ANTI-APOPTOTIC AND PRO-ANGIOGENIC PROPERTIES OF ENDOGENOUS THYROID HORMONE AND ITS ... ·...
Transcript of ANTI-APOPTOTIC AND PRO-ANGIOGENIC PROPERTIES OF ENDOGENOUS THYROID HORMONE AND ITS ... ·...
ANTI-APOPTOTIC AND PRO-
ANGIOGENIC PROPERTIES OF
ENDOGENOUS THYROID
HORMONE AND ITS ACTION ON
P-GLYCOPROTEIN MAY BLUNT
RESPONSE TO CONVENTIONAL
CHEMOTHERAPY
Paul J. Davis, MD
Albany Medical College and
Pharmaceutical Research
Institute, Albany College of
Pharmacy and Health
Sciences, Albany, NY USA
MECHANISMS OF THYROID
HORMONE ACTION
• Thyroid hormone (L-thyroxine, T4;
3,5,3’-triiodo-L-thyronine, T3) acts
via genomic and nongenomic
mechanisms. The genomic
pathway depends primarily upon
formation of intranuclear complexes
of T3 with the nuclear thyroid
hormone receptor proteins (TRs).
• The nongenomic pathways include
actions initiated at a cell surface
receptor for T4 and T3 on the
extracellular domain of integrin
avb3.
O CH2-CH-COOH
NH2
I - -
I - - I
- -
I - -
3’
5’ 5
3
Thyroxine (T4)
O CH2-CH-COOH
NH2
I - -
I - -
I - -
3’
5’ 5
3
3,5,3’-Triiodothyronine (T3)
HO
Integrin
STAT1a
TRb1, ER
PLC PKC
TRb1 ERa
T4
TRb1 p300 NCoR
mRNA
Trip230
MAPK (ERK1/ERK2)
Cytoplasm
T3
TRa
STAT1a, p53
T4
TR∆a1
rT3
TRb1
PI 3-K-
Akt/PKB
aV β3
T3
mRNA
Extracellular matrix
proteins, e.g.,
laminin, vitronectin
p53
SIGNAL TRANSDUCTION
TRb1
ER
STAT1a
a
a
p53
T3
T3
T3
MAPK (ERK1/ERK2)
Na+/H+
antiporter
Amino acid
transporter
Protein trafficking
Serine phosphorylation of nuclear proteins
Specific gene transcription
Activity of ion transporters
Nongenomic Actions of Thyroid Hormone
Initiated at the Cell Surface
Integrin avb3 is primarily expressed
by dividing cells and thus
concentrated and activated in
cancer cells and rapidly-dividing
endothelial cells that serve cancers.
Usually viewed as a sensing
mechanism for extracellular matrix
(ECM) proteins and critical to cell-
matrix and cell-cell interactions that
underlie tissue organization, avb3
has recently been appreciated to
bind small molecules and to contain
the cell surface receptor for thyroid
hormone.
As a prototypic small molecule
ligand of integrin avb3, thyroid
hormone importantly alters
transcription of differentially-
regulable genes important to
cancer cell function and
angiogenesis—and a) regulates
integrin vascular growth factor
receptor function, b) alters the state
of the actin cytoskeleton, c)
changes intracellular protein
trafficking and d) controls the
function of plasma membrane
NHE1 and Na,K-ATPase.
Thyroid hormone and cancer cell
proliferation
Time (h)
_
2 4 6 24 24 1
_ _ _ _ + ICI (3 nM)
MCF-7 cells: 3H-thymidine
uptake
- F
old
In
cre
ase
in
3H
-th
ym
idin
e In
co
rpo
ratio
n
0
10
20
30
40
10-10
M E2
10-7
M T4
Control
* *
* *
37 kDa-
MCF-7 cells: PCNA
PD + + _ _
+
T4 + + _ _ _ _
T4-A + + _ _ _ _
_
- F
old
In
cre
ase
in
I.O
.D.
0
1
2
3
Thyroid hormone and breast cancer
cell proliferation
ICI = ICI 182,780,
fulvestrant
T4 (10-7 M) _ + _ + _ +
- NCoR
IP: anti-integrin αv
Blot: anti-NCoR
Blot: anti-pSTAT1
- pSTAT1
Blot: anti-SMRT
Blot: anti-p300
Blot: anti-pERK1/2
C siRNA scRNA | | | |
- SMRT
- pERK1
- pERK2
- p300
- Lamin B
T4 (10-7 M) _ + _ + _ +
C siRNA scRNA | | | |
Fig. 3
61 kDa -
37 kDa -
82 kDa -
180 kDa -
180 kDa -
- R
ela
tive I
.O.D
.
*
* *
*
* *
* *
* *
*
* * *
0
2
4
6
8
10
12
14
16
NCoR
SMRT
P300
pSTAT1
pERK1/2
* *
OVCAR-3 cells
A
NCI-H522 cells
Blot: anti-PCNA
- PCNA 37 kDa -
T3 (M) _
10-9 10-8 10-7 10-6
B
Thymidine Incorporation
C
T4 (M)
T3 (M)
_ _
_ _ _ _ 10-8 10-7 10-6
10-9 10-8 10-7
CP
M
x 1
04
_ _
0
5
10
15
20
25
30NCI-H522 cells
NCI-H510A cells
Blot: anti-PCNA
- PCNA
- Lamin-B
37 kDa -
61 kDa -
T4 (M) _
10-9 10-8 10-7 10-6
NCI-H522 cells
- Lamin-B 61 kDa -
*
*
*
*
*
*
*
*
*
*
* *
*
- GAPDH
- Integrin α
- Integrin β
D
H510A, small cell lung carcinoma cells
H522, NSCLC cells
RT-PCR
NCI-H510A cells
ICI (nM)
T3 (10-7 M)
_
_
T4 (10-7 M) _
_ _
_ _ _ _
_ + +
_ _ _ + + + +
20 2 20 20 2
Blot: anti-PCNA - PCNA
37 kDa -
37 kDa -
- pERK1 - pERK2
Blot: anti-pERK1/2
Blot: anti-phosphoER-a (118)
62 kDa -
IP: anti-ER-α
Blot: anti-ER-α
- pER-a
62 kDa -
Thymidine Incorporation
- ER-a
0
5
10
15
20
ICI (nM)
T3 (10-7 M)
_
_
T4 (10-7 M) _
_ _
_ _ _
_ +
_ _ + + +
20 20 20
B
A
CP
M X
10
4
-a
0
1
2
3
4
5
6
pER
pERK1/2
PCNA
-Rela
tive
I.
O.D
.
61 kDa -
- Lamin-B
*
* *
+ +
* *
+
* *
* *
*
+ + +
+
* *
* *
ICI = ICI 182,780,
0
1
2
3
4
5pERK1/2
PCNA
pTyr-p85-PI 3-K
p85-PI 3-K
0
1
2
3
4
5
6
pERK1/2
PCNA
U87MG (GBM) cells
Blot: anti-pERK1/2
Nucleus
Blot: anti-PCNA
Blot: anti-pTyrp85-PI 3-K
Cytosol
T4 (M) _ 10-9 10-8 10-7 10-6
- pERK1 - pERK2
- PCNA
- pTyr-p85
48 kDa -
37 kDa -
37 kDa -
84 kDa -
T3 (10-7 M)
Rela
tive
I.O
.D.
-PI 3-K
T3 (M) _ 10-9 10-8 10-7 10-6
Blot: anti-pTyrp85-PI 3-K
Cytosol
10-10
Blot: anti-p85-PI 3-K
- p85-PI 3-K
Blot: anti-pERK1/2
Nucleus
Blot: anti-PCNA
- pERK1 - pERK2
- PCNA
84 kDa -
84 kDa -
48 kDa -
37 kDa -
37 kDa -
Rela
tive
I.
O.D
.
- pTyr-p85
-PI 3-K
T3 T4
Fig. 1
In vitro stimulation of cell proliferation
(PCNA), activation of ERKs, PI3K by
thyroid hormone analogues
Hypothyroid
Median Survival: 10.1 mos
Non-hypothyroid
Median Survival: 3.1 mos
Hercbergs AA et al, Anticancer Res, 2003
Spontaneous or medically-induced
hypothyroidism has been shown to
favorably affect the clinical courses
of GBM (Cleveland Clinic), breast
cancer (MD Anderson Cancer
Center), renal cell carcinoma (TKI
therapy at multiple centers) an
head-and neck cancers (Cleveland
Clinic).
Thyroid hormone has anti-apoptotic
activity in cancer cells
RV = resveratrol
O CH2-CH-COOH
NH2
I - -
I - - I
- -
I - -
3’
5’ 5
3
Thyroxine (T4)
O CH2-CH-COOH
NH2
I - -
I - -
I - -
3’
5’ 5
3
3,5,3’-Triiodothyronine (T3)
HO
O CH2--COOH
I - -
I - - I
- -
I - -
3’
5’ 5
3
Tetrac
HO
Low-grade thyromimetic within cells
TH antagonist at integrin avb3 TH
receptor
Resveratrol
pERK1/2 activation
PD98059
Cytosol
Nucleus
pSer15-p53 NS-398
Apoptosis
Thyroid Hormone
pERK1/2 activation
PD98059
X
Cell Proliferation
[COX-2]-pERK1/2
complexing
Tetrac
Thyroid hormone inhibits induction by
RV of Ser-15 phosphorylation of p53
Thyroid hormone is pro-angiogenic
by a variety of mechanisms. This is
relevant to support by the hormone
of cancer-related vascularization
and to vascularity of nonmalignant
conditions, such as skin diseases.
PBS
A
T4 T4 + Tetrac
B
Summary of effects of T4, T4-agarose
and tetrac on angiogenesis
Treatment Angiogenesis Index
PBS 67 9
T4 (0.1 nM) 156 16**
Tetrac (0.1 M) 76 9
T4 + tetrac 66 6
T4-agarose (total, 0.1 M) 194 28**
T4-agarose + tetrac 74 7
C PBS T4-ag T4-ag + Tetrac
Angiogenesis in the CAM
Data represent mean ± SD, n=8; *p<0.01, indicating significant
stimulation of angiogenesis. PBS, phosphate-buffered saline.
A
B
b-FGF (1.0 µg/ml)
PBS (Control)
DITPA (0.01 µM) VEGF (2.0 µg/ml)
T4 (0.1 µM) T4-agarose (0.1 µM)
Mean Vessel Branch
Treatment Points ± SD
PBS (Control) 87 ± 9
T4 (0.1 μM) 148 ± 7*
T4-agarose (0.1 μM) 167 ± 8*
DITPA (0.01 μM) 134 ± 11*
DITPA (0.1 μM) 170 ± 9*
VEGF (2.0 μg/ml) 168 ± 10*
b-FGF (1.0 μg/ml) 174 ± 8*
PBS T4 (total, 0.1M)
T4+ LM609(10g)
Inhibitory Effect of avb3 MAB (LM609) on T4-
stimulated Angiogenesis in the CAM Model
CAM Treatment # of branch pts ± SEM % Inhibition ± SEM
PBS 73 ± 8
T4(0.1uM) 170 ± 16 0
T4+LM609(10ug) 109 ± 9 64 ± 9
By a variety of mechanisms, thyroid
hormone, specifically T4, has been
shown to influence the activity and
abundance of P-glycoprotein (P-gp;
MDR1; ABCB1), a plasma
membrane efflux pump that serves
to shorten intracellular retention
time of traditional chemotherapeutic
agents that are ligands for the
protein (doxorubicin, etoposide,
paclitaxel, etc.). Thus, thyroid
hormone may support
chemoresistance.
MDR1
EGFR
transcription
transcription
Plasma
Membrane
Cytoplasm
Na+, K+ -
ATPase
Na+/H+
antiporter P-glycoprotein EGFR
Chemotherapeutic
agent efflux EGF
β3 β3 β3
K+
Na+
H+
Na+
Results of
T4 action Increased EGF
input to P-gp
[Na+]i
Ca2+
pHi
pHe
+ Calmodulin
T4
Bold blue arrows indicate stimulatory
action of tetrac/Nanotetrac on Na+,
K+ - ATPase, Na+/H+ antiporter and
EGF receptor
T4
SUMMARY
There are multiple implications of
all of these functions of thyroid
hormone recently recognized to
occur on cancer cells and on
angiogenesis.
• Normal thyroid function may
support tumor cell proliferation and
limit effectiveness of
chemotherapy.
• Induced or spontaneous
hypothyroidism may impede cancer
cell function.
SUMMARY 2
• It is desirable to have a specific
pharmacologic antagonist of thyroid
hormone actions at integrin avb3 (=
tetrac, Nanotetrac).
• The affinity of the thyroid hormone
receptor on avb3 is higher for T4
than T3; at physiological concen-
trations, free T4 supports tumor cell
proliferation and cancer cell sur-
vival pathway gene transcription.
• Nonmalignant, hypervascular skin
disorders, such rosacea, may be
thyroid hormone-supported.
COLLABORATORS
Shaker A. Mousa, PhD Albany
Hung-Yun Lin, PhD Albany
Heng-Yuan Tang, MA Albany
Thangirala Sudha, PhD Albany
Faith B. Davis, MD Albany
Murat Yalcin, DVM, PhD Turkey
Sandra Incerpi, PhD Italy
Osnat Ashur-Fabian, PhD Israel
VEGF
Tetrac _ 2.5 1.0
+ + +
A
B
Effect of tetrac on tube formation by human
dermal microvascular endothelial cells (HDMEC).
In this 3-dimensional human microvascular endothelial cell sprouting assay,
cells were mixed with gelatin-coated Cytodex-3 beads and the mixture suspended in
endothelial basal medium (EBM) with 15% normal human serum, mixed and cultured
overnight in a CO2 incubator. The “EC-beads” were then placed in a fibrinogen solution and
thrombin added. After polymerization of the fibrin, EBM and 20% human serum were added
and samples incubated for 24-48 h.
Int Angiol, 2006
Green: phosphoER-a
Red: Nucleoprotein
Control
NCI-H522 cells
Tetrac
Tetrac
+
T4
L-T4
A
B
- ER-a
- GAPDH
ER-a gene expression ER-a gene expression
Re
lative
I.
O.D
.
-0.1
0.4
0.9
1.4
1.9
2.4
Con ICI T₄ ICI + T₄
Rela
tive B
ound D
NA
NCI-H-522 cells
C
0
2
4
6
NCI-H510A NCI-H522
ER-α
IP: anti-integrin-αv
T₄
IgG
- ER-α Promoter
_ +
- Input Control
* *
+ +
Nucleosome ELISA
0
1
2
3
4
5
6
7
BHP 18-21 cells
FTC 236 cells
RV (𝜇M) _ _
_ _ _ 1 10 1 10
+ + +
-Fo
ld I
ncre
ase
in
nucle
osom
e c
onte
nt
A
0
5
10
15
BHP 2-7 cells
BHP 18-21 cells
RV (10 𝜇M)
T4 (10-7 M)
Tetrac (10-7 M) _ _ _ _ + + + +
_
_
_ _ _ + + + +
_ _ _ + + + +
-Fo
ld I
ncre
ase in
nucle
osom
e c
onte
nt
B Nucleosome ELISA
T4 (10-7 M)
5
10
15
20
25
EC
Mig
ration (
RF
U)
x 1
03
Effects of L-T4 or T4 Nanoparticles (NP) on
Endothelial Cell Migration toward Vitronectin (VN)
Cha
mb
er
Upper
Lower
0
0
0 T4 T4-NP
VN VN VN
Boyden apparatus