GW583340

The overexpression of ATP binding cassette (ABC) transporters often leads to the development of multidrug resistance (MDR) and results in a suboptimal response to chemotherapy. Previously, we reported that lapatinib (GW572016), a human epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinase inhibitor (TKI), significantly reverses MDR in cancer cells by blocking the efflux function of ABC subfamily B member 1 (ABCB1) and ABC subfamily G member 2 (ABCG2). In the present study, we conducted in vitro experiments to evaluate if GW583340 and GW2974, structural analogues of lapatinib, could reverse ABCB1- and ABCG2-mediated MDR. Our results showed that GW583340 and GW2974 significantly sensitized ABCB1 and ABCG2 overexpressing MDR cells to their anticancer substrates. GW583340 and GW2974 significantly increased the intracellular accumulation of [(3)H]-paclitaxel in ABCB1 overexpressing cells and [(3)H]-mitoxantrone in ABCG2 overexpressing cells respectively. In addition, GW583340 and GW2974 significantly inhibited ABCG2-mediated transport of methotrexate in ABCG2 overexpressing membrane vesicles. There was no significant change in the expression levels of ABCB1 and ABCG2 in the cell lines exposed to 5μM of either GW583340 or GW2974 for 3 days. In addition, a docking model predicted the binding conformation of GW583340 and GW2974 to be within the transmembrane region of homology modeled human ABCB1 and ABCG2. We conclude that GW583340 and GW2974, at clinically achievable plasma concentrations, reverse ABCB1- and ABCG2-mediated MDR by blocking the drug efflux function of these transporters. These findings may be useful in developing combination therapy for cancer treatment with EGFR TKIs.

Overexpression of epidermal growth factor receptors (ErbB) is frequently seen in inflammatory breast cancer (IBC). Treatment with ErbB1/2-targeting agents (lapatinib) mediates tumor apoptosis by downregulating ErbB1/2 phosphorylation and downstream survival signaling. In this study, using carboxy-H(2)DCFDA, DHE, and MitoSOX Red to examine changes in hydrogen peroxide radicals, cytoplasmic and mitochondrial superoxide, respectively, we observed that GW583340 (a lapatinib-analog) increases reactive oxygen species (ROS) in two models of IBC (SUM149, SUM190) that are sensitive to ErbB1/2 blockade. This significant increase in ROS levels was similar to those generated by classical oxidative agents H(2)O(2) and paraquat. In contrast, minimal to basal levels of ROS were measured in a clonal population of GW583340-resistant IBC cells (rSUM149 and rSUM190). The GW583340-resistant IBC cells displayed increased SOD1, SOD2, and glutathione expression, which correlated with decreased sensitivity to the apoptotic-inducing effects of GW583340, H(2)O(2), and paraquat. The ROS increase and cell death in the GW583340-sensitive cells was reversed by simultaneous treatment with a superoxide dismutase (SOD) mimic. Additionally, overcoming the high levels of antioxidants using redox modulators induced apoptosis in the GW583340-resistant cells. Taken together, these data demonstrate a novel mechanism of lapatinib-analog-induced apoptosis and indicate that resistant cells have increased antioxidant potential, which can be overcome by treatment with SOD modulators.