U.S. patent application number 15/415883 was filed with the patent office on 2017-05-25 for use of nobiletin in cancer treatment.
The applicant listed for this patent is Macau University of Science and Technology. Invention is credited to Sen-ling FENG, Liang LIU, Wen-zhe MA, Ying XIE, Xiao-jun YAO, Zhong-wen YUAN.
Application Number | 20170143668 15/415883 |
Document ID | / |
Family ID | 54339072 |
Filed Date | 2017-05-25 |
United States Patent
Application |
20170143668 |
Kind Code |
A1 |
MA; Wen-zhe ; et
al. |
May 25, 2017 |
USE OF NOBILETIN IN CANCER TREATMENT
Abstract
The present invention discloses a pharmaceutical composition of
treating multidrug resistance cancer, comprising a citrus
methoxyflavone and a chemotherapeutic drug, in which the citrus
methoxyflavone is nobiletin. A method of treating multidrug
resistance cancer comprising administrating citrus methoxyflavone
and a chemotherapeutic drug is also disclosed.
Inventors: |
MA; Wen-zhe; (Macau, CN)
; FENG; Sen-ling; (Macau, CN) ; YAO; Xiao-jun;
(Macau, CN) ; YUAN; Zhong-wen; (Macau, CN)
; LIU; Liang; (Macau, CN) ; XIE; Ying;
(Macau, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Macau University of Science and Technology |
Macau |
|
CN |
|
|
Family ID: |
54339072 |
Appl. No.: |
15/415883 |
Filed: |
January 26, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14848358 |
Sep 9, 2015 |
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15415883 |
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62198137 |
Jul 29, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/352 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/337 20130101;
A61K 2300/00 20130101; A61K 45/06 20130101; A61K 31/704 20130101;
A61K 31/366 20130101; A61K 31/352 20130101; A61K 31/337 20130101;
A61K 31/704 20130101; A61P 35/00 20180101 |
International
Class: |
A61K 31/366 20060101
A61K031/366; A61K 31/704 20060101 A61K031/704; A61K 31/337 20060101
A61K031/337 |
Claims
1. A pharmaceutical composition for treating multidrug resistance
cancer, comprising a citrus methoxyflavone and a chemotherapeutic
drug.
2. The pharmaceutical composition of claim 1, wherein said citrus
methoxyflavone can inhibit function of ABCB1 transporter such that
intracellular accumulation of said chemotherapeutic drug is
increased.
3. The pharmaceutical composition of claim 1, wherein said citrus
methoxyflavone is nobiletin.
4. The pharmaceutical composition of claim 1, wherein said
chemotherapeutic drug is selected from the group consisting of
paclitaxel, docetaxel, doxorubicin and daunorubicin.
5. The pharmaceutical composition of claim 1, wherein the multidrug
resistance cancer is paclitaxel-resistant cancer.
6. The pharmaceutical composition of claim 5, wherein the
paclitaxel-resistant cancer is paclitaxel-resistant non-small cell
lung cancer or paclitaxel-resistant ovarian cancer.
7. A method of treating multidrug resistance cancer, comprising
administrating a pharmaceutically effective amount of a citrus
methoxyflavone and a chemotherapeutic drug to a subject in need
thereof.
8. The method of claim 7, wherein said citrus methoxyflavone can
inhibit function of ABCB1 transporter such that intracellular
accumulation of said chemotherapeutic drug is increased.
9. The method of claim 7, wherein the citrus methoxyflavone is
nobiletin.
10. The method of claim 7, wherein the chemotherapeutic drug is
selected from the group consisting of paclitaxel, docetaxel,
doxorubicin and daunorubicin.
11. The method of claim 7, wherein the multi-drugs resistance
cancer is paclitaxel-resistant cancer.
12. The method of claim 11, wherein the paclitaxel-resistant cancer
is paclitaxel-resistant non-small cell lung cancer or
paclitaxel-resistant ovarian cancer.
13. A method of enhancing the efficacy of a chemotherapeutic drug
to treat multidrug resistance cancer, comprising: (a) administering
said chemotherapeutic drug to the subject; and (b) applying a
citrus methoxyflavone.
14. The method of claim 13, wherein said citrus methoxyflavone can
inhibit function of ABCB1 transporter such that intracellular
accumulation of said chemotherapeutic drug is increased.
15. The method of claim 13, wherein the citrus methoxyflavone is
nobiletin.
16. The method of claim 13, wherein the chemotherapeutic drug is
selected from the group consisting of paclitaxel, docetaxel,
doxorubicin and daunorubicin.
17. The method of claim 13, wherein the multi-drugs resistance
cancer is paclitaxel-resistant cancer.
18. The method of claim 17, wherein the paclitaxel-resistant cancer
is paclitaxel-resistant non-small cell lung cancer or
paclitaxel-resistant ovarian cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit under 35 U.S.C. .sctn.119(e)
of U.S. Provisional Application having Ser. No. 62/198,137 filed 29
Jul. 2015, which is hereby incorporated by reference herein in its
entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The hard copy of the sequence listing submitted herewith and
the corresponding computer readable form are both incorporated
herein by reference in their entireties.
FIELD OF INVENTION
[0003] This invention relates to a citrus methoxyflavone and the
use thereof for treating cancer.
BACKGROUND OF INVENTION
[0004] Multidrug resistance (MDR) is a major reason for the
clinical failure of many forms of chemotherapy. In the past few
decades, a number of different mechanisms were found to mediate the
development of MDR, and among which the most important were those
associated with the overexpression of various ATP binding cassette
(ABC) transport proteins. Permeability glycoprotein 1 (abbreviated
as P-gp), also known as multidrug resistance protein 1 (MDR1) or
ATP-binding cassette sub-family B member 1 (ABCB1), is the most
extensively studied ABC transporter protein, which is significantly
elevated in drug-resistant tumors and pumps out various anticancer
drugs, such as taxanes, anthracyclines, vinca alkaloids, and
epipodophyllotoxins. Since 1981, P-gp inhibitors have been
intensively studied as potential MDR reversers. However, while
several P-gp inhibitors were found among the available drugs, they
have the disadvantage of toxicity and poor drug interaction
profiles. Therefore, new and more effective compounds with low
toxicity and fewer side effects are desirable.
SUMMARY OF INVENTION
[0005] In the light of the foregoing background, the present
invention, in one aspect, is a pharmaceutical composition for
treating multidrug resistance cancer, including a citrus
methoxyflavone and a chemotherapeutic drug.
[0006] In an exemplary embodiment of the present invention, the
citrus methoxyflavone can inhibit function of ABCB1 transporter
such that intracellular accumulation of the chemotherapeutic drug
is increased. In an exemplary embodiment of the present invention,
the citrus methoxyflavone is nobiletin. In an exemplary embodiment,
the chemotherapeutic drug is paclitaxel, docetaxel, doxorubicin or
daunorubicin. In another exemplary embodiment, the multidrug
resistance cancer is paclitaxel-resistant cancer. In a further
embodiment, the paclitaxel-resistant cancer is paclitaxel-resistant
non-small cell lung cancer or paclitaxel-resistant ovarian
cancer.
[0007] According to another aspect of the present invention, it
provides a method of treating multidrug resistance cancer,
including administrating a pharmaceutically effective amount of a
citrus methoxyflavone and a chemotherapeutic drug to a subject in
need thereof.
[0008] In an exemplary embodiment of the present invention, the
citrus methoxyflavone can inhibit function of ABCB1 transporter
such that intracellular accumulation of the chemotherapeutic drug
is increased. In an exemplary embodiment of the present invention,
the citrus methoxyflavone is nobiletin. In an exemplary embodiment,
the chemotherapeutic drug is paclitaxel, docetaxel, doxorubicin or
daunorubicin. In another exemplary embodiment, the multidrug
resistance cancer is paclitaxel-resistant cancer. In a further
embodiment, the paclitaxel-resistant cancer is paclitaxel-resistant
non-small cell lung cancer or paclitaxel-resistant ovarian
cancer.
[0009] In a further aspect, the present invention is a method of
enhancing the efficacy of a chemotherapeutic drug in treating
multidrug resistance cancer, including (a) administering the
chemotherapeutic drug to the subject; and (b) applying a citrus
methoxyflavone.
[0010] In an exemplary embodiment of the present invention, the
citrus methoxyflavone can inhibit function of ABCB1 transporter
such that intracellular accumulation of the chemotherapeutic drug
is increased. In an exemplary embodiment of the present invention,
the citrus methoxyflavone is nobiletin. In an exemplary embodiment,
the chemotherapeutic drug is selected paclitaxel, docetaxel,
doxorubicin or daunorubicin. In another exemplary embodiment, the
multidrug resistance cancer is paclitaxel-resistant cancer. In a
further embodiment, the paclitaxel-resistant cancer is
paclitaxel-resistant non-small cell lung cancer or
paclitaxel-resistant ovarian cancer.
[0011] In a further aspect, the present invention is a method of
sensitizing ABCB1-ovexpressing cells to chemotherapeutic drug in
the treatment of multidrug resistance cancer, comprising the
administration of nobiletin to a subject in need thereof.
[0012] In an exemplary embodiment of the present invention, the
citrus methoxyflavone can inhibit function of ABCB1 transporter
such that intracellular accumulation of the chemotherapeutic drug
is increased. In an exemplary embodiment of the present invention,
the citrus methoxyflavone is nobiletin. In an exemplary embodiment,
the chemotherapeutic drug is selected paclitaxel, docetaxel,
doxorubicin or daunorubicin. In another exemplary embodiment, the
multidrug resistance cancer is paclitaxel-resistant cancer. In a
further embodiment, the paclitaxel-resistant cancer is
paclitaxel-resistant non-small cell lung cancer or
paclitaxel-resistant ovarian cancer.
BRIEF DESCRIPTION OF FIGURES
[0013] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0014] FIG. 1 is the chemical structure of nobiletin.
[0015] FIGS. 2A to 2F demonstrates multidrug resistance in
paclitaxel (PTX)--resistant ovarian cancer cells (A2780/T) and the
parental sensitive cells (A2780). The cells were treated with
various concentrations of PTX (FIG. 2A) and doxorubicin (DOX) (FIG.
2B) for 48 hours. Cell growth was determined using the
Sulforhodamine B (SRB) assay. The expressions of ABCB1 transporter
in A2780 and A2780/T cells were analyzed at level of both MDR1 mRNA
by RT-qPCR (FIG. 2C) and P-gp protein level by Western blotting
(FIG. 2D). (##: Significantly different from A2780 cells with
P<0.01). Protein expression levels after normalized relatively
to that of .beta.-actin. Cytotoxicity of nobiletin alone in pairs
of A2780/T or A2780 cells was analyzed (FIG. 2E). Cytotoxicity of
nobiletin alone in pairs of A549 or A549/T cells was analyzed (FIG.
2F).
[0016] FIGS. 3A to 3G show the effect of nobiletin on reversing
ABCB1-mediated resistance. Cells were treated with the indicated
drugs for 48 hours and subjected to SRB assay. Nobiletin reduces
the IC.sub.50 of paclitaxel in resistant cancer cells (A2780/T)
(FIG. 3B) but not in drug sensitive cells (A2780) (FIG. 3A). The
cells were treated with paclitaxel in the presence or absence of
nobiletin for 12 days. Colony numbers were counted after Giemsa
staining using the software of Quantity one-Colony counting (FIG.
3C). Nobiletin reduces the IC.sub.50 of doxorubicin in resistant
cancer cells (A2780/T) (FIG. 3D). Nobiletin reduces the IC.sub.50
of paclitaxel in paclitaxel-resistant human non-small cell lung
cancer (NSCLC) cancer cells (A549/T). Nobiletin does not affect the
IC.sub.50 of doxorubicin in sensitive cancer cells (A2780) (FIG.
3F). Nobiletin does not affect the IC.sub.50 of paclitaxel in
paclitaxel-sensitive human non-small cell lung cancer (NSCLC)
cancer cells (A549) (FIG. 3G). IC.sub.50 values are represented as
mean.+-.SD of three independent experiments performed in
triplicate. ## or **, P<0.01, ### or ***, P<0.001,
significantly different from those obtained in the absence of
nobiletin.
[0017] FIGS. 4A and 4B show the effect of nobiletin on the
apoptosis and cell cycle of MDR cancer cells induced by PTX. Effect
of nobiletin on the percentage of annexin V-FITC-positive staining
in A2780/T cells treated with 0.94 .mu.M PTX using flow cytometry
(FIG. 4A). Effect of nobiletin on DNA-ploidy flow cytometric
analysis of A2780/T cells treated with 0.94 .mu.M PTX (FIG. 4B).
Different concentrations of nobiletin were added to A2780/T cells
with PTX for 48 h. The data is representative of three different
experiments and are shown as mean.+-.SD (n=3). ## or **, P<0.01,
### or ***, P<0.001, significantly different from those obtained
in the absence of nobiletin
[0018] FIGS. 5A to 5D show the effect of nobiletin on intracellular
accumulation of doxorubicin (DOX) and flutax-2 (F-tax, a
fluorescent taxol derivative) in drug-resistant ovarian cancer
cells. A2780 cells or A2780/T Cells treated with 5 .mu.M DOX (with
results shown in FIGS. 5A and 5B) or 5 .mu.M F-tax (with results
shown in FIGS. 5C and 5D) for 8 hours in the absence or presence of
4.5 .mu.M nobiletin, and 20 .mu.M quinidine (positive control) as
indicated. Intracellular DOX and F-tax accumulation were observed
with a florescence microscope (with results shown in FIGS. 5A and
5C) and evaluated by measuring florescence with flow cytometry
(with results shown in FIGS. 5B and 5D) as described below, for
example Section 2.6. The experiments were repeated for at least 3
times, presented are representative images
[0019] FIGS. 6A and 6B show that nobiletin increases the adsorption
and inhibits the efflux ratio of DOX in Caco-2 cells. FIG. 6A shows
the effect of nobiletin on the directional transport of DOX (10
.mu.M) across Caco-2 cell monolayers, whereas FIG. 6B illustrates
the effects of nobiletin on the efflux ratio of DOX (10 .mu.M) in
Caco-2 cell monolayers. Data represents the mean.+-.SD of three
individual determinations. .quadrature. AP.fwdarw.BL transport,
.box-solid. BL.fwdarw.AP transport. ## or **, P<0.01, ### or
***, P<0.001, significantly different from those obtained in the
absence of nobiletin
[0020] FIGS. 7A and 7B show that nobiletin was tested at a range of
concentrations for its capacity to stimulate P-gp ATPase activity
and to inhibit 200 .mu.M verapamil-stimulated P-gp ATPase activity.
EC.sub.50 measurements for stimulating P-gp ATPase activity by
nobiletin were shown in FIG. 7A, whereas IC.sub.50 measurements for
inhibiting 200 .mu.M verapamil-stimulated P-gp ATPase activity by
nobiletin were shown in FIG. 7B. Luminescence was read on a
luminometer and data was analyzed as described below, for example
Section 2.9
[0021] FIGS. 8A to 8C show the effects of the combinational
treatment of paclitaxel and nobiletin on ABCB1 expression and
AKT/ERK/Nrf2 pathway in ABCB1 overexpressing ovarian cancer cells.
A2780/T cells or A2780 cells were treated with nobiletin at various
concentrations for 48 hours. (FIG. 8A) The MDR1 mRNA level was
determined by RT-PCR. Equal amounts of total lysate were loaded and
detected by Western blot as shown in FIGS. 8B and 8C. Combination
treatment of paclitaxel and nobiletin did not influence either MDR1
mRNA or P-gp expression levels, but unregulated the p53 expression
and reduced the Nrf2 as well as the phosphorylation of AKT/ERK. The
experiments were performed three times
[0022] FIGS. 9A to 9C illustrate the docking analysis of nobiletin
with human ABCB1 homology model. FIG. 9A shows the cartoon style of
the homology model of human ABCB1 in which the binding poses of
QZ59-RRR (PDB: 4M2S) (green) and nobiletin (orange) are shown in
site 1. FIG. 9B shows the interactions between nobiletin and the
surrounding residues. The red dotted line represents hydrogen bond
between atoms. FIG. 9C shows a two dimensional interaction sketch
between nobiletin and its binding site residues of human ABCB1.
Residues are shown as colored bubbles, cyan indicates polar and
green indicates hydrophobic residues.
[0023] FIGS. 10A and 10B show the effect of transporter inhibitor
on the paclitaxel sensitivity of resistant cells. Quinidine (MDR1
inhibitor) (FIG. 10A) but not MK571 (MRPs inhibitor) or KO143 (BCRP
inhibitor) (FIG. 10B) reduces the IC.sub.50 of paclitaxel in
resistant cancer cells (A2780/T). Cells were treated with the
indicated drugs for 48 hours and subjected to SRB assay. **,
P<0.01, ***, P<0.001, Student's t-test (n=3) or one-way ANOVA
(n=3).
[0024] FIGS. 11A to 11D show the inhibition effect of nobiletin on
the directional transport and the efflux ratio of Rho123 (10 .mu.M)
(FIGS. 11A and 11B) or Flutax-2 (FIGS. 11C and 11D) across Caco-2
cell monolayers. Data represent the mean.+-.SD of three individual
determinations. .quadrature. AP.fwdarw.BL transport, .box-solid.
BL.fwdarw.AP transport. ## or **, P<0.01, ### or ***,
P<0.001, significantly different from those obtained in the
absence of nobiletin.
[0025] FIGS. 12A to 12D show the quantitative diagnostic graphics
for the synergistic effect between nobiletin (N) and paclitaxel (T)
generated by the computer simulation. FIG. 12A shows the fraction
affected (Fa)-Dose plot for nobiletin (N), paclitaxel (T) and
combination (C). FIG. 12B shows the Fa-CI plot (Chou-Talalay plot).
FIG. 12C shows the classic isobologram whereas FIG. 12D shows the
Fa-DRI plot (Chou-Martin plot) for the constant ratio combination
design. CI (combination index)<1 represents synergism; CI=1
indicates additive effect, and CI>1 represents antagonism.
DRI>1 represents reduced dose and reduced toxicity.
[0026] FIG. 13 shows the effects of nobiletin alone on ABCB1
expression and AKT/ERK/Nrf2 pathway in ABCB1 overexpressing ovarian
cancer cells. A2780/T cells or A2780 cells were treated with
nobiletin at various concentrations for 48 hours. Equal amounts of
total lysate were loaded and detected by Western blot. Nobiletin
alone did not influence ABCB1, Nrf2, total and phosphorylation of
AKT/ERK expression levels. The experiments were performed three
times.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] As used herein and in the claims, "comprising" means
including the following elements but not excluding others.
[0028] Recently studies showed that up-regulation of PI3K/AKT, ERK
and Nrf2 pathways are associated with resistance to multiple
chemotherapeutic drugs. Antitumor drugs are known to inhibit these
signaling pathways and consequently induce tumor cell sensitive to
chemotherapy drugs. Therefore, identification of the inhibitors
that potently inhibit the activation of AKT/ERK and Nrf2-dependent
response is desired to develop inhibitors to treat chemoresistant
cancer. Preferably the desired MDR reverser is safe and
non-toxic.
[0029] Nobiletin, the structure of which is shown in FIG. 1, is a
non-toxic dietary polymethoxylated flavone and presents in some
citrus fruits such as Citrus depressa (shiikuwasa) and Citrus
sinensis (oranges). It was reported to exhibit multiple biological
effects such as anti-inflammatory, anti-tumor, and neuroprotective
properties. As a potent chemo-preventive agent, nobiletin inhibited
the growth of several prostate cancer cell lines with IC.sub.50
values around 100 .mu.M by significantly increasing G.sub.0/G.sub.1
phase arrest. Moreover, it was found that nobiletin (20 .mu.M)
could increase the uptake of [.sup.3H] vinblastine in Caco-2 cells
and in ABCB1 transfected cell line LLC-GA5-COL300 by 3- and
1.8-fold, respectively, indicating the potential P-gp inhibition
effect of nobiletin.
[0030] The inventors performed a series of experiments to
investigate the reversal effect of nobiletin on ABCB1
overexpression cancer cell lines to chemotherapeutic agents.
Nobiletin at achievable nontoxic plasma concentrations (0.5 to 9
.mu.M) significantly inhibits the ABCB1 overexpressing MDR cancer
cell lines by inhibiting the AKT/ERK/Nrf2 pathways and modulating
the ABCB1 function, and has the potential for use in combination
therapies to treat MDR.
[0031] 1. Materials and Methods
[0032] 1.1 Reagents and Cell Culture
[0033] Nobiletin was purchased from Dalian Meilun Biology
Technology Co., Ltd, and the structure and purity was confirmed by
LC-MS in our lab. Flutax-2 was purchased from Life Technologies.
Paclitaxel (PTX) and doxorubicin (DOX), verapamil (Vrp), quinidine
(QND), 5-fluorouracil, docetaxel, dounorubicin and other chemicals
were purchased from Sigma-Aldrich (St. Louis, Mo.). Stock solutions
of nobiletin (40 mM), DOX (40 mM) and PTX (80 mM) were prepared in
dimethyl sulfoxide (DMSO) and appropriate working concentrations
were prepared in cell culture medium immediately before use. The
Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine
serum, penicillin and streptomycin were obtained from Life
Technologies Inc. (Grand Island, N.Y.). The DMSO, RNase A,
leupeptin, aprotinin, phenyl methyl sulfonyl fluoride, Triton X-100
and propidium iodide (PI) were purchased from Sigma-Aldrich Co. (St
Louis, Mo.). ERK 1/2 and actin antibodies were purchased from Santa
Cruz Biotechnology, USA; P-gp and P53 antibodies were purchased
from Calbiochem and Abcam; other antibodies such as AKT, P-AKT, and
P-ERK1/2 were purchased from Cell Signaling Technology, Inc.
[0034] Human ovarian cancer cells A2780 and its PTX-resistant cell
line A2780/T, human non-small cell lung cancer (NSCLC) A549 and its
PTX-resistant cell line A549/T were generously provided by
Professor Zhi-Hong Jiang (Macau University of science and
technology, Macau). Cells were grown as monolayers in RPMI-1640
medium supplemented with 10% fetal bovine serum (GIBCO, Paisley,
Scotland) at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere.
The indicated concentration of paclitaxel (0.94 .mu.M) was added to
the culture medium to maintain drug resistance for A2780/T and
A549/T. The mRNA level of P-gp didn't changed significantly after
grown in drug-free culture medium for 10 days for both resistant
cell lines. The human colon carcinoma cell line Caco-2 was
purchased from the ATCC, and cells at passage numbers 25-35 were
used for the assays.
[0035] 1.2 Cell Cytotoxicity Assay
[0036] Sulphorhodamine B (SRB) assays were used for cell density
determination, based on sensitive measure of total cellular
protein, which perform similarly compared with other proliferation
assays such as MTT assay. Briefly, cells were seeded into flat
bottomed 96-well plates at an initial density of 7.5.times.10.sup.3
per well before treatment. Cells were exposed to varying
concentrations of nobiletin (9, 4.5, 1.5 and 0.5 .mu.M) and
combined them with varying concentrations of PTX (1 .mu.M to 0.03
nM with 3.16 fold diluted, 10 .mu.M to 0.3 nM with 3.16 fold
diluted, 100 .mu.M to 3 nM with 3.16 fold diluted respectively) to
test whether this combination can enhance the growth inhibition of
MDR cancer cells. After removing the medium, cells were fixed in
10% trichloroacetic acid for 1 h at 4.degree. C. and then washed
with water five times. 0.4% SRB dissolved in 1% v/v acetic acid was
added and incubated 30 min for staining. The cells were quickly
washed with 1% acetic acid and left to dry overnight. The protein
bound SRB was solubilized by adding 200 .mu.l 10 mM Tris buffer per
well and was measured at wavelengths 490 nm using a plate reader
(Spectra MAX 250; Molecular Devices, Sunnyvale, Calif.). The
optical density of SRB in each well is directly proportional to the
cell number. The degree of resistance was estimated by comparing
the IC.sub.50 (concentration of 50% inhibition) for the MDR cells
to that of parent sensitive cells, while, the degree of reversal of
MDR was calculated by dividing the IC.sub.50 for cells with the
chemotherapeutic drugs in the absence of nobiletin by that obtained
in the presence of nobiletin.
[0037] 1.3 Colony Formation Assay
[0038] For the colony formation assays, A2780/T or A549/T cells
(200 cells/well) in 6-well plates were treated with culture medium
(containing 0.94 .mu.M PTX) or with nobiletin in different
concentrations (containing 0.94 .mu.M PTX) for 12 days. The A2780/T
cells were trypsinized and plated in fresh culture medium at a
density of 600 cells/9.6 cm.sup.2 plate. Subsequently, the cells
were fixed with 70% ethanol and stained with crystal violet (0.5%
in ethanol). The plates were rinsed with phosphate buffered saline
(PBS), and the colony numbers were counted using the software of
Quantity one-Colony counting.
[0039] 1.4 Cell Cycle Analysis
[0040] A2780/T cells were harvested 24 hours, 48 hours, or 72 hours
after treatment and washed twice with ice-cold PBS. The cells were
fixed and permeabilized with 70% ice-cold ethanol overnight at
4.degree. C. or 2 h at -20.degree. C. After one additional wash in
PBS, cells were stained with a staining solution containing
propidium iodide (PI) (50 .mu.l/ml) and RNase A (250 .mu.g/ml) for
30 min at room temperature. They were then pelleted, washed and
suspended in PBS to a final concentration of 1.times.10.sup.6/ml
and analyzed by flow cytometry BD FACS Aria (BD Biosciences, San
Jose, Calif.).
[0041] 1.5 Apoptosis Analysis by Annexin-V/PI Double-Staining
Assay
[0042] After treatment, 1.times.10.sup.6 cells were collected,
washed and suspended in 100 .mu.l of binding buffer (10 mM
N-2-hydroxyethylpiperazine-N,-2-ethanesulfonic acid/NaOH, 140 mM
NaCl, 2.5 mM CaCl2, pH 7.4). Apoptotic cells were identified by
double supravital staining with 5 .mu.l recombinant FITC
(fluorescein isothiocyanate)-conjugated Annexin-V and 5 .mu.l PI
(50 .mu.g/ml). The cells were stained for 15 min at room
temperature in the dark, and analyzed by fluorescence-activated
cell sorting cater-plus flow cytometry. Data acquisition and
analysis were performed in BD FACS Aria with FlowJo software.
[0043] 1.6 Combination Index in Nobiletin Combination Studies
[0044] The synergistic therapeutic effect for the combination of
nobiletin and PTX was evaluated using the Chou-Talalay Method.
"Combination index" (CI) was calculated by this method to
quantitatively depict synergism (CI<1), additive (CI=1), or
antagonism (CI>1) effect. Briefly, drug resistant A2780/T cells
were exposed to a serially diluted mixture of nobiletin
(IC.sub.50=31.62 .mu.M) and PTX (IC.sub.50=2.51 .mu.M) for 48
hours. The 2-fold serial dilution with several concentration points
above and below its IC.sub.50 value was used for evaluating
cytotoxicity of combination by SRB method as above description.
With the use of CalcuSyn software v. 2.1 (Bio-soft), synergy is
further refined as synergism (combination index=0.3-0.7), strong
synergism (combination index=0.1-0.3), and very strong synergism
(combination index<0.1).
[0045] 1.7 Intracellular Accumulation of Doxorubicin and
Flutax-2
[0046] 1.7.1 Fluorescence Microscopy Observation
[0047] A2780 or A2780/T cells (5.times.10.sup.6) were cultured on
the cover glass (ISO LAB 20.times.20 mm) DOX (5 .mu.M), or flutax-2
(1 .mu.M) (active fluorescent taxoids) alone or in combination with
nobiletin (4.5 .mu.M) was added and incubated for 8 h. After
treatment, cells were fixed in 4 wt % formaldehyde (Sigma-Aldrich).
Nuclear DNA was stained with 1 .mu.g/mL blue-fluorescent DAPI (1
mg/mL in H.sub.2O stock solution; Invitrogen D1306). One drop of
fluorescent preservation solution (fluorsave reagent, CALBIOCHEM)
was added before observation. Imaging was carried out for comparing
the intracellular accumulation of DOX and flutax-2 with a
Fluorescence Microscopy (Leica DM2500, Leica, Germany).
[0048] 1.7.2 Flow Cytometry Analysis
[0049] Flutax-2 (1 .mu.M) and DOX (5 .mu.M) was added to A2780 or
A2780/T cells and incubated with or without nobiletin (4.5 .mu.M)
for 8 h. Cells were detached, re-suspended in 500 .mu.l of PBS
after washed twice with cold PBS, and analyzed by flow cytometry
(BD FACS Aria, BD Biosciences, San Jose, Calif.). Excitation and
emission wavelengths (nm) used for DOX and flutax-2 were as
follows: 480 to 585; and 496 to 524. Quinidine (QND, 20 .mu.M), a
known ABCB1 inhibitor, was used as a positive control.
[0050] 1.8 Transport Assay in Caco-2 Monolayer Model
[0051] The Caco-2 cell line was seeded on Millipore Millicell
plates and formed a confluent monolayer over 21 days prior to the
experiment. The integrity of the cell monolayers was checked by
measuring the transepithelial electrical resistance (TEER) before
and after the transport experiments using a WPI EVOM2 Epithelial
voltohmmeter fitted with STX2 chopstick electrodes (World Precision
Instruments, Sarasota, Fla., USA). On day 21, the transport assay
included apical-to-basolateral (A.fwdarw.B) and
basolateral-to-apical (B.fwdarw.A) transport rate determinations
for rhodamin123 (5 .mu.M), flutax-2 (1 .mu.M) and DOX (10 .mu.M) in
Caco-2 cell line was carried out over a 2 hour time period.
Briefly, samples (100 .mu.L) were collected from apical/basolateral
side of Caco-2 cell monolayer at predetermined times of 30, 60, 90,
and 120 min, and immediately detected for the fluorescence
intensity in 96 well black plate (Corning; Cat. 3603) using a
microplate reader (infinite M200 PRO, TECAN, Switzerland). For
inhibition studies, bidirectional transport of target compound was
conducted in Caco2 cell monolayer with nobiletin added in both
apical and basolateral chambers. Quinidine (QND) was used as potent
control inhibitors of P-gp.
[0052] The apparent permeability coefficients (Papp) were
calculated as
P app = Q t .times. 1 C 0 A ##EQU00001##
[0053] Where dQ/dt (mM/sec) is the rate of permeation of compound
across the cells, Co (mM) is the donor compartment concentration at
time zero and A (cm.sup.2) is the area of the cell monolayer. The
decrease in Efflux Ratio (ER=Papp (B to A)/Papp (A to B)) in the
presence of nobiletin and putative inhibitor QND was determined to
assess their relative inhibitory potency to transporter P-gp.
[0054] 1.9 ABCB1 ATPase Activity Assay
[0055] The impact of nobiletin on P-gp ATPase activity was
estimated by Pgp-Glo.TM. assay systems (Promega, USA). The
inhibitory effects of nobiletin were examined against a
verapamil-stimulated ABCB1 ATPase activity. Sodium orthovanadate
(Na.sub.3VO.sub.4) was used as an ABCB1 ATPase inhibitor. Following
manufacture's instruction, 0.25 mM Na.sub.3VO.sub.4, 0.5 mM
verapamil, or nobiletin in various concentrations were incubated
with assay buffer, 25 .mu.g recombinant human ABCB1 membranes and 5
mM MgATP at 37.degree. C. for 40 min. For examination the
inhibitory effects of nobiletin against verapamil-stimulated P-gp
ATPase activity, then 200 .mu.M verapamil was added with nobiletin
together. Luminescence was initiated by ATP detection buffer. The
plate (white opaque 96-well, corning, USA) was further incubated at
room temperature for 20 min to develop luminescent signal, and was
read with luminometer (infinite M200 PRO, TECAN, Switzerland). The
changes of relative light units (ARLU) were determined by comparing
Na.sub.3VO.sub.4-treated samples with nobiletin only or nobiletin
and verapamil combination-treated samples, and hence, the ATP
consumed was calculated by comparing to a standard curve.
[0056] 1.10 RT-PCR Analysis
[0057] RT-PCR was performed to evaluate MDR1 mRNA expression. mRNA
from cell lysates were purified by binding to poly(dT) magnetic
beads (Life technologies) and reverse transcribed by using
SuperScript II (Life technologies). Standard quantitative RT-PCR
was performed in duplicates at least two to three times by using
SYBR Green (Molecular Probes) protocols on the ViiA.TM. 7 Real-Time
PCR System (Life technologies). The primer sequences:
5'-GAGAGATCCTCACCAAGCGG-3' (SEQ ID NO:1) and
3'-CGAGCCTGGTAGTCAATGCT-5' (SEQ ID NO:2) for MDR1, and
5'-AGAAGGCTGGGGCTCATTTG-3' (SEQ ID NO:3) and
3'-AGGGGCCATC-CACAGTCTTC-5' (SEQ ID NO:4) for control gene
eukaryotic translation initiation factor (TIF). RT-PCR data were
normalized by measuring average cycle threshold (Ct) ratios between
candidate genes and control gene TIF.
[0058] 1.11 Western Blot Analysis
[0059] The total cellular samples were harvested and rinsed twice
with ice-cold PBS buffer. Cell extracts were lysed in RIPA buffer
(50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride and
EDTA) containing protease inhibitor cocktails (Roche Life Science,
USA). Protein concentration was determined using the BCA protein
assay kit. Equal amounts of cell lysates were resolved by SDS-PAGE
and subsequently electrophoretically transferred onto PVDF
membranes (Millipore, Darmstadt, Germany). After blocking in
tris-buffered saline containing 0.1% of Tween20 (TBST) with 5%
(w/v) skim milk (Nestle Carnation, New Zealand) for 2 h at room
temperature, the membranes were incubated with primary and
secondary antibodies and subsequently visualized with an enhanced
chemiluminescence detection kit (Thermo Scientific.TM.
SuperSignal.TM. West Pico Chemiluminescent Substrate, USA).
.beta.-Actin was used as the loading control for the experimental
data analysis.
[0060] 1.12. Molecular Modeling--ABCB1
[0061] In order to figure out the exact binding site for nobiletin,
we used homology modeling and molecular docking to study the
interaction between human P-Glycoprotein and nobiletin.
[0062] Human P-glycoprotein (ABCB1) was thought to have four sites
interacting with the inhibitors, so we rebuilt the four sites using
Prime v2.1 in Maestro 9.0 (Schrodinger, Inc., New York, N.Y.,
2009). The 3D structures of ABCB1 from the mouse was selected as
the templates: The complex structure cocrystallized with QZ59-RRR
(PDB: 4M2S) for site 1, the complex structure cocrystallized with
QZ59-SSS (PDB: 4M2T) for site 2, the apo structure (PDB: 3G5U) for
site 3 and site 4. The ligands from the complex templates were
retained and used to define the site 1 and site 2 in the homology
structures. The site 3 was defined by residues contributing to
verapamil binding and the site 4 was defined by two residues which
were common to the other three sites.
[0063] All the docking calculations for four sites were performed
in the Induced Fit Docking module (Schrodinger, Inc., New York,
N.Y., 2009) and the pose was ranked by the XP mode of Glide program
v5.5 (Schrodinger, Inc., New York, N.Y., 2009). Then the pose with
the highest docking were selected for further conformational
analysis.
[0064] 1.13 Statistical Analysis
[0065] Statistical analysis was carried out using Student's t-test
or one-way analysis of variance with Microsoft Excel 2010, and the
level of significance was set at a P value of <0.05(*), <0.01
(**) or <0.001(***). Data was expressed as the mean.+-.SD.
[0066] 2. Results
[0067] 2.1 Demonstration of Multidrug Resistance in Cell Line
Model
[0068] The IC.sub.50 values of several anti-cancer drugs in
parental line (A2780) and stably paclitaxel-resistant cell line
(A2780/T) were determined. The mean IC.sub.50 values for PTX and
DOX were 501-fold and 158-fold greater in A2780/T cells than that
of A2780, as shown in FIG. 2A and FIG. 2B, which confirmed that
this cell line exerted much higher tolerance than the parental
sensitive cell line. In FIG. 2C and FIG. 2D, RT-qPCR and Western
blot analysis confirmed that the MDR1 gene and P-gp protein in the
A2780/T were all significantly increased (p<0.01).
[0069] 2.2 Sensitizing of ABCB1-Overexpressing Cells to
Chemotherapeutic Agents by Nobiletin
[0070] Firstly, the intrinsic cytotoxicity of nobiletin in A2780
and A2780/T was measured by the SRB assay. Nobiletin have similar
IC.sub.50 for both A2780 and A2780/T (without adding 0.94 .mu.M PTX
to culture medium), as shown in FIG. 2E. Notably, the results
showed that nobiletin at 9 .mu.M, showed no obvious cytotoxic
effect to both cell lines, and more than 90% cells were viable.
Based on this result, nobiletin was tested in the reversal assays
at a maximum concentration of 9 .mu.M.
[0071] Next, we tested whether nobiletin could reverse the MDR of
A2780/T cells. Treatment with nobiletin significantly decreased the
IC.sub.50 of PTX and DOX in A2780/T cell in a
concentration-dependent manner, as shown by the shift in the
cytotoxicity curves to the left in FIGS. 3B and 3D. Specifically,
as shown in Table 1, treatment with 0.5, 1.5, 4.5, and 9 .mu.M
nobiletin reduced the IC.sub.50 of PTX in A2780/T cells by 3.0-,
18.5-, 163.5-, and 432.9-fold, respectively. The IC.sub.50 of DOX
was reduced 1.58-, 3.16-, 5.39- and 15.92-fold after combination
treatment with 0.5, 1.5, 4.5, and 9 .mu.M nobiletin, respectively.
Meanwhile, nobiletin, at tested concentrations, had no effect on
the IC.sub.50 of PTX and DOX in parental non-resistant A2780 cells,
as shown in FIGS. 3A and 3F. Moreover, at concentration of 4.5
.mu.M, nobiletin also reduced IC.sub.50 values of docetaxel and
daunorubicin with reversal fold of 15.8 and 13.6, respectively,
whereas it also slightly decreased the IC.sub.50 values of
5-fluorouracil (non-substrate of ABCB1) with reversal fold of 6.3
as shown in Table 1.
TABLE-US-00001 TABLE 1 Nobiletin reverses the ABCB1-mediated drug
resistance to 5- fluorouracil, docetaxel and doxorubicin in A2780/T
cells. A2780/T Drug IC.sub.50 .+-. SD (.mu.M) fold reversal
Paclitaxel 2.67 .+-. 0.22 1.00 +0.5 .mu.M N 0.90 .+-. 0.15*** 3.00
+1.5 .mu.M N 0.15 .+-. 0.02*** 18.47 +4.5 .mu.M N 0.02 .+-.
0.004*** 163.46 +9 .mu.M N 0.006 .+-. 0.0002*** 432.98 Docetaxel
17.9 .+-. 2.89 1.00 +0.5 .mu.M N 8.97 .+-. 1.46* 2.05 +1.5 .mu.M N
3.57 .+-. 0.58** 5.01 +4.5 .mu.M N 1.13 .+-. 0.18*** 15.84
Doxorubicin 5.97 .+-. 0.49 1.00 +0.5 .mu.M N 3.77 .+-. 0.30** 1.58
+1.5 .mu.M N 1.89 .+-. 0.16*** 3.16 +4.5 .mu.M N 1.13 .+-. 0.18***
5.39 +9 .mu.M N 0.38 .+-. 0.04*** 15.92 Daunorubicin 11.91 .+-.
0.97 1.00 +0.5 .mu.M N 9.46 .+-. 0.77 1.26 +1.5 .mu.M N 4.49 .+-.
0.73* 2.70 +4.5 .mu.M N 0.89 .+-. 0.15** 13.58 5-Fluorouracil
159.19 .+-. 18.30 1.00 +0.5 .mu.M N 132.2 .+-. 23.78 1.21 +1.5
.mu.M N 79.78 .+-. 9.18* 1.99 +4.5 .mu.M N 26.14 .+-. 1.77*
6.08
TABLE-US-00002 TABLE 2 Nobiletin reverses the ABCB1-mediated drug
resistance to 5- fluorouracil, docetaxel and doxorubicin in A549/T
cells. A549/T Drug IC50 (.mu.M) Fold reversal Paclitaxel 1.89 .+-.
0.15 1.00 +0.5 .mu.M N 0.75 .+-. 0.06* 2.51 .+-. 0.01 +1.5 .mu.M N
0.34 .+-. 0.02** 5.65 .+-. 0.8 +4.5 .mu.M N 0.09 .+-. 0.01*** 22.33
.+-. 3.6 +9 .mu.M N 0.03 .+-. 0.004*** 62.88 .+-. 0.98 Docetaxel
13.36 .+-. 1.09 1.00 +0.5 .mu.M N 8.43 .+-. 0.69* 1.59 .+-. 0.01
+1.5 .mu.M N 2.67 .+-. 0.22** 5.05 .+-. 0.82 +4.5 .mu.M N 0.71 .+-.
0.11** 19.18 .+-. 4.59 Doxorubicin 5.97 .+-. 0.49 1.00 +0.5 .mu.M N
4.23 .+-. 0.35 1.42 .+-. 0.23 +1.5 .mu.M N 2.67 .+-. 0.22* 2.25
.+-. 0.37 +4.5 .mu.M N 1.19 .+-. 0.10** 5.01 .+-. 0.01 Daunorubicin
10.07 .+-. 1.63 1.00 +0.5 .mu.M N 5.97 .+-. 0.49* 1.68 .+-. 0.14
+1.5 .mu.M N 2.84 .+-. 0.46* 3.55 .+-. 0.01 +4.5 .mu.M N 0.71 .+-.
0.13** 14.17 .+-. 0.04 5-Fluorouracil 133.57 .+-. 10.86 1.00 +0.5
.mu.M N 106.10 .+-. 8.63 1.26 .+-. 0.01 +1.5 .mu.M N 66.95 .+-.
5.44* 1.99 .+-. 0.01 +4.5 .mu.M N 16.82 .+-. 1.36** 7.94 .+-.
0.02
[0072] In another ABCB1-overexpressing non-small cell human lung
cancer cell line A549/T, which is also PTX-resistant, and its
parental cells A549, it was observed that similar reversal effects
of nobiletin to PTX. The intrinsic cytotoxicity of nobiletin in
A549 and A549/T was also measured by the SRB assay. Nobiletin have
similar IC.sub.50 for both A549 and A549/T (without adding 0.94
.mu.M PTX to culture medium), as shown in FIG. 2F. Notably, the
results showed that nobiletin at 9 .mu.M demonstrated no obvious
cytotoxic effect to both cell lines, and more than 90% cells were
viable. Based on this result, nobiletin was tested in the reversal
assays at a maximum concentration of 9 .mu.M.
[0073] In FIG. 3E, the treatment of nobiletin on A549 cell line at
0.5, 1.5, 4.5, and 9 .mu.M significantly decreased the IC.sub.50 of
PTX with reversal fold of 2.51, 5.65, 22.33 and 62.88,
respectively.
[0074] Moreover, at concentration of 4.5 .mu.M, nobiletin also
reduced IC.sub.50 values of paclitaxel, docetaxel and daunorubicin
with reversal fold of 22.33, 19.18 and 14.17, respectively, whereas
it also slightly decreased the IC.sub.50 values of doxorubicin and
5-fluorouracil (non-substrate of ABCB1) with reversal fold of 5.01
and 7.94, respectively, as shown in Table 2.
[0075] In order to determine whether the drug inhibition effect is
related to the specific transport protein ABCB1, the inventors also
tested the effect of quinidine (QND, an inhibitor of P-gp), MK571
(an inhibitor of MRPs) and KO143 (an inhibitor of BCRP) on
inhibiting A2780/T cells to low-dose PTX-induced death. The
fold-reversal of QND at concentration of 0.24, 0.72 and 2.16 .mu.M
to PTX was 3.25, 43.15, and 252.83, respectively, in A2780/T cells,
as shown in FIG. 10A. But, QND showed an intimal cytotoxicity at
concentration of 6.48 .mu.M with only 40% cells survived. Moreover,
MK571 and KO143 had no effects on the IC.sub.50 of PTX which
demonstrate that the reversal effect is special to ABCB1
transporter, as shown in FIG. 10B.
[0076] Moreover, the long term reversal effects of nobiletin on
ABCB1 mediated MDR to PTX were evaluated using colony formation
assays. Complete inhibition of colony formation can be achieved
with the combination of 0.94 .mu.M PTX with different
concentrations of nobiletin, whereas no inhibition was observed for
either 9 .mu.M nobiletin or 0.94 .mu.M PTX alone, as shown in FIG.
3C. Taken together, these results indicated that the combination of
nobiletin and PTX elicits significantly higher cytotoxic response
in ABCB1 overexpression MDR cancer cells.
[0077] In short, the results of this study suggest that nobiletin
significantly sensitizes ABCB1-overexpressing cells to
chemotherapeutic drugs that are substrates of ABCB1.
[0078] 2.3 Potentiating PTX Induced Apoptosis in Resistant A2780/T
Cells by Nobiletin
[0079] The inventors next investigated whether nobiletin increased
the PTX-induced apoptosis in A2780 and A2780/T cells using double
staining method. Consistent with its ability to inhibit cell
growth, treatment with 0.5, 1.5, 4.5, and 9 .mu.M nobiletin could
significantly increase apoptosis induced by 0.94 .mu.M PTX in a
concentration-dependent manner, as shown in FIG. 4A. The inventors
found that treatment with only 0.5 .mu.M nobiletin could boost the
apoptosis induced by PTX (0.94 .mu.M) to a similar degree as that
of 2.51 .mu.M PTX (IC.sub.50). While single treatment of 9 .mu.M
nobiletin or 0.94 .mu.M PTX alone did not show apoptosis
induction.
[0080] To further confirm these results, the inventors examined the
well-established biochemical markers of cell cycle arrest and
apoptosis: p53. Consistent with cell growth inhibition and
apoptosis, treatment of PTX in combination with nobiletin resulted
in accumulation of p53 in treated cells. The results were shown in
FIG. 8B.
[0081] 2.4 Arrest of Resistant Cells in G2/M-Phase by Nobiletin-PTX
Combination
[0082] In this study was, the inventors investigated whether the
effect of nobiletin causing G2/M cell cycle arrest is related to
their observed synergistic effect between nobiletin and PTX. The
results of this study were illustrated in FIGS. 4A and 4B.
[0083] Asynchronously growing A2780/T cells and its sensitive
parental cell line A2780, treated with PTX in absence and presence
of nobiletin, were examined for their cell cycle progression by
flow cytometry. In untreated control, the percentage of A2780 cells
in G.sub.0/G.sub.1-, S- and G.sub.2/M-phases were 71.6%, 7.76% and
18.27%, respectively, while the percentage of A2780/T cells in
G.sub.0/G.sub.1-, S- and G.sub.2/M-phases were 66.13%, 6.25% and
24.77%, respectively. For A2780 cells, single exposure (24, 48, and
72 hours) with PTX (0.01 .mu.M) resulted in G2 arrest, manifested
by an increased G2-M content (31.9%, 63.2% and 80.07%), and
decreased G1 phase content (46.47%, 15.57% and 5.53%, respectively)
as shown in FIG. 4B.
[0084] In the absence of nobiletin treatment, there were 72% G1
phase and 17% G2 phase cells incubated with 0.94 .mu.M PTX, whereas
this distribution significantly shifted to 9.6% G1 and 75.77% G2
phase cells after treatment of nobiletin at 9 .mu.M in combination
with 0.94 .mu.M PTX as further illustrated in FIG. 4B. This pattern
was evidenced after 24 h and persisted over the 72 h of treatment
(N.B. such data was not shown in FIG. 4). Also shown in FIG. 4B, a
notable G2/M arrest was observed even with the lowest concentration
of nobiletin tested (0.5 .mu.M). Thus, while A2780/T cells were
remarkably resistant to 0.94 .mu.M PTX, which means that PTX alone
has no cell cycle effect thereon, the combination of nobiletin with
PTX was found to greatly increase the proportion of G2/M arrested
cells to above 75%. However nobiletin of 9 .mu.M alone had no
effect on the cell cycle of A2780/T.
[0085] 2.5 Evaluation of Combinational Effects of Nobiletin and
PTX
[0086] The combinational cytotoxic effect of nobiletin with PTX in
A2780/T cells was further evaluated using the Median Effect methods
described by T-C Chou and P. Talalay. The combination index (CI)
values calculated at 50% (ED.sub.50) and 90% (ED.sub.90) cell kill
were 0.013 and 5.14.times.10.sup.-5 as shown in Table 3, indicating
very strong synergistic cytotoxic effect (CI<0.1) for
combinations of nobiletin (denoted as `N` in Table 3) with PTX in
the ABCB1-overexpressing A2780/T cells. With CalcuSyn simulation,
an ED.sub.50 is produced by 35.96 .mu.M nobiletin or 4.20 .mu.M PTX
in A2780/T cells, but a combination of agents will produce this
ED.sub.50 at 0.022 .mu.M PTX with 0.286 .mu.M nobiletin, a 200-fold
decrease for the ED.sub.50 dose of PTX (Table 3). The quantitative
diagnostic graphics for the synergistic effect between nobiletin
(N) and paclitaxel (T) were shown in FIGS. 12A to 12D.
TABLE-US-00003 TABLE 3 The calculated CI for combination of
nobiletin and paclitaxel as well as the simulated synergism dose at
Fa 0.5 (ED50) Data for Fa = 0.5 CI value Dose N (.mu.M) Dose PTX
(.mu.M) N 35.9574 PTX 4.20446 N + PTX 0.01334 0.28572 0.02268
[0087] CI analyses of the effects of nobiletin in combination with
paclitaxel are shown. The CI values were plotted as a function of
the particular inhibitory effect. CI values<1 represent a
synergistic combination, CI values equal to 1 indicate an additive
effect whereas CI values>1 represent antagonistic combinations.
It can be concluded from the table that PTX was significantly
reduced in nobiletin treated A2780/T cells.
[0088] In short, this evaluation study confirms the synergistic
effect in the combinational use of nobiletin and PTX in cancer
treatment.
[0089] 2.6 Increase of the Intracellular Accumulation of DOX and
Flutax-2 by Nobiletin
[0090] The above results proved that nobiletin have a significant
effect on reversing ABCB1-mediated MDR. At present, the mechanism
of this phenomenon is unknown. Therefore, the inventors conducted
assays to examine the effect of nobiletin on the accumulation of
DOX, and Flutax-2 (a fluorescent taxol derivative) in A2780 cells
and their corresponding ABCB1-overexpressing A2780/T cells.
[0091] The inventors studied the effect of nobiletin on the
intracellular accumulation of DOX and Flutax-2 using fluoresce
microscope and flow cytometry analysis. The intracellular
accumulation of DOX and Flutax-2 were significantly higher in A2780
than that in A2780/T, as shown in FIGS. 5A to 5D. When the
drug-resistant cells were treated with 4.5 .mu.M nobiletin or 20
.mu.M QND (positive control), the intracellular accumulation of DOX
(as shown in FIG. 5A), and Flutax-2 (as shown in FIG. 5C) were
higher than that in untreated A2780/T. In contrast, nobiletin alone
had no effect on DOX and Flutax-2 levels in the parental A2780
cells. With flow cytometry analysis, the enhanced intracellular
accumulation of DOX, or Flutax-2 by nobiletin were further
confirmed as shown in FIGS. 5B and 5D.
[0092] Taken together, these results showed that nobiletin
significantly increased the intracellular accumulation of
chemotherapeutic drugs in ABCB1-overexpressing cells, thus
increasing the cytotoxicity to these MDR cells. In other words,
nobiletin is shown to enhance the efficacy of DOX or PTX in cancer
treatment.
[0093] 2.7 Inhibition of the Efflux Activity of ABCB1 Transporter
in Caco-2 Cells by Nobiletin
[0094] Human colorectal carcinoma Caco-2 cells are widely used as
an in vitro model for predicting human drug absorption and efflux
activity of transporters. To further confirm the effect of
nobiletin on P-gp function, the inventors evaluated the
concentrations of the P-gp substrates Rho 123, DOX, and Flutax-2 in
the presence or absence of nobiletin using the Caco-2 monolayer
model.
[0095] Two hours after administration, the values of P.sub.app
(A-B) of DOX (as shown in FIG. 6A), Rho 123 (as shown in FIGS. 11A
and 11B), and Flutax-2 (as shown in FIGS. 11C and 11D) was
increased in the presence of nobiletin in a dose-dependent fashion;
moreover, the efflux ratio (the ratio between the P.sub.app from
the BL to the AP side and that from the AP to the BL side) was
decreased in an nobiletin concentration dependent manner. As shown
in FIG. 6B, the efflux ratio of DOX was decreased about 3.23-,
3.75- and 4.6-fold with nobiletin concentration of 0.5, 1.5, 4.5
.mu.M. Intriguingly, the inhibitory effect of nobiletin at 4.5
.mu.M was stronger than that of QND (20 .mu.M) which was used as
positive control.
[0096] These results were in agreement with the notion that
nobiletin increased Rho 123, DOX, and Flutax-2 accumulation in
resistant ABCB1-overexpressing cells by inhibiting ABCB1
transporter.
[0097] In short, the decrease of efflux ratio of DOX in the
presence of nobiletin suggested that nobiletin is shown to increase
absorption of DOX into Caco-2 cells by effecting the ABCB1
transporter function.
[0098] 2.8 Activation of the ATPase Activity of ABCB1 by
Nobiletin
[0099] The efflux function of ABCB1 has a close relationship with
ATP hydrolysis. Therefore, the inventors measured ABCB1-mediated
ATP hydrolysis with different concentrations of nobiletin. As shown
in FIG. 7A, nobiletin stimulated the ATPase activity of ABCB1 in a
dose-dependent manner, with EC.sub.50 of 5.88 .mu.M and a maximal
stimulation of 3-fold of the basal activity, suggesting that
nobiletin affected the ATPase activity of ABCB1 and might interact
at the drug-substrate-binding site as a substrate of ABCB1.
[0100] To characterize inhibition effect of nobiletin on P-gp
ATPase activity, the inventors also examined the effects of
nobiletin on verapamil stimulated P-gp ATPase activity. Verapamil
is sometimes referred as a P-gp inhibitor because as a substrate
for transport it inhibits P-gp activity with other substrates by
interfering with their transport in a competitive mode. FIG. 6B
showed the reduction of 200 .mu.M verapamil-stimulated ATPase
activity by nobiletin with an IC.sub.50 value of 7.17 .mu.M,
indicating nobiletin is a P-gp ATPase inhibitor.
[0101] 2.9 Mechanism of Reversal of ABCB1-Mediated MDR by
Nobiletin
[0102] The reversal of ABCB1-mediated MDR can be achieved either by
reducing ABCB1 expression or by inhibiting the function of ABCB1
transporter. Therefore, the inventors investigated the effect of
nobiletin on the expression of ABCB1 at both mRNA and protein
level.
[0103] At the selected concentrations used in the reversal assays,
nobiletin did not significantly alter the expression of MDR1 mRNA,
as shown in FIG. 8A, or protein level of ABCB1, as shown in FIG. 8B
in A2780/T cells. These findings revealed that the MDR reversal
effect of nobiletin was not due to the inhibition of ABCB1
expression. Therefore, the result suggested that the inhibition of
ABCB1 transporter function appears to be the mechanism of
sensitization of ABCB1-overexpressing MDR cells by nobiletin could
be, and such inhibition leads to an increase in intracellular
accumulation of chemotherapeutic drugs.
[0104] 2.10 Inhibition of the Phosphorylation of AKT/ERK/Nrf2 by
Nobiletin-PTX Combination
[0105] Moreover, nobiletin was reported to inhibit phosphorylation
of AKT and phosphorylation of ERK2 in HGF-treated liver cancer
HepG2 cells. Considering the up-regulation of PI3K/AKT and MAP
kinase/ERK pathways in resistance MDR cancer cells, hence, the
inventors examined the effect of nobiletin on the expression of the
total and phosphorylated AKT and ERK in A2780/T cells.
[0106] After treatment with PTX and nobiletin for 48 h, there was
significant inhibitory effect on phosphorylated AKT and ERK, but
not on total AKT and ERK (as shown in FIG. 8B), indicating the
inhibition of PI3K/AKT and MAP kinase/ERK pathways by the
combinational treatment. Moreover, there was a significant decrease
for the phosphorylated AKT/ERK level after treatment with 50 .mu.M
nobiletin as shown in FIG. 8B. However nobiletin alone at reversal
concentrations had no effect on the expression of the total and
phosphorylated AKT and ERK as shown in FIG. 13. These results
indicated that enhanced cytotoxic response by co-treatment with
nobiletin and PTX in ABCB1 overexpression MDR cancer cells is
associated with inhibition of PI3K/AKT and MAP kinase/ERK
pathways.
[0107] Nuclear factor E2-related factor 2 (Nrf2) is a transcription
factor that upregulates expression of a battery of genes to combat
oxidative and electrophilic stress. Recent studies reveal that
activation of the Nrf2 overexpression enhances chemoresistance,
whereas blockade of Nrf2 inhibits a variety of cancer cells. In
this study, the inventors observed a remarkably higher level of
Nrf2 in A2780/T cells as compared with A2780 cells as shown in FIG.
8C. Nobiletin in combination with 0.94 .mu.M PTX reduced the
protein level of Nrf2 in a dose-dependent manner. These results
clearly demonstrate that nobiletin is a potent small-molecular
inhibitor of Nrf2.
[0108] 2.11 Molecular Docking Simulation of Nobiletin within the
Drug Binding Cavity of ABCB1
[0109] To understand the binding mechanism of nobiletin to homology
model 28 of human ABCB1 at molecular level, the inventors performed
glide docking using ABCB1-QZ59-RRR (site-1), ABCB1-QZ59-SSS
(site-2), ABCB1-verapamil (site-3), and site common to above three
sites (site-4) and ATP binding site. According to the docking
result, the poses of nobiletin was only accommodated to site 1 with
Docking score (Kcal/mol) at -9.216. There were no poses suitable
for nobiletin to other three sites. Thus, site 1 was the only
rational site for nobiletin.
[0110] As shown in FIG. 9A, the binding site of nobiletin was
partially superposed with the binding site of QZ59-RRR known as
Site 1. The ring-a of nobiletin substituted with four methoxyl
groups was mainly engaged in hydrophobic contacts with Tyr307,
Phe303, Tyr310, Phe335, Leu339, Leu336, Leu332. The methoxy of
ring-a and the carbonyl of ring-b formed hydrogen bonds with Tyr
307 and Gln725 respectively, which also appeared in the binding of
vardenafil and tadalafil. As for ring-c, the hydrophobic contacts
with Phe732, Phe971, Ser970, Ile78, Met75, Tyr953, Val71 kept the
conformation stable.
[0111] 3. Discussion
[0112] Traditional chemotherapy drugs such as PTX remain the
cornerstone of tumor therapy, but the occurrence of drug resistance
has been a major obstacle leading to the failure of tumor
treatment. A number of different mechanisms were found to mediate
the development of MDR, including overexpression of ABC
transporters, activation of PI3K/AKT, MAP kinase/ERK and Nrf2
pathways. ABCB1 (P-gp) has been demonstrated to be an essential MDR
transporter along with some relatives of the ABC family
transporters (like ABCG2, ABCC1 and ABCC10) for several major
chemotherapeutic drugs. In the past thirty years, great efforts
have been made to search for the ABCB1 inhibitors. The three
generation of ABC modulators such as quinine, verapamil,
cyclosporine-A, tariquitor, PSC 833, LY335979, and GF120918
required high doses to reverse MDR and were associated with adverse
effects. Currently, discoveries of more efficacious, non-toxic and
less expensive compounds from natural products to reverse MDR are
gaining increasing interests.
[0113] The inventors found that nobiletin was found to restore the
cytotoxicity of PTX in ABCB1-expressing A2780/T cells. Pilot
studies in small-animal and human clinical trials indicated
nobiletin had a favorable safety profile without adverse events and
significant effects in reducing total cholesterol, improving blood
lipid profile. The peak plasma level of nobiletin was .about.2.5
.mu.M in healthy subjects after single dose of Sytrinol (containing
1053 mg of total polymethoxylated flavones) and -22.5 .mu.M in rats
after single administration of 50 mg/kg of body weight by gavage.
Upon considering the appropriate structure, safety, multiple modes
of action and outstanding activity based on inventors' primary
screening data, the inventors investigated if nobiletin could
inhibit the ABCB1-overexpression MDR cancer cells to
chemotherapeutic agents and the underlying mechanisms.
[0114] In this study, nobiletin at non-cytotoxic concentrations
significantly increased the sensitivity of ABCB1 overexpressing
A2780/T, and A549/T cell lines to chemotherapeutic agents such as
DOX, PTX, docetaxel and dounorubicin, whereas it cannot potentiate
the effect of these substrate drugs on parental cells as shown in
FIG. 3A. A significant decrease in the IC.sub.50 values of PTX
(about 432 fold) was observed for the first time by co-treatment
with PTX and nobiletin. In accordance with that of the cytotoxicity
assay, it was found that nobiletin remarkably enhanced the
intracellular accumulation of DOX and flutax-2 in drug resistant
cells but not parental sensitive cells, indicating nobiletin might
affect the ABCB1 function as shown in FIGS. 4A and 4B. Moreover, it
was demonstrated that nobiletin could inhibit the efflux activity
of ABCB1 transporter in Caco-2 monolayer cell model, which was
shown in FIGS. 5A to 5D. Importantly, the concentrations of
nobiletin used in this study were lower than the maximal plasma
concentration (22.5 .mu.M) obtained in vivo pharmacokinetic study
of nobiletin.
[0115] It has been reported that PTX exerts cytotoxicity by
inhibiting mitotic progression and arresting cells in mitosis (G2/M
phase), while nobiletin also could induce apoptosis and block the
cell cycle arrested at G2 phase. Thus, the inventors investigated
the contribution of nobiletin to the observed enhanced cytotoxicity
in MDR cells after co-treatment with both PTX and nobiletin. In
this study, nobiletin at non-cytotoxic concentrations promoted cell
apoptosis induced by paclitaxel as shown in FIG. 4A in a
p53-dependent manner as shown FIG. 8B. Moreover, upon co-treatment
with nobiletin and PTX, a notable reduction in the fraction of cell
in the G0/G1, and a significant accumulation of cells in the G2/M
phase (>75%) were found for A2780/T cells; the result was shown
in FIG. 4B. These data suggested the boosted cytotoxicity after
co-treatment of nobiletin and PTX could be due to the intracellular
accumulation of PTX. In addition, the combination studies indicated
that nobiletin is a very strong synergist for enhancing the
anti-tumor effect of PTX in MDR cancer cell lines, and it was
predicted that 0.286 .mu.M nobiletin could bring a 200-fold
decrease on the ED.sub.50 of PTX. In short, the inventor concludes
that nobiletin decreased the transporter activity of ABCB1, thus
enhancing the intracellular drug concentration, and the finding
that nobiletin enhanced the overall cytotoxicity of these drugs is
consistent with the higher intracellular drug accumulation.
[0116] As energy used by ABCB1 transporter comes from ATP
hydrolysis, the inventors also investigated the ATPase activity of
ABCB1 transporter to confirm their previous assumption. As the
activity of ATPase was stimulated by nobiletin in a concentration
dependent manner, nobiletin might potentially be a substrate of
ABCB1. Moreover, verapamil-stimulated ATPase activity was reduced
by nobiletin. Therefore, it may competitively bound to the
substrate-binding site of ABCB1, leaving little room for other
agents to bind to the transporter, which resulted in decreased
activity of ABCB1 transporter. The MDR reversal effect can be
achieved either by reducing ABCB1 expression or by inhibiting the
efflux ability of ABCB1 transporter. Therefore, the inventors also
examined the effect of nobiletin on the expression of MDR1 mRNA and
ABCB1 protein. However, nobiletin did not affect the ABCB1
expression in both mRNA and protein levels at the reversal
concentrations as shown in FIGS. 8A to 8C.
[0117] Moreover, previous pre-clinical and clinical evidence
suggested that the PI3K/AKT, MAPk/ERK and Nrf2 signaling pathways
were associated with resistance to multiple chemotherapeutic drugs.
Inactivating the AKT/ERK and Nrf2 signaling pathway renders MDR
cancer cells more sensitive to drugs such as paclitaxel,
doxorubicin, 5-fluorouracil, etc. As nobiletin has been
demonstrated with an inhibition effect on the phosphorylation of
AKT and ERK, therefore, the inventors evaluated the effect of
nobiletin on AKT/ERK phosphorylation in A2780/T cells using Western
Blot analysis. As shown in FIG. 8B, nobiletin-PTX co-treatment
reduced phosphorylated AKT/ERK, which indicated that the inhibition
of AKT/ERK also accounts for the inhibiting effect of nobiletin in
MDR-cancer cells. Moreover, co-treatment of nobiletin-PTX
significantly suppressed Nrf2 expression in A2780/T cells in which
the A2780/T cells have a significantly higher level of Nrf2 than
that of A2780 cells. These findings may not only be helpful for
illustrating the multiple mechanisms behind the reversal effect of
nobiletin, but also helpful for explaining the reversal effect of
nobiletin to 5-fluorouracil which is not a P-gp substrates.
Literature has demonstrated that the inhibition of Nrf2 expression
could go through PI3K/AKT and ERK signaling pathway. Thus,
mechanistically, nobiletin inhibits the MDR cancer cells to
chemotherapeutic agents could through significantly reduced Nrf2
expression and down-regulated PI3K-Akt and ERK pathway.
[0118] In order to further study the interaction between nobiletin
and ABCB1 transporter, the inventors conducted the docking analysis
with human ABCB1 homology model. The predicted binding conformation
of nobiletin within the large hydrophobic drug binding cavity
(Site-1) of human ABCB1 shows the major contributions of
hydrophobic interactions as shown in FIGS. 9A to 9C. The methoxyl
and aromatic ring are important for interaction with the
drug-binding cavity of ABCB1 transporters. Overall, docking
simulation will be useful for understanding ligand-protein
interactions and for future optimizing derivatives.
[0119] In conclusion, this study provided the first evidence that
nobiletin significantly reversed ABCB1 mediated MDR by inhibiting
the efflux function of ABCB1 transporter and suppressing the
chemoresistance related AKT/ERK/Nrf2 pathways. As a very strong
synergist, nobiletin promoted cell apoptosis as well as G2/M cell
cycle arrest induced by PTX and reduced EC.sub.50 value of PTX. In
addition, the reversal effect of nobiletin was independent of
inhibiting ABCB1 expression. Given the broad-spectrum organ safety
of nobiletin which has been demonstrated in laboratory animals in
vivo, this invention suggests that nobiletin as combination therapy
may be a good candidate for studies in vivo and could be a
clinically useful drug to reverse ABCB1-medicated drug resistance
in cancer therapy.
[0120] The exemplary embodiments of the present invention are thus
fully described. Although the description referred to particular
embodiments, it will be clear to one skilled in the art that the
present invention may be practiced with variation of these specific
details. Hence this invention should not be construed as limited to
the embodiments set forth herein.
[0121] For example, one skilled in the art could appreciate that to
achieve the synergistic effect mentioned in Section 2.5, the
nobiletin could be applied together with PTX, or before/after PTX
treatment.
Sequence CWU 1
1
4120DNAArtificial SequenceForward primer for MDR1 1gagagatcct
caccaagcgg 20220DNAArtificial SequenceReverse primer for MDR1
2cgagcctggt agtcaatgct 20320DNAArtificial SequenceForward primer
for TIF 3agaaggctgg ggctcatttg 20420DNAArtificial SequenceReverse
primer for TIF 4aggggccatc cacagtcttc 20
* * * * *