The effects of artesunate on the expression of EGFR and ABCG2 in A549 human lung cancer cells and a xenograft model

Abstract

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Clinical and laboratory studies have suggested that multi-targeting approaches against neoplastic cells could help to increase patient survival and might reduce the emergence of cells that are resistant to single-target inhibitors. Artesunate (ART) is one of the most potent and rapidly acting antimalarial agents known, and it also exerts a profound cytotoxic activity toward cancer cells and reverses multi-drug resistance. In the present study, we found that artesunate inhibited NSCLC A549 cell growth and proliferation, induced apoptosis and suppressed tumor growth in a dose-dependent manner in A549 cells and a mouse xenograft model. Furthermore, artesunate down-regulated the expression of epidermal growth factor receptor (EGFR), Akt and ATP-binding cassette subfamily G member 2 (ABCG2) at the mRNA and protein levels in vitro and in vivo. In conclusion, artesunate is an effective anti-cancer drug that may enhance the effectiveness of other anticancer drugs and may reverse multi-drug resistance by suppressing the transcription of ABCG2, which inhibits drug efflux.

Figure 1

Cytotoxicity of artesunate (ART) in human non-small cell lung cancer cell A549 cells. (A) The growth inhibition curves of A549 cells exposed to the indicated concentrations of ART for 24, 48, 72 and 96 h and the mean values from three independent cell viability MTT assays are depicted; (B) The percentage of apoptotic cells was determined by flow cytometric analysis following cell staining with Annexin V-FITC and PI. The cells were treated with varying concentrations of ART at 37 °C for 48 h. The data shown represent the means ± SE obtained from three independent experiments. * p < 0.05 compared with the control group.

Figure 2

ART down-regulates the EGFR and ABCG2 mRNA and protein levels in A549 cells. (A) Cells were treated with varying concentrations of ART for 48 h and then harvested. The EGFR and ABCG2 mRNA levels were detected using real-time RT-PCR; (B) The expression of EGFR, p-EGFR Akt, p-Akt and ABCG2 was measured by western blot analysis. A549 cells were treated with varying concentrations of ART for 48 h, then β-actin expression was used as an internal control to determine the expression levels of proteins. Significant down-regulation of all proteins was observed. The blots are representative of three independent experiments. The data represent the means ± SE from three independent experiments performed in triplicate. * p < 0.05 compared with the control group.

Figure 3

ART suppresses tumor growth and EGFR and ABCG2 mRNA levels in A549 xenografts. (A) The effect of ART on the mean tumor volume in A549 human non-small cell lung cancer xenograft mice was explored. The mice were treated daily with oral doses of ART at 0, 60 and 120 mg·kg⁻¹ for 14 days. The tumor size was measured 3 times per week. The volumes presented are the means from 5 mice; (B) The body weights of the ART-treated mice were similar to that of saline controls; (C) The expression of EGFR and ABCG2 mRNA was measured in A549 xenografts. The mice were administered daily oral doses of 60 mg·kg⁻¹ ART, 120 mg·kg⁻¹ ART or distilled water. The EGFR and ABCG2 mRNA expression levels were measured using real-time RT-PCR. * p < 0.05 compared with the control group.

Figure 4

The expression of ABCG2, Akt, p-Akt and p-EGFR proteins was examined in untreated and ART-treated (60 mg·kg⁻¹ and 120 mg·kg⁻¹) xenografts. Photographs of immunohistochemical staining for ABCG2, Akt, p-Akt and p-EGFR (×40) are shown. A distinct yellow stain can be observed in the membrane and cytoplasm. The images presented are representative of three independent experiments. The data are the means ± SE from three independent experiments performed in triplicate. * p < 0.05 compared with the control group. Bars, 100 μm.