1. Growth Inhibitory Effects of ZD1839 (‘Iressa’) on Human Bladder Cancer Cell Lines Growth Inhibitory Effects of ZD1839 (‘Iressa’) on Human Bladder Cancer Cell Lines Axel Meye*, Ulrike Fiedler, Kathrin Kunert, Andres Melchior, Susanne Füssel, Manfred P. Wirth Department of Urology, Technical University Dresden, Germany * email: axel.meye@mailbox.tu-dresden.de; ‘Iressa’ is a trade mark of the AstraZeneca group of companies. BLADDER CARCINOMA CELL LINES AND METHODS Human bladder cells and culturing All four investigated cell lines EJ 28 (von Andres ???), J82 (ATCC #CRL ???), HT-1376 (ATCC #CRL ???) and 5637 (ATCC #CRL ???) were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cells were propagated as recommended by the supplier and without addition of antibiotics. In former studies we determined for each cell type the optimal cell number for plating experiments in 96 wells EGF, and EGFR inhibitor ZD1839 (‚IRESSA‘) treatment Purified recombinant human EGF was purchased from R&D Systems (Wiesbaden, Germany). Clinical grade ZD1839 (Iressa) was provided by AstraZeneca Pharmaceutical GmbH (Plankstadt, Germany). The IUPAC name of the EGFR inhibitor is 4-(3-chloro-4-fluorophenyl- amino)-7-methoxy-6-(3-(4-morpholi-nyl)propoxy)quinazoline. WST-1 assay, proliferation and apoptosis tests, and cell cycle analysis in vitro To determine the proliferation and viability of cells a colorimetric WST-1 assay (Cell Proliferation Reagent WST-1; Boehringer Mannheim, Mannheim, Germany) based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells was applied according to the manufacturs protocols. Briefly, cells were analysed 3, 6 and 9 days after seeding of defined cell numbers (for treatment experiments with ZD1839: EJ28 with 400 cells/well, 5637 with 1000 cells/well, J82 with 600 cells/well, amd HT1376 with 700 cells/well) in 96-well plates as indicated. So, cells were washed in PBS, incubated with 20 µl WST-1 reagent for two hours at 37 °C (5% CO 2 ) per 96-well. The detection was carried out at 450 nm at a Microplate reader (Fa). To analyse the cell cycle approximately 5x 10 5 cells were in 70% ethanol and stored at 4 °C until measurement. The DNA content was measured through incorporation of propidium iodide using a standard protocol (CycleTEST PLUS DNA Reagent Kit; Cell Cycle Becton Dickinson, Heidelberg, Germany). DNA distribution of the cell populations after descriebed treatment procedures was analysed by flow cytometry in a FACScan (Becton Dickinson). The rate of apoptosis was determined by the ApoAlert Caspase-3 colorimetric assay (Clontech, Heidelberg, Germany). Briefly, cells were seeded into 6-well paltes. After appropiate treatment, the cells were washed once with PBS, and x mL lysis buffer was added. After an incubation step (30 min), the supernatant was recovered and assayed as described by the manufacturs. The data for evaluation of apoptosis and cell cycle analysis represent two independent experiments, respectively. DISCUSSION Treatment with EGFR inhibitors could cause a cytotoxic effect generally with cell cycle arrest at the G1 checkpoint (Fan and Mendelsohn 1998). However, there are also studies describing an induction of apoptosis in several tumor types after EGFR blockade (Tortora et al. 1999, Wu et al. 1995, Ciardiello et al. 2000). Recent results of Cardiello et al. (2000) indicate an supra-additive, growth inhibitory effect of low doses ZD1839 in combination with various cytotoxic agents in vitro, including an increase of apoptotic cell death by approximately 2-3.5-fold. Moreover, recent results indicate that ZD1839 does not require high levels of intratumoral expression as determined for different tumor types (including prostate carcinoma, lung carcinoma, and vulvar carcinoma xenografts) and combinational treatment schedules with other cytotoxic chemotherpeutics (Sirotnak et al. 2000). The data presented clearly indicate the potential of EGFR inhiibition in therapeutic concepts for EGFR-overexpressed TCC. As described recently for other tumor types (Ciardiello et al. 2000, Sirotnak et al. 2000) we are now testing the antiproliferative activity of ZD1839 in combination with relevant cytotoxic agents for advanced bladder cancers (gemcitabine, cisplatin, taxol). Preliminary results indicate an additive effect especially for gemcitabine treatment in combination with ‚IRESSA‘ REFERENCES Bue P, et al. (1998) Int J Cancer 76: 189-93. Cardillo MR et al. (2000) Ciardiello F, et al. G (2000a) Clin Cancer Res. 6: 2053-63. Ciardiello F. (2000b) Drugs 60 (Suppl 1):25-32 (2000). Hammond L, et al. (1999) Proc Am Soc Clin Oncol 18: 1500. Lenferink AE, et al. (2000) Proc Natl Acad Sci USA 97: 9609-14. Liukkonen T, et al. (1999) Eur Urol 36: 393-400. Mendelsohn J, and Fan Z (1997) J Natl Cancer Inst 89: 341-3. Mellon K et al. (1995) J Urol 153: 919-25. Mellon JK, et al. (1996) J Urol 155: 321-6. Messing EM (1990) Cancer Res 50: 2530-7. Nakanishi K et al. 1996 Neal DE, et al. (1990) Cancer 65: 1619-25. Ruck A, and Paulie S (1997) Anticancer Res. 17: 1925-31. Sirotnak FM, et al. (2000) Clin Cancer Res 6:4885-92 (2000). Strawn LM, and Shawver LK (1998) Exp Opin Invest Drugs 7: 553- 73. Theodorescu D, et al. (1998) Int J Cancer 78: 775-82. Theodorescu D, et al. (1998) Cell Growth Differ 9: 919-28. Tortora G et al. Clin Cancer res 5: 875-81 (1999) Turkeri LN, et al. (1998) Urology 51: 645-9. Woodburn JR, et al. (1997) Proc Am Soc Clin Oncol 38: 4251. SUMMARY: Elevated levels of the human epidermal growth factor receptor (EGFR) are associated with various cancers and correlate with migration, radioresistance, chemotherapy resistance and selective growth advantages for metastatic cells. Bladder cancer cells frequently exhibit an increased number of functional EGFR in comparison to normal urothelium. ZD1839 (‘Iressa’) is an orally active selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that shows reversible antitumor activity in a broad range of established carcinoma cell lines and tumour xenografts (Ciardiello et al. Clin Cancer Res 2000; 6:2053- 63). The present study evaluated the antiproliferative activity of ZD1839 on bladder cancer cells in vitro. Four cell lines (EJ28, 5637, J82, HT-1376) derived from human transitional cell carcinoma (TCC) with different EGFR expression levels were investigated. The proliferation activity (WST-1 assay; 3, 6 and 9 days after treatment) was determined after stimulation with EGF and treatment with ZD1839. Furthermore, we evaluated the effects of ZD1839 on cell cycle distribution (FACS analyses) and induction of apoptosis. All four cell lines treated with ZD1839 showed a dose-dependent and specific inhibition of EGF- stimulated tumour cell proliferation. The specific IC50 for ZD1839 in each cell line (HT1376 EJ28 >5637/J82). Under confluent culture conditions cells with a low viability showed a higher resistance to the same ZD1839 concentration as determined for cells growing in the log phase. Initial data on proliferation rates of HT- 1376 cells 3-14 days after ZD0473 treatment indicated that the inhibitory effect of ZD1839 was reversible at relatively low concentrations (0.025-0.1 µM). As expected, cells stimulated with EGF had a lower proportion of cell population in G1 and G2/M phase in comparison to untreated cells. Additional treatment with ZD1839 abolished the EGF-induced alterations of cell cycle distribution. Simultaneously, ZD1839-treated HT-1376 and EJ28 cells showed an increased rate of apoptosis in comparison to the untreated control. The data presented clearly indicate the potential for EGFR/TK inhibition as a therapeutic approach to TCC where EGFR drive is important. As described recently for other tumour types [Ciardiello et al. 2000] we are now testing the antiproliferative activity of ZD1839 in combination with relevant cytotoxic agents for advanced bladder cancers (gemcitabine, cisplatin, paclitaxel). Fig. 3mdm2-AS-Kondo-transfection as long-time incubation and the effect on clonogenic survival for US8-93 cells [Transfection conditions: AS-ODNs without LFA for the whole incubation time period, each 1,5 ml OM-I- Volumen, after 12 days three-fold colony formation tests (each 250 cells) for different incubation times 10/36/50 h), 45-71% plating efficacy of untreated cells (control)] Fig. 2 Cell-associated fluorescence in LMS6-93 (red)/US8-93 cells (green) mediated by LFA-complexed 5’-FITC-ODNs (2 µg+ 8 LFA) [Incubation of complexes with cells for 2h or 8 h, FACS analyses after 24 h (left & middle) or 48 h (right)] Fig. 1Specific [average]. INTRODUCTION Importance of EGFR in bladder cancer biology Bladder cancer cells frequently exhibit an increased number of functional EGFR in comparison to normal urothelium. EGFR plays an important role in bladder cancer motility (Theodorescu et al. 1998a and 1998b). In human bladder cancer upregulation of EGFR often is correlated with increasing malignancy (Neal et al. 1990, Messing 1990, Mellon et al. 1995, and 1996, Bue et al. 1998, Turkeri et al. 1998, Luikkonen et al. 1999). Antiproliferative Activity of the specific EGFR tyrosine kinase inhibitor ZD1839 One of the intensively studied tyrosine kinases is the plasma membran glycoprotein EGFR. The different tumor biological activites of the EGFR in proliferation, angiogenesis and metastases together with the association of tumor patients with an EGFR overexpression with poor prognosis the broad strategy to inhibit EGFR in cancer therapy (Fry 1999, Woodburn 1999). ZD1839 (‘Iressa’) is an orally active selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that shows reversible antitumor activity in a broad range of established carcinoma cell lines and tumour xenografts (Ciardiello et al. 2000a, Sirotnak et al. 2000). The present study evaluated the antiproliferative activity of ZD1839 on bladder cancer cells in vitro. Four cell lines (EJ28, 5637, J82, HT-1376) derived from human transitional cell carcinoma (TCC) with different EGFR expression levels were investigated. RESULTS The detailed results of this study are represented in the figures and they can be summarized as Influence of ZD1839 on Cell cycle of HAT-1376 cells Fig. 4Western-blot for MDM2 after transfection using a monoclonal anti-Mdm2-antibody (19E3, 1:100)and the ECL detection system (30 µg total protein) Hybridisation procedure was carried out as described previously [Meye et al.1998] Control 4µg SE 4µg AS 8µg AS (10h) +16 µg LFA +16 µg LFA +16µg LFA