nanotetrac targets the thyroid hormone receptor on integrin v 3 to promote apoptosis, disrupt cell...

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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

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