Transcript Paul J. Davis

NANOTETRAC TARGETS THE
THYROID HORMONE RECEPTOR
ON INTEGRIN avb3 TO
PROMOTE APOPTOSIS,
DISRUPT CELL DEFENSE
PATHWAYS AND BLOCK
ANGIOGENESIS
Paul J. Davis, MD
Shaker A. Mousa, PhD
Albany Medical College;
Pharmaceutical Research
Institute, Albany College of
Pharmacy and Health
Sciences, Albany, NY, USA
X
B Alberts text
The thyroid hormone receptor on
the extracellular domain of avb3
binds L-thyroxine (T4), 3,5,3’triiodo-L-thyronine (T3) and
tetraiodothyroacetic acid (tetrac), a
deaminated T4 analogue. Tetrac
blocks binding of T4 and T3 to the
integrin and has an array of anticancer and anti-angiogenic
properties at avb3 that are
independent of its inhibition of T4and T3-binding to the integrin.
-I
-
-I
3’
HO
3
NH2
CH2-CH-COOH
O
5’
5
-I
-
-I
-
Thyroxine (T4)
-I
-
-I
3’
3
CH2-CH-COOH
O
5’
NH2
5
-I
-
3,5,3’-Triiodothyronine (T3)
-I
-
-I
3’
HO
3
CH2--COOH
O
5’
5
-I
-
-I
-
Tetrac
Because unmodified tetrac is a
thyromimetic within cells, we reformulated the analogue by
covalently binding it via a linker to
200 nm PLGA (poly[lactic-coglycolic acid]) nanoparticles which
limit its activities to the cell surface
hormone-tetrac receptor on integrin
avb3.
O
I
HO
O
I
O
O
I
N
H
O
N
H
PLGA
nanoparticle
N
H
N
H
I
O
O
I
N
O
OH
I
I
H
N
O
I
I
H
I
O
O
I
I
Nanotetrac
Four-to-eight tetrac moieties covalently
bound via a linker to a 200 nm nanoparticle
O
OH
At the integrin, Nanotetrac
regulates via signal transducing
kinases the expression of a variety
of cancer-relevant and angiogenesis-relevant genes. Nanotetrac
disrupts crosstalk between avb3
and adjacent vascular growth factor
receptors (VEGFR, bFGFR,
PDGFR, EGFR). Nanotetrac
blocks radiation-induced activation
of the integrin and controls plasma
membrane ion transporters, e.g.,
NHE1, important to pHi and pHe.
ACTING AT THE INTEGRIN,
NANOPARTICULATE TETRAC
INDUCES A BLUEPRINT OF ANTICANCER GENE EXPRESSION
• DR of apoptosis inhibitors XIAP,
MCL1, upregulation (UR) of proapoptotic CASP2, BCL2L14
• DR of catenin genes, UR of
CBY1, a nuclear inhibitor of
catenin activity
• Downregulation (DR) of 21 of 23
differentially-regulated protooncogenes and 8 of 9 cyclins
• UR of thrombospondin (THBS1),
an angiogenesis inhibitor, DR of
CTSL1, progenitor endothelial
cell recruiter
Cell Cycle, 2009
Fig. 3B
Pro-apoptosis
Cell Cycle 2009
Fig. 2A
Anti-apoptosis
Cell Cycle, 2009
Fig. 7
Effect of Tetrac and Tetrac Nanoparticles on
Human Non-Small Cell Lung Carcinoma
Xenograft Volume
Left side
Mean Tumor Volume (mm3) ± SEM
700
Control
Tetrac 1 mg/kg (every 3rd day)
600
Tetrac Nano 1 mg/kg (every 3rd day)
500
400
300
200
100
-10
-5
0
5
Days
10
15
20
15
20
Right side
Mean Tumor Volume (mm3) ± SEM
1000
Control
Tetrac 1 mg/kg (every 3rd day)
Tetrac Nano 1 mg/kg (every 3rd day)
800
600
400
200
-10
-5
0
5
Days
10
Lung Cancer, 2012
Subcutaneous treatment effect on xenograft tumor (MPanc96-luc)
after 19 days
IVIS images
Control (PBS)
Tetrac (1 mg/kg)
Nanotetrac (1 mg/kg)
MPanc96-luc cells injected March 7th, 2012
Subcutaneous daily treatment started: March 12th, 2012
IVIS: March 30th, 2012
Effect of Nanotetrac on human
glioblastoma U87MG xenografts in nude mice
(16-day dose/implant study)
0.06
Tumor weight
0.05
Tumor weight (gm)
0.04
0.03
0.02
0.01
0
Control
(Vehicle)
Void NP (3)
Tetrac (3 µg) Tetrac (10 µg) Nanotetrac (3
µg)
Treatment groups
Treatment
(per implant)
Tumor weight
(gm.)
Control (Vehicle)
0.0423
Void NP
0.0417
Tetrac (3 µg)
0.0422
Tetrac (10 µg)
0.0125
Nanotetrac (3 µg)
0.0120
Nanotetrac (10 µg)
0.0089
Nanotetrac
(10 µg)
Nanotetrac has been effective
against xenografts of human
glioma/glioblastoma, breast,
prostate, pancreatic, kidney,
(NSCLC and non-small cell) lung,
colon and thyroid cells.
• The anti-angiogenic properties of
Nanotetrac involve vascular growth
factor gene expression (VEGFA,
bFGF), growth factor receptor gene
expression (EGFR), interaction of
growth factors (VEGF, bFGF,
PDGF, EGF) with their specific cell
surface receptors, release of
vascular growth factors (bFGF),
expression of the cancer cell
THBS1 (TSP1) gene and
endothelial cell motility. This is a
broadly-based set of antiangiogenic actions.
Effect of tetrac (10 mg/kg, i.p. daily) or
Nanotetrac (1.0 mg/kg, i.p. daily) on
pancreatic tumor angiogenesis
PANC-I RIGHT SIDE
3.0
Hemoglobine (mg/ml)
2.5
2.0
1.5
1.0
0.5
0.0
Control
Tetrac
Tetrac Nano
Tetrac
Tetrac Nano
PANC-I LEFT SIDE
3.0
Hemoglobine (mg/ml)
2.5
2.0
1.5
1.0
0.5
0.0
Control
Inhibitory effect of avb3 MAB (LM609) on T4stimulated angiogenesis in the CAM model
PBS
T4 (total, 0.1mM)
CAM Treatment
PBS
T4(0.1uM)
T4+LM609(10ug)
T4+ LM609(10mg)
# of branch pts ± SEM % Inhibition ± SEM
73 ± 8
170 ± 16
109 ± 9
0
64 ± 9
Table 1 Inhibition of activities of pro-angiogenic factors in the CAM
assay by Nanoterac (NT) (2 µg/CAM)
Treatment
PBS control
Void PLGA nanoparticle
T3 (6.5 ng/mL)
T3 + NT
T4 (100 nM)
T4 (100 nM) + NT
LPS + NT
Bradykini (5 µg/mL)
Bradykini + NT
Angiotensin II (5 µg/mL)
Angiotensin II + NT
VEGF (2 µg/mL)
VEGF + NT
bFGF (1 µg/mL)
bFGF + NT
bFGF + VEGF + TNF-α
bFGF + VEGF + TNF-α + NT
Materials and Methods CAM assay was performed in duplicate X3 by our
peviously published method [23]. P values by ANOVA compared single and
multiple agent-containing samples with control or samples with and without
Nanotetrac (NT). All comparisons were significant at least at P < 0.01.
PBS phosphate-buffered saline, PLGA poly[lactic-co-glycolic acid], LPS
lipopolysaccharide, VEGF vascular endothelial growth factor, bFGF basic
fibroblast growth factor, TNF-α tumor necrosis factor-α.
Cell Cycle, 2009
Fig. 5
Anti-angiogenesis
Human medullary thyroid carcinoma cells
VEGFA
mRNA expression
THBS1
Control
Tetrac
NP
Tetrac
Control Tetrac NP Tetrac
THBS1, thrombospondin 1
VEGFA, vascular endothelial growth factor A
Effect of Tetrac on Cancer Cell Implants
on Angiogenesis
in the CAM Model
Control
Increased vascular
proliferation
+ Tetrac (1 µM)
Decreased vascular
proliferation
H1299 Bronchogenic carcinoma
(1 x 106 cells/CAM)
Actions of tetrac initiated nongenomically at avb3
culminate in a complex of genomic and nongenomic
effects on proliferation.
Summary
• Integrin avb3 contains a cell
surface receptor for T4, T3.
• Tetrac and nanoparticulate tetrac
inhibit agonist hormone action at
the receptor and are probes for
contributions of the receptor.
• In vitro, T4, T3 act at the integrin
receptor via ERK1/2 to support
tumor cell proliferation (breast,
glioma, head-and-neck, thyroid,
ovary, pancreas, kidney, lung,
prostate) and angiogenesis;
tetrac and Nanotetrac block
these actions.
Summary 2
• Acting via the cell surface
receptor on avb3, Nanotetrac
coherently modulates expression
of multiple genes relevant to
tumor cell survival.
• Receptor-initiated actions of
thyroid hormone analogues also
include modulation of crosstalk
between the integrin and nearby
vascular growth factor receptors
and crosstalk with nuclear
hormone receptor proteins, such
as ERa.
Summary 3
• Nanotetrac also blocks tumor cell
repair of radiation-induced doublestrand DNA breaks
• Nanotetrac prolongs intracellular
residence time of doxorubicin,
etoposide and cisplatin, apparently
via NHE1 and consequent
alterations in pHi and pHe that
affect P-glycoprotein and organic
cation transporter function.
COLLABORATORS
Shaker A. Mousa, PhD
Hung-Yun Lin, PhD
Heng-Yuan Tang, MA
Thangirala Sudha, PhD
Faith B. Davis, MD
Murat Yalcin, DVM, PhD
Sandra Incerpi, PhD
Osnat Ashur-Fabian, PhD
Albany
Albany
Albany
Albany
Albany
Turkey
Italy
Israel
Hypothyroid
Median Survival: 10.1 mos
Non-hypothyroid
Median Survival: 3.1 mos
Hercbergs AA et al, Anticancer Res, 2003
Angiogenesis in the CAM
A
PBS
B
C
PBS
T4
T4-ag
T4 + Tetrac
T4-ag + Tetrac
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 mM)
76  9
T4 + tetrac
66  6
T4-agarose (total, 0.1 mM) 194  28**
T4-agarose + tetrac
74  7
HDMEC cells
Angiopoietin-1
5
4
3
2
1
0
Tetrac (µM)
VEGF
(50 ng/ml)
B
Normalized Gene Expression
Normalized Gene Expression
A
0
1
3
Angiopoietin-2
10
8
6
4
2
0
Tetrac (µM)
VEGF
(50 ng/ml)
0
1
3
Ang-2 primes vascular endothelium for
action of vascular growth factors
A.
x 100000
Total Cell Counts
Effect of Tetrac and Nano Tetrac on
Cell Proliferation in CV-1 cells
7
6
5
4
3
2
B.
Total Cell Counts
Millions
Effect of Tetrac and Nano Tetrac on
Cell Proliferation in 293T cells
20
15
10
5
0
CV-1, monkey kidney fibroblast
293T, human kidney epithelial cell
Fig. 9
*
Treatment
Tumor
weight (gm)
PBS (Control)
0.123 *
Nanotetrac (1mg/kg)
0.066
Nanotetrac (1mg/kg)
(microfluidizer)
0.073
Control (PBS)
Tetrac (0.1 mg/kg)
Nanotetrac (0.1 mg/kg)
MPanc96-luc cells injected March 7th, 2012
Intra tumor treatment (once a week) started: March 12th, 2012
IVIS: March 30th, 2012
Effect of tetrac (10 mg/kg, i.p. daily) or
tetrac PLGA nano (1.0 mg/kg, i.p. daily) on
human pancreatic cancer xenografts
PANC-1 Right Side
450
Control
Tetrac
Tetrac Nano
Tumor volume mm
3
400
350
300
250
200
150
100
50
-15
-10
-5
0
5
10
15
5
10
15
Time (day)
PANC-1 Left Side
Tumor volume mm
3
350
Control
Tetrac
Tetrac Nano
300
250
200
150
100
-15
-10
-5
0
Time (day)
Integrins are 24 heterodimeric
structural proteins of the plasma
membrane that are critical to cellcell and cell-ECM matrix protein
interactions. Integrin avb3 binds
protein molecules—vitronectin,
fibronectin, osteopontin—and
recently has been appreciated to
bind small molecules. The latter
include thyroid hormone and its
analogues, resveratrol and
dihydrotestosterone.
U87MG (GBM) cells
T3
T4
Nucleus
Blot: anti-pERK1/2
48 kDa -
- pERK1
- pERK2
37 kDa -
48 kDa -
- pERK1
- pERK2
37 kDa -
Blot: anti-PCNA
37 kDa -
Nucleus
Blot: anti-pERK1/2
Blot: anti-PCNA
- PCNA
37 kDa -
Cytosol
Blot: anti-pTyrp85-PI 3-K
Blot: anti-p85-PI 3-K
5
pERK1/2
PCNA
pTyr-p85-PI 3-K
p85-PI 3-K
4
3
pERK1/2
PCNA
4
3
2
1
0
- p85-PI 3-K
84 kDa -
Relative I.O.D.
Relative I.O.D.
5
- pTyr-p85
-PI 3-K
84 kDa -
- PCNA
6
Cytosol
Blot: anti-pTyrp85-PI 3-K
- pTyr-p85
-PI 3-K
84 kDa -
T4 (M)
2
1
_
10-9 10-8 10-7 10-6
T3 (10-7 M)
0
T3 (M)
_
10-10 10-9 10-8 10-7 10-6
In vitro stimulation of cell proliferation
(PCNA), activation of ERKs, PI3K by
thyroid hormone analogues
Fig. 1
Tetrac-induced Radiosensitization
Index of double-stranded DNA breaks