1 / 33

Paul J. Davis, MD

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

mccraney
Download Presentation

Paul J. Davis, MD

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

  2. 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.

  3. - - - - - - - I I I I I I I - - - - - - - NH2 3’ 3 HO CH2-CH-COOH O 5’ 5 Thyroxine (T4) NH2 3’ 3 CH2-CH-COOH HO O 5’ 5 3,5,3’-Triiodothyronine (T3)

  4. Na+/H+ antiporter PLC PKC Amino acid transporter PROTEIN TRAFFICKING PI 3-K- Akt/PKB Nongenomic Actions of Thyroid Hormone Initiated at the Cell Surface T4 T3 Extracellular matrix proteins, e.g., laminin, vitronectin Integrin V β3 rT3 T4 SIGNAL T3 Plasma membrane TRANSDUCTION MAPK (ERK1/ERK2) T3 TR∆a1 TRa STAT1a TRb1 p53 TRb1, ER a MAPK TRb1 SERINE PHOSPHORYLATION (ERK1/ERK2) a ER STAT1a, p53 STAT1a TRb1 ERa p53 Trip230 TRb1 p300 NCoR Nucleus Gene Cytoplasm Transcription mRNA mRNA Protein Synthesis T3 T3 Protein trafficking Serine phosphorylation of nuclear proteins Specific gene transcription Activity of ion transporters

  5. 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.

  6. 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.

  7. Thyroid hormone and cancer cell proliferation

  8. - Fold Increase in I.O.D. MCF-7 cells: PCNA MCF-7 cells: 3H-thymidine uptake * 37 kDa- * Control 3H-thymidine Incorporation - Fold Increase in * * _ _ _ _ T4 + + Time (h) 1 2 4 6 24 24 _ _ _ _ + + T4-A _ _ _ _ _ + ICI (3 nM) _ _ _ PD + + + Thyroid hormone and breast cancer cell proliferation ICI = ICI 182,780, fulvestrant

  9. OVCAR-3 cells IP: anti-integrin αv Blot: anti-NCoR - NCoR Blot: anti-SMRT - SMRT 180 kDa - Blot: anti-p300 * * * * - p300 180 kDa - * * Blot: anti-pSTAT1 - Relative I.O.D. * - pSTAT1 82 kDa - Blot: anti-pERK1/2 * - pERK1 - pERK2 37 kDa - * * * * * * * * - Lamin B 61 kDa - | | | | C siRNA scRNA | | | | C siRNA scRNA _ _ _ _ _ _ + + + T4 (10-7 M) + + + T4 (10-7 M) Fig. 3

  10. A B Blot: anti-PCNA Blot: anti-PCNA NCI-H522 cells NCI-H522 cells 37 kDa - 37 kDa - - PCNA - PCNA - Lamin-B - Lamin-B 61 kDa - 61 kDa - _ _ T3 (M) 10-9 10-8 10-7 10-6 T4 (M) 10-9 10-8 10-7 10-6 C D Thymidine Incorporation * - Integrin β * * * - Integrin α * * CPM x 104 * * - GAPDH * * * * * NCI-510A NCI-H522 OVCAR- 3 _ _ _ _ T4 (M) 10-8 10-7 10-6 _ _ _ _ RT-PCR T3 (M) 10-8 10-7 10-9 H510A, small cell lung carcinoma cells H522, NSCLC cells

  11. A NCI-H510A cells IP: anti-ER-α Blot: anti-phosphoER-a (118) - pER-a 62 kDa - Blot: anti-ER-α - ER-a 62 kDa - Blot: anti-pERK1/2 - pERK1 - pERK2 37 kDa - Blot: anti-PCNA 37 kDa - - PCNA - Lamin-B 61 kDa - * * -a * * -Relative I.O.D. * * * * + + + + * + * _ _ _ ICI (nM) 20 2 20 2 20 _ _ _ _ _ + + + T3 (10-7 M) _ _ _ _ _ + + + T4 (10-7 M) B ICI = ICI 182,780, Thymidine Incorporation * * * * CPM X 104 + + _ _ _ ICI (nM) 20 20 20 _ _ _ _ + + T3 (10-7 M) _ _ _ _ + + T4 (10-7 M)

  12. U87MG (GBM) cells T3 T4 Nucleus Blot: anti-pERK1/2 Nucleus 48 kDa - - pERK1 Blot: anti-pERK1/2 - pERK2 37 kDa - 48 kDa - - pERK1 - pERK2 Blot: anti-PCNA 37 kDa - Blot: anti-PCNA 37 kDa - - PCNA 37 kDa - - PCNA Cytosol Blot: anti-pTyrp85-PI 3-K - pTyr-p85 84 kDa - Relative I.O.D. -PI 3-K Blot: anti-p85-PI 3-K - p85-PI 3-K Cytosol 84 kDa - Blot: anti-pTyrp85-PI 3-K - pTyr-p85 84 kDa - -PI 3-K _ Relative I.O.D. T4 (M) 10-8 10-6 10-9 10-7 T3 (10-7 M) _ T3 (M) 10-8 10-6 10-10 10-9 10-7 In vitro stimulation of cell proliferation (PCNA), activation of ERKs, PI3K by thyroid hormone analogues Fig. 1

  13. Hypothyroid Median Survival: 10.1 mos Non-hypothyroid Median Survival: 3.1 mos Hercbergs AA et al,Anticancer Res, 2003

  14. 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).

  15. Thyroid hormone has anti-apoptotic activity in cancer cells

  16. RV = resveratrol

  17. - - - - - - - - - - - I I I I I I I I I I I - - - - - - - - - - - NH2 3’ 3 HO CH2-CH-COOH O 5’ 5 Thyroxine (T4) NH2 3’ 3 CH2-CH-COOH HO O 5’ 5 3,5,3’-Triiodothyronine (T3) 3’ 3 HO CH2--COOH O 5’ 5 Tetrac Low-grade thyromimetic within cells TH antagonist at integrin avb3 TH receptor

  18. pERK1/2 pERK1/2 activation activation Thyroid Hormone Resveratrol Tetrac αvβ3 αvβ3 PD98059 PD98059 Cytosol X Plasma membrane pSer15-p53 NS-398 Cell Proliferation Apoptosis Nucleus [COX-2]-pERK1/2 complexing Thyroid hormone inhibits induction by RV of Ser-15 phosphorylation of p53

  19. 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.

  20. PBS T4-ag T4-ag + Tetrac Angiogenesis in the CAM A PBS T4 T4 + Tetrac B C Summary of effects of T4, T4-agarose and tetrac on angiogenesis TreatmentAngiogenesis 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

  21. A PBS (Control) T4 (0.1 µM) T4-agarose (0.1 µM) DITPA (0.01 µM) VEGF (2.0 µg/ml) b-FGF (1.0 µg/ml) 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* B Data represent mean ± SD, n=8; *p<0.01, indicating significant stimulation of angiogenesis. PBS, phosphate-buffered saline.

  22. Inhibitory Effect of avb3 MAB (LM609) on T4-stimulated Angiogenesis in the CAM Model T4 (total, 0.1mM) PBS T4+ LM609(10mg)

  23. 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.

  24. Na+, K+ - ATPase Na+/H+ antiporter P-glycoprotein EGFR Chemotherapeutic agent efflux T4 EGF T4 pHe K+ Na+ Plasma Membrane αv αv αv β3 β3 β3 Integrin Integrin Integrin H+ Na+ Cytoplasm [Na+]i pHi Results of T4 action Ca2+ Increased EGF input to P-gp + Calmodulin Nucleus Bold blue arrows indicate stimulatory action of tetrac/Nanotetrac on Na+, K+ - ATPase, Na+/H+ antiporter and EGF receptor transcription MDR1 transcription EGFR

  25. 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.

  26. 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.

  27. 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

  28. A 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. B + + + VEGF _ 1.0 2.5 Tetrac Int Angiol, 2006

  29. A ER-a gene expression ER-a gene expression - ER-a Relative I.O.D. - GAPDH NCI-H522 NCI-H510A C B NCI-H522 cells IP: anti-integrin-αv Green: phosphoER-a - ER-αPromoter Red: Nucleoprotein - Input Control _ + Control T₄ IgG L-T4 * * Tetrac + + Tetrac + T4

  30. A Nucleosome ELISA • Fold Increase • in nucleosome content _ _ RV (𝜇M) 1 10 1 10 _ _ _ + + + T4 (10-7 M) B Nucleosome ELISA • Fold Increase in nucleosome content _ _ _ _ + + + + Tetrac (10-7 M) _ _ _ _ + + + + RV (10 𝜇M) _ _ _ _ + + + + T4 (10-7 M)

  31. 25 20 15 10 5 Effects of L-T4 or T4 Nanoparticles (NP) on Endothelial Cell Migration toward Vitronectin (VN) EC Migration (RFU) x 103 T4 T4-NP 0 0 Upper Chamber 0 VN VN VN Lower Boyden apparatus

More Related