Apoptotic effects of the docetaxel→OSI-774 combination in non-small cell lung carcinoma (NSCLC) cells

Abstract

7143 Background: The epidermal growth factor receptor (EGFR) has been shown to signal through both the mitogen-activated protein kinase (MAPK) and protein kinase B (AKT) pathways, each of which has been shown to be abnormally activated in NSCLC. OSI-774 (Tarceva) is an EGFR tyrosine kinase inhibitor with anti-cancer activity in clinical trials. Docetaxel (Txt), an antimicrotubule agent, is used extensively in NSCLC. We previously reported that in vitro the sequence of Txt →OSI-774 resulted in the greatest proportion of sub-G1 cells compared to the alternate sequence of OSI-774→Txt (Gumerlock, ASCO 2003). Here we hypothesized that this is an apoptotic response that persists, resulting in long-term differences from these alternate administrations. Methods: The NSCLC cell line Calu1 was used as a model because it is EGFR positive and p53-null like the majority of advanced NSCLC tumors. Cells were treated with single doses of OSI-774 (1μM, 24hrs), Txt (50nM, 18hrs), or in these sequences: OSI-774→Txt→drug-free media for 72hrs or Txt→OSI-774→drug-free media. Cleaved poly-ADP-ribose polymerase (PARP) and Caspase-3 apoptosis marker proteins were assessed by Western blotting. Results: The greatest cell death was observed in the Txt→OSI-774→media sequence, while the cells treated with the OSI-774→Txt→media sequence resumed proliferation by 72hrs post-treatment. Cleaved PARP and Caspase-3 were detected in the sequence of Txt→OSI-774, and with simultaneous treatment, but not in the sequence of OSI-774→Txt. Further, cleaved PARP and Caspase-3 persisted to 72hrs after the Txt→OSI-774 treatment. These data support the previous results on sub-G1 cells, and molecularly demonstrate an apoptotic response. Conclusions: 1) The sequence of Txt→OSI-774 showed the greatest levels of apoptosis in NSCLC cells that persisted to at least 72hrs post-treatment. 2) This effect ocurred in p53-null cells, suggesting a potential role in tumor types with high frequencies of disrupted p53. These data form the basis for an ongoing phase I trial examining two regimens of intermittent EGFR blockade with OSI-774 plus Txt. (CM17101, CA62505, HHMI, Aventis, Genentech) Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aventis Oncology Aventis