Depleted aldehyde dehydrogenase 1A1 (ALDH1A1) reverses cisplatin resistance of human lung adenocarcinoma cell A549/DDP

Abstract

Background: Cisplatin is the standard first-line chemotherapeutic agent for the treatment of non-small cell lung cancer (NSCLC). However, resistance to chemotherapy has been a major obstacle in the management of NSCLC. Aldehyde dehydrogenase 1A1 (ALDH1A1) overexpression has been observed in a variety of cancers, including lung cancer. The purpose of this study was to investigate the effect of ALDH1A1 expression on cisplatin resistance and explore the mechanism responsible.

Methods: Reverse transcriptase-PCR was applied to measure the messenger RNA expression of ALDH1A1, while Western blot assay was employed to evaluate the protein expression of ALDH1A1, B-cell lymphoma 2, Bcl-2-like protein 4, phospho-protein kinase B (p-AKT) and AKT. A short hairpin RNA was used to knockdown ALDH1A1 expression. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the effect of ALDH1A1 decrease on cell viability. The cell apoptotic rate was tested using flow cytometry assay.

Results: ALDH1A1 is overexpressed in cisplatin resistant cell line A549/DDP, compared with A549. ALDH1A1 depletion significantly decreased A549/DDP proliferation, increased apoptosis, and reduced cisplatin resistance. In addition, the phosphoinositide 3-kinase (PI3K) / AKT pathway is activated in A549/DDP, and ALDH1A1 knockdown reduced the phosphorylation level of AKT. Moreover, the combination of ALDH1A1-short hairpin RNA and PI3K/AKT pathway inhibitor LY294002 markedly inhibited cell viability, enhanced apoptotic cell death, and increased cisplatin sensitivity.

Conclusion: These results suggest that ALDH1A1 depletion could reverse cisplatin resistance in human lung cancer cell line A549/DDP, and may act as a potential target for the treatment of lung cancers resistant to cisplatin.

Keywords: Aldehyde dehydrogenase 1A1; apoptosis; cisplatin resistance; non-small cell lung cancer; proliferation.

Figure 1

Inhibitory concentration (IC)50 and aldehyde dehydrogenase 1A1 (ALDH1A1) expression level of cisplatin resistant cells, A549/DDP, and its parent cells, A549. (a) A549 and A549/DDP were treated with various concentrations of cisplatin (0, 20, 40, 80, 160, 320 μM). (b) IC50 of A549 and A549/DDP to cisplatin. (c) Reverse transcriptase‐qualitative PCR was used to explore ALDH1A1 messenger RNA expression levels in A549, A549/DDP, PC9 and PC9/GR cells; β‐actin was used as an internal control. (d) Western blot to evaluate ALDH1A1 protein expression in A549 and A549/DDP cells; β‐actin was used as an internal control. P < 0.05 was considered significant (*P < 0.05, **P < 0.01).

Figure 2

Aldehyde dehydrogenase 1A1 (ALDH1A1)‐short hairpin (sh)RNA inhibits ALDH1A1 expression, cell proliferation, and cisplatin resistance in A549/DDP cells. (a) Cells were collected for reverse transcriptase‐qualitative PCR to test ALDH1A1 expression. (b) 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay to measure cell proliferation ability. (c) MTT assay to evaluate the effect of ALDH1A1 decrease on cisplatin resistance. (d) Western blot assays to detect B‐cell lymphoma 2 (BCL‐2), Bcl‐2‐like protein 4 (BAX), phospho‐protein kinase B (p‐AKT), AKT and ALDH1A1 expression. (e) Inhibitory concentration (IC)50 values of cells transfected with scramble shRNA or shRNA. (f) Cell viability markedly declined when treated with LY294002. (#, compared with scramble shRNA group; &, compared with shRNA + dimethyl sulfoxide (DMSO) group; #, & P < 0.05; ##, && P < 0.01.)

Figure 3

The phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT) signal pathway is associated with cisplatin resistance and aldehyde dehydrogenase 1A1 (ALDH1A1) knockdown could inhibit AKT phosphorylation. (a,b) 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay to measure the effect of LY294002 on cisplatin sensitivity and inhibitory concentration (IC)50 of A549/DDP cells with decreased ALDH1A1 expression. (c,d) Flow cytometry analysis of non‐treatment, scramble short hairpin (sh)RNA, shRNA, shRNA + dimethyl sulfoxide (DMSO), shRNA + LY group. (#, compared with scramble shRNA group; &, compared with shRNA + DMSO group; #, & P < 0.05; ##, && P < 0.01.)