Redox NSAIDs and VEGF

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• Redox NSAIDs and VEGF
• Terry Moody, Ph.D.
• NCI Center for Cancer Research
• Bldg. 31, Rm. 4A48
• 301-451-9451
• moodyt_at_mail.nih.gov

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Lung Cancer kills over 150,000 patients in the
U.S. annually.

• There are 45 Million current smokers and 45
  Million ex-smokers in the U.S.
• Lung cancer is comprised of the neuroendocrine
  tumor small cell lung cancer (SCLC) and the
  epithelial tumor non-small cell lung cancer
  (NSCLC)
• NSCLC is associated with elevated levels of COX-2

3

• Indomethacin, which blocks COX-1 and COX-2,
  prevents lung adenoma formation in A/J mice.

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The A/J mouse develops lung adenomas after
carcinogen administration.
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COX-2 immunostaining inthe A/J mouse lung.
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Lung compartments and COX-2.

• Bronchus-epithelial cells show intense staining
  with moderate staining in the muscle but not
  cartilage.
• Bronchioles-Moderate staining in epithelial
  cells.
• Alveoli-Moderate staining in type 2 cells.
• Adenoma-scattered cellular staining.

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• S-NSAIDs and cancer cells
• ?Effects on PGE2
• ?Effects on proliferation

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COX-1 is expressed in lung cancer cells. Hida et
al., (1998) Anticancer Res. 18775.
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• EGF causes increases COX-2 expression in NSCLC
  cells.

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Novel NSAIDs reduce PGE2 in colon cancer cells.

• Addition PGE2, pg
• None 90
• Asp-NO, 1 ug/ml 16
• ACS2, 1 ug/ml 24
• ACS15, 1 ug/ml 16
• ACS18, 1 ug/ml 58
• The mean value of 4 determinations is indicated
  using supernatant from HT-29 cells incubated with
  20 uM arachidonic acid for 5 min by ELISA.

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S-NSAIDs and COX-2 inhibitors reduce HT-29 PGE2

• Addition PGE2
• None 100
• ACS18, 0.1 ug/ml 96
• ACS18, 1 ug/ml 59
• DuP-697, 0.1 ug/ml 56
• DuP-697, 1 ug/ml 27
• The mean value of 4 determinations each repeated
  in duplicate is shown

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• PGE2 binds to EP2-Rs which are present in lung
  cancer cell lines

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3H-PGE2 binds with high affinity to NSCLC
membranes. Casibang and Moody, (2002) Lung
Cancer 3633.
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• PGE1, PGE2, PGF2a and AH6809 bind with high
  affinity to NCI-H157 membranes.
• Compound IC50, uM
• Arachidonic acid gt10
• AH6809 5 0.7
• PGD2 gt10
• PGE1 0.2 .03
• PGE2 0.04 .01
• PGF2a 2 0.2
• PGG2 gt10
• PGI2 gt10

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• The EP2 receptor is coupled to adenylylcyclase.
• ?PGE2 is an agonist which increases the cAMP in
  lung cancer
• ?AH6809 is an antagonist which reversibly blocks
  the receptor

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EP2 receptor antagonists block the increase in
cAMP caused by PGE2.
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• VEGF expression in lung cancer cells is increased
  by many agents. Casibang et al. (2001), Lung
  Cancer 31203.

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VEGF mRNA is increased by PGE2 in a PKA-dependent
manner

• Addition Relative VEGF mRNA
• None 100 5
• PGE2, 1 uM 200 17
• EGF, 0.1 ug/ml 185 16
• H89, 50 uM 104 3
• PGE2 H89 110 6
• The mean value S.D. of 4 determinations is
  indicated p lt 0.05,

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VEGF is secreted from NCI-H157 cells.

• Addition VEGF, pg/ml
• None 1023 57
• PGE2, 1 uM 1240 76
• Forskolin, 50 uM 1365 106
• The mean value S.D. of 4 determinations is
  indicated p lt 0.05,

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Novel NSAIDs reduce cancer cellular
growth HT29 H1299 MCF7 Colon NSCLC
BreastACS2 100 40 30ACS15 40 25 50ACS18 5
0 50 20Diclofenac 50 10 30 Sulindac 30 40
50Asp-NO 15 15 20The mean IC50 (ug/ml) is
indicated using the MTT assay .
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Novel NSAIDs inhibit NSCLC colony formation

• Addition IC50, ug/ml
• Asp-NO 5
• ACS2 20
• ACS15 7
• ACS18 8
• The mean value of 3 determinations is indicated
  for NCI-H1299 colonies

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• ACS2 inhibits lung cancer colony growth

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• EGFR LUNG
• COX-2 CANCER CELL
• PGE2 SIGNAL
• EP2-R TRANSDUCTION
• Adenylyl cyclase
• Protein kinase A
• CREB phosphorylation
• Altered VEGF expression

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• TGF a EGFR EGFR
• Release
• COX-2 TRANSACTIVATION
• Protease
• Activation
• PGE2 BY PGE2
• Src
• Activation EP2-R
• Adenylyl cyclase
• Protein kinase A
• CREB phosphorylation

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Some NSCLC patients, who have failed
chemotherapy, respond to tyrosine kinase
inhibitors

• In the IDEAL-1 and IDEAL-2 clinical trials, 250
  mg of gefitinib caused an objective response in
  approximately 50 of the patients.
• Tumor responsiveness was not associated with EGFR
  expression but rather EGFR genetic mutations.
• EGFR mutations occurred in exons 18 through 21 of
  the tyrosine kinase domain, such as G719S or
  L858R.

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Conclusions1. S-NSAIDs reduce PGE2 levels. In
HT-29 cells DuP-697, which inhibits COX-2,
strongly reduces PGE2 concentrations. These
results suggest that S-NSAIDs are primarily
inhibiting COX-2 in colon cancer cells.2.
S-NSAIDs inhibit the proliferation of breast
cancer, colon cancer and NSCLC cells.The CRBF is
starting to investigate the effects of S-NSAIDS
in animal models of cancer3. It remains to be
determined if S-NSAIDs alter VEGF production in
and secretion from cancer cells.
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AcknowledgmentsNCI NIDDKM. Espey M.
BernaL. Ridnour R.T. JensenC. Switzer CTG
pharmaD. Wink P. Del Soldato