Transcript γ-Tocotrienol - Asia Pharma 2016

Potential application of gamma-tocotrienol as
a novel chemosensitizer in gastric cancer
Dr. Gautam Sethi
Associate Professor
Dept of Pharmacology
Yong Loo Lin School of Medicine
National University of Singapore
Singapore
1
Global Variation in Cancer Incidence and Mortality
50
New York Times
2
BAD LUCK AND CANCER CONNECTION!
3
Cancer is a Preventable Disease That Requires
Major Lifestyle Changes
Pharmaceutical Research, 2008; 25, 2097-2116; Clin Cancer Res. 2009 Jan 15;15(2):425-30.
4
5
Different faces of inflammation and its role in tumorigenesis
Stress
Environmental pollutants
Viruses
(Chemical, physical,and
psychological)
(Cigarette smoke, Diesel)
(HTLV1, HPV, HCV,
HBV, EBV )
Bacteria
Food Factors
(e.g; Helicobacter pylori )
(Grill, Fried, red meat)
Acute
inflammation
• Innate Immunity
• Humoral immunity
• Immune surveillance
Reactive oxygen species
Tumor necrosis factor
Interleukin-1
Interleukin-6
Interleukin-8
Interleukin-18
Nuclear Factor-B
STAT3
AP-1
Hypoxia-inducible factor
Cyclooxygenase-2
PI3K/AKT
Inducible nitric oxidesynthase
Matrix metalloproteinase-9
Chemokines
Therapeutic inflammation
Biochem Pharmacol. 2006 Nov 30;72(11):1605-21.
Chronic
inflammation
• Tumor cell survival
• Tumor cell proliferation
• Tumor cell invasion
• Tumor angiogenesis
• Tumor metastasis
• Tumor chemoresistance
• Tumor radioresistance
Pathological inflammation
6
•
Gastric cancer is the second leading cause of cancer death in both sexes worldwide
(736,000 deaths, 9.7% of the total). The highest mortality rates are estimated in
Eastern Asia (28.1 per 100,000 in men, 13.0 per 100,000 in women), the lowest in
Northern America (2.8 and 1.5 respectively). High mortality rates are also present in
both sexes in Central and Eastern Europe, and in Central and South America.
•
There are marked geographic variations in gastric cancer incidence, with the highest
rates in Japan, China and South America
7
8
Treatment options
• SURGERY: The most common postoperative complication is tumor
recurrence. Many patients present with distant metastases or direct
invasion of organs, obviating the possibility of complete resection.
Surgical procedures such as wide local excision, total gastrectomy, or
gastrointestinal bypass may be performed to allow oral intake of
food and alleviate pain.
• CHEMOTHERAPY: Not very successful due to number of toxic side
effects/chemoresistance. One study revealed recurrence rates of up
to 80 percent in patients undergoing surgical resection alone,
suggesting a need to continue investigation of adjuvant therapies to
chemotherapy.
•
RADIOTHERAPY: Only a modest survival advantage has been shown.
The adverse effects caused by radiation therapy include
gastrointestinal toxicity from dose-limiting structures surrounding the
stomach.
9
What is NF-B?
p50 p65
IBa
Ubiquitination &
Phosphorylation
Nuclear
B enhancer
p50 p65
Activator
import
p50 p65
IBa
Degradation
IBa
Nucleus
Trends Pharmacol Sci. 2009;30:313-321.
Cytoplasm
10
11
NF-κB is constitutively activated in advanced gastric
carcinoma patients and correlated to the survival time
of patients after chemotherapy.
FOLFOX: Folinic acid + 5-FU+ Oxaliplatin
12
1313
14
Trends Pharmacol Sci. 2009;30:313-321.
14
Identification of novel
pharmacological
agents that can
overcome
chemoresistance
15
Vitamin E Family
Vitamin E
Tocopherol (TP)
Tocotrienol (T3)
α- T3
β- T3
γ- T3
δ- T3
Tocotrienols are reported to:
 Anti-Metabolic: Lower cholesterol
: Lower triglyceride
 Anti-Cancers: Inhibit of cancer cell cycle,
Inflammation Induce apoptosis in cancer cells
Inhibit proliferation of cancer stem
cells to delay cancer relapse
α- TP
β- TP
γ- TP
δ- TP
Tocopherols are reported to:
 Neutralize metabolic and UV-induced reactive
oxygen species (ROS)
 Protect neuron from death due to ROS
 Maintain skin cell membranes intact from ROS
16
Sources of Vitamin E
TOCOTRIENOLS
TOCOPHEROLS
Source : 2010 Soya & Oilseed Bluebook
17
Science of Tocotrienol & Chronic Diseases
[J Atheroscler Thromb. 2010 Oct 27]
Obesity
[Chem Biol Inter. 2010 Dec]
[Pig. Cells 2010 Oct]
Cholesterols
Diabetes
[Am J Cardiol. 2006 Sep ]
Neuro
Protection
Skin
Whitening
[Stroke. 2011 Aug]
Tocotrienols
Liver
Cancer
Pancreatic
Cancer
[Mol Cancer Ther. 2011 Oct 4]
[Int J Cancer. 2011 Mar 11]
Prostate
Cancer
Breast
Cancer
[BJP. 2011 Jul-Aug]
[Cell Prolif. 2011 Oct 4]
18
Hypothesis!
• Whether γ-tocotrienol can overcome
chemoresistance in gastric cancer through
modulation of pro-inflammatory NF-κB
signaling cascade.
19
In Silico Proteomics Analysis
The predictive analysis was run by Cellworks, (California) using information available in the public
domain. To Cellworks’ best knowledge, information and belief, no proprietary or confidential
information of any third party has been used in running the studies. The predictive data were generated
using Cellworks’ proprietary Oncology technology asset and is shared for discussion purposes.
NF-κ Inhibition Study Data
21
Effect of NF-κB Inhibition on key biomarkers
associated with GC progression
22
O.D. (570nm)
γ-Tocotrienol inhibited the proliferation of different gastric cancer cells in a
dose and time dependent manner
1
0.8
0.6
0.4
0.2
0
SNU16
SNU5
AGS
MKN 45
γ-toco (µM)
0
10
25
50
0 24 48 72 0 24 48 72 0 24 48 72
Time (h)
0 24 48 72
23
γ-Tocotrienol induced apoptosis in gastric cancer SNU16 cells in a dosedependent manner
45
*
40
35
30
25
0μM
10μM
20
15
10
5
0
0
10
25
50
(24 h)
25µM
50μM
* = p<0.05
24
γ-Tocotrienol potentiates the apoptotic effects of capecitabine
in gastric cancer cells
20
γ-Toco+
Cape
Cape
0
γ-Toco
10
40
30
20
10
0
γ-Toco+
Cape
30
Control
% Sub G1
40
*
50
Cape
*
50
60
γ-Toco
60
γ-Toco+ Cape
Capecitabine
Control
γ-Toco+ Capecitabine
Capecitabine
γ-Toco
Untreated
% Inhibition of esterase activity
γ-Toco
Untreated
25
Constitutive NF-κB activation in different gastric cancer cell lines
Optical density (450 nm)
p65 DNA binding activity
0.4
*
0.3
*
0.2
0.1
MKN45
AGS
SNU16
A293
0
* = p<0.05
(TransAMNF-kB p65 transcription factor assay kit (ActiveMotif, Carlsbad, CA, USA)
26
γ-Tocotrienol inhibited constitutive and capecitabine-induced NF-κB activation in
gastric cancer cells in a dose-dependent manner
0.4
0.4
MKN45
Optical density (450 nm)
p65 DNA binding activity
SNU16
0.3
*
0.3
*
0.2
*
0.2
*
*
0.1
*
0.1
0
0
10
25
50
γ-Tocotrienol (µM)/12 h
0
-
+
-
10
25
50
(γ-Toco, µM)
-
-
+
+
+
+
(Cape, 25 µM)
27
γ-Tocotrienol suppresses various anti-apoptotic, metastatic and
angiogenic gene products in gastric cancer cell lines
28
-Tocotrienol potentiates the antitumor effects of capecitabine
in vivo using athymic (nu/nu) mouse model
γ
γ
29
-Tocotrienol potentiates the effects of capecitabine to
inhibit the growth of gastric cancer in nude mice
30
*
0.35
*
0.3
0.25
0.2
0.15
0.1
0.05
γ-Toco + Cape
Cape
γ-Toco
0
VC
Optical density (450 nm)
p65 DNA binding activity
Effects of -tocotrienol and capecitabine on NF-κB
activation in gastric tumor tissue samples
31
Combination of -tocotrienol and capecitabine was effective in
downregulating the overexpression of various gene products involved
in proliferation, survival, and invasion in gastric cancer tumor tissues
32
γ-tocotrienol in combination with capecitabine inhibited
the expression of proliferative biomarker Ki67 in GC
tumor tissues
33
γ-tocotrienol in combination with capecitabine inhibited
the expression of angiogenic biomarker CD31 in GC tumor
tissues
34
First evidence that gamma-tocotrienol inhibits the
growth of human gastric cancer and chemosensitizes it
to capecitabine in a xenograft mouse model through the
modulation of NF-κB pathway
Manu KA, Shanmugam MK, Ramachandran L, Li F, Fong CW, Kumar AP, Tan P, Sethi G
Clinical Cancer Research, 2012; 18: 2220-2229
35
Angiogenesis
•
•
•
•
•
Angiogenesis is the formation of new blood vessels from preexisting vessels.
It is a normal process in growth and development, as well as
in wound healing.
However, it is also a fundamental step in the transition of
tumors from a dormant state to a malignant state.
Once a tumor has grown to ~3mm diameter, it requires its
own blood supply to stay alive and grow.
Tumor angiogenesis is essential for
1. supplying nutrients and oxygen and removing waste
products
2. dissemination of the tumour cells to distant sites
3. growth of metastatic colony at new site
• Angiogenesis is tightly controlled by the balance of two
sets of counteracting factors; angiogenic activators and
inhibitors.
The 4 major steps of endothelial cells in angiogenesis:
1. Breaking through of the basal lamina that envelopes existing blood
vessels
2. Migration toward a source signal
3. Proliferation
Menzel-Severing, Eye (2012) 26, 2–12
4. Formation of tubes
Effect of γ-tocotrienol on VEGF induced migration of HUVECs
VEGF
+
-
+
+
VEGF
γ-tocotrienol
VEGF +
γ-tocotrienol
HUVECs (70 µl; 5×105 cells/ml) were added into the two reservoirs of the culture insert
(IBIDI GmbH). After 12 hours, the insert was gently removed creating a gap of ~500
µm. The cells were treated with 50 μM γ-tocotrienol for 12 h before being exposed to
10ng/mL VEGF for 24 h. Width of wound was measured at time zero and 24h of
incubation. The representative photographs showed the same area at time zero and
after 24 h of incubation. Columns, mean; bars, SD. *, p < 0.05.
Effect γ-tocotrienol on VEGF induced invasion of HUVECs
VEGF
VEGF + γ-tocotrienol
VEGF
VEGF +
γ-tocotrienol
HUVECs (5×104) were seeded in the top-chamber of the Matrigel coated
polycarbonate membrane containing inserts. After pre-incubation with or without 50
µM γ-tocotrienol for 12 h, transwell chambers were placed into the wells of a 24-well
plate, in which we had added Medium 200 containing 10 ng/mL VEGF. After incubation
for 24h, cell invasion was analyzed. Columns, mean; bars, SD. *, p < 0.05.
Effect on tube formation of HUVECs
Control
VEGF
VEGF + γ-tocotrienol (25 μM)
VEGF + γ-tocotrienol (50 μM)
HUVECs were pretreated with various dilutions of γ-tocotrienol for 12 h and then
seeded onto the Matrigel layer in 24-well plates at a density of 5 × 104 cells in Medium
200 with or without VEGF. After 6 h, tubular structure of endothelial cells was
photographed using an inverted microscope.
Effect on VEGF-induced microvessel sprouting from rat thoracic aorta
Control
VEGF
VEGF + γ-tocotrienol (25μM)
VEGF + γ-tocotrienol (50μM)
Aortas isolated from Sprague-Dawley rats were cut into 1 mm rings, randomized into Growth
Factor Reduced Matrigel-coated wells and further sealed with 100 μl of Matrigel. Medium 200
with and without VEGF along with different dilutions of γ-tocotrienol was added to the wells and
incubated for 6 days. Columns, mean; bars, SD. *, p < 0.05.
Photograph
Effect on angiogenesis in Matrigel plugs implanted in mice
VEGF
VEGF +
γ-tocotrienol (10 μg)
VEGF +
γ-tocotrienol (20 μg)
H & E stain
Control
Matrigel (0.5 mL) containing 100 ng VEGF and 20 units of heparin with or without 10
or 20 μg of γ-tocotrienol were injected subcutaneously into the ventral area of
C57/BL/6 mice (n=5 per group). After 6 days, the mice were euthanized and intact
Matrigel plugs from all groups of mice were removed. The matigel plugs were
photographed and then fixed with 10% neutral buffered formalin and paraffin sections
were prepared and used for H&E staining to identify the formation and infiltration of
new microvessels.
γ-Tocotrienol suppressed the activation of VEGFR2, and the
AKT/mTOR signaling pathway in HUVECs
A.
B.
VEGF (50ng/ml)
0
0
1
2
4
6
h (γ-Tocotrienol)
VEGF (50ng/ml)
0
0
1
2
4
6
h (γ-Tocotrienol)
◄ pTyr1175-VEGFR2
◄ pSer473-AKT
◄ β-actin
◄ AKT
◄ pSer2448-mTOR
◄ mTOR
◄ pThr389 p70S6K1
◄ p70S6K1
◄ pThr389/Ser424 -p70S6K1
◄ p70S6K1
◄ β-actin
A, γ-tocotrienol suppressed the activation of VEGFR2 induced by VEGF in a time
dependent manner. The activation status of VEGFR2 was tested by western blot
analysis and probed with anti–phosphorylated VEGFR2 antibody. The same blot was
stripped and reprobed with β-actin antibody to verify equal protein loading. B, γtocotrienol inhibited the activation of AKT/mTOR signaling pathway in endothelial
cells. Proteins from different treatments were probed with phospho-specific antibodies.
γ-Tocotrienol induced apoptosis and inhibited the
AKT/mTOR pathway in HCC cells
A.
C.
0
1
2
4
6
h (γ-Tocotrienol)
◄ pSer473-AKT
◄ AKT
◄ pSer2448-mTOR
◄ mTOR
◄ pThr389 p70S6K1
B.
0
6
12
24
h (γ-Tocotrienol)
◄ PARP
◄ Cleaved PARP
◄ β-actin
◄ p70S6K1
◄ pThr389/Ser424 p70S6K1
◄ S6K1
◄ β-actin
A, γ-Tocotrienol reduced the viability of HCCLM3 cells in a dose dependent manner.
Cell viability was quantified by MTT assay. Columns, mean; bars, SD. *, p < 0.05. B,
HCCLM3 cells were treated with 50 μM γ-tocotrienol for indicated times, whole-cell
extracts were prepared, subjected to western blot analysis against PARP antibody. C,
γ-tocotrienol suppressed the phosphorylation of mTOR signaling pathway kinases in
HCCLM3 cell. HCCLM3 cells were treated with γ-tocotrienol for the indicated times, and
whole-cell extracts were prepared, and subjected to western blot analysis against
phospho-specific antibodies.
45
46
47
48
Conclusion!
Pro-inflammatory transcription factors
Can be considered As Ideal Targets to
Discover Therapeutics for Treatment of
cancer
49
Acknowledgment(s)
Cancer Pharmacology Members
Dr. Muthu K Shanmugam (Post-doc)
Dr. Wang Hong(Post-doc)
Dr. Siveen KS (Research Assistant)
Mr. Li Feng (Graduate student)
Ms. Radhamani Kannaiyan (Graduate student)
Ms. Lalitha Ramachandran (Graduate student)
Ms. Alamelu Nachiyappan (Graduate student)
Ms. Jingwen Zhang (Graduate student)
Ms. Xiaoyun Dai (Graduate student)
Ms. Fan Lu (Lab manager)
Collaborators
Prof. Kam Man Hui (NCCS)
Prof. Patrick Tan (DUKE-NUS)
Dr. Alan Prem Kumar (CSI)
Prof. John Luk (China)
Grant Support
National Medical Research Council, Singapore
Academic Research Fund Tier 1 and 2, Ministry of Education, Singapore
50