β-elemene enhances the antitumor activity of erlotinib by inducing apoptosis through AMPK and MAPK pathways in TKI-resistant H1975 lung cancer cells

Abstract

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) significantly improve the outcome of non-small-cell lung cancer (NSCLC) patients with EGFR mutations, however, most TKI-treated patients will develop resistance to TKIs. β-elemene, extracted from Curcuma aromatica Salisb., has been widely used to treat various malignant tumors, including TKI-resistant NSCLC, but, the effects and the molecular mechanisms remain unclear. In this study, the NCI-H1975 cell line harboring double mutations L858R/T790M was treated with varying concentrations of β-elemene and/or erlotinib. The effects of β-elemene on cell proliferation, migration, apoptosis, and the expression of relevant proteins of NCI-H1975 cells were evaluated. The results revealed that β‑elemene significantly inhibited the growth, colony formation capacity, wound healing ability of NCI-H1975 cells, and improved the sensitivity of NCI-H1975 cells to erlotinib. Compared with erlotinib alone, β-elemene plus erlotinib significantly promoted the apoptosis of NCI-H1975 cells, accompanied by the down-regulated expression of P-mTOR, P-EGFR, CHOP proteins and up-regulated expression of P-AMPKα and Bax proteins. Taken together, these findings demonstrate that β-elemene suppresses the proliferation and migration of TKI-resistant H1975 cells, and enhances the antitumor activity of erlotinib by inducing apoptosis through AMPK and MAPK pathways in TKI-resistant H1975 lung cancer cells, indicating that β-elemene is a promising anti-cancer therapeutic candidate for TKI-resistant NSCLC.

Keywords: AMPK; EGFR-mutated; NSCLC; TKI-resistant; apoptosis; mechanisms; β-elemene.

Figure 1

The chemical structure of β-elemene.

Figure 2

(A) Time- and dose-dependent growth inhibitory effects of β-elemene on NCI-H1975 cells. The cells were incubated with β-elemene for 24, 48, and 72 hrs, and the cell viability was determined by MTT assay. Data are representative of at least three independent experiments and expressed as mean ± SD; *p < 0.05, **p < 0.01, versus the cells treated for 48 hrs or 72 hrs; ^#p < 0.05, ^##p < 0.01, versus the cells treated for 48 hrs. (B) Time- and dose-dependent growth inhibitory effects of erlotinib on NCI-H1975 cells. The cells were incubated with β-elemene for 24, 48, and 72 hrs, and the cell viability was determined by MTT assay. Data are representative of at least three independent experiments and expressed as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. (C) Compared with β-elemene alone, erlotinib combined with β-elemene significantly inhibited the growth of NCI-H1975 cells. The cells were incubated with indicated concentrations of β-elemene with or without 2 μmol/L erlotinib for 72 hrs, and the cell viability was determined by MTT assay. Data are representative of at least three independent experiments and expressed as mean ± SD; *p < 0.05, **p < 0.01, erlotinib plus β-elemene versus β-elemene alone. (D) β-elemene significantly increased the sensitivity of NCI-H1975 cells to erlotinib. The cells were incubated with indicated concentrations of erlotinib with or without 30 µg/mL β-elemene for 72 hrs, and the cell viability was determined by MTT assay. Data are representative of at least three independent experiments and expressed as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, erlotinib plus β-elemene versus erlotinib alone.

Figure 3

Scratch wound healing assay of NCI-H1975 cells treated with β-elemene (0, 10, 20, 30, 40 and 50 µg/mL) and monitored for 24 hrs.

Figure 4

Colony formation of NCI-H1975 cells treated with β-elemene (0, 20, 40, 60, and 80 µg/mL) and monitored for 14 days. Representative photomicrographs of crystal violet-stained colonies were depicted.

Figure 5

(A) NCI-H1975 cells were treated with β-elemene (0,10,20,30,40,50 µg/mL) for 24 hrs. (B) NCI-H1975 cells were treated with β-elemene (30 µg/mL) and/or Erlotinib (2 µmol/L) for 24 hrs. The expression of proteins was detected by western blot. Each experiment was repeated at least three times. Results are expressed as mean ± SD (n=3, * p <0.05, ** p <0.01, *** p <0.001).

Figure 6

(A) NCI-H1975 cells were treated with β-elemene at different concentrations (0, 20, 30, 40, and 50 µg/mL) for 24 hrs. Cell apoptosis was measured by flow cytometry using Annexin V-FITC/PI staining. (B) Statistical analysis of the cell apoptosis rate at 24 hrs. All data are presented as mean ±SD (n=3, * p <0.05, ** p <0.01, *** p <0.001). (C) Annexin V-FITC/ PI staining and flow cytometry analysis of the effect of β-elemene on apoptosis of NCI-H1975 cells. (D)NCI-H1975 cells were treated with β-elemene (30 µg/mL) and/or erlotinib (2 µmol/L) for 24 hrs. The expression of proteins was detected by Western blot. FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 7

NCI-H1975 cells were treated with β-elemene (30 µg/mL) and/or Erlotinib (2 µmol/L) for 24 hrs. The expression of CHOP and Bax were detected by Western blot. Each experiment was repeated at least three times. Results are expressed as mean ± SD (n=3, * p <0.05, ** p <0.01, *** p <0.001).