CYTOKINES, ANGIOGENESIS AND TUMOR GROWTH

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CYTOKINES, ANGIOGENESIS AND TUMOR GROWTH

• Leon Bernal-Mizrachi
• Washington University - Barnes/Jewish hospital

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• What is theAngiogenetic switch
• Which Cytokines and mechanism are associated
  with angiogenesis
• TNF alfa
• IL-1b
• Prostaglandin E2
• Cancer model examples

3
Angiogenetic switch hypothesis

• The lost of tumor dormancy is accompanied by
  alterations in the angiogenic balance- Taturo et
  al.

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Angiogenetic switch - Methodology

• Cell lines used for animal inoculation cultured
• ST-2 Gastric Ca
• MG-63 OS
• p53-/-SAOS-2 OS
• Lines Cloned vector C-ha-ras (EJ bladder Ca) or
  VEFG 165 Vector
• Cell lines were transfected with green
  fluorescence protein before inoculation
• Injected in the dorsum of immunodeficient mice
  (SICD)

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Effect of VEGF or Activated C-ha-ras Trasfection
on VEGF Protein Levels and Tumor development
Taturo U.,et al FASEB J. 2002
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Quantification of Proliferation, Apoptosis and
Angiogenesis of Transfected Tumor Cells


































Taturo U.,et al FASEB J. 2002
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Cytokines - Angiogenesis
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Induction of production of VEGF by IL -1a in
human PBMNCs
VEGF production of PBMNCs in serum free medium
PBMNCs 4 ng/ml of IL-1a
PBMNCs 4ng/ml IL-1a Cycloheximine

• Isolated PBMNCs of
• healthy donors /- IL-1
• inmmunohistochemical
• stain for vWF

Petri S. et al. FASEB J.2002
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IL 1a promotion of angiogenesis in vivo via
VEGFR-2.
IL-1a Anti-VEGFR-2 13 5 v.e/f
PBS 12 4 v.e/ f
IL-1a Anti-VEGFR-1 46 11 v.e/f
IL-1a 59 18 v.e./ f
Petri S. et al. FASEB J.2002
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Inhibition of IL-1 Suppresses Neovascularization
in Rat Models of Corneal Angiogenesis

• Insertion of paper disc VEGF
• or bFGF
• Disc placed 1.8 mm from blood
• vessel of lateral limbus
• IL-1ra given .05-1-1.5-5 ng/ml
• starting the day of surgery
• Evaluated by transilumination,
• using three criterias
• Disc distance from limbal v.
• Number of vessel crossing
• midline
• Blood vessel area

0.1 BSA negative control
VEGF control
VEGF systemic IL-1ra
Coxon a. et al. Arthr. and Rheu.2002
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Insitu hybridization for CD31, IL-1RI and IL-1RII
Coxon a. et al. Arthr. and Rheu.2002
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Other mechanism implicated in the angiogenesis
induced by IL-1

• Nitric oxide mediates IL-1 gene expression
• of matrix metalloproteinases and BFGF
• (Sasaki K. et. al. J Biochem.1998)
• Cyclooxygenase-1 And -2 Expression In
• Synovial Tissues By IL-1 (Crofford LJ, et
• al.J Clin Inv 1994)

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Activation of Ekt Through TNFR-2 in HUVEC Cells
Pan S. et al Molc Cel Biol.2002
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Angiogenesis Mediated by TNFR-2 / Etk Pathway

• Tube Formation Assay
• Etk-DK/TNF inhibitory effect
• Etk-SK/NTF stimulatory
  effect

• Quantification of tube formation

Pan S. et al Molc Cel Biol.2002
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PGE2 accelerates avb3-mediated cell adhesion and
spreading

• Three enzyme-linked immunosorbent assay plates
• Fibronectin coat
• Gelatin coat
• Vitronectin coat
• Supernatant was removed
• Plates stained with crystal violet (0.5) and
  quantified by optical density
• Spread cells were counted in 3 high power fields

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HUVEC express functional PEG2 receptors EP2 and
EP4

• Expression of EP1,EP2,EP3 and EP4 mRNA in HUVEC
  was analyzed by RT PCR .
• HUVEC plated on gelatin in the presence of PEG1,2
  or butaprost.
• Attached cells were fixed and revealed by crystal
  violet staining

Relative cell adhesion (O.D)
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Increase in cAMP levels results from Cox-2
dependent vb3-mediated adhesion
A.PEG2 modulates avb3-mediated HUVEC cell
adhesion via cAMP B. avb3-mediated HUVEC cell
adhesion to gelatin but not to fibronectin
depends on cAMP production C.8brcAMP induce
adhesion and promote spreading , effect not
blocked by NS398 or U46619
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Prostaglandin E2 and angiogenesis

• PEG2 accelerates avb3-mediated cell adhesion and
  spreading of endothelial cells
• HUVEC express functional PEG2 receptors EP2 and
  EP4
• Increase in cAMP levels results from Cox-2
  dependent avb3-mediated adhesion
• Rac activation and avb3 -dependent HUVEC adhesion
  and migration depend on PKA
• Rac mediates cAMP induced spreading

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Nitric oxide synthase and lung tumorigenesis

• Wild type and INOS KO mice were injected ip
  with1g/kg of urethane q w for 7w
• tumors were enumerated and measured on week 16th
• iNOS deficiency did not affect tumor incidence
  but did affect tumor number

80
P lt0.0001
Kisley LR. Canc res.62.Dec.2002
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No Compensatory Up-regulation of eNOS and nNOS by
nNOS Deficiency

• Lungs were inflated with formalin and embedded in
  parafin. 4 mM sections immunohistochemial
  analysis
• Rehydrated 10 goat antirabbit ecNOS, iNOS and
  nNOS

Kisley LR. Canc res.62.Dec.2002
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VEGF concentration in iNOS deficient mice

• Tumor cells and clara cells had a much greater
  VEGF expression than normal cells
• iNOS -/- had lower VEGF values compared to wild
  type

Plt0.05
54
Kisley LR. Canc res.62.Dec.2002
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Other Tumor Progressions in Which NO and VEGF
Have Also Been Implicated

• Lung colonization after tail injection of
  melanoma cells is reduced, and tumor express less
  VEGF in mice lacking iNOS. (Wang B.Int J.
  Can.91.2001, Shi Q. Canc Res.2000 )
• Tumors derived from injected colon AC cells that
  transgenically overexpress iNOS grow faster and
  more vascularized than tumors from parental cell
  lines (Jenckins D.C. Proc Natl Aca Sci.92.1995)

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Angiogenesis sustains tumor dormancy in patients
with breast cancer treated with adjuvant
chemotherapy

• Evaluate the relationship of tumor
  vascularization with the behavior of recurrence
  in time
• Vascularization of the primary was assessed by
  Anti cd31 immunohistochemial stain
• Intratumoral microvessel density at the vascular
  hot spot of the tumor

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Angiogenesis sustains tumor dormancy in patients
with breast cancer treated with adjuvant
chemotherapy

• Mesh plot of the hazard of recurrence in time
  (Rgt axis) as function of MV counts (Lft axis
  -CD31)
• curves of the hazar function of
• recurrence in time for microvs counts
• a 55mv/fld
• b 82mv/fld
• c 120mv/fld

Risk associated with highly vascularized cancers