Inhibition of Cancer cells by Iressa, an EGFR Tyrosine Kinase Inhibitor

1. Inhibition of Cancer cells by Iressa, an EGFR Tyrosine Kinase Inhibitor Presenters Naser Shams and Jim McCarthy

2. Terms to Know • Epithelial - skin and other lining tissues • Endothelial - epithelial tissue lining blood vessels • Angiogenesis - creation of blood vessels • bFGF - basic Fibroblast Growth Factor • EGF - Epidermal Growth Factor • TGF-α - Transforming Growth Factor α • VEGF - Vascular Endothelial Growth Factor • EGFR - EGF Receptor (type of RTK) • Kinase - enzyme used in phosphorylation • TKI - Tyrosine Kinase Inhibitor

3. Receptor Tyrosine Kinase

4. Specific Modes of Cancer Proliferation

5. Epithelial Cancer

6. Drugs and Cell Types • ZD1839 (Iressa) - EGFR inhibitor • Paclitaxel - mitotic inhibitor used in cancer chemotherapy • colon cancer: GEO, SW480, and CaCo2 • ovarian cancer: OVCAR-3 • breast cancer: ZR-75-1 and MCF-7 ADR

7. Experiment 1 • Purpose: Test various human cancer cell lines for the expression of EGFR. • Provide a baseline for GF expression post treatment • Cancer cells analyzed: • GEO, SW480, CaCO2------colon cancer • Zr-75-1 & MCF-7 ADR-----breast cancer • OVCAR-3---------------------ovarian cancer • Kato 3 & N87----------------gastric cancer

8. Experiment 1: Overview • All cells maintained in pH 7.4 at 37° treated with an antibiotics • Western Blot used to analyze EGFR expression

9. Western Blot Method • The western blot (sometimes called the protein immunoblot) is a widely accepted analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. • It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by size • Proteins are transferred to membrane where they are stained with antihuman EGFR antibodies

10. Western Blot Overview

11. Gel Electrophoresis used in Western Blot Method

12. Western Blot: EGFR Expression in various Cancer cells---- • All cancer cells expressed EGFR----EGFR path- way active in all cancer cells. • Qualitatively, breast cancer cells showed highest expression Lane 1: Geo cells (colon cancer) Lane 2: ZR-75-1 (breast cancer) Lane 3: N87 (gastric cancer) Lane 4: MCF—7 ADR (breast cancer) Lane 5: SW480 (colon cancer)

13. EGFR expression in cancer cells

14. Experiment 2: Overview All cells maintained as in Experiment 1 TGF-α, bFGF secretion levels determined by ELISA Method Cancer cells were treated for 5 days with different concentrations of Iressa Concentrations: 0.01µM to 1.0µM

15. ELISA (Enzyme-Linked Immuno Sorbent Assay) Test Coat plate with capture antibody Add samples and standard Add detection antibody Add biotin enzyme Add substrate for color development

16. Percentage TGF-α versus Control

17. Percentage bFGF versus Control

18. Conclusions from Experiments • Reduced bFGF and TGF-α • Iressa effective for all cell lines studied • Signal Transduction Interference as Therapy • TKI Tied with Chemotherapy Treatment Improves Overall Response

19. Other Vital Components to Beating Cancer

20. Healthy Life-Style Habits

21. Questions?

22. Baseline GF Values

23. Tumor Size Versus Treatment