Abstract B262: Tyrosine kinase inhibitors induce rapid cellular alterations in breast cancer cells in vitro

Abstract

Abstract Purpose: Alterations in tyrosine kinase (TK) expression or functionality have been linked to tumor growth, and the unraveling of TK pathways has led to the discovery of novel anticancer drugs. The aims of this study were to evaluate cytotoxic and multidrug resistance mechanisms, such as Ca2+ homeostasis and efflux pump activity, of three tyrosine kinase inhibitors (TKIs), lapatinib, sorafenib and gefitinib, in two breast cancer cell lines. Methods: Two human breast cancer cell lines, Estrogen Receptor (ER) positive MCF-7 and ER negative BRC-230 were exposed to the investigated drugs for 20, 40, 60, 120 and 240 min. Cytotoxicity, cytosolic Ca2+ levels, extrusion pump activity and mitochondrial membrane depolarization were assessed by flow cytometry. Expression of drug efflux genes was determined by RT-PCR, and protein expression of drug targets by Western blot. Results: MCF-7 cells did not express EGFR, p-EGFR, Her-2 or p-Her-2, while BRC-230 were positive for EGFR and p-EGFR only. ERK1/2 was equally expressed in both cell lines, but only BRC-230 were positive for p-ERK1/2. MEK and p-MEK were expressed in both cell lines, albeit more intensively in BRC-230. As expected, based on their drug target expression profiles, gefitinib and lapatinib were found to induce cytocidal effects and mitochondrial membrane depolarization in BRC-230 only, while sorafenib caused apoptosis and loss of mitochondrial potential in both cell lines. All three drugs triggered a rapid and biphasic increase in cytosolic [Ca2+] (after a 20 min exposure), and a decline in xenobiotic extrusion pump activity that was independent of efflux gene downregulation. The thapsigargin-induced depletion of endoplasmic reticulum Ca2+ blocked the increase in cytosolic Ca2+, but had no effect on multidrug transporter activity or on mitochondrial membrane depolarization. Conclusions: Gefitinib, lapatinib and sorafenib induced a rapid increase in cytosolic [Ca2+] and mitochondrial membrane depolarization, in addition to blocking drug transporter activity, independently of the expression of specific drug targets and of drug-induced cytotoxicity. These data suggest that TKIs may elicit different molecular mechanisms to those previously reported in the literature. Further research of these pathways is warranted in order to identify new, more effective strategies for cancer treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B262.