Download PTX (5 µg/ml)

Resveratrol modulates expression of ABC transporters
in non-small lung cancer cells: Molecular docking and
gene expression studies
S. KARTHIKEYAN and S.L. HOTI
Regional Medical Research Centre (ICMR),
Nehru Nagar- 590010, Belgaum, Karnataka, India.
1
BACKGROUND
Lung cancer
•
Comprising 17% of the total new cancer cases and
23% of the total cancer deaths in males.
•
Among females, lung cancer is the fourth most
commonly diagnosed cancer.
•
Among males, cancers of sites associated with use
of tobacco are the most frequent.
Lung Cancer Fact Sheet, 2014
2
ESTIMATED NEW CANCER CASES & DEATHS, 2014
•
•
One million lung cancer deaths were observed as per
2012 survey.
In 2020, the figure is projected at 1.5 million.
ACS. Cancer Facts and Figures, 2014
Carbone, 1997
3
NON SMALL CELL LUNG CANCER (NSCLC)
•
One of the frequent tumors in the elderly.
•
Constitutes 75-80% of all lung cancers.
•
Late diagnosis of the tumor.
•
NSCLC cells are relatively resistant to
chemo-radiotherapy.
4
Cancer Multidrug Resistance
Cancer cells resist treatments with anticancer drugs.
A major mechanism of cancer multidrug resistance is the
reduced accumulation of drugs due to
decreased uptake and increased efflux.
5
ATP-binding cassettes (ABC) drug transporters
Decreased drug uptake
TMD
NBD
Increased drug efflux
ABC transporters are implicated in the development
of multidrug resistance (MDR) in cancer cells.
• P-glycoprotein (P-gp; ABCB1),
• Multidrug resistant-associated protein
(MRP1; ABCC1)
• Breast cancer resistant protein
(BCRP; ABCG2; MXR).
6
ABC inhibitors as chemosensitizers
The first three generation ABC inhibitors (varapamil, cyclosporin-A, tariquitor)
shows undesirable side effects.
Unfortunately, progress in finding a potent, selective P-gp inhibitor to modulate ABC
transporters and restore drug sensitivity in multidrug-resistant cancer cells has
been slow and challenging.
Candidate drug should ideally be selective, potent and relatively non-toxic.
Utilizing natural products for the development of the next generation inhibitors
will be a novel approach.
7
Resveratrol
Trans-3,4',5-trihydroxystilbene
(C14H12O3), Mol. wt. 228.25
• Resveratrol (RSV) naturally occurring polyphenolic phytoalexin
• Synthesized by a grapes, berries and peanuts etc.
• Its beneficial effects against many diseases such as cancer,
cardiovascular disease, inflammatory disease, and platelet
aggregation.
8
Molecular interaction of RSV (CID:445154) with TMD region of P-gp (PDB ID: 3G61)
(a)
(b)
(c)
(d)
Docking score
Glide score
Hydrogen bond score
-8.343297
-8.3433
-1.21375
The values of docking score, glide score and hydrogen bond score indicates RSV possess
binding affinity with TMD region. Thereby, it may inhibit ABC transporters function. Hence,
in this study, we have chosen RSV as ABC inhibitor to enhance paclitaxel efficacy.
9
Paclitaxel
Paclitaxel (MW 853.906)
• Isolated from Taxes brevifolia.
• Potent anticancer agent.
• Paclitaxel binds to the β subunit of tubulin. Blocking cells at late G2
mitotic phase of cell cycle.
• Regular PTX administration develops MDR in lung cancer patients.
Taxes brevifolia
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Chemosensitizing potential of Resveratrol in NCI-H460 cells
Study model:
•NCI-H460 was obtained from NCCS, Pune.
•These cell lines are developed from NSCLC.
•NSCLCs cells are relatively insensitive to chemotherapy.
•Hence, NCI-H460 was chosen for the cancer multidrug
resistance study.
Photographs shows morphology of PTX resistant NSCLC cell line
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Fig. 1 Selection of optimum doses of RSV and PTX
(B)
120
120
100
100
% cell death
% cell death
(A)
80
60
40
80
60
40
20
20
0
0
0
5
10
15
RSV concentration (µg/ml)
IC50/24 h= 10 µg/mL
20
0
0.5
1
2.5
5
7.5
10
PTX concentration (µg/ml)
IC50/24 h= 5 µg/mL
Effect of (A) RSV and (B) PTX on NCI-H460 cytotoxicity upon 24 h incubation
period. Values are given as means  S.D. of six experiments in each group.
12
Experimental Scheme
RSV (10 µg/ml)
Control
PTX (5 µg/ml)
RSV (10 µg/ml) + PTX (5 µg/ml)
NCI-H460 cells were treated with RSV 1 h before
PTX exposure and incubated for 24hr
Cytotoxicity, ROS
generation and
Apoptotic
morphological
changes
Resveratrol
+
Paclitaxel
Membrane
transport function
ABC transporters
expression
Cell cycle
progression
13
Fig. 2 Effect of RSV, PTX and RSV-PTX on cytotoxicity in
NCI-H460 cells
(A)
Control
(B)
RSV (10 µg/ml)
120
Cell survival rate,%
a
100
80
60
b
b
40
20
c
0
PTX (5 µg/ml)
RSV (10 µg/ml) +PTX (5 µg/ml)
Microscopic images show the purple colored crystal
formation, which is an indication of cell viability
Values are given as means  SD of six experiments in each group.
Values not sharing a common marking (a, b, c,..) differ significantly at P ≤ 0.05 (DMRT).
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Fig. 3 Effect of RSV-PTX on membrane transport function in NCI-H460 cells
Inhibitor
Control
RSV
PTX
RSV - PTX
NCI-H460 cells were incubated with RSV, PTX and RSV-PTX.
The level of fluorescence intensity of rhodamine123 was analyzed by flow cytometry
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Fig. 4 Effect of RSV-PTX on cell cycle progression in NCI-H460 cells
Control
RSV (10 µg/ml)
b
60
c
a
Cell count (% of total cell)
a
50
c
b
40
b
30
c
.d
20
a
ab
ab
ab
10
a ababab
0
SubG1
G0/G1
G2/M
S
Phase of cell cycle
Control
PTX (5 µg/ml)
RSV
PTX
RSV-PTX
RSV (10 µg/ml) +PTX (5 µg/ml)
Effect of RSV-PTX on cell cycle and apoptosis (a) NCI-H460 cells were treated with RSV, PTX or RSV-PTX. Only
RSV + PTX were able to increase the proportion of cells in G2/M as compared to RSV alone or PTX alone
treated cells. (b) The bar graphs show the cell cycle distribution and the percentage of cells in each phase of
the cell cycle. Percentage of total cells was obtained by using the CellQuest software.
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Fig. 5 Effect of RSV-PTX on the expression of P-gp in NCI-H460 cells
A. Western blot analysis
1
2
3
1.2
4
1
P-gp
β-actin
Lane 1: Control;
Lane 2: RSV;
Expression levels of
P-gp/β-actin
Lanes
B. Band intensity by densitometry
a
0.8
b
0.6
0.4
c
0.2
d
0
Lane 3: PTX;
Lane 4: RSV +PTX
Control
RSV
PTX
RSV-PTX
The graph represents the P-gp quantification values normalized to β-actin levels
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Fig. 6 Effect of RSV-PTX on mRNA expression patterns of ABCB1 and
ABCB4 in NCI-H460 cells
B. relative gene expression
A. mRNA expression by qRT-PCR
ABCB1/
MDR1
Relative mRNA expression
1.2
ABCBI
a
1
0.8
b
0.6
c
0.4
0.2
d
0
Control
1.2
Relative mRNA expression
ABCB4/
MDR3
RSV
PTX
RSV-PTX
ABCB4
a
1
b
0.8
c
0.6
0.4
d
0.2
0
Control
RSV
The graph represents the quantification results normalized to 18S rRNA
PTX
RSV-PTX
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Fig. 7 Effect of RSV-PTX on mRNA expression patterns of LRP and
ABCC1 in NCI-H460 cells
B. Relative gene expression
A. mRNA expression by qRT-PCR
LRP/
MVP
Relative mRNA expression
1.2
LRP
a
1
b
0.8
c
0.6
0.4
d
0.2
0
Control
1.2
Relative mRNA expression
ABCC1/
MRP1
RSV
PTX
RSV-PTX
ABCC1
a
1
0.8
b
0.6
c
0.4
0.2
d
0
Control
RSV
The graph represents the quantification results normalized to 18S rRNA
PTX
RSV-PTX
19
Fig. 8 Effect of RSV-PTX on mRNA expression patterns of ABCC2 and
ABCC3 in NCI-H460 cells
ABCC2/
MRP2
B. Relative gene expression
Relative mRNA expression
A. mRNA expression by qRT-PCR
ABCC2
1.2
a
1
0.8
b
0.6
c
0.4
d
0.2
0
Control
RSV
ABCC3
/MRP3
Relative mRNA expression
1.2
PTX
RSV-PTX
ABCC3
a
1
0.8
b
0.6
c
0.4
d
0.2
0
control
RSV
The graph represents the quantification results normalized to 18S rRNA
PTX
RSV-PTX
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Conclusion
1. RSV-PTX combination show superior anti-proliferation
effect in NCI-H460 cells.
2. RSV treatment inhibit ABC transporters function and
thereby may enhance more PTX accumulation inside the
NCI-H460 cells.
3. RSV-PTX combination enhances G2/M apoptotic cell death.
4. RSV, PTX and RSV-PTX combination down-regulates various
ABC transporters expression in NCI-H460 cells
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Recommendations
The RSV-PTX combination showed better cell death efficacy. This
may be due to the additive or synergistic effect of RSV with
PTX.
Future directions
A number of additional experiments are underway.
The anticancer efficacy of RSV and PTX combination will be tested in human
tumor xenograft bearing BALB/c nude mice.
NCI-H460 cells grown in vitro will be implanted sub-cuataneously under the
shoulder in the BALB/c nude mice.
Tumor volume, body weight, Ex vivo tissue imaging and immunofluorescence
studies will be employed to understand the tumour regression in RSV-PTX
treated animals
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ACKNOWLEDGEMENTS
This work was supported by research grant from the Indian
Council of Medical Research (ICMR), Ministry of Health and
Family Welfare, Government of India, New Delhi to
Dr. S. Karthikeyan.
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