Resveratrol modulates expression of ABC transporters in non-small lung cancer cells: Molecular docking and gene expression studies

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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.
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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.

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Lung Cancer Fact Sheet, 2014
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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.

Carbone, 1997
ACS. Cancer Facts and Figures, 2014
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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.

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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.
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ATP-binding cassettes (ABC) drug transporters
Decreased drug uptake
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).

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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.
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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 bene?cial effects against many diseases such
  as cancer, cardiovascular disease, in?ammatory
  disease, and platelet aggregation.

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Molecular interaction of RSV (CID445154) 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.
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Paclitaxel

Paclitaxel (MW 853.906)

• Isolated from Taxes brevifolia.
• Potent anticancer agent.
• Paclitaxel binds to the ß 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
(A)
(B)
IC50/24 h 10 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.
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Experimental Scheme
RSV (10 µg/ml)
PTX (5 µg/ml)
Control
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
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Fig. 2 Effect of RSV, PTX and RSV-PTX on
cytotoxicity in NCI-H460 cells
(A)
(B)
Control
RSV (10 µg/ml)
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
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
b
a
RSV (10 µg/ml)
Control
PTX (5 µg/ml)
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
B. Band intensity by densitometry
Lanes 1 2 3 4
P-gp
ß-actin
Lane 1 Control Lane 2 RSV
Lane 3 PTX Lane 4 RSV PTX
The graph represents the P-gp quantification
values normalized to ß-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
ABCB4/MDR3
The graph represents the quantification results
normalized to 18S rRNA
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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
ABCC1/ MRP1
The graph represents the quantification results
normalized to 18S rRNA
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Fig. 8 Effect of RSV-PTX on mRNA expression
patterns of ABCC2 and ABCC3 in NCI-H460 cells
B. Relative gene expression
A. mRNA expression by qRT-PCR
ABCC2/MRP2
ABCC3/MRP3
The graph represents the quantification results
normalized to 18S rRNA
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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.
THANK YOU
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