Ubiquitin ligase Cbl-b is involved in icotinib (BPI-2009H)-induced apoptosis and G1 phase arrest of EGFR mutation-positive non-small-cell lung cancer

Abstract

Epidermal growth factor receptor (EGFR) is one of the most promising targets for non-small-cell lung cancer (NSCLC). Icotinib, a highly selective EGFR tyrosine kinase inhibitor (EGFR-TKI), has shown promising clinical efficacy and safety in patients with NSCLC. The exact molecular mechanism of icotinib remains unclear. In this study, we first investigated the antiproliferative effect of icotinib on NSCLC cells. Icotinib significantly inhibited proliferation of the EGFR-mutated lung cancer HCC827 cells. The IC50 values at 48 and 72 h were 0.67 and 0.07 μ M, respectively. Flow cytometric analysis showed that icotinib caused the G1 phase arrest and increased the rate of apoptosis in HCC827 cells. The levels of cyclin D1 and cyclin A2 were decreased. The apoptotic process was associated with activation of caspase-3, -8, and poly(ADP-ribose) polymerase (PARP). Further study revealed that icotinib inhibited phosphorylation of EGFR, Akt, and extracellular signal-regulated kinase. In addition, icotinib upregulated ubiquitin ligase Cbl-b expression. These observations suggest that icotinib-induced upregulation of Cbl-b is responsible, at least in part, for the antitumor effect of icotinib via the inhibition of phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways in EGFR-mutated NSCLC cells.

Figure 1

Identification of EGFR mutation in HCC827 and A549 cells. Exon 19 del mutation was detected in the HCC827 cell line.

Figure 2

Icotinib inhibited human NSCLC cell proliferation. HCC827 and A549 cells were exposed to icotinib (1 nM–100 μM) for 48 h, and cell viability was determined by MTT assay. Data are the mean ± SD of at least three independent experiments performed in triplicate.

Figure 3

Icotinib-induced G1 phase cell cycle arrest in HCC827 cells. (a) Cells were exposed to 0.01 and 0.1 μmol/L icotinib for 24 h, and the cell cycle was analyzed by flow cytometry after staining with propidium iodide. (b) Expression of cell cycle proteins cyclins D1, A, and E was analyzed by Western blotting. β-Actin was used as the internal control. Data are representative of one of three independent experiments.

Figure 4

Icotinib-induced apoptosis in HCC827 cells. (a) and (b) Cells were exposed to 0.01 and 0.1 μmol/L icotinib for 24 h, and the cell cycle was analyzed by flow cytometry after staining with propidium iodide. (c) Expression of PARP and caspase-3 and -8 was analyzed by Western blotting. β-Actin was used as the internal control. Data are representative of one of three independent experiments.

Figure 5

Icotinib regulated phosphorylation of EGFR, Akt, and ERK. Western blot analysis of EGFR, Akt, and ERK protein expression in HCC827 cells treated with 0.01–1 μmol/L icotinib. β-Actin was used as the internal control.

Figure 6

Icotinib upregulated expression of Cbl-b. Cells were exposed to 0.01–1 μmol/L icotinib for 24 h, and expression of Cbl-b proteins was analyzed by Western blotting. β-Actin was used as the internal control.