serine.
[0032] In the polypeptide having the activity of inhibiting
autophagy of the present invention, X3 may be glycine, asparagine,
or threonine.
[0033] The polypeptide having the activity of inhibiting autophagy
of the present invention inhibits autophagy in a cell by reducing
the expression of at least one protein selected from the group
consisting of p-AMPK, ATG5-ATG12, Beclin1, p-Beclin1, LC3-I, and
LC3-II.
[0034] According to an embodiment, as a result of confirming the
association of proteins involved in the autophagy mechanism between
non-small cell lung cancer cell lines, it was confirmed that the
expression of p-AMPK, ATG5, and ATG5-ATG12 increased in non-small
cell lung cancer cell lines resistant to anticancer agents and the
expression of p62 protein was decreased, compared to non-small cell
lung cancer cell lines.
[0035] p-AMPK is a factor that increases tumor growth and
resistance to anticancer agents caused by autophagy, ATG5 is a
tumor-promoting factor, and ATG5-ATG12 is an essential factor in
charge of the formation of autophagosomes in autophagy, and thus
the increase in the expression of p-AMPK, ATG5, and ATG5-ATG12
means that the polypeptide according to the present invention is
involved in resistance of cancer cells (see Example 1 and FIGS. 1
to 3A and 3B). That is, according to an embodiment of the present
invention, the increase in the expression of p-AMPK, ATG5, and
ATG5-ATG12 means the involvement of resistance of cancer cells (see
Example 1 and FIGS. 1 to 3A and 3B).
[0036] As a result of treatment of the polypeptide of the present
invention according to concentrations on the non-small cell lung
cancer cell line resistant to anticancer agents, it was confirmed
that the expression of Beclin1, p-Beclin1, ATG5-ATG12, LC3-I, and
LC3-II proteins, which are known as initiators of autophagy, was
decreased. These results suggest that the polypeptide according to
the present invention can inhibit promotion of tumor growth and
reduce resistance to anticancer agents by inhibiting autophagy via
reduction of expression of the proteins which are known as
activators of the autophagy (see Example 2 and FIGS. 4 to 6).
[0037] In the polypeptide having the activity of inhibiting
autophagy of the present invention, the inhibition of autophagy in
a tumor cell may inhibit autophagy in a cell by increasing the
expression of p-mTOR or p62 protein.
[0038] According to an embodiment of the present invention, as a
result of confirming the effect of inhibiting autophagy, antitumor
effect, and effect of inhibiting resistance anticancer agents of
the polypeptide of the present invention, it was confirmed that
when the polypeptide of the present invention was introduced into
mice according to concentrations, the concentration of tumor tissue
decreased in a concentration-dependent manner (see Example 4 and
FIG. 8A, 8B and 8C).
[0039] Additionally, as a result of confirming the expression
levels of the factors involved in autophagy in the tumor tissue, it
was confirmed that expression of p-mTOR and p62 proteins, which
play the role of inhibiting autophagy, was increased, whereas the
expression of p-AMPK, Beclin1, and p-Beclin1 proteins, which are
activators of autophagy, were decreased.
[0040] These results suggest that the polypeptide of the present
invention can reduce the promotion of tumor growth and reduce
resistance to anticancer agents by inactivating p-AMPK, Beclin1,
and p-Beclin1 which induce autophagy, by increasing the factors
that inhibit autophagy (see Example 4 and FIG. 9).
[0041] In the polypeptide having the activity of inhibiting
autophagy of the present invention, the polypeptide may be labeled
with any one labeling material selected from the group consisting
of chromogenic enzymes, radioisotopes, chromophores, luminescent
materials, fluorescers, magnetic resonance imaging (MRI) materials,
super paramagnetic particles, and ultrasuper paramagnetic
particles, however, it does not necessarily mean that the
polypeptide can be labeled with a labeling material.
[0042] According to another aspect of the present invention, the
present invention provides a composition for inhibiting autophagy
in a cell containing the polypeptide represented by General Formula
below as an active ingredient:
X3-X1-T-X1-K-X2 [General Formula]
[0043] in which, in General Formula of a sequence above,
[0044] T is threonine,
[0045] K is lysine;
[0046] X1 is at least one amino acid selected from the group
consisting of alanine, glycine, isoleucine, leucine, methionine,
proline, and valine;
[0047] X2 is at least one amino acid selected from the group
consisting of alanine, threonine, cysteine, asparagine, proline,
glutamine, and serine; and
[0048] X3 is at least one amino acid selected from the group
consisting of alanine, glutamine, threonine, serine, asparagine,
and glycine, or is absent.
[0049] According to still another aspect of the present invention,
the present invention provides a pharmaceutical composition for
preventing or treating a neoplastic disease containing the
polypeptide represented by General Formula below as an active
ingredient:
X3-X1-T-X1-K-X2 [General Formula]
[0050] in which, in General Formula of a sequence above,
[0051] T is threonine,
[0052] K is lysine;
[0053] X1 is at least one amino acid selected from the group
consisting of alanine, glycine, isoleucine, leucine, methionine,
proline, and valine;
[0054] X2 is at least one amino acid selected from the group
consisting of alanine, threonine, cysteine, asparagine, proline,
glutamine, and serine; and
[0055] X3 is at least one amino acid selected from the group
consisting of alanine, glutamine, threonine, serine, asparagine,
and glycine, or is absent.
[0056] In the pharmaceutical composition for preventing or treating
a neoplastic disease of the present invention, the neoplastic
disease may be one selected from the group consisting of lung
cancer, liver cancer, colon cancer, pancreatic cancer, stomach
cancer, breast cancer, ovarian cancer, kidney cancer, thyroid
cancer, parathyroid cancer, esophageal cancer, prostate cancer,
brain cancer, skin cancer, osteosarcoma, soft tissue sarcoma,
glioma, lymphoma, nasopharyngeal cancer, larynx cancer, adrenal
gland cancer, colon carcinoma, ureteral cancer, gallbladder cancer,
bladder cancer, testis cancer, uterine cervical cancer, endometrial
cancer, choriocarcinoma, head and neck cancer, malignant melanoma,
leukemia, multiple myeloma, chronic myeloid leukemia,
neuroblastoma, and aplastic anemia, and preferably lung cancer or
breast cancer, but the neoplastic disease is not limited
thereto.
[0057] In the pharmaceutical composition for preventing or treating
a neoplastic disease of the present invention, the composition may
be used as a pharmaceutical composition for diseases that can be
prevented or treated by inhibiting autophagy, and preferably as a
pharmaceutical composition for preventing or treating a neoplastic
disease.
[0058] According to still another aspect of the present invention,
the present invention provides a pharmaceutical composition for
inhibiting resistance to anticancer agents containing the
polypeptide represented by General Formula below as an active
ingredient:
X3-X1-T-X1-K-X2 [General Formula]
[0059] in which, in General Formula of a sequence above,
[0060] T is threonine,
[0061] K is lysine;
[0062] X1 is at least one amino acid selected from the group
consisting of alanine, glycine, isoleucine, leucine, methionine,
proline, and valine;
[0063] X2 is at least one amino acid selected from the group
consisting of alanine, threonine, cysteine, asparagine, proline,
glutamine, and serine; and
[0064] X3 is at least one amino acid selected from the group
consisting of alanine, glutamine, threonine, serine, asparagine,
and glycine, or is absent.
[0065] In the pharmaceutical composition for inhibiting resistance
to anticancer agents of the present invention, the composition may
have resistance to any anticancer agent used to treat a neoplastic
disease, and preferably have resistance to at least one anticancer
agent selected from the group consisting of erlotinib, celastrol,
cisplatin, docetaxel, osimertinib, taxol, pemetrexed, and
tamoxifen.
[0066] In the pharmaceutical composition for inhibiting resistance
to anticancer agents of the present invention, the composition may
inhibit resistance to anticancer agents by inhibiting
autophagy.
[0067] According to an embodiment, as a result of treating PC9-ER,
H1975, and MCF7.sup.R (i.e., cancer cell lines having resistance to
various anticancer agents) with the polypeptide of the present
invention, it was confirmed that cell growth was inhibited (see
FIGS. 6, 11A, 11B, 14A, 14B and 14C).
[0068] These results suggest that the polypeptide of the present
invention can inhibit (reduce) resistance to anticancer agents in
cancer cell lines, which have resistance to anticancer agents, by
inhibiting autophagy.
[0069] The pharmaceutical composition according to the present
invention may be provided in a pure form of the polypeptide or by
formulating it into a suitable form together with a
pharmaceutically acceptable carrier. As used herein, the term
"pharmaceutically acceptable" refers to a non-toxic composition
that does not normally cause allergic reactions such as
gastrointestinal disorders, dizziness, etc. when it is
physiologically acceptable and administered to humans. Examples of
the carrier may include all kinds of solvents, dispersion media,
oil-in-water or water-in-oil emulsions, aqueous compositions,
liposomes, microbeads and microsomes, biodegradable nanoparticles,
etc., but the pharmaceutically acceptable carrier is not limited
thereto.
[0070] Meanwhile, the pharmaceutical composition according to the
present invention may be formulated together with a
pharmaceutically acceptable carrier according to administration
routes. The administration routes according to the present
invention may include oral or parenteral administration, but the
administration routes are not limited thereto. Examples of the
parenteral administration routes may include various routes such as
transdermal, nasal, peritoneal, muscular, subcutaneous, or
intravenous, but the parenteral administration routes are not
limited thereto.
[0071] In a case where the pharmaceutical composition according to
the present invention is orally administered, the pharmaceutical
composition of the present invention may be formulated in the form
of powders, granules, tablets, pills, sugar-coated tablets,
capsules, solutions, gels, syrups, suspensions, wafers, etc.
together with a pharmaceutically acceptable carrier for oral
administration according to methods known in the art, but the
formulation type is not limited thereto.
[0072] The pharmaceutical composition of the present invention may
be formulated according to methods known in the art so as to
provide rapid, sustained, or delayed release of an active
ingredient after administration to mammals.
[0073] The pharmaceutical composition formulated by the above
methods may be administered in an effective amount via various
routes including oral, transdermal, subcutaneous, intravenous, or
muscular administration. As used herein, the term "effective
amount" refers to an amount of substance that enables the tracking
of diagnosis or therapeutic effect when administered to a
patient.
[0074] The dosage of the pharmaceutical composition according to
the present invention may be appropriately selected according to
the administration route, subject to be treated, disease to be
treated and its severity, age, sex, body weight, individual
difference, and disease state. Preferably, the pharmaceutical
composition containing the polypeptide of the present invention may
vary depending on the severity of the disease, and conventionally,
the active ingredient content may be repeatedly administered
several times a day with an effective dose of 1 mg to 1,000 mg per
single administration based on adults.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] FIGS. 1 to 3A and 3B show the results according to Example 1
of the present invention.
[0076] FIGS. 4 to 6 show the results according to Example 2 of the
present invention.
[0077] FIG. 7 shows the results according to Example 3 of the
present invention.
[0078] FIGS. 8A, 8B, 8C, and 9 show the results according to
Example 4 of the present invention.
[0079] FIGS. 10, 11A, and 11B show the results according to Example
5 of the present invention.
[0080] FIGS. 12 to 14C show the results according to Example 6 of
the present invention.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0081] Hereinafter, the present invention will be described in more
detail with reference to Examples. Since these Examples are for
illustrating the present invention only, the scope of the present
invention is not construed as being limited by these Examples.
EXAMPLE 1
Confirmation of Autophagy Mechanism in Cancer Cell Lines
[0082] The cancer cell line (PC9) used in the present invention was
cultured in a minimum essential medium in a conditioned incubator
(37.degree. C.) supplemented with 10% fetal bovine serum and
antibiotics. The cancer cell line (PC9-ER) was constructed to have
resistance by exposing to erlotinib as an anticancer agent for a
long period of time.
[0083] In order to examine whether proteins with a mechanism
controlling autophagy are involved in the cancer cell line (PC9)
and the cell line (PC9-ER), which has resistance to anticancer
agents, the expression levels of AMP-activated protein kinase
(AMPK), phosphorylated AMPK (p-AMPK), autophagy-related genes 5
(ATG5), ATG5-ATG12, and p62 proteins were compared by western blot
method, and the results are shown in FIG. 1.
[0084] As a result, as can be seen in FIG. 1, it was confirmed that
the cell line (PC9-ER), which has resistance to anticancer agents,
showed an increase of the expression levels of p-AMPK, ATG5, and
ATG5-ATG12 compared to those of the PC9 cell line, and it was also
confirmed that the expression level of p62 protein, which is an
autophagy-inhibiting molecule, was lowered.
[0085] Additionally, the PC9 cell line and the PC9-ER cell line
were subjected to immunofluorescence staining.
[0086] Specifically, the PC9 cell line and the PC9-ER cell line
were fixed at room temperature using 4% formaldehyde. Then, the
cells were reacted at room temperature for 30 minutes so as to
prevent inaccurate binding to antibodies using PBS containing 0.1%
BSA, and allowed to react with LC3 antibodies (cell signaling) at
4.degree. C. for 24 hours. After removing the antibodies, the cells
were washed with PBS and allowed to react with secondary antibodies
(Thermo Fisher) labeled with Alexa 488 fluorescence at room
temperature for 1.5 hours. After the reaction, the cells from which
antibodies were removed were washed with PBS and stained with DAPI
(Thermo Fisher) for 1 minute. The fluorescent staining was
confirmed by confocal microscopy and the results are shown in FIG.
2.
[0087] As a result, as can be seen in FIG. 2, it was confirmed that
the expression level of LC3 puncta was higher in the PC9-ER cell
line compared to that in the PC9 cell line.
[0088] Additionally, the MTT assay was performed so as to confirm
the resistance of the PC9 cell line and the PC9-ER cell line to
anticancer agents.
[0089] Specifically, an equal number of the PC9 cell line and the
PC9-ER cell line were cultured in a 96-well cell culture plate,
treated with an anticancer agent subjected to a 1/10-fold serial
dilution for 24 hours, and confirmed using MTT. As an experiment to
utilize the principle that NADH present in the mitochondria of a
living cell reacts with MTT to form formazan, formazan was
dissolved in DMSO and its absorbance was measured at 570 nm and
thereby the growth activity of the cells was confirmed. The results
are shown in FIG. 3A and 3B.
[0090] As a result, as can be seen in FIG. 3A and 3B, it was
confirmed that the PC9-ER cell line, in which the mechanism
controlling autophagy was further activated, had higher resistance
to docetaxel and taxol (i.e., known anticancer agents) compared to
that of PC9 cell line.
EXAMPLE 2
Confirmation of Effect of Polypeptide in Autophagy Control
[0091] An attempt was made to confirm the inhibitory effects of
SPTPKT (polypeptide 1) and QTATAKA (polypeptide 2) against
autophagy, based on the results that the expression of ATG5 and
ATG5-ATG12 proteins which are known to be involved in autophagy in
the cancer cell line (PC9-ER).
[0092] Specifically, the PC9-ER cell line was treated with each of
the polypeptide 1 and the polypeptide 2 and the expression levels
of Beclin1, p-Belclin1, ATG5-ATG12, LC3 I, and LC3 II (i.e.,
proteins associated with autophagy mechanism) were compared by
western blot method, and the results are shown in FIG. 4.
[0093] As a result, as can be seen in FIG. 4, it was confirmed that
the expression levels of Beclin1 and p-Beclin1, which are known as
autophagy initiators, and ATG5-ATG12, LC3 I, and LC3 II proteins,
which are autophagy-activating molecules, were lowered when the
PC9-ER cell line was treated with the polypeptide 1 and the
polypeptide 2, respectively.
[0094] Additionally, the cells were subjected to immunofluorescence
staining after treating the PC9-ER cell line with the polypeptide
2.
[0095] Specifically, 24 hours after treating the PC9-ER cell line
with the polypeptide 2, the cells were fixed at room temperature
using 4% formaldehyde. Then, the cells were reacted at room
temperature for 30 minutes so as to prevent inaccurate binding to
antibodies using PBS containing 0.1% BSA, and allowed to react with
LC3 antibodies (cell signaling) at 4.degree. C. for 24 hours. After
removing the antibodies, the cells were washed with PBS and allowed
to react with secondary antibodies (Thermo Fisher) labeled with
Alexa 488 fluorescence at room temperature for 1.5 hours. After the
reaction, the cells from which antibodies were removed were washed
with PBS and stained with DAPI (Thermo Fisher) for 1 minute. The
fluorescent staining was confirmed by confocal microscopy and the
results are shown in FIG. 5.
[0096] As a result, as can be seen in FIG. 5, it was confirmed that
the expression level of LC3 puncta was lowered in the PC9-ER cell
line.
[0097] Additionally, the PC9-ER cell line was treated with the
polypeptide 1, polypeptide 2, and GNTITIKT (polypeptide 3),
respectively, and the MTT assay was performed so as to confirm the
resistance of the PC9-ER cell line to anticancer agents.
[0098] Specifically, the PC9-ER cell line was plated on a 96-well
cell culture plate and treated with each of the polypeptides (1 to
3) for 24 hours. Then, the cells were treated for with an
anticancer agent subjected to a 1/10-fold serial dilution for 24
hours and confirmed using MTT. As an experiment to utilize the
principle that NADH present in the mitochondria of a living cell
reacts with MTT to form formazan, formazan was dissolved in DMSO
and its absorbance was measured at 570 nm and thereby the growth
activity of the cells was confirmed. The results are shown in FIG.
6.
[0099] As a result, as can be seen in FIG. 6, it was confirmed that
each of the polypeptides (1 to 3) treatment reduced resistance to
erlotinib (Tarceva), which is an anticancer agent, in the PC9-ER
cell line.
[0100] These results suggest that the polypeptide 1, polypeptide 2,
and polypeptide 3 have an inhibitory effect against autophagy in
the cancer cell line having resistance to anticancer agents.
EXAMPLE 3
Confirmation of Intracellular Expression of Polypeptides
[0101] As in the method of immunofluorescence staining method
according to Example 1 of the present invention, the polypeptide 2
was labeled with green fluorescence (fluorescein isothiocyanate;
FITC) and transfected into the PC9-ER cell line according to a
known method. The results are shown in FIG. 7.
[0102] As a result, as can be seen in FIG. 7, it was confirmed that
FITC-polypeptide 2 is delivered into the cells in a time-dependent
manner.
EXAMPLE 4
Confirmation of Inhibitory Effect of Polypeptides Against Tumor
Formation
[0103] An attempt was made to confirm the in-vivo anticancer action
of the polypeptide 2 based on the results that the polypeptide 2
showed an anticancer action through autophagy in in-vivo
condition.
[0104] Specifically, athymic nude mice (BALB/C nude, 5 to 6-week
old females) were subcutaneously injected on the side with the
PC9-ER cell line (2.times.10.sup.6 cells/mouse) to induce tumor.
When the tumor volume reached about 150 mm.sup.3, the polypeptide 2
was injected a total of 5 times through the caudal vein and the
changes in the tumor size were examined. The tumor volume was
directly measured using a caliper and calculated by the following
equation (length.times.width.times.height.times.0.5). In order to
confirm the inhibitory effect of the polypeptide 2 against tumor
formation, the peptide (at concentrations of 0 .mu.g/mouse, 50
.mu.g/mouse, 100 .mu.g/mouse, and 200 .mu.g/mouse) was injected 5
times through the caudal vein from day 25 at 3 day intervals. On
the 41.sup.st day, the tumors were separated and the sizes of the
tumor volumes were measured, and western blot was performed using
each labeled antibody. The results are shown in FIGS. 8A, 8B, 8C,
and 9.
[0105] As a result, as can be seen in FIG. 8A, 8B, 8C, it was
confirmed that the polypeptide 2 significantly reduced the size of
tumors.
[0106] Additionally, as can be seen in FIG. 9, the tumor tissue
showed a phenomenon that the expression of p-mTOR and p62 proteins
increased in a concentration-dependent manner, and the expression
of p-AMPK, Beclin1, and p-Beclin1 proteins decreased in a
concentration-dependent manner.
[0107] These results show the anticancer action of the polypeptide
2 by the inhibition of the autophagy mechanism.
EXAMPLE 5
Confirmation of Effect of Polypeptides on Controlling Autophagy
[0108] To examine the effect of anticancer activity of the
polypeptide 1 and the polypeptide 2 through autophagy controlling
proteins in H1975, which is a different non-small cell lung cancer
cell line from those in Examples 1 to 4, western blot was performed
using each of the labeled antibody in H1975 cells. The results are
shown in FIG. 10.
[0109] As a result, as can be seen in FIG. 10, it was confirmed
that when treated with the polypeptide 1 and the polypeptide 2,
respectively, the expression of p-AMPK, which is known to be
involved in cancer controlling mechanism, and those of p-Beclin1,
ATG5, ATG5-ATG12, LC3 I, and LC3 II, which are proteins involved in
autophagy controlling mechanism, were lowered.
[0110] Additionally, after the treatment with the polypeptide 1,
polypeptide 2, and polypeptide 3, respectively, the MTT assay was
performed so as to confirm the resistance to osimertinib
(Tagrisso), an anticancer agent.
[0111] Specifically, an equal number of cells of the H1975 cell
line were plated into a 96-well cell culture plate and treated with
the polypeptides (1 to 3) for 24 hours. Then, the cells were
treated with osimertinib (Tagrisso), an anticancer agent, which was
subjected to a 1/10-fold serial dilution, for 24 hours and were
confirmed using MTT. As an experiment to utilize the principle that
NADH present in the mitochondria of a living cell reacts with MTT
to form formazan, formazan was dissolved in DMSO and its absorbance
was measured at 570 nm and thereby the growth activity of the cells
was confirmed. The results are shown in FIGS. 11A and 11B.
[0112] As a result, as can be seen in FIGS. 11A and 11B, it was
confirmed that each of the polypeptides (1 to 3) reduced the
resistance to osimertinib (Tagrisso), an anticancer agent.
[0113] These results show that the polypeptide 1, polypeptide 2,
and polypeptide 3 have anticancer activity through the inhibition
of autophagy.
[0114] Additionally, it was confirmed that when the cells were
treated with polypeptide 2 at different concentrations, the
resistance to osimertinib (Tagrisso), an anticancer agent, was
reduced in a concentration-dependent manner.
EXAMPLE 6
Confirmation of Effect of Anticancer Activity of Polypeptides
[0115] MCF7R cancer cell line having resistance to anticancer
agents was constructed to have resistance by exposing to celastrol
for a long period of time
[0116] To examine the effects of the polypeptide 1 and the
polypeptide 2 on the proteins of autophagy controlling mechanism,
western blot was performed using each labeled antibody in the MCF7R
cell line. The results are shown in FIG. 12.
[0117] As a result, as can be seen in FIG. 12, it was confirmed
that when treated with the polypeptide 1 and the polypeptide 2,
respectively, the expression level of p62 protein, which is an
autophagy inhibiting molecule, was increased, whereas the
expression levels of p-Beclin1, ATG5, ATG5-ATG12, LC3 I, and LC3 II
proteins, which are autophagy activating molecules, were
reduced.
[0118] Additionally, after the treatment with the polypeptide 2 in
the MCF7R cell line, the immunofluorescence staining was
performed.
[0119] Specifically, after treating the polypeptide 2 on the MCF7R
cell line for 24 hours, the cells were fixed at room temperature
using 4% formaldehyde. Then, the cells were reacted at room
temperature for 30 minutes so as to prevent inaccurate binding to
antibodies using PBS containing 0.1% BSA, and allowed to react with
LC3 antibodies (cell signaling) at 4.degree. C. for 24 hours. After
removing the antibodies, the cells were washed with PBS and allowed
to react with secondary antibodies (Thermo Fisher) labeled with
Alexa 488 fluorescence at room temperature for 1.5 hours. After the
reaction, the cells from which antibodies were removed were washed
with PBS and stained with DAPI (Thermo Fisher) for 1 minute. The
fluorescent staining was confirmed by confocal microscopy and the
results are shown in FIG. 13.
[0120] As a result, as can be seen in FIG. 13, it was confirmed
that when the polypeptide 2 was treated on the MCF7.sup.R cell
line, the expression level of LC3 puncta was lowered.
[0121] Additionally, after the treatment in the MCF7R cell line
with the polypeptide 1, polypeptide 2, and polypeptide 3,
respectively, the MTT assay was performed so as to confirm the
resistance to anticancer agent.
[0122] Specifically, an equal number of cells of the H1975 cell
line were plated into a 96-well cell culture plate and treated with
the polypeptides (1 to 3) for 24 hours. Then, the cells were
treated with an anticancer agent, which was subjected to a
1/10-fold serial dilution, for 24 hours and were confirmed using
MTT. As an experiment to utilize the principle that NADH present in
the mitochondria of a living cell reacts with MTT to form formazan,
formazan was dissolved in DMSO and its absorbance was measured at
570 nm and thereby the growth activity of the cells was confirmed.
The results are shown in FIGS. 14A, 14B and 14C.
[0123] As a result, as can be seen in FIGS. 14A, 14B and 14C, it
was confirmed that each of the polypeptides (1 to 3) reduced
resistance to various anticancer agents, such as celastrol,
cisplatin, docetaxel, etc., in the MCF7.sup.R cell line.
ADVANTAGEOUS EFFECTS OF THE INVENTION
[0124] As described above, the polypeptide according to the present
invention has an effect of preventing or treating a neoplastic
disease by inhibiting autophagy and it is also effective in
inhibiting anticancer agent resistance.
* * * * *