Etomidate inhibits cell proliferation and induces apoptosis in A549 non-small cell lung cancer cells via downregulating WWP2

Abstract

Etomidate (ETO) is a commonly used intravenous anesthetic that has been reported to exert a tumor suppressive effect in several types of cancer. The present study aimed to investigate the effect of ETO on cell proliferation and apoptosis in non-small cell lung cancer (NSCLC) cells and elucidate its potential mechanism of action. Therefore, Cell Counting Kit-8 assay was performed to evaluate the effect of different concentrations of ETO (0, 1, 2 or 3 µg/ml) on A549 cell viability. In addition, the possible interaction between ETO and WW domain containing E3 ubiquitin protein ligase 2 (WWP2) was predicted using the STITCH database. Additionally, a stable WWP2-overexpressing A549 cell line was constructed by transfecting A549 cells with the pcDNA3.1-WWP2 plasmid. Cell proliferation and apoptosis were assessed using colony formation and TUNEL assays, respectively. The mRNA and protein expression levels of the apoptosis-related proteins Bcl-2, Bax, caspase 3 and cleaved-caspase 3 were determined by reverse transcription-quantitative PCR and western blotting. In addition, the expression and phosphorylation levels of proliferation-associated genes (PCNA and Ki-67) and proteins in the PI3K/Akt pathway were analyzed by western blotting. The results showed that treatment with ETO attenuated the cell viability and proliferation of A549 cells. ETO also promoted cell apoptosis and decreased the expression of the anti-apoptotic protein Bcl-2, whilst increasing that of pro-apoptotic proteins Bax and cleaved caspase 3 in a dose-dependent manner. Furthermore, ETO was found to negatively regulate the expression of WWP2, such that WWP2 overexpression reversed the potentiating effects of ETO on cell apoptosis. In addition, ETO promoted the expression of PTEN and reduced the phosphorylation levels of the PI3K/AKT pathway-related proteins. These effects aforementioned could also be reversed by WWP2 overexpression. Therefore, data from the present study suggest that ETO can attenuate the progression of NSCLC through by the PI3K/AKT pathway, specifically by targeting WWP2. These findings may provide a novel target for the treatment of NSCLC.

Keywords: WW domain-containing E3 ubiquitin protein ligase 2; apoptosis; etomidate; non-small cell lung cancer; proliferation.

Figure 1

ETO attenuates A549 cell proliferation. (A) BESA-2B cells were treated with different concentrations of ETO (0, 1, 2 or 3 µg/ml) for 24 h, before cell viability was measured by Cell Counting Kit-8 assay. (B) A549 cells were treated with different concentrations of ETO (0, 1, 2 or 3 µg/ml) for 24 h before cell viability was measured the Cell Counting Kit-8 assay. (C) mRNA and (D) protein expression levels of Ki67 and PCNA in A549 cells were detected by reverse transcription-quantitative PCR and western blotting assays, respectively. (E) Cell proliferation was assessed using colony formation assay. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. 0 µg/ml ETO. ETO, etomidate; PCNA, proliferating cell nuclear antigen.

Figure 2

ETO induces apoptosis in A549 cells. (A) A549 cells were treated with different concentrations of ETO (0, 1, 2 or 3 µg/ml) for 24 h, before cell apoptosis was evaluated by TUNEL assay (magnification, x200), (B) the results of which were quantified. (C) mRNA expression levels of Bcl-2 and Bax were determined by reverse transcription-quantitative PCR. (D) Protein expression levels of Bcl-2, Bax, cleaved caspase 3 and caspase 3 were detected by western blot analysis. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. 0 µg/ml ETO group. ETO, etomidate.

Figure 3

WWP2 overexpression abrogates the inhibitory effects of ETO on A549 cell proliferation. (A) The interaction between ETO and WWP2 was predicted using the STITCH database. A549 cells were treated with 3 µg/ml ETO for 24 h, before the (B) mRNA and (C) protein expression levels of WWP2 were measured by RT-qPCR and western blot analyses, respectively. (D and E) A549 cells were transfected with pcDNA-WWP2 to overexpress WWP2. (D) The mRNA and (E) protein expression levels of WWP2 were measured by RT-qPCR and western blot analyses, respectively. (F-I) A549 cells overexpressing or not overexpressing WWP2 were treated with 3 µg/ml ETO for 24 h. (F) Cell viability was assessed using the Cell Counting Kit-8 assay. (G) mRNA and (H) protein levels of Ki67 and PCNA were determined by RT-qPCR and western blot assays, respectively. (I) Cell proliferation was assessed and quantified by colony formation assay. ^**P<0.01 and ^***P<0.001 vs. Control; ^##P<0.01 and ^###P<0.001 vs. ETO + ov-NC. WWP2, WW domain containing E3 ubiquitin protein ligase 2; ETO, etomidate; PCNA, proliferating cell nuclear antigen; ov-NC, overexpression with negative control vector; RT-qPCR, reverse transcription-quantitative PCR.

Figure 4

WWP2 overexpression abrogates the potentiating effects of ETO on A549 cell apoptosis. A549 cells overexpressing or not WWP2 were treated with 3 µg/ml ETO for 24 h. (A) Cell apoptosis was evaluated by TUNEL assay (Magnification, x200), (B) which was quantified. (C) mRNA levels of Bcl-2 and Bax were detected by reverse transcription-quantitative PCR. (D) Protein expression levels of Bcl-2, Bax, cleaved caspase 3 and caspase 3 were determined by western blot analysis. ^***P<0.001 vs. Control. ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. ETO + ov-NC. WWP2, WW domain containing E3 ubiquitin protein ligase 2; ETO, etomidate; ov-NC, overexpression with negative control vector.

Figure 5

ETO upregulates PTEN and inhibits the activation of the PI3K/ATK pathway, which were reversed by WWP2 overexpression. A549 cells overexpressing or not overexpressing WWP2 were treated with 3 µg/ml ETO for 24 h. The (A) mRNA and (B) protein expression levels of PTEN were determined by reverse transcription-quantitative PCR and western blot analysis, respectively. (C) Protein levels of p-AKT/AKT and PI3K were detected by western blot analysis. ^***P<0.001 vs. Control. ^###P<0.001 vs. ETO + ov-NC. ETO, etomidate; WWP2, WW domain containing E3 ubiquitin protein ligase 2; ov-NC, overexpression with negative control vector.