Inhibition of the epidermal growth factor receptor in bladder cancer cells treated with the DNA-damaging drug etoposide markedly increases apoptosis.
OBJECTIVE
To investigate the effect of the epidermal growth factor receptor (EGFR) on the induction of apoptosis by the chemotherapeutic agent etoposide (VP16), and to examine the effect of combining VP16 with gefitinib to see if the cell-survival mechanism can be prevented.


METHODS
The bladder cancer cell lines RT4 and T24, representing low- and high-malignancy grades respectively, were treated with VP16 (10 or 50 microM) and the level of apoptosis determined using a commercial kit. EGFR receptor activity was determined by western blotting using antibodies against phosphorylated EGFR. The EGFR was either activated by heparin-binding (HB)-EGF (1 nM) or inhibited with the specific EGFR inhibitor gefitinib (1 or 5 microM). The pan-caspase inhibitor Z-VAD (30 microM) was used to test the involvement of caspase activity.


RESULTS
Treatment of T24 bladder cancer cells with VP16 (50 microM) for 48 h induced phosphorylation of the EGFR and activation of the EGFR prevented the apoptosis induced by VP16. Thus, treatment of T24 cells with 50 microM VP16 for 48 h resulted in 19% apoptosis. However, activation of the EGFR with HB-EGF (1 nM) with VP16 (50 microM) significantly reduced the level of apoptosis by 25% (P < 0.05) showing that activating the EGFR has a cell-survival function. Inhibiting the EGFR with gefitinib (5 microM) blocked the VP16-induced activation of the EGFR. Combined treatment with gefitinib and VP16 resulted in 45% apoptotic cells, i.e. more than double the percentage of apoptotic cells with VP16 alone. This was found in both T24 and RT4 cells. Gefitinib used alone (1 and 5 microM) generated no apoptosis in the cells. Treatment of T24 cells with Z-VAD showed that apoptosis induced by both VP16 alone and VP16 with gefitinib was caspase-mediated.


CONCLUSIONS
These results suggest that activation of the EGFR induced a cell-survival function when bladder cancer cells were treated with the DNA-damaging drug VP16, and that combined treatment with VP16 and the EGFR inhibitor gefitinib might improve the efficacy of treatment.
Munk
Mathias
M
Department of Clinical Biochemistry, Aarhus University Hospital NBG, Aarhus, Denmark.
Memon
Ashfaque Ahmed
AA
Nexo
Ebba
E
Sorensen
Boe Sandahl
BS
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D005047:Etoposide / Q000008:administration & dosage; D000077156:Gefitinib; D006801:Humans; D011799:Quinazolines / Q000008:administration & dosage; D016896:Treatment Outcome; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000473:pathology
D011799:Quinazolines; D005047:Etoposide; D066246:ErbB Receptors; D000077156:Gefitinib