Interferon-α reduces the gefitinib sensitivity of human non-small cell lung cancer

Abstract

Aim of the study: Many studies have shown that interferon-α (IFN-α) enhances the antiproliferative effect of gefitinib in some solid tumours. We aimed to determine the effect of combining IFN-α with gefitinib in human non-small cell lung cancer (NSCLC) cell lines (A549, H1299, HCC827) with different EGFR and K-Ras gene statuses.

Material and methods: An MTT assay was used to assess cell proliferation. Apoptosis was detected by an Annexin V/propidium iodide assay using flow cytometry, and western blotting was used to determine the expression of epidermal growth factor receptor/phosphorylated epidermal growth factor receptor (EGFR/p-EGFR) and signal transducers and activators of transcription 3/phosphorylated signal transducers and activators of transcription 3 (STAT3/p-STAT3).

Results: There was an additive interaction when gefitinib was combined with IFN-α in all cell lines; however, there was antagonism when gefitinib followed IFN-α pretreatment in three cell lines. Notably, IFN-α pretreatment significantly reduced the gefitinib sensitivity of HCC827 cells. Surprisingly, while IFN-α inhibited STAT3 phosphorylation in cell lines, gefitinib could do so.

Conclusions: The results might confirm the hypothesis that IFN-α induces gefitinib sensitivity of NSCLC, and IFN-α inhibits phosphorylation of STAT3, which may be dependent on EGFR signal activation playing a role in the reduction of gefitinib sensitivity after IFN-α treatment in NSCLC cell lines.

Keywords: STAT3; antagonism; gefitinib; interferon-α; non-small cell lung cancer.

Fig. 1

Evaluation of the antiproliferative effect of IFN-α in NSCLC cell lines. Cell growth is expressed as the percentage of control for each concentration point. Points, mean of quadruplicates; bars, SE. *, p < 0.05

Fig. 2

Additive/antagonistic antiproliferative effect of IFN-α/gefitinib combination on human NSCLC cell lines. A) Cell survival was determined by MTT assay. B) The CIs defining the growth inhibitory effects of the combination treatment of gefitinib and IFN-α. Combinations were antagonistic when CIs > 1.0, and additive when CIs = 0.9–1.0. Points, mean of three individual treatments. MT, mutation type

Fig. 3

IFN-α decreased antiproliferation of gefitinib. A) The antagonistic effect of IFN-α pretreatment on the antiproliferative effect of gefitinib on human NSCLC cell lines was evaluated by MTT assay. B) Apoptosis in the cell lines was analysed by flow cytometry for Annexin V/ PI staining. The results (top and bottom right panels) are expressed as the percentage of the apoptotic cells. Data are reported as the mean ± SE of three determinations, p < 0.05 for gefitinib vs. IFN-α→gefitinib

Fig. 4

IFN-α and gefitinib inhibit STAT3 activation. A) p-EGFR/EGFR and p-STAT3/STAT3 were analysed by western blot. The whole protein from H1299 and HCC827 cells treated for 48 hours with IFN-α (10⁴ IU/ml) and gefitinib (10 nM) alone with IFN-α was extracted. B) The expression levels were analysed by the ratio of optical density with β-actin. STAT3 activation was inhibited by IFN-α and gefitinib in both cell lines, p < 0.05