BLADDER CARCINOMA CELL LINES AND METHODS Human bladder cells and culturing

1. Growth Inhibitory Effects of ZD1839 (‘Iressa’) on Human Bladder Cancer Cell LinesAxel Meye*, Ulrike Fiedler, Kathrin Kunert, Andres Melchior, Susanne Füssel, Manfred P. WirthDepartment of Urology, Technical University Dresden, F. R. of Germany* email: axel.meye@mailbox.tu-dresden.de; ‘Iressa’ is a trade mark of the AstraZeneca group of companies. INTRODUCTION & OBJECTIVES 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 intratumoral EGFR overexpression) with poor prognosis implies the broad strategy to inhibit EGFR in cancer therapy (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. 1,4 HT-1376 1,2 1,0 0,8 OD 450nm 0,6 0,4 0,2 negative cells with only EGF 0.01 0.05 0.1 0.5 1 control 1,5% FCS ZD1839 concentration [µM] Fig. 1: Effect of different ZD1839 concentrations (0.1-1 µM) on proliferation of HT-1376 cells. [Standard deviations are indicated. As controls served only medium (without cells), cells with FCS (1.5%) and EGF-stimulated cells (5 ng/mL) after 3 days (blue columns), 6 days (red colums) and 9 days (yellow colums).] BLADDER CARCINOMA CELL LINES AND METHODS Human bladder cells and culturing The cell lines J82 (ATCC #HTB-1), HT-1376 (ATCC #CRL-1472) and 5637 (ATCC #HTB-9) originated from human bladder cancers obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA), the EJ28 cell line was a gift [Block et al. 1993]. The cells were propagated as recommended by the supplier and without addition of antibiotics. For EGF stimulation only reduced percentage of FCS (1% for EJ28 cells, 1.5% for J82 and HT1376, and 2% for 5637) was added to the culture medium. First experiments were carried out to determine the optimal EGF concentration for each cell type (to mediate a efficient EGFR stimulation): EJ28 for 1 ng/mL EGF, HT-1376 for 5 ng/mL EGF (data not shown). EGF, and EGFR inhibitor ZD1839 (‚IRESSA‘) treatment Purified recombinant human EGF was purchased from R&D Systems (Wiesbaden, Germany). Clinical grade ZD1839 (‚Iressa‘) was kindly provided by AstraZeneca Pharmaceutical GmbH (Plankstadt, Germany). 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% CO2) per 96-well. The detection was carried out at 450 nm at a conventional microplate reader. To analyse the cell cycle approximately 5x 105 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 plates. After appropiate treatment, the cells were washed once with PBS, and lysis buffer was added. After an incubation step for 30 min, the supernatant was recovered and assayed as described by the manufactures. The data for evaluation of apoptosis and cell cycle analysis represent two independent experiments, respectively. • RESULTS • The detailed results of this study are represented in the figures and tables. They can be summarized as following: • dose-dependent and specific inhibition of EGF-stimulated tumour cell proliferation for all for TCC lines (Fig. 1 & 2) under optimized culturing conditions (minimal FCS-c & optimal EGF-c) • cell-type specific IC50 for ZD1839 (HT-1376<EJ28<5637<J82) correlated to the EGFR protein level evaluated by Western blotanalysis (HAT-1376>EJ28 >5637/J82; data not shown) (Melchior et al., manuscript in preparation). • under confluent culture conditions tumour cells with a low viability showed a higher resistance to the same ZD1839 concentration as determined for cells growing in the log phase (data not shown). • initial data on proliferation rates of HT-1376 cells 3-14 days after treat-ment indicated that the inhibitory effect of ‘IRESSA’ was reversible at relatively low concentrations (0.025-0.1 µM) (Fig 3). • As expected, cells stimulated with EGF had a lower proportion of cell population in G1 and G2/M phase in comparison to untreated cells (Table 1a). Additional treatment with ZD1839 abolished the EGF-induced alterations of cell cycle distribution in HT-1376 (Table 1b). • Simultaneously, ZD1839-treated HT-1376 and EJ28 cells showed an increased rate of apoptosis (9.9% and 14.7%, respectively) in comparison to the untreated control (Table 2) Table 1 a) Influence of EGF stimulation experiments on cell cycle phase distribution for HT13-76 cells (standard deviations are indicated) b) Addition of ZD1839 to EGF-stimulated cells results in a reblockade of at the G1 arrest only in HT-1376 (with a high EGFR expression level), whereas the ZD1839 treatment for EJ28 has no remarkebly influence on the EGF-induced cell cycle alterations. 1,3 EJ28 2.d 1,2 3.d 1,1 4.d 6.d 1,0 0,9 0,8 OD 450 nm 0,7 Table 2 Difference in apoptosis rate in cell lines EJ28 and HT-1376 treated with EGF alone or with EGF and ZD1839 (specific concentrations of both are indicated). Both cell types showed a ZD1839-induced specific increase in apoptosis after 3 days in comparison to control (only EGF stimulation). Results of caspase assay (ApoAlert Caspase-3-colorimetric assay) were calculated as difference in caspase activity (PNA per hour [nmol]). 0,6 0,5 0,4 0,3 CONCLUSIONS Treatment with EGFR inhibitors could cause a cytotoxic effect generally with cell cycle arrest at the G1 checkpoint. However, there are also studies describing an induction of apoptosis in several tumor types after EGFR blockade (Tortora et al. 1999, Ciardiello et al. 2000a). The data presented clearly indicate the potential for EGFR/TK inhibition as a therapeutic approach to TCC where EGFR drive is important. Recent results of Cardiello et al. (2000a) 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, Sirotnak et al. (2000) found 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 chemotherapeutics. As described in the latter two studies we started in testing the antiproliferative activity of ZD1839 in combination with relevant cytotoxic agents for advanced bladder cancers (gemcitabine, cisplatin, taxol). Preliminary results indicate an specific additive effect especially for gemcitabine treatment in combination with ‚IRESSA‘ in in vitro studies with TCC cell lines. negative cells with only EGF 0.01 0.05 0.1 0.5 1 control 1% FCS ZD1839 concentration [µM] REFERENCES Block T et al. (1993) Urol Res 21: 217-21. 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). Liukkonen T, et al. (1999) Eur Urol 36: 393-400. 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. Neal DE, et al. (1990) Cancer 65: 1619-25. Ruck A, Paulie S (1997) Anticancer Res. 17: 1925-31. Sirotnak FM, et al. (2000) Clin Cancer Res 6:4885-92 (2000). Strawn LM, Shawver LK (1998) Exp Opin Invest Drugs 7: 553-73. Theodorescu D, et al. (1998a) Int J Cancer 78: 775-82. Theodorescu D, et al. (1998b) 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. Fig. 2: Effect of different inhibitor concentrations (0.1-1 µM) on proliferation of EJ28 cells [Standard deviations are indicated. As controls served only medium (without cells), cells with FCS (1%) and EGF-stimulated cells (1 ng/mL) after 2 days (green), 3 days (blue), 4 days (purple), and 6 days (red)] Treatment with ZD1839 for 3 days Treatment with ZD1839 for 6 days . Fig. 3: Long term viability test showing the reversibility of the EGFR blockade by ZD1839 in HT1376 cells After 3 days (left) or 6 (right) days the medium containig EGFRI was removed and replaced by medium with minimal FCS, Viability was measured after next 17 to 23 days. At relative low concentrations (0.025-0.1 µM) an increase of viability of about 40% could be determined.