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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@mail.nih.gov
 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
Indomethacin, which blocks COX-1 and
    COX-2, prevents lung adenoma
       formation in A/J mice.

                         Indomethacin reduces adenoma number in A/J mice
                   30

                                                                                 None
                   25
                                                                                 U
  Adenoma number




                                                                                 U+I
                   20

                   15

                   10

                    5

                    0
                        0.5   1     1.5    2        2.5     3   3.5   4    4.5
                                               Time, months
The A/J mouse develops lung
 adenomas after carcinogen
       administration.
COX-2 immunostaining in
  the A/J mouse lung.
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.
S-NSAIDs and cancer cells
●Effects on PGE2
●Effects on proliferation
COX-1 is expressed in lung
cancer cells. Hida et al., (1998);
     Anticancer Res. 18:775.
EGF causes increases COX-2
 expression in NSCLC cells.
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.
     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
 PGE2 binds to EP2-Rs
which are present in lung
    cancer cell lines
3H-PGEbinds with high affinity to
      2
NSCLC membranes. Casibang and
                         Moody, (2002) Lung Cancer 36:33.

                                                                                   PGF2a
                                       PGE binds with high affinity to membranes
                                           2                                       PGD2
                           120                                                     PGE1
                                                                                   PGE2

                           100
% Specific H-PGE bound




                            80
                   2




                            60
3




                            40



                            20



                             0
                                 -10      -9       -8       -7        -6     -5     -4

                                                        [PG], Log M
 PGE1, PGE2, PGF2α and AH6809 bind
    with high affinity to NCI-H157
             membranes.
Compound           IC50, ųM
Arachidonic acid   >10
AH6809             5 + 0.7
PGD2               >10
PGE1               0.2 + .03
PGE2               0.04 + .01
PGF2α              2 + 0.2
PGG2               >10
PGI2               >10
 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
    EP2 receptor antagonists block the
   increase in cAMP caused by PGE2.


                       AH6809 antagonizes cAMP production
             80


             70

                                     None
             60
                                     + 10 uM AH6809

             50
cAMP, fmol




             40


             30


             20


             10


              0
                  -9          -8        -7            -6    -5

                                   [PGE , Log M]
                                       2
 VEGF expression in lung cancer
cells is increased by many agents.
Casibang et al. (2001), Lung Cancer 31:203.
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 < 0.05, *
  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 < 0.05, *
Novel NSAIDs reduce cancer cellular growth
           HT29       H1299       MCF7
           Colon      NSCLC         Breast
ACS2 100        40                30
ACS15      40         25               50
ACS18      50         50               20
Diclofenac 50         10               30
Sulindac 30           40               50
Asp-NO     15         15               20
The mean IC50 (ug/ml) is indicated using the MTT
assay      .
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
 ACS2 inhibits lung
cancer colony growth
     EGFR        LUNG

    COX-2       CANCER CELL

     PGE2       SIGNAL

      EP2-R       TRANSDUCTION

   Adenylyl cyclase

   Protein kinase A

 CREB phosphorylation

Altered VEGF expression
 TGF α            EGFR                  EGFR
Release

                  COX-2            TRANSACTIVATION
Protease
Activation
                   PGE2                   BY PGE2
   Src
   Activation       EP2-R

                Adenylyl cyclase


                Protein kinase A


             CREB phosphorylation

          Altered VEGF expression
  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.
Conclusions
1. 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
cancer
3. It remains to be determined if S-NSAIDs
alter VEGF production in and secretion from
cancer cells.
Acknowledgments

NCI               NIDDK
M. Espey          M. Berna
L. Ridnour        R.T. Jensen
C. Switzer        CTG pharma
D. Wink           P. Del Soldato

				
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posted:9/13/2012
language:English
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