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NANOTETRAC TARGETS THE THYROID HORMONE RECEPTOR
ON INTEGRIN v3 TO PROMOTE APOPTOSIS,
DISRUPT CELL DEFENSE PATHWAYS AND BLOCK
ANGIOGENESIS
Paul J. Davis, MDShaker A. Mousa, PhD
Albany Medical College; Pharmaceutical Research Institute, Albany College of
Pharmacy and Health Sciences, Albany, NY, USA
B Alberts text
X
The thyroid hormone receptor on the extracellular domain of v3 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 anti-cancer and anti-angiogenic properties at v3 that are independent of its inhibition of T4- and T3-binding to the integrin.
O CH2-CH-COOH
NH2
I--
I-- I--
I--3’
5’ 5
3
Thyroxine (T4)
OHO 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
Because unmodified tetrac is a thyromimetic within cells, we re-formulated the analogue by covalently binding it via a linker to 200 nm PLGA (poly[lactic-co-glycolic acid]) nanoparticles which limit its activities to the cell surface hormone-tetrac receptor on integrin v3.
H
NH
O
HN
O
NH
ONH
HN
OO
I
IOI
I
O
O OI
IOI
I
OH
NH
OO
I
I OI
I
HO PLGA nanoparticle
Nanotetrac
Four-to-eight tetrac moieties covalentlybound via a linker to a 200 nm nanoparticle
At the integrin, Nanotetrac regulates via signal transducing kinases the expression of a variety of cancer-relevant and angiogen-esis-relevant genes. Nanotetrac disrupts crosstalk between v3 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 ANTI-CANCER GENE EXPRESSION
• DR of apoptosis inhibitors XIAP, MCL1, upregulation (UR) of pro-apoptotic CASP2, BCL2L14
• DR of catenin genes, UR of CBY1, a nuclear inhibitor of catenin activity
• Downregulation (DR) of 21 of 23 differentially-regulated proto-oncogenes and 8 of 9 cyclins
• UR of thrombospondin (THBS1), an angiogenesis inhibitor, DR of CTSL1, progenitor endothelial cell recruiter
Fig. 3B
Pro-apoptosis
Cell Cycle, 2009
Fig. 2A
Anti-apoptosis
Cell Cycle 2009
Fig. 7
Cell Cycle, 2009
Days-10 -5 0 5 10 15 20
200
400
600
800
1000
ControlTetrac 1 mg/kg (every 3rd day)Tetrac Nano 1 mg/kg (every 3rd day)
Right side
Days-10 -5 0 5 10 15 20
100
200
300
400
500
600
700
Control
Tetrac 1 mg/kg (every 3rd day)
Tetrac Nano 1 mg/kg (every 3rd day)
Left side
Mea
n T
umor
Vol
ume
(mm
3)
± S
EM
Effect of Tetrac and Tetrac Nanoparticles on Human Non-Small Cell Lung Carcinoma Xenograft
VolumeM
ean
Tum
or V
olum
e (m
m3)
± S
EM
Lung Cancer, 2012
Subcutaneous treatment effect on xenograft tumor (MPanc96-luc)
after 19 daysIVIS images
Control (PBS) Tetrac (1 mg/kg) Nanotetrac (1 mg/kg)
MPanc96-luc cells injected March 7th, 2012Subcutaneous daily treatment started: March 12th, 2012IVIS: March 30th, 2012
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
Effect of Nanotetrac on human glioblastoma U87MG xenografts in nude mice
(16-day dose/implant study)
Tumor weight
0
0.01
0.02
0.03
0.04
0.05
0.06
Control(Vehicle)
Void NP (3) Tetrac (3 µg) Tetrac (10 µg) Nanotetrac (3µg)
Nanotetrac(10 µg)
Treatment groups
Tum
or w
eigh
t (gm
)
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 anti-angiogenic actions.
Effect of tetrac (10 mg/kg, i.p. daily) or Nanotetrac (1.0 mg/kg, i.p. daily) on
pancreatic tumor angiogenesisH
emog
lobi
ne (
mg/
ml)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Control Tetrac Tetrac Nano
PANC-I RIGHT SIDE
Hem
oglo
bine
(m
g/m
l)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Control Tetrac Tetrac Nano
PANC-I LEFT SIDE
PBS T4 (total, 0.1M)
T4+ LM609(10g)
Inhibitory effect of v3 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
Table 1 Inhibition of activities of pro-angiogenic factors in the CAM assay by Nanoterac (NT) (2 µg/CAM)
Treatment
PBS controlVoid PLGA nanoparticleT3 (6.5 ng/mL)T3 + NTT4 (100 nM)
LPS + NTBradykini (5 µg/mL)Bradykini + NTAngiotensin II (5 µg/mL)Angiotensin II + NTVEGF (2 µg/mL)VEGF + NTbFGF (1 µg/mL)bFGF + NTbFGF + VEGF + TNF-αbFGF + 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.
T4 (100 nM) + NT
Fig. 5
Anti-angiogenesis
Cell Cycle, 2009
THBS1
mRN
A e
xpre
ssio
n
Control Tetrac NP
Tetrac Control Tetrac NP Tetrac
VEGFA
THBS1, thrombospondin 1VEGFA, vascular endothelial growth factor A
Human medullary thyroid carcinoma cells
Effect of Tetrac on Cancer Cell Implants on Angiogenesis in the CAM Model
+ Tetrac (1 µM)
Control
Increased vascular proliferation
Decreased vascular proliferation
H1299 Bronchogenic carcinoma (1 x 106 cells/CAM)
Actions of tetrac initiated nongenomically at v3culminate in a complex of genomic and nongenomiceffects on proliferation.
• Integrin v3 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
Summary 2
• Acting via the cell surface receptor on v3, 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 ER.
Summary 3
• Nanotetrac also blocks tumor cell repair of radiation-induced double-strand 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 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
Hypothyroid
Median Survival: 10.1 mos
Non-hypothyroid
Median Survival: 3.1 mos
Hercbergs AA et al, Anticancer Res, 2003
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
0
2
4
6
8
10
0 1 30
1
2
3
4
5
Nor
mal
ized
Gen
e Ex
pres
sion Angiopoietin-1 Angiopoietin-2
Nor
mal
ized
Gen
e Ex
pres
sion
0 1 3Tetrac (µM)VEGF
(50 ng/ml)
Tetrac (µM)VEGF
(50 ng/ml)
A B
Ang-2 primes vascular endothelium for action of vascular growth factors
HDMEC cells
Fig. 9
Tota
l Ce
ll C
ou
nts
2
3
4
5
6
7
x 10
0000
Effect of Tetrac and Nano Tetrac on Cell Proliferation in CV-1 cells
0
5
10
15
20
Mill
ions
Tota
l Ce
ll C
ou
nts
Effect of Tetrac and Nano Tetrac on Cell Proliferation in 293T cells
A.
B.
CV-1, monkey kidney fibroblast293T, human kidney epithelial cell
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, 2012Intra tumor treatment (once a week) started: March 12 th, 2012IVIS: March 30th, 2012
Time (day)
-15 -10 -5 0 5 10 15
Tum
or v
olum
e m
m3
100
150
200
250
300
350ControlTetracTetrac Nano
PANC-1 Left Side
Time (day)
-15 -10 -5 0 5 10 15
Tum
or v
olum
e m
m3
50
100
150
200
250
300
350
400
450ControlTetracTetrac Nano
PANC-1 Right Side
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
Integrins are 24 heterodimeric structural proteins of the plasma membrane that are critical to cell-cell and cell-ECM matrix protein interactions. Integrin v3 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.
0
1
2
3
4
5pERK1/2PCNApTyr-p85-PI 3-Kp85-PI 3-K
0
1
2
3
4
5
6
pERK1/2PCNA
U87MG (GBM) cells
Blot: anti-pERK1/2Nucleus
Blot: anti-PCNA
Blot: anti-pTyrp85-PI 3-KCytosol
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)
Rel
ativ
e I
.O.D
.
-PI 3-K
T3 (M) _ 10-9 10-8 10-7 10-6
Blot: anti-pTyrp85-PI 3-KCytosol
10-10
Blot: anti-p85-PI 3-K
- p85-PI 3-K
Blot: anti-pERK1/2Nucleus
Blot: anti-PCNA
- pERK1- pERK2
- PCNA
84 kDa -
84 kDa -
48 kDa -
37 kDa -
37 kDa -
Rel
ativ
e
I.O.D
.
- pTyr-p85-PI 3-K
T3 T4
Fig. 1
In vitro stimulation of cell proliferation(PCNA), activation of ERKs, PI3K bythyroid hormone analogues
Index of double-stranded DNA breaks
Tetrac-induced Radiosensitization